Question: 0-dimensional biomaterials show inductive properties.

Title: Electrical, optical, and structural properties of indium-tin-oxide thin films for organic light-emitting devices Content: High-quality indium–tin–oxide (ITO) thin films (200–850 nm) have been grown by pulsed laser deposition (PLD) on glass substrates without a postdeposition annealing treatment. The structural, electrical, and optical properties of these films have been investigated as a function of target composition, substrate deposition temperature, background gas pressure, and film thickness. Films were deposited from various target compositions ranging from 0 to 15 wt % of SnO2 content. The optimum target composition for high conductivity was 5 wt % SnO2+95 wt % In2O3. Films were deposited at substrate temperatures ranging from room temperature to 300 °C in O2 partial pressures ranging from 1 to 100 mTorr. Films were deposited using a KrF excimer laser (248 nm, 30 ns full width at half maximum) at a fluence of 2 J/cm2. For a 150-nm-thick ITO film grown at room temperature in an oxygen pressure of 10 mTorr, the resistivity was 4×10−4 Ω cm and the average transmission in the visible range (400–700 nm) was 85%. For a 170-n...

Title: Biological properties of herpes simplex virus 2 replication-defective mutant strains in a murine nasal infection model. Content: We used a mouse nasal model of herpes simplex virus 2 (HSV-2) infection to examine the biological properties of HSV-2 wild-type (wt), TK-negative, and replication-defective strains in vivo. Nasal septa tissue is the major site of wt viral replication post intranasal (i.n.) inoculation. The HSV-2 strain 186 syn(+)-1 wt virus caused lethal encephalitis at doses of 10(4) PFU and above per nostril, and at lower doses no neurons in the trigeminal ganglia were positive for the latency-associated transcript, indicating a lack of latent infection. The 186DeltaKpn TK-negative mutant virus replicated in nasal septa tissue but showed low-level replication in trigeminal ganglia at only one timepoint. In situ hybridization of trigeminal ganglia showed that the number of LAT-positive neurons was proportional to the inoculum dose from 10(3) to 10(6) PFU per nare. The replication-defective mutant virus 5BlacZ showed no replication in nasal septa tissue and no persistence of viral DNA at the inoculation site or the trigeminal ganglia. Nevertheless, inoculation of 5BlacZ or the double-mutant dl5-29 at distal sites reduced acute replication and latent infection of 186DeltaKpn following intranasal challenge. This infection model provides a biological system to test the properties of HSV-2 strains and shows that replication-defective mutant strains do not persist at sites of inoculation or in sensory ganglia but can induce immune protection that reduces the latent viral load of a challenge virus.

Title: A three-dimensional cell biology model of human hepatocellular carcinoma in vitro Content: We established an in vitro 3-D model of metastatic hepatocellular carcinoma (HCC) by culturing MHCC97H cells on molecular scaffolds within a rotating wall vessel bioreactor. Morphological and biochemical analyses revealed that the 3-D HCC model mirrored many clinical pathological features of HCC in vivo, including cancer cell morphology, tissue ultrastructure, protein production and secretion, glucose metabolism, tissue-specific gene expression, and apoptosis. Xenografts into livers of nude mice resulted in tumorigenesis and distant metastasis. This 3-D HCC spheroid is a promising model for HCC tumor biology, anticancer drug screening, and for the establishment of HCC animal models.

Title: PHENIX: a comprehensive Python-based system for macromolecular structure solution Content: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

Title: GSDS 2.0: an upgraded gene feature visualization server Content: UNLABELLED : Visualizing genes' structure and annotated features helps biologists to investigate their function and evolution intuitively. The Gene Structure Display Server (GSDS) has been widely used by more than 60 000 users since its first publication in 2007. Here, we reported the upgraded GSDS 2.0 with a newly designed interface, supports for more types of annotation features and formats, as well as an integrated visual editor for editing the generated figure. Moreover, a user-specified phylogenetic tree can be added to facilitate further evolutionary analysis. The full source code is also available for downloading. AVAILABILITY AND IMPLEMENTATION Web server and source code are freely available at http://gsds.cbi.pku.edu.cn. CONTACT gaog@mail.cbi.pku.edu.cn or gsds@mail.cbi.pku.edu.cn SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Title: Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis. Content: The recent development of microarray technology has led statisticians and bioinformaticians to develop new statistical methodologies for comparing different biological samples. The objective is to identify a small number of differentially expressed genes from among thousands. In quantitative proteomics, analysis of protein expression using two-dimensional gel electrophoresis shows some similarities with transcriptomic studies. Thus, the goal of this study was to evaluate different data analysis methodologies widely used in array analysis using different proteomic data sets of hundreds of proteins. Even with few replications, the significance analysis of microarrays method appeared to be more powerful than the Student's t test in truly declaring differentially expressed proteins. This procedure will avoid wasting time due to false positives and losing information with false negatives.

Title: Large deformation of red blood cell ghosts in a simple shear flow. Content: Red blood cells are known to change shape in response to local flow conditions. Deformability affects red blood cell physiological function and the hydrodynamic properties of blood. The immersed boundary method is used to simulate three-dimensional membrane-fluid flow interactions for cells with the same internal and external fluid viscosities. The method has been validated for small deformations of an initially spherical capsule in simple shear flow for both neo-Hookean and the Evans-Skalak membrane models. Initially oblate spheroidal capsules are simulated and it is shown that the red blood cell membrane exhibits asymptotic behavior as the ratio of the dilation modulus to the extensional modulus is increased and a good approximation of local area conservation is obtained. Tank treading behavior is observed and its period calculated.

Title: Periosteal bone formation--a neglected determinant of bone strength. Content: Life forms that have low body mass can hunt for food on the undersurface of branches or along shear cliff faces quite unperturbed by gravity. For larger animals, the hunt for dinner and the struggle to avoid becoming someone else's meal require rapid movement against gravity. This need is met by the lever function of long bones, three-dimensional masterpieces of biomechanical engineering that, by their material composition and structural design, achieve the contradictory properties of stiffness and flexibility, strength and lightness.1 Material stiffness results from the encrusting of the triple-helical structure of collagen type I with hydroxyapatite crystals, which confers . . .

Title: Collagen/Polypropylene composite mesh biocompatibility in abdominal wall reconstruction. Content: BACKGROUND Intraperitoneal placement of polypropylene mesh leads to extensive visceral adhesions and is contraindicated. Different coatings are used to improve polypropylene mesh properties. Collagen is a protein with unique biocompatibility and cell ingrowth enhancement potential. A novel acetic acid extracted collagen coating was developed to allow placement of polypropylene mesh in direct contact with viscera. The authors' aim was to evaluate the long-term influence of acetic acid extracted collagen coating on surgical aspects and biomechanical properties of polypropylene mesh implanted in direct contact with viscera, including complications, adhesions with viscera, strength of incorporation, and microscopic inflammatory reaction. METHODS Forty adult Wistar rats were divided into two groups: experimental (polypropylene mesh/acetic acid extracted collagen coating) and control (polypropylene mesh only). Astandardized procedure of mesh implantation was performed. Animals were killed 3 months after surgery and analyzed for complications, mesh area covered by adhesions, type of adhesions, strength of incorporation, and intensity of inflammatory response. RESULTS The mean adhesion area was lower for polypropylene mesh/acetic acid extracted collagen coating (14.5 percent versus 69.9 percent, p < 0.001). Adhesion severity was decreased in the experimental group: grades 0 and 1 were more frequent (p < 0.04 and p < 0.002, respectively) and grade 3 was less frequent (p < 0.0001). An association between adhesion area and severity was found (p < 0.0001). Complications, strength of incorporation, and intensity of inflammatory response to the mesh were similar. CONCLUSIONS Visceral adhesions to polypropylene mesh are significantly reduced because of acetic acid extracted collagen coating. The collagen coating does not increase complications or induce alterations of polypropylene mesh incorporation.

Title: KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases Content: High-throughput experimental technologies often identify dozens to hundreds of genes related to, or changed in, a biological or pathological process. From these genes one wants to identify biological pathways that may be involved and diseases that may be implicated. Here, we report a web server, KOBAS 2.0, which annotates an input set of genes with putative pathways and disease relationships based on mapping to genes with known annotations. It allows for both ID mapping and cross-species sequence similarity mapping. It then performs statistical tests to identify statistically significantly enriched pathways and diseases. KOBAS 2.0 incorporates knowledge across 1327 species from 5 pathway databases (KEGG PATHWAY, PID, BioCyc, Reactome and Panther) and 5 human disease databases (OMIM, KEGG DISEASE, FunDO, GAD and NHGRI GWAS Catalog). KOBAS 2.0 can be accessed at http://kobas.cbi.pku.edu.cn.

Title: Microenvironment rigidity modulates responses to the HER2 receptor tyrosine kinase inhibitor lapatinib via YAP and TAZ transcription factors. Content: Stiffness is a biophysical property of the extracellular matrix that modulates cellular functions, including proliferation, invasion, and differentiation, and it also may affect therapeutic responses. Therapeutic durability in cancer treatments remains a problem for both chemotherapies and pathway-targeted drugs, but the reasons for this are not well understood. Tumor progression is accompanied by changes in the biophysical properties of the tissue, and we asked whether matrix rigidity modulated the sensitive versus resistant states in HER2-amplified breast cancer cell responses to the HER2-targeted kinase inhibitor lapatinib. The antiproliferative effect of lapatinib was inversely proportional to the elastic modulus of the adhesive substrata. Down-regulation of the mechanosensitive transcription coactivators YAP and TAZ, either by siRNA or with the small-molecule YAP/TEAD inhibitor verteporfin, eliminated modulus-dependent lapatinib resistance. Reduction of YAP in vivo in mice also slowed the growth of implanted HER2-amplified tumors, showing a trend of increasing sensitivity to lapatinib as YAP decreased. Thus we address the role of stiffness in resistance to and efficacy of a HER2 pathway-targeted therapeutic via the mechanotransduction arm of the Hippo pathway.

