Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome.

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.

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome.

Retroviruses exploit a variety of host proteins to assemble and release virions from infected cells. To date, most studies that examined possible interacting partners of retroviral Gag proteins focused on host proteins that localize primarily to the cytoplasm or plasma membrane. Given the recent findings that several full-length Gag proteins localize to the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings that reveal previously unknown host processes. In this study, we systematically compared nuclear factors identified in published HIV-1 proteomic studies which had used a variety of experimental approaches. In addition, to contribute to this body of knowledge, we report results from a mass spectrometry approach using affinity-tagged (His6) HIV-1 and RSV Gag proteins mixed with nuclear extracts. Taken together, the previous studies-as well as our own-identified potential binding partners of HIV-1 and RSV Gag involved in several nuclear processes, including transcription, splicing, RNA modification, and chromatin remodeling. Although a subset of host proteins interacted with both Gag proteins, there were also unique host proteins belonging to each interactome dataset. To validate one of the novel findings, we demonstrated the interaction of RSV Gag with a member of the Mediator complex, Med26, which is required for RNA polymerase II-mediated transcription. 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.

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

IMPORTANCE: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

Influence of HIV-1 genomic RNA on the formation of Gag biomolecular condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) and the HIV-1 pr55 Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yield self-assembling BMCs having HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 + T cell nuclear lysates led to the formation of larger BMCs as compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggests that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

HIV-1 Gag colocalizes with euchromatin histone marks at the nuclear periphery.

The retroviral Gag protein of human immunodeficiency virus type 1 (HIV-1) plays a central role in the selection of unspliced viral genomic RNA for packaging into new virions. Previously, we demonstrated that full-length HIV-1 Gag undergoes nuclear trafficking where it associates with unspliced viral RNA (vRNA) at transcription sites. To further explore the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to examine the timing of HIV-1 entry into the nucleus. We also aimed to determine more precisely Gag's subnuclear distribution to test the hypothesis that Gag would be associated with euchromatin, the transcriptionally active region of the nucleus. We observed that HIV-1 Gag localized to the nucleus shortly after its synthesis in the cytoplasm, suggesting that nuclear trafficking was not strictly concentration-dependent. Furthermore, we found that HIV-1 Gag preferentially localized to the transcriptionally active euchromatin fraction compared to the heterochromatin-rich region in a latently-infected CD4+ T cell line (J-Lat 10.6) treated with latency-reversal agents. Interestingly, HIV-1 Gag was more closely associated with transcriptionally-active histone markers near the nuclear periphery, where the HIV-1 provirus was previously shown to integrate. Although the precise function of Gag's association with histones in transcriptionally-active chromatin remains uncertain, together with previous reports, this finding is consistent with a potential role for euchromatin-associated Gag molecules to select newly transcribed unspliced vRNA during the initial stage of virion assembly. Importance: The traditional view of retroviral assembly posits that HIV-1 Gag selection of unspliced vRNA begins in the cytoplasm. However, our previous studies demonstrated that HIV-1 Gag enters the nucleus and binds to unspliced HIV-1 RNA at transcription sites, suggesting that genomic RNA selection may occur in the nucleus. In the present study, we observed nuclear entry of HIV-1 Gag and co-localization with unspliced viral RNA within 8 hours post-expression. In CD4+ T cells (J-Lat 10.6) treated with latency reversal agents, as well as a HeLa cell line stably expressing an inducible Rev-dependent provirus, we found that HIV-1 Gag preferentially localized with histone marks associated with enhancer and promoter regions of transcriptionally active euchromatin near the nuclear periphery, which favors HIV-1 proviral integration sites. These observations support the hypothesis that HIV-1 Gag hijacks euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized genomic RNA for packaging.

Biocompatibility and biodistribution of matrix-bound nanovesicles in vitro and in vivo.

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses. STATEMENT OF SIGNIFICANCE: Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications.

Biocompatible, Multi-Mode, Fluorescent, T2 MRI Contrast Magnetoelectric-Silica Nanoparticles (MagSiNs), for On-Demand Doxorubicin Delivery to Metastatic Cancer Cells.

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.

Measuring Attitude toward Chemistry, Biology, and Math at a Hispanic-Serving Institution.

