Biology subjects

Hwang, S.

Publications and source records attributed to Hwang, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Therapeutic AIBP expression contributes to synaptic preservation and vision restoration against glaucomatous neuroinflammation

Lowering intraocular pressure (IOP) is the only approved treatment for glaucoma, yet retinal ganglion cell (RGC) degeneration and vision loss often progress despite reducing IOP. Apolipoprotein A-I binding protein (AIBP) stabilizes ATP-binding cassette subfamily A member 1 (ABCA1) and promotes cholesterol efflux, thus controlling cholesterol-rich lipid rafts and restraining lipid raft-dependent Toll-like receptor 4 (TLR4) inflammatory signaling. Here we show that AAV2-mediated expression of human AIBP (hAIBP) preserves inner retinal synaptic integrity and visual function in experimental glaucoma without altering IOP. In DBA/2J mice, a single intravitreal injection of AAV2-hAIBP at 5 months protected RGCs and preserved pattern electroretinogram and pattern visual evoked potential responses at 10 months, even though IOP was indistinguishable from empty-vector controls. AAV2-hAIBP preserved CaMKII and synapsin phosphorylation, restored synaptophysin and PSD95 in the inner plexiform layer, and reduced the expression of TLR4, complement C1q, IBA1, IL-1b, and IL-6 induction in Muller glia and microglia. Comparable protection was observed in microbead-induced ocular hypertension in C57BL/6 mice, both when the AAV2-hAIBP was delivered before microbead injection and when it was delivered after IOP elevation was established. Elevated IOP reduced endogenous retinal AIBP and ABCA1 expression, and AAV2-hAIBP restored both. Protection required the TLR4-binding domain, as an AIBP mutant lacking this domain failed to preserve RGCs. Conversely, AIBP knockout retinas exhibited increased CaMKII phosphorylation and reduced synaptophysin in the absence of elevated IOP. These findings identify synaptic preservation through the AIBP-TLR4 axis as an IOP-independent therapeutic mechanism and support AAV2-hAIBP as a candidate gene therapy for glaucoma.

neuroscience↗

bioRxiv2026

AtomWeaver: Multi-Component Flow Matching with a Structured Geometric Prior Facilitates Non-Canonical Peptide Design

Fixed-backbone sequence discovery, or inverse folding, is a critical recurring task in the development of new polypeptide therapeutics. Once promising backbones are established for a target pocket, computational inverse folding methods greatly help accelerate generation of candidate sequences. Such methods are mature for the traditional case of limiting to the fixed twenty-letter canonical vocabulary; however, they cannot access the broader space of non-canonical amino acids (NCAAs). This design constraint is exacerbated for peptide binders, a fast-growing modality that readily incorporates NCAAs, though in practice non-canonical design frequently depends on laborious medicinal-chemistry campaigns. An extension of inverse folding to NCAAs is thus critical to accelerating design of novel therapeutic peptides. AtomWeaver uses a joint all-site, atom-level generative scheme that does not restrict side-chain categorical assignment by either predetermined or co-resolving residue identity. Conditioned only on a fixed peptide backbone and its target protein, its multi-component flow guides side-chain atoms as unlabeled points in R3 from a nested shell prior to a variable-count final atom cloud. Identity is then read by matching each predicted cloud against a reference library of canonical and non-canonical templates. Since identity is decided only at decode time, the addressable vocabulary is a property of the library rather than of the trained weights: a new NCAA costs one reference structure and no retraining, and the model can select residues it was never prompted for and never saw in training. On a deep mutational scan of two peptide-target systems, AtomWeaver's canonical readout shows high observed mean agreement with experimental values among the compared inverse-folding methods. In the mixed canonical-noncanonical setting that canonical-only baselines cannot support at all, it likewise retains ranking signal across both systems. AtomWeaver also displayed self-consistent designs on de novo binder backbones, with the highest interface confidence among compared methods. Notably, it reached these metrics while achieving broad empirical coverage of our 300-residue vocabulary, including four non-canonical types never visible in training. AtomWeaver thus serves canonical and non-canonical peptide design alike, while transforming residue vocabulary to an expandable inference-time choice.

