Biology subjects

Robinson, K.

Publications and source records attributed to Robinson, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Complex-trait dissection with Evolve-and-Resequence: Selection for improved flight performance in laboratory fruit fly populations

Evolve-and-Resequence studies combine experimental evolution with molecular sequencing, enabling associations between complex trait phenotypes and evolved genomic changes. For this, a targeted complex trait is subjected to artificial laboratory selection in genetically variable populations. Using populations of fruit flies Drosophila melanogaster from the DSPR background, we have conducted a short-term selection study on a highly complex trait : flight performance. We selected for improved flight performance using a custom-built flight tunnel system, with light as a motivator and airflow as a resistive factor. As a result, flight performance significantly improved in selected populations compared to controls. We also observed signatures of evolution in flight related traits, such as activity level and wing shape, and in life-history related traits such as adult dry mass and fecundity. We used whole genome pooled sequencing on the selected and control populations, revealing significantly differentiated genomic sites. Gene ontology and enrichment analyses revealed the evolution of pathways involved in response to stimuli, growth, and development. We identified several genes of interest (e.g., Sdc, dally). The range of evolved phenotypes and associated genomic changes show that flight performance is an ideal complex trait for future Evolve-and-Resequence studies.

evolutionary biology↗

bioRxiv2026

Conservation of transcriptional regulatory networks in zebrafish and human periderm facilitates identification of GRHL1 as an orofacial cleft risk gene

Most heritable risk for orofacial clefts (OFC) remains unassigned to specific genes or loci. IRF6 and GRHL3, two established OFC risk genes, encode transcription factors (TFs) essential for the differentiation of the periderm, a transient embryonic tissue required for secondary palate fusion. To identify novel risk candidates, we modeled the zebrafish periderm transcriptional regulatory network (TRN). Using single-cell multiome sequencing (RNA-seq and ATAC-seq) from shield-stage embryos, we inferred TF-to-target gene connections by integrating correlated gene expression with TF binding site predictions within chromatin elements open in periderm cells. We generated sets of gold-standard edges by conducting RNA-seq on TF-depleted embryos and ChIP-seq/CUT&RUN on wild-type embryos and used them to benchmark model performance. Within the top-performing model, zebrafish periderm modules are strongly preserved in human embryonic periderm and orthologs of human OFC-associated genes have higher centrality and edge-sum scores than non-associated genes. Functional validation confirmed the networks predictive power: depleting high-centrality TFs, including grhl1, klf6a, tead3b, and klf17, disrupted periderm differentiation in sensitized embryos. Moreover, analysis of whole-genome sequencing data from 2,415 OFC trios identified 15 individuals with rare or de novo GRHL1 variants, four of which introduced premature stop codons. This study establishes GRHL1 as a novel OFC risk gene and highlights the power of cross-species gene regulatory network analysis to prioritize candidates for rare variants in complex structural birth defects.

developmental biology↗

bioRxiv2025

Common Phenomenal and Neural Substrate Geometry in Visual Motion Perception.

