Biology subjects

Ziemann, M.

Publications and source records attributed to Ziemann, M..

17 recordsLinked to original sources

bioRxiv2026

Tissue-specific divergence in sex-biased DNA methylation across the lifespan

BackgroundAgeing is a sex-specific process characterised by a progressive decline in physiological integrity. DNA methylation represents a primary epigenetic hallmark of ageing, yet sex-specific patterns of epigenetic ageing within and across tissues remain poorly understood. This study aims to address these gaps through an integrated analysis of sex-moderated epigenetic ageing across eight human tissues. MethodsA total of 137 DNA methylation datasets comprising over 36,000 individuals aged 10-114 years were analysed using a meta-analytic workflow to identify age-associated differentially methylated positions (aDMPs) and regions (aDMRs), meta-regression to assess sex moderation, and pathway enrichment analyses to interpret functional relevance. FindingsIndividual tissues displayed distinct age-related methylation trajectories, but some DMP sites showed consistent hyper- or hypomethylation across tissues. Across tissues, we identified 68,630 aDMPs (10%) robustly associated with ageing. Age-associated changes at the regional level were less common, with only 80 robust age-associated aDMRs detected across tissues, representing 0.09% of analysed regions. Sex moderation was observed for only 16 aDMPs (0.002%), indicating that sex effects on age-associated DNA methylation are largely tissue-specific rather than shared across tissues. InterpretationOur findings indicate that age-associated DNA methylation changes predominantly occur at isolated CpG sites rather than extended genomic regions and are strongly dependent on tissue and genomic context. The minimal overlap of sex-moderated methylation signals across tissues suggests that age-related sex differences at the epigenetic level are more likely attributable to tissue- and cell-type-specific variation rather than to broadly conserved epigenetic mechanisms shared across tissues. FundingThis study was funded by an Australian Research Council (ARC) Discovery project (DP200101830). Severine Lamon was funded by an ARC Future Fellowship (FT210100278). Nir Eynon was funded by NHMRC Investigator Grant (APP1194159), and a Hevolution/AFAR New Investigator Award in Aging Biology and Geroscience Research. Mandhri D. Abeysooryia was supported by an Australian Government Research Training Program (RTP) Scholarship. Research in context Evidence before this studyDNA methylation is widely recognised as a central epigenetic hallmark of ageing. Previous research has demonstrated that some age-related methylation changes are conserved across tissues, forming the basis of pan-tissue epigenetic clocks. Most studies to date have primarily examined age effects in isolation. Although biological sex influences ageing trajectories and susceptibility to nearly all age-related diseases, sex-moderated epigenetic ageing has received limited investigation. Specifically, pan-tissue clocks, including GrimAge and PhenoAge, are "sex-aware" but were trained and validated in mixed-sex cohorts, limiting their capacity to disentangle tissue-specific sex effects. Consequently, it remains unclear whether sex-moderated epigenetic ageing signals are shared across tissues or are tissue-specific. Added value of this studyThis study provides a large-scale, comprehensive multi-tissue analysis of sex-moderated epigenetic ageing, integrating 137 DNA methylation datasets across eight human tissues and more than 36,000 male and female individuals spanning the lifespan. Our findings show that while age-associated methylation changes are widespread at the CpG level, sex-moderated effects are rare and largely tissue-specific, with minimal overlap across tissues. Implications of all the available evidenceTogether, the available evidence indicates that epigenetic ageing is predominantly driven by shared, conserved age-related methylation changes, whereas sex differences in epigenetic ageing are modest and context dependent. These sex-related effects are more likely to reflect tissue- and cell-type-specific variation rather than widespread, shared mechanisms. This underscores the need to develop sex-specific epigenetic clocks and to conduct longitudinal cohort and intervention studies to more precisely characterise sex-specific dynamics of epigenetic ageing across tissues.

genetics↗

bioRxiv2026

Characterisation of vaginal Lactobacillus isolates from South African women towards the development of a biotherapeutic to optimise the vaginal microbiome

