Biology subjects

Sun, S.

Publications and source records attributed to Sun, S..

At least 145 records · Page 8Linked to original sources

bioRxiv2024

SUMO Inhibition Plus CD40 Agonism Increases Anti-Tumor Immunogenicity Through Interferon Mediated Macrophage Activation

Resistance to immunotherapy is a cardinal feature of pancreatic ductal adenocarcinoma (PDAC). Inhibition of Small Ubiquitin-like MOdifier (SUMO), a post-translational modification with important immune regulatory functions, augments responsiveness to immunotherapy in non-PDAC models via pro-immunogenic effects on myeloid cells, cancer cells, and T-cells. Recently, it has been reported that SUMO inhibition has direct immunogenic effects on PDAC. Here, we report that the novel combination of SUMO inhibition with a small molecule, TAK-981, plus antibody-mediated CD40 agonism improves survival in an aggressive orthotopic mouse model of PDAC by enhancing anti-tumoral immunogenicity. This combination amplifies CD8+ T-cell tumor infiltration and induces significant changes among macrophages. TAK-981 also leads to enhanced cancer specific MHC-I expression both in vitro and in vivo by augmenting interferon signaling. We show that the improvement in survival is mediated by macrophages. Our findings show that SUMO inhibition complements CD40 agonism to enhance immune activity in PDAC via interferon signaling, improving survival in an aggressive pre-clinical model of PDAC and translating previous findings to a characteristically immunosuppressive and highly aggressive solid malignancy.

cancer biology↗

bioRxiv2024

Essential genes encoded by the mating-type locus of the human fungal pathogen Cryptococcus neoformans

Fungal sexual reproduction is controlled by the mating-type (MAT) locus. In contrast to a majority of species in the phylum Basidiomycota that have tetrapolar mating-type systems, the opportunistic human pathogen Cryptococcus neoformans employs a bipolar mating-type system, with two mating types (a and ) determined by a single MAT locus that is unusually large ([~]120 kb) and contains more than 20 genes. While several MAT genes are associated with mating and sexual development, others control conserved cellular processes (e.g. cargo transport and protein synthesis), of which five (MYO2, PRT1, RPL22, RPL39, and RPO41) have been hypothesized to be essential. In this study, through genetic analysis involving sporulation of heterozygous diploid deletion mutants, as well as in some cases construction and analyses of conditional expression alleles of these genes, we confirmed that with the exception of MYO2, both alleles of the other four MAT genes are indeed essential for cell viability. We further showed that while MYO2 is not essential, its function is critical for infectious spore production, faithful cytokinesis, adaptation for growth at high temperature, and pathogenicity in vivo. Our results demonstrate the presence of essential genes in the MAT locus that are divergent between cells of opposite mating types. We discuss possible mechanisms to maintain functional alleles of these essential genes in a rapidly-evolving genomic region in the context of fungal sexual reproduction and mating-type evolution. IMPORTANCESexual reproduction is essential for long-term evolutionary success. Fungal cell type identity is governed by the MAT locus, which is typically rapidly evolving and highly divergent between different mating types. In this study, we show that the a and alleles of four genes encoded in the MAT locus of the opportunistic human fungal pathogen C. neoformans are essential. We demonstrate that a fifth gene, MYO2, which had been predicted to be essential, is in fact dispensable for cell viability. However, a functional MYO2 allele is important for cytokinesis and fungal pathogenicity. Our study highlights the need for careful genetic analyses in determining essential genes, which is complementary to high-throughput approaches. Additionally, the presence of essential genes in the MAT locus of C. neoformans provides insights into the function, maintenance, and evolution of these fast-evolving genomic regions.

genetics↗

bioRxiv2024

A TROP2/Claudin Program Mediates Immune Exclusion to Impede Checkpoint Blockade in Breast Cancer

