Biology subjects

Kim, E.

Publications and source records attributed to Kim, E..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Second Boost of Omicron SARS-CoV-2 S1 Subunit Vaccine Induced Broad Humoral Immune Responses in Elderly Mice

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death from the disease. However, repeated homologous boosters, while considered a solution for severe forms of the disease caused by new SARS-CoV-2 variants in elderly individuals and immunocompromised patients, cannot provide complete protection against breakthrough infections. This highlights the need for alternative platforms for booster vaccines. In our previous study, we assessed the boost effect of the SARS-CoV-2 Beta S1 recombinant protein subunit vaccine (rS1Beta) in aged mice primed with an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) via subcutaneous injection or intranasal delivery, which induced robust humoral immune responses (1). In this follow-up study, we demonstrated that a second booster dose of a non-adjuvanted recombinant Omicron (BA.1) S1 subunit vaccine with Toll-like receptor 4 (TLR4) agonist RS09 (rS1RS09OM) was effective in stimulating strong S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants in 100-week-old mice. Importantly, the second booster dose elicits cross-reactive antibody responses, resulting in ACE2 binding inhibition against the spike protein of SARS-CoV-2 variants, including Omicron (BA.1) and its subvariants. Interestingly, the levels of IgG and neutralizing antibodies correlated with the level of ACE2 inhibition in the booster serum samples, although Omicron S1-specific IgG level showed a weaker correlation compared to Wuhan S1-specific IgG level. Furthermore, we compared the immunogenic properties of the rS1 subunit vaccine in young, middle-aged, and elderly mice, resulting in reduced immunogenicity with age, especially an impaired Th1-biased immune response in aged mice. Our findings demonstrate that the new variant of concern (VOC) rS1 subunit vaccine as a second booster has the potential to offer cross-neutralization against a broad range of variants and to improve vaccine effectiveness against newly emerging breakthrough SARS-CoV-2 variants in elderly individuals who were previously primed with the authorized vaccines.

immunology↗

bioRxiv2024

A gene-rich mitochondrion with a unique ancestral protein transport system

Mitochondria originated from an ancient endosymbiotic event involving an alphaproteobacterium1-3. Over time, these organelles reduced their gene content massively, with most genes being transferred to the host nucleus before the last eukaryotic common ancestor (LECA)4. This process has yielded varying gene compositions in modern mitogenomes, including the complete loss of this organellar genome in some extreme cases5-14. At the other end of the spectrum, Jakobids harbor the largest mitogenomes, encoding 60-66 proteins8. Here, we introduce the mitogenome of Mantamonas sphyraenae, a protist from the deep-branching CRuMs supergroup15,16. Remarkably, it boasts the most gene-rich mitogenome outside of jakobids, by housing 91 genes, including 62 protein-coding ones. These include rare homologs of the four subunits of the bacterial-type cytochrome c maturation system I (CcmA, CcmB, CcmC, and CcmF), alongside a unique ribosomal protein S6. During the early evolution of this organelle, gene transfer from the proto-mitochondrial endosymbiont to the nucleus became possible thanks to systems facilitating the transport of proteins synthesized in the host cytoplasm back to the mitochondrion. In addition to the universally found eukaryotic protein import systems, jakobid mitogenomes were reported to uniquely encode the SecY transmembrane protein of the bacterial Type II secretion system; its evolutionary origin was however unclear. The Mantamonas mitogenome not only encodes SecY but also SecA, SecE, and SecG, making it the sole eukaryote known to house a complete mitochondrial Sec translocation system. Furthermore, our phylogenetic and comparative genomic analyses provide compelling evidence for the alphaproteobacterial origin of this system, establishing its presence in LECA.

evolutionary biology↗

bioRxiv2024

Parsnp 2.0: Scalable Core-Genome Alignment for Massive Microbial Datasets

MotivationSince 2016, the number of microbial species with available reference genomes in NCBI has more than tripled. Multiple genome alignment, the process of identifying nucleotides across multiple genomes which share a common ancestor, is used as the input to numerous downstream comparative analysis methods. Parsnp is one of the few multiple genome alignment methods able to scale to the current era of genomic data; however, there has been no major release since its initial release in 2014. ResultsTo address this gap, we developed Parsnp v2, which significantly improves on its original release. Parsnp v2 provides users with more control over executions of the program, allowing Parsnp to be better tailored for different use-cases. We introduce a partitioning option to Parsnp, which allows the input to be broken up into multiple parallel alignment processes which are then combined into a final alignment. The partitioning option can reduce memory usage by over 4x and reduce runtime by over 2x, all while maintaining a precise core-genome alignment. The partitioning workflow is also less susceptible to complications caused by assembly artifacts and minor variation, as alignment anchors only need to be conserved within their partition and not across the entire input set. We highlight the performance on datasets involving thousands of bacterial and viral genomes. AvailabilityParsnp is available at https://github.com/marbl/parsnp