HIV remains among the worlds most serious healthcare challenges, with adolescent girls and young women in sub-Saharan Africa at particularly high risk of infection. Bacterial vaginosis (BV) is a key risk factor for HIV acquisition, however current treatment strategies are limited. Optimal vaginal Lactobacillus spp. protect against BV and HIV, largely through immunoregulatory and antimicrobial activities mediated in part by lactic acid. Towards the development of a Lactobacillus-containing live biotherapeutic for African women, we sampled 181 vaginal Lactobacillus isolates from 25 BV-negative South African women. Fifty isolates were selected for evaluation of inflammatory responses using vaginal epithelial cells, D- and L-lactate and lactic acid production and culture acidification. Aside from a single Lactobacillus salivarius strain, L. crispatus isolates acidified the culture media the most and produced the most D- and L-lactic acid. Inflammatory cytokine responses to Lactobacillus strains were variable, with L. crispatus eliciting the lowest levels of cytokine production. When all properties were evaluated collectively, L. crispatus strains exhibited the most desirable biotherapeutic characteristics. Whole genome sequence analysis of ten L. crispatus isolates showed that the majority were more closely related to one another than to isolates from other geographical regions. This supports the need for live biotherapeutics to be tailored for the population of intended use. No antimicrobial resistance elements were detected, while putative bacteriocins and intact prophage sequences were identified in all isolates. L. crispatus isolates displayed characteristics essential for optimal live biotherapeutic performance, however additional analysis is required to determine the functionality of identified putative prophages. ImportanceHIV is a leading cause of morbidity and mortality in sub-Saharan Africa, where adolescent girls and young women are three times more likely to acquire HIV than their male counterparts. A key risk factor for HIV is bacterial vaginosis (BV), a condition characterised by the loss of beneficial Lactobacillus species and increased abundance of non-optimal, inflammatory bacteria. Although BV affects approximately 25% of women in sub-Saharan Africa, effective therapeutics are lacking. Live biotherapeutics containing optimal Lactobacillus spp. represent a promising strategy to improve BV treatment outcomes and reduce HIV infection risk. We isolated 181 vaginal Lactobacillus spp. from 25 BV-negative South African women and characterized 50 selected isolates. This led to the identification of live biotherapeutic candidates for African women with distinct genomes compared to isolates from other geographical regions. This study contributes to current knowledge of the characteristics that should be considered when screening novel isolates for this purpose.

microbiology↗

bioRxiv2026

Genomic Isomerism and Environmental Adaptation in Cyanophages Infecting Freshwater Cyanobacteria

Cyanophages infecting multicellular, heterocyst-forming cyanobacteria play diverse pivotal roles in freshwater ecosystems, yet their infection strategies and genomic adaptability remain poorly understood. Here, we isolated and characterized three Caudoviricetes cyanophages (A-Lf14, A-Alj1 and A-Hlh1) specifically infecting the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. Super-resolution microscopy revealed heterogeneous infection outcomes even among adjacent cells within the same filament, highlighting host-level defense variability. Comparative genomics placed these phages within a cluster that includes the previously isolated phages A-1(L) and N1, and revealed a conserved [~]30 kb invertible genomic region flanked by inverted repeats (IR). This region exists as two stable isomers simultaneously within phage populations, a previously unreported genomic plasticity trait in cyanophages. Consistent with such plasticity, infection kinetics were modulated by light and nitrogen availability, indicating a resource-responsive infection strategy. The novel phages encode an alkaline phosphatase of non-cyanobacterial origin, which was highly upregulated during infection indicating a role in phosphate acquisition in phosphate-limited waters. In contrast, they lack a tnpB gene present in A-1(L), which is identical in sequence to five genes in the host genome. We detected protein-coding potential for both strands of tnpB and upregulated transcription during infection, consistent with a role in this process. The 347 residues protein encoded on the tnpB reverse strand exhibited only limited similarity to other proteins or folding potential, underlining its novelty. Our work illustrates how genomic isomerism, accessory genes, and environmental sensing collectively drive functional diversification in cyanophages, providing insights into phage-host coevolution and the impact of phages on cyanobacterial blooms in dynamic freshwater ecosystems.