Title: Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Content: We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.

Title: Noninvasive Imaging beyond the Diffraction Limit of 3D Dynamics in Thickly Fluorescent Specimens Content: Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

Title: Characterization of biochemical properties of Bacillus subtilis RecQ helicase. Content: RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability.

Title: Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles Content: Although humans have been exposed to airborne nanosized particles (NSPs; < 100 nm) throughout their evolutionary stages, such exposure has increased dramatically over the last century due to anthropogenic sources. The rapidly developing field of nanotechnology is likely to become yet another source through inhalation, ingestion, skin uptake, and injection of engineered nanomaterials. Information about safety and potential hazards is urgently needed. Results of older biokinetic studies with NSPs and newer epidemiologic and toxicologic studies with airborne ultrafine particles can be viewed as the basis for the expanding field of nanotoxicology, which can be defined as safety evaluation of engineered nanostructures and nanodevices. Collectively, some emerging concepts of nanotoxicology can be identified from the results of these studies. When inhaled, specific sizes of NSPs are efficiently deposited by diffusional mechanisms in all regions of the respiratory tract. The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph circulation to reach potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and heart. Access to the central nervous system and ganglia via translocation along axons and dendrites of neurons has also been observed. NSPs penetrating the skin distribute via uptake into lymphatic channels. Endocytosis and biokinetics are largely dependent on NSP surface chemistry (coating) and in vivo surface modifications. The greater surface area per mass compared with larger-sized particles of the same chemistry renders NSPs more active biologically. This activity includes a potential for inflammatory and pro-oxidant, but also antioxidant, activity, which can explain early findings showing mixed results in terms of toxicity of NSPs to environmentally relevant species. Evidence of mitochondrial distribution and oxidative stress response after NSP endocytosis points to a need for basic research on their interactions with subcellular structures. Additional considerations for assessing safety of engineered NSPs include careful selections of appropriate and relevant doses/concentrations, the likelihood of increased effects in a compromised organism, and also the benefits of possible desirable effects. An interdisciplinary team approach (e.g., toxicology, materials science, medicine, molecular biology, and bioinformatics, to name a few) is mandatory for nanotoxicology research to arrive at an appropriate risk assessment.

Title: Nonlinear Elasticity in Biological Gels Content: Unlike most synthetic materials, biological materials often stiffen as they are deformed. This nonlinear elastic response, critical for the physiological function of some tissues, has been documented since at least the 19th century, but the molecular structure and the design principles responsible for it are unknown. Current models for this response require geometrically complex ordered structures unique to each material. In this Article we show that a much simpler molecular theory accounts for strain stiffening in a wide range of molecularly distinct biopolymer gels formed from purified cytoskeletal and extracellular proteins. This theory shows that systems of semi-flexible chains such as filamentous proteins arranged in an open crosslinked meshwork invariably stiffen at low strains without the need for a specific architecture or multiple elements with different intrinsic stiffnesses.

Title: Getting started with yeast. Content: Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

Title: Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Content: Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 12 μm).

Title: Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties. Content: It has been proposed that epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features, and that the presence of EMT characteristics in claudin-low breast tumors reveals their origin in basal stem cells. It remains to be determined, however, whether EMT is an inherent property of normal basal stem cells, and if the presence of a mesenchymal-like phenotype is required for the maintenance of all their stem cell properties. We used nontumorigenic basal cell lines as models of normal stem cells/progenitors and demonstrate that these cell lines contain an epithelial subpopulation ("EpCAM+," epithelial cell adhesion molecule positive [EpCAM(pos)]/CD49f(high)) that spontaneously generates mesenchymal-like cells ("Fibros," EpCAM(neg)/CD49f(med/low)) through EMT. Importantly, stem cell/progenitor properties such as regenerative potential, high aldehyde dehydrogenase 1 activity, and formation of three-dimensional acini-like structures predominantly reside within EpCAM+ cells, while Fibros exhibit invasive behavior and mammosphere-forming ability. A gene expression profiling meta-analysis established that EpCAM+ cells show a luminal progenitor-like expression pattern, while Fibros most closely resemble stromal fibroblasts but not stem cells. Moreover, Fibros exhibit partial myoepithelial traits and strong similarities with claudin-low breast cancer cells. Finally, we demonstrate that Slug and Zeb1 EMT-inducers control the progenitor and mesenchymal-like phenotype in EpCAM+ cells and Fibros, respectively, by inhibiting luminal differentiation. In conclusion, nontumorigenic basal cell lines have intrinsic capacity for EMT, but a mesenchymal-like phenotype does not correlate with the acquisition of global stem cell/progenitor features. Based on our findings, we propose that EMT in normal basal cells and claudin-low breast cancers reflects aberrant/incomplete myoepithelial differentiation.

Title: On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis. Content: Mesenchymal stem cells (MSCs) display substantial cell-to-cell variation. This heterogeneity manifests among donors, among tissue sources, and within cell populations. Such pervasive variability complicates the use of MSCs in regenerative applications and may limit their therapeutic efficacy. Most conventional assays measure MSC properties in bulk and, as a consequence, mask this cell-to-cell variation. Recent studies have identified extensive variability amongst and within clonal MSC populations, in dimensions including functional differentiation capacity, molecular state (e.g. epigenetic, transcriptomic, and proteomic status), and biophysical properties. While the origins of these variations remain to be elucidated, potential mechanisms include in vivo micro-anatomical heterogeneity, epigenetic bistability, and transcriptional fluctuations. Emerging tools for single cell analysis of MSC gene and protein expression may yield further insight into the mechanisms and implications of single cell variation amongst these cells, and ultimately improve the clinical utility of MSCs in tissue engineering and regenerative medicine applications. This review outlines the dimensions across which MSC heterogeneity is present, defines some of the known mechanisms that govern this heterogeneity, and highlights emerging technologies that may further refine our understanding and improve our clinical application of this unique cell type.

Title: The effects of prion protein proteolysis and disaggregation on the strain properties of hamster scrapie. Content: Native mammalian prions exist in self-propagating strains that exhibit distinctive clinical, pathological and biochemical characteristics. Prion strain diversity is associated with variations in PrP(Sc) conformation, but it remains unknown precisely which physical properties of the PrP(Sc) molecules are required to encipher mammalian prion strain phenotypes. In this study, we subjected prion-infected brain homogenates derived from three different hamster scrapie strains to either (i) proteinase K digestion or (ii) sonication, and inoculated the modified samples into normal hamsters. The results show that the strain-specific clinical features and neuropathological profiles of inoculated animals were not affected by either treatment. Similarly, the strain-dependent biochemical characteristics of the PrP(Sc) molecules (including electrophoretic mobility, glycoform composition, conformational stability and susceptibility to protease digestion) in infected animals were unaffected by either proteolysis or sonication of the original inocula. These results indicate that the infectious strain properties of native prions do not appear to be altered by PrP(Sc) disaggregation, and that maintenance of such properties does not require the N-domain (approximately residues 23-90) of the protease-resistant PrP(Sc) molecules or protease-sensitive PrP(Sc) molecules.

Title: In situ regulation of DC subsets and T cells mediates tumor regression in mice. Content: Vaccines are largely ineffective for patients with established cancer, as advanced disease requires potent and sustained activation of CD8(+) cytotoxic T lymphocytes (CTLs) to kill tumor cells and clear the disease. Recent studies have found that subsets of dendritic cells (DCs) specialize in antigen cross-presentation and in the production of cytokines, which regulate both CTLs and T regulatory (Treg) cells that shut down effector T cell responses. Here, we addressed the hypothesis that coordinated regulation of a DC network, and plasmacytoid DCs (pDCs) and CD8(+) DCs in particular, could enhance host immunity in mice. We used functionalized biomaterials incorporating various combinations of an inflammatory cytokine, immune danger signal, and tumor lysates to control the activation and localization of host DC populations in situ. The numbers of pDCs and CD8(+) DCs, and the endogenous production of interleukin-12, all correlated strongly with the magnitude of protective antitumor immunity and the generation of potent CD8(+) CTLs. Vaccination by this method maintained local and systemic CTL responses for extended periods while inhibiting FoxP3 Treg activity during antigen clearance, resulting in complete regression of distant and established melanoma tumors. The efficacy of this vaccine as a monotherapy against large invasive tumors may be a result of the local activity of pDCs and CD8(+) DCs induced by persistent danger and antigen signaling at the vaccine site. These results indicate that a critical pattern of DC subsets correlates with the evolution of therapeutic antitumor responses and provide a template for future vaccine design.

Title: Hypoxic heterogeneity in human tumors: EF5 binding, vasculature, necrosis, and proliferation. Content: We evaluated the levels and distribution of hypoxia in 31 human tumors using fluorescent immunohistochemical detection of binding by the 2-nitroimidazole, EF5. Hypoxia was found to be a heterogeneous property of human tumors. Necrosis was usually found adjacent to the highest level of binding in an individual patient's tumor. However, hypoxia often occurred without necrosis. In the group of tumors studied, the most common relationship between blood vessels (PECAM/CD31) and EF5 staining was consistent with diffusion-limited hypoxia; acute hypoxia occurred infrequently. Within a given patient's tumor, there was an inverse correlation between regions of proliferation (Ki-67) and regions of hypoxia. Again, however, when these parameters were examined in a group of patients, the absence of proliferation did not predict the presence of hypoxia. The relationships between hypoxia and other biologic endpoints are complex, but, within a given tumor's spatial relationships, they are in accord with known physiologic principles. Thus, our data emphasize that the relationships between hypoxia and other biologic parameters vary between patients. Necrosis, proliferation, and blood vessel distribution cannot predict the level or presence of hypoxia in an individual patient's tumor.