bioinformatics↗

bioRxiv2024

Cannabis sativa (Hemp) seed-derived peptides WVYY and PSLPA modulate the Nrf2 signaling pathway in human keratinocytes

Cannabis sativa (Hemp) seeds are used widely for cosmetic and therapeutic applications, and contain peptides with substantial therapeutic potential. Two key peptides, WVYY and PSLPA, extracted from hemp seed proteins were the focal points of this study. These peptides have emerged as pivotal contributors to the various biological effects of hemp seed extracts. Consistently, in the present study, the biological effects of WVYY and PSLPA were explored. We confirmed that both WVYY and PSLPA exert antioxidant and antibacterial effects and promote wound healing. We hypothesized the involvement of the nuclear factor erythroid 2- related factor 2 (Nrf2) signaling pathway in these observed effects, given that Nrf2 is reported to be a central player in the regulation of these observed effects. Molecular-level investigations unequivocally confirmed the role of the Nrf2 signaling pathway in the observed effects of WVYY and PSLPA, specifically their antioxidant effects. Our study highlights the therapeutic potential of hemp seed-derived peptides WVYY and PSLPA, particularly with respect to their antioxidant effects, and provides a nuanced understanding of their effects. Further, our findings can facilitate the investigation of targeted therapeutic applications and also underscore the broader significance of hemp extracts in biological contexts.

cell biology↗

bioRxiv2024

Song Torrent: A modular, open-source 96-chamber audio and video recording apparatus with optogenetic activation and inactivation capabilities for Drosophila

BackgroundO_LIMany Drosophila species use acoustic communication during courtship and studies of these communication systems have provided insight into neurobiology, behavioral ecology, ethology, and evolution. C_LIO_LIRecording Drosophila courtship sounds and associated behavior is challenging, especially at high throughput, and previously designed devices are relatively expensive and complex to assemble. C_LI ResultsO_LIWe present construction plans for a modular system utilizing mostly off-the-shelf, relatively inexpensive components that provides simultaneous high-resolution audio and video recording of 96 isolated or paired Drosophila individuals. C_LIO_LIWe provide open-source control software to record audio and video. C_LIO_LIWe designed high intensity LED arrays that can be used to perform optogenetic activation and inactivation of labelled neurons. C_LIO_LIThe basic design can be modified to facilitate novel study designs or to record insects larger than Drosophila. C_LIO_LIFewer than 96 microphones can be used in the system if the full array is not required or to reduce costs. C_LI ImplicationsO_LIOur hardware design and software provide an improved platform for reliable and comparatively inexpensive high-throughput recording of Drosophila courtship acoustic and visual behavior and perhaps for recording acoustic signals of other small animals. C_LI

animal behavior and cognition↗

bioRxiv2024

Spatial characterization of interface dermatitis in cutaneous lupus reveals novel chemokine ligand-receptor pairs that drive disease