BackgroundImmune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. In triple-negative breast cancer (TNBC), an aggressive and generally immune-rich subtype, an immune-cold microenvironment predicts poor prognosis due to a limited response to chemotherapy and immune checkpoint inhibitors. This study aimed to identify mechanisms regulating immune infiltration in TNBC. MethodsWe performed spatial transcriptomic analysis comparing immune-enriched versus immune-cold treatment-naive TNBCs. Functional analyses, including loss-of-function and reconstitution experiments, were conducted to investigate the role of Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of anti-cancer Antibody Drug Conjugates (ADCs), in promoting TNBC progression. A humanized TROP2 syngeneic TNBC model was used to assess the effects of TROP2-targeting in combination with anti-PD1 therapy. Additionally, data from patients treated with immune checkpoint blockade were used to test hypotheses from the preclinical findings. ResultsWe reveal that TROP2 controls barrier-mediated immune exclusion in TNBC through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and predicts poor outcomes in TNBC. We demonstrate that TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. We show that TROP2 targeting via hRS7, the antibody component of the ADC Sacituzumab govitecan (SG), enhances the anti-PD1 response and improves T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. ConclusionsThis study defines a new mechanism of barrier-mediated immune exclusion in cancer controlled by TROP2-dependent tight junctions. This mechanism drives tumor progression but can be targeted via TROP2-directed therapy to activate anti-tumor immunity and enhance immunotherapy response. What is already known on this topicBarrier-mediated immune exclusion is emerging as a mechanism of immune evasion in cancer such as triple-negative breast cancer (TNBC), but its molecular underpinnings remain poorly understood. TROP2 is a surface glycoprotein targeted by antibody-drug conjugates such as Sacituzumab govitecan, yet its functional role beyond drug delivery has been unclear. What this study addsThis study identifies TROP2 as a key regulator of tight junction-mediated immune exclusion in TNBC, independent of its intracellular signaling function. TROP2 promotes an immune-cold tumor microenvironment by enforcing mechanical barriers that limit T cell infiltration. How this study might affect research, practice or policyThese findings establish TROP2 as a functional driver of immune evasion and provide a mechanistic rationale for combining TROP2-targeting therapies with immune checkpoint inhibitors to overcome resistance in immune-excluded tumors.

cancer biology↗

bioRxiv2024

Molecular mechanism of Na+/H+ antiporting in NhaA

Sodium-proton antiporter NhaA of Escherichia coli is a paradigm to investigate the mechanistic basis of the fundamental Na+/H+ exchange in cells. However, all existing crystal structures of NhaA are inward-facing (IF) and the putative outward-facing (OF) structures are still unsolved by experiment, limiting a complete understanding of the transport cycle where Lys300 plays a key role in both structural stability and transport function. Here, we report a set of regular molecular dynamics (MD) simulations that start from the structure predicted by an artificial intelligence method that generates function-relevant alternative conformations. It is found that NhaA rapidly relaxes into either the IF or OF conformation. Further-more, neutralization of Lys300 allows two sodium ions bound to the reaction cavity, which is associated with enhanced conformational sampling. Based on these observations, we propose a sodium-coupled mechanism of Na+/H+ antiporting.

biophysics↗

bioRxiv2024

The impact of transplant location on the gut microbiome and resistome in patients undergoing hematopoietic stem cell transplantation at home versus in the hospital

