CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma.

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with αCTLA-4, but not αPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ T cells but not CD8+ T cells. αCTLA-4 treatment increased frequencies of intratumoral IFNγ-producing CD4+ T cells, and IFNγ blockade negated the therapeutic impact of αCTLA-4. The anti-tumor activity of CD4+ T cells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ T cells interacted directly with microglia, promoting IFNγ-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary for tumor suppression. Thus, αCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ T cell-microglia circuit wherein IFNγ triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ T cell response.

Allopolyploid subgenome identification and implications for evolutionary analysis.

Whole-genome duplications (WGDs) are widespread genomic events in eukaryotes that are hypothesized to contribute to the evolutionary success of many lineages, including flowering plants, Saccharomyces yeast, and vertebrates. WGDs generally can be classified into autopolyploids (ploidy increase descended from one species) or allopolyploids (ploidy increase descended from multiple species). Assignment of allopolyploid progenitor species (called subgenomes in the polyploid) is important to understanding the biology and evolution of polyploids, including the asymmetric subgenome evolution following hybridization (biased fractionation). Here, I review the different methodologies used to identify the ancestors of allopolyploid subgenomes, discuss the advantages and disadvantages of these methods, and outline the implications of how these methods affect the subsequent evolutionary analysis of these genomes.

TAM Kinases Promote Necroptosis by Regulating Oligomerization of MLKL.

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.

PB-LKS: a python package for predicting phage-bacteria interaction through local K-mer strategy.

Bacteriophages can help the treatment of bacterial infections yet require in-silico models to deal with the great genetic diversity between phages and bacteria. Despite the tolerable prediction performance, the application scope of current approaches is limited to the prediction at the species level, which cannot accurately predict the relationship of phages across strain mutants. This has hindered the development of phage therapeutics based on the prediction of phage-bacteria relationships. In this paper, we present, PB-LKS, to predict the phage-bacteria interaction based on local K-mer strategy with higher performance and wider applicability. The utility of PB-LKS is rigorously validated through (i) large-scale historical screening, (ii) case study at the class level and (iii) in vitro simulation of bacterial antiphage resistance at the strain mutant level. The PB-LKS approach could outperform the current state-of-the-art methods and illustrate potential clinical utility in pre-optimized phage therapy design.

DCiPatho: deep cross-fusion networks for genome scale identification of pathogens.

Pathogen detection from biological and environmental samples is important for global disease control. Despite advances in pathogen detection using deep learning, current algorithms have limitations in processing long genomic sequences. Through the deep cross-fusion of cross, residual and deep neural networks, we developed DCiPatho for accurate pathogen detection based on the integrated frequency features of 3-to-7 k-mers. Compared with the existing state-of-the-art algorithms, DCiPatho can be used to accurately identify distinct pathogenic bacteria infecting humans, animals and plants. We evaluated DCiPatho on both learned and unlearned pathogen species using both genomics and metagenomics datasets. DCiPatho is an effective tool for the genomic-scale identification of pathogens by integrating the frequency of k-mers into deep cross-fusion networks. The source code is publicly available at https://github.com/LorMeBioAI/DCiPatho.

SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin.

BACKGROUND: Efferocytosis is an activity of macrophages that is pivotal for the resolution of inflammation in hypertension. The precise mechanism by which macrophages coordinate efferocytosis and internalize apoptotic cardiomyocytes remains unknown. The aim of this study was to determine whether SIRT3 (sirtuin-3) is required for both apoptotic cardiomyocyte engulfment and anti-inflammatory responses during efferocytosis. METHODS: We generated myeloid SIRT3 knockout mice and FXN (frataxin) knock-in mice carrying an acetylation-defective lysine to arginine K189R mutation (FXNK189R). The mice were given Ang II (angiotensin II) infusion for 7 days. We analyzed cardiac macrophages' mitochondrial iron levels, efferocytosis activity, and phenotype both in vivo and in vitro. RESULTS: We showed that SIRT3 deficiency exacerbated Ang II-induced downregulation of the efferocytosis receptor MerTK (c-Mer tyrosine kinase) and proinflammatory cytokine production, accompanied by disrupted mitochondrial iron homeostasis in cardiac macrophages. Quantitative acetylome analysis revealed that SIRT3 deacetylated FXN at lysine 189. Ang II attenuated SIRT3 activity and enhanced the acetylation level of FXNK189. Acetylated FXN further reduced the synthesis of ISCs (iron-sulfur clusters), resulting in mitochondrial iron accumulation. Phagocytic internalization of apoptotic cardiomyocytes increased myoglobin content, and derived iron ions promoted mitochondrial iron overload and lipid peroxidation. An iron chelator deferoxamine improved the levels of MerTK and efferocytosis, thereby attenuating proinflammatory macrophage activation. FXNK189R mice showed improved macrophage efferocytosis, reduced cardiac inflammation, and suppressed cardiac fibrosis. CONCLUSIONS: The SIRT3-FXN axis has the potential to resolve cardiac inflammation by increasing macrophage efferocytosis and anti-inflammatory activities.