What is a possible physical substrate of the qualitative aspects of consciousness (qualia)? Answering this question is a central goal of consciousness research. Due to their subjective and ineffable nature, finding a quantitative way to characterise qualia from verbal description has thus far proven elusive. To overcome the challenge of expressing subjective experience, recent structural and relational approaches have been proposed from mathematics. Yet, as far as we know, no attempts have been made to evaluate the relationship between a certain structure of qualia and the structure of a candidate underlying physical substrate. Towards this ambitious goal of linking the structures of qualia and physical, we set out to make an empirical first step by focusing on experienced dissimilarity of visual motion in human participants and stimulus-evoked neural population response geometry recorded from mouse primary visual cortex. From human participants (N=171), we obtained dissimilarity ratings of visual motion experiences induced by 48 stimuli, spanning across 8 directions and 6 spatial frequencies. Analysis revealed a human dissimilarity structure that was not well captured by a simple monotonic function of physical motion-direction difference alone nor by a pure orientation-symmetry account. From nine individual mice, we recorded single-neuron activity (n=751) with optical imaging in both awake and lightly anaesthetised conditions (isoflurane 0.6-0.8%). From neuron population responses to a similar set of motion stimuli, we computed a distance matrix that is comparable to our human dissimilarity matrix. Quantitative analyses show structural commonalities between a human dissimilarity structure and mouse neural structure, where a categorical organisation of stimulus direction best explained both. These commonalities were similar in awake and anaesthetised recordings, suggesting that this coarse V1 geometry is relatively insensitive to this type of anaesthesia; future work combining behaviour with causal intervention is required to relate such neural structures to conscious experience. Finally, we list several empirical factors that can be improved to promote our qualia structure approach in the future. Graphical AbstractSimilarity structures derived from a pairwise similarity rating task in humans revealed a mismatch between physical stimuli and subjective experience of similarity of visual motion: participants robustly rated their experience of opposite direction stimuli as similar. In mice, we used 2-photon Ca2+ imaging to record the activity of c.80 neurons per mouse in the primary visual cortex (V1) of nine mice. By computing a correlation matrix that records the similarity of responses to the same pairwise comparisons as in humans across all neurons, we can then represent neuronal distance. This revealed that mice neurons also exhibited the same dissociation between physical stimuli and responses as in humans at lower spatial frequencies. By comparing these dissimilarity and distance matrices using linear mixed effects modelling, we showed that a categorical model, not direction or orientation, best explains both human and mice data. Ultimately, this study is a methodological proof-of-concept for comparing structures of experience to candidate structures of a possible neural substrate in order to constrain their candidacy. O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/679834v2_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@768e3forg.highwire.dtl.DTLVardef@174e501org.highwire.dtl.DTLVardef@166cf75org.highwire.dtl.DTLVardef@be1a6e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIVisual motion feels more similar for opposite than diagonal directions C_LIO_LIPairwise similarity ratings reveal structure of visual motion experience C_LIO_LIHuman motion experience and mouse visual cortex share a common geometry C_LIO_LIMouse visual cortex codes motion similarly when awake and lightly anaesthetised C_LI

neuroscience↗

bioRxiv2025

Anesthesia Induces Shifts in Spatial Frequency Preference in the Primary Visual Cortex

This study examined the impact of general anesthesia on visual processing in the mouse primary visual cortex (V1). By directly comparing visual responses in the same layer 2/3 neurons between the awake and anesthetized states, we found that isoflurane anesthesia significantly shifts the preferred spatial frequency (SF) of neurons to lower values while leaving orientation tuning intact. This downward SF shift impaired the ability to encode fine visual details (high-SF) but not coarse information (low-SF). The shift was observed across all recorded cell types--including excitatory, somatostatin-expressing (SOM), and parvalbumin-expressing (PV) inhibitory neurons--though their respective magnitudes and underlying patterns were distinct. Furthermore, modulation of response gain and tuning sharpness was observed exclusively in SOM neurons. These results highlight the specific effects of anesthesia, which vary with visual feature and neuron type. These findings suggest that reduced sensory perception during anesthesia is linked to a degradation in spatial resolution at the cellular level.