microbiology↗

bioRxiv2025

The dynamic role of HLA proteins on compositional alterations of T-cell repertoires in inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD), which is characterised by genetic predispositions and dysregulated immune responses, is rapidly emerging as a global health challenge. Genetic variations in the human leukocyte antigen (HLA) region are strongly associated with IBD; nonetheless, the functional consequences of this variation on the composition of T-cell receptors remain poorly understood. MethodsWe conducted comprehensive CDR3-QTL mapping using T-cell receptor beta (TRB) repertoires paired with HLA allotypes from 1,973 individuals, including 1,201 individuals with IBD and 772 healthy controls (HCs), to explore the role of the HLA allelic variants on TRB composition. Using network analyses, we defined key CDR3 motifs of public clones that were linked to risk HLA alleles for chronic inflammatory diseases. ResultsWe identified novel sites within both HLA class I and class II proteins that were strongly linked to TRB amino acid composition - cdr3QTLs, in both HCs and individuals with IBD. Those sites in HLA-DRB1 and HLA-DQ had stronger effects on CDR3 composition than did the disease in the IBD cohort. In HCs, but not in UC or CD, the strongest HLA signals that affected expanded clones, overlapped with primary CD risk loci from GWAS, e.g., DRB1 site 70 and DQA1 site 25. The strongest CD-specific effects on TRB composition were found in HLA-B, especially at sites that modulate viral responses (e.g., HLA-B sites 9, 67). Finally, the main risk HLA alleles for chronic inflammatory diseases clustered together based on the physicochemical properties of residues mapped to cdr3QTLs, suggesting that risk alleles might exert similar effects on the TRB repertoire. ConclusionStructurally, the main cdr3QTLs in both HLA class I/II are located in peptide-binding sites or sites contacting TCRs, highlighting their direct and antigen-mediated influences on TRB repertoires. Our findings suggest that cdr3QTLs in HLA class I exert IBD-specific effects on the TRB composition, influencing the dysregulated T cell responses implicated in IBD pathogenesis, possibly on the earlier stages of T cell development. While HLA class II cdr3QTLs show universal effects and strong associations with T-cell receptors, irrespective of disease.

immunology↗

bioRxiv2025

Fast and Furious: Metabolic Pathways Fuelling Devil Facial Tumour Disease

Devil Facial Tumour Diseases (DFTD), threatening Tasmanian devils, consist of two distinct transmissible cancers, DFT1 and DFT2, with differing origins and geographic spread. We investigated the metabolic differences between DFT1 and DFT2, examining cell viability, metabolic outputs, and bulk gene expression. Using both DFT1 and DFT2 cell lines and biopsies, we found that glycolysis, oxidative phosphorylation, glutamate metabolism and fatty acid synthesis are all essential for the survival of both tumour types. However, DFT2 exhibited higher rates of glycolysis and lactate generation compared to DFT1. This coincided with elevated ATP production, cholesterol biosynthesis and ROS generation, as well as an increased reliance on fatty acid metabolism. Furthermore, DFT2 is less metabolically adaptable than DFT1, being unable to switch to oxidative phosphorylation as DFT1 can when required. These metabolic changes in DFT2, in conjunction with its higher growth rate, suggests a more aggressive cancer phenotype than DFT1. Our findings highlight distinct metabolic adaptations in DFT2 that may contribute to its competitive advantage.

cancer biology↗

bioRxiv2025

Analysis of tracrRNAs reveals subgroup V2 of type V-K CAST systems

CRISPR-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in TnsB and TnsC proteins. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0.

microbiology↗

bioRxiv2025

Skeletal muscle mitochondria contain nuclear-encoded RNA species prior to and following adaptation to exercise training in rats

Skeletal muscle mitochondria adaptation to exercise training is mediated by molecular factors that are not fully understood. Mitochondria import over 1000 proteins encoded by the nuclear genome, but the RNA population resident within the organelle is generally thought to be exclusively encoded by the mitochondrial genome. However, recent in vitro evidence suggests that specific nuclear-encoded miRNAs and other non-coding RNAs (ncRNAs) can reside within the mitochondrial matrix. Whether these are present in mitochondria of skeletal muscle tissue, and if this affected by endurance training - a potent metabolic stimulus for mitochondrial adaptation - remains unknown. Rats underwent four weeks of moderate intensity treadmill exercise training, then humanely killed and tissues collected for molecular profiling. Mitochondria from gastrocnemius skeletal muscle were isolated by immunoprecipitation, further purified, then the resident RNA was sequenced to assess the mitochondrial transcriptome. Exercise training elicited typical transcriptomic responses and functional adaptations in skeletal muscle, including increased mitochondrial respiratory capacity. We identified 24 nuclear-encoded, coding or non-coding RNAs in purified mitochondria, in addition to 50 nuclear-encoded miRNAs that met a specified abundance threshold. Although none were differentially expressed in the exercise vs control group at FDR<0.05, exploratory analyses suggested that the abundance of 3 miRNAs were altered (p<0.05) in mitochondria isolated from trained compared with sedentary skeletal muscle. We report the presence of a specific population of nuclear-encoded RNAs in the mitochondria isolated from rat skeletal muscle tissue, which could play a role in regulating exercise adaptations and mitochondrial biology. KEY POINTSO_LIMitochondria are a central metabolic and signalling hub, particularly in tissues with high energy demand such as skeletal muscle. Their ability to adapt to stimuli such as exercise training is directly related to improved metabolic health. Understanding the factors that regulate these adaptive processes is therefore essential. C_LIO_LIMitochondria contain RNA encoded by their own genome (mtDNA); typically are not thought to contain nuclear-encoded RNA. C_LIO_LIHere, we report that mitochondria from rat skeletal muscle do indeed contain a select population of nuclear-encoded protein coding and non-coding RNAs, including 3 microRNAs whose expression tended to be altered by exercise training. C_LIO_LIThese findings suggest that mitochondria-localised nuclear-encoded RNAs may play a role in mediating beneficial adaptive responses to exercise. C_LI