bioengineering↗

bioRxiv2026

Re-evaluation of the report of Culex quinquefasciatus in the Republic of Korea: Molecular evidence of cryptic hybridisation within the Culex pipiens complex

The Culex pipiens complex comprises globally distributed mosquito species that serve as vectors for various arboviruses and filariasis. Female mosquitoes within the Cx. pipiens complex are morphologically very similar, making species identification based on morphologically characteristics challenging. To date, Cx. pallens and Cx. pipiens f. molestus have been reported to inhabit the Republic of Korea (ROK). Conversely, Cx. quinquefasciatus, predominantly distributed in tropical regions, has not been documented in the country since a single record in the 1950s. Recently, however, Cx. quinquefasciatus was reported from the Jeju region of the ROK based on molecular identification. This study aimed to re-evaluate the occurrence of Cx. quinquefasciatus in the ROK by conducting molecular identification using the ace-2 and CQ11 markers on Cx. pipiens complex specimens collected nationwide, including from Jeju, where Cx. quinquefasciatus had previously been reported. Our analysis identified only Cx. quinquefasciatus-Cx. pallens hybrid individuals among the Cx. pipiens complex specimens from the Jeju region, highlighting the need to re-evaluate the analytical methods used in previous studies that reported the presence of pure Cx. quinquefasciatus. These findings provide essential evidence for future surveillance of the distribution, introduction, and establishment of Cx. quinquefasciatus and underscore the need for continuous monitoring.

evolutionary biology↗

bioRxiv2026

Orthogonal CRISPR screens and human liver chimeric mice identify hepatitis B virus host factors

Hepatitis B virus (HBV) chronically infects approximately 250 million people worldwide, and reliable curative therapies are lacking. A broader understanding of viral-host interactions could accelerate efforts to find new host-centric therapeutic targets. However, inefficient cell culture systems and limited replication markers compatible with pooled screening have precluded the widespread use of genetic perturbation screens. Here, we performed the first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection and integrated these results with two orthogonal pooled screens to identify host factors. We selected 72 genes for a multi-step assessment that included arrayed validation assays using both HBV infection and pgRNA transfection. We then independently tested thirteen genes using high-efficiency bulk KO experiments to guide further investigations of both antiviral and proviral factors. In both KO and siRNA-mediated knockdown experiments, depletion of the top antiviral factor, EXOC1, enhanced multiple HBV replication markers, and transcriptomic analysis revealed activation of hypoxia- and HIF-1 gene signatures. Three proviral factors, IRF2, WDR48, and ZCCHC14, were investigated in vivo using a human liver chimeric mouse model, which demonstrated that ZCCHC14 KO greatly reduced HBV replication and spread. Together, these complementary in vitro and in vivo platforms expand the catalog of HBV host factors and provide a scalable framework for host target discovery.

microbiology↗

bioRxiv2026

Community Curation of Microbial Metabolites Enables Biological Insights of Metabolomics Data

Microbial metabolites play a critical role in regulating ecosystems, including the human body and its microbiota. However, understanding the physiologically relevant role of these molecules, especially through liquid chromatography tandem mass spectrometry (LC-MS/MS)-based untargeted metabolomics, poses significant challenges and often requires manual parsing of a large amount of literature, databases, and webpages. To address this gap, we established the Collaborative Microbial Metabolite Center knowledgebase (CMMC-KB), a platform that fosters collaborative efforts within the scientific community to curate knowledge about microbial metabolites. The CMMC-KB aims to collect comprehensive information about microbial molecules originating from microbial biosynthesis, drug metabolism, exposure-related molecules, food, host-derived molecules, and, whenever available, their known activities. Molecules from other sources, including host-produced, dietary, and pharmaceutical compounds, are also included. By enabling direct integration of this knowledgebase with downstream analytical tools, including molecular networking, we can deepen insights into microbiota and their metabolites, ultimately advancing our understanding of microbial ecosystems.

