Biology subjects

Cox, K.

Publications and source records attributed to Cox, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Reference-free clustering as an epidemiological tool for Mycobacterium tuberculosis lineage typing

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) is widely used in the epidemiological investigation of recent transmission events, resulting in high-resolution strain typing. Accurate and rapid strain typing is essential for informing outbreak investigations and guiding tuberculosis control strategies. However, the gold-standard reference-guided SNP-calling pipeline currently used for strain typing relies on computationally intensive reference-mapping approaches, making it challenging to perform in many high-burden, resource-limited settings, where simplified and scalable genomic tools are urgently needed. To address these limitations, we explored reference-free methods for medium resolution epidemiology, namely Mtb strain (lineage) typing, using a dataset of 535 complete genomes spanning the human- and animal-adapted lineages. Illumina paired-end reads were simulated from each complete genome, assembled, and analysed using three reference-free, k-mer-based tools: MASH, PopPUNK, and SKA2 (Split K-mer Analysis). Genetic distances were generated for each method and compared with a ground truth lineage assignment from with TB Profiler. Our results demonstrated that reference-free methods can effectively distinguish Mtb lineages, with SKA2 showing the most promising performance across all datasets. SKA2 consistently recovered lineage and sub-lineage structure with high accuracy, demonstrating strong potential as an alternative to traditional WGS workflows. These findings highlight the utility of reference-free methods, particularly SKA2, for enabling accessible, scalable, and rapid Mtb strain typing, while supporting genomic epidemiology with low computational resources.

bioinformatics↗

bioRxiv2025

Deletion or Targeted Blockade of FcγRIIb (CD32b) Impairs α-Syn Propagation In-Vivo

Parkinsons disease (PD), the most common neurodegenerative movement disorder, is characterised by pervasive deposition of alpha-synuclein (-Syn) aggregates and the death of dopaminergic neurons. Fc gamma receptor IIb (Fc{gamma}RIIb or CD32b), the sole inhibitory Fc{gamma}R in humans (h) and mice (m), serves as a molecular conduit for intercellular -Syn transmission in-vitro. Here, we demonstrate that Fc{gamma}RIIb facilitates -Syn propagation and neurotoxicity in-vivo using the pre-formed fibril (PFF) -Syn model in mice. Genetic ablation of mFc{gamma}RII attenuated PFF -Syn-induced Lewy pathology, dampened associated neuroinflammatory responses, and preserved nigrostriatal dopaminergic neurons. Furthermore, pharmacological blockade of hFc{gamma}RIIb using clinically-related monoclonal antibodies mitigated acute-phase -Syn pathology in hFc{gamma}RIIb-transgenic mice following PFF challenge. Collectively, our results indicate that Fc{gamma}RIIb is a mediator of -Syn propagation in-vivo and highlight it as a tractable therapeutic target against -synucleinopathies like PD.

neuroscience↗

bioRxiv2025

Evaluating a SNP calling pipeline for Mycobacterium leprae

Mycobacterium leprae is the causative agent of leprosy, a persisting disease characterised by skin lesions and peripheral nerve numbness. Attempts to sequence the genome have been challenged by its inability to be cultured on artificial media. Advances in DNA extraction and sequencing technologies have enabled molecular epidemiology to be introduced for M. leprae. However, a validated SNP calling pipeline does not currently exist. A ground truth of pairwise SNP differences was first created using Minimap2 and three complete genomes of M. leprae. Using simulated reads from each of these genomes and the others as a reference, we evaluated the precision and recall of short-read SNP calling using Snippy. Approximately 80% of SNPs were called with false positives only due to ambiguous bases in certain genomes. Repeat region masking was found to be unnecessary for M. leprae SNP calling, unlike for M. tuberculosis. We find that SNP calling from short reads is robust and highly accurate from M. leprae, showing promise as a tool for molecular epidemiology studies to increase case detection and inform leprosy control strategies.

genomics↗

bioRxiv2025

Gastric cancer treatment target identified from an accelerated Helicobacter-induced gastric cancer mouse model

