Biology subjects

Okeoma, C. M.

Publications and source records attributed to Okeoma, C. M..

8 recordsLinked to original sources

bioRxiv2025

Cargo and biological properties of extracellular vesicles released from human adenovirus type 4-infected lung epithelial cells

Extracellular vesicles (EVs) are rapidly gaining recognition as critical mediators of inter-cellular communication during viral infections. To contribute to fill the gap in knowledge regarding the role of EVs in adenovirus infection, we used human adenovirus type 4 of species Mastadenovirus exoticum (HAdV-E4), a prevalent respiratory and ocular pathogen, and characterized the cargo and biological properties of EVs released by HAdV-E4-infected A549 lung epithelial cells at a pre-lytic stage of infection. Using immunocapture-based isolation and multi-omics approaches, we found that infection profoundly alters the EV uploaded proteome and small non-coding RNA repertoire. Mass spectrometry identified 268 proteins unique to EVs purified from infected cells (AdV-EVs), with enrichment in pathways supporting vesicle trafficking and viral protein translation, and importantly also a few virus-encoded proteins. Small RNA transcriptome analysis showed differential uploading in AdV-EVs of various small non-coding RNAs, including snoRNAs, as well as the presence of virus associated RNAs I and II. Notably, AdV-EVs contained viral genomic DNA and were capable of initiating productive infection upon delivery to naive cells in the absence of detectable viral particles. Our data suggest that EVs released during HAdV-E4 infection may serve as vehicles for non-lytic viral dissemination and highlight their possible role in intra-host dissemination.

microbiology↗

bioRxiv2025

Identification of HIV Tat and NF-κB binding proteins associated with semen-derived extracellular vesicles

Semen-derived extracellular vesicles (SEVs) have been shown to inhibit transactivation of the long terminal repeat (LTR) in human immunodeficiency virus type 1 (HIV-1, or HIV) and, hence, viral replication by blocking the interaction of the viruss transcriptional activator Tat and host transcription factors NF-{kappa}B and Sp1. The ability of SEVs to regulate the activities of transcription factors suggests that SEVs may contain transcription activators and repressors. Here, we identified host proteins in human SEVs that interacted with the Tat and NF-{kappa}B subunit p65. Integrative network and pathway enrichment analyses of these complexes revealed associations with an array of biological functions regulating genome transcription. In particular, several proteins in SEVs could bind to both Tat and NF-{kappa}B: the scaffolding and cell signaling regulatory protein AKAP9, the G protein signaling regulator ARHGEF28, the small nuclear RNA processor INTS1, the epigenetic reader BRD2, and the transcription elongation inhibitor NELFB. NF-{kappa}B p65-bound NELFB also interacted with HEXIM1, another transcription elongation inhibitor, suggesting that SEVs may inhibit HIV propagation through networks of transcriptional regulation and repression. One Sentence SummaryProteins in vesicles shed from human semen may repress HIV by targeting transcription factors.

molecular biology↗

bioRxiv2025

Rigorous process for isolation of gut-derived extracellular vesicles and the effect on latent HIV

AimExtracellular particles (EPs) are produced/secreted by cells from all domains of life and are present in all body fluids, brain, and gut. EPs consist of extracellular vesicles (EVs) made up of exosomes, microvesicles, and other membranous vesicles; and extracellular condensates (ECs) that are non-membranous carriers of lipid-protein-nucleic acid aggregates. The purity of EVs|ECs, which ultimately depends on the isolation method used to obtain them is critical, particularly EVs|ECs from the gastrointestinal (GI) tract that is colonized by a huge number of enteric bacteria. Therefore, identifying GI derived EVs|ECs of bacterial and host origin may serve as a window into the pathogenesis of diseases and as a potential therapeutic target. MethodsHere, we describe the use of high-resolution particle purification liquid chromatography (PPLC) gradient-bead-column integrated with polyvinylpolypyrrolidone (PVPP)-mediated extraction of impurities to isolate GI-derived EPs. Results and ConclusionPVPP facilitates isolation of pure and functionally active, non-toxic EVs ColEVs from colonic contents. ColEVs are internalized by cells and they activate HIV LTR promoter. In the absence of PVPP, ColEVs have a direct reductive potential of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance in a cell-free system. Assessment of the origin of ColEVs reveals that they are composed of both bacteria and host particles. This protocol requires [~]12 hours (5 hours preprocessing, 7 hours isolation) to complete and should be used to purify EVs from sources contaminated with microbial agents to improve rigor. Additionally, this protocol provides a robust tool for researchers and clinicians investigating GI-derived EVs and the translational use of GI-derived EVs for diagnostic and therapeutic use. HighlightO_LIColEVs but not ColECs are present in colonic content (GI tract) and can be isolated with gradient or single bead PPLC column. C_LIO_LIColEVs isolated without PVPP are toxic to cells and they have a direct reductive potential of MTT. Addition of PVPP treatment in the isolation protocol results in clean and non-toxic ColEVs that transactivate the HIV LTR promoter. C_LI