bioinformatics↗

bioRxiv2026

A novel imprinting cluster at the porcine CRSP complex locus defines a species-specific imprinted domain

BackgroundGenomic imprinting is an epigenetic phenomenon that results in parent-of-origin-specific gene expression and has been extensively characterized in mice and humans. However, in pigs, imprinting has been investigated primarily through analyses of orthologs of known imprinted genes in mice and humans. The objective of this study was to examine DNA methylation status and gene expression at a porcine locus containing newly identified imprinted calcitonin receptor-stimulating peptide (CRSP)-encoding genes, to compare orthologous loci in mice and humans, and to investigate a potential underlying mechanism. ResultsAnalyses of differentially methylated regions (DMRs) between porcine parthenogenetic embryos and biparental controls revealed multiple parental DMRs at a locus we term the CRSP complex locus, which harbors CRSP-encoding genes that likely arose through gene duplication. In contrast, orthologous genomic intervals in mice and humans exhibited unmethylated promoters and lacked evidence of imprinting. Consistently, CRSP-encoding genes in pigs showed parent-of-origin-specific monoallelic expression, whereas genes within the orthologous locus in mice and humans were biallelically expressed. Further analysis indicated that porcine CRSP promoters are embedded within oocyte-expressed alternative transcripts and co-occurred with DNA methylation, suggesting a transcription-dependent imprinting mechanism. ConclusionsOur comparative analyses identified CRSP-encoding genes at the porcine CRSP complex locus as novel imprinted genes, indicating species-specific evolution of this imprinted domain. The results further suggest that lineage-specific gene duplication may have contributed to the emergence of imprinting at this locus.

genomics↗

bioRxiv2025

Spectral tuning and signaling of diverse and most red sensitive visual rhodopsins that drive high colour discrimination in Mantis Shrimp eyes

Stomatopod crustaceans (mantis shrimps) possess extraordinary eyes with unmatched visual opsin diversity, but their potential for broad colour detection remains unclear. Here, we report that seventeen opsins, mostly from the midband eye region, are involved in colour sensing and signaling via Gq/Gi-coupling and describe the underlying mechanisms of spectral tuning. Two mid-wavelength opsins preferentially absorb blue light and a majority of long-wavelength opsins absorb in the green range, but three absorb light beyond 600 nm, representing exquisite far-red-sensitive animal opsins. Several blue and red opsins exhibit photoreversibility with opposite spectral shifts explained by retinal binding interactions, revealing unrecognised photochemical flexibility in stomatopod photoreception. Row-specific spectral sensitivity results from combining carotenoid filters with multiple individually-tuned rhodopsins, altogether extending broad vision to the far-red spectrum.

biophysics↗

bioRxiv2025

Systematic comparison of colocalization methods using protein quantitative trait loci

Colocalization is frequently performed as a step to triage findings from genetic investigations linking molecular and disease data. However, the reliability and consistency of the various colocalization methods is not well understood. In particular, it is unclear whether a non-colocalization result should be taken as a definitive sign that two traits do not share a genetic cause in the region of interest, or merely as suggestive evidence. We use protein QTL data to benchmark four colocalization methods (coloc, coloc-SuSiE, prop-coloc, and colocPropTest), considering whether the methods conclude there is colocalization between datasets representing associations with the same protein. We consider a baseline scenario in which the associations come from the same dataset split in half at random, and scenarios in which the associations are with the same protein, but measured on different platforms or in different populations. In the baseline scenario, all methods report colocalization for the majority of proteins. In other scenarios, methods do not consistently report colocalization, they often report non-colocalization, and they often disagree. In the worst-case scenario, colocalization was only agreed by all four methods for 20% of proteins, despite our experiment being constructed to select for cases where colocalization is expected. This suggests caution in performing and interpreting colocalization analyses is warranted.

genetics↗

bioRxiv2025

Isolation of Pure Disease Specific Aging Trajectories in Spatial Transcriptomics via the Delta Delta Method

