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An, D.

Publications and source records attributed to An, D..

At least 19 recordsLinked to original sources

How sparse can a fly-brain model be? Connection strength, wiring placement, network state and task jointly determine function-preserving compression of the Drosophila connectome

Whole-brain connectomes are increasingly used as wiring diagrams for spiking simulations and as structural priors for artificial networks, usually after an ad hoc synapse-count threshold whose effect on validated function has never been measured. We define function-preserving sparsification - removing edges under a fixed budget while preserving validated behaviour and population responses - and study it in the leaky integrate-and-fire whole-brain model of Shiu et al. (2024). Six operators - weak-synapse pruning, uniform and weight-proportional random removal, degree-preserving rewiring, weight shuffling and a task-aware criterion - were compared at identical edge budgets across three validated circuits (sugar-evoked proboscis extension, its bitter suppression, grooming pathway specificity), silent and spontaneously active network states, and multiple seeds. Weak-synapse pruning preserves 79% of feeding output with 6% of edges, with a cliff at k* of approximately 9 synapses, whereas random, degree-preserving and weight-shuffled controls at the same budget abolish output, and weight-proportional random sampling survives only while it retains the strong-edge set: function resides in the conjunction of wiring placement and strength, not in degree statistics or synaptic mass. The cliff is task-dependent - grooming specificity collapses below k = 2 through disinhibition. In the active state failure changes character: output over-excites instead of vanishing, and bitter suppression is lost progressively from k = 5 and reverses at k = 31. A task-aware criterion preserves its training stimulus at 0.4% of edges but loses held-out inhibition first. These results provide a first benchmark for connectome sparsification and show that what must be kept depends on task and network state.

neuroscience↗

Electrostatics and Local Aromatic Residues Govern Lipid Binding and Membrane Penetration of Synaptotagmin C2 Domains

Synaptotagmins (Syts) are Ca{superscript 2}-sensing exocytosis regulators whose tandem C2 domains interact with phosphoinositides and membranes to trigger neurotransmitter and hormone release. Although Ca{superscript 2} binding is known to enhance C2 domain-membrane interactions, the sequence determinants governing lipid binding and membrane penetration across Syt isoforms remain incompletely understood. Here, we performed MARTINI coarse-grained molecular dynamics simulations of isolated C2A and C2B domains from eight Ca{superscript 2}-sensing Syt isoforms (Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9, and Syt10) interacting with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing plasma membranes. To systematically modulate electrostatic properties, we introduced partial and full charge-flip mutations at conserved acidic residues within the calcium-binding loops (CBLs). By integrating simulations across multiple isoforms and charge states, we sought to identify the dominant sequence determinants governing membrane interactions. We found that PIP2 binding to both, CBLs and polybasic patches (PBs), is associated with loop net charge, yielding correlations > 0.95 across all isoforms. However, membrane penetration is not sufficiently explained by loop net charge alone. The local phenylalanines additionally increase membrane penetration independent of loop net charge. Together, these findings establish a comprehensive electrostatic-aromatic framework where loop net charge governs PIP2 binding, whereas loop net charge and local phenylalanine enrichment jointly govern membrane penetration across Syt C2 domains.

biophysics↗

Why are so many fusogens rod-shaped?

