Biology subjects

Chen, S.

Publications and source records attributed to Chen, S..

At least 523 records · Page 29Linked to original sources

bioRxiv2023

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

genomics↗

bioRxiv2023

Comparative Phylogenomics and Phylotranscriptomics Provide Insights into the Genetic Complexity of Nitrogen Fixing Root Nodule Symbiosis

Plant root nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. It is widely accepted that nodulation originated through the assembly of modules recruited from existing functions, such as mycorrhizal symbiosis, polar growth, and lateral root development. Because nodulating species are scattered within the NFNC, the number of times nodulation has evolved or has been lost has been a matter of considerable speculation. This interesting evolutionary question has practical implications concerning the ease with which nodulation might be engineered in non-nodulating crop plants. Nodulating species share many commonalities, due either to divergence from a common ancestor over 100 million years ago or to convergence or deep homology following independent origins over that same time period. In either case, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation--what must be acquired or cannot be lost for a functional symbiosis--and what the latitude is for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here we present new information across the spectrum of nodulating groups. We find no evidence for convergence at the level of amino acid residues or gene family expansion across the NFNC. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor, NIN, as a master regulator of nodulation, and identify key mutations affecting its function across the NFNC. We find that nodulation genes are over-represented among orthologous gene groups (OGs) present in the NFNC common ancestor, but that lineage-specific OGs play major roles in nodulation. We identified over 900,000 conserved noncoding elements (CNEs), of which over 300,000 were unique to NFNC species. A significant proportion of these are associated with nodulation-related genes and thus are candidates for transcriptional regulators.

evolutionary biology↗

bioRxiv2023

Duckweed evolution: from land back to water

Terrestialization is supposedly an important evolutionary process plant experience. However, directions of land back to water acquired little attention. Here we integrate multiproxy evidence to elucidate the evolution of duckweed. Three genera of duckweed show chronologically gradient degeneration in roots structure and stomatal function and decrease in lignocellulose content, accompanied by gradual contraction in relevant gene numbers and/or decline in transcription. The gene numbers in the main phytohormonal pathway are also gradually decreased. The co-action of auxin and rhizoid development gene causes a gradual decrease in adventitious roots. The significant expansion of the flavonoid pathway is also related to the adaptation of duckweed to floating growth. This study reconstructs the evolution history of duckweeds from land back to water, reverse to that of early land plants. SummaryWith terrestrialization being the popularly acknowledged plant evolutionary process, little is known about the evolution of higher plant from land back to water. Here we integrate multiproxy evidence to elucidate the gradual reverse evolution of duckweed. Three genera of duckweed show chronologically gradient degeneration in the structure of roots, the function of stomata, and decrease in lignocellulose content, accompanied by gradient contraction in relevant gene numbers and/or decline in transcript expression. The gene numbers in the main phytohormonal pathway are also gradually decreased. The co-action of auxin and rhizoid development gene causes a gradual decrease in adventitious roots. The significant expansion of the flavonoid pathway is also highly related to the adaptation of duckweed to floating growth. Our study combined with the fossil evidence reconstruct the evolution history of duckweeds from land back to water, reverse to that of early land plants. This study reconstructed the process of how a land plant returns to water, a reverse evolutionary approach which is different from what we studied in textbook about plant terrestrialization. This finding could be helpful for us to deeply and widely understand the adaptation of plant to the environment, and to expand and deepen the knowledge of evolution theory.

evolutionary biology↗

bioRxiv2023

Genipin Crosslinks the Extracellular Matrix to Rescue Developmental and Degenerative Defects, and Accelerates Regeneration of Peripheral Neurons