physiology↗

bioRxiv2024

The transcriptomic signature of age and sex is not conserved in human primary myocytes

BackgroundHuman primary muscle cell (HPMC) lines derived from skeletal muscle biopsies are potentially powerful tools to interrogate the molecular pathways underlying fundamental muscle mechanisms. HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment, or at concentrations that do not mirror physiological levels. To address the assumption that HPMCs are valid models of age and sex-specificity in human muscle research, we examined to what extent HPMC lines retain their source phenotype in culture. MethodsBiopsies from the vastus lateralis muscle were collected from ten males aged 18-30, ten females aged 18-30 and ten males aged 60-75 recruited from a general, healthy population. A portion of the muscle was used for the establishment of 30 individual HMPC lines. The remaining sample was immediately snap frozen and stored for further analysis. RNA was extracted from muscle tissue samples and their corresponding, fully differentiated HMPCs and analysed using RNA Sequencing. To compare their transcriptomic signature, principal component analysis (PCA), differential expression analysis, single-cell deconvolution and pathway enrichment analysis were conducted in R. ResultsA comparison of the transcriptomic signature of 30 human muscle biopsies and their corresponding HPMCs indicated a near-complete lack of retention of the genes and pathways differentially regulated in vivo when compared to their in vitro equivalent, with the exception of several genes encoded on the Y-chromosome. ConclusionsThe diversity of resident cell populations in muscle tissue and the lack of sex- and age-dependent circulating factors in the cellular milieu likely contribute to these observations, which call for caution when using HPMCs as an experimental model of human muscle sex or age.

cell biology↗

bioRxiv2024

Direction-aware functional class scoring enrichment analysis of Infinium DNA methylation data

Infinium Methylation BeadChip arrays remain one of the most popular platforms for epigenome-wide association studies, but tools for downstream pathway analysis have their limitations. Functional class scoring (FCS) is a group of pathway enrichment techniques that involve the ranking of genes and evaluation of their collective regulation in biological systems, but the implementations described for Infinium methylation array data do not retain direction information, which is important for mechanistic understanding of genomic regulation. Here, we evaluate several candidate FCS methods that retain directional information. According to simulation results, the best-performing method involves the mean aggregation of probe limma t-statistics by gene followed by a rank-ANOVA enrichment test using the mitch package. This method, which we call "LAM", outperformed an existing over-representation analysis method in simulations, and showed higher sensitivity and robustness in an analysis of real lung tumour-normal paired datasets. Using matched RNA-seq data we examine the relationship of methylation differences at promoters and gene bodies with RNA expression at the level of pathways in lung cancer. To demonstrate the utility of our approach, we apply it to three other contexts where public data were available. Firstly, we examine differential pathway methylation associated with chronological age. Secondly, we investigate pathway methylation differences in infants conceived with in vitro fertilisation. Lastly, we analyse differential pathway methylation in 19 disease states, identifying hundreds of novel associations. These results show LAM is a powerful method for the detection of differential pathway methylation as compared to existing methods. A reproducible vignette is provided to illustrate how to implement this method.

genomics↗

bioRxiv2022

Amyloid beta 42 alters cardiac metabolism and impairs cardiac function in obesity