Molecular fusogens catalyze membrane fusion for many basic biological processes. In eukaryotic cells, SNARE proteins drive membrane fusion for trafficking and for exocytic release in many contexts, from neurotransmission to enzymatic digestion, while other fusogens mediate cell-cell fusion for organ formation, placental development and gamete fusion. Enveloped viruses use glycoprotein fusogens for host cell entry and delivery of the viral genome. Despite this breadth of roles, a structural feature shared by many of these fusogens is their rod shape, conserved across the SNARE superfamily, the class I and II fusogen superfamilies and the class III fusogen family. Here we used highly coarse-grained molecular dynamics (MD) simulations to examine the collective behavior of rod-like fusogens on the microscopically long timescales of physiological membrane fusion. Rod-generated entropic forces maintained a cleared fusion site, squeezed and hemifused the membranes, and then expanded and ruptured the hemifused connection to yield fusion. More fusogens generated higher entropic forces and faster fusion, consistent with electrophysiological measurements at neuronal synapses. The required fusogenic feature was the rod shape, since simulated SNARE complexes, class II EFF-1 fusogens, and model rod-shaped complexes entropically drove fusion along similar pathways, whereas globular complexes failed. Thus, rod-like fusogens are optimally shaped generators of entropic forces that drive membrane fusion. These results suggest a universal rod-based fusion mechanism may have been the evolutionary driver of structural convergence among major classes of eukaryotic and viral fusogens. SignificanceLarge classes of eukaryotic and viral membrane fusion complexes are rod-shaped, a common structural feature despite a multiplicity of functions, from neurotransmission to organogenesis to cell entry. Here we used coarse-grained molecular dynamics to access the millisecond timescales of physiological fusion. Simulations revealed a universal mechanism whereby, regardless of structural details, rod-shaped fusogens generate entropic forces that drive fusion. This suggests the mechanism was a major evolutionary driver of structural convergence and multiple transmissions between eukaryotic hosts and viruses. Rod-mediated fusion is faster with more fusogens, explaining why release at presynaptic nerve terminals is enhanced or suppressed depending on the number of active SNARE fusogens. Thus, modulation of SNARE numbers and fusogenicities may represent a significant mechanism of synaptic plasticity.

biophysics↗

OriGene: A Self-Evolving Virtual Disease Biologist Automating Therapeutic Target Discovery

Here, we present OriGene, a self-evolving multi-agent system that functions as a virtual disease biologist, systematically identifying original and mechanistically grounded therapeutic targets at scale. OriGenes architecture integrates over 600 specialized tools through a Model Context Protocol (MCP), enabling it to reason across diverse data modalities including genomics, protein networks, pharmacology, clinical records and literature evidence, to generate and prioritize target discovery hypotheses. We implemented a strategy combining a knowledge graph-based Tool RAG with an advanced agent selection mechanism to enable dynamic, context-aware tool deployment. Through a self-evolving framework, OriGene continuously integrates human and experimental feedback to iteratively refine its core thinking templates, tool composition, and analytical protocols, thereby enhancing both accuracy and adaptability over time. To comprehensively evaluate its performance, we established TRQA, an original benchmark comprising over 1,900 expert-level question-answer pairs spanning a wide range of diseases and target classes. OriGene consistently outperforms human experts, leading research agents, and state-of-the-art large language models in accuracy, recall, and robustness, particularly under conditions of data sparsity or noise. Critically, OriGene nominated previously underexplored therapeutic targets for liver (GPR160) and colorectal cancer (ARG2), which demonstrated significant anti-tumor activity in patient-derived organoid and tumor fragment models mirroring human clinical exposures. These findings demonstrate OriGenes potential as a scalable and adaptive platform for AI-driven discovery of mechanistically grounded therapeutic targets, offering a new paradigm to accelerate drug development.

bioinformatics↗

An in vivo CRISPR screen unveils top target genes to efficiently improve CAR-T cell persistence in a solid tumour model of lung adenocarcinoma

CAR-T cell therapies are revolutionizing the treatment of refractory and relapsed haematological malignancies, but many patients do not exhibit long-term responses, and these therapies are less effective against solid tumors. Poor persistence of CAR-T cells in patients is associated with therapeutic failure, highlighting the need to identify strategies promoting in vivo expansion. Here, we developed an in vivo competitive screening method to identify genes whose inactivation confers a selective advantage to CAR-T cells. Inactivation of 50 genes in a heterogeneous population of T cells expressing an EGFR-targeting CAR revealed that disruption of REGNASE-1, SOCS1, PTPN2, and P16/NK4A conferred a selective advantage to CAR-T cells in human lung tumor-bearing mice. Consistently, inactivation of these genes improved tumor eradication by CAR-T cells. Interestingly disruption of other genes, described to improve CAR-T cell function in other contexts, had a negative impact in this orthotopic lung tumor model. Further evaluation of long-term effects in a subcutaneous model, highlighted SOCS1 ablation as the most promising strategy for in vivo CAR-T cell amelioration. These results support the importance of evaluating CAR-T cell editing strategies in tumor-specific models and highlight the versatility of our screening approach as a pre-clinical tool for context-specific studies on CAR-T cells amelioration.

immunology↗

Syntaxin 1A Transmembrane Domain Palmitoylation 1 Induces a Fusogenic Conformation