Title: Biochemical Properties of Highly Neuroinvasive Prion Strains Content: Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrP(Sc). Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrP(Sc) over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrP(Sc) over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS.

Title: Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures. Content: A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Title: Fructosamine Is a Useful Indicator of Hyperglycaemia and Glucose Control in Clinical and Epidemiological Studies – Cross-Sectional and Longitudinal Experience from the AMORIS Cohort Content: CONTEXT Fructosamine is a glycemic biomarker which may be useful for indication and control of diabetes respectively. OBJECTIVE The objective of the study was to evaluate fructosamine as an indicator of hyperglycaemia and glucose control in subjects with diabetes. DESIGN, SETTING & PATIENTS From the AMORIS cohort, subjects with serum glucose, fructosamine and HbA1c from the same examination were studied cross-sectionally and longitudinally (n = 10,987; 5,590 overnight-fasting). The guidelines of the American Diabetes Association were followed for classification of prediabetes and diabetes. Separate analyses were performed in patients with a newly detected or a known diagnosis of type 1 or type 2 diabetes respectively. RESULTS All three biomarkers were strongly correlated. With regard to the association between fructosamine and HbA1c Pearson linear correlation coefficients in the range of 0.67-0.75 were observed in fasting and non-fasting subjects with type 1 or type 2 diabetes. Analyses of glucose control in fasting patients with type 2 diabetes having all three biomarkers measured at three separate occasions within on average 290 days of the index examination showed similar trends over time for glucose, fructosamine and HbA1c. Discrimination of subjects with and without diabetes across the range of fructosamine levels was good (area under curve (AUC) 0.91-0.95) and a fructosamine level of 2.5 mmol/L classified subjects to diabetes with a sensitivity of 61% and a specificity of 97%. CONCLUSIONS Fructosamine is closely associated with HbA1c and glucose respectively and may be a useful biomarker of hyperglycaemia and glucose control in clinical and epidemiological studies.

Title: Microbial engineering for the production of advanced biofuels Content: Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure. However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism. Such engineering is not based on just one specific feedstock or host organism. Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production. Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.

Title: Human milk proresolving mediators stimulate resolution of acute inflammation Content: Human milk contains nutrients and bioactive products relevant to infant development and immunological protection. Here, we investigated the proresolving properties of milk using human milk lipid mediator isolates (HLMIs) and determined their impact on resolution programs in vivo and with human macrophages. HLMIs reduced the maximum neutrophil numbers (14.6±1.2 × 106–11.0±1.0 × 106 cells per exudate) and shortened the resolution interval (Ri; 50% neutrophil reduction) by 54% compared with peritonitis. Using rigorous liquid-chromatography tandem-mass spectrometry (LC-MS-MS)-based lipid mediator (LM) metabololipidomics, we demonstrated that human milk possesses a proresolving LM-specialized proresolving mediator (LM-SPM) signature profile, containing SPMs (e.g. resolvins (Rv), protectins (PDs), maresins (MaRs), and lipoxins (LXs)) at bioactive levels (pico-nanomolar concentrations) that enhanced human macrophage efferocytosis and bacterial containment. SPMs identified in human milk included D-series Rvs (e.g., RvD1, RvD2, RvD3, AT-RvD3, and RvD4), PD1, MaR1, E-series Rvs (e.g. RvE1, RvE2, and RvE3), and LXs (LXA4 and LXB4). Of the SPMs identified in human milk, RvD2 and MaR1 (50 ng per mouse) individually shortened Ri by ∼75%. Milk from mastitis gave higher leukotriene B4 and prostanoids and lower SPM levels. Taken together, these findings provide evidence that human milk has proresolving actions via comprehensive LM-SPM profiling, describing a potentially novel mechanism in maternal–infant biochemical imprinting.

Title: Interchangeability of Biosimilars: A European Perspective Content: Many of the best-selling ‘blockbuster’ biological medicinal products are, or will soon be, facing competition from similar biological medicinal products (biosimilars) in the EU. Biosimilarity is based on the comparability concept, which has been used successfully for several decades to ensure close similarity of a biological product before and after a manufacturing change. Over the last 10 years, experience with biosimilars has shown that even complex biotechnology-derived proteins can be copied successfully. Most best-selling biologicals are used for chronic treatment. This has triggered intensive discussion on the interchangeability of a biosimilar with its reference product, with the main concern being immunogenicity. We explore the theoretical basis of the presumed risks of switching between a biosimilar and its reference product and the available data on switches. Our conclusion is that a switch between comparable versions of the same active substance approved in accordance with EU legislation is not expected to trigger or enhance immunogenicity. On the basis of current knowledge, it is unlikely and very difficult to substantiate that two products, comparable on a population level, would have different safety or efficacy in individual patients upon a switch. Our conclusion is that biosimilars licensed in the EU are interchangeable.

Title: Purification and activation properties of UreD-UreF-urease apoprotein complexes. Content: In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S. Park, M.B. Carr, and R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S. Park and R.P. Hausinger, J. Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein. These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2. The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins. Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand.

Title: Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat. Content: BACKGROUND The electrical activity of the atrioventricular node (AVN) is functionally heterogeneous, but how this relates to distinct cell types and the 3-dimensional structure of the AVN is unknown. To address this, we have studied the expression of Na(V)1.5 and other Na+ channel isoforms in the AVN. METHODS AND RESULTS The rat AVN was identified by Masson's trichrome staining together with immunolabeling of marker proteins: connexin40, connexin43, desmoplakin, atrial natriuretic peptide, and hyperpolarization-activated and cyclic nucleotide-gated channel 4. Na+ channel expression was investigated with immunohistochemistry with isoform-specific Na+ channel antibodies. Na(V)1.1 was distributed in a similar manner to Na(V)1.5. Na(V)1.2 was not detected. Na(V)1.3 labeling was present in nerve fibers and cell bodies (but not myocytes) and was abundant in the penetrating atrioventricular (AV) bundle and the common bundle but was much less abundant in other regions. Na(V)1.5 labeling was abundant in the atrial and ventricular myocardium and the left bundle branch. Na(V)1.5 labeling was absent in the open node, penetrating AV bundle, AV ring bundle, and common bundle but present at a reduced level in the inferior nodal extension and transitional zone. Na(V)1.6 was not detected. CONCLUSIONS Our findings provide molecular evidence of multiple electrophysiological cell types at the AV junction. Impaired AV conduction as a result of mutations in or loss of Na(V)1.5 must be the result of impaired conduction in the AVN inputs (inferior nodal extension and transitional zone) or output (bundle branches) rather than the AVN itself (open node and penetrating AV bundle).

Title: Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes. Content: Defective hematopoiesis supporting capacity of bone marrow (BM) stroma has been implicated in the pathophysiology of myelodysplastic syndromes (MDS). The aim of this study is to explore whether the BM stroma progenitors, namely the mesenchymal stem cells (MSCs), are primarily affected in MDS by evaluating the reserves, the functional properties, as well as the cytogenetic characteristics, in comparison to BM hematopoietic cells, in patients with de novo MDS (n = 13). The number, differentiation potential toward adipocytes/chondrocytes/osteoblasts and immunosuppressive function in terms of inhibition of mitogen-induced T-cell proliferation did not differ significantly between patient and normal (n = 20) MSCs. Patient MSCs did not show any aberrations in the production of proinflammatory or growth-promoting cytokines and did not harbor the cytogenetic abnormalities present in hematopoietic cells. Occasional patient and normal MSC cultures, however, developed irrelevant chromosomal alterations (trisomies 5 and 7) with uncertain pathophysiologic significance. Compared to controls, patient MSCs displayed impaired proliferative and clonogenic potential through passages that might represent a nonspecific abnormality associated with the chronic inflammatory process present in patients' BM. These data suggest that BM MSCs from MDS patients do not belong to the abnormal clone and do not represent the main cellular source contributing to the inflammatory marrow microenvironment.

Title: Three-dimensional chemical imaging of skin using stimulated Raman scattering microscopy Content: Abstract. Stimulated Raman scattering (SRS) microscopy is used to generate structural and chemical three-dimensional images of native skin. We employed SRS microscopy to investigate the microanatomical features of skin and penetration of topically applied materials. Image depth stacks are collected at distinct wavelengths corresponding to vibrational modes of proteins, lipids, and water in the skin. We observed that corneocytes in stratum corneum are grouped together in clusters, 100 to 250 μm in diameter, separated by 10- to 25-μm-wide microanatomical skin-folds called canyons. These canyons occasionally extend down to depths comparable to that of the dermal–epidermal junction below the flat surface regions in porcine and human skin. SRS imaging shows the distribution of chemical species within cell clusters and canyons. Water is predominately located within the cell clusters, and its concentration rapidly increases at the transition from stratum corneum to viable epidermis. Canyons do not contain detectable levels of water and are rich in lipid material. Oleic acid-d34 applied to the skin surface lines the canyons down to a depth of 50 μm below the surface of the skin. This observation could have implications on the evaluation of penetration profiles of bioactive materials measured using traditional methods, such as tape-stripping.

Title: Investigating survival prognosis of glioblastoma using evolutional properties of gene networks Content: In recent years, there has been widespread interest and a large number of publications on the application of graph theory techniques into constructing and analyzing biologically-informed gene networks from cancer cell line data sets. Current research efforts have predominantly looked at an overall static, topological, representation of the network, and have not investigated the application of graph theoretical techniques to evolutionary investigations of cancer. A number of these studies have used graph theory metrics, such as degree, betweenness, and closeness centrality, to identify important hub genes in these networks. However, these have not fully investigated the importance of genes across the different stages of the disease. Previous human glioblastoma publications have identified four subtypes of glioblastoma in adults, based on signature genes. In one such publication, Verhaak et al. found that the subtypes correspond to a narrow median survival range, from 11.3 months for the most aggressive subtype, to 13.1 months for the least aggressive one. In this work, we present an evolutionary graph theory study of glioblastoma based on survival data categorization, confirming genes associated with different survival times identified using established graph theory metrics. The work is extending the application of graph theory approaches to evolutionary studies of cancer cell line data.