This work describes the evaluation of the Attitude toward the Subject of Chemistry Inventory (ASCI), as well as two modifications (one for measuring attitude toward math and one for measuring attitude toward biology), for college students at a Hispanic Serving Institution. Instrument reliability was tested via multiple administrations of the instruments, and confirmatory factor analysis supported a two-factor structure similar to an existing model of a revised version of the ASCI for all three instruments. The similar factor structure of the three instruments, coupled with interviews with students, provide validity evidence for the instruments and support an interpretation that one of the subscales aligns with a cognitive aspect of attitude while the other subscale aligns with an affective aspect. The results of these instruments indicate that students have a more positive attitude toward biology than either chemistry or math, and more positive affective attitude than cognitive attitude for all three subjects, although student attitudes show little change with respect to biology, chemistry, or math during a typical semester. However, major perturbations, such as switching to remote instruction midsemester, can lead to small but significant increases and decreases in attitude.

A liquid fraction of extracellular matrix inhibits glioma cell viability in vitro and in vivo.

Suppressive effects of extracellular matrix (ECM) upon various cancers have been reported. Glioblastoma multiforme has poor prognosis and new therapies are desired. This work investigated the effects of a saline-soluble fraction of urinary bladder ECM (ECM-SF) upon glioma cells. Viability at 24 hours in 1, 5, or 10 mg/mL ECM-SF-spiked media was evaluated in primary glioma cells (0319, 1015, 1119), glioma cell lines (A172, T98G, U87MG, C6), and brain cell lines (HCN-2, HMC3). Viability universally decreased at 5 and 10 mg/mL with U87MG, HCN-2, and HCM3 being least sensitive. Apoptosis in 0319 and 1119 cells was confirmed via NucView 488. Bi-weekly intravenous injection of ECM-SF (120 mg/kg) for 10 weeks in Sprague-Dawley rats did not affect weight, temperature, complete blood count, or multi-organ histology (N = 5). Intratumoral injection of ECM-SF (10 uL of 30 mg/mL) at weeks 2-4 post C6 inoculation in Wistar rats increased median survival from 24.5 to 51 days (hazard ratio for death 0.22) and decreased average tumor volume at time of death from 349 mm3 to 90 mm3 over 10 weeks (N = 6). Mass spectrometry identified 2,562 protein species in ECM-SF, parent ECM, and originating tissue. These results demonstrate the suppressive effects of ECM on glioma and warrant further study.

Tissue response, macrophage phenotype, and intrinsic calcification induced by cardiovascular biomaterials: Can clinical regenerative potential be predicted in a rat subcutaneous implant model?

The host immune response to an implanted biomaterial, particularly the phenotype of infiltrating macrophages, is a key determinant of biocompatibility and downstream remodeling outcome. The present study used a subcutaneous rat model to compare the tissue response, including macrophage phenotype, remodeling potential, and calcification propensity of a biologic scaffold composed of glutaraldehyde-fixed bovine pericardium (GF-BP), the standard of care for heart valve replacement, with those of an electrospun polycarbonate-based supramolecular polymer scaffold (ePC-UPy), urinary bladder extracellular matrix (UBM-ECM), and a polypropylene mesh (PP). The ePC-UPy and UBM-ECM materials induced infiltration of mononuclear cells throughout the thickness of the scaffold within 2 days and neovascularization at 14 days. GF-BP and PP elicited a balance of pro-inflammatory (M1-like) and anti-inflammatory (M2-like) macrophages, while UBM-ECM and ePC-UPy supported a dominant M2-like macrophage phenotype at all timepoints. Relative to GF-BP, ePC-UPy was markedly less susceptible to calcification for the 180 day duration of the study. UBM-ECM induced an archetypical constructive remodeling response dominated by M2-like macrophages and the PP caused a typical foreign body reaction dominated by M1-like macrophages. The results of this study highlight the divergent macrophage and host remodeling response to biomaterials with distinct physical and chemical properties and suggest that the rat subcutaneous implantation model can be used to predict in vivo biocompatibility and regenerative potential for clinical application of cardiovascular biomaterials.

HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites.

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.

Host macrophage response to injectable hydrogels derived from ECM and α-helical peptides.

