Metabolomics approaches and applications in prostate cancer research.
Prostate cancer is a leading cause of cancer deaths in men worldwide. Although prostate-specific antigen (PSA) has been extensively used as a serum biomarker to detect prostate cancer, this screening method has suffered from a lack of specificities and sensitivities. The successful prevention and treatment of prostate cancer relies on the early and accurate detection of the disease; therefore, more sensitive biomarkers are urgently needed. Prostate has long been known to exhibit unique metabolite profiles, fortunately, metabolomics, the study of all metabolites produced in the body, can be considered most closely related to a patient's phenotype. It may provide clinically useful biomarkers applied toward identifying metabolic alterations in prostate cancer and has introduced new insights into the pathology of prostate cancer. This advanced bioanalytic method may now open door for diagnostics. Metabolomics has a great and largely potential in the field of disease, and the analysis of the cancer metabolome to identify novel biomarkers and targets can now be undertaken in many research laboratories. In this review, we take a closer look at the metabolomics in the field of prostate cancer and highlight the interesting publications as references for the interested reader.


[Mitosis in the free-living flagellate Bodo saltans strain PS+ (Kinetoplastidea, Bodonida)].
The mitosis in the free-living flagellate Bodo saltans Ps+ with prokaryotic cytobionts in perinuclear space has been studied. The nuclear division in B. saltans Ps+ occurs by closed mitosis type without condensation of chromosomes. Two spatially separated mitotic spindles begin to form consistently at the initial stages of nuclear division. The spindle including about 20 microtubules appears first and later the second spindle with half the number of microtubules comes at the angle of 30-40 degrees. Both spindles rest their ends against the inner nuclear membrane and form 4 distinct poles. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one are associated with 2 pairs of kinetochores. The divergence of the kinetochores towards the poles occurs independently in each spindle. The equatorial phase is not revealed in B. saltans Ps+. The poles of both spindles unite in pairs at the elongation phase of mitosis and form the integrated bipolar structure. At this stage of the nuclear division, the kinetochores reach the poles of subspindles and become indistinguishable. Then the nucleus takes the shape of a dumbbell. The inner nuclear membranes of just formed nuclei have layers of condensed chromatin characteristic of the interphase nuclei of kinetoplastidea. The daughter nuclei separate at the phase of reorganization. There are 1-2 prokaryotic endocytobionts in the perinuclear space of the interphase nuclei in B. saltans Ps+. The symbionts multiply during mitosis and their number reaches more than 20 specimens par nucleus.


Singapore telcos pick Nokia, Ericsson over Huawei to build main 5G networks - 80mph
https://www.reuters.com/article/us-singapore-telecoms-5g/singapore-telcos-pick-nokia-ericsson-over-huawei-to-build-main-5g-networks-idUSKBN23V1PG
======
mytailorisrich
Singapore tends to have good relations with China. I'm sure everyone will have
a piece of the cake


GOP Congress must remember why it was elected

"I've never seen anything quite like it," said a political-insider friend of mine as we sipped coffee.

"What is it?" I said.

"Several members of the Republican-controlled House and Senate are suffering from a rare form of memory loss. Until recently, I fear, they completely forgot why they were elected."

"Explain, please."

"When Republicans took over the House in 2011, they did so because the majority of Americans were fed up with ObamaCare, wasteful spending and a federal government that was running amok. They wanted the Republican House to thwart President Obama's policies."

"I remember the 2010 election well," I said.

"During the 2014 election, the Republicans took back the Senate because the majority of American people were fed up with ObamaCare, wasteful spending and a federal government that was running amok. They wanted the Republican Senate to work with the Republican House to overturn Obama's polices."

"There was a sense of great hope at that time," I said.

"Then in 2016, despite several 'expert' predictions that Hillary Clinton would become America's first female president, Donald Trump was elected. That's because the people in most U.S. states wanted to repeal and replace ObamaCare, get federal spending under control and overturn Obama's policies — including many executive orders that many considered presidential overreach at best and unconstitutional at worst."

"Well, Trump has been successful overturning Obama's executive orders, but he hasn't enjoyed much success passing big bills through the Republican House and Senate."

"That is correct, and I fear it is due to mass memory loss. You see, too many Republicans seem to have forgotten that they had promised to repeal ObamaCare, but repeal efforts failed repeatedly."

"That's frustrating to the voters who put Republicans in power."

"Trump has been struggling to get any big bills through Congress, including his infrastructure bill, despite the fact that Republicans own the White House, House and Senate. I figure they have one chance to wake up and remember why they are there or we are all cooked."

"What is their one chance?

"They must come together to give the country its first comprehensive tax reform since President Reagan reformed the tax code, and they'd better do it this year!"

"Hear, hear! How are they doing?"

"A cynic like me is afraid to get too hopeful, but it would appear they are finally making real headway on real tax reform."

"I'm afraid to be hopeful, too. What does their plan include?"

"Investor's Business Daily reports that the Republican reform plan includes reducing America's outdated corporate tax rates, which are among the highest in the world. They plan a top corporate tax-rate reduction to 20 percent, which would unleash significant economic growth, create new jobs and increase wages for millions of Americans."

"Bring it on."

"The reform plan would introduce other commonsense measures to reduce rates for small businesses, while allowing them to write off business costs faster. As it is now, they have to depreciate many investments over many years - thus, it takes them many years to recoup their costs through tax savings. Such a simple move would spur significant investment and growth, benefiting everyone."

"Bring that on, too."

"Tax filing would be simplified for everyone, with fewer brackets and increased standard deductions and child credits. Democrats are rolling out tired old lines about the rich getting huge tax breaks while the middle class gets the shaft, but it is just not so. The fact is, the real commonsense reform that Republicans are working on will hopefully return our country to a massive 5- or 6-percent growth rate, which will benefit us all. But it will only happen IF Republicans overcome mass memory loss and remember why they were elected in the first place!"


Actinomycetospora rishiriensis sp. nov., isolated from a lichen.
An actinomycete, strain RI109-Li102(T), was isolated from a lichen sample obtained from Rishiri Island in Japan. Cells of strain RI109-Li102(T) were Gram-positive, aerobic and non-motile and formed bud-like spore chains. The isolate grew with 0-3 % (w/v) NaCl, at pH 5-9 and at 10-30 °C (optimum 30 °C). The whole-cell hydrolysate contained meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone was MK-8(H(4)) and the diagnostic phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were iso-C(16 : 0) and iso-C(16 : 1) H. Comparative 16S rRNA gene sequence analysis revealed that strain RI109-Li102(T) was most closely related to Actinomycetospora corticicola 014-5(T) (99.0 % rRNA gene sequence similarity) and Actinomycetospora chiangmaiensis YIM 0006(T) (98.4 %). However, DNA-DNA hybridization assays, as well as physiological and biochemical analyses, showed that strain RI109-Li102(T) could be differentiated from its closest phylogenetic relatives. It is proposed that strain RI109-Li102(T) ( = NBRC 106356(T) = KCTC 19782(T)) be classified as the type strain of a novel species, with the name Actinomycetospora rishiriensis sp. nov.


Hamas has crticized US President George W. Bush for dropping any commitment to a fixed timetable for the creation of a Palestinian state and warned against pressure to disarm "resistance" factions.

Hamas stated Friday that Bush broke his promise during his Thursday's talks with Palestinian leader Mahmud Abbas at the White House. "Bush said he wanted to see a Palestinian state, but without a timetable," Hamas spokesman Sami Abu Zuhri told ,i>AFP in Gaza City. "He went back on his last promise," Abu Zuhri noted.

On Thursday, Bush said he believed his two-state vision was possible but acknowledged he did not know if a Palestinian state would be created by the time he left office in January 2009.

The Hamas spokesman also slammed Bush's call for Abbas to crack down on "armed gangs". "Bush focused on putting pressure on the Palestinian Authority to confront the factions. This is dangerous," Zuhri warned.


Schools face new pay disclosure requirements

Saturday

Sep 26, 2009 at 12:01 AMSep 26, 2009 at 4:13 PM

llinois school officials have extra things to contend with this academic year besides test scores and unruly students. They also must comply with a couple of new laws requiring them to more readily provide information about educators' salaries and other forms of compensation.

ADRIANA COLINDRES

Illinois school officials have extra things to contend with this academic year besides test scores and unruly students. They also must comply with a couple of new laws requiring them to more readily provide information about educators' salaries and other forms of compensation.

The lawmakers who pushed the pair of measures say they were motivated by a desire for greater transparency in government. Some also said they were inspired by news reports about how some Chicago-area school boards were quietly awarding large perks to top administrators.

"We were finding that a number of superintendents would be given huge bonuses and huge boosts on their final salaries, and that would increase their pensions - sometimes doubling it, almost," said Rep. Monique Davis, a Chicago Democrat who sponsored one of the plans.

Sen. Kirk Dillard, a Hinsdale Republican who co-sponsored one of the new laws, said the public needs to know "the total cost of a superintendent."

"I have a sneaking suspicion that some school districts - not all -- hide the true perks and compensation package for their superintendents," he said. "Teachers are not as much of a mystery, and they don't have all the perks that administrators have."

The two laws impose different sets of rules, and they have different implementation dates.

Under the plan previously known as Senate Bill 2270, every school district must post on its Internet site a compensation report for every employee who holds an administrative certificate and who works in that capacity. The report must include such details as base salary, bonuses, pension contributions, the cost of health insurance and any other form of compensation.

The report must be posted by Oct. 1 of every year, starting on Thursday.

Under House Bill 2235, every school board must report the base salary and benefits of the school superintendent, all administrators and teachers by July 1, starting in 2010. The information, including vacation and sick days, bonuses and retirement enhancements, will be sent to the State Board of Education.

Public universities and community colleges must report similar information about their administrators, faculty members and instructors to the Illinois Board of Higher Education.

The State Board of Education already makes salary data for educators available to the public through Freedom of Information Act requests, said spokeswoman Mary Fergus.

Information about public university employees' salaries presently may be obtained through the individual institutions. Once that data is reported to the Board of Higher Education, the agency hopes to post it on the Web, said spokesman Don Sevener.

The new laws have generated a mixed reaction from the education community.

A spokesman for the Illinois Education Association, a teachers' union, said disclosing information about compensation "is not an issue that makes all of our members happy."

"Some people are comfortable with it. Others are not," said spokesman Charles McBarron. "On the other hand, I think everybody understands the concept of public employees and tax dollars."