Neurotransmitter release is triggered by the fusion of synaptic vesicles with the plasma membrane, orchestrated by SNARE proteins Synaptobrevin 2 (Syb2), Syntaxin 1A (Stx1A), and SNAP25. Recent experimental studies showed that Stx1A palmitoylation of C271/C272 promotes spontaneous neurotransmitter release. However, the mechanistic role of SNARE transmembrane domain (TMD) palmitoylation in membrane fusion remains unclear. To investigate the structural and functional implications of TMD palmitoylation, we employed coarse-grained molecular dynamics simulations with the MARTINI force field. In simulations of individual SNAREs and of SNAP-25/Stx1A (t-SNARE) complexes in a membrane the palmitoyl chains of Syb2 and Stx1A localize to the membrane midplane, with Stx1A palmitoyl chains bending toward the extracellular leaflet. Non-palmitoylated Stx1A assumed a conformation where the SNARE domain was lying flat, adhering to the intracellular surface of the membrane. Stx1A dual palmitoylation induced dramatic changes, reducing the tilt of its TMD and stabilizing a more upright conformation of its SND. This conformation resembles the Stx1A conformation in a s Stx1A-SNAP25 t-SNARE complex, providing a potential mechanistic explanation of how Stx1A TMD palmitoylation facilitates early steps in SNARE complex formation and thus promotes spontaneous release. In simulations of the late steps of layers 5 to 8 SNARE complex zippering in a system of 4 SNARE complexes bridging a 10-nm nanodisc and a planar membrane, FPs spontaneously opened after a few hundred nanoseconds, preceded by distal leaflet lipid transfer and followed by FP flickering conductance before FP closure. At this stage, Stx1A TMD palmitoylation delayed lipid transfer and FP formation and decreased FP flicker open times, whereas the palmitoylation of Syb2 did not affect fusion pore dynamics. These findings suggest that after facilitation of priming before FP opening, Stx1A TMD palmitoylation, directly affects FP dynamics. These results highlight the essential role of SNARE TMD palmitoylation at multiple stages of neurotransmitter release. Statement of SignificanceSynaptic vesicle fusion is critical for neurotransmitter release, enabling neuron-to-neuron communication at synapses. Post-translational modifications, such as palmitoylation, are known to influence this process. Using MARTINI coarse-grained molecular dynamics simulations, we examined the impact of SNARE transmembrane domain (TMD) palmitoylation on SNARE protein conformation and fusion dynamics. Stx1A palmitoylation reduces its TMD tilting and changes its SNARE domain conformation, facilitating SNARE complex formation. In fusion pore (FP) simulations, Stx1A palmitoylation delayed FP opening, decreased FP flicker open times, and shortened FP conductance flicker durations by direct interactions with the FP. Interestingly, dual palmitoylation of Stx1A and Syb2 restored flickering duration but decreased FP opening probability within 4 s, suggesting a nuanced role of TMD palmitoylation in modulating neurotransmitter release.

biophysics↗

SNARE complex assembly and disassembly dynamics in response to Ca2+ current activation in live cells

