Biology subjects

Allen, J.

Publications and source records attributed to Allen, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Restoring and sustaining human postmortem retinal light responses with scalable methods for testing degenerative disease therapies

Neuro- and retinal degenerative diseases, including Alzheimers, age-related macular degeneration, stroke, and central retinal artery occlusion, rob millions of their independence. Studying these diseases in human retinas has been hindered by the immediate loss of neuronal activity postmortem. While recent studies restored limited activity in postmortem CNS tissues, synchronized neuronal transmission >30 minutes postmortem remained elusive. Our study overcomes this barrier by reviving and sustaining light signal transmission in human retinas recovered up to four hours and stored 48 hours postmortem. We also establish infrared-based ex vivo imaging for precise sampling, a closed perfusion system for drug testing, and an ex vivo ischemia-reperfusion model in mouse and human retina. This platform enables testing of neuroprotective and neurotoxic effects of drugs targeting oxidative stress and glutamate excitotoxicity. Our advances question the irreversibility of ischemic injury, support preclinical vision restoration studies, offer new insights into treating ischemic CNS injuries, and pave the way for transplantation of human donor eyes. TeaserReviving light signaling in postmortem human retinas challenges the irreversibility of ischemic injury and advances research to restore vision.

neuroscience↗

bioRxiv2024

Aerobic capacity and exercise mediate protection against hepatic steatosis via enhanced bile acid metabolism.

High cardiorespiratory fitness and exercise show evidence of altering bile acid (BA) metabolism and are known to protect or treat diet-induced hepatic steatosis, respectively. Here, we tested the hypothesis that high intrinsic aerobic capacity and exercise both increase hepatic BA synthesis measured by the incorporation of 2H2O. We also leveraged mice with inducible liver-specific deletion of Cyp7a1 (LCyp7a1KO), which encodes the rate-limiting enzyme for BA synthesis, to test if exercise-induced BA synthesis is critical for exercise to reduce hepatic steatosis. The synthesis of hepatic BA, cholesterol, and de novo lipogenesis was measured in rats bred for either high (HCR) vs. low (LCR) aerobic capacity consuming acute and chronic high-fat diets. HCR rats had increased synthesis of cholesterol and certain BA species in the liver compared to LCR rats. We also found that chronic exercise with voluntary wheel running (VWR) (4 weeks) increased newly synthesized BAs of specific species in male C57BL/6J mice compared to sedentary mice. Loss of Cyp7a1 resulted in fewer new BAs and increased liver triglycerides compared to controls after a 10-week high-fat diet. Additionally, exercise via VWR for 4 weeks effectively reduced hepatic triglycerides in the high-fat diet-fed control male and female mice as expected; however, exercise in LCyp7a1KO mice did not lower liver triglycerides in either sex. These results show that aerobic capacity and exercise increase hepatic BA metabolism, which may be critical for combatting hepatic steatosis. HighlightsO_LIRats with intrinsic high aerobic capacity have more significant reductions in de novo lipogenesis and increased cholesterol and bile acid synthesis on a high-fat diet compared to rats with low aerobic capacity. C_LIO_LIChronic exercise increases hepatic bile acid synthesis in mice. C_LIO_LILoss of Cyp7a1 blunts the capacity for exercise to increase bile acid synthesis and treat hepatic steatosis in male and female mice fed a high-fat diet. C_LI

physiology↗

bioRxiv2024

AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone

Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet(R) followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) [~] 9 M. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.

biochemistry↗

bioRxiv2024

PROPEL: a scalable model for postbaccalaureate training to promote diversity in the biomedical workforce

Promoting diversity in the scientific workforce is crucial for harnessing the potential of available talent and ensuring equitable access to Science, Technology, Engineering, Mathematics, and Medicine (STEM-M) careers. We have developed an innovative program called Post-baccalaureate Research Opportunity to Promote Equity in Learning (PROPEL) that provides scientific and career development training for postbaccalaureate scholars from historically excluded backgrounds in STEM-M fields with an interest in pursuing a PhD or MD/PhD degree. Our program is distinct from other postbaccalaureate programs in that scholars are hired by individual labs rather than funded centrally by the program. This funding mechanism removes the idea that central funding is necessary to encourage faculty to train diverse scholars and allows the program to scale dynamically according to the needs of the scientific community. The PROPEL program started in 2020 with six scholars and has since grown to an enrollment of over 100, making it the largest postbaccalaureate program for biomedical research in the country. Here, we describe the program structure and curriculum, our strategy for recruitment, the enrollment trends; the program demographics; metrics of scholar engagement; and outcomes for scholars who completed the program in 2023. Our experience demonstrates the strong demand from both scholars and faculty for programming of this type and describes the feasibility of implementation.