W2V-repeated index: Prediction of enhancers and their strength based on repeated fragments.

Enhancers are crucial in gene expression regulation, dictating the specificity and timing of transcriptional activity, which highlights the importance of their identification for unravelling the intricacies of genetic regulation. Therefore, it is critical to identify enhancers and their strengths. Repeated sequences in the genome are repeats of the same or symmetrical fragments. There has been a great deal of evidence that repetitive sequences contain enormous amounts of genetic information. Thus, We introduce the W2V-Repeated Index, designed to identify enhancer sequence fragments and evaluates their strength through the analysis of repeated K-mer sequences in enhancer regions. Utilizing the word2vector algorithm for numerical conversion and Manta Ray Foraging Optimization for feature selection, this method effectively captures the frequency and distribution of K-mer sequences. By concentrating on repeated K-mer sequences, it minimizes computational complexity and facilitates the analysis of larger K values. Experiments indicate that our method performs better than all other advanced methods on almost all indicators.

Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction.

Meiosis is the cellular program by which a diploid cell gives rise to haploid gametes for sexual reproduction. Meiotic progression depends on tight physical and functional coupling of recombination steps at the DNA level with specific organizational features of meiotic-prophase chromosomes. The present study reveals that every step of this coupling is mediated by a single molecule: Asy2/Mer2. We show that Mer2, identified so far only in budding and fission yeasts, is in fact evolutionarily conserved from fungi (Mer2/Rec15/Asy2/Bad42) to plants (PRD3/PAIR1) and mammals (IHO1). In yeasts, Mer2 mediates assembly of recombination-initiation complexes and double-strand breaks (DSBs). This role is conserved in the fungus Sordaria However, functional analysis of 13 mer2 mutants and successive localization of Mer2 to axis, synaptonemal complex (SC), and chromatin revealed, in addition, three further important functions. First, after DSB formation, Mer2 is required for pairing by mediating homolog spatial juxtaposition, with implications for crossover (CO) patterning/interference. Second, Mer2 participates in the transfer/maintenance and release of recombination complexes to/from the SC central region. Third, after completion of recombination, potentially dependent on SUMOylation, Mer2 mediates global chromosome compaction and post-recombination chiasma development. Thus, beyond its role as a recombinosome-axis/SC linker molecule, Mer2 has important functions in relation to basic chromosome structure.

AFITbin: a metagenomic contig binning method using aggregate l-mer frequency based on initial and terminal nucleotides.

BACKGROUND: Using next-generation sequencing technologies, scientists can sequence complex microbial communities directly from the environment. Significant insights into the structure, diversity, and ecology of microbial communities have resulted from the study of metagenomics. The assembly of reads into longer contigs, which are then binned into groups of contigs that correspond to different species in the metagenomic sample, is a crucial step in the analysis of metagenomics. It is necessary to organize these contigs into operational taxonomic units (OTUs) for further taxonomic profiling and functional analysis. For binning, which is synonymous with the clustering of OTUs, the tetra-nucleotide frequency (TNF) is typically utilized as a compositional feature for each OTU. RESULTS: In this paper, we present AFIT, a new l-mer statistic vector for each contig, and AFITBin, a novel method for metagenomic binning based on AFIT and a matrix factorization method. To evaluate the performance of the AFIT vector, the t-SNE algorithm is used to compare species clustering based on AFIT and TNF information. In addition, the efficacy of AFITBin is demonstrated on both simulated and real datasets in comparison to state-of-the-art binning methods such as MetaBAT 2, MaxBin 2.0, CONCOT, MetaCon, SolidBin, BusyBee Web, and MetaBinner. To further analyze the performance of the purposed AFIT vector, we compare the barcodes of the AFIT vector and the TNF vector. CONCLUSION: The results demonstrate that AFITBin shows superior performance in taxonomic identification compared to existing methods, leveraging the AFIT vector for improved results in metagenomic binning. This approach holds promise for advancing the analysis of metagenomic data, providing more reliable insights into microbial community composition and function. AVAILABILITY: A python package is available at: https://github.com/SayehSobhani/AFITBin .

iProbiotics: a machine learning platform for rapid identification of probiotic properties from whole-genome primary sequences.