ObjectivesHome-based hematopoietic stem cell transplantation (HCT) is a novel approach that has the potential to improve outcomes, however, the impact of transplant location on the gut microbiome remains uncharacterized. We hypothesized that patients randomized to undergo home HCT would have higher gut taxonomic diversity and lower antimicrobial resistance (AMR) gene abundance compared to those undergoing standard hospital HCT. MethodsWe identified 28 patients enrolled in Phase II randomized trials of home (n=16) v. hospital (n=12) HCT at Duke and performed shotgun metagenomic sequencing of stools to compare taxonomic and AMR gene composition between groups. We performed a secondary analysis of patients from each group transplanted at an outpatient infusion clinic with those who underwent standard inpatient HCT ("outpatient" v. "inpatient"). ResultsNo significant differences in duration of hospitalization were found in those randomized to home v. hospital HCT. Taxonomic and AMR gene - and {beta}-diversity were comparable. In contrast, secondary analyses demonstrated that patients from both home and hospital groups transplanted at an outpatient infusion clinic spent significantly less time in the hospital and demonstrated higher taxonomic -diversity and differential {beta}-diversity compared to standard inpatient HCT, although AMR gene -diversity did not differ, and comparisons were confounded by both differences in transplant type and use of antibiotics. ConclusionsRandomization by transplant location did not impact the gut microbiota to the same extent as the duration of hospitalization, although secondary analyses were heavily confounded. Even when taxonomic differences were observed, AMR genes were similar between groups.

microbiology↗

bioRxiv2024

Interpretable PROTAC degradation prediction with structure-informed deep ternary attention framework

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional ligands that form ternary complexes with Protein Of Interests (POIs) and E3 ligases, exploiting the ubiquitin-proteasome system to degrade disease-associated proteins, promising to drug the undruggable. While PROTAC research primarily relies on costly and time-consuming wet experimental approaches, deep learning offers a promising avenue to accelerate development and reduce expenses. However, existing deep learning methods for PROTAC degradation prediction often overlook the significance of hierarchical molecular representation and protein structural information, hindering effective data modeling. Moreover, their black-box nature limits the interpretability of computational outcomes, failing to provide intuitive insights into substructure interactions within the PROTAC system. This study introduces PROTAC-STAN, a structure-informed deep ternary attention network (STAN) framework for interpretable PROTAC degradation prediction. PROTAC-STAN represents PROTAC molecules across atom, molecule, and property hierarchies and incorporates structure information for POIs and E3 ligases using a protein language model infused with structural data. Furthermore, it simulates interactions among three entities at the atom and amino acid levels via a novel ternary attention network tailored for the PROTAC system, providing unprecedented insights into the degradation mechanism. By integrating hierarchical PROTAC molecule representation, structural embedding of POI and E3 ligase, and ternary attention network modeling interactions, our approach substantially improves prediction accuracy by 10.95% while enabling significant model interpretability via atomic and residue level visualization of molecule and complex. Experiments on the refined public PROTAC dataset demonstrate that PROTAC-STAN outperforms state-of-the-art baselines in overall performance. The excellent performance of PROTAC-STAN is anticipated to establish it as a foundational tool in future research on PROTAC-related drugs, thereby accelerating the development of this field.

bioinformatics↗

bioRxiv2024

Novel environment exposure drives temporally defined and region-specific chromatin accessibility and gene expression changes in the hippocampus

The interaction of mammals with a novel environment (NE) results in structural and functional changes in multiple brain areas, including the hippocampus. This experience-dependent circuit reorganization is driven in part by changes in gene expression however, the dynamic sensory experience-driven chromatin states and the diverse cell type specific gene expression programs that are regulated by novel experiences are not well described. We employed single- nucleus multiomics (snRNA- and ATAC-seq) and bulk RNA-seq of the hippocampal DG, CA3, and CA1 regions to characterize the temporal evolution of cell-type-specific chromatin accessibility and gene expression changes that occur in 14 different cell types of the hippocampus upon exposure of mice to a novel environment. We observe strong hippocampal regional specificity in excitatory neuron chromatin accessibility and gene expression as well as great diversity in the inhibitory neuron and non-neuronal transcriptional responses. The novel environment-regulated genes in each cell type were enriched for genes that encode secreted factors, and cell-type-specific expression of their cognate receptors identified promising candidates for the modulation of learning and memory processes. Our characterization of the effect of novel experience on chromatin revealed thousands of cell-type-specific changes in chromatin accessibility. Coordinated analysis of chromatin accessibility and gene expression changes within individual cell types identified Fos/AP-1 as a key driver of novel experience-induced changes in chromatin accessibility and cell-type-specific gene expression. Together, these data provide a rich resource of hippocampal chromatin accessibility and gene expression profiles across diverse cell types in response to novel experience, a physiological stimulus that affects learning and memory.