Helicobacter pylori (H. pylori) infection and consequent inflammation leads to gastric cancer (GC). Despite the prevalence of this bacterium and availability of genomic data, targeted therapies for GC are still early in development. Previously in our accelerated Helicobacter-induced gastric cancer mouse model we identified several differentially expressed genes (DEGs), including PSMB8 (proteasome subunit beta type 8, also called LMP7); one of the immune subunits of the immunoproteasome, which has been associated with disease severity in multiple cancers. We observed elevated expression of PSMB8 in our accelerated gastric cancer model, in the human gastric cancer cell line (MKN45), and in gastric cancer patient samples. Moreover, we identified carfilzomib as a potential drug that targets PSMB8. Therefore, to test its efficacy against gastric cancer, nude mice were subcutaneously implanted with MKN45 derived tumors and treated with carfilzomib, alone or in combination with 5-fluorouracil (5-FU), the standard care drug. The effectiveness of drug treatment was measured by tumor growth, cell proliferation, and apoptosis. We observed that carfilzomib retarded tumor growth, inhibited cell proliferation and induced apoptosis. These results strongly suggest that carfilzomib has a robust anti-tumor activity and is a suitable drug candidate for targeted therapy in gastric cancer.

cancer biology↗

bioRxiv2025

The Freshwater Sounds Archive

Freshwater ecosystems are full of underwater sounds produced by amphibians, aquatic arthropods, reptiles, plants, fishes, and methane bubbles escaping from the sediment. Although much headway has been made in recent years investigating the overall soundscapes of various freshwater ecosystems around the world, there remains a significant knowledge gap in our collective inability to accurately and reliably link recorded sounds with the species that produced them. Here, we present The Freshwater Sounds Archive, a new global initiative, which seeks to address this knowledge gap by collating species-specific freshwater sound recordings into a publicly available database. By means of metadata collection, we also present a snapshot of the species studied, the recording equipment, and recording parameters used by freshwater ecoacousticians globally. In total, 61 entries were submitted to the archive between the 4th of March 2023 and the 30th of April 2025, representing 16 countries and 6 continents. The most numerous taxonomic group was arthropods (29 entries), followed by fishes (14 entries), amphibians (10 entries), macrophytes (7 entries), and a freshwater mollusk (1 entry). The majority of the submissions were from European countries (27 entries), of which the United Kingdom was the most represented with 14 entries. The next most represented region was North America (11 entries), followed by South America (8 entries), Oceania and Asia (5 entries each), Africa (3 entries), and the Middle East and Central America with 1 entry each. The global south, polar regions, and areas with an elevation >500 m (asl) were underrepresented. The field of freshwater ecoacoustics to date has largely focused on the analysis of sound types due to a current lack of knowledge of species-specific sounds. The Freshwater Sounds Archive presents an opportunity to move beyond the sound type approach, and towards an approach with higher taxonomic resolution, ultimately resulting in species-specific descriptions. Furthermore, The Freshwater Sounds Archive will provide freshwater ecoacousticians with one of the main tools required to start creating annotated training datasets for machine learning models from soundscape recordings by referring to known species sounds present in the archive. In the long-term, this will result in the automatic detection and classification of species-specific freshwater sounds from soundscape recordings, such as indicator, invasive, and endangered species.

ecology↗

bioRxiv2025

Age-Associated Decline in Autophagy Pathways in Retinal Pigment Epithelium and Protective Effects of Topical Trehalose in Light-induced Outer Retinal Degeneration in Mice

Age is a primary risk factor for chronic conditions, including age-related macular degeneration (AMD). Impairments in autophagy processes are implicated in AMD progression, but the extent of autophagys contribution and its therapeutic potential remain ambiguous. This study investigated age-associated transcriptomic changes in autophagy pathways in the retinal pigment epithelium (RPE) and evaluated the protective effects of topical trehalose, an autophagy-enhancing small molecule, against light-induced outer retinal degeneration in mice. Transcriptomic analysis of human RPE/choroid and mouse RPE revealed consistent downregulation of autophagy pathways with age, alongside variable changes as AMD severity progressed. Given the age- and AMD-associated perturbation of autophagy pathways, we examined trehalose treatment in vitro, which enhanced autophagic flux and restored mitochondrial respiratory function in primary murine RPE cells exposed to oxidative stress. In vivo, topical trehalose improved autophagy-lysosome activity in mouse RPE, demonstrated by elevated LC3B turnover and SQSTM1/p62 degradation. Furthermore, trehalose eyedrops protected mice from light-induced damage to the RPE and photoreceptors, preserving outer nuclear layer thickness, RPE morphology, and junctional F-actin organization. Taken together, the data support that age-related decline and severe dysregulation in autophagy contributed to AMD progression. By restoring autophagic flux, topical trehalose demonstrates therapeutic potential to address early autophagy-related pathological changes in AMD.