The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the population suffer nerve degeneration or peripheral damage. For example, over 40% of patients with diabetes or undergoing chemotherapy develop peripheral neuropathies. Despite this, there are major gaps in the knowledge of human PNS development and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder that specifically affects the PNS making it an ideal model to study PNS dysfunction. FD is caused by a homozygous point mutation in ELP1 leading to developmental and degenerative defects in the sensory and autonomic lineages. We previously employed human pluripotent stem cells (hPSCs) to show that peripheral sensory neurons (SNs) are not generated efficiently and degenerate over time in FD. Here, we conducted a chemical screen to identify compounds able to rescue this SN differentiation inefficiency. We identified that genipin, a compound prescribed in Traditional Chinese Medicine for neurodegenerative disorders, restores neural crest and SN development in FD, both in the hPSC model and in a FD mouse model. Additionally, genipin prevented FD neuronal degeneration, suggesting that it could be offered to patients suffering from PNS neurodegenerative disorders. We found that genipin crosslinks the extracellular matrix, increases the stiffness of the ECM, reorganizes the actin cytoskeleton, and promotes transcription of YAP-dependent genes. Finally, we show that genipin enhances axon regeneration in an in vitro axotomy model in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system, CNS). Our results suggest genipin can be used as a promising drug candidate for treatment of neurodevelopmental and neurodegenerative diseases, and as a enhancer of neuronal regeneration. One sentence summaryGenipin rescues the developmental and degenerative phenotypes of the peripheral neuropathy familial dysautonomia and enhances neuron regeneration after injury.

neuroscience↗

bioRxiv2023

Artificially cultivated duckweed: a high-efficiency starch producer

The increasing demand for starch has been a social struggle. We report a new technology that efficiently produces starch from duckweed. Although Landoltia punctata has a dramatic contraction of gene families, its starch content and productivity reached 72.2% (dry basis) and 10.4 g m-2 d-1 in 10 days, equivalent to a yield of 38.0 t ha-1 y-1 under nutrient limitation and CO2 elevation treatment. Meanwhile, we also examined the mechanism of duckweeds high starch accumulation. This is exhibited in the regulation of DNA methylation and transcription factors as well as the significantly up-regulated transcription levels and the increased enzyme activities of key genes in starch biosynthesis. Meantime, while nitrogen redistribution was enhanced, sucrose biosynthesis and transportation, and lignocellulose biosynthesis were all reduced. These alterations led to a reduction in lignocellulose and protein content and ultimately an increase in an accumulation of starch in the chloroplast. This work demonstrates duckweeds potential of being a highly efficient starch producer.

plant biology↗

bioRxiv2023

Multiomic Analysis of Intercellular Communication through Tumor-Stroma Tunnels in Breast Cancers

Estrogen receptor-positive (ER+) breast cancer commonly disseminates to bone marrow, where interactions with mesenchymal stromal cells (MSCs) shape disease trajectory. We modeled these interactions with tumor-MSC co-cultures and used an integrated transcriptome-proteome-network-analyses workflow to identify a comprehensive catalog of contact-induced changes. Conditioned media from MSCs failed to recapitulate genes and proteins, some borrowed and others tumor-intrinsic, induced in cancer cells by direct contact. Protein-protein interaction networks revealed the rich connectome between borrowed and intrinsic components. Bioinformatics prioritized one of the borrowed components, CCDC88A/GIV, a multi-modular metastasis-related protein that has recently been implicated in driving a hallmark of cancer, growth signaling autonomy. MSCs transferred GIV protein to ER+ breast cancer cells (that lack GIV) through tunnelling nanotubes via connexin (Cx)43-facilitated intercellular transport. Reinstating GIV alone in GIV-negative breast cancer cells reproduced [~]20% of both the borrowed and the intrinsic gene induction patterns from contact co-cultures; conferred resistance to anti-estrogen drugs; and enhanced tumor dissemination. Findings provide a multiomic insight into MSC[->]tumor cell intercellular transport and validate how transport of one such candidate, GIV, from the haves (MSCs) to have-nots (ER+ breast cancer) orchestrates aggressive disease states.

cancer biology↗

bioRxiv2023

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beauty composite system

Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC (mRNA AAV-Sleeping-Beauty Joint Engineering of Stable Therapeutic Immune Cells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo. This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

bioengineering↗

bioRxiv2023

Perturbomics of tumor-infiltrating NK cells

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.

immunology↗

bioRxiv2023

CTLA-4 tail fusion enhances CAR-T anti-tumor immunity

Chimeric antigen receptor (CAR) T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here, utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CT), we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen, and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior anti-tumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

immunology↗

bioRxiv2023

Immunogenetic metabolomics revealed key enzymes that modulate CAR-T metabolism and function

