Biology subjects

Harris, J.

Publications and source records attributed to Harris, J..

At least 19 recordsLinked to original sources

bioRxiv2026

A novel proviral mechanism for the ISG, Shiftless, during human coronavirus infection

As highly pathogenic human coronaviruses continue to emerge from zoonotic sources, understanding how host intrinsic immune responses control infection is critical for preventing severe disease. The interferon (IFN) pathway induces antiviral effectors, including the interferon-stimulated gene (ISG) Shiftless (SHFL), which restricts -1 programmed ribosomal frameshifting during SARS-CoV-2 infection. Here, we investigate a novel proviral mechanism for SHFL using loss-of-function and gain-of-function approaches. Despite its established antiviral activity, SHFL enhanced infection by SARS-CoV-2 and the seasonal coronavirus HCoV-229E by increasing cellular susceptibility to infection. SHFL further promoted coronavirus infection by reducing cellular sensitivity to IFN, potentially through modulation of JAK/STAT signaling. Finally, we demonstrate that SHFL-mediated enhancement of baseline viral susceptibility is partially dependent on the domain previously implicated in its antiviral inhibition of -1 programmed ribosomal frameshifting. In contrast, SHFL-mediated regulation of IFN signaling is independent of this domain. Together, these findings establish SHFL as a dual-function ISG with distinct proviral and antiviral activities, highlighting the complex roles of innate immune effectors during coronavirus infection.

microbiology↗

bioRxiv2026

Two decades of Novel Ecosystems: Literature analysis of topics, trends and connectivity

1. As human-driven climate change and other anthropogenic forces produce widespread impacts on biodiversity and ecosystem functioning, many ecosystems are shifting into unprecedented, novel states. The Novel Ecosystem Concept (NEC), formalized in 2006, was developed as an alternative framework for managing "novel ecosystems", human-altered systems that are challenging to restore to their historical state. In the years since, the NEC has been proposed as a broader lens for ecosystem management under ongoing global change. 2. We evaluated how the uptake and potential of the NEC have been realized over time using a bibliometric and linguistic toolkit. Using bibliometric data from the Web of Science, we clustered 40,914 applied ecology papers published between 2006 and 2025 into twenty research topics based on language use. We quantified the volume of NEC research within each topic, rates of publication over time, and cross-topic connectivity proxied by citations. 3. We found NEC research in most of the topics of applied ecology, particularly invasion biology, ecosystem services, soil research and studies of freshwater, urban and marine systems. In contrast, several major conservation-focused topics, including protected areas, threatened species, and human-wildlife interactions, contained little research with an explicit link to novel ecosystems. Publication rates increased across all topics, although growth slowed over time. At the same time, NEC research became more focused into ecosystem-specific topics, especially marine ecosystems. NEC research across different topics was well-connected by citations, with papers more likely to cite each other when they used similar language. 4. Overall, the NEC does not appear to have expanded beyond its origins in restoration to become a broadly applied framework across conservation and ecosystem management. Instead, the concepts behind the NEC have been integrated into restoration and management frameworks grappling with climate and other anthropogenic change, while "novelty" is emerging as a powerful quantitative variable for measuring ecological change over space and time. Our results highlight the potential value of bibliometric and linguistic tools to deconstruct the ecological literature to inform what we can, or should, do to intervene for benefit of nature and people.

ecology↗

bioRxiv2026

Chromatin compartments define distinct modes of action and biological roles of the pioneer Pax7

Pioneer transcription factors direct cell differentiation by remodeling closed chromatin to deploy new enhancers. It remains unclear how the chromatin context shapes their action. During pituitary development, the pioneer PAX7 opens thousands of enhancers and specifies the melanotrope cell fate. We report that PAX7 acts in both the A (active) and B (inactive) genomic compartments, engaging two distinct closed states: H1-enriched chromatin in A, and H3K9me2- and lamin-rich chromatin in B. Whereas A-compartment enhancers open in a single, cell-division-independent step, B-compartment opening requires cell division and triggers domain-wide lamin dissociation coupled with B-to-A compartment shift. The two modes involve distinct biological outputs: A-compartment action modulates broadly expressed genes, whereas B-compartment action drives de novo expression of cell-type-specific developmental genes central to melanotrope identity.

