Biology subjects

Hunter, C.

Publications and source records attributed to Hunter, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Impact of RNA modification and IL-12 boosts on mRNA-LNP vaccine-elicited CD8+ T cell responses

Development of effective HIV-1 vaccines requires strategies for inducing both CD8+ T cell and neutralizing antibody responses. Nucleoside-modified mRNA-lipid-nanoparticles (LNP) induce more potent antibody responses than unmodified mRNA-LNP vaccines, while unmodified mRNA is typically employed for CD8+ T cell-inducing vaccines. Here, we compared the abilities of differing ratios of unmodified versus modified mRNA expressing a SIV conserved-region CD8+ T cell immunogen co-administered with IL-12-encoding mRNA-LNP during boosting to induce CD8+ T cell and humoral responses in non-human primates, assessing responses in ChAdOx1-primed controls in parallel. Serial priming and boosting with fully-unmodified mRNA elicited higher-magnitude, more CD8-biased T cell responses than fully-modified mRNA, that were durably maintained after the IL-12-encoding mRNA boosts. Inclusion of 30% nucleoside-modified mRNA enhanced antibody titers, suggesting potential benefits to a mixed-composition vaccine.

immunology↗

bioRxiv2026

Fetal-intrinsic antiviral mechanisms emerge over the course of gestation

Congenital viral infections have variable effects on pregnancy outcomes with implications for maternal and fetal health. However, the maternal and fetal immune mechanisms that emerge over the course of gestation to determine protective or pathological outcomes remain poorly understood. Here, we use the emerging congenital pathogen Oropouche virus (OROV) to examine gestational stage-dependent differences in maternal and fetal outcomes in a mouse model of congenital infection. Pregnant mice (dams) infected during early gestation resist severe OROV disease, whereas mid-gestation-infected dams succumb to infection. In contrast, fetal pathology is substantial following early gestation infection but limited following infection during mid-gestation, revealing discordant maternal and fetal susceptibility across gestation. Mid-gestation fetal tissues effectively restricted vertical transmission compared to early gestation fetal tissues, corresponding with reduced fetal pathology. Moreover, both placental and fetal tissue cleared OROV RNA over the course of infection, independent of gestational stage, and failure to clear viral RNA was associated with severe fetal pathology. Spatial analysis of early gestation implantation sites further revealed distinct regional susceptibility to OROV infection across the maternal-fetal interface. We identified potential instances of placental-independent vertical transmission via direct fetal contact with highly infected regions of the contralateral maternal uterus. Finally, we uncovered an unexpected mechanism by which type I interferon signaling contributes to inter-fetal immune crosstalk to restrict both OROV vertical transmission and pathology. Together, these findings establish the fetus as an active participant in antiviral defense and reveal previously unrecognized mechanisms by which fetal-intrinsic antiviral immune responses limit congenital viral infection and disease.

