Introduction
============

The ability to silence a single target gene with minimal toxicity and few off-target effects (OTEs) would offer the medical community new tools to combat a wide variety of diseases. Several hybridization-based technologies have been tested over the past 30 years that utilize synthetic oligonucleotides to suppress expression of a specific target gene. In broad terms, these technologies are based on a gene being targeted either by small interfering RNAs (siRNAs) which exploit the natural cellular RNA interference machinery^[@bib1],[@bib2]^ or by an antisense oligonucleotide (ASO) that can function *via* a variety of mechanisms, including RNase H-mediated cleavage of RNA, steric hindrance of mRNA translation, splice site switching, and miRNA antagonists.^[@bib3],[@bib4],[@bib5]^ ASOs and siRNAs have struggled to produce commercially viable therapeutics, in spite of initial excitement and large-scale investment^[@bib6]^ presenting an opportunity for alternative gene-silencing technologies.

U1 Adaptors are a recently invented gene-silencing technology that exploits the natural ability of the U1 small nuclear ribonucleoprotein (snRNP) splicing factor to inhibit gene-specific polyA site activity of the target gene, a regulated nuclear pre-mRNA processing step obligatory for nearly all RNA Polymerase II genes.^[@bib7],[@bib8],[@bib9],[@bib10],[@bib11],[@bib12]^ A U1 Adaptor is a synthetic oligonucleotide (typically 28--33 nucleotides) comprised of a 5′ target domain (TD), which binds to the target pre-mRNA, and a 3′ U1 domain (U1D), which binds to the 5′-end of the U1 small nuclear RNA subunit of U1 snRNP.^[@bib7],[@bib8]^ Tethering of the U1 snRNP to a target pre-mRNA blocks maturation leading to reduced levels of mature mRNA. The U1D sequence is common to all U1 Adaptors and is defined by the U1 snRNP; design and chemical modification patterns have already been optimized.^[@bib7]^ In contrast, the TD sequence is target-specific and hence unique to each U1 Adaptor. Like all other gene knockdown technologies, empiric testing is required for site selection and optimization. Extensive medicinal chemistry studies have been done in ASO and siRNA systems to find chemical modifications that improve nuclease stability, enhance potency, and reduce OTEs.^[@bib3],[@bib5],[@bib13],[@bib14],[@bib15],[@bib16],[@bib17]^ Unlike siRNAs or RNase H-mediated ASOs, U1 Adaptors do not interact or function with any cellular enzymes (such as RNase H, Dicer, Argonaut 2, etc.) and thus can be made entirely using modified components, such as 2′-O-Methyl RNA (2′OMe) or locked nucleic acids (LNAs), with or without phosphorothioate (PS)-modified internucleotide linkages.^[@bib7]^ All oligonucleotide-based silencing technologies have associated toxicities and U1 Adaptors are no exception, as evidenced by a recent report that showed significant OTEs when used at high doses.^[@bib18]^ In a previous report, use of U1 Adaptors at a lower dose gave effective and specific silencing with few OTEs.^[@bib7]^

Here, we present the first report of *in vivo* use of U1 Adaptors by targeting two human genes to suppress growth of human melanoma cells in a mouse xenograft model system. The antiapoptotic human B-cell lymphoma 2 (*BCL2*) gene has been a frequent target in studies using ASO and RNA interference technologies^[@bib19],[@bib20],[@bib21],[@bib22],[@bib23],[@bib24]^ and plays a role in many cancers, including melanoma^[@bib25],[@bib26],[@bib27],[@bib28],[@bib29]^ that has extraordinary intrinsic resistance to apoptotic cell death commonly induced by anticancer drugs, in part due to elevated levels of BCL2.^[@bib28],[@bib29]^ We chose a human melanoma xenograft mouse system for its proven track record in predicting efficacy in clinical trials as evidenced by riluzole that has gone on to show efficacy in phase 0 and 2 human trials.^[@bib30]^ Our second target, the metabotropic glutamate receptor 1 (*GRM1*) gene has only recently been established as an important factor in melanoma as well as other cancers.^[@bib31],[@bib32],[@bib33]^ Using our novel tumor-specific dendrimer delivery vehicle, we demonstrate that very low doses of anti-*BCL2* or anti-*GRM1* U1 Adaptors are sufficient to reduce growth/progression of human melanoma xenografts with little apparent toxicity. These results offer proof-of-concept that U1 Adaptors are an effective gene-silencing therapeutic platform that can suppress tumor growth using doses far lower than expected based on published experience using other oligonucleotide-based methods.^[@bib34]^ These results also lay a foundation for exploiting U1 Adaptors to target other genes as well as a wide variety of other human disorders.

Results
=======

U1 Adaptor silencing of *BCL2 in vitro*
---------------------------------------

Twelve anti-human-*BCL2* U1 Adaptors were screened for functional potency in C8161 melanoma cells (**Supplementary Figure S1a,b**). Two U1 Adaptors, BCL2-A and BCL2-B showed strong activity in reducing *BCL2* mRNA levels (**Supplementary Figure S1b,c**) and were used in subsequent studies. LNA- and 2′OMe-modified variants of BCL2-A were compared for activity in C8161 cells (**[Figure 1a](#fig1){ref-type="fig"}**, **Supplementary Figure S2a**) and the LNA-modified variant BCL2-A~L2~ showed the highest silencing activity at both the protein (western immunoblots) and mRNA (reverse transcription-quantitative PCR) levels (**[Figure 1b](#fig1){ref-type="fig"}** lanes 5--6, **Supplementary Figure S2b**). To demonstrate that the silencing activity of BCL2-A~L2~ is mediated *via* a U1 Adaptor mechanism, C8161 cells were transfected with matching control U1 Adaptors having either the inactivated TD or U1D by mutation or by unlinking the TD and U1D into two separate molecules called "half-Adaptors" (**[Figure 1a](#fig1){ref-type="fig"}**). In all cases, these negative controls failed to reduce BCL2 protein (**[Figure 1b](#fig1){ref-type="fig"}**) or mRNA (**Supplementary Figure S2b**) levels, strongly supporting a U1 Adaptor-based silencing mechanism, e.g., one where the U1 Adaptor oligonucleotide must tether the target pre-mRNA to the U1 snRNP. Modification of BCL2-A with three PS linkages at each end to improve exonuclease resistance (BCL2-A~ps~) or a fluorescent label (Cy3-BCL2-A) did not alter potency compared with the parent compound (**Supplementary Figure S2c**).

Development of a tumor-targeting dendrimer nanoparticle
-------------------------------------------------------

The cyclic RGD pentapeptide (RGD) was chosen as a tumor-targeting ligand because it is small, easy to conjugate, and specifically binds the α5β3 splice variant of an integrin cell surface receptor that is overexpressed in a wide variety of cancer cells, including C8161.^[@bib35],[@bib36]^ The particle was based on a generation 5 (G5) polypropyleneimine (PPI) dendrimer that was previously used to successfully deliver nucleic acid cargos in mouse xenograft models of human tumors.^[@bib37],[@bib38],[@bib39],[@bib40],[@bib41]^ As RGD binds to its receptor with much higher affinity as a dimer,^[@bib42],[@bib43]^ the RGD targeting ligand was coupled to the PPI G5 dendrimer in a final 2:1 molar ratio to give RGD-G5 (**[Figure 1c](#fig1){ref-type="fig"}**). Transfection of C8161 cells *in vitro* with a BCL2-A:RGD-G5 complex demonstrated that RGD-G5 was active as a delivery vehicle, having potency comparable to transfection of BCL2-A with the cationic lipid reagent Lipofectamine 2000 (LF2000) (**[Figure 1d](#fig1){ref-type="fig"}**, **Supplementary Figure S3a--c**). Surprisingly, an anti-*BCL2* Dicer-substrate siRNA that was effective using LF2000 transfection did not suppress *BCL2* mRNA levels when used with the RGD-G5 dendrimer, suggesting this delivery vehicle is not compatible with siRNA class reagents (**[Figure 1d](#fig1){ref-type="fig"}**). Use of RGD to PPI G5 coupling ratios of 4:1 and 8:1 resulted in reduced efficacy *in vitro* (**Supplementary Figure S4**).

BCL2-A is active *in vivo* and uses a U1 Adaptor mechanism
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Mice bearing established C8161 subcutaneous xenografts were administered BCL2-A:RGD-G5 or control complexes in phosphate-buffered saline (PBS) *via* intravenous (iv) tail vein injection. Pilot studies (data not shown) established that effective tumor suppression could be achieved using 1.7 µg of the U1 Adaptor and a 1:1.3 Adaptor:RGD-G5 stoichiometry. Dynamic light scattering measurements indicated the particle size of the injected complex (\~3.2 µmol/l U1 Adaptor and \~4 µmol/l RGD-G5 in 1x PBS) was 193 nm with a polydispersion index = 0.18 with little difference in size for different U1 Adaptor sequences (**Supplementary Figure S5**). Mice were given biweekly injections of various formulations for 3 weeks and the tumors were evaluated (**[Figure 2a](#fig2){ref-type="fig"}**). Tumor-bearing treatment group 2 (TG2) mice that were dosed with a 1.7 µg BCL2-A:2.4 µg RGD-G5 complex (68 µg Adaptor/kg) per injection exhibited \~70% tumor reduction compared with TG1, the vehicle control group (*P* = 0.004). No significant tumor suppression was seen with the two control U1 Adaptor cohorts, which were treated with either a TD-mutant BCL2-A (TG3) or a U1D-mutant BCL2-A (TG4). Thus disruption of either the TD or the U1D resulted in loss of activity, strongly arguing for a U1 Adaptor mechanism of action. The TG6 mice were dosed with a 1.7 µg BCL2-A:2.4 µg G5 complex, where G5 matches the RGD-G5 vehicle but lacks the RGD ligand. This cohort did not show reduction in tumor mass, demonstrating the importance of the targeting ligand. Use of a fivefold lower dose (0.34 µg BCL2-A:0.48 µg RGD-G5 complex) resulted in no inhibition of tumor growth (TG5), establishing that \>0.34 µg Adaptor/dose was needed for tumor suppression.

Protein and RNA extracted from tumors of day 22 TG1-4 mice demonstrated that only the BCL2-A:RGD-G5--treated mice had reduced BCL2 protein (**[Figure 2b](#fig2){ref-type="fig"}**) and mRNA (**Supplementary Figure S6a**). Others have shown that therapeutic-based BCL2 silencing in xenograft mice by either ASOs or siRNAs increases the rate of apoptosis.^[@bib19],[@bib20],[@bib21],[@bib23]^ Immunohistochemical analysis shown in **[Figure 2c](#fig2){ref-type="fig"}** demonstrated that BCL2-A--treated, but not the control animals, showed an increased number of positive, activated Caspase 3 stained cells. Furthermore, immunohistochemical analysis using the cell proliferation marker, Ki67, showed that BCL2-A--treated, but not the control animals, had a reduced number of actively proliferating cells (**[Figure 2d](#fig2){ref-type="fig"}**). Both visual and histopathological inspection of several organs including liver, kidney, spleen, heart, brain, and lung from the TG1-TG6 mice showed no evidence for tissue damage or toxicity (data not shown). None of the mice exhibited overt signs of toxicity such as lethargy, not eating or drinking, loss of body weight or ulceration of the transplanted tumor.