BackgroundChemokines play critical roles in the recruitment and activation of immune cells in both homeostatic and pathologic conditions. Here, we examined chemokine ligand-receptor pairs to better understand the immunopathogenesis of cutaneous lupus erythematosus (CLE), a complex autoimmune connective tissue disorder. ObjectivesOur objectives were to (1) characterize the cellular and proteomic constitution of interface dermatitis in CLE using blister biopsies, (2) map chemokine:ligand receptor pairs that govern recruitment of immune cells to form interface dermatitis in CLE, and (3) perform unbiased analyses in tandem on different clinical subtypes to identify novel genes and proteins underlying discoid versus subacute CLE. MethodsWe used suction blister biopsies to measure cellular infiltrates with spectral flow cytometry in the interface dermatitis reaction, as well as 184 protein analytes in interstitial skin fluid using 96-plex immunoassay targeted proteomics. Flow and 96-plex immunoassay data concordantly demonstrated significant increases in T cells and antigen presenting cells (APCs). We also performed spatial transcriptomics and spatial proteomics of punch biopsies using digital spatial profiling (DSP) technology on CLE skin and healthy margin controls to examine discreet locations within the tissue. ResultsSpatial and 96-plex immunoassay data confirmed elevation of interferon (IFN) and IFN-inducible CXCR3 chemokine ligands. Comparing involved versus uninvolved keratinocytes in CLE samples revealed upregulation of essential inflammatory response genes in areas near interface dermatitis, including AIM2. 96-plex immunoassay data confirmed upregulation of Caspase 8, IL-18 which is the final product of AIM2 activation, and induced chemokines including CCL8 and CXCL6 in CLE lesional samples. Chemotaxis assays using PBMCs from healthy and CLE donors revealed that T cells are equally poised to respond to CXCR3 ligands, whereas CD14+CD16+ APC populations are more sensitive to CXCL6 via CXCR1 and CD14+ are more sensitive to CCL8 via CCR2. ConclusionsTaken together, our data map a pathway from keratinocyte injury to lymphocyte recruitment in CLE via AIM2-Casp8-IL-18-CXCL6/CXCR1 and CCL8/CCR2, and IFNG/IFNL1-CXCL9/CXCL11-CXCR3, and identify potential novel biomarkers of disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/574422v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1e86dbeorg.highwire.dtl.DTLVardef@7d0208org.highwire.dtl.DTLVardef@1108aa8org.highwire.dtl.DTLVardef@ff255f_HPS_FORMAT_FIGEXP M_FIG C_FIG Model of chemokine systems governing recruitment of immune cell subsets to form interface dermatitis in cutaneous lupus. A. Summary of fresh tissue blister biopsy studies demonstrating increases in HLADR+ cells in nonlesional lupus biopsies as assessed by flow cytometry, and increased CXCL6 and CCL8 as assessed by 96 plex immunoassay. Lesional lupus biopsies also demonstrated significant increases in T cells and CXCL9/10/11 production. B. Model of chemokine-directed formation of interface dermatitis in cutaneous lupus. 1. Whole transcriptome atlas (WTA) digital spatial profiling (DSP) revealed increased AIM2 in keratinocytes proximal to inflammation, which is reported to be induced by environmental triggers including UV light and toxins. We also noted increased Caspase 8 (Casp8) and IL18 at the protein level, which can be induced downstream of AIM2. Chemokines including CXCL6 and CCL8 can be induced downstream of IL18, explaining how CCL8 and CXCL6 might be induced. 2. Recruitment of myeloid cell first responders by CCL8 and CXCL6. CD14+CD16+ myeloid cells, which were recently described in nonlesional lupus skin, express CXCR1 and migrate towards CXCL6, whereas CD14+CD16-myeloid cells express more CCR2 and migrate towards CCL8. 3. The CXCR3 ligands CXCL9/10/11 are expressed by keratinocytes, but more strongly in CD45+ immune cell and T cell regions of interest (ROIs). 4. T cells express CXCR3 and migrate towards CXCL9 and to a greater extent CXCL11. 5. The recruited HLADR+ myeloid populations and T cells contribute to formation of interface dermatitis. Thus, we propose a model in which keratinocyte/myeloid crosstalk can reinforce chemokine systems to optimally recruit lymphocytes and other immune cells to form interface dermatitis. Created with Biorender.com. Plain language summaryLupus skin rashes arise during flares, after exposure to medications or sunlight, or in response to other triggers of inflammation. To understand how white blood cells enter the skin to cause these rashes, we used new technologies to look at proteins that attract them into the skin. We found proteins that are expressed by skin cells in lesions that can recruit specific types of white blood cells that are thought to be the initiators of skin rashes. Once in the skin, these and other white blood cells can make additional proteins that bring in more and more cells. We hope that our findings will be used to test new topical treatments for lupus and other autoimmune skin rashes.