neuroscience↗

bioRxiv2025

Methanotrophy Under Extreme Alkalinity in a Serpentinizing System

Serpentinization produces hyperalkaline, H2- and CH4-rich fluids that support microbial life in extreme conditions and serve as analogs for ocean worlds such as Enceladus. While methane production in these systems has been well studied, methane consumption--especially under high pH--remains poorly understood. Here, we present isotopic, geochemical, and genomic evidence for hyperalkaliphilic (pH > 11) methanotrophy in the Samail Ophiolite in Oman. Using models that account for fluid mixing and gas exsolution, we identify {delta}13CH4 enrichment that cannot be explained by abiotic processes alone. The enrichment of 13CH4 co-occurs with methanotroph 16S rRNA gene sequences, particularly in fluids formed by mixing CH4-rich, anoxic fluids with oxidant-rich surface waters. Shotgun metagenomics reveals a metagenome-assembled genome affiliated with Methylovulum, encoding a complete methane oxidation pathway, multiple carbon assimilation routes, and Na+/H+ antiporters--adaptations likely enabling growth above pH 11. Methanotroph diversity and abundance peak in mixed fluids but are suppressed at total ammonia nitrogen concentrations >20 M. Anaerobic methane-oxidizing archaea (ANME) may also contribute to CH4 oxidation in the deep subsurface. Our findings highlight the viability of methanotrophy under extreme alkaline conditions and provide a framework for interpreting {delta}13CH4 signals in serpentinizing environments on Earth and beyond.

microbiology↗

bioRxiv2025

Cell Cycle Arrest of a "Zippering" Epithelial Cell Cluster Shapes the Face and is Disrupted in Craniofacial Disorders

Facial features identify individuals, but the mechanisms shaping the human face remain elusive. Orofacial clefting (OFC), the most common craniofacial abnormality, results from failed fusion of the facial prominences that is in part caused by persistence of the cephalic epithelium. Here we uncover the identity, behaviors, and molecular blueprints of a novel craniofacial epithelial population, the Zippering Lambda (ZL), which mediates prominence fusion and is characterized by cell cycle arrest in mouse and human embryos. Remarkably, cell cycle is unleashed in the ZL of Pbx1/2 and p63 mutant mice with OFC. Intersection of ZL-enriched genes with human OFC whole-genome sequencing datasets identifies ZFHX3 variants in affected individuals and cephalic epithelial Zfhx3 deletion causes murine OFC. ZFHX3 and PBX1 genetically interact and synergistically regulate cell cycle inhibitor genes within a complex in embryonic faces. Collectively, we deconstruct new mechanisms that pattern the face, connecting cell cycle arrest to developmental tissue fusion.

developmental biology↗

bioRxiv2025

Precision in Spinal Cord Injury Research: A Novel Electromagnetic Impactor for a Consistent Porcine Model

PurposeReplicating spinal cord injury (SCI) in large animals is necessary for evaluating therapeutics for potential human translation, yet there is currently no commercial, standardized device for inducing SCI. We present the fabrication and testing of a custom impactor device for producing repeatable contusion SCI in porcine models. MethodsWe first designed and built the device. Mechanical modeling was subsequently utilized to calibrate our benchtop testing setup. Benchtop verification was performed to measure impact force post calibration. We then used the device to generate a contusion SCI model in 2 pigs and the results were compared to an uninjured pig. Intraoperative ultrasound was used to visualize a hematoma in the injured spinal cord. Hematoxylin-eosin (H&E) and Massons trichrome staining were used to confirm injury presence on ex vivo spinal cord samples. ResultsMechanical modeling forces matched benchtop impact forces within 1.4 N, indicating successful calibration of the testing setup. Our device demonstrated repeatability and the potential for modulating injury severity on the benchtop. Impactor forces were demonstrated across a range from 12.8 to 67.6 N, with variability remaining within 0.2 to 0.7 N standard deviation. The device induced two contusion injuries of different severity in vivo, confirmed by intraoperative ultrasound imaging and post-excision histology of the spinal cord. ConclusionOur impactor device is a major advancement towards producing repeatable and titratable contusions in large animal SCI models.

bioengineering↗

bioRxiv2025

Plakophilin-2 Coordinates Energy Metabolism and Contractility in Cardiomyocytes, Revealing Its Roles beyond Desmosomes