neuroscience↗

bioRxiv2024

Targeting the 3D genome by anthracyclines for chemotherapeutic effects

The chromatin is folded into three-dimensional (3D) structures, and aberrant 3D chromatin folding has been implicated in cancer. We performed ATAC-seq and TOP2A ChIP-seq to assess the potential effects of various anthracycline drugs on the chromatin architecture. We found that specific anthracycline variants selectively disrupt chromatin looping anchors by interfering with CTCF binding, suggesting an additional therapeutic mechanism of anthracycline drugs targeting the 3D genome. Hi-C experiments in K562 cells treated with anthracycline drugs revealed widespread disruption of 3D chromatin organization, including altered long-range regulation at the Myc locus. Furthermore, AML patients treated with anthracycline drugs exhibited changes in chromatin structures near possible looping anchors, which were associated with distinct clinical outcomes. Together, our findings indicate that anthracycline drugs function as potent and selective epigenomic modulators, with the capacity to further target the 3D genome to exert anticancer effects, highlighting their potential for personalized therapy in tumors with aberrant 3D chromatin architecture. SignificanceChromatin structure plays a crucial role in regulating gene expression and maintaining cellular function. Aberrant chromatin 3D organization is a common feature in cancer. In this study, we investigate how anthracycline chemotherapy affects chromatin architecture using state-of-the-art genomic profiling techniques, including ATAC-seq, TOP2A ChIP-seq, CTCF ChIP-seq, and Hi-C. We show that certain anthracycline variants disrupt chromatin looping by interfering with CTCF binding, thereby altering the spatial genome organization. This disruption leads to changes in the regulation of associated genes, as exemplified by the Myc locus, and is associated with distinct clinical outcomes in AML patients. These findings highlight the potential of anthracycline drugs as therapeutics that target both the epigenome and 3D chromatin architecture, a promising strategy in personalized therapy.

cancer biology↗

bioRxiv2024

A Multi-Modal Genomic Knowledge Distillation Framework for Drug Response Prediction

Precision oncology utilizes genomic data to tailor treatment to individuals. Cancer drug sensitivity studies can predict the response levels of different drugs for the same cultured cancer cell line, which is beneficial for personalized medicine. Recent studies have demonstrated that integrating multi-modal genomic data, e.g., gene expression, mutation, copy number alteration, methylation, can provide comprehensive knowledge and improve drug response prediction. Although multimodal genomic profiles are generally available from public datasets, only gene expression data is commonly used in clinical settings. In this study, we propose a framework for privileged information knowledge distillation to transfer knowledge from a multi-modal genomic teacher network, using only gene expression for inference. Specifically, we train a teacher network by feature re-weighting based on inter-modality dependencies and align the inter-sample correlations through our proposed relation-aware differentiation distillation. Experiments on the Genomics of Drug Sensitivity in Cancer (GDSC) dataset demonstrate that our framework improves drug response prediction by about 6% compared to the baseline and outperforms state-of-the-art methods. Transferable studies performed on missing GDSC data and clinical datasets further confirm the feasibility of our model for predicting drug responses using only gene expression data.

bioinformatics↗

bioRxiv2024

Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture

ABSTRACTSynaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter enables monitoring of proteins in live microscopy, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 here as a highly-efficient dye for SNAP-tag labeling of extracellular epitopes, and of intracellular proteins in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal- and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for the analysis of presynaptic nanoarchitectural dynamics, with a potential for wide adoption.

neuroscience↗

bioRxiv2024

Endosomal hyper-acidification via proton-activated chloride channel deletion in neurons impairs AMPA receptor endocytosis and LTD