immunology↗

bioRxiv2024

Hidden route of protein damage through confined oxygen gas

Oxidative modifications can severely impair protein structure, fold, and function, closely linked to human aging and diseases. Conventional oxidation pathways typically involve the free diffusion of reactive oxygen species (ROS), followed by chemical attacks on the protein surface. Here, we report a hidden route of protein oxidative damage, which we refer to as O2-confinement oxidation pathway. This pathway starts with the initial trapping of dissolved molecular oxygen (O2) within protein cavity spaces, followed by interaction with photosensitizing tryptophan residues. The trapped O2 is then converted to singlet oxygen (1O2), a powerful ROS, through spin-flip electron transfer mechanism under blue light. The generated 1O2 within the protein ultimately attacks the protein core residues through constrained diffusion, accelerating the oxidative damage. This alternative photooxidation pathway through the initial O2 trapping would bypass the antioxidant defense systems which target freely-diffusing ROS, constituting an additional layer of protein oxidative damage in cells and tissues.

biochemistry↗

bioRxiv2023

Detecting Features of Protein Structure through their Mediator-Accessible Redox Activities

Protein function relies on sequence, folding, and post-translational modification, and molecular measurements are commonly used to reveal these structural features. Here, we report an alternative approach that represents these molecular features as readily measurable electronic patterns and validate this experimental approach by detecting structural perturbations commonly encountered during protein biomanufacturing. Specifically, we studied a monoclonal antibody standard (NISTmAb) and focused on the electronic detection of variants that have undergone interchain-disulfide bond reduction and methionine oxidation. Electronic detection of these structural perturbations is based on mediated electrochemical probing (MEP) that discerns patterns associated with the antibodys mediator-accessible redox activity. We demonstrate that MEP can rapidly (within minutes) and quantitatively transduce the proteins structural features into robust electronic signals that can enable bioprocess monitoring and control. More broadly, the ability to transduce information of a proteins molecular structure into a more convenient electronic domain offers fundamentally new opportunities to apply the power of microelectronics and real-time data analytics to chemical and biological analysis.

bioengineering↗

bioRxiv2023

Fourth dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-lasting Humoral Responses in mice

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skins immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU+Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

immunology↗

bioRxiv2023

Adeno-to-squamous transition drives resistance to KRAS inhibition in LKB1 mutant lung cancer

KRASG12C inhibitors including adagrasib and sortorasib have shown clinical promise in targeting KRASG12C-mutated lung cancers, however, most patients eventually develop drug resistance. In lung adenocarcinoma patients with co-occurring KRASG12C and STK11/LKB1 mutations, we found a high squamous gene signature at baseline significantly correlated with poor adagrasib response. Through integrative studies of Lkb1-deficient KRASG12Cand KrasG12D lung cancer mouse models and/or organoids treated with KRAS inhibitors, we found tumor cells invoked a lineage plasticity program: adeno-to-squamous transition (AST) that mediated resistance to KRAS inhibition. Transcriptomic and epigenomic analyses revealed {Delta}Np63 drives AST and modulates response to KRAS inhibition. We identified an intermediate high-plasticity cell state with distinct gene expression program marked by Krt6a upregulation. Notably, higher KRT6A expression at baseline correlated with shorter overall survival in KRAS-mutant patients receiving adagrasib. These data support the role of AST in KRAS inhibitor resistance and provide predictive biomarker for KRAS-targeted therapies in lung cancer.

cancer biology↗

bioRxiv2023

Encyclopaedia of DNA polymerases localized in organelles: Contribution of genes from diverse bacteria including the proto-mitochondrion.

DNA polymerases (DNAPs) synthesize DNA from deoxyribonucleotides in a semi-conservative manner and serve as the core of DNA replication and repair machineries. In eukaryotic cells, there are two genome-containing organelles, mitochondria and plastids, that were derived from an -proteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNAPs that localize and work in them to maintain their genomes. The evolution of organellar DNAPs has yet to be fully understood because of two unsettled issues. First, the diversity of organellar DNAPs has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNAPs that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNAPs known to date show no phylogenetic affinity to those of the extant -proteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNAP sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNAPs were further examined experimentally. The results presented here suggest that the diversity of organellar DNAPs has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed two mitochondrial DNAPs, POP and a candidate of the direct descendant of the proto-mitochondrial DNAP, rdxPolA, identified in this study.

evolutionary biology↗

bioRxiv2023

Redox-enabled Electronic Interrogation and Feedback Control of Hierarchical and Networked Biological Systems

