Biology subjects

Sharp, B.

Publications and source records attributed to Sharp, B..

11 recordsLinked to original sources

bioRxiv2025

Efficient and reliable measles reprogramming platform for the generation of human iPSC

We previously established the Measles virus (MeV) vector for reprogramming somatic cells into induced pluripotent stem cells (iPSCs); however, efficiency was limited to 0.2%. Here, we present the next generation that reprograms with an average efficiency of up to 2.3% and with similar or superior efficiency to the Sendai system. Twenty of the ninety iPSC isolated clones were amplified and analyzed. All clones showed a strong induction of endogenous pluripotency-associated markers SSEA-4, TRA-1-81, TRA-1-60, and NANOG. Analysis of the N mRNA transcript over passages showed rapid elimination of the vector from the iPSC at or before passage four. The pluripotency propensity was further analyzed using spontaneous and guided differentiation into three germ layers. All clones showed similar ability to differentiate into hematopoietic, pancreatic, and neuronal progenitor cells. In conclusion, this study shows that this new MeV reprogramming vector has a comparable or higher reprogramming efficiency than currently available systems and offers faster vector elimination from iPSCs. It uses a lower multiplicity of transduction as it uses a single vector versus multiple vectors. Finally, MeV produces iPSCs that can differentiate into multiple cell types. This makes MeV an efficient reprogramming platform for iPSC generation from patient samples.

cell biology↗

bioRxiv2025

Genome-Wide Annotation of Promoter-Enhancer Interactions via Chromatin Loops in the Hybrid Rat Diversity Panel

Despite the prominence of laboratory rats in behavioral neuroscience and complex trait genetics, a significant gap exists in the functional rat genomics database that explains the regulation of gene expression at the genome level. To address this, we analyzed genome-wide Hi-C data from the frontal cortex of ten strains in the Hybrid Rat Diversity Panel. While originally generated to improve the rat genome assembly, these data provided a unique opportunity to characterize the regulatory landscape of the adult rat brain. We identified an average of 5,899 {+/-} 1,997 (STD) loops per sample and integrated these with over 3 million curated CTCF binding sites, which serve as architectural anchors for chromatin loops. Our multi-stage filtering workflow identified 15,085 unique, high-confidence regulatory interactions throughout the genome. As a validation of our approach, we observed that genes with the highest loop counts, including Foxo1, Gja1, and Spry2, were significantly enriched for developmental processes, reflecting the role of 3D genome structure in maintaining adult neuronal identity. The resulting resource addresses a critical deficiency in rat functional genomics and provides a foundation for dissecting the genetic architecture of complex traits.

genomics↗

bioRxiv2025

Susceptibility of bovine respiratory and mammary epithelial cells to avian and mammalian derived clade 2.3.4.4b H5N1 highly pathogenic avian influenza viruses

Zoonotic transmission of avian influenza viruses into mammals is relatively rare due to anatomical differences in the respiratory tract between species. Recently, clade 2.3.4.4b highly pathogenic H5N1 avian influenza viruses were detected circulating in North American cattle. Sporadic transmission between cattle, humans, and other animals proximal to cattle or after consuming products from infected cattle has occurred, but thus far there is no evidence of human-to-human transmission. However, the virus has the potential to adapt to the mammalian respiratory tract with every transmission event that occurs, making it crucial to understand cellular and species tropism of the H5N1 2.3.4.4b viruses. We compared viral kinetics of clade 2.3.4.4b viruses isolated from birds and mammals in respiratory epithelial cells derived from cattle, human, swine, and ferret. We found that avian derived viruses could replicate in swine cells only, yet mammalian derived strains could replicate efficiently in all tracheal and nasal epithelial cells tested. Interestingly, only bovine mammary epithelial cells (MEC) and swine respiratory epithelial cells were permissive to both avian and mammalian derived strains, possibly due to increased sialic acid expression on bovine MEC compared to bovine tracheal epithelial cells (TEC). However, sialic acid expression differed between dairy and beef cows: TEC derived from a dairy cow had increased expression of 2,3 sialic acid receptors compared to TEC from a beef-dairy cow cross. This study highlights the ability of clade 2.3.4.4b H5N1 viruses derived from mammals but not wild birds to infect the respiratory epithelium of other mammalian hosts.