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Mutations in the desmosome gene Plakophilin-2, PKP2, lead to reduction in PKP2 protein and collapse of desmosomes that is known to compromise contractility and electrical stability of cardiomyocytes. Our previous studies demonstrated the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2 and revealed profound changes in mRNA signatures of metabolic enzymes that were reversed by the gene replacement approach. In this study, we used PKP2-deficient mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to identify changes in steady-state metabolite levels associated with impaired lipid homeostasis, glycolysis, and glucose oxidation. These metabolic phenotypes align with human ARVC metabolic data and reflect an intrinsic impairment of cellular energy metabolism. Here we showed for the first time that these intracellular metabolic defects were associated specifically with poor contractility of cardiomyocytes. AAV9:PKP2 restored contractility, improved electrophysiological properties and Ca2+ transients. In contrast, we observed that treating PKP2-deficient cardiomyocytes pharmacologically with small molecule metabolic enhancers improved contractility but not electrophysiological properties and Ca2+ transients, suggesting differential sensitivity of structure-mediated functions in response to metabolic perturbance. Our study modeled and revealed a direct intracellular connection between compromised PKP2 function and metabolic impairment. We proposed that an increased risk of decoupling energy-responsive contractility from less energy-responsive electrical activities can be a new arrhythmogenic mechanism, potentially responsible for exercise-triggered cardiac adversity in ARVC disease development and progression.

cell biology↗

bioRxiv2025

Gene expression patterns of the developing human face at single cell resolution reveal cell type contributions to normal facial variation and disease risk

Craniofacial development gives rise to the complex structures of the face and involves the interplay of diverse cell types. Despite its importance, our understanding of human-specific craniofacial developmental mechanisms and their genetic underpinnings remains limited. Here, we present a comprehensive single-nucleus RNA sequencing (snRNA-seq) atlas of human craniofacial development from craniofacial tissues of 24 embryos that span six key time points during the embryonic period (4-8 post-conception weeks). This resource resolves the transcriptional dynamics of seven major cell types and uncovers distinct major cell types, including muscle progenitors and cranial neural crest cells (CNCCs), as well as dozens of subtypes of ectoderm and mesenchyme. Comparative analyses reveal substantial conservation of major cell types, alongside human biased differences in gene expression programs. CNCCs, which play a crucial role in craniofacial morphogenesis, exhibit the lowest marker gene conservation, underscoring their evolutionary plasticity. Spatial transcriptomics further localizes cell populations, providing a detailed view of their developmental roles and anatomical context. We also link these developmental processes to genetic variation, identifying cell type-specific enrichments for common variants associated with facial morphology and rare variants linked to orofacial clefts. Intriguingly, Neanderthal-introgressed sequences are enriched near genes with biased expression in cartilage and specialized ectodermal subtypes, suggesting their contribution to modern human craniofacial features. This atlas offers unprecedented insights into the cellular and genetic mechanisms shaping the human face, highlighting conserved and distinctly human aspects of craniofacial biology. Our findings illuminate the developmental origins of craniofacial disorders, the genetic basis of facial variation, and the evolutionary legacy of ancient hominins. This work provides a foundational resource for exploring craniofacial biology, with implications for developmental genetics, evolutionary biology, and clinical research into congenital anomalies.

developmental biology↗

bioRxiv2024

Conserved effectors underpin the virulence of liverwort-isolated Pseudomonas in divergent plants

Plant pathogenic Pseudomonas species naturally antagonize a diverse range of flowering plants. While emerging research demonstrates that isolates belonging to the P. syringae species complex colonize diverse hosts, the extent to which these bacteria naturally infect non-flowering plants like the model liverwort Marchantia polymorpha remains unclear. Here, we identify natural associations between Pseudomonas viridiflava and the liverwort Marchantia polymorpha. Pseudomonas bacteria isolated from diseased liverworts in the wild successfully re-infected M. polymorpha in pure culture conditions, producing high in planta bacterial densities and causing prominent tissue maceration. Comparative genomic analysis of Marchantia-associated P. viridiflava identified core virulence machinery like the type-III secretion system (T3SS) and conserved effectors (AvrE and HopM1) that were essential for liverwort infection. Disease assays performed in Nicotiana benthamiana further confirmed that liverwort-associated P. viridiflava infect flowering plants in an effector-dependent manner. Our work highlights P. viridiflava as an effective broad host pathogen that relies on conserved virulence factors to manipulate evolutionarily divergent host plants.