genomics↗

bioRxiv2026

Spatial and Molecular Progression of Neural Progenitor Cells in the Developing Human Dentate Gyrus

The large diversity of neuronal and glial cell types in the human brain is underpinned by foundational cell populations known as neural progenitor cells (NPCs). The dentate gyrus (DG) of the hippocampus, a key structure in learning and memory, maintains a tightly organized NPC population into adulthood across many mammalian species. However, the emergence, organization and persistence of NPCs in the human hippocampus remain poorly characterized. Reports of NPCs in the juvenile, adult, and aged periods have been variable, reflecting differences in identification criteria and highlighting the need for a unified framework across development. In this study, we provide a spatial and molecular map of the developmental trajectory of NPCs in the human DG, combining multimodal transcriptomic analysis within a neuroanatomical context. At mid-gestation, we observed changes in the structural and cellular arrangement of the hippocampus, coinciding with the emergence of a multicellular NPC layer within the DG, herein named the granular-hilar progenitor zone (GHPZ). Neurogenic transcriptomic signatures in the GHPZ were diminished by early infancy, coinciding with a reduction in NPC number as they progressed toward an astrocytic program. At childhood, the GHPZ dissolved with only sparse radial NPCs remaining in the DG. Lastly, we validated WNT signaling pathway-associated genes as NPC identity markers in the developing human DG, observing a decline in their expression after infancy. Our study defines the steep decline of NPCs from gestation to the postnatal period, identifies their progression to an astrocytic nature, and sets the molecular blueprint for NPC identification in the human DG. HighlightsO_LIMultimodal mapping of neural progenitor cells from gestational to postnatal stages in the human hippocampus C_LIO_LIFormation of the granular-hilar progenitor zone within the dentate gyrus at mid-gestation C_LIO_LINeurogenic potential declines sharply from the prenatal period to childhood, with radial glia cells progressively acquiring astrocytic features C_LIO_LIDevelopmental modulation of the WNT signaling pathway accompanies radial glia cell transitions C_LI

neuroscience↗

bioRxiv2026

Microbial Influenced Corrosion: A Novel Initiator of In Vivo Spine Rod Fracture

ABSTRACTSpine rod catastrophic failure/rod fracture after implantation is estimated to occur in [~]10% of all cases worldwide with this rate remaining consistent for more than 20 years. To accommodate the spine mechanical environment, metal alloys such as austenitic stainless steels, cobalt-chromium-molybdenum alloys, and /{beta} type titanium alloys such are used as they have a high resistance to fatigue failure. The work presented herein addresses the gap in understanding about what happens in a laboratory environment versus what is happening in human patients that results in metal leaching into tissues as well as a shortened lifespan of a spine rod in a patient population that is not capable of breaking these spine rods from purely mechanical means. Eighty-five (N=85) patients (51 female - 62.9 {+/-} 13.5 years old, 34 male - 62.7 {+/-} 10.9 years old) who had spine revision surgery due to patient mechanical issues, such as loss of sagittal or coronal balance, pain, pedicle screw loosening, hardware associated infection were included in this study. All explanted rods had optical indications of surface modification that were not the result of mechanical damage, e.g. gouges, scratches, notches. The presence of Ti6Al4V rods increases the presence of local tissue concentrations of elemental Ti (190.98 {+/-} 207.84 g/g (ppm)) in all patients over the amount that would normally be present in patients who never had any titanium based orthopedic device implants (spinous muscle, 13.12 {+/-} 11.43 g/g). PQS, HHQ, and NHQ ToF-SIMS molecular signals were found co-located to corrosion pits as well with AHLs and palmitic acids indicating microbial presence and metabolism in all patients. This suggests that MIC can cause in vivo pitting corrosion resulting on rod failure.

bioengineering↗

bioRxiv2026

Genomic imprinting of the metabolic regulator gene Klf14 is regulated by a paternal sub-TAD anchored at Mest and a shared enhancer in mice.