pathology↗

bioRxiv2024

Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton

Xanthomonas spp. employ transcription activator-like effectors (TALEs) to promote pathogenicity by activating host susceptibility (S) genes. Cotton GhSWEET10 is an S gene targeted by a TALE in an early isolate of Xanthomonas citri pv. malvacearum (Xcm), but not by recent field Xcm isolates. To understand the pathogenicity shift in Xcm and its adaptation to cotton, we assembled the whole genome and the TALE repertoire of three recent Xcm Texas field isolates. A newly evolved TALE, Tal7b, activated different GhSWEET genes, GhSWEET14a and GhSWEET14b. Simultaneous activation of GhSWEET14a and GhSWEET14b resulted in pronounced water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identified a pectin lyase as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9-based gene editing supported the function of GhSWEETs as S genes in cotton bacterial blight and the promise of disrupting the TALE-binding site in these genes to control the disease. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes simultaneously to promote pathogenicity.

plant biology↗

bioRxiv2024

Resistance patterns in drug-adapted cancer cell lines reflect complex evolution in clinical tumors

BackgroundHere, we introduce a novel set of triple-negative breast cancer (TNBC) cell lines consisting of MDA-MB-468, HCC38, and HCC1806 and their sublines adapted to cisplatin, doxorubicin, eribulin, paclitaxel, gemcitabine, or 5-fluorouracil. MethodsThe cell lines were characterized by whole exome sequencing and the determination of drug-response profiles. Moreover, genes harbouring resistance-associated mutations were investigated using TCGA data for potential clinical relevance. ResultSequencing combined with TCGA-derived patient data resulted in the identification of 682 biomarker candidates in the pan-cancer analysis. Thirty-five genes were considered the most promising candidates because they harboured resistance-associated variants in at least two resistant sublines, and their expression correlated with TNBC patient survival. Exome sequencing and response profiles to cytotoxic drugs and DNA damage response inhibitors identified revealed remarkably little overlap between the resistant sublines, suggesting that each resistance formation process follows a unique route. All of the drug-resistant TNBC sublines remained sensitive or even displayed collateral sensitivity to a range of tested compounds. Cross-resistance levels were lowest for the CHK2 inhibitor CCT241533, the PLK1 inhibitor SBE13, and the RAD51 recombinase inhibitor B02, suggesting that CHK2, PLK1, and RAD51 are potential drug targets for therapy-refractory TNBC. ConclusionsWe present novel preclinical models of acquired drug resistance in TNBC and many novel candidate biomarkers for further investigation. The finding that each cancer cell line adaptation process follows an unpredictable route reflects recent findings on cancer cell evolution in patients, supporting the relevance of drug-adapted cancer cell lines as preclinical models of acquired resistance.

cancer biology↗

bioRxiv2023

ATAC and SAGA histone acetyltransferase modules facilitate transcription factor binding to nucleosomes in an acetylation independent manner

Transcription initiation involves the coordination of multiple events, starting with activators binding specific DNA target sequences, which recruits transcription co-activators to open chromatin and enable binding of general transcription factors and RNA polymerase II to promoters. Two key human transcriptional coactivator complexes, ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), target genomic loci to increase promoter accessibility. To better understand the function of ATAC and SAGA histone acetyltransferase (HAT) complexes, we used in vitro biochemical and biophysical assays to characterize human ATAC and SAGA HAT module interactions with nucleosomes and how a transcription factor (TF) coordinates these interactions. We found that ATAC and SAGA HAT modules bind nucleosomes with high affinity, independent of post-translational modifications (PTMs) and TFs. ATAC and SAGA HAT modules directly interact with the VP16 activator domain and a TF containing this domain enhances HAT module acetylation activity. Surprisingly, ATAC and SAGA HAT modules increase TF binding to its DNA target site within the nucleosome by an order of magnitude independent of histone acetylation. Altogether, our results reveal synergistic coordination between HAT modules and a TF, where ATAC and SAGA HAT modules: (i) acetylate histones to open chromatin, and (ii) facilitate TF targeting within nucleosomes independently of their acetylation activity.

molecular biology↗

bioRxiv2023

Replenishing Age-Related Decline of IRAK-M Expression in Retinal Pigment Epithelium Attenuates Outer Retinal Degeneration