Disentangling normal aging from disease driven transcriptional change remains a major obstacle for spatial genomics. We introduce the Delta-Delta ({Delta}{Delta}) Method, a contrastive trajectory framework that resolves a four-dimensional progression (genes x cell types x brain regions x time) by subtracting the wild type (WT) aging trajectory from the transgenic (TG) trajectory to yield a pure disease trajectory ({Delta}{Delta}log2FC). The method is platform agnostic, integrates with common spatial transcriptomics workflows, and outputs direction and speed of change summaries, enriched pathways, and region and cell type specific maps. In a demonstration using G2-3 -synuclein TG mice and age matched WT controls at 6 and 10 months across hippocampus and midbrain, {Delta}{Delta} uncovered opposite regional dynamics in glutamatergic neurons and a convergent enrichment of RNA splicing pathways, corroborated by alternative splicing analyses. By explicitly modeling time while controlling for aging within each region and cell type, the {Delta}{Delta} Method isolates disease specific molecular programs that are obscured in conventional bulk or single cell analyses, and provides a generalizable framework for trajectory aware mechanistic target prioritization in neurodegeneration and other progressive conditions.

bioinformatics↗

bioRxiv2025

Rapid, accurate long- and short-read mapping to large pangenome graphs with vg Giraffe

1We previously introduced Giraffe, a short-read-to-pangenome graph mapper available in the vg pangenomics toolkit. Giraffe was fast and accurate for mapping short reads to human-scale pangenomes, but struggled with long reads. Long reads present a unique challenge to pangenome mapping algorithms due to their length and error profile, which allow them to take more topologically complex paths through the pangenome graph and increase the possible search space for the algorithm. We present updates to Giraffe that allow it to quickly and accurately map long reads to pangenome graphs. For both short and long reads, Giraffe mapping to a pangenome containing data from more than 450 human haplotypes, generated by the Human Pangenome Reference Consortium, is comparable in speed to linear mappers to human reference genomes; Giraffe is also over an order of magnitude faster than GraphAligner, the current state-of-the-art long-read-to-pangenome mapper. Its alignments produce similar or improved small and structural variant calling results, compared to those from commonly used graph-based and linear mappers. We additionally demonstrate using Giraffes long read alignments in a pangenome-guided assembly workflow, which is capable of producing more contiguous local assemblies than Hifiasm in our test regions.

bioinformatics↗

bioRxiv2025

Integrative analysis of gene networks associated with adipose and muscle traits in Hanwoo steers

ObjectiveThis study aims to characterize tissue-specific expression patterns in Hanwoo steers by identifying co-expression modules, functional pathways, and hub genes related to fat and muscle traits using Weighted Gene Co-expression Network analysis (WGCNA). MethodsRNA-Seq data were generated from three muscle tissues (longissimus muscle, tenderloin, and rump) and two fat tissues (back fat and abdominal fat) collected from six 30-month-old Hanwoo steers. Quality control of raw sequencing reads was performed using FastQC, and trimmed reads were aligned to the bovine reference genome (ARS-UCD1.3) using HISAT2. We also identified a gene co-expression network via WGCNA using normalized gene expression values. Modules were defined based on topological overlap and correlated with tissue-specific expression patterns. Modules with a significant association (p < 0.05) were used for functional enrichment based on Gene Ontology (GO) and KEGG pathways, as well as Protein-Protein Interaction Network analysis. ResultsA total of seven co-expression modules were identified by WGCNA and labeled in distinct colors (yellow, blue, red, brown, turquoise, green, black). Among them, the yellow and blue modules were positively associated with back fat, while the turquoise and green modules showed a negative correlation with abdominal fat. Additionally, the turquoise or green module was positively correlated with longissimus and rump tissues, indicating distinct gene expression patterns between fat and muscle. ConclusionThis study identified key co-expression modules and hub genes associated with muscle and fat metabolism. Notably, ARPC5 (blue module) was involved in lipid metabolism and energy storage, whereas AGPAT5 (turquoise module) was linked to maintaining muscle cell structure and function. These findings reveal biological mechanisms for tissue-specific gene regulation, providing targets for enhancing meat quality in Hanwoo.

bioinformatics↗

bioRxiv2025

Clinical and molecular characterisation of primary refractoriness to atezolizumab plus bevacizumab in patients with unresectable hepatocellular carcinoma.