KLF14 acts as a master regulator of gene expression in adipose tissue and variants at the human KLF14 gene show strong and reproducible association with type 2 diabetes and metabolic syndrome. Risk alleles are only pathologic when maternally inherited, consistent with the observation that KLF14/Klf14 is a maternally-expressed imprinted gene in human and mouse. However, how genomic imprinting is regulated at this important locus is currently unknown. In both species, KLF14/Klf14 is located [~]200 kb away from the paternally imprinted gene MEST/Mest, which is regulated by a maternal gametic differentially methylated region (gDMR) at its CpG island (CGI) promoter. Although the Klf14 CGI is kept unmethylated in most tissues, maternally-inherited DNA methylation (DNAme) marks are paradoxically required for Klf14 expression in mouse. Here, we show that Mest and Klf14 reside within the same topologically associating domain (TAD) in both mouse embryonic stem cells (ESCs) and differentiated cells, defined by sites of biallelic CTCF binding at the boundaries. Using allele-specific 4C-seq in F1 hybrid ESCs, we show that CTCF binding specifically to the unmethylated Mest gDMR generates a paternal allele-specific sub-TAD encompassing Klf14. We further show through CRISPR-Cas9-mediated mutagenesis, that deletions of the paternal Mest promoter region in ESCs as well as in vivo in mutant mice, results in both loss of Mest expression and acquisition of biallelic expression at Klf14. By analyzing epigenetic marks and chromatin looping in the Klf14-expressing pituitary cell line AtT-20, we identify a putative enhancer element shared by Mest and Klf14, providing a mechanistic model for the regulation of Klf14 imprinting. This work defines a new role for the Mest maternally methylated gDMR, revealing that it exerts long-range effects via allele-specific modulation of TAD structures and consequently act as an imprinting control region (ICR) in the regulation of Klf14 imprinting. Conservation of this TAD organisation at the human locus suggests that a similar regulatory mechanism operates at KLF14.

developmental biology↗

bioRxiv2026

Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex.

Astrocytes are highly abundant in the mammalian brain and coordinate with neurons and other glial cells to regulate neural circuit structure, function, and blood brain barrier integrity among many to maintain proper brain homeostasis. Astrocytes perform most of these functions owing to their highly complex morphologies and hundreds of thousands of fine processes that are important in contacting neuronal synapses and other glial cells. In fact, the morphological complexity of astrocytes is regulated by the presence and activity of neurons and helps establish astrocyte territory/tiling in a non-overlapping pattern; however, the mechanisms of astrocyte tiling are not well characterized. Using a human astrocyte-mouse neuron coculture system, we previously showed that sphingosine-1-phosphate receptor 1 (S1PR1) regulates astrocyte morphogenesis in a neuronal contact dependent manner. In this study, we find that S1PR1, in vivo, regulates astrocyte morphogenesis in a cortical layer specific manner. Using astrocyte-specific S1PR1 knock out mouse models and adenoassociated viral labeling methods, we show that S1PR1 is crucial in establishing competition driven astrocyte tiling and morphogenesis in the developing brain. Furthermore, we show that JAK-STAT3 signaling regulates neuronal contact induced expression of S1PR1 in cocultured astrocytes. These studies therefore uncover a lipid signaling receptor as a major regulator of astrocyte morphogenesis and tiling in murine cortical layers.

neuroscience↗

bioRxiv2026

Directional variation and method-specific detection patterns in offshore bat migration: implications for wind farm mitigation