Individual teachers who were contacted in the past few days said they recognize that they're public employees and that taxpayers provide the dollars used to compensate them.

"I don't see a problem," said Bob Darling, who teaches at Peoria Richwoods High School.

But he said salary numbers for teachers can be misleading because they don't spell out how many hours teachers work or what extra duties they handle.

Davis said it shouldn't be difficult for school districts to put together the information.

"I don't foresee any obstacles because all school systems, no matter how poor they are, they have to use computer technology," she said.


Introduction {#sec1}
============

Insect-pathogen association can be seen as an evolutionary arms race where pathogen evolves to optimize host infection and its dispersion; in turn, the host (insect) activates immune response involving humoral and cellular reaction to defense against the infection. Being devoid of acquired immunity, insects developed efficient innate immune system (IIS) ([@ref18]) which rely on various mechanisms: such as phagocytosis, activation of proteolytic cascades including coagulation and melanization as well as production of different antimicrobial peptides (AMPs) ([@ref40]; [@ref2]; [@ref5]). AMPs involved in insect humoral defense reactions and were rapidly secreted into the hemolymph upon microbial infection ([@ref26]). These AMPs can serve as novel alternatives to traditional antibiotics for the treatment of pathogenic microorganisms due to their broad spectrum of activity, less potential for resistance development, and capability to modulate the host immune response ([@ref31]; [@ref30]).

To date, a large number of insect AMPs have been identified and classified into four groups according to their basic structure, including the linear α-helical (cecropin and moricin), glycine-rich (attacin and gloverin), proline-rich (drosocin, and lebocin), and cysteine-rich peptides (insect defensin and drosomycin) ([@ref3]; [@ref7]; [@ref53]). A broad range of AMPs including cecropins, moricins, gloverins, attacins, lebocins, and defensins have been reported in Lepidopteran insects ([@ref38]; [@ref9]). Moricins are important α-helical cationic AMPs which play an important role in insect humoral defense reactions. This type of peptide was initially isolated from the hemolymph of *E. coli* immunized *Bombyx mori* larvae (Bm-moricin A1) and contains 42-amino-acid peptide ([@ref14]). Later on, Bm-moricin2 and other moricin-like variants were also reported in the *B. mori* genome ([@ref52]; [@ref9]). Recently, several moricin genes were identified in *Galleria mellonella* ([@ref6]) and *Manduca sexta* ([@ref57]), and another moricin analogues peptide was isolated from *Spodoptera litura* ([@ref35]) and *Helicoverpa armigera* ([@ref49]). Previous studies reported that the AMP moricin has a high activity against Gram-positive and Gram-negative but is less active against yeasts. Moricin has been shown to be membrane active as it affects the permeability of cytoplasmic membranes ([@ref14]). In addition, an artificial moricin gene was synthesized and expressed in *Escherichia coli* ([@ref15]; [@ref43]), and it was confirmed that the physical and biological characteristics of the recombinant moricin were identical with their natural counterparts. The recombinant moricin efficiently suppressed the growth of methicillin-resistant *Staphylococcus aureus* ([@ref15]). But in-depth studies of the interaction between moricin with the fungal pathogen are not yet performed.

The black yeast-like fungi *Aureobasidium pullulans*, can produce yeast-like hyaline conidia as well as a filamentous growth form ([@ref47]). It is also known as an important producer of the biodegradable extracellular polysaccharide (EPS) pullulan ([@ref55]). Despite the biotechnological significance, *A. pullulans* is considered as an opportunistic human pathogen and causes illness in immunocompromised patients ([@ref8]). It also causes a variety of localization as well as rare systemic infections in humans ([@ref16]) including diseases such as catheter-related septicemia ([@ref20]), fungemia, nosocomial infection ([@ref1]), abscess in the spleen, peritonitis ([@ref10]), pneumonia, meningitis, corneal ulcer ([@ref23]), invasive pulmonary infection, scleral infection ([@ref36]), cutaneous infection ([@ref24]) and asthma ([@ref34]). Its increased indoor concentrations have also been correlated to various health symptoms ([@ref44]). Therefore, a potential antimicrobial agent is needed to control this opportunistic human pathogen.

In the present study, we report the characterization of a new antibacterial peptide moricin (Px-Mor) from the lepidopteran insect, *P. xylostella*. Furthermore, we investigated the antimicrobial activity and mechanism of the purified recombinant Px-Mor against the mycelia of the opportunistic human pathogen *A. pullulans*.

Materials and Methods {#sec2}
=====================

Insects and Microorganisms {#sec3}
--------------------------

The larvae of *P. xylostella* were collected from the vegetable fields of South China Agricultural University. The insects were reared on cabbage mustard at 25 ± 2°C, 60--70% RH for a 16:8 h light/dark photoperiod. Microorganisms including Gram-positive bacteria (*Staphylococcus aureus*, *Bacillus cereus*, and *B. subtilis*); Gram-negative bacteria (*E. coli DH5α*, *E. coli K12D31*, *Pseudomonas fluorescent*, and *Salmonella choleraesuis*); and fungi (*A. pullulans*, *Aspergillus flavus*, *Botrytis cinerea*, *Peronophythora litchi*, *Aspergillus fumigatus*, and *Fusarium oxysporum*) were obtained from the Research Institute of Microbiology, Guangzhou, China. *E. coli* DH5a, BL21 (DE3), and *B. thuringiensis* were grown in LB broth at 37°C until they reached the mid-log (2--7 × 10^5^CFU/ml) phage. Whereas *A. pullulans*, was grown on potato dextrose agar (PDA) plates and incubated at 26 ± 2°C for 10 days.

RNA Extraction and Reverse Transcription {#sec4}
----------------------------------------

Total RNA was extracted from the composite sample of four larvae (each instar), four pupae and four adults of *P. xylostella* using Trizol reagent following the manufacturer's protocol. First-strand cDNA was synthesized by oligo-dT~18~ primer and Super-script III Reverse Transcriptase (TaKaRa, Japan) in reaction volume of 20 μl. The reaction was incubated at 42°C for 60 min, and then was maintained at 70°C for 15 min. The resultant cDNA was used as the template for ORF sequence amplification. The total RNA, which was extracted from the 4^th^ instar larvae of *P. xylostella* after immunization with heat-inactivated *B. thuringiensis*, was used to synthesize 5′- and 3′-cDNA. The 5′- and 3′-cDNA were obtained by using SMARTer RACE 5′/3′Kit (Clontech, USA) according to the manufacturer's protocol. Prior to RNA extraction from different tissues, hemocyte, fat body, Malpighian tubule and midgut were isolated from the 4^th^ instar larvae which were induced by PBS, and *E. coil*, heat-inactivated and active *B. thuringiensis*. The synthetic cDNA of these samples was served as a template for real time PCR.

Cloning the Full-Length cDNA of Px-Mor {#sec5}
--------------------------------------

Px-Mor unigene sequences, which were obtained from *P. xylostella* transcriptome, were verified through RT-PCR. PCR was performed with forward primer Moricin F:5′-CTTCCACTTGCTGATGCTGGCGC-3′ and reverse primer Moricin R:5′-CTGTTCCGCACGTGGTTGTACAC-3′ by the following reactions: 94°C 5 min; 94°C, 30 s, 59°C, 30 s, 72°C, 30 s, 30 cycles; 72°C, 8 min. Amplified PCR products were detected on a standard 1.0% (w/v) agarose gel and purified by E.Z.N.A.™ Gel Extraction kit (OMEGA, USA). The purified PCR product was cloned into pMD18-T vector (TaKaRa, Japan) as per the manufacturer's instructions. Both 5′- and 3′ Rapid Amplification of cDNA Ends reactions were primed with the gene specific primer and the universal primer mix (UPM) supplied in SMARTer RACE 5′/3′Kit (Clontech, USA). The nested gene specific primers, which contained two 5′-GSP and two 3′-GSP, were designed based on the verified unigene sequences of Moricin. In 5′-RACE reaction, the two 5′-GSP, which consisted of 5′-Moricin R1: 5′-GCGTTGACGTTGACCTTGGGCGCGG-3′ and 5′-Moricin R2: 5′-GCAGCGTCATGGCCGCCAGCGCCAGC-3′, were the reverse primers, while the UPM was the forward primer. In 3′-RACE, the two 3′-GSP, which contained 3′-Moricin F1:5′-CGCGCCCAAGGTCAACGTCAACGCC-3′ and 3′-Moricin F2: 5′-GGGACG GCGCATGAAGTGTACAACC-3′, were the forward primers. The RACE-PCR was carried out by the touchdown PCR program as follows: after denaturation for 2 min at 94°C, 5 cycles at 94°C for 30 s, 70°C for 30 s and 72°C for 1 min were used, continuing with 30 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 1 min, and finally 72°C for 8 min. Nested PCR reactions were performed using the 50 times diluted product of the first PCR under the conditions described above with each gene specific primers and NUP (UPM short) supplied in the kit. Generated fragments of the 5′- and 3′-RACE-PCR were cloned into pMD18-T vector (TaKaRa, Japan).

Sequences Analysis of Moricin {#sec6}
-----------------------------

The obtained cDNA sequence were blasted to NCBI through the Internet to estimate the degree of homology to known antibacterial peptides. Other reported Lepidoptera Moricin sequences were retrieved from the GenBank database[^1^](#fn001){ref-type="fn"} for phylogenetic analysis. The signal peptide was analyzed by SignalP[^2^](#fn002){ref-type="fn"} and the theoretical isoelectric point (pI) was calculated by the compute pI software[^3^](#fn003){ref-type="fn"}. The predicted molecular weight, and charges of the protein at pH 7.00 were estimated using Protein Calculator v3.3[^4^](#fn004){ref-type="fn"}. The sequences were aligned in Clustal X. 2.0[^5^](#fn005){ref-type="fn"}. The phylogenetic tree was generated using the neighbor-joining method in MEGA 6.0 ([@ref45]). The secondary and tertiary structure was predicted by the homology modeling method on Phyre 2[^6^](#fn006){ref-type="fn"}.