plant biology↗

bioRxiv2024

Precise Lineage Tracking Using Molecular Barcodes Demonstrates Fitness Trade-offs for Ivermectin Resistance in Nematodes

A fundamental tenet of evolutionary genetics is that the direction and strength of selection on individual loci varies with the environment. Barcoded evolutionary lineage tracking is a powerful approach for high-throughput measurement of selection within experimental evolution that to date has largely been restricted to studies within microbial systems, largely because the random integration of barcodes within animals is limited by physical and molecular protection of the germline. Here, we use the recently developed TARDIS barcoding system in Caenorhabditis elegans (Stevenson et al., 2023) to implement the first randomly inserted genomic-barcode experimental evolution animal model and use this system to precisely measure the influence of the concentration of the anthelmintic compound ivermectin on the strength of selection on an ivermectin resistance cassette. The combination of the trio of knockouts in neuronally expressed GluCl channels, avr-14, avr-15, and glc-1, has been previously demonstrated to provide resistance to ivermectin at high concentrations. Varying the concentration of ivermectin in liquid culture allows the strength of selection on these genes to be precisely controlled within populations of millions of individuals, yielding the largest animal experimental evolution study to date. The frequency of each barcode was determined at multiple time points via sequencing at deep coverage and then used to estimate the fitness of the individual lineages in the population. The mutations display a high cost to resistance at low concentrations, rapidly losing out to wildtype genotypes, but the balance tips in their favor when the ivermectin concentration exceeds 2nM. This trade-off in resistance is likely generated by a hindered rate of development in resistant individuals. Our results demonstrate that C. elegans can be used to generate high precision estimates of fitness using a high-throughput barcoding approach to yield novel insights into evolutionarily and economically important traits.

evolutionary biology↗

bioRxiv2023

In vivo PSC differentiation as a platform to identify factors for improving the engraftability of cultured muscle stem cells

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

cell biology↗

bioRxiv2023

'Drifting' Buchnera genomes track the microevolutionary trajectories of their aphid hosts

Evolution of Buchnera-aphid host symbioses is often studied among species at macroevolutionary scales. Investigations within species offer a different perspective about how eco-evolutionary processes shape patterns of genetic variation at microevolutionary scales. Our study leverages new and publicly available whole-genome sequencing data to study Buchnera-aphid host evolution in Myzus persicae, the peach potato aphid, a globally invasive and polyphagous pest. Across 43 different asexual, clonally reproducing isofemale strains, we examined patterns of genomic covariation between Buchnera and their aphid host and considered the distribution of mutations in protein-coding regions of the Buchnera genome. We found Buchnera polymorphisms within aphid strains, suggesting the presence of genetically different Buchnera strains within the same clonal lineage. Genetic distance between pairs of Buchnera samples was positively correlated to genetic distance between their aphid hosts, indicating shared evolutionary histories. However, there was no segregation in the genetic variation of both M. persicae and Buchnera with plant host (Brassicaceae and non-tobacco Solanaceae) and no associations between genetic and geographic distance at global or regional spatial scales. Abundance patterns of non-synonymous mutations were similar to synonymous mutations in the Buchnera genome, and both mutation classes had similar site frequency spectra. We hypothesize that a predominance of neutral processes results in the Buchnera of M. persicae to simply drift with the evolutionary trajectory of their aphid hosts. Our study presents a unique microevolutionary characterisation of Buchnera-aphid host genomic covariation across multiple aphid clones. This provides a new perspective on the eco-evolutionary processes generating and maintaining polymorphisms in a major pest aphid species and its obligate primary endosymbiont.