While the above data support that BCL2-A\'s tumor suppression activity is likely to be mediated through reducing *BCL2* mRNA levels, it did not rule out a possible contribution of an extracellular mechanism such as impaired tumor vascularization, a possible side effect of RGD targeting.^[@bib35],[@bib36],[@bib42],[@bib43]^ This was addressed by showing that C8161 cells transfected with BCL2-A:RGD-G5 *ex vivo* before implantation led to significant tumor suppression (**[Figure 2e](#fig2){ref-type="fig"}**). The results of this *ex vivo* experiment strongly suggest that tumor suppression detected *in vivo* is primarily if not completely mediated through an intracellular mechanism and also demonstrate a long-lasting effect as the single *ex vivo* transfection of BCL2-A led to tumor suppression persisting at least through 23 days *in vivo*. C8161 cells transfected with RGD-G5:Adaptor complexes all had a similar doubling time and apoptosis rate (data not shown) independent of whether the Adaptor was capable of silencing BCL2 indicating *in vitro* results do not always predict *in vivo* activities.

Two chemically modified variants of BCL2-A were studied (BCL2-A~ps~ with terminal PS modification and BCL2-A~L2~ with LNA residues in the TD) in an attempt to increase potency *in vivo* (**[Figure 3a](#fig3){ref-type="fig"}**). Although BCL2-A~ps~ (TG9) gave significant tumor suppression as compared with the vehicle control (TG7), it was less active than BCL2-A (TG8). BCL2-A~L2~ (TG13) showed tumor suppression equal to BCL2-A, an observation that contrasts with its superior activity *in vitro*, further demonstrating that *in vitro* results do not always translate to similar outcomes *in vivo*. Others have also found unpredictable behavior of LNA backbone modifications.^[@bib16],[@bib17]^ The other data in **[Figure 3a,b](#fig3){ref-type="fig"}** demonstrated that a 2:1 RGD:PPIG5 coupling ratio was superior to a 4:1 ratio (compare TG8 with TG10); further, a 1:4 Adaptor:RGD-G5 stoichiometry was less effective than a 1:1.3 stoichiometry (compare TG16 with TG18); a minimally effective dose for BCL2-A was 0.85 µg/injection (34 µg Adaptor/kg).

To further support that *BCL2* is indeed the therapeutic target and that the observed tumor suppression was not due to sequence-specific OTEs, we analyzed a second anti-*BCL2* Adaptor (BCL2-B) which targets a different site of the *BCL2* gene. BCL2-B also was effective in reducing tumor mass, albeit with lower potency than BCL2-A (**[Figure. 3c](#fig3){ref-type="fig"}**), and used a U1 Adaptor mechanism as single domain mutant BCL2-B Adaptors were not active *in vivo* (**[Figure 3d](#fig3){ref-type="fig"}**). Notably, **[Figure 3d](#fig3){ref-type="fig"}** used an (RGD)~x2~-containing G5 PAMAM dendrimer (RGD-PAMAM-G5) in place of the PPI core found in RGD-G5 as we wanted to demonstrate that our results were not dependent on a particular class of dendrimer. Indeed a head-head comparison of RGD-G5 and RGD-PAMAM-G5 found they had comparable tumor suppression activity *in vivo* (**Supplementary Figure S6b**). Dynamic light scattering measurements indicated the particle size of the injected complex (\~3.2 µmol/l U1 Adaptor and \~4 µmol/l RGD-PAMAM-G5 in 1x PBS) was 200 nm with a polydispersion index = 0.147 with little difference in size for different U1 Adaptor sequences (**Supplementary Figure S4c**). We also demonstrated that BCL2-A\'s tumor suppression activity is not peculiar to C8161 xenografts as BCL2-A also suppressed UACC903 xenograft tumor growth (**Supplementary Figure S6c**). As compared with C8161, UACC903 is a more aggressive and genotypically distinct melanoma that harbors a V600E *B-RAF* mutation found in 70% of all melanomas (C8161 has a wild-type *B-RAF*).^44,45^

Anti-*GRM1* Adaptors also suppress melanoma tumor growth at low dose
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Ectopic expression of *GRM1* in melanocytes is sufficient to induce melanocytic cell transformation *in vitro* and spontaneous melanoma development *in vivo*; short hairpin RNA-based silencing of *GRM1* inhibited tumor cell growth *in vitro* and *in vivo*.^[@bib31],[@bib46]^ Screening anti-human--*GRM1* Adaptors in C8161 cells *in vitro* identified three candidate U1 Adaptors that reduced human GRM1 at the protein (**[Figure 4a,b](#fig4){ref-type="fig"}**) and mRNA (**Supplementary Figure S7a--d**) levels. Treatment of C8161 xenograft mice with these anti-*GRM1* U1 Adaptors decreased tumor growth and reduced *GRM1* protein levels in day 21 excised xenograft tumors compared with the vehicle control (**[Figure 4c](#fig4){ref-type="fig"}**). To demonstrate mechanism of action, the two most potent anti-*GRM1* Adaptors (GRM1-A~ps~ and GRM1-B~ps~) underwent additional analysis as follows, GRM1-B~ps~ uses a U1 Adaptor mechanism as repeating the GRM1-B~ps~ treatment with matching TD-mutant or U1D-mutant control U1 Adaptors gave no tumor suppression (**[Figure 4d](#fig4){ref-type="fig"}**) and no reduction of *GRM1* at the protein and mRNA levels as compared with RGD-G5--treated (vehicle only) control mice (**[Figure 4e](#fig4){ref-type="fig"}**). TD and U1D mutant Adaptor control xenograft experiments for GRM1-A~ps~ also demonstrated that the reduced tumor volumes were again mediated by a U1 Adaptor mechanism (**[Figure 5a](#fig5){ref-type="fig"}**). Tumor suppression was lost when RGD was replaced with RAD, a well-validated inactive sequence variant of RGD that fails to bind integrin receptors, once again demonstrating that tumor targeting is required. Finally, testing the variant RGD-G5/SPDP vehicle that has a short 10 Å non-PEG--containing linker (in contrast to the 50 Å PEG-linker employed in the original RGD-G5 dendrimer) showed significant reduction in tumor volume, demonstrating that RGD-G5\'s linker length and composition were not critical for activity.

It was previously shown that short hairpin RNA-based silencing of *GRM1* in human melanoma cell xenografts leads to reduced levels of phosphorylated AKT (pAKT).^[@bib46],[@bib47]^ Excised GRM1-A~ps~ U1 Adaptor-treated day 17 tumor samples also showed decreased levels of pAKT (**Supplementary Figure S8**). Immunohistochemical analysis of the excised day 17 tumors from the anti-*GRM1* U1 Adaptor-treated xenografts demonstrated increased activated Caspase 3 and decreased Ki67 staining (**[Figure 5b,c](#fig5){ref-type="fig"}**), similar to the previous observation using anti-*BCL2* U1 Adaptors. Both GRM1-A~ps~ and GRM1-B~ps~ U1 Adaptors were also able to suppress tumor progression of UACC903 xenografts, broadening their scope of action to melanoma with mutated B-RAF (**Supplementary Figure S9a**). In contrast to previous observations using BCL2-A, the presence of PS end-modifications on the anti-*GRM1* U1 Adaptors resulted in superior *in vivo* activity compared with the same oligonucleotides with phosphodiester end linkages (**Supplementary Figure S9b**).

No toxicity or immune stimulation was observed in U1 Adaptor-treated C57BL6 mice
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It was important to examine the effects of U1 Adaptor administration in immune-competent mice (C57BL6) to directly assess whether the U1 Adaptor antitumor effects could in part be based on stimulation of the innate immune system, a complication observed using other gene-silencing therapeutics.^[@bib48],[@bib49],[@bib50]^ Although a variety of receptors will recognize various forms of DNA and RNA, no receptors are known that recognize 2′OMe RNA or LNA residues, making it unlikely that the U1 Adaptors employed in the present study would trigger immune activation. BCL2-A and GRM1-A~ps~ U1 Adaptor:RGD-G5 complexes were injected into C57BL6 mice; interleukin-12 (IL-12) and interferon-α (IFN-α) levels were measured 4 hours post-injection. Baseline levels of both cytokines were seen for all mice except those in the positive control cohort which were administered poly(I:C), a known toll-like receptor 3 (TLR3) agonist (**[Figure 6a](#fig6){ref-type="fig"}** and **Supplementary Figure S10**).^[@bib51]^ In a similarly designed but independent experiment, baseline levels of IL-12 and interferon-α were also observed 24 hours post-injection (**Supplementary Figure S11a,b**). C57BL6 mice were given the standard 3-week U1 Adaptor treatment regimen (68 µg Adaptor/kg; two times/week) and serum levels of alkaline phosphatase and alanine transaminase were measured to assess for evidence of hepatic injury; both enzymes remained at basal levels throughout the study period (**[Figure 6b,c](#fig6){ref-type="fig"}**). These same serum samples also contained basal levels of IL-12 and IFN-α levels indicative of no immune stimulation even after 3 weeks of biweekly treatments (data not shown). Six organs from the same mice (liver, kidneys, spleen, heart, brain, and lungs) were excised, stained, and analyzed by the Rutgers-UMDNJ Molecular Histopathological Facility Core. No tissue damage was detected and only rare foci of inflammatory cells were seen in all organ samples. Thus no evidence for toxicity was found for any treatment group.

Discussion
==========

U1 Adaptors were previously shown to be effective triggers of gene silencing in mammalian cells *in vitro* but their applicability for *in vivo* use was heretofore untested. The present study offers proof-of-concept that U1 Adaptors can be effective in suppressing expression of targeted genes and slow tumor growth *in vivo* when combined with a suitable delivery vehicle. Success was observed using the same xenograft mouse system and human cell lines (C8161 and UACC903) that proved predictive for riluzole in preclinical studies that later went on to show efficacy in phase 0 and 2 human trials.^[@bib30]^ The broad utility of the U1 Adaptor approach is underscored by the fact we successfully targeted two genotypically different melanoma cell lines (BRAF wild-type and BRAF V600E mutant) and two different cancer-implicated genes, *BCL2* and *GRM1*. The technology also proved robust as our very first *in vivo* experiment and all subsequent experiments, encompassing \~108 treatment groups totaling over 800 mice, exhibited a very high potency for both genes, suppressing tumor progression at doses as low as 34--68 µg Adaptor/kg. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to function *via* a U1 snRNP-based mechanism of action. Suitable negative controls for mechanism of action include use of full-length U1 Adaptors with mutations in either the TD or the U1D or half-Adaptors (isolated TD or U1D) to show that both functional domains must reside on the same molecule.^[@bib7]^ The present study employed all of these mechanistic controls and demonstrated that an intact U1 Adaptor oligonucleotide with functional (*i.e.,* non-mutated) sequences in both the TD and U1D is required to achieve tumor suppression and a gene suppression effect. These controls further support a U1 mechanism of action in both the *in vitro* and *in vivo* studies.