Lactic acid bacteria consortia are commonly present in food, and some of these bacteria possess probiotic properties. However, discovery and experimental validation of probiotics require extensive time and effort. Therefore, it is of great interest to develop effective screening methods for identifying probiotics. Advances in sequencing technology have generated massive genomic data, enabling us to create a machine learning-based platform for such purpose in this work. This study first selected a comprehensive probiotics genome dataset from the probiotic database (PROBIO) and literature surveys. Then, k-mer (from 2 to 8) compositional analysis was performed, revealing diverse oligonucleotide composition in strain genomes and apparently more probiotic (P-) features in probiotic genomes than non-probiotic genomes. To reduce noise and improve computational efficiency, 87 376 k-mers were refined by an incremental feature selection (IFS) method, and the model achieved the maximum accuracy level at 184 core features, with a high prediction accuracy (97.77%) and area under the curve (98.00%). Functional genomic analysis using annotations from gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Rapid Annotation using Subsystem Technology (RAST) databases, as well as analysis of genes associated with host gastrointestinal survival/settlement, carbohydrate utilization, drug resistance and virulence factors, revealed that the distribution of P-features was biased toward genes/pathways related to probiotic function. Our results suggest that the role of probiotics is not determined by a single gene, but by a combination of k-mer genomic components, providing new insights into the identification and underlying mechanisms of probiotics. This work created a novel and free online bioinformatic tool, iProbiotics, which would facilitate rapid screening for probiotics.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

Reference-free inferring of transcriptomic events in cancer cells on single-cell data.

BACKGROUND: Cancerous cells' identity is determined via a mixture of multiple factors such as genomic variations, epigenetics, and the regulatory variations that are involved in transcription. The differences in transcriptome expression as well as abnormal structures in peptides determine phenotypical differences. Thus, bulk RNA-seq and more recent single-cell RNA-seq data (scRNA-seq) are important to identify pathogenic differences. In this case, we rely on k-mer decomposition of sequences to identify pathogenic variations in detail which does not need a reference, so it outperforms more traditional Next-Generation Sequencing (NGS) analysis techniques depending on the alignment of the sequences to a reference. RESULTS: Via our alignment-free analysis, over esophageal and glioblastoma cancer patients, high-frequency variations over multiple different locations (repeats, intergenic regions, exons, introns) as well as multiple different forms (fusion, polyadenylation, splicing, etc.) could be discovered. Additionally, we have analyzed the importance of less-focused events systematically in a classic transcriptome analysis pipeline where these events are considered as indicators for tumor prognosis, tumor prediction, tumor neoantigen inference, as well as their connection with respect to the immune microenvironment. CONCLUSIONS: Our results suggest that esophageal cancer (ESCA) and glioblastoma processes can be explained via pathogenic microbial RNA, repeated sequences, novel splicing variants, and long intergenic non-coding RNAs (lincRNAs). We expect our application of reference-free process and analysis to be helpful in tumor and normal samples differential scRNA-seq analysis, which in turn offers a more comprehensive scheme for major cancer-associated events.

Persistence of Basal Ganglia Oscillatory Activity During Tremor Attenuation by Movement in Parkinson's Disease Patients.

BACKGROUND: One of the characteristics of parkinsonian tremor is that its amplitude decreases with movement. Current models suggest an interaction between basal ganglia (BG) and cerebello-thalamo-cortical circuits in parkinsonian tremor pathophysiology. OBJECTIVE: We aimed to correlate central oscillation in the BG with electromyographic activity during re-emergent tremor in order to detect changes in BG oscillatory activity when tremor is attenuated by movement. METHODS: We performed a prospective, observational study on consecutive parkinsonian patients who underwent deep brain stimulation surgery and presented re-emergent tremor. Coherence analysis between subthalamic nucleus/globus pallidus internus (STN/GPi) tremorous activity measured by microrecording (MER) and electromyogram (EMG) from flexor and extensor wrist muscles during rest, posture, and re-emergent tremor pause was performed during surgery. The statistical significance level of the MER-EMG coherence was determined using surrogate data analysis, and the directionality of information transfer between BG and muscle was performed using entropy transfer analysis. RESULTS: We analyzed 148 MERs with tremor-like activity from 6 patients which were evaluated against the simultaneous EMGs, resulting in 296 correlations. Of these, 26 presented a significant level of coherence at tremor frequency, throughout rest and posture, with a complete EMG stop in between. During the pause, all recordings showed sustained MER peaks at tremor frequency (±1.5 Hz). Information flows preferentially from BG to muscle during rest and posture, with a loss of directionality during the pause. CONCLUSIONS: Our results suggest that oscillatory activity in STN/GPi functionally linked to tremor sustains firing frequency during re-emergent tremor pause, thus suggesting no direct role of the BG circuit on tremor attenuation due to voluntary movements. © 2024 International Parkinson and Movement Disorder Society.