immunology↗

bioRxiv2026

A PERK/FOXO1 AXIS LINKS DNA DAMAGE TO FIBROBLAST SURVIVAL IN DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS

ObjectiveDiffuse cutaneous systemic sclerosis (dcSSc) is a life-limiting fibrotic disease. We and others have shown that dcSSc fibroblasts accumulate numerous somatic mutations associated with senescence-like features; however, the mechanism(s) enabling their survival remain unclear. MethodsSkin biopsies were obtained from lesional tissues from dcSSc (n=10), dcSSc treated with autologous hematopoietic stem cell transplantation (ASCT, n=8) or 7 age/sex-matched healthy controls. Primary dermal fibroblasts were generated from biopsies. Spatial RNA sequencing, immunoblotting, confocal microscopy, and functional assays were used to mechanistically delineate signaling pathways linking DNA-damage with fibroblast survival. ResultsdcSSc fibroblasts demonstrated increased pH2AX DNA double-strand-break foci yet remained apoptosis resistant. These cells displayed features of metabolic-stress remodeling, including mitochondrial hyperpolarization, increased reactive oxygen species production, and enhanced mitochondrial biogenesis. Spatial transcriptomics and subsequent biochemical analyses identified activation of a PERK/ATF4/FOXO1 axis, characterized by PERK phosphorylation, selective ATF4 translation, FOXO1 nuclear translocation, and induction of downstream antioxidant and metabolic programs. In contrast, fibroblasts from post-ASCT patients exhibited normalization of DNA-damage markers and mitochondrial parameters without ATF4/FOXO1 activation. Pharmacologic inhibition of either PERK or FOXO1 selectively restored mitochondrial-dependent apoptosis in dcSSc fibroblasts, demonstrating that this axis is required for their survival following extensive genomic injury. ConclusiondcSSc fibroblasts persist despite substantial genomic injury by engaging a PERK/ATF4/FOXO1 metabolic-adaptation program that suppresses mitochondrial-dependent apoptosis. This survival axis is not present after ASCT. Targeting PERK or FOXO1 restores apoptosis selectively in dcSSc fibroblasts, highlighting its potential use as a therapeutic target for eliminating pathogenic senescence-like fibroblasts in dcSSc. HighlightsO_LIBoth ex-vivo skin and in-vitro primary dermal fibroblasts derived from dcSSc patients have a higher frequency of intrinsic DNA damage signals and senescence-associated features; yet they evade mitochondrial-dependent apoptosis. C_LIO_LIPathogenic dcSSc fibroblasts rewire their metabolism, characterized by mitochondrial hyperpolarization and elevated ROS. C_LIO_LISpatial transcriptomics and functional analyses reveal a PERK/ATF4/FOXO1 stress-adaptation axis that drives fibroblast survival in dcSSc. C_LIO_LIThis maladaptive survival program characterized by increased genotoxic stress, and mitochondrial remodelling is absent in post-ASCT fibroblasts. C_LIO_LITargeting PERK or FOXO1 selectively sensitizes dcSSc fibroblasts to apoptosis revealing a potential promising therapeutic strategy in dcSSc. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/706443v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1b3f791org.highwire.dtl.DTLVardef@42548aorg.highwire.dtl.DTLVardef@bc4ce1org.highwire.dtl.DTLVardef@5b4d48_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Diverse infections transcriptionally reprogram the intestinal epithelium and epithelial-immune cell interactions

The distal small intestine plays vital roles in host physiology by regulating nutrient and fluid homeostasis. Despite being impacted in Crohns disease and a major target for a range of infections, we know relatively little about the complexity of cellular responses and cell-cell communication in the ileum during infection. Single cell and spatial transcriptomics have emerged as powerful technologies to study tissue heterogeneity in the gut, but these tools have focused on the large intestine, in part due to the accessibility of this tissue for biopsies and its importance in cancer. Here we present GutPath, an atlas of over 500,000 single cells with RNA and protein expression profiles for 91 cell states in the ileum across diverse infectious archetypes. We show that GutPath accurately captures established immune responses to infection while revealing pathogen-specific responses in enterocytes. To highlight the discovery potential of this atlas, we identify a novel enterocyte cell state present during Yersinia pseudotuberculosis infection that is spatially linked to bacterial load and tissue pathology. GutPath establishes a much-needed resource for the immunology community that will accelerate the study of the transcriptional diversity of cellular landscapes in the small intestine.

immunology↗

bioRxiv2025

Polysialic acid is a versatile marker for retinal Müller glia in common vertebrate model organisms and systems