RT-qPCR of Px-Mor {#sec7}
-----------------

Assessment of the mRNA expression level of Px-Mor during the developmental stages in 4^th^ instar *P. xylostella* larvae was carried out after the induction of inactivated *E. coil*, *B. thuringiensis*, *A. pullulans*, and PBS (as bacteria control), Tween-80 (as fungi control). Fat body and hemocytes were isolated from the inactivate pathogen induced *P. xylostella* larvae after 0, 6, 12, 18, 24, 30, 36, 42, and 48 h followed by wash three times using 1 × PBS buffer. The primers for RT-qPCR amplification (F: 5′-TTTGATGCTGGCTCTGGT GG-3′ R: 5′-GCCCTTCTTGAGTGCGTTGA-3′) were designed according to ORF of moricin genes. The final reaction mixture contained 1 μl of each primer, 12.5 μl of SYBR® Green PCR Master Mix, and 2 μl cDNA. The reaction were subjected to: 95°C for 3 min followed by 39 cycles at 95°C for 10 s, 58°C for 30 s, and ending with 95°C for 10 s. The partial fragment of the muscle-actin gene (NCBI accession no. AB282645) was served as an internal control and amplified from the same cDNAs with the Actin qF/qR primers. Melting curve analysis was applied to all reactions to ensure homogeneity of each reaction product. Three biological replications (*n* = 3) were performed for the each reaction and the2^−ΔΔCt^ method was used to measure the relative transcription levels.

Construction of Expression Vectors pMTHisAPx-Mor {#sec8}
------------------------------------------------

The cDNA sequence encoding mature Px-Mor was amplified by PCR using primers, i.e., Px-Mor F:CCG***AGATCT***ATGAGATTCTTGCACTTGCTGATGCTGGCG and Px-Mor R:GCC***ACCGGT***CCCCTGGTTCCTGTTCCGCACGT. The purified PCR fragments were digested with Bgl II and AgeI enzymes and ligated into the Bgl II /AgeI-digested expression vector pMT/BiP/V5-HisA (Invitrogen, USA) and transformed into the competent cells DH5α. The recombinant expression plasmid pMTV5HisAPx-Mor was confirmed by DNA sequencing. The positive plasmid was transient transfected into *Drosophila Schneider* S2 and the expression was further confirmed by SDS-PAGE and western blot analysis. For transfection assay, *Drosophila Schneider* S2 cells were maintained at 28°C in incubator and seeded overnight in serum-free medium, and Lipofectamine 2000 (Invitrogen, USA) was used for transfection based on the protocol. In order to generate stable S2 cells expressing Px-Mor, the recombinant expression vectors pMTHisAPx-Mor was transfected into S2 (5 × 10^6^ cells/ml). The pCoBlast plasmid was used for stable selection. Calcium Phosphate Transfection kit (Invitrogen, USA) was used for the transfection of the S2 cells. The cells were further transferred into a selection medium containing 10 μg/ml blasticidin. The blasticidin-resistant clones appeared in about 2 weeks and were further expanded in selection medium to express the Px-Mor in S2 cells with the induction of 500 μM copper sulfate. These cultures were harvested after 7 days cultivation. The media and cell lysate were obtained by centrifugation at 800 rpm for 3 min. And the expression protein was analyzed by SDS-PAGE and western blot.

Expression and Purification of Recombinant Protein Px-Mor {#sec9}
---------------------------------------------------------

Large scale expression of Px-Mor were carried out by expanding the stably transfected S2 cells in large suspension cultures. In 150-cm^2^ flasks, cell culture medium was collected every 24 h after copper sulfate (final concentration 500 μM) induction. The cell culture medium was collected and combined 10 days collection, and centrifuged with 3,000 rpm 10 min at 4°C, the debris was removed and cell free medium was collected. This cell free medium was then thoroughly dialyzed multiple times using an 3 kDa molecular weight cut off, membrane tubing into a buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 1% glycerol to completely remove the copper ions. About 1 ml His Mag TM Agarose Bead (Novagen, USA) was used to purify the protein. Fractions were analyzed by Tricine-SDS-PAGE. All the fractions containing Px-Mor were collected, concentrated with PEG2000, dialyzed in ddH~2~O.

Synthesis of Px-Mors and Melittin {#sec10}
---------------------------------

According to the mature peptides amino acids, Px-Mor, Px-Mor113/114, Px-Mor115, and melittin were synthesized using the solid phase method and standard 9-fluorenyl methoxy carbonyl chemistry and purified to \>95% purity using reverse-phase high-pressure liquid chromatography at the Peptide Synthesis Core Facility of the Qiangyao Biotechnology Company (Shanghai, China). The C-terminus was amidated to prevent degradation and improve the activity of the proteins.

Antimicrobial Activity Assays {#sec11}
-----------------------------

The antimicrobial activity of the purified recombinant Px-Mor was tested against several GPB, GNB, and fungi. The MICs was determined with the reference to the Clinical and Laboratory Standards Institute guidelines ([@ref50]). The minimal growth inhibition concentration ([@ref46]) was analyzed using a liquid growth inhibition assay and expressed as the lowest concentration of the peptide at which no growth was found, while the activity was determined by monitoring the absorbance at 494 nm.

Hemolytic Assay {#sec12}
---------------

The hemolytic activity of the recombinant protein Px-Mor and synthesized Px-Mor, Px-Mor113/114, Px-Mor115, and Melittin were performed using human red blood cells (RBCs) isolated from heparinized blood ([@ref22]). The HBCs were prepared from freshly collected human blood (4 ml) by centrifugation at 1,500 rpm for 10 min at 4°C. The cells were washed three times with 0.01MPBS (NaCl 0.8 g/L, KCl 0.2 g/L, KH~2~PO~4~ 0.2 g/L, and Na~2~HPO~4~ 1.15 g/L, pH 7.2) and suspended as 10% suspension in hematocrite solution. The human HBCs were incubated with dissolved in PBS protein (purified recombinant protein Px-Mor and synthesized Px-Mor, Px-Mor113/114, Px Mor115, and Melittin) for 1 h at 37°C. The concentrations of tested proteins were: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μM. The samples were centrifuged at 3,500 rpm for 5 min and the absorbance of the supernatant was measured at 414 nm with an ELISA plate reader. Zero hemolysis and 100% hemolysis were determined in PBS and Triton X-100, respectively.

Scanning Electron Microscopy and Transmission Electron Microscopy {#sec13}
-----------------------------------------------------------------

The electron microscopy was used to investigate recombinant moricin interacting with *A. pullulans* mycelia ([@ref34]). The mycelia were incubated with 30 μM recombinant proteins moricin at 28°C for 5 days, and then collected (0.5 cm × 0.5 cm) followed by a twice wash with 0.1 M phosphate buffer. The suspension were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 3 h, and washed three times with 0.1 M phosphate buffer followed by dehydration in ascending series of ethanol (50, 70, 80, 90, and 100%, 15 min each). Samples were dried for few seconds in room temperature and mounted on the SEM stubs using double-sided carbon tape. Dried samples were sputtered with the gold and analyzed with the SEM under Quanta 200 FEG at high-vacuum mode. For TEM analysis *A. pullulans* spores (1 × 10^6^ conidia/ml) were treated with 30 μM Px-Mor at 37°C for 30 min. Finally the supernatant was collected using centrifugation (5,000 *g* for 5 min) and cold glutaraldehyde (0.5%, w/v, in 0.1 M sodium cacodylate buffer, pH 7.4) was added and incubated for 2 h at 4°C.

Data Analysis {#sec14}
-------------

Figures were made with the GraphPad Prism 7.0. All the statistical analysis was conducted using the SPSS software; data were analyzed using Student's *t*-test for significant differences between the treatment groups and the control. One-way analysis of variation (ANOVA) followed by Tukey's multiple comparisons was used to compare the relative expression of Px-Mor transcript in different development stages or tissues.

Results {#sec15}
=======

Sequence Characteristics of Full-Length Px-Mor {#sec16}
----------------------------------------------

The RT-PCR approach performed with a pair of primers designed from *P. xylostella* transcriptome identified the cDNA fragment (Moricin). By RACE-PCR strategies, a full-length cDNA, encoding antimicrobial peptide gene Moricin, was cloned from *P. xylostella*. The nucleotide and deduced amino acid sequences are shown in [Figure 1](#fig1){ref-type="fig"}. The full-length cDNA of Moricin was 482 bp. The open-reading frame (ORF) is 198 bp in length and encodes a precursor with 65 amino acid residues, which we have named Px-Mor. The name was assigned according to the high identity of this protein with proteins from lepidopteran insects in BLAST searches. The sequence for Px-Mor have been deposited in GenBank under the accession number KF960047. The deduced amino acid sequence for moricin possesses a putative signal sequence of 23 amino acid residues. The predicted mature Px-Mor consists of 42 amino acid residues with a molecular mass of 4.393 kDa and a pI of 11.17. The secondary structure comprised α-helix and turns and its tertiary structure are shown in [Supplementary Figure S1](#SM1){ref-type="supplementary-material"}.

![Nucleotide and deduced amino acid sequences of *Px-Mor* cDNAs from the diamondback moth, *Plutella xylostella* (GenBank: No.KF960047). The stop codons are indicated by an asterisk. Putative signal peptides at the N-termini are underlined.](fmicb-10-02211-g001){#fig1}

We aligned the amino acid sequence of Px-Mor with moricins from *B. mori* and 14 other species of Lepidoptera. Px-Mor displayed high identities with moricin-like peptide A from *G. mellonella* and moricin-like peptide C4 from *Danaus plexippus*, sharing 64% identity, and 63% identity, respectively. A phylogenetic tree was constructed by neighbor joining method in [Figure 2](#fig2){ref-type="fig"}, which indicated homology of Px-Mor with several Lepidopteran's moricins. Px-Mor showed maximum homology with moricins from other lepidopteran insects, and was more close to *G. mellonella*, *D. plexippus*, and *B. mori*, which suggested that they might possess similar functions ([Figure 2](#fig2){ref-type="fig"}). We also aligned the amino acid sequence of Px-Mor with those predicted from the genome of *P. xylostella*. These uncharacterized proteins, which were referred to as Px010113, Px010114, and Px010115, showed 92.21 to 95.38 (based on the premature peptide) or 95.24 to 97.62 (based on the mature peptides) of the identities to Px-Mor ([Figure 2B](#fig2){ref-type="fig"}). The secondary structure and peptide characteristics of the Px-Mor analogues are shown in [Supplementary Table S1](#SM2){ref-type="supplementary-material"}.