O_LICurtailment of wind farms effectively reduces collision mortality in bats. Implementing this measure in offshore wind farms requires knowledge on the spatiotemporal occurrence and environmental predictors of migration over sea. In bats, such information can be obtained through acoustic monitoring and individual tracking. However, these techniques provide seemingly contradictory insights into migration patterns. C_LIO_LIWe used a Bayesian capture-recapture state-space model to investigate how environmental predictors influence spring departure decisions of Nathusius pipistrelle Pipistrellus nathusii migrating over the North Sea. The model was applied to both acoustic and tracking data, enabling comparable analyses across methods and incorporating uncertainty in migration dates of tracked bats. Additionally, we examined nightly offshore bat occurrence to further explore differences in movement patterns detected by the two techniques. C_LIO_LIWind conditions at 200 m above sea level were identified as key driver of Nathusius pipistrelle spring migration. In May-June, most bats migrated from the United Kingdom under westerly and northwesterly tailwinds. Tracked individuals flew in stronger supportive winds than acoustically recorded bats, which were also detected under crosswinds and headwinds. In March-April, acoustic detections occurred mainly during strong southerly winds, suggesting that early-season migrants largely consisted of individuals migrating over the European mainland and drifted northwards onto the North Sea by strong crosswinds. C_LIO_LIAcoustic detectors primarily recorded bats that landed on offshore platforms, likely because they were unable to cross the North Sea in a single flight due to less favorable wind conditions, or because they departed from more inland locations. In contrast, tracking data mainly represented bats that successfully crossed the North Sea in a non-stop flight under moderate supportive tailwinds. C_LIO_LISynthesis and applications: Combining observation techniques improves our understanding of bat migration patterns. Additionally, acoustic monitoring can capture migration from different geographic origins. Current mitigation measures for offshore wind farms at the North Sea rely solely on acoustic data, likely overlooking the part of the population that crosses over sea with optimal wind support. Acoustic and tracking data are therefore complementary rather than contradictory, and both methods should be used together when developing mitigation measures. C_LI

ecology↗

bioRxiv2026

Gene-drug interactions identify genomic loci that enhance statin effectiveness in lowering LDL cholesterol.

Hyperlipidemia, and high low-density lipoprotein cholesterol (LDL-c) in particular, is a risk factor for cardiovascular disease, including atherosclerosis, myocardial infarction, and stroke. Nearly 200 million people worldwide take HMG-CoA reductase inhibitors, commonly known as statins, to lower their LDL-c. If statins interfere with the genetic pathways that endogenously increase the risk for hyperlipidemia, gene-statin interactions may identify genomic variants, and thereby individuals with those genotypes, that are particularly sensitive to these medications. We performed a series of genome-wide gene-statin interaction analyses in the UK Biobank for LDL-c and two related lipids: high-density lipoprotein cholesterol (HDL-c) and triglycerides (TG). We identified five genome-wide significant gene-statin interactions for LDL-c, two interactions for HDL-c, and four interactions for TG. Importantly, only the SNP-based heritability of LDL-c was reduced by statin use. Using data from All of Us, we replicated all five significant gene-statin interaction loci for LDL-c in the European-like ancestry sample, two loci in the Americas-like ancestry sample, and one locus in the African-like ancestry sample. We also identified fifteen loci that remained associated with LDL-c despite statin treatment, highlighting potential additional genetic targets for drug development, enhancement, and repurposing. These loci include gene-targets for the recently developed hyperlipidemia drug class (PCSK9 inhibitors) validating our approach to finding new treatments. These results are an important step towards personalized medicine for patients with hyperlipidemia. Author SummaryHigh cholesterol raises the risk of heart attacks and strokes and nearly 200 million people worldwide take statins to lower it. While statins work for nearly everyone, they work better for some people than others. We examined how genetic differences enhance the effectiveness of statin medication as a step toward enhancing personalized medicine for those with high cholesterol. By analyzing genetic and health data from about 390,000 people, we found that statins primarily disrupt the link between genes and LDL or "bad" cholesterol levels. Across the genome, statins reduce the impact of genes on LDL-c levels, but not other blood lipids like HDL-c or triglycerides. For LDL-c specifically, we found five regions where genetic differences increase the effectiveness of statins. People with the protective genetic variants are expected to see greater cholesterol reduction with statin use. We confirmed four of the five findings in European-like ancestry samples, with partial replication in Americas-like and African-like ancestry samples (two variants and one variant, respectively). We also found 15 genomic regions where cholesterol stays high despite statin treatment. These genomic regions could be targets for new or enhanced cholesterol medications. In fact, a newer drug class targets one of these regions supporting our approach to finding new treatments.