A SNAP25 based FRET construct named SCORE (SNARE COmplex REporter) has revealed a transient FRET increase that specifically occurred at fusion sites preceding fusion events by tens of milliseconds and presumably reflects vesicle priming. The FRET increase lasts for a few seconds until it is reversed. In those experiments, the FRET increase was found to be localized to areas <0.5 {micro}m2 at sites of transmitter release as detected amperometrically using electrochemical detector arrays. Due to the localization to such small areas, it was unknown if the reversal of the FRET increase is due to local dispersion of high-FRET SCORE copies leaving the site after fusion and exchange with surrounding low-FRET copies, or if it reflects disassembly of the high-FRET complexes. To resolve this question, we performed whole-cell patch clamp pulse stimulation experiments, imaging the entire footprint of the cells in Total Internal Reflection Fluorescence (TIRF) excitation mode such that diffusional exchange between high-FRET and low-FRET copies does not produce a net FRET change. We show here that pulse stimulation of calcium currents results in FRET ratio transients with a time course very similar that related to fusion events. By comparing the kinetics of the FRET ratio decay with analytical and numerical diffusion simulation results, we show that the experimentally observed kinetics cannot be explained by diffusional exchange and conclude that the SCORE FRET ratio transients reflect incorporation of SCORE in SNARE complexes followed by SNARE complex disassembly. Experiments using Synaptobrevin 2/Cellubrevin double knock-out mouse embryonal chromaffin cells showed no pulse induced FRET change, indicating that the vSNARE is required for the incorporation of SCORE (or SNAP25 in wild type cells) in the SNARE complex during priming. Statement of SignificanceIn chromaffin cells, SNAP25-based FRET constructs (SCORE) revealed transient FRET increases within <0.5 {micro}m2 areas, preceding individual fusion events that reversed within seconds. It remained unknown whether this reversal stems from high-FRET complex disassembly or diffusion-mediated exchange with low-FRET complexes. Here, we performed whole-cell patch-clamp pulse stimulation with TIRF microscopy, imaging large [~]30 {micro}m2 areas of the cell footprint. Calcium currents induced FRET transients with the same decay time constant of [~]1.5 s, significantly shorter than the time scale of diffusion. The SCORE FRET ratio thus reports in real time the dynamics of SNRE complex assembly and disassembly in live cells. Using Synaptobrevin 2/Cellubrevin double knock-out mouse chromaffin cells we show that vSNAREs are required for SNAP25 incorporation into SNARE complexes during priming.

biophysics↗

Performance evaluation of structural variation detection using DNBSEQ whole-genome sequencing

DNBSEQ platforms have been widely used for variation detection, including single-nucleotide variants and short insertions and deletions, comparable to Illumina. However, the performance of structural variations (SVs) detection using DNBSEQ platforms remains unclear. We assessed SV detection using 40 tools on eight DNBSEQ and two Illumina NA12878 whole-genome sequencing datasets. We confirmed that the performance of SV detection on DNBSEQ and Illumina platforms is consistent when using the same tools, with high consistency in metrics of number, size, precision, and sensitivity. Furthermore, we integrated representative SV sets for both DNBSEQ (4,785 SVs) and Illumina (6,797 SVs) platforms. We found high consistency between the DNBSEQ and Illumina SV sets in terms of genomic characteristics, including repetitive regions, GC distribution, difficult-to-sequence regions, and gene features, indicating the value of both platforms in understanding repetitive regions and the genomic context of SVs. Our study provides a benchmark resource for further studies of SVs using DNBSEQ platforms.

genomics↗

A regulator controls both DNA damage response and anti-phage defense networks in Moraxellaceae

DNA damage chemicals, including many antibiotics, often induce prophage induction and phage outbreaks in microbial communities, a major threat to bacterial survival. Moraxellaceae contains clinically highly relevant strains with outstanding antibiotic and radio resistance, yet their cellular-level regulation in DNA damage response and anti-phage defense is largely unknown. Here, we identified a WYL family protein that had replaced the ubiquitous SOS system in evolution and directly regulated a transcriptional network of both DNA repair genes and anti-phage defense genes under DNA damage stress. This study depicts a mechanism that how bacteria maintain immunity to phages without compromising on antibiotic resistance, and sheds light on controlling the resistance of Moraxellaceae strains in clinical practice.

microbiology↗

SLC13A2-transported citrate is a metabolic signal for PKM2 acetylation and degradation to suppress tumor growth

Background and aimsMetabolic reprogramming represents as a pivotal hallmark for cancer, but TCA cycle in tumorigenesis and progression has long been neglected. Solute carrier (SLC) transporters mediate the transport of TCA cycle intermediates across membrane, but their functions in cancer pathogenesis remains unclear. Approaches & ResultsUsing integrated analysis of solute carrier (SLC) transporters for TCA cycle intermediates, we found that SLC13A2 was consistently downregulated in hepatocellular carcinoma (HCC) cells and liver tissues from human patients and heterogeneous mouse models. Adeno-associated virus (AAV)-transduced liver-specific knockout or overexpression of SLC13A2 promoted or ameliorated HCC progression in the primary mouse model, demonstrating that SLC13A2 served as a protective factor during HCC pathogenesis. SLC13A2 inhibited HCC cell proliferation by decreasing mitochondrial function via suppressed oxygen consumption and ATP production. Combined with metabolic flux analysis, we found that SLC13A2 imported citrate, which secreted acetyl-CoA as a precursor for the acetylation of pyruvate kinase muscle isozyme M2 (PKM2), which led to its protein degradation. Decreased activity of pyruvate kinase depleted pyruvate for the TCA cycle, thus inhibiting amino acid synthesis and nucleotide metabolism. Additionally, a decrease in nuclear PKM2 protein transduced to reprogrammed gene transcription for cell proliferation and metabolism which is required for tumor growth. ConclusionsThis study revealed that citrate transported by SLC13A2 acts as a signal to disrupt metabolic homeostasis for tumor growth and suggests potential drug targets for HCC therapy. graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/591017v2_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@80c95eorg.highwire.dtl.DTLVardef@124750corg.highwire.dtl.DTLVardef@809ab4org.highwire.dtl.DTLVardef@12cbe74_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Mitochondrial metabolism sustains CD8+ T cellmigration for an efficient infiltration into solid tumors