We enable microelectronic devices to interrogate biologys molecular communication, perform computations, and in real time control biological systems, including at several hierarchical levels: proteins, cells, and cell consortia. A key driver is establishing electronic access to and from biologys native redox networks. First, redox-mediated electro-biofabrication facilitates facile assembly of biological components onto microelectronic systems, then electrode-actuated redox allows digital programming of enzyme activity, and further, redox-mediated electrogenetics facilitates closed-loop electronic control of cellular function. Specifically, we show algorithm-based feedback control of enzyme activity, cellular genetic circuits (via eCRISPR) and cell consortia behavior, all enabled by electronic data transfer. We further demonstrate electronic switching of cell-cell quorum sensing communication from one autoinducer network to another, creating an electronically controlled "bilingual" cell. We suggest these methodologies will not only help us to better understand biological systems, but design and control those currently unimagined.

synthetic biology↗

bioRxiv2023

Investigating Brain Alterations in the Dp1Tyb Mouse Model of Down Syndrome

Down syndrome (DS) is one of the most common birth defects and the most prevalent genetic form of intellectual disability. DS arises from trisomy of chromosome 21, but its molecular and pathological consequences are not fully understood. In this study, we compared Dp1Tyb mice, a DS model, against their wild-type (WT) littermates of both sexes to investigate the impact of DS-related genetic abnormalities on the brain phenotype. We performed in vivo whole brain magnetic resonance imaging (MRI) and hippocampal 1H magnetic resonance spectroscopy (MRS) on the animals at 3 months of age. Subsequently, ex vivo MRI scans and histological analyses were conducted post-mortem. Our findings unveiled distinct neuroanatomical and biochemical alterations in the Dp1Tyb brains. Dp1Tyb brains exhibited a smaller surface area and a rounder shape compared to WT brains. Regional volumetric analysis revealed significant changes in 26 out of 72 examined brain regions, including the medial prefrontal cortex and dorsal hippocampus. These alterations were consistently observed in both in vivo and ex vivo imaging data. Additionally, high-resolution ex vivo imaging enabled us to investigate cerebellar layers and hippocampal subregions, revealing selective areas of decrease and remodelling in these structures. An analysis of hippocampal metabolites revealed an elevation in glutamine and the glutamine/glutamate ratio in the Dp1Tyb mice compared to controls, suggesting a possible imbalance in the excitation/inhibition ratio. This was accompanied by the decreased levels of taurine. Histological analysis revealed fewer neurons in the hippocampal CA3 and DG layers, along with an increase in astrocytes and microglia. These findings recapitulate multiple neuroanatomical and biochemical features associated with DS, enriching our understanding of the potential connection between chromosome 21 trisomy and the resultant phenotype.

neuroscience↗

bioRxiv2023

Self-assembling Gn head ferritin nanoparticle vaccine provides full protection from lethal challenge of Dabie Bandavirus in aged ferrets

Dabie Bandavirus (DBV), previously known as Severe Fever with Thrombocytopenia Syndrome (SFTS) Virus, induces a characteristic thrombocytopenia with a mortality rate ranging from 12% to as high as 30%. The sero-prevalence of DBV in healthy people is not significantly different among age groups, but clinically diagnosed SFTS patients are older than [~]50 years, suggesting that age is the critical risk factor for SFTS morbidity and mortality. Accordingly, our immune-competent ferret model demonstrates an age (>4 years old)-dependent DBV infection and pathogenesis that fully recapitulates human clinical manifestation. To protect the aged population from DBV-induced SFTS, vaccine should carry robust immunogenicity with high safety profile. Previous studies have shown that glycoproteins Gn/Gc are the most effective antigens for inducing both neutralizing antibody (NAb)- and T cell-mediated immunity and, thereby, protection. Here, we report the development of a protein subunit vaccine with 24-mer self-assembling ferritin (FT) nanoparticle to present DBV Gn head region (GnH) for enhanced immunogenicity. Anion exchange chromatography and size exclusion chromatography readily purified the GnH-FT nanoparticles to homogeneity with structural integrity. Mice immunized with GnH-FT nanoparticles induced robust NAb response and T-cell immunity against DBV Gn. Furthermore, aged ferrets immunized with GnH-FT nanoparticles were fully protected from DBV challenge without SFTS symptoms such as body weight loss, thrombocytopenia, leukopenia, and fatality. This study demonstrates that DBV GnH-FT nanoparticles provide an efficient vaccine efficacy in mouse and aged ferret models and should be an outstanding vaccine candidate targeted for the aged population against fatal DBV infection. ImportanceDabie Bandavirus (DBV) is an emerging tick-borne virus that causes Severe Fever with Thrombocytopenia Syndrome (SFTS) in infected patients. Human SFTS symptoms progress from fever, fatigue, and muscle pain to the depletion of white blood cells and platelets with fatality rates up to 30%. The recent spread of its vector tick to over 20 states in the United States increases the potential for outbreaks of the SFTS beyond the East Asia. Thus, the development of vaccine to control this rapidly emerging virus is a high priority. In this study, we applied self-assembling ferritin (FT) nanoparticle to enhance the immunogenicity of viral Gn head domain as a vaccine target. Mice immunized with the GnH-FT nanoparticle vaccine induced potent antibody responses and cellular immunity. Immunized aged-ferrets were fully protected from the lethal challenge of DBV. Our study describes the GnH-FT nanoparticle vaccine candidate that provides protective immunity against the emerging DBV infection.