microbiology↗

bioRxiv2024

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity--either from LAIV vaccination and/or from Cal09 infection--showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

microbiology↗

bioRxiv2024

Effects of Oral Exposure to HPAI H5N1 Pasteurized in Milk on Immune Response and Mortality in Mice

In March 2024, there was the first reported outbreak of a highly pathogenic avian H5N1 influenza (HPAI) clade 2.3.4.4b virus in dairy cows in the United States. Since then, there have been several spillover events to cats, poultry, and humans. Multiple reports have discovered infectious virus in raw milk from infected dairy cows. Infectious virus can also last over a period on milking machine surfaces as a potential route of spread in cattle and contamination in raw milk. While the U.S. Food and Drug Administration has cleared commercial pasteurized milk as safe for consumption given the lack of infectious virus, there have been numerous reports that up to 30 percent of commercial milk tested were positive for HPAI H5N1 influenza virus genome copies. This is not necessarily unique to the HPAI H5N1 virus, as retrospective studies have identified H1N1 and H3N2 seropositivity in cows linked to decreased milk production. However, it is unknown how repeat exposure to the remaining viral proteins and genomic material in pasteurized milk modulates immune responses once ingested. We developed a successful in-house pasteurization protocol that inactivated high viral loads of the pandemic H1N1 strain A/California/04/2009 (Cal09) or bovine-derived HPAI H5N1 (A/bovine/Ohio.B24OSU-439/2024) viruses in raw milk. Mice were administered this milk daily for five days and rechallenged with each respective virus. We found that repeated oral exposure to inactivated virus was not sufficient to prevent or accelerate mortality from lethal challenge of HPAI H5N1, though it did result in a [~]0.5 log10 reduction viral titers in the brain and delayed clinical signs. In contrast, oral gavage of mice with pre-existing immunity to H1N1 influenza virus with virus pasteurized in milk were protected from morbidity and mortality upon bovine H5N1 viral challenge. These findings suggest that ingestion of inactivated HPAI H5N1 has limited potential health risk and does not prevent protective immune history-mediated responses to lethal infection.

microbiology↗

bioRxiv2023

Maternal immunization with distinct influenza vaccine platforms elicits unique antibody profiles that impact the protection of offspring

Pregnant women and infants are considered high-risk groups for increased influenza disease severity. While influenza virus vaccines are recommended during pregnancy, infants cannot be vaccinated until at least six months of age. Passive transfer of maternal antibodies (matAbs) becomes vital for the infants protection. Here, we employed an ultrasound-based timed-pregnancy murine model and examined matAb responses to distinct influenza vaccine platforms and influenza A virus (IAV) infection in dams and their offspring. We demonstrate vaccinating dams with a live-attenuated influenza virus (LAIV) vaccine or recombinant hemagglutinin (rHA) proteins administered with adjuvant resulted in enhanced and long-lasting immunity and protection from influenza in offspring. In contrast, a trivalent split-inactivated vaccine (TIV) afforded limited protection in our model. By cross-fostering pups, we show the timing of antibody transfer from vaccinated dams to their offspring (prenatal versus postnatal) can shape the antibody profile depending on the vaccine platform. Our studies provide information on how distinct influenza vaccines lead to immunogenicity and efficacy during pregnancy, impact the protection of their offspring, and detail roles for IgG1 and IgG2c in the development of vaccine administration during pregnancy that stimulate and measure expression of both antibody subclasses.

microbiology↗

bioRxiv2023

The beta 6 Integrin Negatively Regulates TLR7-Mediated Epithelial Immunity via Autophagy During Influenza A Virus Infection

Integrins are essential surface receptors that sense extracellular changes to initiate various intracellular signaling cascades. The rapid activation of the epithelial-intrinsic {beta}6 integrin during influenza A virus (IAV) infection has been linked to innate immune impairments. Yet, how {beta}6 regulates epithelial immunity remains undefined. Here, we identify the role of {beta}6 in mediating the Toll-like receptor 7 (TLR7) through the regulation of intracellular trafficking. We demonstrate that deletion of the {beta}6 integrin in lung epithelial cells significantly enhances the TLR7-mediated activation of the type I interferon (IFN) response during homeostasis and respiratory infection. IAV-induced {beta}6 facilitates TLR7 trafficking to lysosome-associated membrane protein (LAMP2a) components, leading to a reduction in endosomal compartments and associated TLR7 signaling. Our findings reveal an unappreciated role of {beta}6-induced autophagy in influencing epithelial immune responses during influenza virus infection.