Unchecked, chronic inflammation is a constitutive component of age-related diseases, including age-related macular degeneration (AMD). Here we identified interleukin-1 receptor-associated kinase (IRAK)-M as a key immunoregulator in retinal pigment epithelium (RPE) that declines with age. Rare genetic variants of IRAK-M increased the likelihood of AMD. IRAK-M expression in RPE declined with age or oxidative stress and was further reduced in AMD. IRAK-M-deficient mice exhibited increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M disrupted RPE cell homeostasis, including compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of AAV-expressing IRAK-M rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in IRAK-M- deficient mice. Our data support that replenishment of IRAK-M expression may redress dysregulated pro-inflammatory processes in AMD, thereby treating degeneration. One Sentence SummaryIRAK-M is a protective molecule and promising therapeutic target for macular degeneration

immunology↗

bioRxiv2023

Genome-wide methylome stability and parental effects in the worldwide distributed Lombardy poplar

BackgroundDespite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome. ResultsWe present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH-context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offsprings phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment. ConclusionsGenome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as bio-markers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.

genomics↗

bioRxiv2023

SOS2 regulates the threshold of mutant EGFR-dependent oncogenesis

Son of Sevenless 1 and 2 (SOS1 and SOS2) are RAS guanine nucleotide exchange factors (RasGEFs) that mediate physiologic and pathologic RTK-dependent RAS activation. Here, we show that SOS2 modulates the threshold of epidermal growth factor receptor (EGFR) signaling to regulate the efficacy of and resistance to the EGFR-TKI osimertinib in lung adenocarcinoma (LUAD). SOS2 deletion sensitized EGFR-mutated cells to perturbations in EGFR signaling caused by reduced serum and/or osimertinib treatment to inhibit PI3K/AKT pathway activation, oncogenic transformation, and survival. Bypass RTK reactivation of PI3K/AKT signaling represents a common resistance mechanism to EGFR-TKIs; SOS2 KO reduced PI3K/AKT reactivation to limit osimertinib resistance. In a forced HGF/MET-driven bypass model, SOS2 KO inhibited HGF-stimulated PI3K signaling to block HGF-driven osimertinib resistance. Using a long term in situ resistance assay, a majority of osimertinib resistant cultures exhibited a hybrid epithelial/mesenchymal phenotype associated with reactivated RTK/AKT signaling. In contrast, RTK/AKT-dependent osimertinib resistance was markedly reduced by SOS2 deletion; the few SOS2 KO cultures that became osimertinib resistant primarily underwent non-RTK dependent EMT. Since bypass RTK reactivation and/or tertiary EGFR mutations represent the majority of osimertinib-resistant cancers, these data suggest that targeting SOS2 has the potential to eliminate the majority of osimertinib resistance. One sentence summarySOS2 modulates the threshold of EGFR-PI3K signaling to regulate the efficacy of and resistance to osimertinib.

cancer biology↗

bioRxiv2022

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells (DTPs) within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 or KSR1 both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid initiating cells (SICs) in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA co-mutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA- mutated and PIK3CA wildtype cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS- mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying co-mutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations. Significance StatementWe provide an experimental framework for evaluating both adaptive and acquired resistance to RAS pathway-targeted therapies and demonstrate how targeting specific RAS pathway signaling intermediates SOS1 or KSR1 enhanced effectiveness of and prevented resistance to MEK inhibitors in KRAS-mutated cancer cells with genotypic precision. The contribution of either effector was dependent upon the mutational landscape: SOS1 inhibition synergized with trametinib in KRASG12/G13-mutated cells expressing WT PI3K but not in KRASQ61-mutated cells or if PIK3CA is mutated. KSR1 deletion inhibited MEK/ERK complex stability and was effective in cells that are unresponsive to SOS1 inhibition. These data demonstrate how a detailed understanding of functional dependencies imposed both by allele specific KRAS mutations and specific co-mutations facilitates the optimization of therapeutic combinations.

cancer biology↗

bioRxiv2022

Immune Contexture of Isocitrate Dehydrogenase stratified Human Gliomas

The brain tumor immune microenvironment (TIME) continuously evolves during glioma progression, but only a limited view of a highly complex glioma associated immune contexture across isocitrate dehydrogenase mutation (IDH) classified gliomas is known. Herein, we present an unprecedentedly comprehensive view of myeloid and lymphoid cell type diversity based on our single cell RNA sequencing and spectral cytometry-based interrogation of tumor-associated leukocytes from fifty-five IDH stratified primary and recurrent human gliomas and three non-glioma brains. Our analyses revealed twenty-two myeloid and lymphoid cell types within and across glioma subtypes. Glioma severity correlated with microglial attrition concomitant with a continuum of invading monocyte-derived microglia-like and macrophages amongst other infiltrating conventional T and NK lymphocytes and unconventional mucosa associated invariant T (MAIT) cells. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). Furthermore, we identified phagocytosis and antigen presentation gene modules enriched in Triggering receptor expressed on myeloid (TREM)-2+ cells as a putative anti-glioma axis. Accelerated glioma growth was observed in Trem2 deficient mice implanted with CT2A glioma cells affirming the anti-glioma role of TREM2+ myeloid cells. In addition to providing a comprehensive landscape of glioma-specific immune contexture, our investigations discover TREM2 as a novel immunotherapy target for brain malignancies.

