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T cells are integral players in the adaptive immune system that readily adapt their metabolism to meet their energetic and biosynthetic needs. A major hurdle to understand physiologic T-cell metabolism has been the differences between in vitro cell culture conditions and the complex in vivo milieu. To address this, we have developed a protocol that merges traditional immunology infection models with whole-body metabolite infusion and mass-spectrometry-based metabolomic profiling to assess T-cell metabolism in vivo. In this protocol, pathogen-infected mice are infused via the tail vein with an isotopically labeled metabolite (2–6 h), followed by rapid magnetic bead isolation to purify T-cell populations (<1 h) and then stable isotope labeling analysis conducted by mass spectrometry (~1–2 d). This procedure enables researchers to evaluate metabolic substrate utilization into central carbon metabolic pathways (i.e., glycolysis and the tricarboxylic acid cycle) by specific T-cell subpopulations in the context of physiological immune responses in vivo. This protocol describes how to rapidly isolate a specific cell type from mouse blood or tissues for mass-spectrometry-based metabolomics. Mice are infused with isotopically labeled compounds, and cells are isolated using antibody-coated magnetic beads.

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism. Metabolite extraction with organic solvents is assumed to remove/denature proteins. Here, the authors uncover a vast landscape of >1,000 proteins in metabolite extracts. These proteins can retain catalytic activity and drive post-extraction metabolite changes, obscuring biological interpretation.

Mineral fluorescence under different portions of the visible and invisible light spectrum has a long history of scientific study. In our study of marine sediments from the Georgia Bight, we have utilized the blue portion of the light spectrum in the 445 nanometer (nm) range. The use of fluorescence has proven very useful in microscopic analyses of carbonate minerals. While the sediment prism of the inner-to-mid continental shelf in the southeastern Atlantic is predominantly siliceous, the dissolution and deterioration of marine shell contribute a significant amount to the fabric of any sediment sample. Together with carbonate minerals such as dolomite, eroded from basement rock and redeposited on the shelf, a potentially robust fluorescent response was expected and observed in samples. In marine sediments, blue light illumination has produced an easily observed fluorescent response in both underwater and in laboratory settings. This fluorescence can be attributed to carbonate minerals—calcite/aragonite. Feldspars are major accessory minerals in the sediment prism of the Georgia Bight, and much of the observed fluorescence in our samples can be attributed to their presence. To identify specific minerals responsible for any observed fluorescence, X-ray diffraction and energy dispersive spectroscopy were utilized. This combined methodology of luminescent excitation, X-ray diffractometry and spectroscopy has produced the results reported herein.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects. After infection with SARS-CoV-2 a diverse set of symptoms remain or develop longer term in a condition termed long-CoVID, yet an accurate and tractable model has remained elusive. Here the authors present a non-human primate model of long-CoVID and show persistent hyperglycemia following acute infection.

Finding prehistoric sites, on the sea floor off the United States, has proven to be a difficult task. Over four decades of innovative attempts have produced few discoveries. This discussion outlines the difficulties in finding submerged prehistory on the sea floor, examines current methodology(ies), and presents a new methodology that demonstrates promise based on research conducted over the past decade. The purpose of this discussion is to outline the problems facing archaeologists searching the sea floor for archaeological sites. It is less of a critique of past efforts and methodologies used in those attempts. Without those efforts, a reason for developing a different methodological approach would be unnecessary. Any “one size fits all” methodology is limiting, and the scant number of marine archaeological sites located off the shores of the United States can attest to the need for practitioners to continue refining their methods. This is especially the case on the continental shelf of the eastern United States, where sediment cover is often meters, or several meters, thick. This opportunistic methodology developed on the continental shelf of the Georgia Bight, western Atlantic Ocean, has located intact paleo-landforms with in situ, palaeoecological remains thereby offering promise for future archaeological discoveries.

ObjectivesStudies in several sub-Saharan geographies conducted early in the COVID-19 pandemic suggested little impact on contraceptive behaviours. Initial results may mask widening disparities with rising poverty, and changes to women’s pregnancy desires and contraceptive use amid prolonged health service disruptions. This study examined trends in contraceptive behaviours in four sub-Saharan African settings 1 year into the pandemic.DesignNationally and regionally representative longitudinal surveys.SettingBurkina Faso, Kenya, Democratic Republic of Congo (Kinshasa) and Nigeria (Lagos).ParticipantsWomen aged 15–49 years with sample size ranging from 1469 in Nigeria to 9477 in Kenya.Outcome measuresFertility preferences, contraceptive use and unintended pregnancies measured before COVID-19 (November 2019 to January 2020) and during COVID-19 (November 2020 to January 2021).AnalysisWe described population-level and individual-level changes by socioeconomic characteristics using generalised equation modelling. We used logistic regression models to identify factors related to contraceptive adoption and discontinuation and to experiencing an unintended pregnancy.ResultsAt the population level, we found no change in women’s exposure to unintended pregnancy risk, alongside 5–9 percentage point increases in contraceptive prevalence in Burkina Faso, Kenya and Lagos. Reliance on provider-dependent methods dropped by 2 and 4 percentage points in Kenya and Burkina Faso, respectively, although these declines were not statistically significant. Between 1.0% and 2.8% of women across sites experienced an unintended pregnancy during COVID-19, with no significant change over time. Individual-level trajectories showed contraceptive adoption was more common than discontinuation in Burkina Faso, Kenya and Lagos, with little difference by sociodemographic characteristics. Women’s COVID-19-related economic vulnerability was unrelated to unintended pregnancy across sites.ConclusionsThis study highlights the resilience of African women across diverse settings in sustaining contraceptive practices amid the COVID-19 pandemic. However, with reports of rising poverty in sub-Saharan Africa, there is continued need to monitor access to essential sexual and reproductive health services.