immunology↗

bioRxiv2023

Detection and Phylogenetic Analysis of Highly Pathogenic A/H5N1 Avian Influenza Clade 2.3.4.4b Virus in Chile, 2022

Since the emergence of the Highly Pathogenic Avian Influenza H5N1 goose/Guangdong (Gs/GD) lineage in China in 1996, these viruses have spread globally. This includes the recent introduction of the Gs/GD lineage 2.3.4.4b in the Americas in 2021, which has affected both wild and domestic bird populations causing high mortality. Since November 2022, reports of mortality in wild birds in numerous South American countries along the Pacific Migratory Flyway have been attributed to this lineage. Through an ongoing longitudinal avian influenza virus (AIV) surveillance, we determined that AIV prevalence remained below 1% in the Lluta river wetland from August through October 2022. However, there was a significant increase in prevalence to 2.6% and 11.7% in November and December respectively, coinciding with the arrival of migratory birds from the Northern Hemisphere. Of the AIV RT-qPCR positive environmental feces samples 7 were identified as A/H5. Sequencing of the COI gene demonstrated that the H5 positive samples were obtained from Peruvian pelican (n=1), Franklins gull (n=1), Gray gull (n=1), Elegant tern (n=2) and Black skimmer (n=2). Full genomes were obtained from 3 samples and the putative cleavage site composition of the HA was polybasic REKRRKR/GLF for all. Phylogenetic analysis of the samples revealed them to belong to the H5 2.3.4.4b clade, closely related to isolates obtained in Peru in late November. The emergence of H5 clade 2.3.4.4b in South America has an immediate impact on public health, animal production and wildlife, hence increased active surveillance is warranted.

genetics↗

bioRxiv2022

Improved metabolic syndrome and timing of weight loss is crucial for influenza vaccine-induced immunity in obese mice

Persons with obesity are at higher risk for developing severe complications upon influenza virus infection making vaccination a priority. Yet, studies demonstrate vaccine responses are less effective in obese hosts. In these studies, we examined how the timing of weight loss influenced influenza vaccine efficacy in male and female diet- induced obese mice. Here, we show weight loss post-vaccination is insufficient to rescue poor vaccine efficacy; however, weight loss occurring pre-vaccination successfully improves outcomes at viral challenge. Pre-vaccination weight loss improved vaccine immunogenicity and restored a functional recall response at challenge. Through tracking sera metabolic biomarkers, we propose the metabolic state at the time of vaccination is predictive of vaccine immunogenicity. Altogether, these findings highlight how timing of host-directed interventions is vital when seeking to improve influenza vaccine immunogenicity in obese hosts.

microbiology↗

bioRxiv2019

Astrovirus Replication Is Inhibited by Nitazoxanide In Vitro and In Vivo

Astroviruses (AstV) are a leading cause of diarrhea especially in the very young, the elderly, and immunocompromised populations. Despite their significant impact on public health, no drug therapies for astrovirus have been identified. In this study we fill this gap in knowledge and demonstrate that the FDA-approved broad-spectrum anti-infective drug nitazoxanide (NTZ) blocks astrovirus replication in vitro with a 50% effective concentration (EC50) of approximately 1.47M. It can be administered up to 8 hours post-infection and is effective against multiple human astrovirus serotypes including clinical isolates. Most importantly, NTZ reduces viral shed and clinical disease (diarrhea) in vivo, exhibiting its potential as a future clinical therapeutic.\n\nImportanceHuman astroviruses (HAstV) are thought to cause between 2 and 9% of acute, non-bacterial diarrhea cases in children worldwide. HAstV infection can be especially problematic in immunocompromised people and infants where the virus has been associated with necrotizing enterocolitis, severe and persistent diarrhea, as well as systemic and often fatal disease. Yet no antivirals have been identified to treat astrovirus infection. Our study provides the first evidence that nitazoxanide may be an effective therapeutic strategy against astrovirus disease.

microbiology↗

bioRxiv2019

Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics

Animals employ multiple and distributed neuronal networks with diverse learning rules and synaptic plasticity dynamics to record distinct temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso & Rubin 2016). Here we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NOs effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

neuroscience↗