genetics↗

bioRxiv2025

Rhizobia independently adapt to soil and legume host environments, but soil conditions influence the abundance of high-quality partners.

Rhizobia live as free-living microorganisms in the soil and in association with legume hosts. Both environments exert selective pressures on rhizobia, influencing the reproductive success of individual strains (e.g., fitness). The soil, a heterogeneous and fluctuating environment, is often overlooked, and little is known about whether selection in the soil influences the outcomes of the rhizobia-legume mutualism. We exposed a mixture of 68 Sinorhizobium meliloti strains to eight soil treatments (temperature, osmotic, and texture perturbations) and selection by two Medicago plant hosts. We found that cold (4{degrees}C) and warm (32{degrees}C) temperatures, as well as salt addition, had the strongest effects on diversity, community composition, or population size. Strain relative fitness was strongly positively correlated among soil treatments, except for cold. Genome-wide association analysis revealed a complex genetic architecture for soil fitness. In contrast, when comparing rhizobial fitness between soil and host environments, we found minimal strain fitness correlations, suggesting independent genetic bases and habitat-specific adaptations. Lastly, by examining the relationship between rhizobial fitness in the soil and their nitrogen-fixing plant benefits, we found that soil selection influenced the relative abundance of high- and low-quality strains; However, whether these effects were positive or negative for the plant was host dependent. Our results suggest that rhizobial evolution in soil and host are largely independent, but soil selection can alter mutualism benefits. IMPORTANCERhizobia-legume mutualism is crucial for introducing nitrogen into agricultural and natural ecosystems, and rhizobia persistence in the soil is an important component of agroecosystems. However, we know little about how individual strains of rhizobia persist and adapt to this environment, especially in the context of the soils spatial and temporal variations (temperature, moisture, and soil texture). We found that rhizobia similarly adapt to abiotic soil conditions but their reproductive success in the soil is independent from their reproductive success in the host. Intriguingly, we found that certain soil conditions increase (or decrease) the relative abundance of more effective nitrogen-fixing strains. Understanding how rhizobia adapt to diverse environments is crucial for developing effective bioinoculants that maintain high persistence in the soil while are also highly competitive to colonize the host and are beneficial to the plant.

ecology↗

bioRxiv2025

Cover crop microbiomes affect legume cash crop growth but not consistently through enriching nitrogen-fixing rhizobia

Harnessing plant-microbe interactions offers a promising path to reduce chemical inputs and enhance crop resilience in agricultural systems. However, microbial inoculants often fail to persist or function consistently across soils, which limits their broad utility. Here, we explore whether legume cover crops can be used to create microbial legacies that improve nodulation and nitrogen fixation in downstream legume cash crops. In a greenhouse experiment, we inoculated four cash crops with rhizosphere and nodule microbiomes derived from different legume cover crops, then used 16S rRNA and nifH amplicon sequencing to profile bacterial and diazotroph communities, respectively. Host identity shaped cover crop rhizosphere and nodule microbial communities, and specific taxa within these communities predicted nodulation and growth in some cash crops. Host specificity varied widely across cash crops, with alfalfa narrowly dependent on a specific symbiont and common bean and fava bean forming more permissive, taxonomically diverse nodule communities. Increased nodulation did not consistently improve biomass, and outcomes in some cash crops depended on more than symbiont compatibility alone. In particular, common bean growth was predicted by both rhizobial and non-rhizobial taxa, while soybean nodulation was shaped by its compatible symbiont as well as a mismatched rhizobial taxon associated with other hosts. Together, these results suggest that cover crops can shape cash crop microbiomes and productivity in host-specific ways, requiring precise symbiont matching in selective hosts but offering more flexible, multi-taxon management opportunities in permissive hosts.