![**(A)** Phylogenetic analysis of the lepidopteran moricin family. Computer-aimed phylogenetic analysis was done by the neighbor-joining method for amino acid sequences among moricin described above. A phylogenetic tree was constructed using Clustal X. Genbank accession numbers of the amino acid sequences used for the comparison are BAC79440 (*S. litura* moricin), AAT3887 (*S. exigua* moricin), BAB13508 (*B. mori* moricin), ADR51149 (*H. armigera* moricin), EHJ70108 (*D. plexippus* moricin), BAA77338 (*B. mori* moricin 2), AAW21269 (*H. puera* moricin II), AAO74637 (*M. sexta* moricin), AEM66445 (*B. mori* moricin B3), AEM66435 (*B. mandarina* moricin B3), ABQ42579 (*G. mellonella* moricin-like peptide C5), EHJ73174 (*D. plexippus* moricin-like peptide C4), ABQ42574 (*G. mellonella* moricin-like peptide B), ABQ42580 (*G. mellonella* moricin-like peptide D), ABQ42575 (*G. mellonella* moricin-like peptide C1), and ABQ42573 (*G. mellonella* moricin-like peptide A). Px010113, Px010114, and Px010115 come from *P. xylostella* genome. **(B)** Comparison of the predicted amino acid sequences of moricins from *P. xylostella*. The alignment of *Px-Mor*, Px010115, Px010114, and Px010113 was conducted using the DNAMAN. Px010115, Px010114, and Px010113 is the amino acid sequence of moricin from *P. xylostella* genome. The same amino acid is remarked with blue box. Before the arrows is the putative signal sequence of 23 amino acid residues and after the arrows is the mature peptide.](fmicb-10-02211-g002){#fig2}

Px-Mor Expression Patterns in *P. xylostella* {#sec17}
---------------------------------------------

To investigate the transcript levels of *Px-Mor* in different developmental stages of *P. xylostella*, a real-time PCR was performed with different cDNAs from egg, larva, prepupa, pupa and adult stages as the template. *Px-Mor* was expressed throughout the life cycle of *P. xylostella* ([Figure 3A](#fig3){ref-type="fig"}). The relative expression levels of *Px-Mor* were highest in fourth instar and prepupa stage, suggesting that moricin played different roles at different developmental stages. The main tissue of *P. xyostella* related to immune response, including hemocyte, midgut, fat body, Malpighian tubule and epidermis, were isolated from the fourth instar larvae of *P. xyostella* to synthesize cDNA templates. To determine the tissue distribution of moricin at mRNA level, RT-qPCR experiments were performed. Moricin was detected in all of the tissues, mainly expressed in hemocyte and fat body ([Figure 3B](#fig3){ref-type="fig"}).

![Spatial and temporal expression difference *Px-Mor* in *P. xylostella*. **(A)** Developmental expression patterns of *Px-Mor* in *P. xylostella* from egg to adult. **(B)** Tissue distribution of *Px-Mor* in the fourth instar larvae of *P. xylostella*. The temporal and spatial expression of *Px-Mor* transcript levels in *P. xylostella* as detected by SYBR green qRT-PCR. Comparison of the level of *Px-Mor* mRNA (relative to β-actin mRNA) was performed by *t* test. Data with different letters are significantly different (*p* \< 0.05) (DMRT) among different developmental stages and tissue distribution.](fmicb-10-02211-g003){#fig3}

The Relative Expression of Px-Mor Upon Infection With Different Microorganisms {#sec18}
------------------------------------------------------------------------------

The fourth instar larvae of *P. xyostella* were infected with heat-inactivated pathogen by microinjection technique. To determine whether heat-inactivated pathogen infection induced an immune response, we respectively extracted RNA of fatbody and hemocyte from larvae 0, 6, 12, 18, 24, 30, 36, 42, and 48 h post infection and probed the expression level of *Px-Mor* with specific primer by RT-qPCR. As shown in [Figure 4](#fig4){ref-type="fig"}, the expression level of *Px-Mor* was differently up-regulated after induction of microorganism in the tissues. The greatest up regulation was detectable in hemocyte and fat body. *Px-Mor* was highly expressed at the time point of 6 h after the infection with heat-inactivated *B. thuringiensis* in fat body ([Figure 4D](#fig4){ref-type="fig"}). In hemocyte, 30 h after *E. coli* infection, the expression level of *Px-Mor* gene rapidly increased to the maximum, which was 23-fold higher than that in the control group ([Figure 4C](#fig4){ref-type="fig"}). The bacteria *B. thuringiensis* can induce a strong expression of moricin in *P. xyostella*, rat two time points in fatbody, one after 12 h of infection and the other after 30 h ([Figure 4B](#fig4){ref-type="fig"}). The fungi *A. pullulans* also induced a strong expression of moricin in *P. xyostella*, which reached a high peak after 6 h infection and reached the maximum after 36 h in both fatbody and hemocyte ([Figures 4E](#fig4){ref-type="fig"},[F](#fig4){ref-type="fig"}). We suspected that *Px-Mor* is mainly expressed in changeable period of metamorphosis of prepupa development, which illustrated that Moricin have certain biological significance during the process of metamorphosis.

![Relative quantification of expression of moricin genes from hemocytes and fat body after injection with different microorganisms. Actin was used as an internal control; PBS and Tween-80 were used as tests control. **(A,B)** The relative expression of moricin after injection with *E. coli* in the hemocyte and fat body of *P. xylostella*. **(C,D)** The relative expression of Px-Mor after injection *B. thuringiensis* in the hemocyte and fat body in *P. xylostella*, respectively. **(E,F)**. The relative expression of moricin after injection with *A. pullulans* in the hemocyte and fat body of *P. xylostella*. Three biological replications (*n* = 3) were conducted, and the 2^−ΔΔCt^ method was used to measure the relative transcription levels. Means with different number letters are significantly different (*p* \< 0.05) (Duncan's Multiple Range Test) among different time after treated with *E. coli*, *B. thuringiensis*, and *A. pullulans*.](fmicb-10-02211-g004){#fig4}

Recombinant Expression and Purification of Px-Mor {#sec19}
-------------------------------------------------

Recombinant protein Px-Mor was produced as a fusion protein with a 6 × His tag. Stable S2 cells containing *Px-Mor* was expressed in S2 cells under the induction of 500 μM copper sulfate for a large scale, and the fusion peptide was purified to homogeneity using Ni^2+^-NTA column. The findings of the purified recombinant Px-Mor by Tricine-SDS-PAGE and western blot revealed a peptide of approximately 4.93 kDa which could strongly cross-react with anti-His antibody ([Figure 5](#fig5){ref-type="fig"}).

![Tricine-SDS-PAGE analysis of the purified recombinant Px-Mor. Lane M, polypeptide SDS-PAGE molecular weight markers; Lane 1, 2 μg purified recombinant Px-Mor was analyzed using Tricine-SDS-PAGE. The proteins was stained with Coomassie brilliant blue.](fmicb-10-02211-g005){#fig5}

Antimicrobial Spectrum of Px-Mors {#sec20}
---------------------------------

Px-Mor peptide from *P. xyostella* was tested against Gram-positive, Gram-negative and fungi. The results clearly illustrate that the peptides exhibit a high activity against the tested microbes ([Table 1](#tab1){ref-type="table"}). The most effective values were recorded from the purified recombinant Px-Mor against *Escherichia coli* K~12~D~31~ and *Escherichia coli* DH5α with MICs of 1.1 and 1.2 μM, respectively. Purified recombinant Px-Mor was also active against *A. pullulans* with the MIC value of 3.5 μM. Compared to the purified recombinant Px-Mor, the synthetic protein, Px-Mor, Px-Mor113/114, and Px-Mor 115 also exhibited a high activity against the tested target microorganisms. Px-Mor, Px-Mor113/114, and Px-Mor115 showed strong activities against *A. pullulans* with the MIC values of 3.6, 3.7, and 6.9 μM, respectively.

######

Antimicrobial activity of AMPs against microorganisms.

  Microorganisms              MIC (μM)
  --------------------------- ---------- ------ ------ -----
  **Gram-positive**
  *Staphylococcus aureus*     2.8        2.7    3.1    4.6
  *Bacillus cereus*           4.5        4.5    3.7    5.7
  *Bacillus subtilis*         6.3        6.4    6.7    8.2
  **Gram-negative**
  *Escherichia coli* DH5α     1.2        1.2    1.3    2.1
  *Escherichia coli* K12D31   1.1        1.0    1.4    1.9
  *Pseudomonas fluorescent*   4.6        4.6    3.2    5.8
  *Salmonella choleraesuis*   5.0        4.9    5.3    4.7
  **Fungi**
  *Aureobasidium pullulans*   3.5        3.6    3.7    6.9
  *Aspergillus flavus*        15         16     11     28
  *Botrytis cinerea*          23         22     25     36
  *Peronophythora litchi*     15.6       16.5   18.3   42
  *Aspergillus fumigatus*     21         20     25     37
  *Fusarium oxysporum*        8.9        8.8    9.8    24

*Data are collected as minimal inhibitory concentrations (MICs)a and expressed in μM*.

Hemolytic Assays of Px-Mors {#sec21}
---------------------------

Hemolytic assay results showed that all the Px-Mor analogues investigated had no hemolytic activity against the human red blood cells (RBCs). It suggested that none of the Px-Mor analogues were observed to be toxic to the human RBCs, comparing with melittin, which lysed 100% of the human RBCs even at a low concentration of 0.78 μM ([Table 2](#tab2){ref-type="table"}).

######

Hemolytic activity of recombinant and synthesized Px-Mor peptides.

  Peptides                      Percentage hemolysis (μM)
  ----------------------------- --------------------------- ----- ----- ----- ----- ----- ----- -----
  Px-Mor-His (recombinant)      0                           0     0     0     0     0     0     0
  Px-Mor113/114 (synthesized)   0                           0     0     0     0     0     0     0
  Px-Mor115 (synthesized)       0                           0     0     0     0     0     0     0
  Px-Mor (synthesized)          0                           0     0     0     0     0     0     0
  Melittin (synthesized)        100                         100   100   100   100   100   100   100

*The hemolytic activity of the peptides was evaluated by determining the hemoglobin release of 8% suspensions of fresh human erythrocytes at 414 nm*.

Effect of Px-Moron *A. pullulans* Morphology {#sec22}
--------------------------------------------

The effect of *Px-Mor* on the morphology of *A. pullulans* was determined using SEM. The treatment of *A. pullulans* mycelia with moricins at 30 μM for 3 h caused differences in the morphology. In the treatment with moricin, mycelia were short. Spores were wrinkled and showed blebbing as compared to the control ([Figure 6](#fig6){ref-type="fig"}). The morphological changes of *A. pullulans* spore after treatment with 30 μM Px-Mor was also examined using TEM. The graph shows that the cell membrane appeared to be wafery and the cellular cytoplasmic contents were dissolved and unclear ([Figure 7](#fig7){ref-type="fig"}). We also observed disruption in the cell membrane of *A. pullulans* spores by Px-Mor.

