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Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology. Graphical Abstract

The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.

There is a need to improve current cancer treatment regimens to reduce systemic toxicity, to positively impact the quality-of-life post-treatment. We hypothesized the negation of off-target toxicity of anthracyclines (e.g., Doxorubicin) by delivering Doxorubicin on magneto-electric silica nanoparticles (Dox-MagSiNs) to cancer cells. Dox-MagSiNs were completely biocompatible with all cell types and are therapeutically inert till the release of Doxorubicin from the MagSiNs at the cancer cells location. The MagSiNs themselves are comprised of biocompatible components with a magnetostrictive cobalt ferrite core (4–6 nm) surrounded by a piezoelectric fused silica shell of 1.5 nm to 2 nm thickness. The MagSiNs possess T2-MRI contrast properties on par with RESOVIST™ due to their cobalt ferrite core. Additionally, the silica shell surrounding the core was volume loaded with green or red fluorophores to fluorescently track the MagSiNs in vitro. This makes the MagSiNs a suitable candidate for trackable, drug nanocarriers. We used metastatic triple-negative breast cancer cells (MDAMB231), ovarian cancer cells (A2780), and prostate cancer cells (PC3) as our model cancer cell lines. Human umbilical vein endothelial cells (HUVEC) were used as control cell lines to represent blood-vessel cells that suffer from the systemic toxicity of Doxorubicin. In the presence of an external magnetic field that is 300× times lower than an MRI field, we successfully nanoporated the cancer cells, then triggered the release of 500 nM of doxorubicin from Dox-MagSiNs to successfully kill >50% PC3, >50% A2780 cells, and killed 125% more MDAMB231 cells than free Dox.HCl. In control HUVECs, the Dox-MagSiNs did not nanoporate into the HUVECS and did not exhibited any cytotoxicity at all when there was no triggered release of Dox.HCl. Currently, the major advantages of our approach are, (i) the MagSiNs are biocompatible in vitro and in vivo; (ii) the label-free nanoporation of Dox-MagSiNs into cancer cells and not the model blood vessel cell line; (iii) the complete cancellation of the cytotoxicity of Doxorubicin in the Dox-MagSiNs form; (iv) the clinical impact of such a nanocarrier will be that it will be possible to increase the current upper limit for cumulative-dosages of anthracyclines through multiple dosing, which in turn will improve the anti-cancer efficacy of anthracyclines.

Background Evolution occurred exclusively under the full spectrum of sunlight. Conscription of narrow regions of the solar spectrum by specific photoreceptors suggests a common strategy for regulation of genetic pathways. Fluorescent light (FL) does not possess the complexity of the solar spectrum and has only been in service for about 60 years. If vertebrates evolved specific genetic responses regulated by light wavelengths representing the entire solar spectrum, there may be genetic consequences to reducing the spectral complexity of light. Results We utilized RNA-Seq to assess changes in the transcriptional profiles of Xiphophorus maculatus skin after exposure to FL (“cool white”), or narrow wavelength regions of light between 350 and 600 nm (i.e., 50 nm or 10 nm regions, herein termed “wavebands”). Exposure to each 50 nm waveband identified sets of genes representing discrete pathways that showed waveband specific transcriptional modulation. For example, 350–400 or 450–500 nm waveband exposures resulted in opposite regulation of gene sets marking necrosis and apoptosis (i.e., 350–400 nm; necrosis suppression, apoptosis activation, while 450–500 nm; apoptosis suppression, necrosis activation). Further investigation of specific transcriptional modulation employing successive 10 nm waveband exposures between 500 and 550 nm showed; (a) greater numbers of genes may be transcriptionally modulated after 10 nm exposures, than observed for 50 nm or FL exposures, (b) the 10 nm wavebands induced gene sets showing greater functional specificity than 50 nm or FL exposures, and (c) the genetic effects of FL are primarily due to 30 nm between 500 and 530 nm. Interestingly, many genetic pathways exhibited completely opposite transcriptional effects after different waveband exposures. For example, the epidermal growth factor ( EGF ) pathway exhibits transcriptional suppression after FL exposure, becomes highly active after 450–500 nm waveband exposure, and again, exhibits strong transcriptional suppression after exposure to the 520–530 nm waveband. Conclusions Collectively, these results suggest one may manipulate transcription of specific genetic pathways in skin by exposure of the intact animal to specific wavebands of light. In addition, we identify genes transcriptionally modulated in a predictable manner by specific waveband exposures. Such genes, and their regulatory elements, may represent valuable tools for genetic engineering and gene therapy protocols.

OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and common primary tumor type in the central nervous system across all age groups; affected adults have a life expectancy of just 14 months. As glioma cells invade the surrounding normal parenchyma they remodel the composition and ultrastructure of the surrounding extracellular matrix (ECM), suggesting that the native (i.e., “normal”) microenvironment is not ideal for their survival and proliferation. Recent reports describe suppressive and/or lethal effects of mammalian ECM hydrogels derived from normal (nonneoplastic) sources upon various cancer types. ECM-based bioscaffolds placed at sites of neoplastic tissue resection in humans have never been reported to facilitate cancer recurrence. The objective of the present research is to evaluate mammalian ECM as a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECM hydrogels from porcine dermis, small intestine, and urinary bladder were produced as described previously. Primary glioma cells were graciously supplied by Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered through ATCC. Cells were plated onto tissue culture plastic at ~60% confluence and allowed to attach for 24 hours before treatment. The saline-soluble fraction (SSF) of ECM was obtained by mixing lyophilized, comminuted ECM with 0.9% saline for 24 hours then filtering the resulting mixture through a 10 kDa molecular weight cutoff column. All assays and kits were followed according to the manufacturer’s instructions. Cell viability was measured via MTT assay (Vybrant ® MTT Cell Proliferation Assay, Invitrogen) and by live/dead staining (LIVE/DEAD ® Cell Imaging Kit, Invitrogen). Time lapse videos were created by taking images every 20 minutes for 18 hours (phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagent was ordered from Biotium. Temozolomide was ordered through Abmole. All in vivo work was conducted according to protocols approved by the University of Pittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS: ECM hydrogels derived from porcine dermis, small intestine, or urinary bladder all decreased the viability of primary glioma cells in vitro, with urinary bladder extracellular matrix (UBM) having the most dramatic effects. The SSF of UBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, was sufficient to recapitulate this detrimental effect upon neoplastic cells in vitro and was used for the remainder of the experiments described herein. In a cell viability assay normalized to the media treatment, non-neoplastic CHME5 and N1E-115 cells scored 103% and 114% after 48 hours when treated with UBM-SSF and 2 primary high-grade glioma cell types scored 17% and 30.5% with UBM-SSF (n=2). Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence of UBM-SSF for 18 hours while most primary glioma cells shriveled and died within this time. Darkfield time-lapse video of wells containing Nucview dye, fluorescent upon cleavage by active caspase-3, confirmed that within 12 hours most primary glioma cells underwent apoptosis while CHME5 and HFF did not. In culture with primary astrocytes, high grade primary glioma cells, and U-87 MG glioma cells for 24 hours, UBM-SSF was found to significantly increase the population of primary astrocytes compared with media ( p <0.05) while decreasing the 2 glioma cell types to approximately one-third as many cells as the media control ( p <0.0001). A dose-response of temozolomide from 0 to 10,000 μM showed that when treating 2 non-neoplastic cell types (CHME5 and HFF) and 2 types of primary glioma cell there was no difference in survivability at any concentration. Contrasted to this, a dose-response of UBM-SSF from 350 to 7000 μg/mL showed that the non-neoplastic cells survived significantly better than the glioma cells at concentrations of 875 μg/mL and upward ( p <0.05). In preliminary animal experiments, large primary glioma tumors in the flanks of athymic nude mice were resected and replaced with either UBM SSF or Matrigel (an ECM product of neoplastic cell origin). After 7 days the resection sites with UBM-SSF had little tumor regrowth if any compared with the dramatic recurrence seen in the Matrigel injection sites (n=2). In a separate survival study comparing PBS to UBM-SSF injections in the flank-resection model, all animals given PBS had to be sacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSF were sacrificed at 15, 24, and 39 days (n=3), indicating a moderate increase in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OF IMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZ in 2005, the standard of care for patients with glioblastoma multiforme has not improved. These findings indicate that non-neoplastic ECM contains potent bioactive regulators capable of abrogating malignancy. Our in vitro data suggest these molecules appear to have no deleterious effect on non-neoplastic cells while specifically inducing apoptosis in glioma cells. Our in vivo data suggest that these molecules may be useful in delaying glioma recurrence, thus resulting in extended lifespan. Delivering soluble fractions of ECM to a tumor site may represent a novel approach to glioma therapy, sidestepping traditional cytotoxic therapies in favor of utilizing putative endogenous anti-tumor pathways.