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we seek to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism, whereby cancer cells suppress T cell function by generating a metabolically unfavorable tumor microenvironment (TME). Specifically, we use an in silico screen to identify ADA and PDK1 as metabolic regulators, in which gene overexpression (OE) enhances the cytolysis of CD19-specific CD8 CAR-T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampens such effect. ADA-OE in CAR-T cells improves cancer cytolysis under high concentrations of adenosine, the ADA substrate and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics in these CAR-Ts reveal alterations of global gene expression and metabolic signatures in both ADA- and PDK1- engineered CAR-T cells. Functional and immunological analyses demonstrate that ADA-OE increases proliferation and decreases exhaustion in -CD19 and -HER2 CAR-T cells. ADA-OE improves tumor infiltration and clearance by -HER2 CAR-T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR-T cells, and reveal potential targets for improving CAR-T based cell therapy. SynopsisThe authors identify the adenosine deaminase gene (ADA) as a regulatory gene that reprograms T cell metabolism. ADA-overexpression (OE) in -CD19 and -HER2 CAR-T cells increases proliferation, cytotoxicity, memory, and decreases exhaustion, and ADA-OE -HER2 CAR-T cells have enhanced clearance of HT29 human colorectal cancer tumors in vivo.

cancer biology↗

bioRxiv2023

Rational design of immune gene therapy combinations via in vivo CRISPR activation screen of tumor microenvironment modulators

The hostile tumor microenvironment (TME) is major challenge for cancer immunotherapies. Here, we design and perform TME-targeted in vivo CRISPR activation (CRISPRa) screens to uncover factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Through adeno-associated virus (AAV) delivery, multiplexed activation of pooled immunoregulatory genes encoding antigen presentation, cytokine, and co-stimulation molecules (APCM) leads to enhanced anti-tumor immunity. APCM screen in metastatic tumors identifies Cd80, Tnfsf14, Cxcl10, Tnfsf18, Tnfsf9, and Ifng as the top immunostimulatory candidates. AAV-mediated delivery of these factors individually or in combination shows anti-tumor efficacy across different cancer models. Further optimization pinpoints Ifng+Tnfsf9+Il12b(Il12/Il23) as a potent therapeutic combination, leading to increased IFN-{gamma}+CD8+ and tissue-resident memory T cells. APCM therapy synergizes with CAR-T cell therapy against human solid tumors in vivo. APCM-based CRISPRa screen and gene activation systems can thus be leveraged for the rapid generation of off-the-shelf immune gene therapies against solid tumors.

immunology↗

bioRxiv2023

Multiplexed repression of immunosuppressive genes as combinatorial cancer immunotherapy

Checkpoint blockade immunotherapy is a potent class of cancer treatment, however, the complex immunosuppressive tumor microenvironment (TME) often requires multi-agent combinations to be effective. Current cancer immunotherapy combination approaches are cumbersome, usually involving one-drug-at-a-time scheme. Here, we devise Multiplex Universal Combinatorial Immunotherapy via Gene-silencing (MUCIG), as a versatile approach for combinatorial cancer immunotherapy. We harness CRISPR-Cas13d to efficiently target multiple endogenous immunosuppressive genes on demand, allowing us to silence various combinations of multiple immunosuppressive factors in the TME. Intratumoral AAV-mediated administration of MUCIG (AAV-MUCIG) elicits significant anti-tumor activity with several Cas13d gRNA compositions. TME target expression analysis driven optimization led to a simplified off-the-shelf MUCIG targeting a four gene combination (PGGC: Pdl1, Galectin9, Galectin3 and Cd47). AAV-PGGC shows significant in vivo efficacy in syngeneic tumor models. Single cell and flow profiling revealed that AAV-PGGC remodeled the TME by increasing CD8+ T cell infiltration and reducing myeloid-derived immunosuppressive cells (MDSCs). MUCIG thus serves as a universal method to silence multiple immune genes in vivo, and can be delivered via AAV as a therapeutic approach.

immunology↗

bioRxiv2023

Cysteine: an ancestral Cu binding ligand in green algae?