immunology↗

bioRxiv2022

Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model

We have developed a novel inducible Huntingtons disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant Huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt ([~]50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow up revealed that the benefits, in all mHtt lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12- months of age and late mHtt lowering was unable to ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefit in humans.

neuroscience↗

bioRxiv2022

HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification

HIV-1 DNA integration into the host chromosomes is carried out by the preintegration complex (PIC). The PIC contains the viral DNA, virally encoded integrase enzyme and other critical viral/host factors. The PIC-associated viral DNA is preferentially integrated into gene bodies of actively transcribing genes. Here, we identify a biochemical mechanism underlying the preference of PIC-mediated viral DNA integration (PIC-VDI). Specifically, we observed that the PIC-VDI into human chromatin is preferred over the genomic DNA. Surprisingly, nucleosome core particles without any histone modifications were not preferred for PIC-VDI when compared to the analogous naked DNA. However, PIC-VDI was markedly enhanced with nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to HIV-1 DNA integration preference. Interestingly, we observed that nucleosomes with flanking linker DNA promoted PIC-VDI in the presence of LEDGF/p75. We also discovered that nucleosomes with linker DNA and H3K36me3 served as the optimal substrate for PIC-VDI. Mapping of the integration sites within these substrates identified preference of specific regions of the nucleosome core DNA for integration. Finally, we provide biochemical and genetic evidence that histone H1 protein, that condenses the chromatin, negatively regulates HIV-1 DNA integration, consistent with the integration preference for open chromatin structure. Collectively, these results identify the role of specific chromatin marks that drive HIV-1 integration preference and define the optimal substrate requirement for efficient DNA integration by the PIC.

microbiology↗

bioRxiv2021

Overcoming the effect of ASH1L haploinsufficiency on stem cells amenability to genome editing and differentiation into the neuronal lineage - a technical report

Genome editing and neuronal differentiation protocols have proliferated in the last decade. Mutations in genes that control pluripotency could lead to a potential obstacle with regards to the survival and differentiation potential of the genome-edited cell lines. Here we describe a protocol for the generation, and differentiation, of cell lines containing CRISPR/Cas9 induced mutations in the histone methyl transferase ASH1L. This chromatin modifier was previously implicated in hematopoietic stem cell pluripotency and is a major genetic risk factor for autism spectrum disorders (ASD). We find that haploinsufficiency of ASH1L leads to decreased NANOG gene expression leading to reduce cell survival and increased spontaneous differentiation. We report a method that provides improved single-cell survival with higher colony formation efficiency in ASH1L mutant stem cells. Additionally, we describe a modified dual-SMAD inhibition neuronal induction methodology that permits the successful generation of human neurons with mutations in ASH1L, in a smaller scale than previously reported methods. With our modified CRISPR-genome editing and neuronal differentiation protocols, it is possible to generate genome-edited stem cells containing mutations in genes that impact pluripotency and could affect subsequent cell lineage specific differentiation. Our detailed technical report presents cost-effective strategies that will benefit researchers focusing on both translational and basic science using stem cell experimental systems.

neuroscience↗

bioRxiv2020

Reintroduced native Populus nigra in restored floodplain reduces spread of exotic poplar species

Exotic Populus taxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exotic Populus taxa on softwood riparian forest development along the river Common Meuse after introducing native Populus nigra and after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonised restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinated P. nigra females, we investigated natural hybridization between frequently planted cultivated poplars and native P. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified as P. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified as P. nigra. For three seedlings (4%), paternity was assigned to a cultivar of P. x canadensis. Almost two decades after reintroducing P. nigra, the constitution of the seed and pollen pools changed in the study area in favour of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programmes of the native P. nigra can vigorously outcompete the exotic gene flows and strongly reduce the impact of exotic Populus taxa on the softwood riparian forest development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