scientific communication and education↗

bioRxiv2024

Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective non-catechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and non-catechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, non-catechol agonists display G protein biased signaling at the D1R and have reduced or absent {beta}-arrestin recruitment. A notable exception was compound (Cmpd) 19, a non-catechol agonist with full efficacy at both D1R-G protein or D1R-{beta}-arrestin pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R-{beta}-arrestin activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The {beta}-arrestin super agonist, A-77636, showed significantly increased D1R endocytosis. Moreover, {beta}-arrestin recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential. Significance StatementThe D1R is a validated therapeutic target and the recently discovered non-catechol D1R agonists have translational potential. We have systematically characterized several structurally distinct D1R agonists including non-catechols with balanced or G protein biased activity. When examined for agonist-induced D1R endocytosis, balanced agonists induced robust D1R endocytosis while G protein biased agonists did not. These results indicate the degree of D1R signaling functional selectivity profoundly impacts receptor endocytosis. This work also independently validates agonist tools to further investigate D1R activation in basic and translational research.

pharmacology and toxicology↗

bioRxiv2024

Biophysical characterization of high-confidence, small human proteins

Significant efforts have been made to characterize the biophysical properties of proteins. Small proteins have received less attention because their annotation has historically been less reliable. However, recent improvements in sequencing, proteomics, and bioinformatics techniques have led to the high-confidence annotation of small open reading frames (smORFs) that encode for functional proteins, producing smORF-encoded proteins (SEPs). SEPs have been found to perform critical functions in several species, including humans. While significant efforts have been made to annotate SEPs, less attention has been given to the biophysical properties of these proteins. We characterized the distributions of predicted and curated biophysical properties, including sequence composition, structure, localization, function, and disease association of a conservative list of previously identified human SEPs. We found significant differences between SEPs and both larger proteins and control sets. Additionally, we provide an example of how our characterization of biophysical properties can contribute to distinguishing protein-coding smORFs from non-coding ones in otherwise ambiguous cases. Why it MattersThe identification of proteins smaller than 100 amino acids has lagged being that of larger proteins in part because their small size makes proteomic and genomic approaches more challenging. However, small proteins can perform significant functions. There are now enough proteins with high confidence of function to enable meaningful comparison with larger proteins, and to determine whether putative small proteins might be identified, based on their biophysical properties. As an example, we found that small proteins often contain transmembrane helices but are relatively devoid of beta sheets, which helped to support the annotation of a putative protein as functional. We also identified a wide range of biological functions and localization for small proteins, including many with disease associations.

biophysics↗

bioRxiv2024

All-trans retinoic acid induces durable tumor immunity in IDH-mutant gliomas by rescuing transcriptional repression of the CRBP1-retinoic acid axis

Diffuse gliomas are epigenetically dysregulated, immunologically cold, and fatal tumors characterized by mutations in isocitrate dehydrogenase (IDH). Although IDH mutations yield a uniquely immunosuppressive tumor microenvironment, the regulatory mechanisms that drive the immune landscape of IDH mutant (IDHm) gliomas remain unknown. Here, we reveal that transcriptional repression of retinoic acid (RA) pathway signaling impairs both innate and adaptive immune surveillance in IDHm glioma through epigenetic silencing of retinol binding protein 1 (RBP1) and induces a profound anti-inflammatory landscape marked by loss of inflammatory cell states and infiltration of suppressive myeloid phenotypes. Restorative retinoic acid therapy in murine glioma models promotes clonal CD4+ T cell expansion and induces tumor regression in IDHm, but not IDH wildtype (IDHwt), gliomas. Our findings provide a mechanistic rationale for RA immunotherapy in IDHm glioma and is the basis for an ongoing investigator-initiated, single-center clinical trial investigating all-trans retinoic acid (ATRA) in recurrent IDHm human subjects.