Tissue engineering materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated. Traditional natural and synthetic materials are superseded by bespoke materials that cross the boundary between these two categories. Here we present hydrogels that are derived from decellularised extracellular matrix and those that are synthesised from de novo α-helical peptides. We assess in vitro activation of murine macrophages to our hydrogels and whether these gels induce an M1-like or M2-like phenotype. This was followed by the in vivo immune macrophage response to hydrogels injected into rat partial-thickness abdominal wall defects. Over 28 days we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface without promoting a foreign body reaction and see no evidence of hydrogel encapsulation or formation of multinucleate giant cells. We also note an upregulation of myogenic differentiation markers and the expression of anti-inflammatory markers Arginase1, IL-10, and CD206, indicating pro-remodelling for all injected hydrogels. Furthermore, all hydrogels promote an anti-inflammatory environment after an initial spike in the pro-inflammatory phenotype. No difference between the injected site and the healthy tissue is observed after 28 days, indicating full integration. These materials offer great potential for future applications in regenerative medicine and towards unmet clinical needs. STATEMENT OF SIGNIFICANCE: Materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated in tissue engineering. Here we present injectable hydrogels derived from decellularised extracellular matrix and de novo designed α-helical peptides. Over 28 days in the rat abdominal wall we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface with no foreign body reaction, no evidence of hydrogel encapsulation and no multinucleate giant cells. Our data indicate pro-remodelling and the promotion of an anti-inflammatory environment for all injected hydrogels with evidence of full integration with healthy tissue after 28 days. These unique materials offer great potential for future applications in regenerative medicine and towards designing materials for unmet clinical needs.

Cytocompatibility and mechanical properties of surgical sealants for cardiovascular applications.

OBJECTIVES: The present study compared physical, mechanical, and biologic characteristics of 4 clinically available surgical sealants for cardiovascular repair. METHODS: BioGlue (Cryolife Inc, Kennesaw, Ga), PreveLeak (Mallinckrodt Pharmaceuticals, St Louis, Mo), Tridyne VS (BD, Franklin Lakes, NJ), and Coseal (Baxter Healthcare Corporation, Westlake Village, Calif) were compared for the following properties: hydrated swelling, cytocompatibility, burst strength, biaxial stretching (elasticity), and in vitro degradation. RESULTS: Sealants showed a wide range of swelling upon hydration. By gravimetric and volumetric measurement, swelling was greatest for Coseal followed by Tridyne VS, BioGlue, and PreveLeak. Tridyne VS was the most cytocompatible based on Alamar Blue assay results, supporting 85% cell survival compared with 36% to 39% survival with the other sealants. All sealants withstood pressure above mean arterial pressure (70-110 mm Hg) and physiologic systolic blood pressure (90-140 mm Hg) in an ex vivo arterial flow burst model; lowest peak pressure at failure was PreveLeak at 235 ± 48 mm Hg, and highest peak pressure at failure was BioGlue at 596 ± 72 mm Hg. Biaxial tensile testing showed no differences in elasticity between ex vivo porcine aorta and carotid arteries and Tridyne VS or Coseal, and BioGlue and PreveLeak were significantly stiffer. In vitro degradation time for Coseal was 6 days and 21 days for Tridyne VS. No degradation was observed in BioGlue or PreveLeak for 30 days. CONCLUSIONS: Although all sealants withstood supraphysiologic arterial pressure, there were differences in characteristics that may be important in clinical outcome. Coseal degradation time was short compared with other sealants, whereas BioGlue and PreveLeak showed a significant compliance mismatch with native porcine carotid artery. Tridyne VS was significantly more cytocompatible than the other 3 sealants.

Waveband specific transcriptional control of select genetic pathways in vertebrate skin (Xiphophorus maculatus).

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.

Exposure to 4100K fluorescent light elicits sex specific transcriptional responses in Xiphophorus maculatus skin.

It has been reported that exposure to artificial light may affect oxygen intake, heart rate, absorption of vitamins and minerals, and behavioral responses in humans. We have reported specific gene expression responses in the skin of Xiphophorus fish after exposure to ultraviolet light (UV), as well as, both broad spectrum and narrow waveband visible light. In regard to fluorescent light (FL), we have shown that male X. maculatus exposed to 4100K FL (i.e. "cool white") rapidly suppress transcription of many genes involved with DNA replication and repair, chromosomal segregation, and cell cycle progression in skin. We have also detailed sex specific transcriptional responses of Xiphophorus skin after exposure to UVB. However, investigation of gender differences in global gene expression response after exposure to 4100K FL has not been reported, despite common use of this FL source for residential, commercial, and animal facility illumination. Here, we compare RNA-Seq results analyzed to assess changes in the global transcription profiles of female and male X. maculatus skin in response to 4100K FL exposure. Our results suggest 4100K FL exposure incites a sex-biased genetic response including up-modulation of inflammation in females and down modulation of DNA repair/replication in males. In addition, we identify clusters of genes that become oppositely modulated in males and females after FL exposure that are principally involved in cell death and cell proliferation.