evolutionary biology↗

bioRxiv2023

Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple non-hematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAK[a], p38 and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAK[a] knockout or pharmacologic inhibitors of ZAK[a] and p38 kinase activities. By surveying a diverse panel of ionophores, we show that the electroneutrality of potassium efflux is essential to activate ZAK[a]-driven RSR, likely because a greater extent of K+ depletion is necessary to activate ZAK[a]-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux and innate immunity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/546021v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1afe96aorg.highwire.dtl.DTLVardef@53c59org.highwire.dtl.DTLVardef@1bd14d1org.highwire.dtl.DTLVardef@a1bccc_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCENigericin is familiar to the inflammasome field as the most robust and commonly used NLRP3 inducer. It has enabled numerous breakthroughs in the field linking NLRP3 activation to potassium efflux. In this manuscript, we report that nigericin activates an alternate inflammasome sensor, NLRP1 in primary human skin, nasal and corneal epithelial cells. NLRP1 activation by nigericin requires K+ efflux-driven ribosome stalling and the ribotoxic stress response (RSR) sensor MAP3K, ZAK[a]. We further identify the key biophysical principles that explain why only a subset of K+ ionophores, exemplified by nigericin, function as super inflammasome agonists that can activate either NLRP1 or NLRP3, depending on cell type. These results reveal an unexpected connection between RSR, potassium ion flux and innate immunity.

immunology↗

bioRxiv2022

The soybean rust pathogen Phakopsora pachyrhizi displays transposable element proliferation that correlates with broad host-range adaptation on legumes

Asian soybean rust, caused by Phakopsora pachyrhizi, is one of the worlds most economically damaging agricultural diseases. Despite P. pachyrhizis impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. We simultaneously sequenced three P. pachyrhizi genomes uncovering a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. The proliferation of TEs within the genome occurred in several bursts and correlates with the radiation and speciation of the legumes. We present data of clear de-repression of TEs that mirrors expression of virulence-related candidate effectors. We can see a unique expansion in amino acid metabolism for this fungus. Our data shows that TEs play a dominant role in P. pachyrhizis genome and have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity of the genome.

microbiology↗

bioRxiv2022

Decreased miRNA-148a-3p expression in skeletal muscle of patients with chronic kidney disease

IntroductionSkeletal muscle wasting is a common complication of chronic kidney disease which leads to a loss of muscle function. The pathogenesis of skeletal muscle wasting is incompletely understood, which is preventing the development of targeted therapeutics. Recent evidence implicates miRNAs in the of skeletal muscle wasting. Our aim was to firstly examine miRNA profiles of CKD human skeletal muscle for the identification of aberrant expression patterns compared to a healthy control (HC) cohort, and secondly, investigate the role these miRNAs may play in inducing or promoting skeletal muscle atrophy using a novel human primary skeletal muscle cell model of CKD skeletal muscle. MethodsFor the comparison between CKD and HC populations, skeletal muscle biopsies were collected from the vastus lateralis of n=15 non-dialysis dependent CKD patients stage 3b-5 CKD patients, and n=15 healthy controls matched for age, gender and physical activity. n=5 biopsies from each group underwent next generation sequencing to obtain complete microRNA profiles in CKD vs HC cohorts, which were then validated in a separate cohort by PCR (N=10 in each group). A causative role in muscle wasting was determined by transfection of key microRNAs into a primary culture model of CKD skeletal muscle and changes in protein degradation determined by L-[3H]- phenylalanine release into the media. ResultsNext Generation Sequencing identified differential expression of 16 miRNAs in skeletal muscle of CKD patients versus controls, and PCR validation confirmed miRNA-148a-3p expression was significantly decreased in CKD patients. The reduced miRNA-148a-3p expression was also maintained in the primary culture model. Upon overexpression of miRNA-148a-3p in CKD myotubes, protein degradation rates were decreased non-significantly (p=0.28) by 16.3% compared to un-transfected CKD cells. ConclusionCKD was associated with a significant reduction in miRNA-148a-3p expression in skeletal muscle compared to non-CKD controls which was retained in our in vitro model. Overexpression of miRNA-148a-3p in primary skeletal myotubes non-significantly decreased muscle protein degradation by 16.3%. In order to determine the importance of miRNA-148a-regulation of protein degradation, a deeper understanding of miRNA-148a-3p targets and their associated pathways with respect to those dysregulated in skeletal muscle wasting is required.

molecular biology↗

bioRxiv2022

Genomic diversity of Helicobacter pylori populations from different regions of the human stomach.