Tumor growth can be suppressed by a variety of routes including downregulation of necessary oncogenes, nutritional starvation (disruption of vascular supply), direct chemical cytotoxicity, or immune activation. In the present study, we found evidence for induction of apoptosis and reduced cell division only in tumor-bearing animals treated with intact (non-mutated) anti-*BCL2* or anti-*GRM1* U1 Adaptors, which correlated with reduction of BCL2 or GRM1 levels in the tumor tissue. Treated animals showed no evidence for either general chemical toxicity from the administered compounds or immune activation. Further, tumor cells treated *ex vivo* with the U1 Adaptors before implantation showed marked growth retardation in animals that never received iv dosing of the U1 Adaptor:RGD-G5 complex, making it unlikely that disrupted vascularization contributed to tumor suppression.

U1 Adaptors are large, highly charged synthetic nucleic acids with a molecular weight of around 11,000 daltons. Molecules of this type do not readily enter most cell types without assistance, so use of an appropriate delivery vehicle is crucial to the success. The need for efficient delivery, one of the primary challenges of the oligonucleotide therapeutic field, continues to inspire production of an ever-widening variety of delivery systems ranging from cationic lipid or polymer-based nanoplexes, to antibody-protein fusion systems, to encapsulation of the oligonucleotide drug in exosomes or bacterial minicells. With the exception of delivery to liver,^[@bib34],[@bib52],[@bib53]^ efficacious siRNA-based targeting using low microgram doses is usually not possible with systemic iv dosing and typically is only seen with local administration (e.g., direct intraocular or intratumoral injection). Use of dendrimers to deliver therapeutic oligonucleotides is relatively under-explored, although a recent report successfully used a PAMAM dendrimer to deliver therapeutic doses of multiple Dicer-substrate siRNAs at single digit microgram levels in a humanized mouse model of HIV1.^[@bib54]^ Although the PPI dendrimer-based system employed here was useful in this proof-of-concept study and exhibited no detectable toxicity, it is clearly prudent to explore the compatibility of other delivery systems with U1 Adaptors. It is important to acknowledge the contribution of ligand targeting for the success we observed using the present dendrimer-based systems. The low-dose efficacy achieved here required the RGD targeting ligand and tumor suppression was lost when the RGD ligand was either omitted from the vehicle or substituted with the related but inactive RAD variant.

To date, most reports targeting *BCL2* with siRNAs and ASOs in tumor-bearing mice have involved injection of a naked oligonucleotide, precluding comparison with the work presented here. Systemic injection in tumor-bearing mice of 10 mg doses of a naked anti-*BCL2* ASO was efficacious leading to phase 3 trials that ultimately failed due to poor efficacy.^[@bib2],[@bib27]^ Systemic injection of a naked anti-*BCL2* siRNA at 200 µg/kg daily for 24 days led to tumor suppression in human Panc1 (pancreatic) mouse xenografts.^[@bib22],[@bib23]^ Of particular interest was a dual functional siRNA that suppressed *BCL2 via* an RNA interference mechanism while activating RIG-1 expression and an immune response through a 5′ end triphosphate group with a combined efficacy at 50 µg doses delivered on days 3, 6, and 9 in a mouse melanoma model in C57BL6 mice.^[@bib55]^ However, the complex chemistry needed to produce triphosphate siRNAs may preclude the scale-up needed for clinical studies. While the RGD ligand used here was chosen for its simplicity and prior success when used at very low doses to image human tumors growing in mice,^[@bib56]^ there are many other tumor-targeting ligands that may prove superior. Although concern has been raised about the nonspecific effects of RGD when used at high doses,^[@bib43]^ this may be largely alleviated at far lower doses such as the ones reported here.

The achievement of proof-of-concept paves the way for future studies including exploration of pharmacodynamic and pharmacokinetic properties as well as expanded dose response studies and the use of other delivery systems/targeting ligands. Previous *in vitro* work found that, unlike siRNAs where the most potent siRNA in a pool appears to dominate the functional response,^[@bib57],[@bib58]^ the combined use of several U1 Adaptors against the same target increased the level of gene suppression.^[@bib7]^ The use of multiple U1 Adaptors against the same target may permit even lower dosing to be used, which could be beneficial if dose-related toxicity or other OTEs were found in a different system. Further, considering that clinical responses are not durable and relapse is a near-certainty for most cancers using monotherapy, the possibility of simultaneous multi-gene targeting with multiple U1 Adaptors to achieve higher levels of tumor cell apoptosis and longer lasting tumor suppression is an attractive option. Combinational therapy combining U1 Adaptors and small molecule chemotherapeutic agents is also expected to provide enhanced therapeutic benefit, an approach already shown to be successful for siRNAs^[@bib23]^ and ASOs.^[@bib21],[@bib24],[@bib27]^

Materials and Methods
=====================

*Xenografts in immunodeficient nude mice.* All animal studies were approved by the Institutional Review Board for the Animal Care and Facilities Committee of Rutgers University. Male nude mice (5 weeks old) were purchased from Taconic (Hudson, NY). Human melanoma cells, C8161 or UACC903, were injected into the dorsal area at 10^6^ cells per site. Unless indicated, all treatment groups comprised 10 mice. Tumors were measured weekly with a Vernier caliper and tumor volume (V; cubed centimeters) was calculated by using the equation *V* = *d *^2^*x*·*D*/2, where *d* (centimeters) and *D* (centimeters) are the smallest and largest perpendicular diameters. Once tumors reached ≈10 mm^3^, the animals were divided randomly into treatment groups so that the mean difference in tumor size between each group was \<10%. Mice were killed after \~3 weeks (for C8161) or \~5 weeks (for UACC903) corresponding to when the tumor volume in the vehicle-treated group had reached the maximum size permitted by the institutional review board. The tumor xenografts were excised for further histological and molecular analyses. *P* values were calculated using Student\'s two-tailed *t*-test.

*Preparation and analysis of RNA, proteins, and source of oligonucleotides.* RNA and protein levels from transfected cells and excised tumors from xenografts were determined by western blotting and reverse transcription-quantitative PCR as previously described,^[@bib7],[@bib31]^ except for the immunoprecipitation (IP) western blot (described below). The quantitative PCR primers, siRNAs, and gene-specific U1 Adaptors were manufactured by Integrated DNA Technologies (IDT, Coralville, IA) except for the LNA-containing Adaptors, which were purchased from Exiqon (Vedbaek, Denmark). The Control U1 Adaptor used is a 2′OMe-modified oligonucleotide not complementary to human transcripts 5′-GUCAGAAAUACA CAAUACCUGCCAGGUAAGUAU derived from a reporter transcript described in ref. ^[@bib7]^. Poly(I:C) (VacciGrade) was purchased from Invivogen (San Diego, CA).

*IPs and western blots.* IP of protein from excised tumors was done as follows with all steps done at 4 °C. Protein G beads (catalog 17-0618-01; GE Healthcare, Pittsburgh, PA) were prepared by washing 3x in TNET (20 mmol/l Tris pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% triton X-100), followed by 1 hour TNET + 1 mg/ml bovine serum albumin, followed by three washes in TNET and resuspended as a 50% slurry in TNET. Each IP sample contained 500 µg of tumor protein diluted to 300 µl with TNET containing protease cocktail inhibitors II, III, and IV (Calbiochem catalog nos. 524625, 539134, and 524628; Merck KGaA, Darmstadt, Germany) diluted as per the manufacturer\'s instructions. To each IP sample in **[Figure 2b](#fig2){ref-type="fig"}**; lanes 3--6, was added 8 µl anti-BCL2 antibody (Invitrogen catalog 13-8800; Life Technologies, Carlsbad, CA) and 1 µl antitubulin antibody (catalog AB-2/DM1A; Abcam, Cambridge, MA), except for lane 2 that had only antitubulin antibody. After gentle rocking for 4 hours, 20 µl protein G bead slurry (10 µl packed beads) was added and the sample gently rocked for four more hours. The beads were gently pelleted, the supernatant aspirated and the beads washed three times in TNET with a 5-minute rocking/wash. After removing the last of the supernatant, the beads were resuspended in laemmli loading buffer, heated and analyzed by 12% western blot. The upper half of the membrane was probed with antitubulin antibody. To avoid interference with the mouse light chain signal that migrates close to the BCL2 band, the lower half of the membrane was probed with anti-BCL2 from rabbit (SAB4300340; Sigma-Aldrich, St Louis, MO).

*Immunohistochemistry.* The Tissue Analytical Services at the Cancer Institute of New Jersey performed all the immunohistochemical staining of excised tumor xenografts to detect changes in the number of apoptotic and proliferating cells using the well-known activated Caspase 3 and Ki-67 markers, respectively. The number of stained cells was quantified with a digital Aperio ScanScopeGL system and ImageScope software (v 10.1.3.2028) (Aperio Technologies, Vista, CA) according to the manufacturer\'s protocol with modifications as described.^[@bib59]^

*Immune response and liver enzyme assays.* IL-12 and IFN-α levels in mouse serum samples were measured using specific ELISA kits from Invitrogen (Life Technologies) and PBL Interferon Source (Piscataway, NJ), respectively. The activities of alanine transaminase and alkaline phosphatase in mice serum were determined using the MaxDiscovery ALT enzymatic assay kit and MaxDiscovery AP enzymatic assay kit (Bioo Scientific, Austin, TX) according to the manufacturer\'s instructions.

*Statistics.* The number of mice used for each experiment was determined with the help from the Biometrics Facility Core at the Cancer Institute of New Jersey. *P* values were determined using unpaired Student\'s two-tailed *t*-test. Xenograft experiments comprised 10 mice/group except in a few cases where 5--8 mice/group were used. With 10 mice per group, a 35% treatment difference between control and tested group can be detected with 80% power at an α-level of 5% (two-sided test). A *P* value of \<0.05 was considered significant.