microbiology↗

bioRxiv2023

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils

The Structural Maintenance of Chromosome (SMC) protein complexes cohesin, condensin and the Smc5/6 complex (Smc5/6) are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6s recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled chromosomal DNA at cohesin-dependent chromosome loop boundaries. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes, and efficiently initiates loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

molecular biology↗

bioRxiv2023

The Janelia Atalanta plasmids provide a simple and efficient CRISPR/Cas9-mediated homology directed repair platform for Drosophila

Homology-directed repair (HDR) is a powerful tool for modifying genomes in precise ways to address many biological questions. Use of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-Cas9 induced targeted DNA double-strand breakage has substantially simplified use of homology-directed repair to introduce specific perturbations in Drosophila, but existing platforms for CRISPR-Cas9-mediated HDR in Drosophila involve multiple cloning steps and have low efficiency. To simplify cloning of HDR plasmids, we designed a new plasmid platform, the Janelia Atalanta (pJAT) series, that exploits recent advances in dsDNA synthesis to facilitate Gateway cloning of gRNA sequences and homology arms in one step. Surprisingly, the pJAT plasmids yielded considerably higher HDR efficiency (approximately 25%) than we have observed with other approaches. pJAT plasmids work in multiple Drosophila species and exhibited such high efficiency that previously impossible experiments in Drosophila, such as driving targeted chromosomal inversions, were made possible. We provide pJAT plasmids for a range of commonly performed experiments including targeted insertional mutagenesis, insertion of phiC31-mediated attP landing sites, generation of strains carrying a germ-line source of Cas9, and induction of chromosomal rearrangements. We also provide "empty" pJAT plasmids with multiple cloning sites to simplify construction of plasmids with new functionality. The pJAT platform is generic and may facilitate improved efficiency CRISPR-Cas9 HDR in a wide range of model and non-model organisms.

genetics↗

bioRxiv2023

Molecular and morphological characterisation of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching orphan lineage that is considered key to better understanding the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to better characterise ancyromonads by integrating environmental surveys with behavioural observation and description of cell morphology, for which sample isolation and culturing is indispensable. We studied 18 ancyromonad strains, including 14 new isolates and 7 new species. Most of them belong to three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas (encompassing 4 species). The remaining three new species belong to the already known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, based on morphological and phylogenetic grounds. For all these new species, we studied their morphology under light and electron microscopy and carried out molecular phylogenetic analyses, including as well 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

microbiology↗

bioRxiv2023

Endocardial HDAC3 is required for myocardial trabeculation

Failure of proper ventricular trabeculation is often associated with congenital heart disease (CHD). Support from endocardial cells, including the secretion of extracellular matrix (ECM) and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of ECM and growth factors is initiated and regulated by endocardial cells. We found that genetic knockout (KO) of histone deacetylase 3 (Hdac3) in the endocardium in mice resulted in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identified significant downregulation of ECM components in Hdac3 KO endocardial cells. Secretome from cultured Hdac3 KO mouse cardiac endothelial cells lacked transforming growth factor {beta}3 (TGF{beta}3) and showed significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which was remarkably rescued by TGF{beta}3 supplementation. Mechanistically, we identified that HDAC3 induced Tgf{beta}3 expression through repressing microRNA (miR)-129-5p. Our findings provide novel insights into the pathogenesis of CHD and conceptual strategies to promote myocardial regeneration.

developmental biology↗