There are epidemiological associations between obesity and type 2 diabetes, cardiovascular disease and Alzheimers disease. While some common aetiological mechanisms are known, the role of amyloid beta 42 (A{beta}42) in these diverse chronic diseases is obscure. Here we show that adipose tissue releases A{beta}42, which is increased from adipose tissue of obese mice and is associated with higher plasma A{beta}42. Increasing circulating A{beta}42 levels in non-obese mice had no effect on systemic glucose homeostasis but had obesity-like effects on the heart, including reduced cardiac glucose clearance and impaired cardiac function. These effects on cardiac function were not observed when circulating levels of the closely related A{beta}40 isoform were increased. Administration of an A{beta} neutralising antibody prevented obesity-induced cardiac dysfunction and hypertrophy. Furthermore, A{beta} neutralising antibody administration in established obesity prevented further deterioration of cardiac function. Multi-contrast transcriptomic analyses revealed that A{beta}42 impacted pathways of mitochondrial metabolism and exposure of cardiomyocytes to A{beta}42 inhibited mitochondrial function. These data reveal a role for systemic A{beta}42 in the development of cardiac disease in obesity and suggest that therapeutics designed for Alzheimers disease could be effective in combating obesity-induced heart failure.

physiology↗

bioRxiv2022

CvkR, a novel MerR-type transcriptional regulator, is a repressor of class 2 type V-K CRISPR-associated transposase systems

CRISPR-associated transposons (CASTs) exist in different groups of bacteria, including certain cyanobacteria, which contain type V-K CAST systems. These systems contain genes encoding Tn7-like transposase subunits and a divergent number of cargo genes. How the activity of these systems is controlled in situ has remained largely unknown but possibly regulatory genes within these elements are prime candidates. Deletion of the respective regulator gene alr3614 in the cyanobacterium Anabaena (Nostoc) sp. PCC 7120 led to the overexpression of CRISPR tracrRNA, precursor crRNAs and mRNAs encoding the Cas12k effector protein (all3613) and Tn7-like transposase subunits. Upon complementation, these same genes were repressed again. DNase I footprinting and electrophoretic mobility shift assays verified the direct interaction between Alr3614 and the promoter of cas12k and identified a widely conserved binding motif. Structural analysis of Alr3614 at 1.5 [A] resolution revealed that it belongs to the MerR-type transcription factor family but with distinct dimerization and effector-binding domains. This protein assembles into a homodimer interacting with DNA through its N-terminal winged helix-turn-helix (wHTH) domain and binds an effector molecule through a C-terminal -helical domain lacking a conserved cysteine. These results identify Alr3614 as a transcriptional repressor of the CAST system in Anabaena sp. PCC 7120. We suggest naming this family of repressors CvkR for Cas V-K repressors, which are at the core of a widely conserved regulatory mechanism that controls type V-K CAST systems.

microbiology↗

bioRxiv2021

Guidelines for reliable and reproducible functional enrichment analysis

Gene set enrichment tests (a.k.a. functional enrichment analysis) are among the most frequently used methods in computational biology. Despite this popularity, there are concerns that these methods are being applied incorrectly and the results of some peer-reviewed publications are unreliable. These problems include the use of inappropriate background gene lists, lack of false discovery rate correction and lack of methodological detail. An example analysis of public RNA-seq reveals that these methodological errors alter enrichment results dramatically. To ascertain the frequency of these errors in the literature, we performed a screen of 186 open access research articles describing functional enrichment results. We find that 95% of analyses using over-representation tests did not implement an appropriate background gene list or did not describe this in the methods. Failure to perform p-value correction for multiple tests was identified in 43% of analyses. Many studies lacked detail in the methods section about the tools and gene sets used. Only 15% of studies avoided major flaws, which highlights the poor state of functional enrichment rigour and reporting in the contemporary literature. We provide a set of minimum standards that should act as a checklist for researchers and peer-reviewers.

bioinformatics↗

bioRxiv2021

Population genomics of Salmonella enterica serovar Weltevreden ST365, an emerging predominant causative agent of diarrheal disease