There is a need to improve current cancer treatment regimens to reduce systemic toxicity, to positively impact the quality-of-life post-treatment. We hypothesized the negation of off-target toxicity of anthracyclines (e.g., Doxorubicin) by delivering Doxorubicin on magneto-electric silica nanoparticles (Dox-MagSiNs) to cancer cells. Dox-MagSiNs were completely biocompatible with all cell types and are therapeutically inert till the release of Doxorubicin from the MagSiNs at the cancer cells location. The MagSiNs themselves are comprised of biocompatible components with a magnetostrictive cobalt ferrite core (4−6 nm) surrounded by a piezoelectric fused silica shell of 1.5 nm to 2 nm thickness. The MagSiNs possess T2-MRI contrast properties on par with RESOVIST™ due to their cobalt ferrite core. Additionally, the silica shell surrounding the core was volume loaded with green or red fluorophores to fluorescently track the MagSiNs in vitro. This makes the MagSiNs a suitable candidate for trackable, drug nanocarriers. We used metastatic triple-negative breast cancer cells (MDAMB231), ovarian cancer cells (A2780), and prostate cancer cells (PC3) as our model cancer cell lines. Human umbilical vein endothelial cells (HUVEC) were used as control cell lines to represent blood-vessel cells that suffer from the systemic toxicity of Doxorubicin. In the presence of an external magnetic field that is 300× times lower than an MRI field, we successfully nanoporated the cancer cells, then triggered the release of 500 nM of doxorubicin from Dox-MagSiNs to successfully kill >50% PC3, >50% A2780 cells, and killed 125% more MDAMB231 cells than free Dox.HCl. In control HUVECs, the Dox-MagSiNs did not nanoporate into the HUVECS and did not exhibited any cytotoxicity at all when there was no triggered release of Dox.HCl. Currently, the major advantages of our approach are, (i) the MagSiNs are biocompatible in vitro and in vivo; (ii) the label-free nanoporation of Dox-MagSiNs into cancer cells and not the model blood vessel cell line; (iii) the complete cancellation of the cytotoxicity of Doxorubicin in the Dox-MagSiNs form; (iv) the clinical impact of such a nanocarrier will be that it will be possible to increase the current upper limit for cumulative-dosages of anthracyclines through multiple dosing, which in turn will improve the anti-cancer efficacy of anthracyclines.