Title: Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1. Content: We have used NMR spectroscopy to determine the solution structure of protein AAH26994.1 from Mus musculus and propose that it represents the first three-dimensional structure of a ubiquitin-related modifier 1 (Urm1) protein. Amino acid sequence comparisons indicate that AAH26994.1 belongs to the Urm1 family of ubiquitin-like modifier proteins. The best characterized member of this family has been shown to be involved in nutrient sensing, invasive growth, and budding in yeast. Proteins in this family have only a weak sequence similarity to ubiquitin, and the structure of AAH26994.1 showed a much closer resemblance to MoaD subunits of molybdopterin synthases (known structures are of three bacterial MoaD proteins with 14%-26% sequence identity to AAH26994.1). The structures of AAH26994.1 and the MoaD proteins each contain the signature ubiquitin secondary structure fold, but all differ from ubiquitin largely in regions outside of this fold. This structural similarity bolsters the hypothesis that ubiquitin and ubiquitin-related proteins evolved from a protein-based sulfide donor system of the molybdopterin synthase type.

Title: Impedance and Interface Properties of Al/Methyl-Red/p-InP Solar Cell Content: An Al/methyl-red/p-InP solar cell was fabricated via solution-processing method and was characterized by using current-voltage (I-V) and capacitance-voltage-frequency (C-V-f) measurements at room temperature. From dark I-V characteristics, the values of ideality factor and barrier height of the device were calculated as 1.11 eV and 2.02, respectively. It has been seen that the device exhibited a good photovoltaic behavior with a maximum open circuit voltage of 0.38 V and short-circuit current of 2.8 nA under only 200 lx light intensity. The barrier height and acceptor carrier concentration values for the Al/methyl-red/p-InP devices were extracted as 1.27 eV and from linear region of its characteristics, respectively. The difference between (I-V) and (C-V) for Al/methyl-red/p-InP device was attributed the different nature of the I-V and C-V measurements. Also, the energy distribution curves of the interface states and their time constants were obtained from the experimental conductance properties of the Al/methyl-red/p-InP structure at room temperature. The interface state densities and their relaxation times of the device have ranged from and s at (1.11-) eV to and s at (0.79-) eV, respectively. It was seen that both the interface state density and the relaxation time of the interface states have decreased with bias voltage from experimental results.

Title: Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2 Content: Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.

Title: Geometry, epistasis, and developmental patterning. Content: Developmental signaling networks are composed of dozens of components whose interactions are very difficult to quantify in an embryo. Geometric reasoning enumerates a discrete hierarchy of phenotypic models with a few composite variables whose parameters may be defined by in vivo data. Vulval development in the nematode Caenorhabditis elegans is a classic model for the integration of two signaling pathways; induction by EGF and lateral signaling through Notch. Existing data for the relative probabilities of the three possible terminal cell types in diverse genetic backgrounds as well as timed ablation of the inductive signal favor one geometric model and suffice to fit most of its parameters. The model is fully dynamic and encompasses both signaling and commitment. It then predicts the correlated cell fate probabilities for a cross between any two backgrounds/conditions. The two signaling pathways are combined additively, without interactions, and epistasis only arises from the nonlinear dynamical flow in the landscape defined by the geometric model. In this way, the model quantitatively fits genetic experiments purporting to show mutual pathway repression. The model quantifies the contributions of extrinsic vs. intrinsic sources of noise in the penetrance of mutant phenotypes in signaling hypomorphs and explains available experiments with no additional parameters. Data for anchor cell ablation fix the parameters needed to define Notch autocrine signaling.

Title: The use of biosimilars in immune-mediated disease: A joint Italian Society of Rheumatology (SIR), Italian Society of Dermatology (SIDeMaST), and Italian Group of Inflammatory Bowel Disease (IG-IBD) position paper. Content: Biological agents are widely used in rheumatology, dermatology and inflammatory bowel disease. Evidence about their efficacy and safety has been strengthened for all those therapeutic indications over the last decade. Biosimilar agents are monoclonal antibodies similar to previously approved biologics. In the European Union, they have been approved for all the indications in the management of immune-mediated inflammatory diseases (IMIDs), although data only in rheumatoid arthritis and ankylosing spondylitis are currently available. Direct evidence on efficacy, safety, and immunogenicity of biosimilars is mandatory in psoriasis, psoriatic arthritis, and inflammatory bowel disease, as well as in children. Based on the current evidence in the literature, we present the joint official position of the Italian Societies of Rheumatology, Dermatology and Inflammatory Bowel Disease on the use of biosimilars in IMIDs.

Title: A versatile zero background T-vector system for gene cloning and functional genomics. Content: With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.

Title: Differential regulation of adherens junction dynamics during apical-basal polarization. Content: Adherens junctions (AJs) in epithelial cells are constantly turning over to modulate adhesion properties under various physiological and developmental contexts, but how such AJ dynamics are regulated during the apical-basal polarization of primary epithelia remains unclear. Here, we used new and genetically validated GFP markers of Drosophila E-cadherin (DE-cadherin, hereafter referred to as DE-Cad) and β-catenin (Armadillo, Arm) to quantitatively assay the in vivo dynamics of biosynthetic turnover and membrane redistribution by fluorescence recovery after photobleaching (FRAP) assays. Our data showed that membrane DE-Cad and Arm in AJs of polarizing epithelial cells had much faster biosynthetic turnover than in polarized cells. Fast biosynthetic turnover of membrane DE-Cad is independent of actin- and dynamin-based trafficking, but is microtubule-dependent. Furthermore, Arm in AJs of polarizing cells showed a faster and diffusion-based membrane redistribution that was both quantitatively and qualitatively different from the slower and exchange-based DE-Cad membrane distribution, indicating that the association of Arm with DE-Cad is more dynamic in polarizing cells, and only becomes stable in polarized epithelial cells. Consistently, biochemical assays showed that the binding of Arm to DE-Cad is weaker in polarizing cells than in polarized cells. Our data revealed that the molecular interaction between DE-Cad and Arm is modulated during apical-basal polarization, suggesting a new mechanism that might be crucial for establishing apical-basal polarity through regulating the AJ dynamics.

Title: Topiramate for smoking cessation: a randomized, placebo-controlled pilot study. Content: INTRODUCTION Topiramate (TOP) blocks glutamate receptors and facilitates GABA (γ-aminobutyric acid) neurotransmission, effects that may facilitate smoking cessation. We compared the effects of behavioral counseling combined with (a) TOP, (b) TOP/nicotine patch (TOP/NIC), or (c) placebo (PLC) for smoking cessation. METHODS We conducted a 10-week randomized trial in which subjects and research personnel were blinded to TOP versus PLC but not to the TOP/NIC patch condition. In groups receiving TOP, the medication dosage was titrated gradually up to 200 mg/day. The smoking quit date (QD) was scheduled after 2 weeks of medication treatment. NIC (21 mg) was started on the QD in subjects randomized to the TOP/NIC condition. The main outcome measure was the end-of-treatment, 4-week continuous abstinence rate (CAR; biochemically confirmed). RESULTS Fifty-seven subjects were randomized to treatment. The 4-week CAR was 1 of 19 (5%) in the PLC group, 5 of 19 (26%) in the TOP group, and 7 of 19 (37%) in the TOP/NIC group (p = .056). Pairwise comparisons showed a difference between TOP/NIC and PLC (p = .042) and a nonsignificant difference between TOP and PLC (p = .18). The PLC group gained 0.37 lb/week, the TOP group lost 0.41 lb/week, and the TOP/NIC group lost 0.07 lb/week (p = .004). Pairwise comparisons showed a difference between TOP and PLC (p < .001) and between TOP/NIC and PLC groups (p = .035). Paresthesia was more frequent in subjects on TOP than PLC (p = .011). CONCLUSIONS TOP, alone or in combination with the NIC, resulted in a numerically higher quit rate than PLC and decreased weight. A larger, PLC-controlled trial is needed to confirm these findings.

Title: Pseudoknots: RNA Structures with Diverse Functions Content: RNA molecules fulfill a diverse set of biological functions within cells, from the transfer of genetic information from DNA to protein, to enzymatic catalysis. Reflecting this range of roles, simple linear strings of RNA—made up of uracil, guanine, cytosine, and adenine—form a variety of complex three-dimensional structures. Just as proteins form distinct structural motifs such as zinc fingers and beta barrels, certain structures are also commonly adopted by RNA molecules. Among the most prevalent RNA structures is a motif known as the pseudoknot. First recognized in the turnip yellow mosaic virus [1], a pseudoknot is an RNA structure that is minimally composed of two helical segments connected by single-stranded regions or loops (Figure 1). Although several distinct folding topologies of pseudoknots exist, the best characterized is the H type. In the H-type fold, the bases in the loop of a hairpin form intramolecular pairs with bases outside of the stem (Figure 1A and and1B).1B). This causes the formation of a second stem and loop, resulting in a pseudoknot with two stems and two loops (Figure 1C). The two stems are able to stack on top of each other to form a quasi-continuous helix with one continuous and one discontinuous strand. The single-stranded loop regions often interact with the adjacent stems (loop 1–stem 2 or loop 2–stem 1) to form hydrogen bonds and to participate in the overall structure of the molecule. Hence, this relatively simple fold can yield very complex and stable RNA structures. Due to variation of the lengths of the loops and stems, as well as the types of interactions between them, pseudoknots represent a structurally diverse group. It is fitting that they play a variety of diverse roles in biology. These roles include forming the catalytic core of various ribozymes [2,3], self-splicing introns [4], and telomerase [5]. Additionally, pseudoknots play critical roles in altering gene expression by inducing ribosomal frameshifting in many viruses [6–9].