*Preparation of RGD-G5 nanoparticles.* PPIG5 (SyMO-Chem, Eindhoven, Netherlands) was diluted to a 10 mmol/l stock in water using HCl to bring the pH to 7.0 and stored in aliquots at −80 °C. cRGD and cRAD (Peptides International, Louisville, KY), and the SM(PEG)~12~ and LC-SPDP linkers (Thermo Scientific Pierce, Rockford, IL) were resuspended and stored as per the manufacturer\'s instructions. Conjugation to make RGD-G5 was as follows: 24 µl of 200 mmol/l SM(PEG)~12~ in DMSO was added to 6.1 ml of 0.25 mmol/l PPIG5 in aqueous buffer containing 50 mmol/l KCl, and incubated for 1 hour at room temperature, (SM(PEG)~12~ was threefold excess over PPIG5). Glycine was then added to a final 3 mmol/l concentration to neutralize any unreacted SM(PEG)~12~ and after 30 minutes the solution brought to 40 mmol/l Tris pH 6.8 and a sixfold excess cRGD added to a final 1.5 mmol/l concentration. After 1 hour of conjugation, excess cysteine was added to neutralize any unreacted groups and the sample extensively dialyzed 48 hours with three changes of buffer against 30 mmol/l Hepes pH 7.9. The final sample was 200 µmol/l RGD-G5 in 30 mmol/l Hepes pH 7.9 and was stored in aliquots at −80 °C. Multiple freeze thawing (\>20) had no detectable effect on activity.

*Preparation of RGD-G5/SPDP, a short, non-PEG--containing linker version of RGD-G5.* RGD-G5/SPDP was prepared as follows: 8 ml of 0.25 mmol/l PPIG5 in 10 mmol/l Hepes pH 7.9, 1 mmol/l EDTA, and 1.5 mmol/l LC-SPDP (added last) was incubated for 2 hours at room temperature. After overnight dialysis with two changes of buffer against 10 mmol/l Hepes pH 7.9, 1 mmol/l EDTA, a pyridine 2-thione release assay was done as per the manufacturer\'s instructions to determine the conjugation efficiency. Then 500 µl of 5 mg/ml cRGD freshly dissolved in 3% acetic acid was added to 7.3 ml of the activated PPIG5 and the reaction proceeded overnight at 4 °C. After overnight dialysis with two changes of buffer against 10 mmol/l Hepes pH 7.9, 1 mmol/l EDTA, a pyridine 2-thione release assay was done and the final samples stored in aliquots at −80 °C.

*Dynamic light scattering.* Particle size of the injected formulation at standard dose either a 1.7 µg (3.2 µmol/l) U1 Adaptor:2.4 µg (4 µmol/l) RGD-G5 complex in 1x PBS or a 1.7 µg (3.2 µmol/l) U1 Adaptor:8 µg (4 µmol/l) RGD-PAMAM-G5 complex in 1x PBS was determined by a DLS apparatus (Malvern Zetasizer; Malvern Instruments, Westborough, MA). Several dilutions of the sample were analyzed by automatic measurement setting comprised of the average of runs, each run having 30 measurements of 5 seconds per measurement.

*Preparation of RGD-PAMAM-G4 and RGD-PAMAM-G5.* PAMAM-G4 and PAMAM-G5 (Dendritech, Midland, MI) were diluted to a 2 mmol/l stock in water using HCl to bring the pH to 7.0 and stored in aliquots at −20 °C. RGD-PAMAM-G4 was prepared as follows: 1 ml of 0.2 mmol/l PAMAM-G4 in 25 mmol/l Hepes pH 7.9, 1 mmol/l EDTA, 150 mmol/l NaCl, and 1.2 mmol/l LC-SPDP (added last) was incubated for 2 hours at room temperature. After overnight dialysis with two changes of buffer against 25 mmol/l Tris pH 8.0, 1 mmol/l EDTA, and 150 mmol/l NaCl, a pyridine 2-thione release assay was done as per the manufacturer\'s instructions to determine the LC-SPDP conjugation efficiency. Then 23.2 µl of 25 mg/ml cRGD freshly dissolved in 3% acetic acid was added to 1.3 ml of the activated PAMAM-G4 and the reaction allowed to proceed overnight at 4 °C. A pyridine 2-thione release assay was done to measure cRGD coupling efficiency. After overnight dialysis with two changes of buffer against 1x PBS, the final samples were stored in aliquots at −80 °C. The same protocol was used to prepare RGD-PAMAM-G5.

*Calculation of the RGD:PPIG5 ratio.* The number of RGD\'s linked to PPIG5 was determined in several ways. First, the colorimetric bicinchoninic acid protein assay (Thermo Scientific Pierce) that measures reduction of Cu^2+^ to Cu^1+^ by the RGD peptide was used. Second, RGD-G5 and RGD-G5/SPDP underwent MALDI-MS analysis using as a matrix 2,4,6-Trihydroxyacetophenone (THAP) in 50% acetonitrile in 50 mg/ml ammonium acetate. Third, the LC-SPDP linker used to make RGD-G5/SPDP permits conjugation efficiency by monitoring pyridine 2-thione release at 343 nm as per the manufacturer\'s instructions. All of these methods led to close agreement that an average of two RGD\'s were coupled to each PPIG5.

*Cell culture and transfection.* C8161 and UACC903 cells were grown in RPMI medium supplemented with 10% fetal bovine serum and antibiotics. Cationic lipid-based transfection with LF2000 was done as previously described.^[@bib7]^ Transfection with RGD-G5 dendrimer was done as follows. For a 6-well plate, a 0.2 ml transfection mix containing 1x PBS pH 7.2 was prepared. RGD-G5 was diluted into 180 µl PBS and then the Dicer-substrate siRNA or U1 Adaptor was added to give a final volume of 0.2 ml in 1x PBS, and the solution gently mixed. After 5 minutes at room temperature, the RGD-G5:oligonucleotide complexes were added to cells that had been overlaid with 1.8 ml of fresh growth media. For larger or smaller scale transfections, the transfection mix was scaled accordingly. After the indicated time, cells were harvested and protein and/or RNA was extracted.

[**SUPPLEMENTARY MATERIAL**](#sup1){ref-type="supplementary-material"} **Figure S1.** *In vitro* silencing activity of anti-*BCL2* U1 Adaptors. **Figure S2.** *In vitro* silencing activity of variants of the BCL2-A Adaptor. **Figure S3.** *In vitro* dose response silencing activity of the BCL2-A Adaptor complexed with the RGD-G5 nanoparticle. **Figure S4.** *In vitro* silencing activity of various dendrimer:BCL2-A Adaptor complexes. **Figure S5.** Particle size of various dendrimer:BCL2-A Adaptor complexes. **Figure S6.** Analysis of BCL2-A activity in C8161 and UACC903 xenografts. **Figure S7.** *In vitro* silencing activity of anti-*GRM1* U1 Adaptors and matching controls. **Figure S8.** Western blot analysis to assess the level of pAKT versus AKT in day 17 tumors from anti-*GRM1* U1 Adaptor-treated mice. **Figure S9.** The anti-*GRM1* Adaptors suppress growth of UACC903 xenografts and terminal PS bonds in the Adaptors are needed for full activity. **Figure S10.** U1 Adaptor treatment does not elicit an immune response at 4 hours. **Figure S11.** U1 Adaptor treatment does not elicit an immune response at 24 hours.

This work was done in Piscataway, New Jersey; Hillsborough, New Jersey; and Coralville, Iowa. This work was supported by the National Institutes of Health SBIR grants R43CA153842 to R.G. and S.C. and 5R44GM085863-03 to M.A.B, and an National Institute of Environmental Health Sciences center grant ES005022. This work was also supported by grants to S.I.G. and S.C. from the QED Proof-of-Concept Program (University City Science Center) and the Busch Biomedical Research Program. We also thank the Rutgers University Summer Undergraduate Research Fellowship (SURF) fellowship program and Aresty Center for supporting the undergraduate research performed by N.H.Z., C.J., N.R.P., S.Z., C.M., and S.A.S. We thank members of the Gunderson and Chen labs, the Rutgers University/University of Medicine and Dentistry New Jersey RNA community, the IDT Molecular Genetics & Biophysics group for advice and comments during the course of this work and Li Gu of the Kathryn Uhrich lab for instructing us on use of their Malvern Zetasizer instrument. S.I.G. and R.G. declare competing financial interests due to part ownership of Silagene Inc. M.A.B. and K.A.L. are employed by Integrated DNA Technologies, Inc., (IDT) which offers oligonucleotides for sale similar to some of the compounds described in the manuscript. IDT is, however, not a publicly traded company and neither author personally owns any shares/equity in IDT. The other authors declared no conflict of interest.

**Supplementary material** {#sup1}
==========================

######

Click here for additional data file.

![***In vitro* silencing activity of anti-*BCL2* U1 Adaptors and a tumor-targeting nanoparticle**. (**a**) Design and sequences of the BCL2-A and BCL2-B Adaptors, backbone-modified variants of BCL2-A and matching controls as well as an anti-*BCL2* DsiRNA used as a positive control. The boxed region is to indicate a full set of matching control Adaptors. (**b**) The BCL2-A~L2~ U1 Adaptor reduces BCL2 protein. At 24 hours post-transfection of the indicated Lipofectamine 2000 (LF2000):Adaptor or LF2000:anti-*BCL2* DsiRNA complexes, total C8161 lysates were prepared followed by western blot. Lanes 3--10 contain 50 µg/well while lane 2 has 17 µg of protein to show the dose response of the western blot signal. Tubulin was used to show equal loading (top panel) and the lower panel was probed for BCL2. Quantitation of the western signals is shown as a BCL2:tubulin ratio with lane 3 set to 1.0. (**c**) Schematic of RGD-G5 (MW \~9,600) that has a 2:1 RGD to PPIG5 conjugation ratio. For simplicity, only some of the 64 free amino groups of PPIG5 are shown. The attachment positions of the SM(PEG)~12~-linker-RGD group are random and are shown here for illustrative purposes. (**d**) The RGD-G5 nanoparticle (right panel) is comparable to LF2000 (left panel) at delivering the BCL2-A Adaptor to C8161 cells as measured by RT-qPCR of *BCL2* mRNA normalized to *HPRT1*. Note Cq values between the LF2000 control and RGD-G5 control were comparable. DsiRNA, Dicer-substrate small interfering RNA; LNA, locked nucleic acid; MW, molecular weight; RT-qPCR, reverse transcription-quantitative PCR.](mtna201324f1){#fig1}