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding "first responder" proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, {gamma}-glutamylcysteine and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ~80-fold, corresponding to ~ 2.8 x 109 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

biochemistry↗

bioRxiv2023

Cas12a/Cpf1 knock-in mice enable efficient multiplexed immune cell engineering

Cas9 transgenic animals have drastically accelerated the discovery of novel immune modulators. But due to its inability to process its own CRISPR RNAs (crRNAs), simultaneous multiplexed gene perturbations using Cas9 remains limited, especially by pseudoviral vectors. Cas12a/Cpf1, however, can process concatenated crRNA arrays for this purpose. Here, we created conditional and constitutive LbCas12a knock-in transgenic mice. With these mice, we demonstrated efficient multiplexed gene editing and surface protein knockdown within individual primary immune cells. We showed genome editing across multiple types of primary immune cells including CD4 and CD8 T cells, B cells, and bone-marrow derived dendritic cells. These transgenic animals, along with the accompanying viral vectors, together provide a versatile toolkit for a broad range of ex vivo and in vivo gene editing applications, including fundamental immunological discovery and immune gene engineering.

genetics↗

bioRxiv2023

High Volume Rate 3-D Ultrasound Imaging Using Fast-Tilting and Redirecting Reflectors

3-D ultrasound imaging has many advantages over 2-D imaging such as more comprehensive tissue evaluation and less operator dependence. Although many 3-D ultrasound imaging techniques have been developed in the last several decades, a low-cost and accessible solution with high imaging volume rate and imaging quality remains elusive. Recently we proposed a new, high volume rate 3-D ultrasound imaging technique: Fast Acoustic Steering via Tilting Electromechanical Reflectors (FASTER), which uses a water-immersible and fast-tilting acoustic reflector to steer ultrafast plane waves in the elevational direction to achieve high volume rate 3-D ultrasound imaging with conventional 1-D array transducers. However, the initial implementation of FASTER imaging only involves a single fast-tilting acoustic reflector, which is inconvenient to use because the probe cannot be held in the regular upright position. Also, conventional FASTER imaging can only be performed inside a water tank because of the necessity of using water for acoustic conduction. To address these limitations of conventional FASTER, here we developed a novel ultrasound probe clip-on device that encloses a fast-tilting reflector, a redirecting reflector, and an acoustic wave conduction medium. The new FASTER 3-D imaging device can be easily attached to or removed from clinical ultrasound transducers, allowing rapid transformation from 2-D to 3-D ultrasound imaging. In vitro B-mode imaging studies demonstrated that the proposed method provided comparable imaging quality (e.g., spatial resolution and contrast-to-noise ratio) to conventional, mechanical-translation-based 3-D imaging while providing a much faster 3-D volume rate (e.g., 300 Hz vs [~]10 Hz). In addition to B-mode imaging, we also demonstrated 3-D power Doppler imaging and 3-D super-resolution ultrasound localization microscopy with the newly developed FASTER device. An in vivo imaging study showed that the FASTER device could clearly visualize the 3-D anatomy of the basilic vein of a healthy volunteer, and customized beamforming was implemented to accommodate the speed of sound difference between the acoustic medium and the imaging object (e.g., soft tissue). These results suggest that the newly developed redirecting reflector and the clip-on device could overcome key hurdles for future clinical translation of the FASTER 3-D imaging technology.

bioengineering↗

bioRxiv2023

Brain-wide neural activity underlying memory-guided movement

Behavior requires neural activity across the brain, but most experiments probe neurons in a single area at a time. Here we used multiple Neuropixels probes to record neural activity simultaneously in brain-wide circuits, in mice performing a memory-guided directional licking task. We targeted brain areas that form multi-regional loops with anterior lateral motor cortex (ALM), a key circuit node mediating the behavior. Neurons encoding sensory stimuli, choice, and actions were distributed across the brain. However, in addition to ALM, coding of choice was concentrated in subcortical areas receiving input from ALM, in an ALM-dependent manner. Choice signals were first detected in ALM and the midbrain, followed by the thalamus, and other brain areas. At the time of movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.