Endosomal homeostasis is critical for neuronal function, including the post-synaptic trafficking of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Dynamic AMPAR trafficking is a major component of synaptic plasticity, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD) and is thought to be required for learning and memory. Dramatic alteration of endosomal pH has been reported to negatively affect synaptic transmission and neural development, but the underlying mechanisms by which pH is involved in AMPAR trafficking are unclear. Here, we show that the proton-activated chloride (PAC) channel localizes to early and recycling endosomes along neuronal dendrites and prevents hyper-acidification of endosomes. To directly measure AMPAR endocytosis, we used a new method to assess LTD using HaloTag-GluA2 and found that the loss of PAC reduces AMPAR internalization during chemical LTD in primary neurons, while AMPAR trafficking in unstimulated cells or during chemical LTP is unaffected. Consistently, pyramidal neuron-specific PAC knockout mice had impaired hippocampal LTD, but not LTP, and performed poorly in the Morris water maze reversal test, exhibiting an inability to adapt to changing environments (also referred to as behavioral flexibility). We conclude that proper maintenance of pH by PAC is important during LTD to regulate AMPAR trafficking in a manner critical for animal physiology and behavior. SummaryThe ability to adapt to changing environments stems from the plasticity of neurons, which can modulate their synaptic strength in response to neural activity. We discovered a novel mechanism by which an endosomal proton-activated chloride channel (PAC) is involved in synaptic weakening, or long-term depression (LTD). To improve tools used to study LTD, we developed a live-cell imaging assay to directly observe -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) endocytosis. PAC-deficient neurons have hyper-acidified endosomes and fail to endocytose AMPARs during LTD. Neuron-specific PAC knockout mice have impaired hippocampal LTD and fail to adapt to changes in their environment. The role of endosomal pH in synaptic function is understudied, and our results provide a novel mechanism whereby PAC can affect synaptic LTD.

neuroscience↗

bioRxiv2024

Mechanism of NPC1L1 mediated 27-hydroxycholesterol metabolisms in the occurrence and development of osteoporosis

Cholesterol metabolism is closely related to the occurrence and development of osteoporosis, but the exact mechanism remains unclear. Niemann-Pick C1-like 1 (NPC1L1) is one of the key cholesterol transporter proteins, however, there are few reports on the functions of NPC1L1 besides regulating cholesterol transport, let alone bone homeostasis. Our preliminary research indicated that NPC1L1 may play a negative regulatory role in osteogenic differentiation. In this study, using in vitro osteogenic differentiation experiment and mouse osteoporosis model, we showed here that NPC1L1 expression was downregulated during osteogenesis, and NPC1L1 knockdown significantly enhanced osteogenic differentiation ability of osteoblasts and delayed progress of osteoporosis. Mechanistically, through RNA sequencing, NPC1L1 was found regulate cholesterol metabolism rather than transportation. It increased 27-Hydroxycholesterol (27-OHC) level through activating 27-hydroxylase (Cyp27a1), resulting in 27-OHC accumulation in osteoblasts and inhibition of osteogenesis. Moreover, C/EBP was proved mediated NPC1L1 promotes production of 27-OHC by Cyp27a1. These findings reveal that NPC1L1 inhibits osteogenesis and promotes osteoporosis via regulate cholesterol metabolism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/615783v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11a62bforg.highwire.dtl.DTLVardef@148fb9aorg.highwire.dtl.DTLVardef@7e486eorg.highwire.dtl.DTLVardef@c53116_HPS_FORMAT_FIGEXP M_FIG NPC1L1 inhibits osteogenic differentiation and promotes the progression of OP through the C/EBP/Cyp27a1/27-OHC axis. C_FIG

molecular biology↗

bioRxiv2024

A one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ( dye shuttle) conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.

pharmacology and toxicology↗

bioRxiv2024

Single-cell analysis of the somatic mutational landscape in human chondrocytes during aging and in osteoarthritis