Title: A core subunit of Polycomb repressive complex 1 is broadly conserved in function but not primary sequence. Content: Polycomb Group (PcG) proteins mediate heritable gene silencing by modifying chromatin structure. An essential PcG complex, PRC1, compacts chromatin and inhibits chromatin remodeling. In Drosophila melanogaster, the intrinsically disordered C-terminal region of PSC (PSC-CTR) mediates these noncovalent effects on chromatin, and is essential for viability. Because the PSC-CTR sequence is poorly conserved, the significance of its effects on chromatin outside of Drosophila was unclear. The absence of folded domains also made it difficult to understand how the sequence of PSC-CTR encodes its function. To determine the mechanistic basis and extent of conservation of PSC-CTR activity, we identified 17 metazoan PSC-CTRs spanning chordates to arthropods, and examined their sequence features and biochemical properties. PSC-CTR sequences are poorly conserved, but are all highly charged and structurally disordered. We show that active PSC-CTRs--which bind DNA tightly and inhibit chromatin remodeling efficiently--are distinguished from less active ones by the absence of extended negatively charged stretches. PSC-CTR activity can be increased by dispersing its contiguous negative charge, confirming the importance of this property. Using the sequence properties defined as important for PSC-CTR activity, we predicted the presence of active PSC-CTRs in additional diverse genomes. Our analysis reveals broad conservation of PSC-CTR activity across metazoans. This conclusion could not have been determined from sequence alignments. We further find that plants that lack active PSC-CTRs instead possess a functionally analogous PcG protein, EMF1. Thus, our study suggests that a disordered domain with dispersed negative charges underlies PRC1 activity, and is conserved across metazoans and plants.

Title: Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Content: BACKGROUND Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. METHODS Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. RESULTS Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. CONCLUSIONS Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.

Title: A three-dimensional human neural cell culture model of Alzheimer’s disease Content: Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

Title: A randomized, placebo-controlled trial of diindolylmethane for breast cancer biomarker modulation in patients taking tamoxifen Content: Diindolylmethane (DIM), a bioactive metabolite of indole-3-carbinol found in cruciferous vegetables, has proposed cancer chemoprevention activity in the breast. There is limited evidence of clinically relevant activity of DIM or long-term safety data of its regular use. A randomized, double-blind, placebo-controlled trial was conducted to determine the activity and safety of combined use of BioResponse DIM® (BR-DIM) with tamoxifen. Women prescribed tamoxifen (n = 130) were randomly assigned oral BR-DIM at 150 mg twice daily or placebo, for 12 months. The primary study endpoint was change in urinary 2/16α-hydroxyestrone (2/16α-OHE1) ratio. Changes in 4-hydroxyestrone (4-OHE1), serum estrogens, sex hormone-binding globulin (SHBG), breast density, and tamoxifen metabolites were assessed. Ninety-eight women (51 placebo, 47 DIM) completed intervention; compliance with treatment was >91%. BR-DIM increased the 2/16α-OHE1 ratio (+3.2 [0.8, 8.4]) compared to placebo (−0.7 [−1.7, 0.8], P < 0.001). Serum SHBG increased with BR-DIM compared to placebo (+25 ± 22 and +1.1 ± 19 nmol/L, respectively). No change in breast density measured by mammography or by MRI was observed. Plasma tamoxifen metabolites (endoxifen, 4-OH tamoxifen, and N-desmethyl-tamoxifen) were reduced in women receiving BR-DIM versus placebo (P < 0.001). Minimal adverse events were reported and did not differ by treatment arm. In patients taking tamoxifen for breast cancer, daily BR-DIM promoted favorable changes in estrogen metabolism and circulating levels of SHBG. Further research is warranted to determine whether BR-DIM associated decreases in tamoxifen metabolites, including effects on endoxifen levels, attenuates the clinical benefit of tamoxifen. Trial Registration: ClinicalTrials.gov NCT01391689.

Title: Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex. Content: TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.

Title: Identification and analysis of ribonuclease P and MRP RNA in a broad range of eukaryotes Content: RNases P and MRP are ribonucleoprotein complexes involved in tRNA and rRNA processing, respectively. The RNA subunits of these two enzymes are structurally related to each other and play an essential role in the enzymatic reaction. Both of the RNAs have a highly conserved helical region, P4, which is important in the catalytic reaction. We have used a bioinformatics approach based on conserved elements to computationally analyze available genomic sequences of eukaryotic organisms and have identified a large number of novel nuclear RNase P and MRP RNA genes. For MRP RNA for instance, this investigation increases the number of known sequences by a factor of three. We present secondary structure models of many of the predicted RNAs. Although all sequences are able to fold into the consensus secondary structure of P and MRP RNAs, a striking variation in size is observed, ranging from a Nosema locustae MRP RNA of 160 nt to much larger RNAs, e.g. a Plasmodium knowlesi P RNA of 696 nt. The P and MRP RNA genes appear in tandem in some protists, further emphasizing the close evolutionary relationship of these RNAs.

Title: Cerebral organoids model human brain development and microcephaly Content: The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype. Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue.

Title: Accuracy of ICD-9-CM codes for identifying cardiovascular and stroke risk factors. Content: OBJECTIVES We sought to determine which ICD-9-CM codes in Medicare Part A data identify cardiovascular and stroke risk factors. DESIGN AND PARTICIPANTS This was a cross-sectional study comparing ICD-9-CM data to structured medical record review from 23,657 Medicare beneficiaries aged 20 to 105 years who had atrial fibrillation. MEASUREMENTS Quality improvement organizations used standardized abstraction instruments to determine the presence of 9 cardiovascular and stroke risk factors. Using the chart abstractions as the gold standard, we assessed the accuracy of ICD-9-CM codes to identify these risk factors. MAIN RESULTS ICD-9-CM codes for all risk factors had high specificity (>0.95) and low sensitivity (< or =0.76). The positive predictive values were greater than 0.95 for 5 common, chronic risk factors-coronary artery disease, stroke/transient ischemic attack, heart failure, diabetes, and hypertension. The sixth common risk factor, valvular heart disease, had a positive predictive value of 0.93. For all 6 common risk factors, negative predictive values ranged from 0.52 to 0.91. The rare risk factors-arterial peripheral embolus, intracranial hemorrhage, and deep venous thrombosis-had high negative predictive value (> or =0.98) but moderate positive predictive values (range, 0.54-0.77) in this population. CONCLUSIONS Using ICD-9-CM codes alone, heart failure, coronary artery disease, diabetes, hypertension, and stroke can be ruled in but not necessarily ruled out. Where feasible, review of additional data (eg, physician notes or imaging studies) should be used to confirm the diagnosis of valvular disease, arterial peripheral embolus, intracranial hemorrhage, and deep venous thrombosis.

Title: The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling Content: Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.

Title: A Novel 3-Hydroxysteroid Dehydrogenase That Regulates Reproductive Development and Longevity Content: Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs) regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.

Title: Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Content: Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.

Title: Kinetic Analysis Reveals the Fate of a MicroRNA following Target Regulation in Mammalian Cells Content: Considerable details about microRNA (miRNA) biogenesis and regulation have been uncovered, but little is known about the fate of the miRNA subsequent to target regulation. To gain insight into this process, we carried out kinetic analysis of a miRNA's turnover following termination of its biogenesis and during regulation of a target that is not subject to Ago2-mediated catalytic cleavage. By quantitating the number of molecules of the miRNA and its target in steady state and in the course of its decay, we found that each miRNA molecule was able to regulate at least two target transcripts, providing in vivo evidence that the miRNA is not irreversibly sequestered with its target and that the nonslicing pathway of miRNA regulation is multiple-turnover. Using deep sequencing, we further show that miRNA recycling is limited by target regulation, which promotes posttranscriptional modifications to the 3' end of the miRNA and accelerates the miRNA's rate of decay. These studies provide new insight into the efficiency of miRNA regulation that help to explain how a miRNA can regulate a vast number of transcripts and that identify one of the mechanisms that impart specificity to miRNA decay in mammalian cells.

Title: Rodent Models of Depression: Neurotrophic and Neuroinflammatory Biomarkers Content: Rodent models are an indispensable tool for studying etiology and progress of depression. Since interrelated systems of neurotrophic factors and cytokines comprise major regulatory mechanisms controlling normal brain plasticity, impairments of these systems form the basis for development of cerebral pathologies, including mental diseases. The present review focuses on the numerous experimental rodent models of depression induced by different stress factors (exteroceptive and interoceptive) during early life (including prenatal period) or adulthood, giving emphasis to the data on the changes of neurotrophic factors and neuroinflammatory indices in the brain. These parameters are closely related to behavioral depression-like symptoms and impairments of neuronal plasticity and are both gender- and genotype-dependent. Stress-related changes in expression of neurotrophins and cytokines in rodent brain are region-specific. Some contradictory data reported by different groups may be a consequence of differences of stress paradigms or their realization in different laboratories. Like all experimental models, stress-induced depression-like conditions are experimental simplification of clinical depression states; however, they are suitable for understanding the involvement of neurotrophic factors and cytokines in the pathogenesis of the disease-a goal unachievable in the clinical reality. These major regulatory systems may be important targets for therapeutic measures as well as for development of drugs for treatment of depression states.