A k-mer-Based Approach for Phylogenetic Classification of Taxa in Environmental Genomic Data.

In the age of genome sequencing, whole-genome data is readily and frequently generated, leading to a wealth of new information that can be used to advance various fields of research. New approaches, such as alignment-free phylogenetic methods that utilize k-mer-based distance scoring, are becoming increasingly popular given their ability to rapidly generate phylogenetic information from whole-genome data. However, these methods have not yet been tested using environmental data, which often tends to be highly fragmented and incomplete. Here, we compare the results of one alignment-free approach (which utilizes the D2 statistic) to traditional multi-gene maximum likelihood trees in 3 algal groups that have high-quality genome data available. In addition, we simulate lower-quality, fragmented genome data using these algae to test method robustness to genome quality and completeness. Finally, we apply the alignment-free approach to environmental metagenome assembled genome data of unclassified Saccharibacteria and Trebouxiophyte algae, and single-cell amplified data from uncultured marine stramenopiles to demonstrate its utility with real datasets. We find that in all instances, the alignment-free method produces phylogenies that are comparable, and often more informative, than those created using the traditional multi-gene approach. The k-mer-based method performs well even when there are significant missing data that include marker genes traditionally used for tree reconstruction. Our results demonstrate the value of alignment-free approaches for classifying novel, often cryptic or rare, species, that may not be culturable or are difficult to access using single-cell methods, but fill important gaps in the tree of life.

Microorganisms Involved in Methylmercury Demethylation and Mercury Reduction are Widely Distributed and Active in the Bathypelagic Deep Ocean Waters.

The ocean's mercury (Hg) content has tripled due to anthropogenic activities, and although the dark ocean (>200 m) has become an important Hg reservoir, concentrations of the toxic and bioaccumulative methylmercury (MeHg) are low and therefore very difficult to measure. As a consequence, the current understanding of the Hg cycle in the deep ocean is severely data-limited, and the factors controlling MeHg, as well as its transformation rates, remain largely unknown. By analyzing 52 globally distributed bathypelagic deep-ocean metagenomes and 26 new metatranscriptomes from the Malaspina Expedition, our study reveals the widespread distribution and expression of bacterial-coding genes merA and merB in the global bathypelagic ocean (∼4000 m depth). These genes, associated with HgII reduction and MeHg demethylation, respectively, are particularly prevalent within the particle-attached fraction. Moreover, our results indicate that water mass age and the organic matter composition shaped the structure of the communities harboring merA and merB genes living in different particle size fractions, their abundance, and their expression levels. Members of the orders Corynebacteriales, Rhodobacterales, Alteromonadales, Oceanospirillales, Moraxellales, and Flavobacteriales were the main taxonomic players containing merA and merB genes in the deep ocean. These findings, together with our previous results of pure culture isolates of the deep bathypelagic ocean possessing the metabolic capacity to degrade MeHg, indicated that both methylmercury demethylation and HgII reduction likely occur in the global dark ocean, the largest biome in the biosphere.

Measuring functional similarity of lncRNAs based on variable K-mer profiles of nucleotide sequences.