![**BCL2-A suppresses tumor growth *in vivo***. (**a**) The design and sequences of the BCL2-A and matching control U1 Adaptors are shown at the top. C8161 xenograft mice were treated when tumor volumes reached \~10 mm^3^, which is denoted as day 1. Each treatment group (TG) mouse was treated two times/week consisting of a tail vein injection of 50 µl 1x PBS containing BCL2-A or matching control U1 Adaptors in complex with the RGD-G5 vehicle or the G5 vehicle that lacked RGD (TG6). The only TG that showed significant tumor suppressive activity was TG2 in comparison with TG1. Note that 1.7 µg Adaptor is \~0.16 nmol, 2.4 µg RGD-G5 is 0.2 nmol. (**b**) Immunoprecipitation (IP)-western blot of day 22 excised tumor samples. Due to the low BCL2 signal in standard western blots using excised tumor lysates in comparison to cultured cells (compare lane 9 with lanes 7--8, 10-11), we performed anti-BCL2 IP in lanes 3--6 before the western blot. Antitubulin IP was also done in lanes 3--6 to internally control for IP efficiency. Lane 2 was antitubulin IP only to show the specificity of the BCL2 IP signals. Lane 1 had 3x less protein than lane 3 to show the dose response of the IP-western blot signals. Levels of BCL2 protein were comparable in TG1, TG3, and TG4 (lanes 3, 5, and 6) whereas TG2 (lane 4) BCL2 levels were barely detectable. Quantitation of the western signals is shown in lanes 3--6 as a BCL2:tubulin ratio with lane 3 set to 1.0. (**c**,**d**) Immunohistochemistry of day 22 excised tumor samples for TG1-4 mice with (**c**) activated Caspase 3 as a measure of cellular apoptosis and (**d**) Ki67 as a measure of proliferation. The entire slide ("Whole Tumor" image, ×10 original magnification) was counted and the percent of positive cells is given below the image. The *P* value was only significant for TG2 as compared with TG1. The "Zoom" images are a representative ×100 original magnification image of the whole tumor. In **c** and **d**, \*\*\**P* \< 0.0001 when compared with the TG1 vehicle control. (**e**) Tumor volumes of *ex vivo*-treated C8161 xenograft mice. One day before tumor cell implantation, C8161 were transfected as per **[Figure 1d](#fig1){ref-type="fig"}** with the following complexes: RGD-G5 as the vehicle control, BCL2-A/TDmt:RGD-G5 as an inactive Adaptor control, or BCL2-A:RGD-G5. After 24 hours, cells were prepared and 1 million cells were subcutaneously implanted into each flank of each mouse. Tumor growth was then monitored for 23 days when the experiment was terminated. \*\*\**P* \< 0.0001 when the tumor volumes of BCL2-A:RGD-G5 were compared with the RGD-G5 vehicle controls. MW, molecular weight.](mtna201324f2){#fig2}

![**Tumor suppression activity of various anti-*BCL2* U1 Adaptors**. (**a**) Tumor volumes of C8161 xenografts are shown with the Adaptor sequences given in **[Figure 1a](#fig1){ref-type="fig"}**. Except for the indicated differences, these tumor suppression experiments and statistics are as described in **[Figure 2a](#fig2){ref-type="fig"}**. The left and right panels are from experiments done at different times. Treatments of TG10 mice matched those of TG8 except the RGD-G5 vehicle had a 4:1 rather than 2:1 RGD:G5 ratio. The bracket is the *P* value of TG10 versus TG8. All other *P* values (\*\*\**P* \< 0.0001 and \*\**P* \< 0.001) were calculated as compared with either the TG7 vehicle control (left panel) or the TG11 vehicle control (right panel). (**b**) Dose response studies for treatment of C8161 xenografts. Except for the indicated differences, these tumor suppression experiments, statistical analysis, and symbols are as in **[Figures 2a](#fig2){ref-type="fig"}** and **[3a](#fig3){ref-type="fig"}**. The bracket is the TG18 versus TG16 *P* value that shows a significant reduction in efficacy when using 3x more RGD-G5 vehicle than U1 Adaptor. All other *P* values (\*\*\**P* \< 0.0001 and \*\**P* \< 0.001) were calculated as compared with the TG14 vehicle control. (**c**) Shows the tumor suppression activity of BCL2-B in C8161 xenografts as compared with BCL2-A. Except for the indicated differences, these experiments are as described in **[Figures 2a](#fig2){ref-type="fig"}** and **[3a](#fig3){ref-type="fig"}**. (**d**) Shown are the design and sequences of the BCL2-B and matching control U1 Adaptors and their *in vivo* tumor suppression activities in C8161 xenograft mice. Except for the indicated differences and the use of 8 µg (0.2 nmol) RGD-PAMAM-G5 nanoparticle in place of 2.4 µg (0.2 nmol) RGD-G5, these experiments and symbols are as described in **[Figures 2a](#fig2){ref-type="fig"}** and **[3a](#fig3){ref-type="fig"}** and the *P* values calculated as compared with the vehicle only control. In **c** and **d**, \*\*\**P* \< 0.0001 and \*\**P* \< 0.001 when compared with the corresponding vehicle control.](mtna201324f3){#fig3}

![**Anti-*GRM1* U1 Adaptors are active *in vitro* and suppresses tumor growth *in vivo***. (**a**) The design and sequences of three anti-*GRM1* U1 Adaptors made of 2′OMe RNA with three phosphorothioate internucleotide linkages at each end. (**b**) Western blot demonstrating the anti-*GRM1* U1 Adaptors reduce GRM1 protein levels in cultured C8161 cells. Transfection and western blotting are as in **[Figure 1b](#fig1){ref-type="fig"}** except C8161 lysates were prepared at 72 hours post-transfection and 50 µg/lane were separated by 8% SDS-PAGE. The transfection and western blots were repeated three times with similar results. (**c**) Shown are the *in vivo* tumor suppression activities of the anti-*GRM1* U1 Adaptors in C8161 xenograft mice. Except for the indicated differences, these experiments are as described in **[Figures 2a](#fig2){ref-type="fig"}** and **[3a](#fig3){ref-type="fig"}** and the *P* values calculated as compared with the vehicle only control. The insert is a western blot of excised day 21 tumor samples with amounts of total tumor protein loaded per lane indicated (note, unlike **[Figure 2b](#fig2){ref-type="fig"}** for *BCL2* where IP-western blot performed, this is a regular western blot). Lanes 1 and 2 contained less protein to show the dose response of the western blot signals. Tubulin was used to show equal loading. (**d**) The design and sequences of the GRM1-B~ps~ and matching control U1 Adaptors and their *in vivo* tumor suppression activities in C8161 xenograft mice. Except for the indicated differences, these experiments are as described in **[Figures 2a](#fig2){ref-type="fig"}** and **[3a](#fig3){ref-type="fig"}** and the *P* value calculated as compared with the vehicle only control. In **c** and **d**, \*\*\**P* \< 0.0001 when compared with the corresponding vehicle control. (**e**) To assess *GRM1* expression, protein and RNA were extracted from excised (as shown in **d**) day 17 tumors and analyzed by RT-qPCR (upper panel) or western blot (lower panel). The experiments were done three times with similar results. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative PCR; TD, target domain; U1D, U1 domain.](mtna201324f4){#fig4}

![**Mechanism of anti-*GRM1* U1 Adaptors**. (**a**) Shown are the design and sequences of the GRM1-A~ps~ and matching control U1 Adaptors and their *in vivo* tumor suppression activities in C8161 xenograft mice. Except for the indicated differences, these experiments and symbols are as described in **[Figures 2a](#fig2){ref-type="fig"}** and **[3a](#fig3){ref-type="fig"}** and the *P* values calculated as compared with the vehicle only control. Treatments for the GRM1-A~ps~:RAD-G5 group matches that of the GRM1-A~ps~:RGD-G5 group except the RGD peptide was replaced with an RAD peptide that has lost binding to the receptor. Treatments for the GRM1-A~ps~:RAD-G5-SPDP linker group matches that of the GRM1-A~ps~:RGD-G5 group except the former nanoparticle had a far shorter linker and non-PEGylated chemistry as described in the text and Materials and Methods section. (**b**,**c**) IHC of excised day 17 tumor samples from **[Figure 5a](#fig5){ref-type="fig"}** mice to assess levels of (**b**) activated Caspase 3 as a measure of apoptosis and (**c**) Ki67 as a measure of proliferation. Symbols and quantitation are as described in **[Figure 2c,d](#fig2){ref-type="fig"}**. In **a**--**c**, \*\*\*P \< 0.0001 when compared with the corresponding vehicle control. IHC, immunohistochemistry; TD, target domain; U1D, U1 domain.](mtna201324f5){#fig5}

![**U1 Adaptor treatment does not elicit immune responses or increase liver enzyme activity in C57BL6 mice**. (**a**) C57BL6 male mice (five mice/group) underwent a single tail vein injection with the standard dose of 1.7 µg Adaptor:2.4 µg RGD-G5 complex or just 2.4 µg RGD-G5 vehicle alone. The first group was not injected. Poly(I:C) was injected as a positive control.^[@bib51]^ At 4 hours post-injection, blood was drawn and IL-12 levels were measured as well as IFN-α levels (**Supplementary Figure S10**). (**b**,**c**) C57BL6 male mice (five mice/group) underwent 3 weeks of standard U1 Adaptor treatment as described in **[Figure 2a](#fig2){ref-type="fig"}** with two times/week dosing with a 1.7 µg Adaptor:2.4 µg RGD-G5 complex or just 2.4 µg RGD-G5 vehicle alone. The first group of mice was not injected. At 48 hours after the final injection, blood was drawn and levels of the (**b**) ALT and (**c**) AP liver enzymes were measured. ALT, alanine transaminase; AP, alkaline phosphatase; IFN-α, interferon-α IL, interleukin.](mtna201324f6){#fig6}


Pre-operative and post-operative psychiatric manifestations in patients with supratentorial meningiomas.
In the present study, we have tried to delineate the nature of psychiatric abnormalities caused by supratentorial meningiomas and the effect of surgery on them. We have tried to find the patient and tumor characters influencing the psychiatric abnormalities and their post-operative outcome. This is a prospective study conducted on patients with supratentorial meningiomas, admitted and operated in neurosurgery department, Nizam's Institute of Medical Sciences, India, from July 2006 to July 2009. We have included fifty-seven patients aged between 15 and 65 years with a clinico-radiological diagnosis of supratentorial meningioma in our study. We later confirmed the diagnosis by histopathological examination of the tumor. We have evaluated the patients for psychiatric manifestations before and after surgery and also analyzed the various clinical and radiological factors influencing the psychiatric status. We have enrolled 57 patients into the study. Frontal group had 22 patients (38.6%), parietal group had 10 patients (17.5%), temporal group had 10 patients (17.5%), occipital group had 6 patients (10.5%), and suprasellar group had 9 patients (15.8%). Twenty patients (35.1%) presented with psychiatric symptoms. The frequency of psychiatric symptoms was highest in the temporal group (60%) followed by the frontal group (45.5%). Frontal convexity meningiomas presented predominantly with depression, basifrontal and sphenoid wing meningiomas presented with mania or depressive symptoms, Suprasellar lesions and temporal convexity lesions presented with organic delusional disorder. Basifrontal meningiomas also caused organic personality disorders. The frequency of psychiatric symptoms was much higher in meningiomas with volume greater than 35cc compared to the smaller ones, in the frontal group. None of the patients developed new psychiatric symptoms after surgery. Among the twenty patients with psychiatric symptoms, 3 (15%) didnot improve, 8 (40%) improved partly and 9 (45%) improved completely. Meningiomas, although extra-axial, cause significant psychiatric symptoms up to 35.1%. Frontal and temporal group of meningiomas have the highest frequency of psychiatric symptoms. The frequency of psychiatric symptoms was significantly higher in meningiomas with volume greater than 35cc compared to the smaller ones, in the frontal group. Surgical excision of meningiomas ameliorates the psychiatric symptoms, either completely or partly, in the majority of the patients.