BackgroundDespite improved outcomes with atezolizumab plus bevacizumab (A+B) in hepatocellular carcinoma (HCC), primary refractoriness (PRef), characterised by early progression or short-lived disease stabilisation following treatment, remains a significant and poorly understood clinical challenge. MethodsWe analysed 1296 patients with HCC and Child-Pugh A liver cirrhosis treated with frontline A+B (AB-real) and validated findings in 645 trial participants recruited to IMbrave150 and GO30140. PRef was defined by Society for the Immunotherapy of Cancer (SITC) criteria as progressive disease in the first 6 months after treatment initiation. Patients who achieved complete response, partial response or stable disease for [≥] 6 months were classified as responders. We performed a multi-parametric analysis of pre-treatment tumour tissue including machine learning-based quantification of tumour-infiltrating lymphocytes, imaging mass cytometry and RNA sequencing (RNAseq) to evaluate differences in the tumour microenvironment (TME) of PRef versus responding patients. We employed conditional inference tree analyses to provide a hierarchical organisation of determinants of PRef. ResultsAmong 677 AB-real and 378 trial patients evaluable by SITC criteria, PRef identified inferior median OS in comparison with responding patients (AB-real: 7.3 vs. 31.5 months, HR 3.7, 95%CI 2.8-8.5, p<0.001; Trials: 10.8 vs. NR, HR 4.6, 95%CI 3.3-6.3, p<0.001). PRef patients exhibited higher baseline systemic inflammation (neutrophil-to-lymphocyte ratio, NLR [≥]3), a distinctively immunosuppressive TME enriched in CD163+ tumour-associated macrophages and a higher Treg/Teff ratio. RNAseq of tumour tissue demonstrated lower intrinsic immunogenicity in PRef samples, characterised by repressed IFN-{gamma} and Teff signatures, with elevated myeloid infiltration. Conditional inference tree analysis identified IFN-{gamma} signature downregulation combined with NLR [≥]3 as the strongest contributor of PRef. ConclusionsPRef to A+B identifies a distinct biological entity characterised by unopposed systemic inflammation, myeloid cell infiltration and T-cell depletion. Targeting myeloid-mediated immunosuppression, particularly in patients with low IFN-{gamma} signature expression and elevated NLR might enhance responsiveness to A+B. HighlightsO_LIPrimary refractoriness to atezolizumab plus bevacizumab in hepatocellular carcinoma, as defined by SITC criteria, is associated with poor clinical outcomes. C_LIO_LITumour microenvironment profiling reveals an immunosuppressive phenotype characterized by high myeloid infiltration, reduced interferon-{gamma} signalling, and T-cell depletion. C_LIO_LIThe combination of systemic inflammation and low IFN-{gamma} signature expression strongly predicts primary refractoriness and may inform therapeutic decision-making. C_LI

cancer biology↗

bioRxiv2025

EvANI benchmarking workflow for evolutionary distance estimation

1Advances in long-read sequencing technology has led to a rapid increase in high-quality genome assemblies. These make it possible to compare genome sequences across the Tree of Life, deepening our understanding of evolutionary relationships. Average nucleotide identity (ANI) is a distance measure that has been applied to species delineation, building of guide trees, and searching large sequence databases. Since computing ANI is computationally expensive, the field has increasingly turned to sketch-based approaches that use assumptions and heuristics to speed this up. We propose a suite of simulated and real benchmark datasets, together with a rank-correlation-based metric, to study how these assumptions and heuristics impact distance estimates. We call this evaluation framework EvANI. With EvANI, we show that ANIb is the ANI estimation algorithm that best captures tree distance, though it is also the least efficient. We show that k-mer based approaches are extremely efficient and have consistently strong accuracy. We also show that some clades have inter-sequence distances that are best computed using multiple values of k, e.g. k = 10 and k = 19 for Chlamydiales. Finally, we highlight that approaches based on maximal exact matches may represent an advantageous compromise, achieving an intermediate level of computational efficiency while avoiding over-reliance on a single fixed k-mer length.