Title: Scaffold-based three-dimensional human fibroblast culture provides a structural matrix that supports angiogenesis in infarcted heart tissue. Content: BACKGROUND We have developed techniques to implant angiogenic patches onto the epicardium over regions of infarcted cardiac tissue to stimulate revascularization of the damaged tissue. These experiments used a scaffold-based 3D human dermal fibroblast culture (3DFC) as an epicardial patch. The 3DFC contains viable cells that secrete angiogenic growth factors and has previously been shown to stimulate angiogenic activity. The hypothesis tested was that a viable 3DFC cardiac patch would stimulate an angiogenic response within an area of infarcted cardiac tissue. METHODS AND RESULTS A coronary occlusion of a branch of the left anterior descending coronary artery was performed by thermal ligation in severe combined immunodeficient mice. 3DFCs with or without viable cells were sized to the damaged area, implanted in replicate mice onto the epicardium at the site of tissue injury, and compared with animals that received infarct surgery but no implant. Fourteen and 30 days after surgery, hearts were exposed and photographed, and tissue samples were prepared for histology and cytochemistry. Fourteen and 30 days after surgery, the damaged myocardium receiving viable 3DFC exhibited a significantly greater angiogenic response (including arterioles, venules, and capillaries) than nonviable and untreated control groups. CONCLUSIONS In this animal model, viable 3DFC stimulates angiogenesis within a region of cardiac infarction and can augment a repair response in damaged tissue. Therefore, a potential use for 3DFC is the repair of myocardial tissue damaged by infarction.

Title: Coordinated actions of SLX1-SLX4 and MUS81-EME1 for Holliday junction resolution in human cells. Content: Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted in vitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.

Title: Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis Content: The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in ∼50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

Title: High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain Content: Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC50 1 µM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain.

Title: Recommendations on how to ensure the safety and effectiveness of biosimilars in Latin America: a point of view Content: The use of biotechnology-derived medicines has significantly increased in recent decades. Although biosimilars undergo rigorous characterization as well as clinical studies to document their safety and effectiveness, they are highly complex molecules and small changes in the purification and production process of a biosimilar can have major implications in its safety and effectiveness profile. In Latin America, regulatory authorities have begun to establish well-described and standardized pathways that permit a biosimilar to gain commercial licensure. In order to be certain that a biosimilar reaches its potential in ordinary clinical use, an intensive post-licensing monitoring system must be established since it is the only means to ascertain the true similarity between the original biologic and its biosimilar. Pharmacovigilance allows national authorities to determine a drug's performance in the marketplace. An effective tracking and pharmacovigilance system for biological medicines has many steps and processes. To aid policy makers in Latin American in addressing the many issues surrounding the establishment of an effective pharmacovigilance system, the Americas Health Foundation convened a group of experts to discuss the topic and develop recommendations for implementation. The group discussed current challenges and gaps in pharmacovigilance in Latin America, paying close attention to the major issues associated with traceability and pharmacovigilance of biosimilars following their approval. The recommendations developed should enable countries to accurately document the safety and performance of a biosimilar as experienced by patients under real-life conditions and have a significant impact on the successful implementation of pharmacovigilance of biosimilars throughout the region.

Title: Cytoscape 2.8: new features for data integration and network visualization Content: UNLABELLED Cytoscape is a popular bioinformatics package for biological network visualization and data integration. Version 2.8 introduces two powerful new features--Custom Node Graphics and Attribute Equations--which can be used jointly to greatly enhance Cytoscape's data integration and visualization capabilities. Custom Node Graphics allow an image to be projected onto a node, including images generated dynamically or at remote locations. Attribute Equations provide Cytoscape with spreadsheet-like functionality in which the value of an attribute is computed dynamically as a function of other attributes and network properties. AVAILABILITY AND IMPLEMENTATION Cytoscape is a desktop Java application released under the Library Gnu Public License (LGPL). Binary install bundles and source code for Cytoscape 2.8 are available for download from http://cytoscape.org.

Title: A unified concept of species and its consequences for the future of taxonomy Content: Contemporary species concepts are diverse. Nonetheless, all share the fundamental idea that species are segments of lineages at the population level of biological organization. They differ in the secondary properties (e.g., intrinsic reproductive isolation, monophyly, diagnosability) that are treated as necessary for considering lineages to be species. A unified species concept can be achieved by interpreting the common fundamental idea of being a separately evolving lineage segment as the only necessary property of species and viewing the various secondary properties either as lines of evidence relevant to assessing lineage separation or as properties that define different subcategories of the species category (e.g., reproductively isolated species, monophyletic species, diagnosable species). This unified species concept has a number of consequences for taxonomy, including the need to acknowledge that undifferentiated and undiagnosable lineages are species, that species can fuse, that species can be nested within other species, that the species category is not a taxonomic rank, and that new taxonomic practices and conventions are needed to accommodate these conclusions. Although acceptance of a unified species concept has some radical consequences for taxonomy, it also reflects a change in the general conceptualization of the species category that has been underway for more than a half-century — a shift from viewing the species category as one member of the hierarchy of taxonomic ranks to viewing it as a natural kind whose members are the units at one of the levels of biological organization. This change is related to a more general shift in the primary concern of the discipline of systematics (including taxonomy), from the utilitarian activity of classifying organisms to the scientific activity of testing hypotheses about lineage boundaries and phylogenetic relationships. The unified species concept is a natural outcome of this conceptual shift and represents the more complete acceptance of the idea that species are one of the fundamental units of biology. As such, the unified species concept is central to the future of taxonomy.

Title: Differential activation of DNA-PK based on DNA strand orientation and sequence bias Content: DNA-PKcs and Ku are essential components of the complex that catalyzes non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Ku, a heterodimeric protein, binds to DNA ends and facilitates recruitment of the catalytic subunit, DNA-PKcs. We have investigated the effect of DNA strand orientation and sequence bias on the activation of DNA-PK. In addition, we assessed the effect of the position and strand orientation of cisplatin adducts on kinase activation. A series of duplex DNA substrates with site-specific cisplatin–DNA adducts placed in three different orientations on the duplex DNA were prepared. Terminal biotin modification and streptavidin (SA) blocking was employed to direct DNA-PK binding to the unblocked termini with a specific DNA strand orientation and cisplatin–DNA adduct position. DNA-PK kinase activity was measured and the results reveal that DNA strand orientation and sequence bias dramatically influence kinase activation, only a portion of which could be attributed to Ku-DNA binding activity. In addition, cisplatin–DNA adduct position resulted in differing degrees of inhibition depending on distance from the terminus as well as strand orientation. These results highlight the importance of how local variations in DNA structure, chemistry and sequence influence DNA-PK activation and potentially NHEJ.

Title: Immune complex relay by subcapsular sinus macrophages and non-cognate B cells drives antibody affinity maturation Content: Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

Title: In vitro substrate specificity of protein tyrosine kinases Content: Synthetic peptides such as P60stc autophosphorylation site peptides and angiotensin are indiscriminately phosphorylated by protein tyrosine kinases. The observation has led to the general belief that protein tyrosine kinases are highly promiscuous, displaying littlein vitro site specificity. In recent years, evidence has been accumulating to indicate that such a belief requires close examination. Synthetic peptides showing high substrate activity for specific groups of protein tyrosine kinases have been obtained. Systematic modification of certain substrate peptides suggests that kinase substrate determinants reside with specific amino acid residues proximal to the target tyrosine. A number of protein kinases have been shown to be regulated by tyrosine phosphorylation at specific sites by highly specific protein tyrosine kinases. These and other selected biochemical studies that contribute to the evolving view ofin vitro substrate specificity of protein tyrosine kinases are reviewed.

Title: Building-in biosafety for synthetic biology. Content: As the field of synthetic biology develops, real-world applications are moving from the realms of ideas and laboratory-confined research towards implementation. A pressing concern, particularly with microbial systems, is that self-replicating re-engineered cells may produce undesired consequences if they escape or overwhelm their intended environment. To address this biosafety issue, multiple mechanisms for constraining microbial replication and horizontal gene transfer have been proposed. These include the use of host-construct dependencies such as toxin-antitoxin pairs, conditional plasmid replication or the requirement for a specific metabolite to be present for cellular function. While refactoring of the existing genetic code or tailoring of orthogonal systems, e.g. xeno nucleic acids, offers future promise of more stringent 'firewalls' between natural and synthetic cells, here we focus on what can be achieved using existing technology. The state-of-the-art in designing for biosafety is summarized and general recommendations are made (e.g. short environmental retention times) for current synthetic biology projects to better isolate themselves against potentially negative impacts.

Title: The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor+ Innate Lymphoid Cells Content: Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-α produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

Title: DNA replication fidelity Content: DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.

Title: A single RNA-dependent RNA polymerase assembles with mutually exclusive nucleotidyl transferase subunits to direct different pathways of small RNA biogenesis. Content: Members of the conserved family of eukaryotic RNA-dependent RNA polymerases (Rdrs) synthesize double-stranded RNA (dsRNA) intermediates in diverse pathways of small RNA (sRNA) biogenesis and RNA-mediated silencing. Rdr-dependent pathways of sRNA production are poorly characterized relative to Rdr-independent pathways, and the Rdr enzymes themselves are poorly characterized relative to their viral RNA-dependent RNA polymerase counterparts. We previously described a physical and functional coupling of the Tetrahymena thermophila Rdr, Rdr1, and a Dicer enzyme, Dcr2, in the production of approximately 24-nucleotide (nt) sRNA in vitro. Here we characterize the endogenous complexes that harbor Rdr1, termed RDRCs. Distinct RDRCs assemble to contain Rdr1 and subsets of the total of four tightly Rdr1-associated proteins. Of particular interest are two RDRC subunits, Rdn1 and Rdn2, which possess noncanonical ribonucleotidyl transferase motifs. We show that the two Rdn proteins are uridine-specific polymerases of separate RDRCs. Two additional RDRC subunits, Rdf1 and Rdf2, are present only in RDRCs containing Rdn1. Rdr1 catalytic activity is retained in RDRCs purified from cell extracts lacking any of the nonessential RDRC subunits (Rdn2, Rdf1, Rdf2) or if the RDRC harbors a catalytically inactive Rdn. However, specific disruption of each RDRC imposes distinct loss-of-function consequences at the cellular level and has a differential impact on the accumulation of specific 23-24-nt sRNA sequences in vivo. The biochemical and biological phenotypes of RDRC subunit disruption reveal a previously unanticipated complexity of Rdr-dependent sRNA biogenesis in vivo.