plant biology↗

bioRxiv2025

Accurate determination of the preferred aggregation number of a micelle-encapsulated membrane protein dimer

The preferred aggregation number of dodeclyphoshocholine (DPC) micelles [Formula] encapsulating dimeric and higher order protein assemblies is difficult to determine via experimental techniques due to uncertainty in dimer geometry and heterogeneity in the conformational ensemble. Dimerization of the Amyloid Precursor Protein transmembrane domain (C99) is a particular step of importance in the production of amyloid-{beta} protein and the amyloid cascade. Molecular dynamics simulations of the C99 dimer and other transmembrane proteins have been performed to compliment micelle-phase protein structure studies. It has often been assumed that the value of [Formula] is the same as that of the pure, empty micelle. Here, we provide a convenient method for testing that assumption, while also accounting for the finite-size effects inherent in computer simulations of micelle self-assembly. Employing large, unbiased, coarsegrained molecular dynamics simulations of DPC and C99 dimer self-assembly, we determined the radius of gyration to be 21.6 {+/-} 2.0 [A] for the micelle-encapsulated dimer, and 16.0 {+/-} 1.0 [A] for the pure DPC micelle. Using these radii of gyration, we performed all-atom simulations of DPC-encapsulated C99 dimers with preferred aggregation numbers of 100 and 54 DPC to test the effect of using an expected versus a naive estimate of aggregation number on the structure of the transmembrane protein dimer. Through atomistic simulations, we determined that the transmembrane dimeric structure displays different characteristics depending on the aggregation number of the micelle, in addition to increased water penetration and micelle defects when the aggregation number is too small.

biophysics↗

bioRxiv2025

Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T cell therapies

Patients with T-cell lymphomas and leukemias have overall poor outcomes due to the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. Development of chimeric antigen receptor (CAR) T-cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective anti-tumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, using anti-CD2 CAR T-cells engineered without CD2 expression (CART2), would support CAR T-cell manufacturability and preclinical efficacy. Optimized CD2-knockout CART2, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T-cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, co-stimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel PD-1:CD2 switch receptor that rescues intracellular CD2 signaling, particularly when PD-L1 is engaged, resulting in improved in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T-cells and provides an effective strategy to overcome CD2-signaling deficiencies.

cancer biology↗

bioRxiv2025

Cationic antimicrobial copolymers reveal immunomodulatory properties in LPS stimulated macrophages in vitro

Antimicrobial polymers, which have emerged as a promising alternative to antibiotics in the fight against antimicrobial resistance, are based on the design of cationic host defence peptides (CHDPs). Being a part of the mammalian innate immune system, CHDPs possess both antimicrobial and immunoregulatory effects to manage bacterial infections. However, the immunomodulatory effects of antimicrobial polymers remain largely unexplored. Within this work, a library of 15 copolymers were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation and their abilities to modulate pro-inflammatory pathways in LPS-activated murine and human macrophages were investigated. We found that two diblock copolymers with cationic units copolymerised with either apolar or hydrophilic comonomers appeared to have anti-inflammatory activity through suppression of the activation of the NF-{kappa}B signalling pathway, scavenging of reactive oxygen species and reduced production of the pro-inflammatory cytokine IL-6. Furthermore, the cationic-apolar copolymer exhibits significant antimicrobial activity against P. aeruginosa. Thus, this promising copolymer holds potential as a dual-action therapeutic, effectively combating bacterial infections while curbing prolonged inflammation and thereby preventing sepsis at the site of infection.