Individuals infected with Helicobacter pylori harbour unique and diverse populations of quasispecies, but diversity between and within different regions of the human stomach and the process of bacterial adaptation to each location are not yet well understood. We applied whole-genome deep sequencing to characterise the within- and between- stomach region genetic diversity of H. pylori populations from paired antrum and corpus biopsies of 15 patients, along with single biopsies from one region of 3 patients, by scanning allelic diversity. We combined population deep sequencing with more conventional sequencing of multiple H. pylori single colony isolates from individual biopsies to generate a unique dataset. Single colony isolates were used to validate the scanning allelic diversity pipelines. We detected extensive population allelic diversity within the different regions of each patients stomach. Diversity was most commonly found within non-coding, hypothetical, outer membrane, restriction modification system, virulence, lipopolysaccharide biosynthesis, efflux systems and chemotaxis-associated genes. Antrum and corpus populations from the same patient grouped together phylogenetically, indicating that most patients were initially infected with a single strain, which then diversified. Single colonies from the antrum and corpus of the same patients grouped into distinct clades, suggesting mechanisms for within-location adaptation across multiple H. pylori isolates from different patients. Recombination was observed both within and between different regions of the same stomach. The comparisons made available by combined sequencing and analysis of isolates and populations enabled comprehensive analysis of the genetic changes associated with H. pylori diversification and stomach region adaptation.

microbiology↗

bioRxiv2022

Sex-specific distribution and classification of Wolbachia infections and mitochondrial DNA haplogroups in Aedes albopictus from the Indo-Pacific

The arbovirus vector Aedes albopictus (Asian tiger mosquito) is common throughout the Indo-Pacific region, where most global dengue transmission occurs. We analysed population genomic data and tested for cryptic species in 160 Ae. albopictus sampled from 16 locations across this region. We found no evidence of cryptic Ae. albopictus but found multiple intraspecific COI haplotypes partitioned into groups representing three Asian lineages: East Asia, Southeast Asia and Indonesia. Papua New Guinea (PNG), Vanuatu and Christmas Island shared recent coancestry, and Indonesia and Timor-Leste were likely invaded from East Asia. We used a machine learning trained on morphologically sexed samples to classify sexes using multiple genetic features and then characterized the wAlbA and wAlbB Wolbachia infections in 664 other samples. The wAlbA and wAlbB infections as detected by qPCR showed markedly different patterns in the sexes. For females, most populations had a very high double infection incidence, with 67% being the lowest value (from Timor-Leste). For males, the incidence of double infections ranged from 100% (PNG) to 0% (Vanuatu). Only 6 females were infected solely by the wAlbA infection, while rare uninfected mosquitoes were found in both sexes. The wAlbA and wAlbB densities varied significantly among populations. For mosquitoes from Torres Strait and Vietnam, the wAlbB density was similar in single-infected and superinfected (wAlbA and wAlbB) mosquitoes. There was a positive association between wAlbA and wAlbB infection densities in superinfected Ae. albopictus. Our findings provide no evidence of cryptic species of Ae. albopictus in the region and suggest site-specific factors influencing the incidence of Wolbachia infections and their densities. We also demonstrate the usefulness of SNPs as sex-specific mosquito markers. The results provide baseline data for the exploitation of Wolbachia-induced cytoplasmic incompatibility (CI) in dengue control.

microbiology↗