cancer biology↗

bioRxiv2024

Impact of Glycan Depletion, Glycan Debranching and Increased Glycan Charge on HIV-1 Neutralization Sensitivity and Immunogenicity

Modifying HIV-1s envelope glycoprotein glycans can impact its neutralization sensitivity. The use of the knock out cell line GnT1-prevents the elaboration of complex-type glycans, and opens up the glycan shield, increasing bNAb vulnerability. Some bNAb precursors can bind to GnT1-trimers, supporting their use in vaccine priming. However, GnT1-trimers express poorly and exhibit very low infectious counts. Here, we describe two other potentially vaccine-relevant glycoengineered trimers, 1) To truncate complex glycans, we used of a cocktail of glycosidases, termed "NGAF3" (Neuraminidase, {beta}-Galactosidase, N-Acetylglucosaminidase and endoglycosidase F3). Like GnT1-trimers, NGAF3 reduced glycan clashes and increased bNAb potency while retaining a closed trimer conformation; 2) Modified by {beta}-1,4-galactosyltransferase 1 (B4GalT1) and {beta}-galactoside -2,6 sialyltransferase 1 (ST6Gal1) during Env biosynthesis. Glycan mass spectrometry revealed that NGAF3 removed glycan heads of 3 of 7 positions on the trimer that are largely occupied by complex glycans. It also revealed a novel B4GalT1 activity to favor glycan precursor conversion to hybrid glycans rather than complex glycans. A comparison to monomeric gp120 revealed that B4GalT1s new activity depends on tight glycan spacing. B4GalT1 affected more glycans than NGAF3 (6 out of 7 glycans), perhaps due to greater accessibility during Env folding rather than after folding. Surprisingly, the N611 glycan was unaffected by either modification. B4GalT1 and ST6Gal1 cooperatively increased the abundance of hybrid glycans and -2,6 hypersialylated termini. This occurred largely by amplifying the abundance of a limited number of hybrid glycan structures that are also present on unmodified trimers. In rabbit vaccinations, B4GalT1+ST6GalT1-modified virus-like particles reduced the frequency and titers of serum NAbs that showed a modest preference for modified glycans. Conversely, chronically HIV-1-infected donor plasma neutralizing antibody titers were 1.7- to 10.8- fold higher against B4GalT1+ST6GalT1-modified pseudovirus. Overall, our data provide tools for heterologous prime, boost and polishing vaccine regimens using modified glycans. AUTHOR SUMMARYAn HIV-1 vaccine remains one of the most significant biomedical challenges today. Vaccines often work by triggering virus fighting antibodies to stave off infection. For HIV-1, this has been exceedingly difficult because the target, called the HIV-1 envelope glycoprotein (Env) is extremely variable and carries a thick sugar coat, protecting it from all but a few rare broadly reactive antibodies (termed bNAbs) that sometimes develop during natural HIV-1 infection. Given these challenges, it is reasonable to propose that any successful vaccine will need to be rationally designed to trigger the rare bNAbs. To meet this goal might require more 3 different vaccine components. First, a vaccine "prime" to trigger rare bNAb proliferation, followed by heterologous "boosts" and "polishing" immunogens to select for bNAbs. In this study, we evaluated two potential immunogens. In one approach, we used cocktails of enzymes to strip Envs sugar coat, in the hope of reducing the barrier to stimulate bNAb precursors. In another, we used excess enzymes to build particular structures on Envs coat. We checked how well these two Env variants were recognized by antibodies. We also checked in fine detail how the sugars were changed at the molecular level. Finally, we immunized rabbits. Our data enrich the number of strategies available to further explore the concept of priming, boosting and polishing vaccine shots.

immunology↗

bioRxiv2024

Ensemble automated approaches for producing high quality herbarium digital records