![SEM analysis of the hypha of *A. pullulans* interacted with Px-Mor. **(A)** Naive *A. pullulans hypha*, **(B)** the hypha of *A. pullulans* interacted with Px-Mor. The SEM analysis showed that *A. pullulans hypha* became perforation and ruptured **(B)** after interacted with moricin from *P. xylostella* as compared to the untreated hypha **(A)**, which had a bright and normal smooth surface.](fmicb-10-02211-g006){#fig6}

![TEM analysis of the hypha of *A. pullulans* interacted with Px-Mor. **(A)** Naive *A. pullulans* hypha; **(B)** the hypha of *A. pullulans* interacted with Px-Mor. The TEM images revealed that there are holes in the mycelium and the cellular cytoplasmic contents were dissolved and became vague **(B)**. The untreated spore **(A)** had a bright and normal smooth surface and the cellular cytoplasmic content was clear.](fmicb-10-02211-g007){#fig7}

Discussion {#sec23}
==========

AMPs are part of the innate immune response and present among all classes of life and show remarkable structural and functional diversity. They possess antimicrobial as well as immunomodulatory properties which make them important candidates for the development of novel therapeutics ([@ref12]; [@ref29]). The present study described the purification and cloning of Px-Mor, which can be classified in the lepidopteran moricin family, from *P. xyostella*. So far, moricins have been found only in Lepidopteran insects, and cDNAs encoding moricins have been identified in *B. mori* ([@ref13]), *M. sexta* ([@ref57]), *S. litura* ([@ref35]), *G. mellonella* ([@ref6]), *H. armigera* ([@ref49]), and *S. exigua* ([@ref25]). The numbers of amino acid residue of signal peptide or mature peptide and the molecular mass of Px-Mor were similar to those of the other AMPs under moricin family. The solution structure of moricin was determined to have a long α-helix of about eight turns along nearly the full length of the molecule except for several N-terminal and C-terminal residues ([@ref17]; [@ref35]; [@ref11]). The N-terminal part of the Px-Mor α-helix (residues 5--22) is an amphipathic section and responsible for the increasement in the permeability of the membrane to kill the bacteria, whereas the C-terminal region (residues 23--36) is a hydrophobic section and plays a critical for the antimicrobial activity of moricin ([@ref17]). This structure is similar to that of cecropins, except lacking a hinge region in moricins ([@ref53]). Hence, Px-Mor can be classified as a cecropin-type antibacterial peptide, which may play a similar role in the effective defense system of insects against invading pathogens. Furthermore, Px-Mor that derives from transcriptome displayed high identities with three moricins deduced from the genome ([@ref54]). Phylogenetic analysis indicated that Px-Mor have a high similarity to the other moricins identified from the Lepidopteran insects and have a strong evolutionary relationship with the moricin peptides from *G. mellonella*, *B. mori*, and *D. plexippus*, suggesting they might have derived from a common ancestral gene.

It was shown that silencing of the AMP *gloverin* reduces hatching rate in *B. mori* ([@ref32]). Similarly, RNAi in *S. exigua gloverin* gene decreases pupation and prolongs larval period ([@ref21]). In this study, Px-Mor mRNA was detected in different developmental stages of diamondback moth and in various tissues of the fourth instar larvae of *P. xyostella*. Px-Mor was highly expressed in fourth instar and prepupa stage, which differentially expressed comparing to other stages. These results suggest that, in addition to have antimicrobial activity, AMP Px-Mor also played a major role in the development. Px-Mor was strongly induced by *B. thuringiensis*, and the expression of Px-Mor reached a peak at 42 h post-infection in the fourth instar larvae, which illustrated that Px-Mor involved in the process of metamorphosis and immune response. In this study, moricin gene expression was observed in the Malpighian tubule as well as in midgut, epidermis, fat body and hemocytes. Px-Mor was mainly expressed in fat body and hemocyte. After induction by *B. thuringiensis*, the expression of Px-Mor reached a high level in the fat body at an earlier stage than in hemocyte. This supported the hypothesis that most of immune genes encoding antibacterial or antifungal peptides are induced by microbial infection, and synthesized predominantly in fat body and then subsequently released into hemolymph ([@ref26]).

Defense against Gram-positive bacteria and natural fungal infection is mediated by the Toll signaling pathway, whereas defense against Gram-negative bacterial infections is associated with the Imd pathway. It has been reported that expression of moricin can be regulated by the Toll pathway, nuclear factor-κB (NF-κB)/Rel, and GATA transcription factors ([@ref56]). [@ref9] identified NF-κB and GATA binding sites in the promoter regions of the moricin ([@ref9]). A 22-bp NF-κB-GATA cis-element associated with the enhanced activity of the AMP in *D. melanogaster* and *M. sexta* was also reported in the previous study ([@ref39]). In the present study, the expression levels of Px-Mor in hemolymph were rapidly increased by the injection of heat-inactivated *B. thuringiensis* and *A. pullulans*. In the other species, the expression of HaMor (moricin from *H. armigera*) was increased after fungal infection ([@ref49]). This implied that infection with *B. thuringiensis and A. pullulans* possibly activates the Toll pathway, which mainly responds to Gram-positive bacteria or fungi and the high expression levels of Toll and IMD pathways genes in pupae might be associated with high expression of AMPs ([@ref51]). This is not surprising as early study showed that the recombinant moricin expressed by *E. coli* has consistent activity with the native peptide ([@ref15]). In our earlier reports we cloned and expressed both native (Mdcec) and recombinant (Mdcec/6His with native N-terminus) *Musca domestica* cecropin A in *P. patoris*. The study found that both Mdcec and Mdcec/6His possess similar antimicrobial property against bacteria. While Mdcec/6His possess higher activity against fungal infection when compared with the native peptide and the results indicated that the 6His-tag has no negative effect of the protein activity ([@ref22]). It was shown that moricin from *B. mori* have a relatively higher activity against Gram-positive bacteria than cecropin B from *Hyalophora cecropia* which displayed a relatively higher activities against Gram-negative bacteria ([@ref19]). In the present study, to facilitate purification of Px-Mor, the C-terminal recombinant protein Px-Mor was also fused to His-tag and expressed in Drosophila S2 cells. Antimicrobial assays demonstrated that both native and recombinant Px-Mor had high activity against target microbes, and showed that the His-tag fused to the C-terminus had a positive effect on the activity. Similar results were found in previous study where His-tag containing recombinant protein showed high antimicrobial activity ([@ref28]). It was also reported that strategically placed histidine allows AMP with pH-dependent antimicrobial activity ([@ref27]). Addition of His-tag may increase the affinity of the recombinant proteins toward the bacterial membranes due to the increase in positive charges in C-terminal position ([@ref46]).

The *widespread use of antifungal agent* during the disease treatment often leads to the development of resistance among the fungal and other microbes which is a major concern mainly for the immunocompromised patients. Thus, the development of novel, safe and efficient antifungal agents is highly needed to combat with the increasing number of resistant fungal isolates ([@ref37]; [@ref41]; [@ref42]; [@ref4]). A wide range of new insect AMPs were reported in the last few years mainly due to the availability of the complete genome and transcriptomic data from diverse insect species and showed potentials against various microbial infections. These AMPs are considered as a potential source of alternative antibiotics to fight against antibiotic resistance ([@ref48]; [@ref33]). The reported native and recombinant Px-Mor in this study showed high activity against the treated microbial pathogens and can be further exploited for the development of new antifungal drugs against the opportunistic fungi *A. pullulans.*

In conclusion, this study described the characteristics of newly identified AMP Px-Mor from *P. xyostella* and evaluates its activity against Gram-positive and Gram-negative bacterial and fungal infection. Px-Mor was synthesized primarily in fat body and then released into hemolymph. Its expression was highest in fat body and hemocyte. The recombinant protein and synthesized Px-Mor analogues had high activity against the opportunistic fungi *A. pullulans*. Our work elucidated the role of Px-Mor role in the immune responses of *P. xylostella* and supported its potential as a topical antimicrobial agent against pathogens including *A. pullulans.*

Data Availability Statement {#sec24}
===========================

The sequence for Px-Mor has been deposited in GenBank under the accession number KF960047.

Author Contributions {#sec25}
====================

XX, AZ, YW, BL, PL, JY, SDM, and FJ performed the experiment. XZ, WJ, and SDM analyzed the data. XX, FJ, and SDM reviewed data, performed statistical analysis, planned and coordinated the study, and wrote the manuscript. All authors revised and approved the final version of the manuscript.

Conflict of Interest {#sec26}
--------------------

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We would like to thank the editor and reviewers for their invaluable comments and suggestions.

**Funding.** This work was supported by grant from the National Natural Science Foundation of China (31572069), Natural Science Foundation of Guangdong Province of China (2018A030313402) and Science and Technology Program of Guangzhou, China (201803020011).

^1^<http://www.ncbi.nlm.nih.gov/>

^2^<http://www.expasy.ch/SingalP>

^3^<http://www.expasy.org/tools/pi-tool.html>

^4^<http://www.scripps.edu/~cdputnam/protcalc.html>

^5^<http://www.ebi.ac.uk/tools/clustalw2>

^6^<http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index>

Supplementary Material {#sec27}
======================

The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2019.02211/full#supplementary-material>

######

Predicted structures of Px-Mor, using Phyre 2. **(A)** Secondary structure. **(B)** Tertiary structure.

######

Click here for additional data file.

######

Structures and characteristics of the Px-Mor analogues.

######

Click here for additional data file.