Downtown Newark is getting another luxury residential tower, an $80 million project that would bring 256 units to the Brick City, according to developer Strategic Development Partners.

The building, named Vibe, will be made up of 60 percent one-bedroom, 20 percent two-bedroom and 20 percent studio apartments. It is slated for the intersection of Halsey and William streets in the Living Downtown Redevelopment Area, Strategic Development Partners said in a news release.

“Vibe will offer a technology-rich environment with abundant lifestyle amenities for millennials, who prefer the energy and convenience of cities like Newark,” Anthony V. Bastardi, Strategic Development Partners’ chairman and CEO, said in a prepared statement. “The location — just minutes from diverse dining options, world-class cultural events, sports and transportation — will make Vibe an enviable address.”

The building will join residential construction projects in the city that include One Theater Square and One Rector Street, the first market-rate residential towers in the city in more than 50 years.

“Vibe represents a significant investment in Newark by a talented and experienced team of redevelopment professionals,” Mayor Ras Baraka said in a statement. “It is yet another exciting sign that this city is a highly desirable location.”

The property will include two restaurants on the ground level, the developer said, joining a number of other new Newark eateries in the area. Amenities for residents will include a rooftop pool and spa, outdoor dining area, fitness center and on-site parking for 156 vehicles, as well as a coffee bar and rooftop lounge.

“We have incorporated resort-style amenities into every detail of Vibe,” Barbara A. Hillier, principal with venture partner Studio Hillier. “Residents will enjoy light and airy living spaces, as well as a menu of highly desired social and recreational opportunities.”

The developer said the project represents the closest new residential development to the Prudential Center, and will sit within walking distance of several other downtown landmarks and minutes from Newark Penn Station.

“This technology-rich environment, coupled with other first-class amenities, distinguishes Vibe as a leading player in Newark’s hottest residential market,” Adrienne Albert, CEO of The Marketing Directors, another venture partner, said in a statement. “This is especially important to millennials, who prefer the convenience of living in the city, with access to cultural and sporting events, as well as transportation, recreational amenities and the latest technologies. Vibe has it all.”

The building’s website will be online soon at VibeNewark.com.


                               COURT OF APPEALS OF VIRGINIA

Present: Judges Kelsey, Haley and Beales
Argued at Chesapeake, Virginia

STEVEN ALLEN RIDDICK
                                                              MEMORANDUM OPINION ∗ BY
v.     Record No. 1102-07-1                                   JUDGE JAMES W. HALEY, JR.
                                                                     MAY 13, 2008
COMMONWEALTH OF VIRGINIA

                   FROM THE CIRCUIT COURT OF THE CITY OF HAMPTON
                               Christopher W. Hutton, Judge

                 Charles E. Haden for appellant.

                 Rosemary V. Bourne, Assistant Attorney General (Robert F.
                 McDonnell, Attorney General, on brief), for appellee.

                                        I. INTRODUCTION

       Steven Allen Riddick appeals his conviction for first-degree murder in the death of

Veronica Collins-Goss from the Circuit Court of the City of Hampton. Riddick argues the circuit

court erred in (1) admitting statements Collins-Goss made to others that she was pregnant and

that Riddick was the father, (2) permitting his probation officer to testify during the guilt phase

of the trial, and (3) failing to strike the first-degree murder charge. We affirm the circuit court.

                                             II. FACTS

       Under settled principles, we review the evidence in the “light most favorable” to the

Commonwealth. Commonwealth v. Hudson, 265 Va. 505, 514, 578 S.E.2d 781, 786 (2003).

That principle requires us to “discard the evidence of the accused in conflict with that of the

Commonwealth, and regard as true all the credible evidence favorable to the Commonwealth and

all fair inferences to be drawn therefrom.” Parks v. Commonwealth, 221 Va. 492, 498, 270

       ∗
           Pursuant to Code § 17.1-413, this opinion is not designated for publication.
S.E.2d 755, 759 (1980) (emphasis and citation omitted). So viewed, the evidence at trial

established as follows.

       In the evening of September 8, 2003, Collins-Goss returned to her apartment alone

around 10:00 p.m. Thereafter, an argument began in her apartment. Khaira Blandford, who

lived below Collins-Goss, “heard a loud argument, two people arguing, a female voice and a

male’s voice arguing, screaming, things being thrown around.” Blandford stated as the argument

intensified, someone turned on loud music, which made the voices more difficult to understand.

At one point and in spite of the loud music, Blandford heard Collins-Goss scream “don’t hurt

me, don’t hurt my baby, don’t hurt your baby, don’t you want your baby.”

       While Blandford initially did not take any action in response to the argument, she became

alarmed and called the police after she “heard an extremely loud thump, as though something

had been thrown against the wall.” Due to the impact, “the pictures and the clock in my

roommate’s room fell off the wall and boxes fell out of the closet.” The police arrived

approximately ten to fifteen minutes later. They knocked on Collins-Goss’ apartment, but no

one answered. The police then waited in their car outside the building for fifteen to twenty

minutes, but since it appeared to them no one was in the apartment, departed. Blandford

knocked on Collins-Goss’ door twice the next day, but received no answer.

       At approximately 2:20 a.m. on September 9, 2003, Officer John D’Busk encountered

Riddick walking in an industrial area of Suffolk, Virginia in the rain and heavy wind. D’Busk

approached Riddick and asked him why he was there. Riddick stated he had been traveling with

a friend in his friend’s car, but the car had broken down and the two of them had separated to

find assistance. Riddick indicated the left front tire was flat. At Riddick’s request, D’Busk took

him to a nearby pay phone. D’Busk then began searching for the vehicle, which he had obtained

a description of from Riddick, and found a vehicle matching the description. The car also had a

                                               -2-
flat left front tire. D’Busk contacted another officer in the area to bring Riddick to the car to

identify it. When Riddick arrived, he denied it was the right car and, in contradiction of his

earlier statement, claimed the car he spoke of did not have a flat tire. He stated his friend was

driving the car, but could not tell D’Busk the name of his friend. Riddick then stated it was not

his friend who was driving, but his friend’s girlfriend and that her name was Monique. The car

was registered to Collins-Goss.

          D’Busk offered Riddick a seat in his patrol car given the inclement weather, which

Riddick accepted. As D’Busk continued to examine the car, Officer Gaines, who transported

Riddick to the scene, saw Riddick take something from his pocket and throw it in his boot.

Gaines removed Riddick from the car and frisked him. Gaines took car keys from Riddick’s

boot. D’Busk asked Riddick whether they were the keys to the car, and Riddick stated the car

belonged to his friend. D’Busk asked whether it belonged to his friend or his friend’s girlfriend,

and Riddick said he meant to say the girlfriend. The keys opened the car. The officers later

freed Riddick when they could not contact Collins-Goss and the vehicle did not register as

stolen.

          Collins-Goss’ body was discovered in her apartment late in the evening of September 10,

2003, by two police officers. They gained entry to the apartment after having a maintenance

worker unlock it.

          Dr. Wendy Gunther performed an autopsy on Collins-Goss. Her testimony revealed that

the victim had suffered multiple injuries. These included blackened eyes, a split lip, and a

broken nose. Her death was caused by “blunt impact . . . trauma” to the rear and side of her

head, resulting in bleeding into and swelling of the brain. This trauma was consistent with being

struck by an iron. In addition, though not a cause of death, physical evidence demonstrated that

the victim had also been strangled.

                                                -3-
       Betty Jane Blankenship, who is a scientist working in DNA testing, testified blood taken

from the crime scene matched the DNA profile of Riddick. Riddick’s DNA was found in a

blood stain on a hamper. It was also located in fingernail clippings taken from Collins-Goss.

Blankenship testified that given the number of samples tested where Riddick’s DNA appeared,

Riddick was the only person in the world whose DNA could produce the matches. Blankenship

further testified Riddick’s DNA from Collins-Goss’ fingernail clippings likely arrived there soon

before she died since a simple hand washing would have eliminated the DNA.

       Police arrested Riddick on September 12, 2003. Riddick denied responsibility for

Collins-Goss’ death during questioning. An examination of Riddick’s body by police pursuant

to a search warrant revealed multiple recent wounds. Detective Curtis Crouch stated Riddick

“had multiple scratches on different and various parts of his body.” The scratches appeared on

the neck, torso, and legs. Crouch further stated Riddick “had one particular wound that was to

one of his fingers that was still oozing. It was not scabbed over.” Riddick volunteered to Crouch

that the injuries occurred during a recent incarceration.

       Collins-Goss had told her mother, Thelma Graham, and two friends, Melissa Grubb and

Anisha Brown, that she was pregnant by Riddick. All three testified to this at trial. The autopsy

confirmed Collins-Goss was pregnant.

       Riddick’s probation officer also testified. Riddick told him on July 16, 2003, and

September 10, 2003, that his address was the same as Collins-Goss’ apartment. An agent of the

apartment complex where Collins-Goss lived testified her apartment was leased to both her and

Riddick.

       A grand jury indicted Riddick for murder on February 2, 2004. Riddick was tried before

a jury, which found him guilty of first-degree murder on September 28, 2006. On September 29,

                                                -4-
2006, the jury recommended Riddick receive a sentence of fifty years incarceration. The court

imposed this sentence. Riddick now appeals.

                                         III. ANALYSIS

       “On appeal, we review the evidence in the light most favorable to the Commonwealth,

granting to it all reasonable inferences fairly deducible therefrom.” Martin v. Commonwealth, 4

Va. App. 438, 443, 358 S.E.2d 415, 418 (1987). We presume the decision of the circuit court to

be correct. Davis v. Commonwealth, 39 Va. App. 96, 99, 570 S.E.2d 875, 876-77 (2002). This

Court will “affirm the conviction unless it is plainly wrong or without evidence to support it.”

Shackleford v. Commonwealth, 262 Va. 196, 209, 547 S.E.2d 899, 906 (2001).

                 A. Whether the Circuit Court Erred in Admitting Hearsay that
                          Collins-Goss Was Pregnant by Riddick

       Riddick first claims the circuit court erred in admitting hearsay statements made by

Collins-Goss that she was pregnant and that Riddick was the father. Riddick claims this violated

his rights under the Confrontation Clause of the Sixth Amendment of the United States

Constitution. He further argues the court should have prohibited the testimony because it did not

come within the state-of-mind exception to hearsay.