The ability of CD8+ T cells to infiltrate solid tumors and reach cancer cells is associated with improved patient survival and responses to immunotherapy. Thus, identifying the factors controlling T cell migration in tumors is critical, so that strategies to intervene on these targets can be developed. Although interstitial motility is a highly energy-demanding process, the metabolic requirements of CD8+ T cells migrating in a 3D environment remain unclear. Here, we demonstrate that the tricarboxylic acid (TCA) cycle is the main metabolic pathway sustaining human CD8+ T cell motility in 3D collagen gels and tumor slices while glycolysis plays a much minor role. Using pharmacological and genetic approaches, we report that CD8+ T cell migration depends on the mitochondrial oxidation of glucose and glutamine, but not fatty acids, and both ATP and ROS produced by mitochondria are required for T cells to migrate. Pharmacological interventions to increase mitochondrial activity improve CD8+ T cells intra-tumoral migration and CAR T cell recruitment into tumor islets leading to better control of tumor growth in human xenograft models. Our study highlights the rationale of targeting mitochondrial metabolism to enhance the migration and antitumor efficacy of CAR T cells in treating solid tumors.

immunology↗

Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production.

Commensal bacteria of the Bacteroidetes phylum are the primary producers of sphingolipids in the gut lumen. These lipids serve dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells (iNKT). Sphingolipid composition is significantly altered in fecal samples of patients with inflammatory bowel disease (IBD). However, the specific mechanisms by which bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remain unclear. In this study, we investigated the impact of bacterial sphingolipids on intestinal inflammation by mono-colonizing mice with Bacteroides fragilis strains that either express or lack sphingolipids during DSS-induced colitis. We discovered that B. fragilis sphingolipids exacerbate intestinal inflammation. Mice mono-colonized with B. fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin-22 (IL-22) by innate lymphoid cell type 3 (ILC3). Consistent with the inhibitory effect of sphingolipids on IL-22 production, mice colonized with B. fragilis lacking sphingolipids showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production following DSS treatment compared to those colonized with B. fragilis producing sphingolipids. Additionally, colitis severity in mice colonized with B. fragilis lacking sphingolipids was exacerbated upon IL-22 blockade. Furthermore, our study reveals that bacterial sphingolipids restrict epithelial IL-18 production following DSS treatment and interfere with IL-22 production by a subset of ILC3 cells expressing both the interleukin-18 receptor (IL-18R) and major histocompatibility complex class II (MHC II). These findings indicate that B. fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL-18 expression, resulting in compromised production of IL-22 by ILC3 cells. HighlightsO_LIB. fragilis-derived sphingolipids exacerbate DSS-induced colitis in mono-colonized C57BL/6 mice. C_LIO_LIB. fragilis-derived sphingolipids constrain ILC3-derived IL-22, leading to reduced colonic epithelial cell proliferation and compromised barrier function. C_LIO_LIB. fragilis-derived sphingolipids restrict epithelial NLRC4 inflammasome activation and IL-18 secretion. C_LIO_LIB. fragilis-derived sphingolipids modulate IL-22 production by IL18R+ MHC II+ ILC3s. C_LI

immunology↗

Efficacy of Parainfluenza Virus 5 (PIV5)-vectored Intranasal COVID-19 Vaccine as a Single Dose Vaccine and as a Booster against SARS-CoV-2 Variants