[^1]: Edited by: Marina Rautenbach, Stellenbosch University, South Africa

[^2]: Reviewed by: Xuesong Luo, Huazhong Agricultural University, China; Pei Liang, China Agricultural University, China

[^3]: ^†^These authors have contributed equally to this work

[^4]: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology


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-2/21440135
Divide -438198082 by 1358.
-322679
Calculate 52 divided by 1215596.
13/303899
What is 5849718 divided by -139279?
-42
Calculate -90313674 divided by 15609.
-5786
-1 divided by 25952169
-1/25952169
Divide -93411200 by 934112.
-100
-75615977 divided by -3
75615977/3
Calculate 402167026 divided by 2.
201083513
64531488 divided by 21
3072928
Divide -183160120 by -1573.
116440
Divide 1 by -7376632.
-1/7376632
Calculate 1249678825 divided by 7495.
166735
Calculate -445681673 divided by 20621.
-21613
35768342 divided by 19
35768342/19
Divide 2 by -948864.
-1/474432
Calculate -2571532 divided by 642883.
-4
Divide 166620115 by -4.
-166620115/4
1737896 divided by 4
434474
Calculate 339887613 divided by -26145201.
-13
Divide 0 by 3040692.
0
Calculate 22202433 divided by 2466937.
9
What is -5912710 divided by -2956355?
2
What is -11018982 divided by 39?
-282538
3814 divided by -107
-3814/107
Divide 1568842 by 45.
1568842/45
What is -357 divided by -9150?
119/3050
Divide 2791569 by -83.
-2791569/83
135619 divided by 4102
135619/4102
-3085240 divided by -11260
274
4497 divided by 4371
1499/1457
What is -25082478 divided by -9?
2786942
Calculate 4059 divided by -514.
-4059/514
What is -8638542 divided by 9?
-959838
Calculate 59891565 divided by -3.
-19963855
Divide -187367656 by 46841914.
-4
Divide -31850085 by -10616695.
3
What is -2891995 divided by -2?
2891995/2
What is 11 divided by -241454?
-11/241454
Calculate -53 divided by -94278.
53/94278
What is -1839 divided by 4130?
-1839/4130
What is -30486 divided by -852?
5081/142
Divide -13 by -3471642.
13/3471642
What is -18719 divided by 13703?
-18719/13703
-102730575 divided by -16745
6135
Calculate 39210654 divided by 127722.
307
10933 divided by -65
-841/5
Calculate 3561583 divided by 5.
3561583/5
Calculate 114 divided by -1737416.
-57/868708
Calculate 13744284 divided by 2.
6872142
Divide 372953600 by 25.
14918144
Calculate -5 divided by -124570633.
5/124570633
1259071 divided by -120
-1259071/120
167928 divided by -1564
-41982/391
What is 2476931 divided by -79901?
-31
Calculate -314437544 divided by -6697.
46952
Divide 0 by -9598217.
0
1001526756 divided by -333842252
-3
3617020 divided by 17
3617020/17
-1391019 divided by -21
66239
What is 43633415 divided by -8726683?
-5
What is 3 divided by 178247961?
1/59415987
Calculate 3486745 divided by -201.
-3486745/201
What is 0 divided by 25011209?
0
Divide 1030449725 by 6.
1030449725/6
What is 40683063 divided by 13561021?
3
233 divided by 20014
233/20014
Divide -8933486 by -4.
4466743/2
What is 3 divided by 15110180?
3/15110180
Calculate -1044 divided by -3764.
261/941
Divide -1 by -7706565.
1/7706565
What is -14281980 divided by 714099?
-20
What is -6440040 divided by -20?
322002
Calculate 5 divided by -280203610.
-1/56040722
Calculate 66881 divided by 410.
66881/410
Divide 4025736 by 792.
5083
Divide -182 by -65644.
91/32822
Divide 66080 by -84.
-2360/3
What is -18712030 divided by 17167?
-1090
Divide -67804941 by -1.
67804941
Calculate 37602264 divided by 21.
1790584
What is 13 divided by 617654?
13/617654
-2490 divided by 44812
-1245/22406
5678 divided by 950
2839/475
What is 7193 divided by -2096?
-7193/2096
5654 divided by -3835
-5654/3835
Calculate -17267520 divided by 32.
-539610
Calculate 3 divided by 1066673.
3/1066673
Calculate 3016 divided by -6.
-1508/3
-37 divided by 70773
-37/70773
What is -4489 divided by 67?
-67
Divide 2916804 by -2508.
-1163
Divide 638649853 by -1.
-638649853
40123 divided by 2270
40123/2270
-11011662 divided by -381
28902
-82917 divided by 9
-9213
What is 54376956 divided by 12?
4531413
Calculate -531 divided by 1194184.
-531/1194184
Calculate -3790989 divided by 2.
-3790989/2
What is -667 divided by 2042556?
-667/2042556
-12 divided by -388071
4/129357
Divide -1178594 by -37.
1178594/37
Divide 1672486053 by -4.
-1672486053


Yesterday, Electronic Arts reported its annual results, posting a net loss of $677 million for the year ended March 31, 2010. While that was a substantial improvement over the $1.09 billion loss it posted for the previous year, it wasn't enough to please investors.

Electronic Arts has a lot going for it, but analysts are starting to tire of its inability to capitalize.

EA shares exchanged hands at more than three times the daily average today, with the sell-off driving the price of the publisher's stocks down nearly 6 percent, shedding $1.09 to close Tuesday's trading at $17.71.

Some analysts were similarly unsatisfied with the publisher's report, but more for its forecast for the coming year (between $0.85 and $1.15 per share) than its latest losses. FBR Capital Markets released a note to investors today, noting that EA "continues to struggle to build the kind of margin expanding franchises necessary to really build the bottom line…In order to meet its targets for the year, the company is going to have to see stronger results in efforts to launch/relaunch franchises like Medal of Honor, Dragon Age, and Dead Space against an extremely competitive environment."

Wedbush analyst Michael Pachter was more upbeat on Electronic Arts, maintaining his "Outperform" rating on the stock and suggesting its forecast for the coming year was "realistically conservative." However, he stated in no uncertain terms that he has misjudged EA's prospects before.

"We have been wrong about this stock for almost five years," Pachter wrote. "Either we're stupid, stubborn, or unlucky, but we've been wrong. The definition of insanity is doing the same thing over and over again, each time hoping for a different result. This time, while we are again hoping for a different result, EA's Q4 report provided some evidence that the company is not doing the same things over and over again: lower headcount in cheaper locations, fewer facilities, fewer games, and a growing digital business. This time, we think that EA is on the right path."


Protest of Hands On Originals draws about 60

More than two dozen people demonstrated Friday afternoon against local T-shirt company Hands On Originals, which has been accused of discriminating against the organizers of Lexington's gay pride festival.

About 60 people attended a demonstration against local T-shirt company, Hands On Originals, which has been accused of discriminating against the organizers of Lexington's gay pride festival.

Those in downtown's Triangle Park held signs and encouraged honks of support, saying they hoped to educate the public about the issue.

"It's been nothing but positive," said Andrew Lucas, one of the organizers of the event. "It was perfectly peaceful, no drama. I'm quite in awe of everything that's going on."

Lucas said about 60 people showed up for the demonstration, and many motorists honked their horns in support as they drove past the park. There was no counterdemonstration, he said.

"We couldn't have asked for it to go any better," he said.

Lexington's Sue Bonner took an hour to attend in the morning, despite her daughter being in town from California.

"I told her to go back to sleep," said Bonner, who is involved with advocacy group Parents, Families and Friends of Lesbians and Gays.

Bonner and others there said they were surprised by the company's actions. The issue is rooted in a complaint filed with the city's Human Rights Commission by the Gay and Lesbian Services Organization, which organizes Lexington's annual gay pride festival in June. The GLSO alleged in its complaint that Hands On Originals declined to produced T-shirts for the event "because we're a Christian organization."

Hands On Originals co-owner Blaine Adamson issued a statement Monday, saying Hands On Originals "both employs and conducts business with people of all genders, races, religions, sexual preferences and national origins.

"However, due to the promotional nature of our products, it is the prerogative of the company to refuse any order that would endorse positions that conflict with the convictions of the ownership," he said.

Adamson has not returned messages and emails since seeking further comment.

The controversy led to stern comments from Lexington Mayor Jim Gray on Wednesday against the company's refusal, as well as a decision by the Fayette County Public Schools to hold off on new orders from Hands On Originals until the complaint is resolved. The school system has no pending orders with Hands On Originals.

The issue has created raucous public debate on talk radio stations and also led to Facebook groups both supporting Hands On Originals' actions and another urging a boycott. Both groups had more than 2,000 members each by Friday afternoon.

As of early afternoon, Friday's demonstration had not yet had any visits from people with opposing views.

In fact, the only incident of the day was a vehicle collision at the corner of Main Street and Broadway where the protest took place.

Three people intending to join the demonstration were turning left on to Main Street toward Victorian Square when they were hit by an oncoming car.

"I just got out of the car and grabbed a sign," said Jenna Watkins, who said there were no injuries. But the vehicle was in bad shape. "Vehicle juices were spewing everywhere," she added.

Also in attendance was Aaron Baker, president of the board of directors of the GLSO. The GLSO did not organize the event.

"It just confirmed our impression that Lexington's a very fair-minded city," Baker said of the positive reactions from passing vehicles.

Baker said the GLSO has received a number of offers in recent days from other printers to handle production of the shirts. The GLSO had intended to print 500, but that's likely changing.

"I think we're going to order a few more now, because I think people will be interested in having the controversial T-shirt," he said. "Of course, it's such a non-controversial T-shirt."

The T-shirt design features a stylized number 5 on the front, with "Lexington Pride Festival" and the event's sponsors on the back.

Baker said the GLSO has not yet discussed in great detail what they feel would lead to a satisfactory conclusion of their complaint.

But in general, he said, "Ultimately, the owners of Hands On Originals need to recognize that discrimination is not OK and need to make a commitment not to continue that."


Q:

Getting 404 when opening Sitecore Media Library

Related to the question
Getting Sitecore 404 for physical files
I was specifically tasked to remove the tilde prefix on the media folder (also mentioned in one of the answer from the question).
I get 404 when opening the media library afterwards like the related question above. So I added /sitecore/shell/Applications/Media/MediaShop AND /sitecore/shell/Applications/Media/Media Folder.aspx to the IgnoreUrlPrefix setting in the web.config
This fixes the issue, but i'm not sure how this fixed the problem, and why removing the tilde on the media folder causes 404 on media library page.
Can someone please explain ?