       In Crawford v. Washington, 541 U.S. 36, 68 (2004), the Supreme Court held that when

the prosecution seeks to admit testimonial hearsay, “the Sixth Amendment demands what the

common law required: unavailability and a prior opportunity for cross-examination.” The Court

held testimonial evidence includes “prior testimony at a preliminary hearing, before a grand jury,

or at a former trial; and to police interrogations.” Id. The Court chose not to address whether

nontestimonial hearsay receives Sixth Amendment protection. Id. at 61. In Davis v.

Washington, 547 U.S. 813, 821 (2006), the Supreme Court made clear that only testimonial

statements implicate the Sixth Amendment. In another portion of the opinion relevant to this

appeal, the Court stated that “statements made unwittingly to a Government informant” and

                                               -5-
“statements from one prisoner to another” “were clearly nontestimonial.” Id. at 825. Relying on

these cases, our Supreme Court recently wrote that “the question whether admission of a hearsay

statement . . . violates the Confrontation Clause turns on whether the statement is ‘testimonial’ in

nature.” Magruder v. Commonwealth, 275 Va. 283, 294-95, 657 S.E.2d 113, 118 (2008).

       The statements at issue in this case, which were casual remarks from Collins-Goss that

she was pregnant and that Riddick was the father, were clearly nontestimonial under these cases.

They, therefore, do not receive Confrontation Clause protection. Indeed, Riddick concedes on

brief the statements are nontestimonial. Riddick’s counsel stated at oral argument he was

unaware of the Supreme Court’s holding in Davis at the time he wrote his brief, but conceded its

strength. We apply Davis, as we are bound to do.

       We next consider whether the statements were admissible under the state-of-mind

exception to the hearsay rule. The Supreme Court of Virginia has held regarding this exception:

                       Generally, statements made by a crime victim that show the
               victim’s state of mind are admissible as an exception to the hearsay
               rule, provided the statements are relevant and probative of some
               material issue in the case. Evidence is relevant if it tends to prove
               or disprove, or is pertinent to, matters in issue.

Clay v. Commonwealth, 262 Va. 253, 257, 546 S.E.2d 728, 730 (2001) (citations omitted). “The

key to the admissibility of evidence showing a victim’s state of mind is thus its relevance to a

material issue in the case. Relevance exists when the evidence has a ‘logical tendency, however

slight, to prove a fact at issue in a case.’” Hodges v. Commonwealth, 272 Va. 418, 436-37, 634

S.E.2d 680, 690 (2006) (quoting Winston v. Commonwealth, 268 Va. 564, 596, 604 S.E.2d 21,

39 (2004) (citations omitted), cert. denied, 546 U.S. 850 (2005)). We review the circuit court’s

decision only for an abuse of discretion. Schneider v. Commonwealth, 47 Va. App. 609, 613-14,

625 S.E.2d 688, 690 (2006).

                                                -6-
       We conclude the circuit court did not abuse its discretion since the statements at issue

here fall within the state-of-mind exception because they tend to prove the identity of

Collins-Goss’ attacker. Riddick has not contested the admissibility of the testimony of Khaira

Blandford that Collins-Goss stated “don’t hurt me, don’t hurt my baby, don’t hurt your baby,

don’t you want your baby.” The person Collins-Goss believed to be the father of her unborn

child represents key evidence in establishing the identity of her assailant. The hearsay testimony

that Collins-Goss believed Riddick to be the father strongly tends to establish Riddick as the

attacker. It is thus relevant “to a material issue in the case” and admissible. 1 Hodges, 272 Va. at

436, 634 S.E.2d at 690.

                   B. Whether the Circuit Court Erred in Permitting Riddick’s
                                 Probation Officer to Testify

       Riddick next contends the circuit court erred by allowing his probation officer to testify.

Riddick argues the presence of the probation officer unfairly portrayed him as someone likely to

commit crime.

       We hold that assuming without deciding Riddick’s argument has merit, at this point in

our analysis any error in this regard was harmless as the Commonwealth presented

overwhelming evidence of Riddick’s guilt. The Supreme Court of Virginia has adopted the test

of the United States Supreme Court for determining non-constitutional harmless error. Billips v.

Commonwealth, 274 Va. 805, 810, 652 S.E.2d 99, 102 (2007). That test has been stated as

follows:

       1
         As noted, Riddick argues the statements made by Collins-Goss were not within the
state-of-mind exception to the hearsay rule. Accordingly, we addressed and rejected that
argument. In addition, however, the Collins-Goss statements can be characterized as not hearsay
at all. Rather, the statements could be relevant circumstantial evidence of her state of mind, but,
not being offered as to the truth of the matter asserted, that is, that she was, in fact, pregnant and
Riddick was, in fact, the father, not hearsay and thus admissible. See Hodges v. Commonwealth,
45 Va. App. 735, 756, 613 S.E.2d 834, 844 (2005), aff’d in part, rev’d in part, 272 Va. 418,
428-29, 634 S.E.2d 680, 685 (2006); 2 McCormick on Evidence § 274 (6th ed. 2006).

                                                -7-
                       “If, when all is said and done, the conviction is sure that the
               error did not influence the jury, or had but slight effect, the verdict
               and the judgment should stand . . . . But if one cannot say, with fair
               assurance, after pondering all that happened without stripping the
               erroneous action from the whole, that the judgment was not
               substantially swayed by the error, it is impossible to conclude that
               substantial rights were not affected. . . . If so, or if one is left in
               grave doubt, the conviction cannot stand.”

Clay, 262 Va. at 260, 546 S.E.2d at 731-32 (quoting Kotteakos v. United States, 328 U.S. 750,

764-65 (1946)). “Under this standard, non-constitutional error is harmless if other evidence of

guilt is so ‘overwhelming’ and the error so insignificant by comparison that we can conclude the

error ‘failed to have any “substantial influence” on the verdict.’” Schwartz v. Schwartz, 46

Va. App. 145, 159, 616 S.E.2d 59, 66 (2005) (quoting United States v. Lane, 474 U.S. 438, 450

(1986)). The Commonwealth has the burden of showing harmless error. Jones v.

Commonwealth, 50 Va. App. 437, 446, 650 S.E.2d 859, 864 (2007).

       The Commonwealth presented overwhelming evidence of Riddick’s guilt. As noted

above, Collins-Goss’ neighbor heard her say shortly before her death that her assailant was the

father of her unborn child. The testimony of Graham, Brown, and Grubb agreed that

Collins-Goss regarded Riddick as the father. Riddick’s DNA was found under Collins-Goss’

fingernails, even though had Collins-Goss simply washed her hands between the time it got there

and her death it likely would have been removed. Riddick’s DNA was also located in a blood

stain on a hamper in Collins-Goss’ apartment. When Officer Crouch photographed Riddick’s

body shortly past midnight on September 13, 2003, he observed multiple scratches on his body,

including a wound that had yet to scab. Finally, when the police encountered Riddick in the

early hours of September 10, 2003, only a couple hours after the attack on Collins-Goss, Riddick

had keys to Collins-Goss’ car but claimed it belonged to someone else and further provided

police with inconsistent statements. The fact finder could consider this evidence of guilt.

                                                -8-
Rollston v. Commonwealth, 11 Va. App. 535, 548, 399 S.E.2d 823, 831 (1991). Thus, we find

that assuming the circuit court committed error, it was plainly harmless.

                    C. Whether the Circuit Court Erred in Declining to Strike
                               the First-Degree Murder Charge

       Finally, Riddick contends the circuit court erred in denying his motion to strike the

first-degree murder charge. He makes two arguments under this heading. First, he contends the

evidence failed to show he acted with the premeditation required for a first-degree murder

conviction. Second, he argues the Commonwealth failed to exclude every reasonable hypothesis

of his innocence and, therefore, urges us to dismiss his conviction. Having already found the

Commonwealth presented overwhelming evidence of Riddick’s guilt, we evaluate only the first

argument.

       When reviewing the sufficiency of the evidence from a jury verdict, we inquire only

whether “‘reasonable jurors could have made the choices that the jury did make. We let the

decision stand unless we conclude no rational juror could have reached that decision.’” Pryor v.

Commonwealth, 50 Va. App. 42, 54, 646 S.E.2d 21, 27 (2007) (quoting Pease v.

Commonwealth, 39 Va. App. 342, 355, 573 S.E.2d 272, 278 (2002) (en banc), aff’d, 266 Va.

397, 588 S.E.2d 149 (2003)). The question is simply if “‘after viewing the evidence in the light

most favorable to the prosecution, any rational trier of fact could have found the essential

elements of the crime beyond a reasonable doubt.’” Maxwell v. Commonwealth, 275 Va. 437,

442, 657 S.E.2d 499, 502 (2008) (quoting Jackson v. Virginia, 443 U.S. 307, 319 (1979)).

       As relevant here, the Code requires that to prove a first-degree murder charge the

Commonwealth must prove a “willful, deliberate, and premeditated killing.” Code § 18.2-32.

‘“The test of murder is malice. Every malicious killing is murder either in the first or second

degree -- the former if deliberate and premeditated, and the latter if not.’” Wooden v.

Commonwealth, 222 Va. 758, 762, 284 S.E.2d 811, 814 (1981) (quoting Jacobs v.

                                                -9-
Commonwealth, 132 Va. 681, 686, 111 S.E. 90, 92 (1922)). The law gives “a prima facie

presumption of malice arising from the mere fact of a homicide, but there is no presumption

therefrom of deliberation and premeditation.” Bradshaw v. Commonwealth, 174 Va. 391, 398, 4

S.E.2d 752, 755 (1939). The Commonwealth has the burden of demonstrating premeditation,

and the defendant has the burden of showing factors reducing the offense below murder. Painter

v. Commonwealth, 210 Va. 360, 364, 171 S.E.2d 166, 170 (1969).

       Premeditation involves “a specific intent to kill.” Rhodes v. Commonwealth, 238 Va.

480, 485, 384 S.E.2d 95, 98 (1989). A person may form this intent only a moment before

committing murder. Coles v. Commonwealth, 270 Va. 585, 590, 621 S.E.2d 109, 112 (2005),

cert. denied, 127 S. Ct. 441 (2006). “It is the will and purpose to kill, not necessarily the interval

of time, which determine the grade of the offense.” Akers v. Commonwealth, 216 Va. 40, 48,

216 S.E.2d 28, 33 (1975).

       The question of whether a defendant killed with premeditation is committed to the jury.

Smith v. Commonwealth, 220 Va. 696, 701, 261 S.E.2d 550, 553 (1980). Factors the jury may

consider include “the brutality of the attack, and whether more than one blow was struck; the

disparity in size and strength between the defendant and the victim; the concealment of the

victim’s body; and the defendant’s lack of remorse and efforts to avoid detection.” Epperly v.

Commonwealth, 224 Va. 214, 232, 294 S.E.2d 882, 892 (1982) (citations omitted). Motive is

“often most persuasive.” Id. at 214, 294 S.E.2d at 893. It is appropriate to consider how these

factors combine. Id. As long as a reasonable jury could have reached the same conclusion as the

jury below, we will affirm. Hudson, 265 Va. at 513, 578 S.E.2d at 785.