molecular biology↗

bioRxiv2024

Berberine: A dual anti-HIV and anti- cervical cancer compound

We report the effects of berberine (BBR), a benzylisoquinoline alkaloid small molecule on inhibition of HIV infection of cervical cancer cells. We used HeLa cell-derived TZM-bl cell line as a model of cervical cancer and HIV infection. BBR significantly inhibits viral and cancer processes, including expression of cell-associated HIV RNA, secretion of HIV reverse transcriptase, and HIV Tat-mediated LTR promoter transactivation. BBR significantly inhibits HIV-induced cancer cell viability and cell clustering. Besides its ability to inhibit HIV-induced cancer cell viability, BBR inhibits migration and matrix invasion of cervical cancer cells that are infected with HIV or treated with HIV Tat protein. The ability of BBR to inhibit cell clustering, collective cell migration, and cell invasion may have an effect in controlling progression of cervical cancer and HIV since collective migration and invasion are strategies for local tissue infiltration, as well as metastatic invasion in epithelial cancers. Interestingly, molecular docking and dynamic stimulation show that BBR binds HIVIIIB Tat amino acid residues through non-covalent interactions that occurs at multiple sites including LYS71, providing mechanistic insights into BBR regulation HIV infection. The results of the present study suggest that BBR has the potential to inhibit HIV infection and comorbid cervical cancer progression. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/624212v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1b88635org.highwire.dtl.DTLVardef@17311d8org.highwire.dtl.DTLVardef@19881c7org.highwire.dtl.DTLVardef@170483a_HPS_FORMAT_FIGEXP M_FIG C_FIG Berberine (BBR), a benzylisoquinoline alkaloid small molecule, inhibits HIV infection of cervical cancer cells, suppresses HIV-induced migration and invasion of the cancer cells. Mechanistically, BBR interferes with Tat-mediated HIV LTR promoter transactivation and expression of different HIV RNA species, expression of early genes (multiply spliced Tat-Rev) and late genes (unspliced Gag-Pol).

molecular biology↗

bioRxiv2024

Epigenetic modulation of NLRP6 inflammasome sensor as a therapeutic modality to reduce necroptosis-driven gastrointestinal mucosal dysfunction in HIV/SIV infection

The epigenetic mechanisms driving persistent gastrointestinal mucosal dysfunction in HIV/SIV infection is an understudied topic. Using reduced-representation bisulfite sequencing, we identified HIV/SIV infection in combination anti-retroviral therapy (cART)-naive rhesus macaques (RMs) to induce marked hypomethylation throughout promoter-associated CpG islands (paCGIs) in genes related to inflammatory response (NLRP6, cGAS), cellular adhesion and proliferation in colonic epithelial cells (CEs). Moreover, low-dose delta-9-tetrahydrocannabinol (THC) administration reduced NLRP6 protein expression in CE by hypermethylating the NLRP6 paCGI and blocked polyI:C induced NLRP6 upregulation in vitro. In cART suppressed SIV-infected RMs, NLRP6 protein upregulation associated with significantly increased expression of necroptosis-driving proteins; phosphorylated-RIPK3(Ser199), phosphorylated-MLKL(Thr357/Ser358), and HMGB1. Most strikingly, supplementing cART with THC effectively reduced NLRP6 and necroptosis-driving protein expression to pre-infection levels. These findings for the first time demonstrate that NLRP6 upregulation and ensuing activation of necroptosis promote HIV/SIV-induced gastrointestinal mucosal dysfunction and that epigenetic modulation using phytocannabinoids represents a feasible therapeutic modality for alleviating HIV/SIV-induced gastrointestinal inflammation and associated comorbidities.