neuroscience↗

bioRxiv2023

Cat LCA-CRX model, homozygous for an antimorphic mutation has a unique phenotype

PURPOSEHuman mutations in the CRX transcription factor are associated with dominant retinopathies often with more severe macular changes. The CRX-mutant cat (Rdy-A182d2) is the only animal model with the equivalent of the critical retinal region for high acuity vision, the macula. Heterozygous cats (CRXRdy/+) have a severe phenotype modeling Leber congenital amaurosis. This study reports the distinct ocular phenotype of homozygous cats (CRXRdy/Rdy). METHODSGene expression changes were assessed at both mRNA and protein levels. Changes in globe morphology and retinal structure were analyzed. RESULTSCRXRdy/Rdy cats had high levels of mutant CRX mRNA and protein. The expression of photoreceptor target genes was severely impaired while there were variable effects on the expression of other transcription factors. The photoreceptor cells remained immature and failed to elaborate outer segments consistent with the lack of retinal function. The retinal layers displayed a progressive remodeling with cell loss but maintained overall retinal thickness due to gliosis. Rapid photoreceptor loss largely occurred in the macula-equivalent retinal region. The homozygous cats developed markedly increased ocular globe length. CONCLUSIONSThe phenotype of CRXRdy/Rdy cats was more severe compared to CRXRdy/+ cats by several metrics. TRANSLATIONAL RELEVANCEThe CRX-mutant cat is the only model for CRX-retinopathies with a macula-equivalent region. A prominent feature of the CRXRdy/Rdy cat phenotype not detectable in homozygous mouse models, was the rapid degeneration of the macula-equivalent retinal region highlighting the value of this large animal model and its future importance in the testing of translational therapies aiming to restore vision.

neuroscience↗

bioRxiv2023

Analyzing genomic alterations involved in fluoroquinolone-resistant development in Staphylococcus aureus

AimRecently, the rise in Staphylococcal infection incidence accompanied by a rise of antibiotic-resistant strains is a major threat to public health. In this study, mechanisms leading to the occurrence of high-level multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) strains after fluoroquinolone (FQ) exposure were investigated. MethodologySerially exposing S. aureus ATCC 29213 to ciprofloxacin (CIP), ofloxacin (OFL), or levofloxacin (LEV) at sub-minimum inhibitory concentrations (sub-MICs) for 12 days was performed to obtain S. aureus -1 strains and culturing for another 10 days without antibiotics to obtain S. aureus-2 strains. The genomic alterations in FQ-exposed strains were reached using whole genome sequencing and target sequencing. The expressions of efflux-related genes, alternative sigma factors, and genes involved in FQ resistance were evaluated using RT-qPCR. ResultsAfter serial FQ exposure, we observed a strong and irreversible increase of MICs to all applied FQs, i.e 32 to 128 times in all S. aureus-1 and remained 16 to 32 times in all S. aureus-2. WGS indicated 10 significant mutations including 2 deletions, 1 insertion, and 7 missense mutations that occur in all S. aureus-1 and -2 but not in initial strain. The FQ target, GrlA, was also mutated (R570H) in all S. aureus-1 and -2 which can partly explain the development of FQ resistance over the FQ exposure. Besides, FQ exposure also resulted in overexpression of genes encoding for (1) efflux pumps and their regulator (norA, norB, norC, and mgrA); (2) alternative sigma factors (sigB and sigS); (3) acetyltransferase (rimI); (4) methicillin resistance (fmtB); and (5) hypothetical protein BJI72_0645. ConclusionThe mutations occurred in the FQ-target sequence were associated with high-level FQ resistance while the activation of efflux pump systems and post-translational proteins played an important role in the emergence of MDR in S. aureus. Author summaryAntimicrobial resistance is a major public health problem worldwide. Multiple studies have been performed to understand how bacteria develops resistance during the antibiotic therapy in vitro and in vivo. Here we revealed how Staphylococcus aureus, a stubborn human pathogen, changed its genome and expression of important genes in responding with sub-MIC exposure to flouroquinolone antibiotics. Mutations were found in the target of flouroquinolones such as GrlA (R570H) and interestingly in some hypothetical regions which may be important for gene expression regulation. We have observed an marked overexpression of genes encoding for (1) efflux pumps and their regulator (norA, norB, norC, and mgrA); (2) alternative sigma factors (sigB and sigS); (3) acetyltransferase (rimI); (4) methicillin resistance (fmtB); and (5) hypothetical protein BJI72_0645 in all exposed strains. Importantly, the expression change still remained when the bacteria were no longer exposed to the antibiotics. This study is important to understand response of S. aureus to flouroquinolone and how it obtains the resistance phenotype under antibiotic exposure.

microbiology↗