One of the slowest steps in digitizing natural history collections is converting labels associated with specimens into a digital data record usable for collections management and research. Recent work has shown a path for semi-automated approaches that can find labels, OCR them and convert the raw OCR text into digital data records. Here we address how raw OCR can be converted into a digital data record via extraction into standardized Darwin Core fields. We first showcase development of a rule-based approach and compare outcomes with a large language model-based approach, in particular ChatGPT4. We next quantified error rates in a set of OCRed labels, determining omission and commission errors for both approaches and documenting other issues. For example, we find that ChatGPT4 will often create field names that are not Darwin Core compliant. Our results suggest that these approaches each have different limitations. Therefore, we developed an ensemble approach that utilizes outputs from both in order to reduce problems from each individual method. An ensemble method reduces issues with field name heterogeneity and strongly reduces information extraction errors. This suggests that such an ensemble method is likely to have particular value for creating digital data records, even for complicated label content, given that longer labels, though more error prone, are still successfully extracted. While human validation is still much needed to ensure the best possible quality, we showcase working solutions to speed digitization of herbarium specimen labels that are likely usable more broadly for all natural history collection types.

bioinformatics↗

bioRxiv2024

Pre-existing Immunity to Influenza Aids Ferrets in Developing Stronger and Broader Vaccine-induced Antibody Responses

Influenza seasons occur annually, building immune history for individuals, but the influence of this history on subsequent influenza vaccine protection remains unclear. We extracted data from an animal trial to study its potential impact. The trial involved 80 ferrets, each receiving either one type of infection or a placebo before vaccination. We quantified the vaccine protection by evaluating hemagglutination inhibition (HAI) antibody titer responses. We tested whether hosts with different infection histories exhibited similar level of responses when receiving the same vaccine for all homologous and heterologous outcomes. We observed that different pre-existing immunities were generally beneficial to vaccine induced responses, but varied in magnitude. Without pre-immunity, post-vaccination HAI titers after the 1st dose of the vaccine were less likely to be above 1:40, and a booster shot was needed. Our study suggests that pre-existing immunity may strengthen and extend the homologous and heterologous vaccine protection.

immunology↗

bioRxiv2024

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody

SUMMARY/ABSTRACTThree coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

molecular biology↗

bioRxiv2023

Genome sequencing and molecular networking analysis of the wild fungus Anthostomella pinea reveal its ability to produce a diverse range of secondary metabolites

BackgroundFilamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level. ResultsIn this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti-Gram-positive effect of the extracts that we observed in antibacterial plate assays. ConclusionsTaken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites.

microbiology↗

bioRxiv2023

3D-intrusions transport active surface microbial assemblages to the dark ocean

Subtropical oceans contribute significantly to global primary production, but the fate of the picophytoplankton that dominate in these low nutrient regions is poorly understood. Working in the subtropical Mediterranean, we demonstrate that subduction of water at ocean fronts generates 3D intrusions with uncharacteristically high carbon, chlorophyll, and oxygen that extend below the sunlit photic-zone into the dark ocean. These contain "fresh" picophytoplankton assemblages that resemble the photic-zone regions where the water originated. Intrusions propagate depth-dependent seasonal variations in microbial assemblages into the ocean interior. Strikingly, the intrusions included dominant biomass contributions from non-photosynthetic bacteria and enrichment of enigmatic heterotrophic bacterial lineages. Thus, the intrusions not only deliver material that differs in composition and nutritional character from sinking detrital particles, but also drive shifts in bacterial community composition, organic matter processing, and interactions between surface and deep communities. Modeling efforts paired with global observations demonstrate that subduction can flux similar magnitudes of particulate organic carbon as sinking export, but is not accounted for in current export estimates and carbon cycle models. Intrusions formed by subduction are a particularly important mechanism for enhancing connectivity between surface and upper mesopelagic ecosystems in stratified subtropical ocean environments that are expanding due to the warming climate.