Muller glia are retinal support cells that play crucial roles in tissue structure, waste management, and repair. A challenge for the field has been to find Muller glia markers that detect non-reactive cells or work well in non-mammalian models. We introduce two novel markers for identifying reactive and non-reactive Muller glia in vertebrate retinas: a modified enzyme lectin (GFP-EndoNDM) and a monoclonal antibody (mAb735). These markers recognize polysialic acid (polySia), which is a highly conserved glycosylation modification in humans and vertebrates. In the retina, polySia is present on Muller glia predominantly in the form of polySia-NCAM. We used GFP-EndoNDM and mAb735 to investigate polySia distribution in Muller glia of fish, amphibians, reptiles, birds, rodents, retinal organoids, and humans. In adult retinas of most species, polySia was localized to the Muller glia and spanned outer to inner retina, with inner plexiform layer (IPL) sublaminae ramifications. Gliosis was also detectable in degenerating murine retinas. Notable species differences were that only outer retinal regions of Muller glia were labelled in adult zebrafish, whereas the outer Muller glia body up to the first IPL sublamina was labelled in adult turquoise killifish. There was no significant retinal polySia labeling in larval zebrafish, but it was present in the brain. Larval turquoise killifish have polySia throughout the retina, similar to other adult vertebrates. Labelling polySia expands the scientific toolbox for Muller glia markers, and offers a versatile way to visualize and monitor structural changes in non-reactive and reactive Muller glia across most vertebrate species. HighlightsO_LIPolySia is a highly conserved Muller glia marker in vertebrate retina and retinal organoids C_LIO_LILabeling Muller glia with markers of polySia, facilitates the analysis of fine Muller glia processes in the IPL sublaminae, whole Muller glia morphology, and degenerative gliosis C_LIO_LIPolySia can be labeled with an engineered lectin conjugate (GFP-EndoNDM) and a commercially-available monoclonal antibody C_LI

cell biology↗

bioRxiv2025

Diverse activity in prefrontal projections promotes temporal control of action

Prefrontal neurons can have diverse activity during cognitive functions like working memory, attention, and timing; however, the importance of this heterogeneity is unclear. Our goal was to better understand the diversity of prefrontal activity through connectivity. We harnessed circuit-specific tools to capture activity within prefrontal projections during interval timing, an elementary cognitive process that requires working memory for temporal rules and attention to the passage of time to estimate a temporal interval of several seconds. We used human electroencephalography and single neuronal recordings in mice to capture prefrontal activity during interval timing, with major patterns characterized by time-dependent ramping (monotonic changes) over a temporal interval. We then leveraged retrograde viruses to interrogate prefrontal cortex (PFC) projections to the mediodorsal thalamus (PFC-MD) and to the dorsomedial striatum (PFC-DMS). We report three novel results. First, circuit-specific calcium fiber photometry revealed that PFC-MD and PFC-DMS activity encoded distinct temporal signals, with PFC-MD projections ramping down and PFC-DMS ramping up to interval timing response times. Second, circuit-specific inactivation revealed that PFC-DMS inactivation disrupted animals internal estimates of time. Third, circuit-specific single-nucleus RNA sequencing of prefrontal projections revealed distinct transcriptomic profiles between PFC-MD and PFC-DMS projections, with enriched genes for cortical layers and neuromodulators, and specific genes such as Cux2, Camk2n1, Htr4, and Foxp2. These data suggest differences in gene expression and connectivity give rise to the diversity of prefrontal activity during interval timing. These findings advance our fundamental understanding of prefrontal function and dysfunction in human disease.

neuroscience↗

bioRxiv2025

Interleukin-27 is antiviral at the maternal-fetal interface.

Congenital viral infections can have severe consequences for pregnancy and fetal outcomes. Remarkably, the fetal-derived placenta serves as a robust barrier to infection through meticulous regulation by immune effectors and a diverse repertoire of cytokines. Yet, the regulatory roles of many cytokines remain undefined at the maternal-fetal interface. Interleukin 27 (IL-27) is a highly expressed cytokine in the placenta whose functional consequence during congenital infection is unknown. Here, we utilized trophoblast organoids (TO) derived from primary human placentas and a mouse model of congenital viral infection to uncover the functional role of IL-27 signaling during pregnancy. We show that TOs constitutively express IL-27 and its receptor, IL27RA, and demonstrate that IL-27 signaling restricts Zika virus (ZIKV) infection of TOs. Through bulk RNA-sequencing of TOs in the absence and presence of IL-27 signaling, we demonstrate IL-27-mediated upregulation of antiviral genes. Finally, we show that IL-27 signaling is critical within the context of congenital murine ZIKV infection, as IL-27 restricts placental ZIKV burdens and protects against pathologic fetal outcomes early in gestation. These findings collectively demonstrate a novel role for IL-27 in the placenta and establish IL-27 as an innate antiviral defense at the maternal-fetal interface during congenital viral infection.