Title: Novel Functional Sets of Lipid-Derived Mediators with Antiinflammatory Actions Generated from Omega-3 Fatty Acids via Cyclooxygenase 2–Nonsteroidal Antiinflammatory Drugs and Transcellular Processing Content: Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with ω-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 ω-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX5 and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as ω-5 and ω-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2–nonsteroidal antiinflammatory drug–dependent oxygenations and cell–cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of ω-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Title: Autoimmunity due to molecular mimicry as a cause of neurological disease Content: One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Title: Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics Content: Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.

Title: Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures Content: Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

Title: Disruption of the 5S RNP–Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia Content: Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2C305F knock-in mice that have a disrupted 5S RNP–Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2C305F reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP–Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP–Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

Title: Platelet activation in type 2 diabetes mellitus. Content: The abnormal metabolic state that accompanies diabetes renders arteries susceptible to atherosclerosis, being capable of altering the functional properties of multiple cell types, including endothelium and platelets. In particular, an altered platelet metabolism and changes in intraplatelet signaling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. A variety of mechanisms may be responsible for enhanced platelet aggregation. Among them, hyperglycemia may represent a causal factor for in vivo platelet activation, and may be responsible for nonenzymatic glycation of platelet glycoproteins, causing changes in their structure and conformation, as well as alterations of membrane lipid dynamics. Furthermore, hyperglycemia-induced oxidative stress is responsible for enhanced peroxidation of arachidonic acid to form biologically active isoprostanes, which represents an important biochemical link between impaired glycemic control and persistent platelet activation. Finally, increased oxidative stress is responsible for activation of transcription factors and expression of redox-sensitive genes leading to a phenotypic switch of endothelium toward an adhesive, pro-thrombotic condition, initial platelet activation, adhesion and subsequent platelet aggregate formation. All this evidence is strengthened by the results of clinical trials documenting the beneficial effects of metabolic control on platelet function, and by the finding that aspirin treatment may even be more beneficial in diabetic than in high-risk non-diabetic patients. Attention to appropriate medical management of diabetic patients will have great impact on long-term outcome in this high-risk population.

Title: Urine microRNAs as biomarkers for bladder cancer: a diagnostic meta-analysis Content: BACKGROUND The diagnostic value of microRNA (miRNA) detection in patients with bladder cancer (BCa) is controversial. We performed a diagnostic meta-analysis to evaluate current evidence on the use of miRNA assays to diagnose BCa. METHODS We systematically searched PubMed, Embase, and Web of Science for studies published before March 31, 2015. The pooled sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and area under the curve (AUC) were calculated to evaluate the overall test performance. Subgroup analyses were used to explore the between-study heterogeneity. Deeks' funnel plot asymmetry test was used to test publication bias. We applied the software of RevMan 5.2 and Stata 11.0 to the meta-analysis. RESULTS A total of 23 studies from nine articles were included in the meta-analysis, with a total of 719 patients and 494 controls. The pooled sensitivity and specificity were 0.75 (95% confidence interval [CI], 0.68-0.80) and 0.75 (95% CI, 0.70-0.80), respectively. The pooled positive likelihood ratio was 3.03 (95% CI, 2.50-3.67); negative likelihood ratio was 0.33 (95% CI, 0.27-0.42); and diagnostic odds ratio was 9.07 (95% CI, 6.35-12.95). The pooled AUC was 0.81 (95% CI, 0.78-0.85). Subgroup analyses indicated that the multiple miRNAs assays and urine supernatant assays showed high accuracies in diagnosing BCa. CONCLUSION The miRNA assays may serve as potential noninvasive diagnostic tool for the detection of BCa. However, the clinical application of miRNA assays for BCa diagnosis still needs further validation by large prospective studies.

Title: A general model to explore complex dominance patterns in plant sporophytic self-incompatibility systems. Content: We developed a general model of sporophytic self-incompatibility under negative frequency-dependent selection allowing complex patterns of dominance among alleles. We used this model deterministically to investigate the effects on equilibrium allelic frequencies of the number of dominance classes, the number of alleles per dominance class, the asymmetry in dominance expression between pollen and pistil, and whether selection acts on male fitness only or both on male and on female fitnesses. We show that the so-called "recessive effect" occurs under a wide variety of situations. We found emerging properties of finite population models with several alleles per dominance class such as that higher numbers of alleles are maintained in more dominant classes and that the number of dominance classes can evolve. We also investigated the occurrence of homozygous genotypes and found that substantial proportions of those can occur for the most recessive alleles. We used the model for two species with complex dominance patterns to test whether allelic frequencies in natural populations are in agreement with the distribution predicted by our model. We suggest that the model can be used to test explicitly for additional, allele-specific, selective forces.

Title: Molecular analysis of circulating tumour cells—biology and biomarkers Content: Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test—and readily amenable to serial sampling—have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch® system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial–mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

Title: Microenvironmental regulation of biomacromolecular therapies Content: There is currently great interest in molecular therapies to treat various diseases, and this has prompted extensive efforts to achieve target-specific and controlled delivery of bioactive macromolecules (for example, proteins, antibodies, DNA and small interfering RNA) through the design of smart drug carriers. By contrast, the influence of the microenvironment in which the target cell resides and the effect it might have on the success of biomacromolecular therapies has been under-appreciated. The extracellular matrix (ECM) component of the cellular niche may be particularly important, as many diseases and injury disrupt the normal ECM architecture, the cell adhesion to ECM, and the subsequent cellular activities. This Review will discuss the importance of the ECM and the ECM–cell interactions on the cell response to bioactive macromolecules, and suggest how this information could lead to new criteria for the design of novel drug delivery systems.

Title: Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics. Content: Decreasing costs of next-generation sequencing (NGS) experiments have made a wide range of genomic questions open for study with nonmodel organisms. However, experimental designs and analysis of NGS data from less well-known species are challenging because of the lack of genomic resources. In this work, we investigate the performance of alternative experimental designs and bioinformatics approaches in estimating variability and neutrality tests based on the site-frequency-spectrum (SFS) from individual resequencing data. We pay particular attention to challenges faced in the study of nonmodel organisms, in particular the absence of a species-specific reference genome, although phylogenetically close genomes are assumed to be available. We compare the performance of three alternative bioinformatics approaches – genotype calling, genotype–haplotype calling and direct estimation without calling genotypes. We find that relying on genotype calls provides biased estimates of population genetic statistics at low to moderate read depth (2–8X). Genotype–haplotype calling returns more accurate estimates irrespective of the divergence to the reference genome, but requires moderate depth (8–20X). Direct estimation without calling genotypes returns the most accurate estimates of variability and of most SFS tests investigated, including at low read depth (2–4X). Studies without species-specific reference genome should thus aim for low read depth and avoid genotype calling whenever individual genotypes are not essential. Otherwise, aiming for moderate to high depth at the expense of number of individuals, and using genotype–haplotype calling, is recommended.

Title: A Protein Inventory of Human Ribosome Biogenesis Reveals an Essential Function of Exportin 5 in 60S Subunit Export Content: The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

Title: Interactive effects of oligofructose and obesity predisposition on gut hormones and microbiota in diet-induced obese rats. Content: OBJECTIVE Oligofructose (OFS) is a prebiotic that reduces energy intake and fat mass via changes in gut satiety hormones and microbiota. The effects of OFS may vary depending on predisposition to obesity. The aim of this study was to examine the effect of OFS in diet-induced obese (DIO) and diet-resistant (DR) rats. METHODS Adult, male DIO, and DR rats were randomized to: high-fat/high-sucrose (HFS) diet or HFS diet + 10% OFS for 6 weeks. Body composition, food intake, gut microbiota, plasma gut hormones, and cannabinoid CB(1) receptor expression in the nodose ganglia were measured. RESULTS OFS reduced body weight, energy intake, and fat mass in both phenotypes (P < 0.05). Select gut microbiota differed in DIO versus DR rats (P < 0.05), the differences being eliminated by OFS. OFS did not modify plasma ghrelin or CB(1) expression in nodose ganglia, but plasma levels of GIP were reduced and PYY were elevated (P < 0.05) by OFS. CONCLUSIONS OFS was able to reduce body weight and adiposity in both prone and resistant obese phenotypes. OFS-induced changes in gut microbiota profiles in DIO and DR rats, along with changes in gut hormone levels, likely contribute to the sustained lower body weights.

Title: Organization of the mitotic chromosome. Content: Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type-specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.

Title: The epithelial inward rectifier channel Kir7.1 displays unusual K+ permeation properties. Content: Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.

Title: From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate Content: The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

Title: Chemical tools for biomolecular imaging. Content: The visualization of biologically relevant molecules and activities inside living cells continues to transform cell biology into a truly quantitative science. However, despite the spectacular achievements in some areas of cell biology, the majority of cellular processes still operate invisibly, not illuminated by even our brightest laser beams. Further progress therefore will depend not only on improvements in instrumentation but also increasingly on the development of new fluorophores and fluorescent sensors to target these activities. In the following, we review some of the recent approaches to generating such sensors, the methods to attach them to selected biomolecules, and their applications to various biological problems.

Title: A 3D Map of the Yeast Kinetochore Reveals the Presence of Core and Accessory Centromere-Specific Histone Content: The budding yeast kinetochore is ~68 nm in length with a diameter slightly larger than a 25 nm microtubule. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1Δ or xrn1Δ mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.