pathology↗

bioRxiv2024

Cannabinoids shift the basal ganglia miRNA m6A methylation profile towards an anti-inflammatory phenotype in SIV-infected Rhesus macaques

BackgroundEpitranscriptomic modifications modulate diverse biological processes like regulation of gene expression, abundance, location and function. N6-methyladenosine (m6A) methylation has been shown to regulate various diseases, including cancer and inflammation. While there is evidence that m6A modification is functionally relevant in neural development and differentiation, the role of m6A modification in HIV neuropathogenesis is unknown. MethodsHere, we used anti-N6-methyladenosine (m6A) antibody immunoprecipitation and microarray profiling to identify m6A modifications in miRNAs in basal ganglia (BG) of Rhesus macaques (RMs) that were uninfected (VEH) and SIV-infected on combination anti-retroviral therapy (cART) and either VEH-treated (VEH/SIV/cART), or THC:CBD-treated (THC:CBD/SIV/cART). Ingenuity pathway analysis was conducted to understand the biological implications of miRNA m6A methylation in HIV neuropathogenesis. Finally, to understand the functional significance of m6A modifications in miRNAs, we overexpressed FAM-labeled wild-type or m6A-modified miR-194-5p in SCC-25 cells and determined its impact on the expression of its target, STAT1, an interferon-stimulated transcription factor known to drive persistent neuroinflammation in several neurodegenerative diseases. ResultsHIV/SIV infection promoted an overall hypomethylated miRNA m6A profile. While the overall hypomethylated m6A profile was not significantly impacted by THC:CBD, specific miRNAs predicted to target proinflammatory genes showed markedly reduced m6A methylation levels compared to the VEH-treated RMs. Additionally, specific BG tissue miRNAs bearing m6A epi-transcriptomic marks were transferred and detected in BG-derived extracellular vesicles. Mechanistically, the DRACH motif in the seed region of miR-194-5p was significantly m6A hypomethylated in THC:CBD/SIV/cART RMs. In SCC-25 cells, unlike wild-type miR-194-5p, transfected m6A-modified miR-194-5p mimics failed to downregulate STAT1 protein expression. Further, compared to VEH/SIV/cART RMs, THC:CBD administration significantly reduced m6A methylation of 44 miRNAs directly involved in regulating CNS network genes. ConclusionsThese results underscore the need for investigating the qualitative, and posttranscriptional modifications in RNA along with the more traditional, quantitative alterations in pathological conditions or in response to disease modifying treatments. Our findings indicate that m6A epitranscriptomic marks in the seed nucleotide region can impair miRNA function and that cannabinoids may preserve it by reducing m6A methylation levels. Finally, these findings provide a novel mechanistic (miRNA m6A hypomethylation) explanation underlying the anti-neuroinflammatory effects of phytocannabinoids in HIV/SIV infection.

neuroscience↗

bioRxiv2024

Extracellular condensates (ECs) are endogenous modulators of HIV transcription and latency reactivation