microbiology↗

bioRxiv2025

Defining neuronal responses to the neurotropic parasite Toxoplasma gondii

A select group of pathogens infect neurons in the brain. Prior dogma held that neurons were "defenseless" against infecting microbes, but many studies suggest that neurons can mount anti-microbial defenses. However, a knowledge gap in understanding how neurons respond in vitro and in vivo to different classes of micro-organisms remains. To address this gap, we compared a transcriptional dataset derived from primary neuron cultures (PNCs) infected with the neurotropic intracellular parasite Toxoplasma gondii with a dataset derived from neurons injected with T. gondii protein in vivo. These curated responses were then compared to the transcriptional responses of PNCs infected with the single stranded RNA viruses West Nile Virus (WNV) or Zika Virus (ZKV). These analyses highlighted a conserved response to infection associated with chemokines (Cxcl10, Ccl2) and cytokines (interferon signaling). However, T. gondii had diminished IFN- signaling in vitro compared to the viral datasets and was uniquely associated with a decrease in neuron-specific genes (Snap25, Slc17a7, Prkcg). These data underscore that neurons participate in infection-induced neuroinflammation and illustrate that neurons possess both pathogen-specific and pathogen-conserved responses. ImportanceThough neurons are commonly the target of pathogens that infect the CNS, few datasets assess the neuronal response to infection. This paucity of data is likely because neurons are perceived to have diminished immune capabilities. However, to understand the role of neurons in neuroinflammation and their immune capabilities, their responses must be investigated. Here we analyzed publicly accessible, neuron-specific datasets to compare neuron responses to a eukaryotic pathogen versus two Orthoflaviviruses. A better understanding of neuron responses to different infections will allow us to develop methods for inhibiting pathways that lead to neuron dysfunction, enhancing those that limit pathogen survival, and mitigating infection-induced damage to the CNS.

microbiology↗

bioRxiv2025

Proteomic Analysis of Breast Cancer Subtypes Identifies Stromal Contributions that Dictate Aggressive Malignant Behavior

Breast cancer manifests as multiple subtypes with distinct patient outcomes and treatment strategies. Here, we optimized proteomic analysis of Formalin-Fixed Paraffin-Embedded (FFPE) specimens from patients diagnosed with five breast cancer subtypes, luminal A, luminal B, Her2, triple negative (TNBC) and metaplastic breast cancers (MBC), and from disease-free individuals undergoing reduction mammoplasty (RM). We identified and quantified [~]6,000 protein groups (with >2 peptides per protein) with significant changes in over 26% of proteins comparing each cancer subtype with control RM. Stringent statistical filters allowed us to deeply mine 576 significant conserved protein changes shared by all subtypes and protein changes unique to each subtype. The most aggressive subtype, MBC, revealed exacerbated stromal stress responses, as illustrated by a collagenolytic extracellular matrix (ECM) and immune participation biased towards neutrophils and eosinophils. Immunostaining of breast tissue sections confirmed differences across subtypes, in particular, a strong upregulation of SERPINH1, neutrophil-specific myeloperoxidase and eosinophil cationic protein in MBC. In summary, we present deep proteomic, digitalized protein abundance profiles, generated from FFPE breast cancer tissues, that revealed significant changes in ECM and cellular proteins. Statement of Significance of the StudyThis study is significant as it discovered deep proteomic signatures for the highly aggressive and malignant metaplastic breast cancer (MCB) which is now considered a fifth subtype based upon its remarkable intra-tumoral heterogeneity that illustrates its unique cell plasticity. To efficiently analyze formalin-fixed paraffin-embedded (FFPE) breast tissues from patients with different breast cancer subtypes and disease-free individuals, we optimized a novel workflow in which we combined paraffinization and Folch extraction. We identified and confidently quantified [~]6,000 protein groups. We were able to find robust changes in extracellular matrix (ECM) with cancer, even though no ECM enrichments were performed. Interestingly, despite the relatively small human cohort size (42 patients), distinct protein signatures emerged throughout all cancer subtypes - common and unique - with remarkable statistical significance for many cancer-relevant proteins and pathways. This Pilot study indicates the hypothesis that an altered stroma can dictate epithelial tumor cell fate. We also observed that MBC was characterized by an especially immunosuppressed tumor environment. We do note the limitation of the relatively small cohort size of our study, and in the future additional patient cohorts will be needed to further validate our findings.