Title: Induction of stem-like cells with malignant properties by chronic exposure of human lung epithelial cells to single-walled carbon nanotubes Content: BACKGROUND Carbon nanotubes (CNT) hold great promise to create new and better products for commercial and biomedical applications, but their long-term adverse health effects are a major concern. The objective of this study was to address human lung cancer risks associated with chronic pulmonary exposure to single-walled (SW) CNT through the fundamental understanding of cellular and molecular processes leading to carcinogenesis. We hypothesized that the acquisition of cancer stem cells (CSC), a subpopulation that drive tumor initiation and progression, may contribute to CNT carcinogenesis. METHODS Non-tumorigenic human lung epithelial cells were chronically exposed to well-dispersed SWCNT for a period of 6 months at the physiologically relevant concentration of 0.02 μg/cm2 surface area dose. Chronic SWCNT-exposed cells were evaluated for the presence of CSC-like cells under CSC-selective conditions of tumor spheres and side population (SP). CSC-like cells were isolated using fluorescence-activated cell sorting and were assessed for aggressive behaviors, including acquired apoptosis resistance and increased cell migration and invasion in vitro, and tumor-initiating capability in vivo. Non-small cell lung cancer cells served as a positive control. RESULTS We demonstrated for the first time the existence of CSC-like cells in all clones of chronic SWCNT-exposed lung epithelial cells. These CSC-like cells, in contrary to their non-CSC counterpart, possessed all biological features of lung CSC that are central to irreversible malignant transformation, self-renewal, aggressive cancer behaviors, and in vivo tumorigenesis. These cells also displayed aberrant stem cell markers, notably Nanog, SOX-2, SOX-17 and E-cadherin. Restored expression of tumor suppressor p53 abrogated CSC properties of CSC-like cells. Furthermore, we identified specific stem cell surface markers CD24low and CD133high that are associated with SWCNT-induced CSC formation and tumorigenesis. CONCLUSIONS Our findings provide new and compelling evidence for the acquisition of CSC-like cells induced by chronic SWCNT exposure, which are likely to be a major driving force for SWCNT tumorigenesis. Thus, our study supports prudent adoption of prevention strategies and implementation of exposure control for SWCNT. We also suggest that the detection of CSC and associated surface markers may provide an effective screening tool for prediction of the carcinogenic potential of SWCNT and related nanoparticles.

Title: A single residue determines the cooperative binding property of a primosomal DNA replication protein, PriB, to single-stranded DNA. Content: PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)25, and (StPriB)25 are proposed.

Title: Determination of chloramphenicol residues in milk by enzyme-linked immunosorbent assay: improvement by biotin-streptavidin-amplified system. Content: A sensitive biotin-streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed for the determination of chloramphenicol residues in milk. The biotin-streptavidin system was applied to enhance the sensitivity. After optimization, the detection limit of the method was found to be 0.042 +/- 0.006 ng mL(-1), which is 8-fold more sensitive than the traditional competitive ELISA using the same antibody and coating antigen. The amplification mechanism of the biotin-streptavidin system and the major factors affecting the sensitivity of detection are discussed. This method was successfully applied to determine the chloramphenicol residues in milk samples with a simple and rapid extraction procedure, and good recoveries (85.66-109.67%) were obtained. The result indicated that the biotin-streptavidin system may be a valuable tool to improve the specific detection of trace veterinary drug residues and could be widely used for routine monitoring of food samples.

Title: Accurate coarse-grained modeling of red blood cells. Content: We develop a systematic coarse-graining procedure for modeling red blood cells (RBCs) using arguments based on mean-field theory. The three-dimensional RBC membrane model takes into account the bending energy, in-plane shear energy, and constraints of fixed surface area and fixed enclosed volume. The coarse-graining procedure is general, it can be used for arbitrary level of coarse-graining and does not employ any fitting parameters. The sensitivity of the coarse-grained model is investigated and its behavior is validated against available experimental data and in dissipative particle dynamics (DPD) simulations of RBCs in capillary and shear flows.

Title: Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Content: We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.

Title: Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes. Content: The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Muller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+ -buffering action of brain astrocytes in a region-specific manner.

Title: Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Content: Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research.

Title: Purification, characterization, and sequence analysis of a potential virulence factor from Porphyromonas gingivalis, peptidylarginine deiminase. Content: The initiation and progression of adult-onset periodontitis has been associated with infection of the gingival sulcus by Porphyromonas gingivalis. This organism utilizes a multitude of virulence factors to evade host defenses as it establishes itself as one of the predominant pathogens in periodontal pockets. A feature common to many other oral pathogens is the production of ammonia due to its protective effect during acidic cleansing cycles in the mouth. Additionally, ammonia production by P. gingivalis has been proposed as a virulence factor due to its negative effects on neutrophil function. In this study, we describe the first purification of a peptidylarginine deiminase (PAD) from a prokaryote. PAD exhibits biochemical characteristics and properties that suggest that it may be a virulence agent. PAD deiminates the guanidino group of carboxyl-terminal arginine residues on a variety of peptides, including the vasoregulatory peptide-hormone bradykinin, to yield ammonia and a citrulline residue. The soluble protein has an apparent mass of 46 kDa, while the DNA sequence predicts a full-length protein of 61.7 kDa. PAD is optimally active at 55 degrees C, stable at low pH, and shows the greatest activity above pH 9.0. Interestingly, in the presence of stabilizing factors, PAD is resistant to limited proteolysis and retains significant activity after short-term boiling. We propose that PAD, acting in concert with arginine-specific proteinases from P. gingivalis, promotes the growth of the pathogen in the periodontal pocket, initially by enhancing its survivability and then by assisting the organism in its circumvention of host humoral defenses.

Title: Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study. Content: CONTEXT The mechanisms that drive progression from fatty liver to steatohepatitis and cirrhosis are unknown. In animal models, obese mice with fatty livers are vulnerable to liver adenosine triphosphate (ATP) depletion and necrosis, suggesting that altered hepatic energy homeostasis may be involved. OBJECTIVE To determine if patients with fatty liver disease exhibit impaired recovery from hepatic ATP depletion. DESIGN Laboratory analysis of liver ATP stores monitored by nuclear magnetic resonance spectroscopy before and after transient hepatic ATP depletion was induced by fructose injection. The study was conducted between July 15 and August 30, 1998. SETTING University hospital. PATIENTS Eight consecutive adults with biopsy-proven nonalcoholic steatohepatitis and 7 healthy age- and sex-matched controls. MAIN OUTCOME MEASURE Level of ATP 1 hour after fructose infusion in patients vs controls. RESULTS In patients, serum aminotransferase levels were increased (P = .02 vs controls); albumin and bilirubin values were normal and clinical evidence of portal hypertension was absent in both groups. However, 2 patients had moderate fibrosis and 1 had cirrhosis on liver biopsy. Mean serum glucose, cholesterol, and triglyceride levels were similar between groups but patients weighed significantly more than controls (P = .02). Liver ATP levels were similar in the 2 groups before fructose infusion and decreased similarly in both after fructose infusion (P = .01 vs initial ATP levels). However, controls replenished their hepatic ATP stores during the 1-hour follow-up period (P<.02 vs minimum ATP) but patients did not. Hence, patients' hepatic ATP levels were lower than those of controls at the end of the study (P = .04). Body mass index (BMI) correlated inversely with ATP recovery, even in controls (R = -0.768; P = .07). Although BMI was greater in patients than controls (P = .02) and correlated strongly with fatty liver and serum aminotransferase elevations, neither of the latter 2 parameters nor the histologic severity of fibrosis strongly predicted hepatic ATP recovery. CONCLUSIONS These data suggest that recovery from hepatic ATP depletion becomes progressively less efficient as body mass increases in healthy controls and is severely impaired in patients with obesity-related nonalcoholic steatohepatitis.

Title: Age-related behaviors have distinct transcriptional profiles in Caenorhabditis elegans. Content: There has been a great deal of interest in identifying potential biomarkers of aging. Biomarkers of aging would be useful to predict potential vulnerabilities in an individual that may arise well before they are chronologically expected, due to idiosyncratic aging rates that occur between individuals. Prior attempts to identify biomarkers of aging have often relied on the comparisons of long-lived animals to a wild-type control. However, the effect of interventions in model systems that prolong lifespan (such as single gene mutations or caloric restriction) can sometimes be difficult to interpret due to the manipulation itself having multiple unforeseen consequences on physiology, unrelated to aging itself. The search for predictive biomarkers of aging therefore is problematic, and the identification of metrics that can be used to predict either physiological or chronological age would be of great value. One methodology that has been used to identify biomarkers for numerous pathologies is gene expression profiling. Here, we report whole-genome expression profiles of individual wild-type Caenorhabditis elegans covering the entire wild-type nematode lifespan. Individual nematodes were scored for either age-related behavioral phenotypes, or survival, and then subsequently associated with their respective gene expression profiles. This facilitated the identification of transcriptional profiles that were highly associated with either physiological or chronological age. Overall, our approach serves as a paradigm for identifying potential biomarkers of aging in higher organisms that can be repeatedly sampled throughout their lifespan.

Title: Identification of a Multicomponent Complex Required for Outer Membrane Biogenesis in Escherichia coli Content: Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.

Title: Lessons learnt from malaria epidemics in the Islamic Republic of Iran. Content: The Islamic Republic of Iran is in the pre-elimination phase of malaria control, but malaria epidemics are still a concern in the south of the country. This retrospective study presents the epidemiological characteristics and predisposing factors of 60 of the malaria epidemics reported in Sistan va Baluchestan province during 2005-09. A zero-truncated negative binomial model was used to investigate the relation between predictor variables and the total number of malaria cases. Malaria epidemics occurred mainly in the southern part of the province, mostly between July and October, peaking in August. Most malaria epidemics were small-scale (68.3% were < 100 cases) and short (51.7% lasted < 1 month). Plasmodium falciparum was present in 46.7% of the epidemics. An increase in the rainfall rate as well as population movements were the most significant predisposing factors. The results may help inform an epidemic investigation and reporting system as the country approaches the malaria elimination phase.