genomics↗

bioRxiv2025

Atomistic Mechanism of Non-Canonical Voltage Gating in TREK K2P Channels

Two-pore-domain K+ (K2P) channels are essential regulators of cellular excitability and respond to various external stimuli that also include membrane potential for most K2P channels. Voltage gating is particularly pronounced in members of the TREK/TRAAK subfamily. This voltage sensitivity was surprising, as K2P channels lack a classical voltage sensing domain. Prior studies have attributed this non-canonical voltage sensing mechanism to the unique ion-flux gating properties of the K2P selectivity filter (SF), where inward currents induce fast inactivation, while depolarization activates the SF. Here, we performed large-scale molecular dynamics (MD) simulations across various voltages to gain an atomistic understanding of ion-flux gating in TREK channels. Our analysis revealed an asymmetric stability difference in the SF, enabling water influx into the filter due to conformational flexibility on the extracellular side. Inward flux inactivation occurs when water entry halts ion permeation, followed by the unbinding of three K+ ions, consistent with gating charge analysis. Additionally, MD simulations of TREK-2 mutants and the TWIK-1 channel, which exhibited increased SF flexibility, showed augmented SF water occupancy, aligning with their electrophysiological phenotypes. Key experimental evidence of this mechanism was provided by electrophysiology measurements, which showed that high extracellular sucrose slowed ion-flux inactivation by reducing water entry into the SF. These findings uncover the atomistic mechanism of voltage gating in TREK K2P channels and pave the way for exploring non-canonical voltage gating mechanisms in other ion channels.

biophysics↗

bioRxiv2025

Endocannabinoids facilitate transitory reward engagement through retrograde gain control

Neuromodulatory signaling is poised to serve as a neural mechanism for gain control, acting as a crucial tuning factor to influence neuronal activity by dynamically shaping excitatory and inhibitory fast neurotransmission. The endocannabinoid (eCB) signaling system, the most widely expressed neuromodulatory system in the mammalian brain, is known to filter excitatory and inhibitory inputs through retrograde, pre-synaptic action. However, whether eCBs exert retrograde gain control to ultimately facilitate reward-seeking behaviors in freely moving mammals is not established. Using a suite of in vivo physiological, imaging, genetic and machine learning-based approaches, we report a fundamental role for eCBs in controlling behavioral engagement in reward-seeking behavior through a defined thalamo-striatal circuit.

neuroscience↗

bioRxiv2024

Structural insights into the opening mechanism of C1C2 channelrhodopsin

Channelrhodopsins, light-gated cation channels, enable precise control of neural cell depolarization or hyperpolarization with light in the field of optogenetics. This study integrates time-resolved serial crystallography and atomistic molecular dynamics (MD) simulations to resolve the structural changes during C1C2 channelrhodopsin activation. Our observations reveal that within the crystal environment, C1C2 predominantly remains in a light-activated state with characteristics of the M390 intermediate. Here, rearrangement of retinal within its binding pocket partially opens the central gate towards the extracellular vestibule. These structural changes initiate channel opening but were insufficient to allow K+ flow. Adjusting protonation states to represent the subsequent N520 intermediate in our MD simulations induced further conformational changes, including rearrangements of transmembrane helices 2 and 7, that opened the putative ion- translocation pathway. This allows spontaneous but low cation but not anion conduction, that matches experiments. Our findings provide critical structural insights into key intermediates of the channel opening mechanism, enhancing our understanding of ion conduction and selectivity in channelrhodopsins at an atomistic level.

biophysics↗

bioRxiv2024

Genomic re-sequencing reveals substantial mutational divergence across genetically engineered strains of model archaea