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has greatly reduced coronavirus disease 2019 (COVID-19)-related deaths and hospitalizations, but waning immunity and the emergence of variants capable of immune escape indicate the need for novel SARS-CoV-2 vaccines. An intranasal parainfluenza virus 5 (PIV5)-vectored COVID-19 vaccine CVXGA1 has been proven efficacious in animal models and blocks contact transmission of SARS-CoV-2 in ferrets. CVXGA1 vaccine is currently in human clinical trials in the United States. This work investigates the immunogenicity and efficacy of CVXGA1 and other PIV5-vectored vaccines expressing additional antigen SARS-CoV-2 nucleoprotein (N) or SARS-CoV-2 variant spike (S) proteins of beta, delta, gamma, and omicron variants against homologous and heterologous challenges in hamsters. A single intranasal dose of CVXGA1 induces neutralizing antibodies against SARS-CoV-2 WA1 (ancestral), delta variant, and omicron variant and protects against both homologous and heterologous virus challenges. Compared to mRNA COVID-19 vaccine, neutralizing antibody titers induced by CVXGA1 were well-maintained over time. When administered as a boost following two doses of a mRNA COVID-19 vaccine, PIV5-vectored vaccines expressing the S protein from WA1 (CVXGA1), delta, or omicron variants generate higher levels of cross-reactive neutralizing antibodies compared to three doses of a mRNA vaccine. In addition to the S protein, the N protein provides added protection as assessed by the highest body weight gain post-challenge infection. Our data indicates that PIV5-vectored COVID-19 vaccines, such as CVXGA1, can serve as booster vaccines against emerging variants. ImportanceWith emerging new variants of concern (VOC), SARS-CoV 2 continues to be a major threat to human health. Approved COVID-19 vaccines have been less effective against these emerging VOCs. This work demonstrates the protective efficacy, and strong boosting effect, of a new intranasal viral-vectored vaccine against SARS-CoV-2 variants in hamsters.

microbiology↗

CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis

Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata. RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

molecular biology↗

Microbial Biotextiles for a Circular Materials Economy

The synthesis and bottom-up assembly of nanocellulose by microbes offers unique advantages to tune and meet key design criteria--rapid renewability, low toxicity, scalability, performance, and degradability--for multi-functional, circular economy textiles. However, development of green processing methods that meet these criteria remains a major research challenge. Here, we harness microbial biofabrication of nanocellulose and draw inspiration from ancient textile techniques to engineer sustainable biotextiles with a circular life cycle. The unique molecular self-organization of microbial nanocellulose (MC) combined with bio-phosphorylation with a lecithin treatment yields a compostable material with superior mechanical and flame-retardant properties. Specifically, treatment of MC with a lecithin-phosphocholine emulsion makes sites available to modulate cellulose cross-linking through hydroxyl, phosphate and methylene groups, increasing the interaction between cellulose chains. The resultant bioleather exhibits enhanced tensile strength and high ductility. Bio-phosphorylation with lecithin also redirects the combustion pathway from levoglucosan production towards the formation of foaming char as an insulating oxygen barrier, for outstanding flame retardance. Controlled color modulation is demonstrated with natural dyes. Life cycle impact assessment reveals that MC bioleather has up to an order of magnitude lower carbon footprint than conventional textiles, and a thousandfold reduction in the carcinogenic impact of leather production. Eliminating the use of hazardous substances, these high performance materials disrupt linear production models and strategically eliminate its toxicity and negative climate impacts, with widespread application in fashion, interiors and construction. Importantly, the biotextile approach developed in this study demonstrates the potential of biofabrication coupled with green chemistry for a circular materials economy.

bioengineering↗

Why are SNAREpins rod-shaped?