cancer biology↗

bioRxiv2025

IL-27 limits HSPC differentiation during infection and protects from stem cell exhaustion

Many inflammatory stimuli can induce progenitor cells in the bone marrow to produce increased numbers of myeloid cells as part of the process of emergency myelopoiesis. These events are associated with trained immunity and have long-term impacts on hematopoietic stem and progenitor cell (HSPC) development but can also compromise their function. While many cytokines support emergency myelopoiesis, less is known about the mechanisms that temper these events. When mice that lack the cytokine IL-27 were infected with Toxoplasma gondii, there was enhanced generation of monocyte progenitors and increased numbers of inflammatory monocytes. In the bone marrow of infected mice there was increased production of IL-27 that localized with HSPCs and a survey of cytokine receptor expression highlighted that HSPCs were uniquely poised to respond to IL-27. Furthermore, the use of in vitro differentiation assays and mixed bone marrow chimeras revealed that HSPCs from IL-27 deficient mice are pre-disposed towards the monocyte lineage. Additional studies highlighted that after infection loss of the IL-27R resulted in reduced HSPC fitness that manifested as reduced proliferative responses and a decreased ability to reconstitute the hematopoietic system. Thus, the ability of IL-27 to act on HSPC provides a regulatory brake on differentiation to limit monocyte induction and preserve HSPC stemness.

immunology↗

bioRxiv2025

Polysialylation is a general feature of immune activation

Sialic acids are critical regulators of immune responses and the sialic acid-Siglec axis has receive much attention recently for it role as a new immune checkpoint. While 2,3- and 2,6-linked sialosides have been well studied, much less is known about the 2,8-linked polysialic acid (polySia), especially in the adaptive immune system. We demonstrate here that polySia is actually found in all major classes of circulating immune cells, including B and T cells, where it is strongly associated with prior antigen exposure. Cell surface expression of polySia could be observed within 24 h of activation of naive T cells but did not correlate with expression of the immune-specific polysialyltransferase ST8Sia4. Finally, we assessed the effects of smoking on polySia levels, and noted substantial increased levels of polySia, particularly on effector T cells and preferentially in males. Significance StatementThe immune system is central to human health and holds the key to treating many chronic diseases. We show for the first time that the immunomodulatory glycan polysialic acid is widespread in the human immune system and associated with immune cell activation. Moreover, sex-dependent increases in polysialic acid could be observed on key populations of immune effector cells in smokers. This study offers insight into how glycans might contribute to sex differences in development and progression of chronic diseases.

immunology↗

bioRxiv2024

IL-27 Stablizes Myc-Mediated Transcription In Memory-Fated, Vaccine-Elicited CD8+ T Cells

Protection from pathogens relies on both humoral (antibody-mediated) and cellular (T cell-mediated) responses. While infections robustly elicit both types of immunity, currently approved vaccine adjuvants largely fail to induce T cell responses on par with that instigated by infections. Our goal was to investigate the transcriptional programming that supports the formation of CD8+ T cells elicited by subunit vaccines compared to those elicited by infections. Our data show that vaccine-elicited T cells represent a transcriptionally unique subset of activated T cells with high proliferative capacity and a memory cell fate. This relies on IL-27 signaling, which stabilizes c-Myc and thereby supports the biomass acquisition necessary for clonal expansion. Collectively, our findings reveal that subunit vaccine-elicited T cells uniquely combine aspects of both memory and effector T cell subsets, and selectively utilize IL-27 signaling to sustain the clonal expansion of cells dedicated to a memory fate. One Sentence SummaryIn contrast to infection, subunit vaccines induce a distinct population of CD8+ T cells with memory fate characteristics which maintain their proliferative capacity during the expansion phase through IL-27 receptor signaling.