Somatic mutation is now recognized as a cause of multiple human diseases other than cancer. Osteoarthritis (OA), a highly prevalent age-related disease, has been associated with increased chromosomal abnormalities in articular cartilage of OA patients. Thus far no systematic attempt has been made to characterize the somatic mutational landscape of chondrocytes during normal aging and in affected cartilage of OA patients. Here we used single-cell whole genome sequencing to quantitatively analyze single-nucleotide variants (SNVs) and small insertions and deletions (InDels) in 100 single chondrocytes isolated from the cartilage of hip femoral heads of 17 subjects aged from 26 to 90 years, including 9 OA patients and 8 non-OA donors. Both SNVs and InDels were found to accumulate with age in chondrocytes with a clock-like mutational signature. Surprisingly, the age-related accumulation rate of these mutations was found to be lower in OA chondrocytes compared with chondrocytes from non-OA control subjects.

genomics↗

bioRxiv2024

Solving the "Blind men and the elephant problem": Additive deep learning of complex high dimensional models from partial faceted datasets

Biological systems are complex networks involving tens of thousands of interacting molecular components, and measurable biological functions are emerging properties of these complex networks. Many quantitative studies in biology attempt to connect biological function with molecular components and genes, in the process developing mechanistic understanding. However, it is challenging to quantify the contribution of all components to the biological function simultaneously, especially at the single cell level. Instead, in typical experiments, only a subset of the variables (or facet) is measured. This makes it difficult to obtain a complete and unbiased understanding of the network and how different components of the network cooperatively contribute to the biological function. In this paper, we explore a machine learning approach to combine different facets of data and obtain a complete picture of the biological system based on conditional distributions from faceted data subsets. Both a polynomial regression approach and a neural network approach are developed and examined with two set of concrete examples: A mechanical spring network system deforming under external forces and a small (8-dimensions) biological network including the cellular senescence marker P53. In the later example, single cell data is collected to validate the machine learning approach. We find that the full system is successfully reconstructed from faceted data in both examples. We further discuss the additive property of the model, where the model predictive accuracy increases with increasing number of simultaneously measured variables (dimension of subsets). Our model provides a systematic and novel approach to integrate different pieces of experimental information to reconstruct complex high dimensional systems, arriving at an unbiased and wholistic model of biological function.

bioinformatics↗

bioRxiv2024

Temporal Dynamics and Adaptive Mechanisms of Microbial Communities: Divergent Responses and Network Interactions

Microbial communities are integral to aquatic ecosystems, playing key roles in biogeochemical cycles, nutrient recycling, and ecosystem functioning. Despite their importance, the temporal dynamics of these communities, especially in response to short-term environmental changes, remain inadequately understood. This study utilizes high-throughput amplicon sequencing and Co-occurrence network analysis to elucidate how prokaryotic and eukaryotic microbial communities dynamically respond to short-term environmental fluctuations, and dynamics of networks. Our results reveal distinct temporal patterns, with eukaryotic communities showing consistent increases in diversity, while prokaryotic communities exhibited more pronounced fluctuations and directional changes. Co-occurrence network analysis further highlighted the differences in community structure between phases of homogenization and heterogenization. Specifically, we observed a shift from a more modular network during the homogenization phase to a more centralized network in the heterogenization phase, resulting in increased network fragility. Environmental factors, such as nutrient levels and climatic conditions, were found to play pivotal roles in shaping these dynamics. This study provides critical insights into the resilience and adaptability of microbial communities, emphasizing the intricate interplay between biodiversity, network structure, and ecosystem stability under varying environmental conditions.

microbiology↗

bioRxiv2024

Distinct evolutionary trajectories following loss of RNA interference in Cryptococcus neoformans