microbiology↗

bioRxiv2024

Rubisco packaging and stoichiometric composition of a native β-carboxysome

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within {beta}-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the {beta}-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in {beta}-carboxysomes, which is instrumental for Rubisco encapsulation and {beta}-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire {beta}-carboxysome. This study and recent findings on the {beta}-carboxysome structure provide insights into the assembly principles and structural variation of {beta}-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

biochemistry↗

bioRxiv2024

Circular RNAs exhibit exceptional stability in the aging brain and serve as reliable age and experience indicators

Circular RNAs (circRNAs) comprise a large class of stable RNAs produced through backsplicing. While circRNAs have been shown to be very stable in cell culture, it is unknown how stable they are in vivo. Interestingly, studies across various animal systems demonstrated that circRNAs levels increase with age in neural tissue. However, the underlying reasons for this age-related accumulation are still unclear. To address these questions, we profiled circRNAs from fly heads at six timepoints across their lifespan. We found that circRNA levels increase linearly with age, independent of changes in mRNA levels, overall transcription, intron retention, or host genes splicing. This indicates that the age-related accumulation is attributed to their extraordinary stability in neural tissue rather than changes in biosynthesis. Furthermore, exposure to environmental stimuli like different temperatures, resulted in the increase but not decrease of circRNAs subsets, further confirming their stability in vivo. This exceptional stability implies that circRNAs can serve as markers of environmental experience. Indeed, flies subjected to a ten-day regimen at 29{degrees}C exhibit higher levels of specific brain circRNAs even six weeks after returning to standard conditions, indicating that circRNAs can reveal past environmental stimuli. Additionally, circRNA half-life measurements revealed values exceeding 20 days, with some showing no degradation over the animals lifetime. These findings demonstrate the extreme stability of circRNAs in vivo and their use as markers for aging, stress and life experiences.

neuroscience↗

bioRxiv2023

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5 stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bio-informatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIF expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen. Author SummaryOxygen levels influence HBV replication and hypoxia inducible factors (HIFs) activate HBV transcription. Long-read sequencing and mapping the HBV transcriptome showed an increased abundance of all viral RNAs under hypoxic conditions that was dependent on N6-methyladenosine modifications. Investigating the oxygen-dependent expression of RNA demethylases identified ALKBH5 as a hypoxic activated gene and silencing its expression showed a key role in regulating HBV and host gene expression under hypoxic conditions.

molecular biology↗

bioRxiv2023

Transmission bottleneck size estimation from de novo viral genetic variation

Sequencing of viral infections has become increasingly common over the last decade. Deep sequencing data in particular have proven useful in characterizing the roles that genetic drift and natural selection play in shaping within-host viral populations. They have also been used to estimate transmission bottleneck sizes from identified donor-recipient pairs. These bottleneck sizes quantify the number of viral particles that establish genetic lineages in the recipient host and are important to estimate due to their impact on viral evolution. Current approaches for estimating bottleneck sizes exclusively consider the subset of viral sites that are observed as polymorphic in the donor individual. However, allele frequencies can change dramatically over the course of an individuals infection, such that sites that are polymorphic in the donor at the time of transmission may not be polymorphic in the donor at the time of sampling and allele frequencies at donor-polymorphic sites may change dramatically over the course of a recipients infection. Because of this, transmission bottleneck sizes estimated using allele frequencies observed at a donors polymorphic sites may be considerable underestimates of true bottleneck sizes. Here, we present a new statistical approach for instead estimating bottleneck sizes using patterns of viral genetic variation that arose de novo within a recipient individual. Specifically, our approach makes use of the number of clonal viral variants observed in a transmission pair, defined as the number of viral sites that are monomorphic in both the donor and the recipient but carry different alleles. We first test our approach on a simulated dataset and then apply it to both influenza A virus sequence data and SARS-CoV-2 sequence data from identified transmission pairs. Our results confirm the existence of extremely tight transmission bottlenecks for these two respiratory viruses, using an approach that does not tend to underestimate transmission bottleneck sizes.

evolutionary biology↗