A:

If you have existing content which was created when the tilde prefix was in place then that existing content in Rich Text Fields in particular still has the tilde prefix being used. You can check this by looking at the Edit HTML or Raw Values of the field, it will be in the format:
<img src="-/media/123A4B6789012CAB34C567AB79CBD084.ashx?h=100%25&w=100%25" style="height: 100%; width:100%;">
In order to change the tilde and still support the old content you need to supplement your media config changes, patch in the changes to the following sections:
<?xml version="1.0"?>
<configuration xmlns:patch="http://www.sitecore.net/xmlconfig/">
  <sitecore>

    <settings>
      <!-- Extensions of media files should match it's type -->
      <setting name="Media.RequestExtension">
        <patch:attribute name="value"></patch:attribute>
      </setting>

      <!-- Change the media prefix from the default -->
      <setting name="Media.MediaLinkPrefix">
        <patch:attribute name="value">-/media</patch:attribute>
      </setting>
    </settings>

    <!-- Media handler prefixes -->
    <customHandlers>
      <handler patch:before="*[@trigger='~/media/']" trigger="-/media/" handler="sitecore_media.ashx" />
    </customHandlers>

    <!-- Support Media Prefixes -->
    <mediaLibrary>
      <mediaPrefixes>
        <prefix value="-/media"/>
        <prefix value="~/media"/>
      </mediaPrefixes>
    </mediaLibrary>

  </sitecore>
</configuration>

Change the prefixes to match whatever you need.
The link will be rendered correctly on the front end site in any case. Note, you have used /media, which media that if there is a piece of content created called media* then the media handler will try to handle it even if it is content, e.g. mysite.com/media/news or mysite.com/news/latest/media/. You may want to add some validation to prevent such items being created.


/*******************************************************************************
 * Copyright (c) 2015-2018 Skymind, Inc.
 *
 * This program and the accompanying materials are made available under the
 * terms of the Apache License, Version 2.0 which is available at
 * https://www.apache.org/licenses/LICENSE-2.0.
 *
 * Unless required by applicable law or agreed to in writing, software
 * distributed under the License is distributed on an "AS IS" BASIS, WITHOUT
 * WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. See the
 * License for the specific language governing permissions and limitations
 * under the License.
 *
 * SPDX-License-Identifier: Apache-2.0
 ******************************************************************************/

package org.datavec.api.transform.sequence.window;

import lombok.Data;
import lombok.EqualsAndHashCode;
import org.datavec.api.transform.ColumnType;
import org.datavec.api.transform.metadata.ColumnMetaData;
import org.datavec.api.transform.metadata.TimeMetaData;
import org.datavec.api.transform.schema.Schema;
import org.datavec.api.transform.schema.SequenceSchema;
import org.datavec.api.writable.LongWritable;
import org.datavec.api.writable.Writable;
import org.joda.time.DateTimeZone;
import org.nd4j.shade.jackson.annotation.JsonIgnoreProperties;
import org.nd4j.shade.jackson.annotation.JsonProperty;

import java.util.ArrayList;
import java.util.List;
import java.util.concurrent.TimeUnit;

/**
 * A windowing function based on time, with potentially overlapping windows. Time for each entry in the sequence is provided by a Time column

 * The overlapping nature of the windowing function allows for things such as a window size of 1 day, produced every hour.

 * Two parameters (and one optional parameter) are necessary to specify how windowing is conducted:
 * - The size of the window (for example, 1 day)

 * - The separation between window periods (for example, 1 hour)

 * - The offet for the start/end time of the windows

 *

 * Example with a window size of 12 hours, with the window separation of 1 hour, we end up with windows as follows:

 * (0:00 to 12:00), (1:00 to 13:00), (2:00 to 14:00) and so on.

 * If the offset was set to 15 minutes, windows would instead be at (0:15 to 12:15), (1:15 to 13:15), (2:15 to 14:15) and so on.

 *

 * Note that the windows generated by this window function need not contain any data - i.e., it can generate empty an empty
 * window if no data occurs in the specified time period.
 *
 * @author Alex Black
 */
@JsonIgnoreProperties({"inputSchema", "offsetAmountMilliseconds", "windowSizeMilliseconds",
                "windowSeparationMilliseconds", "timeZone"})
@EqualsAndHashCode(exclude = {"inputSchema", "offsetAmountMilliseconds", "windowSizeMilliseconds",
                "windowSeparationMilliseconds", "timeZone"})
@Data
public class OverlappingTimeWindowFunction implements WindowFunction {

    private final String timeColumn;
    private final long windowSize;
    private final TimeUnit windowSizeUnit;
    private final long windowSeparation;
    private final TimeUnit windowSeparationUnit;
    private final long offsetAmount;
    private final TimeUnit offsetUnit;
    private final boolean addWindowStartTimeColumn;
    private final boolean addWindowEndTimeColumn;
    private final boolean excludeEmptyWindows;
    private Schema inputSchema;

    private final long offsetAmountMilliseconds;
    private final long windowSizeMilliseconds;
    private final long windowSeparationMilliseconds;

    private DateTimeZone timeZone;

    /**
     * Constructor with zero offset
     *
     * @param timeColumn           Name of the column that contains the time values (must be a time column)
     * @param windowSize           Numerical quantity for the size of the time window (used in conjunction with windowSizeUnit)
     * @param windowSizeUnit       Unit of the time window
     * @param windowSeparation     The separation between consecutive window start times (used in conjunction with WindowSeparationUnit)
     * @param windowSeparationUnit Unit for the separation between windows
     */
    public OverlappingTimeWindowFunction(String timeColumn, long windowSize, TimeUnit windowSizeUnit,
                    long windowSeparation, TimeUnit windowSeparationUnit) {
        this(timeColumn, windowSize, windowSizeUnit, windowSeparation, windowSeparationUnit, 0, null);
    }

    /**
     * Constructor with zero offset, ability to add window start/end time columns
     *
     * @param timeColumn           Name of the column that contains the time values (must be a time column)
     * @param windowSize           Numerical quantity for the size of the time window (used in conjunction with windowSizeUnit)
     * @param windowSizeUnit       Unit of the time window
     * @param windowSeparation     The separation between consecutive window start times (used in conjunction with WindowSeparationUnit)
     * @param windowSeparationUnit Unit for the separation between windows
     */
    public OverlappingTimeWindowFunction(String timeColumn, long windowSize, TimeUnit windowSizeUnit,
                    long windowSeparation, TimeUnit windowSeparationUnit, boolean addWindowStartTimeColumn,
                    boolean addWindowEndTimeColumn) {
        this(timeColumn, windowSize, windowSizeUnit, windowSeparation, windowSeparationUnit, 0, null,
                        addWindowStartTimeColumn, addWindowEndTimeColumn, false);
    }

    /**
     * Constructor with optional offset
     *
     * @param timeColumn           Name of the column that contains the time values (must be a time column)
     * @param windowSize           Numerical quantity for the size of the time window (used in conjunction with windowSizeUnit)
     * @param windowSizeUnit       Unit of the time window
     * @param windowSeparation     The separation between consecutive window start times (used in conjunction with WindowSeparationUnit)
     * @param windowSeparationUnit Unit for the separation between windows
     * @param offset               Optional offset amount, to shift start/end of the time window forward or back
     * @param offsetUnit           Optional offset unit for the offset amount.
     */
    public OverlappingTimeWindowFunction(String timeColumn, long windowSize, TimeUnit windowSizeUnit,
                    long windowSeparation, TimeUnit windowSeparationUnit, long offset, TimeUnit offsetUnit) {
        this(timeColumn, windowSize, windowSizeUnit, windowSeparation, windowSeparationUnit, offset, offsetUnit, false,
                        false, false);
    }

    /**
     * Constructor with optional offset, ability to add window start/end time columns
     *
     * @param timeColumn               Name of the column that contains the time values (must be a time column)
     * @param windowSize               Numerical quantity for the size of the time window (used in conjunction with windowSizeUnit)
     * @param windowSizeUnit           Unit of the time window
     * @param windowSeparation         The separation between consecutive window start times (used in conjunction with WindowSeparationUnit)
     * @param windowSeparationUnit     Unit for the separation between windows
     * @param offset                   Optional offset amount, to shift start/end of the time window forward or back
     * @param offsetUnit               Optional offset unit for the offset amount.
     * @param addWindowStartTimeColumn If true: add a time column (name: "windowStartTime") that contains the start time
     *                                 of the window
     * @param addWindowEndTimeColumn   If true: add a time column (name: "windowEndTime") that contains the end time
     *                                 of the window
     * @param excludeEmptyWindows      If true: exclude any windows that don't have any values in them
     */
    public OverlappingTimeWindowFunction(@JsonProperty("timeColumn") String timeColumn,
                    @JsonProperty("windowSize") long windowSize,
                    @JsonProperty("windowSizeUnit") TimeUnit windowSizeUnit,
                    @JsonProperty("windowSeparation") long windowSeparation,
                    @JsonProperty("windowSeparationUnit") TimeUnit windowSeparationUnit,
                    @JsonProperty("offset") long offset, @JsonProperty("offsetUnit") TimeUnit offsetUnit,
                    @JsonProperty("addWindowStartTimeColumn") boolean addWindowStartTimeColumn,
                    @JsonProperty("addWindowEndTimeColumn") boolean addWindowEndTimeColumn,
                    @JsonProperty("excludeEmptyWindows") boolean excludeEmptyWindows) {
        this.timeColumn = timeColumn;
        this.windowSize = windowSize;
        this.windowSizeUnit = windowSizeUnit;
        this.windowSeparation = windowSeparation;
        this.windowSeparationUnit = windowSeparationUnit;
        this.offsetAmount = offset;
        this.offsetUnit = offsetUnit;
        this.addWindowStartTimeColumn = addWindowStartTimeColumn;
        this.addWindowEndTimeColumn = addWindowEndTimeColumn;
        this.excludeEmptyWindows = excludeEmptyWindows;

        if (offsetAmount == 0 || offsetUnit == null)
            this.offsetAmountMilliseconds = 0;
        else {
            this.offsetAmountMilliseconds = TimeUnit.MILLISECONDS.convert(offset, offsetUnit);
        }

        this.windowSizeMilliseconds = TimeUnit.MILLISECONDS.convert(windowSize, windowSizeUnit);
        this.windowSeparationMilliseconds = TimeUnit.MILLISECONDS.convert(windowSeparation, windowSeparationUnit);
    }

    private OverlappingTimeWindowFunction(Builder builder) {
        this(builder.timeColumn, builder.windowSize, builder.windowSizeUnit, builder.windowSeparation,
                        builder.windowSeparationUnit, builder.offsetAmount, builder.offsetUnit,
                        builder.addWindowStartTimeColumn, builder.addWindowEndTimeColumn, builder.excludeEmptyWindows);
    }