There is a need to improve current cancer treatment regimens to reduce systemic toxicity, to positively impact the quality-of-life post-treatment. We hypothesized the negation of off-target toxicity of anthracyclines (e.g., Doxorubicin) by delivering Doxorubicin on magneto-electric silica nanoparticles (Dox-MagSiNs) to cancer cells. Dox-MagSiNs were completely biocompatible with all cell types and are therapeutically inert till the release of Doxorubicin from the MagSiNs at the cancer cells location. The MagSiNs themselves are comprised of biocompatible components with a magnetostrictive cobalt ferrite core (4–6 nm) surrounded by a piezoelectric fused silica shell of 1.5 nm to 2 nm thickness. The MagSiNs possess T[sub.2] -MRI contrast properties on par with RESOVIST™ due to their cobalt ferrite core. Additionally, the silica shell surrounding the core was volume loaded with green or red fluorophores to fluorescently track the MagSiNs in vitro. This makes the MagSiNs a suitable candidate for trackable, drug nanocarriers. We used metastatic triple-negative breast cancer cells (MDAMB231), ovarian cancer cells (A2780), and prostate cancer cells (PC3) as our model cancer cell lines. Human umbilical vein endothelial cells (HUVEC) were used as control cell lines to represent blood-vessel cells that suffer from the systemic toxicity of Doxorubicin. In the presence of an external magnetic field that is 300× times lower than an MRI field, we successfully nanoporated the cancer cells, then triggered the release of 500 nM of doxorubicin from Dox-MagSiNs to successfully kill >50% PC3, >50% A2780 cells, and killed 125% more MDAMB231 cells than free Dox.HCl. In control HUVECs, the Dox-MagSiNs did not nanoporate into the HUVECS and did not exhibited any cytotoxicity at all when there was no triggered release of Dox.HCl. Currently, the major advantages of our approach are, (i) the MagSiNs are biocompatible in vitro and in vivo; (ii) the label-free nanoporation of Dox-MagSiNs into cancer cells and not the model blood vessel cell line; (iii) the complete cancellation of the cytotoxicity of Doxorubicin in the Dox-MagSiNs form; (iv) the clinical impact of such a nanocarrier will be that it will be possible to increase the current upper limit for cumulative-dosages of anthracyclines through multiple dosing, which in turn will improve the anti-cancer efficacy of anthracyclines.

Semantic segmentation is the task of dividing entire images into non-overlapping regions with per-pixel class labels that correspond to a problem's objects of interest. To do the task well, researchers introduce techniques of extracting object features from the image cues of shape, color, and texture to give models an improved ability to discriminate between classes. Over the decades, researchers have developed both histogram-based and spatial filter-based methods for extracting texture features in segmentation pipelines. Today, however, convolutional filters are the dominant choice. These are best suited for learning ordered spatial relationships in images as opposed to orderless pixel value distributions within textures. Histogram-based features allow us to represent these informative texture features. To leverage the power of histograms in deep architectures, I propose a module that extracts both convolutional and histogram features and embeds these features within the normal deep feature learning pipeline. The proposed module uses a learnable histogram layer in parallel with convolutional feature extraction then recombines their respective outputs through concatenation. Also, there is a lack of research on learnable histogram layers and the impact that initialization can have on such layers' performance. With a synthetic dataset based on Voronoi diagrams, I provide insights into learning behavior and initialization strategies for a learnable histogram layer. Second, I evaluate the proposed module's effectiveness on multiple real-world semantic segmentation datasets, which include the French Land cover from Aerospace ImageRy, Multimodal Material Segmentation, MUUFL-Gulfport, and HyperPRI datasets. Lastly, I discuss strengths and weaknesses of the proposed module, which are shown through results on the related datasets, and I conclude with some ideas for future work on embedding histogram feature learning within deep learning pipelines.

OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and commonprimary tumor type in the central nervous system across all age groups; affectedadults have a life expectancy of just 14 months. As glioma cells invade thesurrounding normal parenchyma they remodel the composition and ultrastructure ofthe surrounding extracellular matrix (ECM), suggesting that the native (i.e.,“normal”) microenvironment is not ideal for their survivaland proliferation. Recent reports describe suppressive and/or lethaleffects of mammalian ECM hydrogels derived from normal (nonneoplastic) sourcesupon various cancer types. ECM-based bioscaffolds placed at sites of neoplastictissue resection in humans have never been reported to facilitate cancerrecurrence. The objective of the present research is to evaluate mammalian ECMas a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECMhydrogels from porcine dermis, small intestine, and urinary bladder wereproduced as described previously. Primary glioma cells were graciously suppliedby Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered throughATCC. Cells were plated onto tissue culture plastic at~60% confluence and allowed to attach for 24 hours beforetreatment. The saline-soluble fraction (SSF) of ECM was obtained by mixinglyophilized, comminuted ECM with 0.9% saline for 24 hours thenfiltering the resulting mixture through a 10 kDa molecular weight cutoff column.All assays and kits were followed according to the manufacturer’sinstructions. Cell viability was measured via MTT assay(Vybrant® MTT Cell Proliferation Assay, Invitrogen)and by live/dead staining(LIVE/DEAD® Cell Imaging Kit, Invitrogen). Timelapse videos were created by taking images every 20 minutes for 18 hours(phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagentwas ordered from Biotium. Temozolomide was ordered through Abmole. All in vivowork was conducted according to protocols approved by the University ofPittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS:ECM hydrogels derived from porcine dermis, small intestine, or urinary bladderall decreased the viability of primary glioma cells in vitro, with urinarybladder extracellular matrix (UBM) having the most dramatic effects. The SSF ofUBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, wassufficient to recapitulate this detrimental effect upon neoplastic cells invitro and was used for the remainder of the experiments described herein. In acell viability assay normalized to the media treatment, non-neoplastic CHME5 andN1E-115 cells scored 103% and 114% after 48 hours whentreated with UBM-SSF and 2 primary high-grade glioma cell types scored17% and 30.5% with UBM-SSF (n=2).Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence ofUBM-SSF for 18 hours while most primary glioma cells shriveled and died withinthis time. Darkfield time-lapse video of wells containing Nucview dye,fluorescent upon cleavage by active caspase-3, confirmed that within 12 hoursmost primary glioma cells underwent apoptosis while CHME5 and HFF did not. Inculture with primary astrocytes, high grade primary glioma cells, and U-87 MGglioma cells for 24 hours, UBM-SSF was found to significantly increase thepopulation of primary astrocytes compared with media(p<0.05) while decreasing the 2 glioma cell types toapproximately one-third as many cells as the media control(p<0.0001). A dose-response of temozolomide from 0to 10,000 μM showed that when treating 2 non-neoplastic cell types(CHME5 and HFF) and 2 types of primary glioma cell there was no difference insurvivability at any concentration. Contrasted to this, a dose-response ofUBM-SSF from 350 to 7000 μg/mL showed that thenon-neoplastic cells survived significantly better than the glioma cells atconcentrations of 875 μg/mL and upward(p<0.05). In preliminary animal experiments, largeprimary glioma tumors in the flanks of athymic nude mice were resected andreplaced with either UBM SSF or Matrigel (an ECM product of neoplastic cellorigin). After 7 days the resection sites with UBM-SSF had little tumor regrowthif any compared with the dramatic recurrence seen in the Matrigel injectionsites (n=2). In a separate survival study comparing PBS to UBM-SSFinjections in the flank-resection model, all animals given PBS had to besacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSFwere sacrificed at 15, 24, and 39 days (n=3), indicating a moderateincrease in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OFIMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZin 2005, the standard of care for patients with glioblastoma multiforme has notimproved. These findings indicate that non-neoplastic ECM contains potentbioactive regulators capable of abrogating malignancy. Our in vitro data suggestthese molecules appear to have no deleterious effect on non-neoplastic cellswhile specifically inducing apoptosis in glioma cells. Our in vivo data suggestthat these molecules may be useful in delaying glioma recurrence, thus resultingin extended lifespan. Delivering soluble fractions of ECM to a tumor site mayrepresent a novel approach to glioma therapy, sidestepping traditional cytotoxictherapies in favor of utilizing putative endogenous anti-tumor pathways.