Persistence of human immunodeficiency virus (HIV) latent reservoir is the major challenge to HIV cure because the latent reservoir is not eliminated by antiretroviral therapy (ART), and they serve as sources for viral rebound upon cessation of ART. Mechanisms regulating viral persistence are not well understood. This study used model systems of post-integration latency to explore the role of basal ganglia (BG) isolated extracellular condensates (ECs) in reprogramming HIV latent cells. We found that BG ECs from uninfected macaques (VEH) and SIV infected macaques (VEH|SIV) activate latent HIV transcription in various model systems. VEH and VEH|SIV ECs significantly increased expression of viral antigen in latently infected cells. Activation of viral transcription, antigen expression, and latency reactivation was inhibited by ECs from the brain of macaques treated with Delta-9-tetrahydrocannabinol (THC) and infected with SIV (THC|SIV). Virus produced by latently infected cells treated with VEH|SIV ECs potentiated cell-cell and cell-free HIV transmission. VEH|SIV ECs also reversed dexamethasone-mediated inhibition of HIV transcription while TNF-mediated reactivation of latency was reversed by THC|SIV ECs. Transcriptome and secretome analyses of total RNA and supernatants from latently infected cells treated with ECs revealed significant alteration in gene expression and cytokine secretion. THC|SIV ECs increased secretion of Th2 and decreased secretion of proinflammatory cytokines. Most strikingly, while VEH/SIV ECs robustly induced HIV RNA in latently HIV-infected cells, long-term low-dose THC administration enriched ECs for anti-inflammatory cargo that significantly diminished their ability to reactivate latent HIV, an indication that ECs are endogenous host factors that may regulate HIV persistence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/613037v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d5e9fborg.highwire.dtl.DTLVardef@f48088org.highwire.dtl.DTLVardef@22696aorg.highwire.dtl.DTLVardef@136c0ae_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIECs isolated from SIV infected macaques (VEH|SIV ECs) is a positive regulator of LTR-dependent HIV transcription and production of infectious viral particles in vitro. C_LIO_LIECs isolated from THC treated SIV infected macaques (THC|SIV ECs) prevents the transcription and reactivation of HIV in latently infected cells and prevents production of viral particles in vitro. C_LIO_LIECs reprogram host transcriptome and secretome in manners that or suppress promote reactivation of latent HIV reservoir. C_LI The above highlights led to the conclusion that while VEH/SIV ECs robustly induced HIV RNA in latently HIV-infected cells, long-term low-dose THC administration enriched ECs for anti-inflammatory cargo that significantly diminished their ability to reactivate latent HIV.

cell biology↗

bioRxiv2024

Lipidomic and Proteomic Insights from Extracellular Vesicles in Postmortem Dorsolateral Prefrontal Cortex Reveal Substance Use Disorder-Induced Brain Changes

Substance use disorder (SUD) significantly increases the risk of neurotoxicity, inflammation, oxidative stress, and impaired neuroplasticity. The activation of inflammatory pathways by substances may lead to glial activation and chronic neuroinflammation, potentially mediated by the release of extracellular particles (EPs), such as extracellular condensates (ECs) and extracellular vesicles (EVs). These particles, which reflect the physiological, pathophysiological, and metabolic states of their cells of origin, might carry molecular signatures indicative of SUD. In particular, our study investigated neuroinflammatory signatures in SUD by isolating EVs from the dorsolateral prefrontal cortex (dlPFC) Brodmanns area 9 (BA9) in postmortem subjects. We isolated BA9-derived EVs from postmortem brain tissues of eight individuals (controls: n=4, SUD: n=4). The EVs were analyzed for physical properties (concentration, size, zeta potential, morphology) and subjected to integrative multi-omics analysis to profile the lipidomic and proteomic characteristics. We assessed the interactions and bioactivity of EVs by evaluating their uptake by glial cells. We further assessed the effects of EVs on complement mRNA expression in glial cells as well as their effects on microglial migration. No significant differences in EV concentration, size, zeta potential, or surface markers were observed between SUD and control groups. However, lipidomic analysis revealed significant enrichment of glycerophosphoinositol bisphosphate (PIP2) in SUD EVs. Proteomic analysis indicates downregulation of SERPINB12, ACYP2, CAMK1D, DSC1, and FLNB, and upregulation of C4A, C3, and ALB in SUD EVs. Gene ontology and protein-protein interactome analyses highlight functions such as cell motility, focal adhesion, and acute phase response signaling that is associated with the identified proteins. Both control and SUD EVs increased C3 and C4 mRNA expression in microglia, but only SUD EVs upregulated these genes in astrocytes. SUD EVs also significantly enhanced microglial migration in a wound healing assay.This study successfully isolated EVs from postmortem brains and used a multi-omics approach to identify EV-associated lipids and proteins in SUD. Elevated C3 and C4 in SUD EVs and the distinct effects of EVs on glial cells suggest a crucial role in acute phase response signaling and neuroinflammation.

neuroscience↗