immunology↗

bioRxiv2024

An IL-12 mRNA-LNP adjuvant enhances mRNA-LNP vaccine induced CD8+ T cell responses

The design of vaccines that induce CD8+ T cell responses has historically been a challenge, but the development of viral vectors or lipid nanoparticles (LNPs) to deliver mRNA that encode for target antigens provide more effective strategies to generate protective CD8+ T cell memory. Because interleukin-12 (IL-12) supports CD8+ T cell expansion and acquisition of effector functions, studies were performed to assess its contribution to the ability of an mRNA vaccine to promote CD8+ T cell responses. In vitro and in vivo, mRNA-LNPs did not stimulate myeloid cell production of IL-12, and the CD8+ T cell response to vaccination with the model antigen ovalbumin (OVA) was IL-12 independent. However, co-administration of IL-12 mRNA-LNPs with OVA mRNA-LNPs enhanced OVA-specific CD8+ T cell expansion, improved acquisition of effector function and resulted in an expanded memory CD8+ T cell pool. These heightened responses were associated with improved protective responses against Listeria monocytogenes-OVA and B16 FO-OVA melanoma. Thus, modification of mRNA vaccine formulations by inclusion of a cytokine mRNA provides a strategy to enhance CD8+ T cell mediated protection.

immunology↗

bioRxiv2024

Dextran sodium sulfate-induced colitis alters the proportion and composition of replicating gut bacteria

The bacteria living in the human gut are essential for host health. Though the composition and metabolism of these bacteria is well described in both healthy hosts and those with intestinal disease, less is known about the activity of the gut bacteria prior to, and during, disease development - especially regarding gut bacterial replication. Here, we use a recently developed single-cell technique alongside existing metagenomics-based tools to identify, track, and quantify the replicating gut bacteria and their replication dynamics in the dextran sodium sulfate mouse model of colitis. We show that the proportion of replicating gut bacteria decreases when mice have the highest levels of inflammation and returns to baseline levels as mice begin recovering. We additionally report significant alterations in the composition of the total replicating gut bacterial community during colitis development. On the taxa level, we observe significant changes in the abundance of taxa such as the mucus-degrading Akkermansia muciniphila and the poorly described Erysipelatoclostridium genus. We further demonstrate that many taxa exhibit variable replication rates during colitis, including A. muciniphila. Lastly, we show that colitis development is positively correlated with increases in the presence and abundance of bacteria predicted to be fast replicators, suggesting that taxa with the potential to replicate quickly may have an advantage during intestinal inflammation. These data support the need for additional research using activity-based approaches to further characterize the gut bacterial response to intestinal inflammation and its consequences for both the host and the gut microbial community at large. ImportanceIt is well known that the bacteria living inside the gut are important for human health. Indeed, the type of bacteria which are present and their metabolism is different in healthy people versus those with intestinal disease. However, less is known about how these gut bacteria are replicating, especially as someone begins to develop intestinal disease. This is especially important as it is thought that the active gut bacteria may be more relevant to health. Here, we begin addressing this gap by using several complementary approaches to characterize the replicating gut bacteria in a mouse model of intestinal inflammation. We reveal which gut bacteria are replicating, and how quickly, as mice develop and recover from inflammation. This work can serve as a model for future research to identify how the active gut bacteria may be impacting health, or why these particular bacteria tend to thrive during intestinal inflammation.

microbiology↗

bioRxiv2023

Mitotic deacetylase complex (MiDAC) recognizes the HIV-1 core promoter to control Tat-activated transcription and latency