SNARE proteins are the core components of the cellular machineries that fuse membranes for neurotransmitter or hormone release and other fundamental processes. Fusion is accomplished when SNARE proteins hosted by apposing membranes form SNARE complexes called SNAREpins, but the mechanism of fusion remains unclear. Computational simulations of SNARE-mediated membrane fusion are challenging due to the millisecond timescales of physiological membrane fusion. Here we used ultra-coarse-grained (UCG) simulations to investigate the minimal requirements for a molecular intracellular fusogen, and to elucidate the mechanisms of SNARE-mediated fusion. We find fusion by simple body forces that push vesicles together is highly inefficient. Inter-vesicle fusogens with different aspect ratios can fuse vesicles only if they are rodlike, of sufficient length to clear the fusogens from the fusion site by entropic forces. Simulations with rod-shaped SNAREpin-like fusogens fused 50-nm vesicles on ms timescales, driven by entropic forces along a reproducible fusion pathway. SNARE-SNARE and SNARE-membrane entropic forces cleared the fusion site and pressed the vesicles into an extended contact zone (ECZ), drove stalk nucleation at the high curvature ECZ boundary, and expanded the stalk into a long-lived hemifusion diaphragm in which a simple pore completed fusion. Our results provide strong support for the entropic hypothesis of SNARE-mediated membrane fusion, and implicate the rodlike structure of the SNAREpin complex as a necessity for entropic force production and fusion.

biophysics↗

A hemifused complex is the hub in a network of pathways to membrane fusion

Membrane fusion is a critical step for many essential processes, from neurotransmission to fertilization. For over 40 years protein-free fusion driven by calcium or other cationic species has provided a simplified model of biological fusion, but the mechanisms remain poorly understood. Cation-mediated membrane fusion and permeation are essential in their own right to drug delivery strategies based on cell-penetrating peptides or cation-bearing lipid nanoparticles. Experimental studies suggest calcium drives anionic membranes to a hemifused intermediate which constitutes a hub in a network of pathways, but the pathway selection mechanism is unknown. Here we develop a mathematical model that identifies the network hub as a highly dynamical hemifusion complex. Multivalent cations drive expansion of this high tension hemifusion interface between interacting vesicles during a brief transient. The fate of this interface determines the outcome, either fusion, dead-end hemifusion or vesicle lysis. The model reproduces the unexplained finding that calcium-driven fusion of vesicles with planar membranes typically stalls at hemifusion, and we show the equilibrated hemifused state is a novel lens-shaped complex. Thus, membrane fusion kinetics follow a stochastic trajectory within a network of pathways, with outcome weightings set by a hemifused complex intermediate. SignificanceCells use multicomponent machineries to fuse membranes for neurotransmitter and hormone release and other fundamental processes. Protein-free fusion using calcium or other multivalent cationic fusogens has long been studied as a simplifying model. Cation-mediated membrane fusion or permeation are key events for a number of current drug delivery strategies. However, the mechanisms determining outcomes are unknown. Here we develop a mathematical model that identifies a dynamic hemifusion complex as the decision hub that stochastically sets the outcome in a network of pathways. Cations transiently grow a high tension hemifusion interface between membrane-enclosed compartments, whose fate governs whether fusion, dead-end hemifusion or vesicle lysis occurs. The model provides a systematic framework to predict outcomes of cationic fusogen-mediated interactions between membrane-enclosed compartments.

biophysics↗

Membrane pore energetics and the pathways to membrane rupture

Biological membranes owe their strength and low permeability to the phospholipid bilayers at their core. Membrane strength is determined by the energetics and dynamics of membrane pores, whose tension-dependent nucleation and growth leads to rupture. Creation of nanoscale membrane pores is central to exocytosis, trafficking and other processes fundamental to life that require breaching of secure plasma or organelle membranes, and is the basis for biotechnologies using drug delivery, delivery of genetic material for gene editing and antimicrobial peptides. A prevailing view from seminal electroporation and membrane rupture studies is that pore growth and bilayer rupture are controlled by macroscopically long-lived metastable defect states that precede fully developed pores. It was argued that defect nucleation becomes rate-limiting at high tensions, explaining the exponential tension-dependence of rupture times [E. Evans et al., Biophys. J. 85, 2342-2350 (2003)]. Here we measured membrane pore free energies and bilayer rupture using highly coarse-grained simulations that probe very long time scales. We find no evidence of metastable pore states. At lower tensions, small hydrophobic pores mature into large hydrophilic pores on the pathway to rupture, with classical tension dependence of rupture times. Above a critical tension membranes rupture directly from a small hydrophobic pore, and rupture times depend exponentially on tension. Thus, we recover the experimentally reported regimes, but the origin of the high tension exponential regime is unrelated to macroscopically long-lived pre-pore defects. It arises because hydrophilic pores cannot exist above a critical tension, leading to radically altered pore dynamics and rupture kinetics.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