    @Override
    public void setInputSchema(Schema schema) {
        if (!(schema instanceof SequenceSchema))
            throw new IllegalArgumentException(
                            "Invalid schema: OverlappingTimeWindowFunction can only operate on SequenceSchema");
        if (!schema.hasColumn(timeColumn))
            throw new IllegalStateException("Input schema does not have a column with name \"" + timeColumn + "\"");

        if (schema.getMetaData(timeColumn).getColumnType() != ColumnType.Time)
            throw new IllegalStateException("Invalid column: column \"" + timeColumn + "\" is not of type "
                            + ColumnType.Time + "; is " + schema.getMetaData(timeColumn).getColumnType());

        this.inputSchema = schema;

        timeZone = ((TimeMetaData) schema.getMetaData(timeColumn)).getTimeZone();
    }

    @Override
    public Schema getInputSchema() {
        return inputSchema;
    }

    @Override
    public Schema transform(Schema inputSchema) {
        if (!addWindowStartTimeColumn && !addWindowEndTimeColumn)
            return inputSchema;

        List<ColumnMetaData> newMeta = new ArrayList<>(inputSchema.getColumnMetaData());

        if (addWindowStartTimeColumn) {
            newMeta.add(new TimeMetaData("windowStartTime"));
        }

        if (addWindowEndTimeColumn) {
            newMeta.add(new TimeMetaData("windowEndTime"));
        }

        return inputSchema.newSchema(newMeta);
    }

    @Override
    public String toString() {
        return "OverlappingTimeWindowFunction(columnName=\"" + timeColumn + "\",windowSize=" + windowSize
                        + windowSizeUnit + ",windowSeparation=" + windowSeparation + windowSeparationUnit + ",offset="
                        + offsetAmount + (offsetAmount != 0 && offsetUnit != null ? offsetUnit : "")
                        + (addWindowStartTimeColumn ? ",addWindowStartTimeColumn=true" : "")
                        + (addWindowEndTimeColumn ? ",addWindowEndTimeColumn=true" : "")
                        + (excludeEmptyWindows ? ",excludeEmptyWindows=true" : "") + ")";
    }

    @Override
    public List<List<List<Writable>>> applyToSequence(List<List<Writable>> sequence) {

        int timeColumnIdx = inputSchema.getIndexOfColumn(this.timeColumn);

        List<List<List<Writable>>> out = new ArrayList<>();

        //We are assuming here that the sequence is already ordered (as is usually the case)

        //First: work out the window to start on. The window to start on is the first window that includes the first time step values
        long firstTimeStepTimePlusOffset = sequence.get(0).get(timeColumnIdx).toLong() + offsetAmountMilliseconds;
        long windowBorder = firstTimeStepTimePlusOffset - (firstTimeStepTimePlusOffset % windowSeparationMilliseconds); //Round down to time where a window starts/ends
        //At this windowBorder time: the window that _ends_ at windowBorder does NOT include the first time step
        // Therefore the window that ends at windowBorder+1*windowSeparation is first window that includes the first data point

        //Second: work out the window to end on. The window to end on is the last window that includes the last time step values
        long lastTimeStepTimePlusOffset =
                        sequence.get(sequence.size() - 1).get(timeColumnIdx).toLong() + offsetAmountMilliseconds;
        long windowBorderLastTimeStep =
                        lastTimeStepTimePlusOffset - (lastTimeStepTimePlusOffset % windowSeparationMilliseconds);
        //At this windowBorderLastTimeStep time: the window that _starts_ this time is the last window to include the last time step

        long lastWindowStartTime = windowBorderLastTimeStep;

        long currentWindowStartTime = windowBorder + windowSeparationMilliseconds - windowSizeMilliseconds;
        long nextWindowStartTime = currentWindowStartTime + windowSeparationMilliseconds;
        long currentWindowEndTime = currentWindowStartTime + windowSizeMilliseconds;
        List<List<Writable>> currentWindow = new ArrayList<>();

        int currentWindowStartIdx = 0;
        int sequenceLength = sequence.size();
        boolean foundIndexForNextWindowStart = false;
        while (currentWindowStartTime <= lastWindowStartTime) {

            for (int i = currentWindowStartIdx; i < sequenceLength; i++) {
                List<Writable> timeStep = sequence.get(i);
                long currentTime = timeStep.get(timeColumnIdx).toLong();

                //As we go through: let's keep track of the index of the first element in the next window
                if (!foundIndexForNextWindowStart && currentTime >= nextWindowStartTime) {
                    foundIndexForNextWindowStart = true;
                    currentWindowStartIdx = i;
                }
                boolean nextWindow = false;
                if (currentTime < currentWindowEndTime) {
                    //This time step is included in the current window
                    if (addWindowStartTimeColumn || addWindowEndTimeColumn) {
                        List<Writable> timeStep2 = new ArrayList<>(timeStep);
                        if (addWindowStartTimeColumn)
                            timeStep2.add(new LongWritable(currentWindowStartTime));
                        if (addWindowEndTimeColumn)
                            timeStep2.add(new LongWritable(currentWindowStartTime + windowSizeMilliseconds));
                        currentWindow.add(timeStep2);
                    } else {
                        currentWindow.add(timeStep);
                    }
                } else {
                    //This time step is NOT included in the current window -> done with the current window -> start the next window
                    nextWindow = true;
                }

                //Once we reach the end of the input sequence: we might have added it to the current time step, but still
                // need to create the next window
                if (i == sequenceLength - 1)
                    nextWindow = true;

                if (nextWindow) {
                    if (!(excludeEmptyWindows && currentWindow.size() == 0))
                        out.add(currentWindow);
                    currentWindow = new ArrayList<>();
                    currentWindowStartTime = currentWindowStartTime + windowSeparationMilliseconds;
                    currentWindowEndTime = currentWindowStartTime + windowSizeMilliseconds;
                    foundIndexForNextWindowStart = false;
                    nextWindowStartTime = currentWindowStartTime + windowSeparationMilliseconds;
                    break;
                }
            }
        }

        return out;
    }

    public static class Builder {
        private String timeColumn;
        private long windowSize = -1;
        private TimeUnit windowSizeUnit;
        private long windowSeparation = -1;
        private TimeUnit windowSeparationUnit;
        private long offsetAmount;
        private TimeUnit offsetUnit;
        private boolean addWindowStartTimeColumn = false;
        private boolean addWindowEndTimeColumn = false;
        private boolean excludeEmptyWindows = false;

        public Builder timeColumn(String timeColumn) {
            this.timeColumn = timeColumn;
            return this;
        }

        public Builder windowSize(long windowSize, TimeUnit windowSizeUnit) {
            this.windowSize = windowSize;
            this.windowSizeUnit = windowSizeUnit;
            return this;
        }

        public Builder windowSeparation(long windowSeparation, TimeUnit windowSeparationUnit) {
            this.windowSeparation = windowSeparation;
            this.windowSeparationUnit = windowSeparationUnit;
            return this;
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Syrian Air Force Strikes Positions of Foreign Militants in Aleppo4.75
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Syrian Air Force Strikes Positions of Foreign Militants in Aleppo

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The Syrian Air Force carried out a series of airstrikes on positions of the so-called Caucasian Battalion in the south-west of Aleppo. At least 8-9 terrorists were killed, and the same number of gunmen was wounded, as well as their military equipment was destroyed.

Positions of the so-called Caucasian Battalion in the south-west of Aleppo, near the Al-Hikma School, south the 1070 Apartments Project, became the target of a series of Syrian airstrikes, a Colonel of the Republican Guard of the Syrian Arab Army (SAA) told the FAN news agency.

According to Syrian military, the attack on the positions of the Caucasian Battalion, which is a part of the so-called ‘First Line’ of the Ahrar al-Sham terrorist group, was extremely successful. The main reason for large losses, suffered by the enemy in the air attack, was a hasty preparation of the battalion to move to the west of the area of the 1070 Apartments Project in order to replenish a defense line of terrorists at a height, adjoined to Minyan quarter.

The airstrike hit the battalion, when the terrorists were loading their pickup trucks with ammunition, weapons and food. After specification of the coordinates by forward air controllers of the Republican Guard, three MiG-21 fighter jets were raised in the air. According to preliminary data, as a result of the attack, three vehicles, one of which was equipped with a turret with the DShK heavy machine gun, and the MAZ heavy truck, captured by the Caucasian Battalion from the SAA during fights for Ramusi living quarter a month ago, were destroyed.

Official sources reported about 17 dead and 22 wounded terrorists, mostly natives of Dagestan, Chechnya and Kyrgyzstan. However, the FAN’s source stressed that most likely the losses are exaggerated, since at the moment, strength of the battalion was not more than 30-37 people. According to the Colonel of the Republican Guard, the real permanent losses of the terrorists due to the attack were around 8-9 people, and about the same number of terrorists were wounded. Anyway, even such seemingly minor losses can radically affect a combat capability of the Ahrar al-Sham on this sector of the front.

Will remind, two days ago, immediately after the end of a truce, the Syrian Armed Forces, supported by the People’s Militia, the Republican Guard and the Russian Aerospace Forces, launched an offensive on the most dangerous sections of the front in the south-western part of the city. During the offensive, the Syrian Army managed to advance to 800-900 meters on several parts of the front, in particular, east and west of the area of the 1070 Apartments Project. However, a defense line of terrorist groups, occupying the area, was not broken by the first series of blows. The Ahrar al-Sham regularly takes attempts to counter-attack, quickly throwing main attack forces from one sector of the front to another one.


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Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation.
The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27Kip1 and p16INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.


Addressing a ceremony in connection with Economic Cooperation Organization (ECO) Day in Islamabad, he said the goal could be achieved by improving connectivity through rail, roads, ports and use of latest technology.

Highlighting the significance of the ECO, Shah Mahmood Qureshi said the organisation gave great importance to the objectives of regional peace and development. He said efforts were required to enhance cooperation amongst the ECO countries at different levels.

He assured that Pakistan would continue to play its role to promote cooperation amongst the regional countries in order to achieve the sustainable development goals.

CPEC road to economic development for whole region: economic affairs minister
In a statement in February this year, former deputy chairman planning commission Sartaj Aziz has said Pakistan was ready to expand mutually beneficial trade, economic cooperation and connectivity in the Shanghai Cooperation Organization (SCO) region.

“Pakistan encourages trade and transit through its territory as it is upgrading infrastructure, roads, railways and ports,” he said.

He said country was open to become conduit for linking Eurasian landmass, China, Russia and Central Asia with the Arabian Sea. – NNI