Because archaea are the evolutionary ancestors of eukaryotes, archaeal molecular biology has been a topic of intense recent research. The hypersaline adapted archaeal species Halobacterium salinarum and Haloferax volcanii serve as important model organisms because facile tools enable genetic manipulation. As a result, the number of strains in circulation among the haloarchaeal research community has increased over the last few decades. However, the degree of genetic divergence and effects on genetic integrity during inter-lab transfers remain unclear. To address this question, we performed whole genome re-sequencing on a cross-section of wild-type, parental, and knockout strains in both model species. Integrating these data with existing repositories of re-sequencing data, we identify mutations that have arisen in a collection of 60 strains, sampled from 2 species across 8 different labs. Independent of sequencing, we construct strain lineages, identifying branch points and significant genetic effects in strain history. Combining this with our sequencing data, we identify small clusters of mutations that definitively separate lab strains. Additionally, an analysis of gene knockout strains suggests that roughly 1 in 3 strains currently in use harbors second-site mutations of potential phenotypic impact. Overall, we find that divergence among lab strains is thus far minimal, though as the archaeal research community continues to grow, careful strain provenance and genomic re-sequencing are required to keep inter-lab divergence to a minimum, prevent the compounding of mutations into fully independent lineages, and maintain the current high degree of reproducible research between lab groups in the haloarchaeal research community. Data SummaryNovel sequencing data for this project was submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and can be found under bioproject accession PRJNA1120443. SRA accessions for previously published sequencing data are available in supplementary table 1. R code for performing analysis and generating figures is available at https://github.com/andrew-soborowski/halophile_genome_resequencing. Impact StatementArchaea are important due to their shared evolutionary history with eukaryotes. As the archaeal research community grows, keeping track of the genetic integrity of archaeal strains of interest is necessary. In particular, routine genetic manipulations and the common practice of sharing strains between labs allow mutations to arise in lab stocks. If these mutations affect cellular processes, they may jeopardize the reproducibility of work between research groups and confound the results of future studies. In this work, we examine DNA sequences from 60 strains across two species of archaea. We identify shared and unique mutations occurring between and within strains. Independently, we trace the lineage of each strain, identifying which genetic manipulations lead to observed off-target mutations. While overall divergence across labs is minimal so far, our work highlights the need for labs to continue proper strain husbandry.

genomics↗

bioRxiv2024

Disruption of mitochondrial folate metabolism leads to mitochondrial DNA leakage and activation of apoptotic or inflammatory pathways

Folate-mediated one-carbon metabolism (FOCM) is required for the biosynthesis of purines, thymidylate (dTMP), and methionine. Maintenance of adequate cellular dTMP levels is essential to preserve the integrity of the nuclear and mitochondrial genomes. Inadequate dTMP production leads to uracil misincorporation into mitochondrial DNA (mtDNA) and impaired mitochondrial function. However, the mechanisms whereby uracil in mtDNA impairs mitochondrial function are uncharacterized. The release of mtDNA into the cytosol acts as a damage-associated molecular pattern (DAMP), resulting in inflammation and cell death. Previously, we reported that disrupting mitochondrial dTMP synthesis through serine hydroxymethyltransferase 2 (Shmt2) heterozygosity increases uracil in mtDNA and impairs mitochondrial function in mice. This study aimed to investigate whether impairment of mitochondrial FOCM through Shmt2 disruption leads to the release of mtDNA into the cytosol. Shmt2+/- MEF cells showed a > 2-fold increase in cytosolic mtDNA leakage compared to Shmt2+/+ cells (p<0.05). There was no significant difference in total mtDNA content between Shmt2+/+ and Shmt2+/- MEF cells. MEFs with decreased Shmt2 expression activated apoptosis by the ratio of cleaved caspase-3 to caspase-3. In addition, SHMT2 knock-out (SHMT2 KO) haploid chronic myeloid leukemia (HAP1) cells also exhibited increased cytosolic mtDNA content compared to the wild-type (WT) HAP1 cells. HAP1 lacking Shmt2 expression activated the cGAS/STING pathway, but suppressed apoptosis, compared to WT HAP1 cells. This study demonstrates that decreased Shmt2 expression leads to cytosolic mtDNA leakage and that the downstream effects of mtDNA leakage vary by cell type.

biochemistry↗