Long non-coding RNAs are a class of essential non-coding RNAs with a length of more than 200 nts. Recent studies have indicated that lncRNAs have various complex regulatory functions, which play great impacts on many fundamental biological processes. However, measuring the functional similarity between lncRNAs by traditional wet-experiments is time-consuming and labor intensive, computational-based approaches have been an effective choice to tackle this problem. Meanwhile, most sequences-based computation methods measure the functional similarity of lncRNAs with their fixed length vector representations, which could not capture the features on larger k-mers. Therefore, it is urgent to improve the predict performance of the potential regulatory functions of lncRNAs. In this study, we propose a novel approach called MFSLNC to comprehensively measure functional similarity of lncRNAs based on variable k-mer profiles of nucleotide sequences. MFSLNC employs the dictionary tree storage, which could comprehensively represent lncRNAs with long k-mers. The functional similarity between lncRNAs is evaluated by the Jaccard similarity. MFSLNC verified the similarity between two lncRNAs with the same mechanism, detecting homologous sequence pairs between human and mouse. Besides, MFSLNC is also applied to lncRNA-disease associations, combined with the association prediction model WKNKN. Moreover, we also proved that our method can more effectively calculate the similarity of lncRNAs by comparing with the classical methods based on the lncRNA-mRNA association data. The detected AUC value of prediction is 0.867, which achieves good performance in the comparison of similar models.

The expression analysis of long noncoding RNAs PCAT-1, PCAT-29, and MER11C in bipolar disorder.

Long non-coding RNAs (lncRNAs) are transcripts with a length of usually more than 200 nucleotides (nt) that have promised functions in varied biological processes. lncRNAs participate in the regulation of differentiation, development, and function of the brain. Thus, their dysregulation might play important roles in the etiology of neurological disorders such as BD. In this study, the expression level of PCAT-1, PCAT-29, and MER11C lncRNAs was evaluated in the blood of BD patients compared to the control group. Peripheral blood mononuclear cells of 50 BD type I patients and 50 healthy individuals were isolated. The RNAs were extracted and cDNA was synthesized. Then, the expression level of the desired lncRNAs was measured through Real-Time PCR. The expression levels of PCAT-29 and MER11C lncRNAs were significantly lower in BD patients compared to controls. However, the expression level of PCAT-1 was not significantly different between these two sets of samples. According to the ROC curve, PCAT-29 and MER11C had significant diagnostic power for the differentiation of BD patients from controls. Taken together, our results indicate dysregulation of two lncRNAs in patients with BD and the possible roles of these lncRNAs in the neuropathology of bipolar disorder.

The TAM Subfamily of Receptor Tyrosine Kinases: The Early Years.

The TAMs are a subfamily of receptor tyrosine kinases (RTKs) comprised of three members, Tyro3, Axl and Mer. Evidence in support of the existence of this subfamily emerged from a screen for novel RTKs performed in the laboratory of Dr. Greg Lemke in 1991. A PCR-based approach to selectively amplify tyrosine kinase-specific genes yielded 27 different tyrosine kinase genes, of which 13 were novel (the "Tyros"). Of these, Tyro3, 7 and 12 were more closely related to each other than to any other kinases and it was proposed that they constituted a novel subfamily of RTKs. Additional support for this hypothesis required determining the complete sequences for these receptor tyrosine kinases. By the end of 1991, full-length sequences for Tyro7 (Axl) revealed a unique extracellular domain organization that included two immunoglobulin-like domains and two fibronectin type III repeats. In 1994, the complete sequences for Tyro12 (Mer) and Tyro3 were shown to have an extracellular region domain structure similar to that of Axl. In 1995, Gas6 and Pros1 were reported as ligands for Tyro3 and Axl, setting the stage for functional studies. The Lemke lab and its many trainees have since played leading roles in elucidating the physiological relevance of the TAMs.

Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.

Local Genomic Surveillance of Invasive Streptococcus pyogenes in Eastern North Carolina (ENC) in 2022-2023.

The recent increase in Group A Streptococcus (GAS) incidences in several countries across Europe and some areas of the Unites States (U.S.) has raised concerns. To understand GAS diversity and prevalence, we conducted a local genomic surveillance in Eastern North Carolina (ENC) in 2022-2023 with 95 isolates and compared its results to those of the existing national genomic surveillance in the U.S. in 2015-2021 with 13,064 isolates. We observed their epidemiological changes before and during the COVID-19 pandemic and detected a unique sub-lineage in ENC among the most common invasive GAS strain, ST28/emm1. We further discovered a multiple-copy insertion sequence, ISLgar5, in ST399/emm77 and its single-copy variants in some other GAS strains. We discovered ISLgar5 was linked to a Tn5801-like tetM-carrying integrative and conjugative element, and its copy number was associated with an ermT-carrying pRW35-like plasmid. The dynamic insertions of ISLgar5 may play a vital role in genome fitness and adaptation, driving GAS evolution relevant to antimicrobial resistance and potentially GAS virulence.