       We conclude a reasonable jury could have found Riddick underwent the necessary

premeditation to commit first-degree murder. Indeed, each of the Epperly factors weighs in

favor of premeditation except for concealing the body and motive. First, the attack was quite

                                                - 10 -
brutal. Collins-Goss died from being beaten in the head. Her injuries were consistent with being

hit in the face with an iron. She suffered two black eyes, a split lip, and a broken nose. She also

showed evidence of having been strangled, along with numerous other minor injuries. Second,

Riddick attacked a pregnant woman. Third, Riddick failed to demonstrate remorse. He denied

responsibility when questioned by police. Finally, Riddick attempted to avoid detection by

telling the police Collins-Goss’ car belonged to someone else even though he had the keys to it.

Taken together, these factors permitted the jury to find Riddick formed a premeditated intent to

kill.

        For the foregoing reasons, we affirm the judgment of the circuit court.

                                                                                Affirmed.

                                               - 11 -


Dame Kiri among new faces at Downton Abbey

Downton Abbey will play host to a number of new faces in season four, including New Zealand soprano Dame Kiri Te Kanawa as well as a drunk, a gentleman with 'history' and the show's first black character.

A new character arrives ... Jessica Brown Findlay as Lady Sybil and Allen Leech as Tom Branson in Downton Abbey

The show will also introduce a black character in the coming season as part of a storyline about race relations in the 1920s.

Casting notes were sent out to actors' agents last month for the role of musician Jack Ross. The description given for Ross is “Male, 25-30. A musician (singer) at an exclusive club in the 20s. He's black and very handsome. A real man (not a boy) with charm and charisma.

The first Christmas special we did of Downton, it was pretty Christmassy. It was the proposal, which was very nice. But that isn't particularly the tradition of these Christmas shows here. In a way, they're supposed to be more dramatic than an ordinary episode of the series.

“Overall he should be a very attractive man with a certain wow factor.”

Ross is expected to be a key character in the fourth series, and should add to the musical mix as “ideally be able to sing brilliantly”.

Executive producer Gareth Neame said "Downton Abbey has seen many great characters visit the house over the years and we couldn't be more thrilled to welcome the new faces that will be joining the regular cast of Downton in series four.

Oscar winner Shirley MacLaine, will also reprise her role as Martha Levinson, the Dowager's nemesis and mother to Cora (Elizabeth McGovern) for the feature-length Christmas special at the end of season four.

Levinson was introduced in the first episodes of season three and was a big hit with fans.

Two major characters will depart over the course of season three, with another destined to disappear between seasons.

Siobhan Finnerman, the actress who fans have grown to love (or to love to hate) as scheming maid Sarah O'Brien, will not return for season four.

Finnerman, 47, told Britain's Daily Mirror: “I'm not doing any more. O'Brien is a thoroughly despicable human being - that was great to play.”

The actress has landed a role in new series The Syndicate, in which she will play a nurse who wins a share of a multimillion-pound Lottery jackpot.

“It is nice to play someone the audience is willing on,” Finnerman said. “Watching the first episode, I was delighted to see how many times I smiled. It's great for the story that starts to unfold. And there's no wig and no corsets, which is good.”

She may not have entirely untangled the corset strings, however, as a spokesperson for Downton Abbey said of the departure: "O'Brien is leaving to do something else, but the door for her character is open."

The foundations for O'Brien's exit are laid in the show's Christmas special and come swiftly on the heels of those of Lady Sybil (Jessica Brown Findlay) halfway through season three (in an episode to air this Sunday in Australia), as well as Matthew Crawley (Dan Stevens).

Stevens has a role in the forthcoming Julian Assange biopic The Fifth Estate, while Findlay has a role in new British series Labyrinth.

The three actors' departures will carry varying degrees of finality, giving weight to comments Fellowes made in an interview with The New York Times regarding writing characters out of the series.

“When an actor playing a servant wants to leave, there isn't really a problem,” Fellowes said. “[The character] gets another job. With members of the family, once they're not prepared to come back for any episodes at all, then it means death.

“So we didn't have any option, really. I was as sorry as everyone else.”

Fellowes acknowledges that the potentially depressing nature of the Christmas special is always likely to be a surprise for international markets.


Thursday, September 5, 2013

11 more days

11 more days hasta la campa. I´m not sure how to feel about that. On one hand, it is going to be awesome in Chile and we will be more free to do what we want and we´ll be teaching actual investigators. But on the other hand, I can still barely squeeze out enough Spanish to have a conversation with Mexicans but Chileans talk ridiculously fast. Right now I don´t feel ready at all but from what I hear, the last week completely changes that. I´m excited to be at the point and I can already feel myself getting there. This week went as usual. Lots of studying, lots of teaching, lots of uncomfortable situations and lots of new Spanish vocabulary. I am proud to say that I can now, with enough thinking, conjugate almost any verb into the future, preterit, imperfect, command and conditional form.

Everyday we play soccer. There are about 3 or 4 other teams (made up of districts) that play against us. It´s indoor soccer and we switch which team is on either when someone makes a goal or after four minutes. Two of the teams are made up of Latinos. And in case you don´t know, every Latino can play soccer as if that is all he does. That isn´t a blanket statement either. Every single Latino that we have played against is better that any gringo here. But that doesn´t stop us from trying and it just makes us better. After playing with the Latinos for a while, we will play a gringo team and destroy them. I´m not very good at all, but I´m definitely getting better. Last Pday we were playing a game and one of the elders in our district went in for a header but missed the ball and got someones elbow instead. It doesn´t sound that bad but he was bleeding like none other. He went into the Doctor and they didn´t have much so they poured water on his head (because they had nothing anti bacterial) then proceeded to give him two stitches without numbing. They took it out and re did it because they didn´t do it right. He´s fine now but it´s just a funny story.

I´ve been saying that our district is the best, but this week we got proof of that. Some of the teachers have been asking us to give pep talks to other districts. We also have people who constantly tell us that they wish they were put with us.

Tomorrow we are having Christmas. We are building a Christmas tree out of palm leaves and we are doing a secret Santa and we are going to sing Christmas songs instead of hymns all day. I´m not sure where the idea came from but i´m excited.

Alright, that's all for now. till next week!

Elder Thompson

a murder of parrots

a sad bus top

The sisters all took this pose in front of the temple so I thought I would join them.


Q:

Union query removing blank rows

I'm trying to union two different results but I'm not sure how to remove the blank values. The paid_date and check_date are sometimes different. What do I need to do to modify the code? I've tried doing MAX but didn't seem to work. Thanks.
Results:

What I want:

WITH PAID AS
(
SELECT PIDATE
,SUM(PAY) AS PAID
,ROW_NUMBER() OVER(ORDER BY PAYDATE DESC) AS ROW_NUM
FROM CLAIM
WHERE PAY<>0
GROUP BY PAYDATE
)
,TOTAL_PAID AS
(
SELECT CONVERT(DATE,CONVERT(VARCHAR(10),PAYDATE)) AS PAID_DATE
,FORMAT(PAID,'C','EN-US') AS PAID
FROM PAID
WHERE ROW_NUM <= 10
)

,CHECKS AS
(
SELECT DISTINCT CHECK_DATE
,SUM(PAYMENT) AS PAYMENT
,ROW_NUMBER() OVER(ORDER BY CHECK_DATE DESC) AS ROW_NUM
FROM CR
GROUP BY CHECK_DATE
)
,TOTAL_CHECKS AS
(
SELECT CONVERT(DATE,CONVERT(VARCHAR(10),CHECK_DATE)) AS CHECK_DATE
,FORMAT(PAYMENT,'C','EN-US') AS PAYMENT
FROM CHECKS
WHERE ROW_NUM <= 10
)

,FINAL AS
(
SELECT PAID_DATE
,PAID
,'' AS CHECK_DATE
,'' AS PAYMENT
FROM TOTAL_PAID

UNION ALL

SELECT '' AS PAID_DATE
,'' AS PAID
,CHECK_DATE
,PAYMENT
FROM TOTAL_CHECKS
)

SELECT *
FROM FINAL

A:

In this scenario, you must not use union. you must use join instead.
Try join two sequence on paid_date = check_date and select proper field from each set.


Sumatriptan overuse in episodic cluster headache: lack of adverse events, rebound syndromes, drug dependence and tachyphylaxis.
This observational study was designed to examine the pattern of sumatriptan use in patients with cluster headache using more than the recommended daily dose of subcutaneously injected (s.c.) sumatriptan. Thirteen patients suffering from episodic cluster headache were asked to record the characteristics of their attacks and drug intake for 1 year. All reported a high daily frequency of attacks (more than 3 per day) and the related overuse of s.c. sumatriptan. The results show that the overall incidence of adverse events among patients receiving sumatriptan injections for the treatment of cluster headache is low. The extended administration of this drug in episodic cluster headache did not result in tolerance problems or tachyphylaxis. Only 4 patients experienced minor adverse events and recovered more slowly than the others. They suffered from migraine without aura and cluster headache, and showed a family history of migraine. Even though they must be viewed with caution, due to the observational nature of the study and the low number of patients included, these results suggest that the profile of sumatriptan may differ in cluster headache compared with migraine.


Mediastinal Bronchogenic Cysts: Demonstration of Fluid-Fluid Level in Bronchoscopic US Imaging.
We report the findings for 2 patients with a fluid-fluid level seen on endobronchial ultrasound (EBUS) images of bronchogenic cysts. The EBUS images demonstrated a well-circumscribed cyst with a fluid-fluid level showing an anechoic upper part and a relatively hyperechoic lower part. A fluid-fluid level on EBUS imaging of a bronchogenic cyst, which can help confirm the cystic nature of the lesion, has not been previously reported. EBUS-based confirmation of these cysts using fluid-fluid levels may help avoid unnecessary aspiration of the lesions.


The hydroxylamine OXANOH and its reaction product, the nitroxide OXANO., act as complementary inhibitors of lipid peroxidation.
The effects of the nitroxide 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO.) and the corresponding hydroxylamine 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) on in vitro lipid peroxidation in rat liver microsomes and reconstituted lipid vesicles were investigated, and compared with those of some commonly used spin trapping agents. OXANO. and OXANOH (10-100 microM) inhibited iron-dependent lipid peroxidation, as did the spin trapping agents (10-100 mM). OXANO. mainly inhibited the rate of peroxidation, but caused only a small delay in the time of onset. OXANOH exerted its effect by delaying the onset of peroxidation in an antioxidant fashion, and also by inhibiting the rate. Higher concentrations of both substances were required to inhibit t-butylhydroperoxide-dependent lipid peroxidation. OXANO. was found to oxidize the ferrous-ADP complex required for initiation of peroxidation, and this is probably the basis of the inhibitory effect of this compound. Since the reaction of OXANO. tends to produce OXANOH and vice versa, either one could inhibit all reactions of lipid peroxidation.