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5 HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis in a model of latency. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control HIV latency, and as new drug targets for HIV cure strategies. Author summaryLatent HIV integrated within the host cell genome poses a major problem for viral eradication. The reactivation of latent HIV depends on host cell transcription factors that are hijacked by the 5 long terminal repeat (LTR) region of the HIV genome to produce viral RNA. At the heart of the LTR of HIV lies a particularly crucial DNA region named the core promoter that is specifically required for the reactivation of HIV by a viral protein named Tat. A significant body of work over more than 30 years has established the specific requirement for the HIV core promoter in Tats control of HIV latency, but the underlying molecular mechanisms have remained elusive. Here, we identify host cell transcription factors that bind selectively to the HIV core promoter to control HIV gene expression. Our data reveal three human proteins that act in a complex to reactivate latent HIV, including one that directly recognizes the HIV core promoter, one that regulates chromatin, and a third that binds to the HIV Tat protein. Our data fill a significant and long-standing gap in the understanding of latency and identify new potential drug targets for HIV cure strategies.

microbiology↗

bioRxiv2023

A quantitative survey of the blueberry (Vaccinium spp.) nectar microbiome: variation between cultivars, locations, and farm management approaches

Microbes in floral nectar can impact both their host plants and floral visitors, yet little is known about the nectar microbiome of most pollinator-dependent crops. In this study, we examined the abundance and composition of the fungi and bacteria inhabiting Vaccinium spp. nectar, as well as nectar volume and sugar concentrations, hypothesizing that nectar traits and microbial communities would vary between plants. We compared wild V. myrsinites with two field-grown V. corymbosum cultivars collected from two organic and two conventional farms. Differences in nectar traits and microbiomes were identified between V. corymbosum cultivars but not Vaccinium species. The microbiome of cultivated plants also varied greatly between farms, whereas management regime had only subtle effects, with higher fungal populations detected under organic management. Nectars were hexose-dominant, and sugars were depleted in nectar with higher cell densities. Bacteria were more common than fungi in blueberry nectar, although both were frequently detected and co-occurred more often than would be predicted by chance. Cosmopolitan blueberry nectar microbes that were isolated in all plants, including Rosenbergiella sp. and Symmetrospora symmetrica, were identified. This study provides the first systematic report of the blueberry nectar microbiome, which may have important implications for pollinator and crop health. One-sentence summaryParallel analysis of blueberry crops and a wild relative offers insight into the impacts of management and domestication on the nectar microbiome O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/556904v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@15f36caorg.highwire.dtl.DTLVardef@63496org.highwire.dtl.DTLVardef@1667a6eorg.highwire.dtl.DTLVardef@eff26e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles

Polysialic acid (polySia) is a long homopolymer consisting of 2,8-linked sialic acid with tightly regulated expression in humans. In healthy adults, it occurs on cell surface glycoproteins in neuronal, reproductive, and immune tissues; however, it is aberrantly present in many cancers and its overexpression correlates with significantly increased metastasis and poor prognosis. Prompted by the observation that the MCF-7 breast cancer cell line contains only intracellular polySia, we investigated the secretion of polySia from MCF-7 cells. PolySia was found predominantly on soluble proteins in MCF-7 conditioned media, but also on extracellular vesicles (EVs), secreted from the cells. Since MCF-7 cells do not express known polysialylated proteins, we developed a robust method for purifying polysialylated proteins that uses a metabolic labelling strategy to introduce a bioorthogonal functionality into polySia. Using this method we identified three previously unknown polysialylated proteins, and found that two of these proteins - AGR2 and QSOX2 - were secreted from MCF-7 cells. We confirmed that QSOX2 found in EV-depleted MCF-7 cell conditioned media was polysialylated. Herein we report the secretion of polysialic acid on both soluble and EV-associated proteins from MCF-7 cancer cells and introduce a new method to efficiently identify polysialylated proteins. These findings have exciting implications for understanding the roles of polySia in cancer progression and metastasis and for identifying new cancer biomarkers.

biochemistry↗

bioRxiv2022

Attenuation of polysialic acid biosynthesis in cells by the small molecule inhibitor 8-keto-sialic acid

Sialic acids are key mediators of cell function, particularly with regards to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here we describe a strategy to block the formation of 2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, can not be elongated. 8-Keto-sialic acid is non-toxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.

biochemistry↗