Psittacine bornavirus type-4 (PaBV-4) causes proventricular dilatation disease and death among diverse birds, most notably caged parrots and related species, with no known cure or vaccine. Infected birds can shed virus in fecal matter, urine, and feather dander but it is unknown how well PaBV-4 survives outside of the host. This study focused on assessing the persistence of PaBV-4 in common environmental situations. The presence of viral RNA was examined in aqueous solutions at varying temperatures and recovery from typical avian husbandry materials (plastic, wood, metal, and cloth). Viral RNA persistence in aqueous samples was found to be 3 weeks at 37 °C, 2 months at 24 °C (room temperature), and 3 months at 4 °C. Viral RNA was also recovered from plastic and metal surfaces up to 72 h after inoculation. Also examined were disinfection protocols comparing coverage versus contact time for a reduction in viral RNA. Complete coverage by the disinfecting agent was more important for preventing recovery of viral RNA. Additionally, PaBV-4 RNA was transferable by paper towel. These results provide the first evidence of the robust nature of PaBV-4 in an aqueous environment and show that cleaning protocols need to be carefully curated to limit possible viral spread.

Transforming growth factor-β signaling (TGF-β) maintains a balanced physiological function including cell growth, differentiation, and proliferation and regulation of immune system by modulating either SMAD2/3 and SMAD7 (SMAD-dependent) or SMAD-independent signaling pathways under normal conditions. Increased production of TGF-β promotes immunosuppression in Human Immunodeficiency Virus (HIV)/Simian Immunodeficiency Virus (SIV) infection. However, the cellular source and downstream events of increased TGF-β production that attributes to its pathological manifestations remain unknown. Here, we have shown increased production of TGF-β in a majority of intestinal CD3−CD20−CD68+ cells from acute and chronically SIV infected rhesus macaques, which negatively correlated with the frequency of jejunum CD4+ T cells. No significant changes in intestinal TGF-β receptor II expression were observed but increased production of the pSMAD2/3 protein and SMAD3 gene expression in jejunum tissues that were accompanied by a downregulation of SMAD7 protein and gene expression. Enhanced TGF-β production by intestinal CD3−CD20−CD68+ cells and increased TGF-β/SMAD-dependent signaling might be due to a disruption of a negative feedback loop mediated by SMAD7. This suggests that SIV infection impacts the SMAD-dependent signaling pathway of TGF-β and provides a potential framework for further study to understand the role of viral factor(s) in modulating TGF-β production and downregulating SMAD7 expression in SIV. Regulation of mucosal TGF-β expression by therapeutic TGF-β blockers may help to create effective antiviral mucosal immune responses.

Angiotensin converting enzyme-2 (ACE2) and associated proteins play a pivotal role in various physiological and pathological events, such as immune activation, inflammation, gut barrier maintenance, intestinal stem cell proliferation, and apoptosis. Although many of these clinical events are quite significant in SIV/HIV infection, expression profiling of these proteins has not been well reported. Considering the different pathological consequences in the gut after HIV infection, we hypothesized that the expression of ACE2 and associated proteins of the Renin-angiotensin system (RAS) could be compromised after SIV/HIV infection. We quantified the gene expression of ACE2 as well as AGTR1/2 , ADAM17 , and TMPRSS2 , and compared between SIV infected and uninfected rhesus macaques ( Macaca mulatta ; hereafter abbreviated RMs). The gene expression analysis revealed significant downregulation of ACE2 and upregulation of AGTR2 and inflammatory cytokine IL-6 in the gut of infected RMs. Protein expression profiling also revealed significant upregulation of AGTR2 after infection. The expression of ACE2 in protein level was also decreased, but not significantly, after infection. To understand the entirety of the process in newly regenerated epithelial cells, a global transcriptomic study of enteroids raised from intestinal stem cells was performed. Interestingly, most of the genes associated with the RAS, such as DPP4 , MME , ANPEP , ACE2 , ENPEP , were found to be downregulated in SIV infection. HNFA1 was found to be a key regulator of ACE2 and related protein expression. Jejunum CD4+ T cell depletion and increased IL-6 mRNA, MCP-1 and AGTR2 expression may signal inflammation, monocyte/macrophage accumulation and epithelial apoptosis in accelerating SIV pathogenesis. Overall, the findings in the study suggested a possible impact of SIV/HIV infection on expression of ACE2 and RAS-associated proteins resulting in the loss of gut homeostasis. In the context of the current COVID-19 pandemic, the outcome of SARS-CoV-2 and HIV co-infection remains uncertain and needs further investigation as the significance profile of ACE2, a viral entry receptor for SARS-CoV-2, and its expression in mRNA and protein varied in the current study. There is a concern of aggravated SARS-CoV-2 outcomes due to possible serious pathological events in the gut resulting from compromised expression of RAS- associated proteins in SIV/HIV infection.