ecology↗

bioRxiv2023

Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization

Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1 envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is more often measured than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy by active or passive immunization and the therapeutic outcome of the latter. HIV-1 CZA97.012, a clone of a Clade C isolate, is neutralized to [~]100% by many NAbs. But here NAb PGT151, directed to a fusion-peptide epitope, was shown to leave a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, was less potent but more effective. We sought explanations of the different persistent fractions by depleting pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased; it decreased by the depleting NAb. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-{beta}19 transition in a defect of the glycan shield on CZA97.012 Env. Affinity-fractionated soluble native-like CZA97.012 trimer showed commensurate antigenic differences in analyses by ELISA and surface plasmon resonance. We then demonstrated glycan differences between PGT151- and PGT145-purified trimer fractions by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-[A] resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. PGT151-purified trimer showed a closed conformation, refuting apex opening as the cause of reduced PGT145 binding. The evidence suggests that differences in binding and neutralization after trimer purification or PV depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants confer antigenic heterogeneity through direct effects on antibody contacts, whereas others act allosterically. Author SummaryNeutralizing antibodies block the entry of HIV-1 into cells and protect against HIV-1 infection in animal models. Therefore, a goal of vaccination is to elicit antibodies that potently neutralize most HIV-1 variants. Such antibodies suppress virus levels when given to HIV-1-infected patients. Their potency is often measured as the concentration that gives 50% or 80% neutralization. But higher degrees of neutralization are needed to protect an organism from infection. And for some antibodies a ceiling is reached, so that even with increased concentrations a constant fraction of infectious virus persists. We studied the carbohydrate moieties on the envelope glycoprotein, which is the sole target for neutralizing antibodies, of one HIV-1 isolate of the most widespread subtype, Clade C, prevalent in Africa and Asia. We show how differences in carbohydrates can contribute to persistent infectivity, because distinct carbohydrates fit different antibodies. With a new three-dimensional structure of the entry-mediating protein from the Clade-C isolate, we illustrate that some carbohydrate differences occur exactly where the antibodies bind, whereas others are located elsewhere and can act indirectly. When we combined two neutralizing antibodies the persistent infectivity shrank. Our results reinforce the need for multiple specificities of neutralizing antibodies in prevention and therapy.

immunology↗

bioRxiv2023

The genome of Litomosoides sigmodontis illuminates the origins of Y chromosomes in filarial nematodes

Heteromorphic sex chromosomes are usually thought to have originated from a pair of autosomes that acquired a sex-determining locus and subsequently stopped recombining, leading to degeneration of the sex-limited chromosome. In contrast, in rhabditid nematodes, sex is determined by an X-chromosome counting mechanism and males are hemizygous for one or more X chromosomes (XX/X0). Some species of filarial nematodes, including important parasites of humans, have heteromorphic XX/XY systems. It has been assumed that sex is determined by a Y-linked locus in these species. However, karyotypic analyses suggested that filarial Y chromosomes are derived from the unfused autosome following an X-to-autosome fusion. Here, we generated a chromosome-level reference genome for Litomosoides sigmodontis, a filarial nematode with the ancestral filarial karyotype and sex determination mechanism (XX/X0). We mapped the assembled chromosomes to the rhabditid nematode ancestral linkage (or Nigon) elements. We found that the L. sigmodontis X chromosome was formed from a fusion of NigonX (the ancestrally X-linked element) and NigonD (ancestrally autosomal) that occurred in the last common ancestor of all filarial nematodes. In the two filarial lineages with XY systems, the X chromosomes were formed from two recent and independent fusions of the ancestral X chromosome with different autosomal Nigon elements. In both lineages, the region shared by the neo-X and neo-Y chromosomes is within the ancestrally autosomal portion of the X, confirming that the filarial Y chromosomes are derived from unfused autosomes. Sex determination in XY filarial nematodes therefore likely continues to operate via the ancestral X-chromosome counting mechanism, rather than via a neo-Y-linked sex-determining locus.

evolutionary biology↗

bioRxiv2023

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays.

molecular biology↗

bioRxiv2023

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations--including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of {beta}2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations. Brief summaryUnbiased drug screens identify targeted therapies as drugs that make lung cancers with driver mutations more vulnerable to macrophage attack.

cancer biology↗

bioRxiv2022

The complete sequence of a human Y chromosome

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure including long palindromes, tandem repeats, and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4, 5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029 base pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, revealing the complete ampliconic structures of TSPY, DAZ, and RBMY gene families; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a prior assembly of the CHM13 genome4 and mapped available population variation, clinical variants, and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

genomics↗