The transcriptional response of skin to fluorescent light exposure in viviparous (Xiphophorus) and oviparous (Danio, Oryzias) fishes.

Differences in light sources are common in animal facilities and potentially can impact experimental results. Here, the potential impact of lighting differences on skin transcriptomes has been tested in three aquatic animal models commonly utilized in biomedical research, (Xiphophorus maculatus (platyfish), Oryzias latipes (medaka) and Danio rerio (zebrafish). Analysis of replicate comparative RNA-Seq data showed the transcriptional response to commonly utilized 4100K or "cool white" fluorescent light (FL) is much greater in platyfish and medaka than in zebrafish. FL induces genes associated with inflammatory and immune responses in both medaka and zebrafish; however, the platyfish exhibit suppression of genes involved with immune/inflammation, as well as genes associated with cell cycle progression. Furthermore, comparative analyses of gene expression data from platyfish UVB exposures, with medaka and zebrafish after exposure to 4100K FL, show comparable effects on the same stress pathways. We suggest the response to light is conserved, but that long-term adaptation to species specific environmental niches has resulted in a shifting of the wavelengths required to incite similar "genetic" responses in skin. We forward the hypothesis that the "genetic perception" of light may have evolved differently than ocular perception and suggest that light type (i.e., wavelengths emitted) is an important parameter to consider in experimental design.

Molecular genetic response to varied wavelengths of light in Xiphophorus maculatus skin.

Xiphophorus fishes represent a model often utilized to study UVB induced tumorigenesis. Recently, varied genetic responses to UVB exposure have been documented in the skin of female and male Xiphophorus, as have differences in UVB response in the skin of different parental species and for interspecies hybrids produced from crossing them. Additionally, it has been shown that exposure to "cool white" fluorescent light induces a shift in the genetic profiles of Xiphophorus skin that is nearly as robust as the UVB response, but involves a fundamentally different set of genes. Given these results and the use of Xiphophorus interspecies hybrids as an experimental model for UVB inducible melanoma, it is of interest to characterize genes that may be transcriptionally modulated in a wavelength specific manner. The global molecular genetic response of skin upon exposure of the intact animal to specific wavelengths of light has not been investigated. Herein, we report results of RNA-Seq experiments from the skin of male Xiphophorus maculatus Jp 163 B following exposure to varied 50nm wavelengths of light ranging from 300-600nm. We identify two specific wavelength regions, 350-400nm (88 genes) and 500-550nm (276 genes), that exhibit transcriptional modulation of a significantly greater number of transcripts than any of the other 50nm regions in the 300-600nm range. Observed functional sets of genes modulated within these two transcriptionally active light regions suggest different mechanisms of gene modulation.

Exposure to fluorescent light triggers down regulation of genes involved with mitotic progression in Xiphophorus skin.

We report RNA-Seq results from skin of X. maculatus Jp 163 B after exposure to various doses of "cool white" fluorescent light (FL). We show that FL exposure incites a genetic transcriptional response in skin nearly as great as observed for UVB exposure; however, the gene sets modulated due to exposure to the two light sources are quite different. Known light responsive genes involved in maintaining circadian cycling (e.g., clock, cry2a, cry1b, per1b, per2, per3, and arntl1a) exhibited expected shifts in transcriptional expression upon FL exposure. Exposure to FL also resulted in down-regulated transcription of many genes involved with cell cycle progression (e.g., cdc20, cdc45, cdca7b, plk1, cdk1, ccnb-3, and cdca7a) and chromosome segregation (e.g., cenpe, cenpf, cenpi, cenpk, cenpo, cenpp, and cenpu; cep70; knstrm, kntc, mcm2, mcm5; smc2). In addition, several DNA replication and recombination repair genes (e.g., pola1, pole, rec52, rad54l, rpa1, and parpbp) exhibit reduced expression in FL exposed X. maculatus skin. Some genes down modulated by FL are known to be associated with DNA repair and human diseases (e.g., atm2, brip1, fanc1, fancl, and xrcc4). The overall suppression of genes involved with mitotic progression in the skin of adult fish is consistent with entry into the light phase of the circadian cycle. Current efforts are aimed at determining specific wavelengths that may lead to differential expression among the many genes affected by fluorescent light exposure.