Salmonella enterica serovar Weltevreden is a recently emerged pathogen, and as such we lack a comprehensive knowledge of its microbiology, genomics, epidemiology and biogeography. In this study, we analyzed 174 novel S. Weltevreden isolates including 111 isolates recovered from diarrheal patients in China between 2006 and 2017. Our results demonstrate that the ST365 clone was the predominant causative agent of the diarrhea-outbreak during this period, as vast majority of the isolates recovered from diarrheal patients belonged to this sequence type (97.37%, 74/76). We also determined the ST365 clone as the predominant sequence type of S. Weltevreden from diarrheal patients globally from previously published sequences (97.51%, 196/201). In order to determine the possible antimicrobial genes and virulence factors associated with S. Weltevreden, we performed whole genome sequencing on our novel isolates. We were able to identify a range of key virulence factors associated with S. Weltevreden that are likely to be beneficial to their fitness and pathogenesis. Furthermore, we were able to isolate a novel 100.03-kb IncFII(S) type virulence plasmid that used the same replicon as pSPCV virulence plasmid. Importantly, we demonstrated through plasmid elimination a functional role for this plasmid in bacterial virulence. These findings are critical to further our knowledge of this high consequence pathogen. ImportanceSalmonella Weltevreden is a newly emerged foodborne pathogen and has caused several outbreaks of diarrheal diseases in some regions in the world. However, comprehensive knowledge of microbiology, genomics, epidemiology and biogeography of this newly emerged pathogen is still lack. In this study, we made an unexpected discovery that S. Weltevreden sequence type (ST) 365 is the causative agent in the diarrhea-outbreak in China and many other regions of the world. We also shown that this sequence type was widely recovered from animal, food, and environmental samples collected in different regions in the world. Importantly, we discovered a novel IncFII(S) type virulence plasmid commonly carried by S. Weltevreden strains of both human, animal, and food origins. These data facilitate future studies investigating the emergence of S. Weltevreden involved in diarrheal outbreaks and the global spread of S. Weltevreden strains.

microbiology↗

bioRxiv2021

Gene name errors: lessons not learned

Erroneous conversion of gene names into other dates and other data types has been a frustration for computational biologists for years. We hypothesized that such errors in supplementary files might diminish after a report in 2016 highlighting the extent of the problem. To assess this, we performed a scan of supplementary files published in PubMed Central from 2014 to 2020. Overall, gene name errors continued to accumulate unabated in the period after 2016. An improved scanning software we developed identified gene name errors in 30.9% of articles with supplementary Excel gene lists; a figure significantly higher than previously estimated. This is due to gene names being converted not just to dates and floating-point numbers, but also to internal date format (five-digit numbers). These findings further reinforce that spreadsheets are ill-suited to use with large genomic data.

genomics↗

bioRxiv2020

HLAssign 2.0: An advanced Graphical User Interface for the analysis of short and long read Human Leukocyte Antigen-typing data

Next Generation Sequencing (NGS) based Human Leukocyte Antigen (HLA) typing has been a challenge due to the polymorphism of the HLA region. Nevertheless, the methods accuracy has increased during the last years and it is now routinely used by many large centers including bone marrow registries. However, challenging HLA genotype compositions exist, which hinder a fully automated analysis. Therefore, HLA typing results are still visually inspected in diagnostics, i.e. the underlying read mappings and phasing information is controlled. Here, we present HLAssign 2.0 that now includes a strict workflow, improved tools for visual inspection and read phasing analysis in the automatic caller. In collaboration with interface design researchers, biologists and informaticians we developed an elaborate graphical user interface for visual evaluation of automated HLA calls for Illumina NGS reads. We also provide tools to preprocess 10x Genomics and PacBio sequencing reads for HLAssign analysis. We benchmarked our automatic caller against STC-seq and xHLA, showing comparable automatic call rates. Additional manual inspection of the automatic results in our GUI assists the user to assign the correct HLA calls and to achieve diagnostic accuracy. HLAssign 2.0 is free for research and commercial use and is available for Windows and MacOS.

bioinformatics↗

bioRxiv2020

Specificities of and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups

The majority of bacteria and archaea possess an RNA-guided adaptive and inheritable immune system against viruses and other foreign genetic elements that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. In some bacteria, multiple instances of cas gene-free (orphan) repeat-spacer arrays exist, while additional instances of arrays that are linked to cas gene cassettes are present elsewhere in the genome. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR-Cas repeat-spacer arrays are present, but only two cas gene cassettes plus a Tn7-associated eleventh array are observed. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support a strong tendency for array fragmentation in multicellular cyanobacteria and disfavor other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our data demonstrate the functional coordination of Cas6 endonucleases with both neighboring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

microbiology↗

bioRxiv2018

Valproic acid attenuates hyperglycemia-induced complement and coagulation cascade gene expression

Atherothrombosis remains the leading cause of morbidity and mortality in patients diagnosed with diabetes mellitus, but the molecular mechanisms underpinning this remain unresolved. As the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is an interest in understanding the mechanisms of hepatic cell activation under hyperglycemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycemia stimulates major changes in chromatin organisation and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA; IUPAC: 2-propylpentanoic acid) is able to reverse some of these metabolic changes. In this study, we used deep transcriptome sequencing to show that VPA attenuates hyperglycemia-induced activation of complement and coagulation cascade genes. These findings reveal a novel mechanism of VPA protection against hyperglycemia, which might improve the therapeutic approaches for diabetes.

molecular biology↗