While increased mutation rates typically have negative consequences in multicellular organisms, hypermutation can be advantageous for microbes adapting to the environment. Previously, we identified two hypermutator Cryptococcus neoformans clinical isolates that rapidly develop drug resistance due to transposition of a retrotransposon, Cnl1. Cnl1-mediated hypermutation is caused by a nonsense mutation in the gene encoding a novel RNAi component, Znf3, combined with a tremendous transposon burden. To elucidate adaptative mechanisms following RNAi loss, two bioinformatic pipelines were developed to identify RNAi loss-of-function mutations in a collection of 387 sequenced C. neoformans isolates. Remarkably, several RNAi-loss isolates were identified that are not hypermutators and have not accumulated transposons. To test if these RNAi loss-of-function mutations can cause hypermutation, the mutations were introduced into a non-hypermutator strain with a high transposon burden, which resulted in a hypermutator phenotype. To further investigate if RNAi-loss isolates can become hypermutators, in vitro passaging was performed. Although no hypermutators were found in two C. neoformans RNAi-loss strains after short-term passage, hypermutation was observed in a passaged C. deneoformans strain with increased transposon burden. Consistent with a two-step evolution, when an RNAi-loss isolate was crossed with an isolate containing a high Cnl1 burden, F1 hypermutator progeny inheriting a high transposon burden were identified. In addition to Cnl1 transpositions, insertions of a novel gigantic DNA transposon KDZ1 ([~]11 kb), contributed to hypermutation in the progeny. Our results suggest that RNAi loss is relatively common (7/387, [~]1.8%) and enables distinct evolutionary trajectories: hypermutation following transposon accumulation or survival without hypermutation. Significance StatementThere is a dearth of antifungal drugs available to treat Cryptococcus neoformans, a human fungal pathogen of global impact. We previously identified natural hypermutators with a loss-of-function mutation in the RNAi machinery and transposon expansion. Here, we identified several novel natural isolates with RNAi defects, none of which are hypermutators or have undergone transposon expansion. Furthermore, we demonstrate that these isolates can lie on a pathway to hypermutation following introduction of a transposon burden. In addition, a novel DNA transposon class was discovered that contributes to antifungal drug resistance. These findings highlight the importance of transposons in driving rapid adaptation in the absence of RNAi and reveal distinct evolutionary trajectories following RNAi loss, a relatively common event in C. neoformans.

genetics↗

bioRxiv2024

A Small Interfering Peptide Potentiates AMPA Receptor Diffusional Trapping and Prevents Social-Isolation-Induced Forgetting of Fear Memory

Synaptic trapping of AMPA receptors (AMPARs) is a key mechanism regulating excitatory synaptic transmission and activity-dependent plasticity underlying learning and memory. Destabilization of synaptic AMPARs is increasingly implicated in cognitive dysfunction across neurological and neuropsychiatric disorders, yet strategies to directly modulate this process in vivo remain limited, constraining both mechanistic insight and therapeutic development. Here we present a peptide-based strategy to enhance AMPAR synaptic trapping by targeting the interaction between transmembrane AMPA receptor regulatory proteins (TARPs) and activity-regulated cytoskeleton-associated protein (ARC/Arg3.1). We designed a 21-amino-acid TAT-fused peptide (TARP-pep) mimicking the TARP C-terminal motif that binds the ARC N-lobe. TARP-pep disrupted the TARP-ARC interaction and increased the stabilization of AMPARs at synaptic surfaces in cultured hippocampal neurons. In vivo, acute intrahippocampal infusion of TARP-pep enhanced perforant path-evoked synaptic transmission in the rat dentate gyrus (DG) and strengthened the interaction between TARPs and postsynaptic density protein 95 (PSD-95), a key mechanism underlying AMPAR anchoring. Consistent with a long-term potentiation (LTP)-like synaptic state, TARP-pep increased basal phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and occluded further chemical LTP (cLTP)-induced increases in phosphorylated CaMKII (p-CaMKII). Notably, a 7-day regimen of daily bilateral DG injections of TARP-pep prevented social isolation (SI)-induced impairment of fear memory in mice. Together, these findings identify the TARP-ARC interaction as a druggable regulator of AMPAR diffusional trapping and highlight synaptic AMPAR stabilization as a promising therapeutic strategy for preserving cognitive function under conditions of circuit vulnerability.

neuroscience↗