Biology subjects

Tang, S.

Publications and source records attributed to Tang, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

The heritability and structural correlates of brain entropy

Brain entropy (BEN) quantifies the temporal complexity of spontaneous neural activity, which has been increasingly recognized as providing important insights into cognitive functions and psychiatric disorders. However, its heritability and structural underpinnings are not well understood. Here, we utilize sample entropy to calculate BEN in healthy young adults using resting-state fMRI from the Human Connectome Project. We show that BEN is significantly heritable in broad brain regions. Heritability estimates exhibit regional variability, with lower heritability in the limbic and subcortical networks than in other networks. We further relate BEN to structural features including surface area (SA), cortical myelination (CM), cortical thickness (CT), and subcortical volumes (SV). We find that BEN is negatively correlated with SA in most cortical regions, but is positively correlated with CM in the prefrontal cortex, lateral temporal lobe, precuneus, lateral parietal cortex, and cingulate cortex. Only a few cortical regions show significant correlations between BEN and CT. By decomposing correlations into genetic and environmental components, we observe that most of BEN-SA correlations are associated with shared genetic and environmental effects. BEN-CM correlations are mainly attributed to shared environmental effects. BEN-CT correlations are partly related to shared environmental effects. We find no significant associations between BEN and SV. Collectively, our work establishes the genetic basis and structural correlates of resting-state fMRI-based BEN, supporting its potential application as an endophenotype for psychiatric disorders.

neuroscience↗

bioRxiv2024

Unicellular cyanobacteria rely on sodium energetics to fix N2

Diazotrophic cyanobacteria can thrive in combined nitrogen (N)-limited environments due to their ability to fix nitrogen gas (N2) from the atmosphere. Despite this, they occur in low abundance in N-limited coastal waters, which represents an ecological paradox1-3. One hypothesis is that this is partly due to elevated salinity (> 10 g/L NaCl), which inhibits cyanobacterial N2 fixation2,3. Here we show that N2 fixation in a unicellular coastal cyanobacterium is not inhibited but rather exclusively dependent on sodium (Na+) ions. In N-deficient environments, both N2 fixation and population growth were significantly inhibited at low NaCl concentrations (< 4 g/L). Additional experiments indicated that sodium energetics, rather than proton energetics, is necessary for N2 fixation, as Na+ deficiency resulted in insufficient ATP supply for N2 fixation. We show that this is due to the non-functioning Na+-coupled ATP synthase, which we found to be likely coupled to anaerobic rather than aerobic respiration. Sequence alignment analysis of the ion-coupling site of the ATP synthase revealed a high prevalence of Na+ energetics in cyanobacteria, with all unicellular N2 fixers capable of Na+ energetics. This suggests a critical role for sodium energetics in cyanobacteria. It also raises the possibility that sodium energetics is not as rare as thought, but that we may have underestimated the prevalence and importance of sodium energetics in other organisms. Finally, the low abundance of diazotrophic unicellular cyanobacteria in coastal waters may be due to insufficient NaCl levels to support N2-fixation during periods of growth-supporting high temperatures. This provides another perspective on the regulation of the oceanic N cycle that needs to be considered in times of global climate change. Changes in current patterns could lead to an overlap of periods optimal for N2 fixation and population growth, likely resulting in dense cyanobacterial blooms.

microbiology↗

bioRxiv2024

Imaging and quantification of prostate cancer-associated bone by polarization-sensitive optical coherence tomography

Prostate cancer frequently metastasizes to bone, leading to a spectrum of osteosclerotic and osteolytic lesions that cause debilitating symptoms. Accurate differentiation of bone tissue or lesion types can provide opportunity for local pathologic investigation, which is critical for understanding the bone metastasis and remains challenging. Current imaging methods lack the ability to directly differentiate tissues based on collagen organization and may induce invasive effect on bone tissues. We introduce Polarization-Sensitive Optical Coherence Tomography (PS-OCT) to investigate normal, osteosclerotic, and osteolytic bone tissues. High-resolution PS-OCT imaging reveals collagen fiber arrangement, enabling nuanced distinction of degree of collagen alignment among different bone tissue types or regions. We present a novel feature named degree of ordered organization (DOO), derived from the multiple contrasts of PS-OCT that can quantitatively evaluate bone samples from different pathologic groups, including control,osteoblastic and osteolytic tissues. The capacity of PS-OCT to differentiate trabecular/lamellar and irregular (woven bone) regions within the same specimen is tested and validated on ex-vivo samples extracted from 13 subjects. Our study is the first time that PS-OCT is applied to metastatic bone disease with the aim of enhancing the understanding of bone-related pathologies, and potentially impacting clinical practice. This work demonstrates that PS-OCT can provide useful insight into bone microstructures, and thus it has potential applications across diverse bone disorders.

biophysics↗

bioRxiv2024

GSDMD pore formation regulates caspase-4 cleavage to limit IL-18 production in the intestinal epithelium

Epithelial inflammasomes induce pyroptosis and release cytokines to defend against cytosolic pathogens. However, pyroptosis in epithelial barriers must be carefully regulated to facilitate elimination of infected cells while limiting widespread pyroptosis to preserve the single cell barrier. How epithelial cells achieve this is unknown. In this study, we describe a novel epithelial caspase regulation mechanism. By examining caspase-4 activation in human epithelial cells, we discovered that GSDMD pore formation serves as a signal to terminate caspase-4 activity thus facilitating epithelial cell expulsion while controlling cytokine secretion. Inhibition of epithelial pyroptosis led to IL-18 hyperproduction, likely as a mechanism to combat increased pathogen burden and initiate a wider immune response. Moreover, we demonstrate that full-length, rather than cleaved caspase-4 is active against IL-18 and propose that GSDMD pore formation facilitates cleavage of caspase-4 to terminate its catalytic activity. By comparing human cells and murine epithelial organoids to immune cells, we show that GSDMD pore mediated inhibition of caspase activity is largely specific to epithelial cells. Overall, these studies characterise a novel, epithelial-specific negative feedback loop that modulates inflammasome activity and challenge the dogma that autocatalytic caspase cleavage is required for caspase activity against substrates. Graphical AbstractIn intestinal epithelial cells, caspase activation simultaneously leads to GSDMD pore formation and IL-18 release. GSDMD pore formation provides a signal to terminate caspase activity and limit cytokine production. In GSDMD deficient cells, lack of an inhibition signal leads to caspase mediated IL-18 hyperproduction. Upon cell death this leads to release of massive amounts of IL-18. Created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/578487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@46f768org.highwire.dtl.DTLVardef@11cf918org.highwire.dtl.DTLVardef@125be35org.highwire.dtl.DTLVardef@ea9ead_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Rapid dexamethasone treatment inhibits LPS-induced cytokine storm in mice

Severe infection-induced cytokine storm is an urgent medical syndrome with high mortality. To date, no therapy is available. This study shows that high concentrations of lipopolysaccharide (LPS) induce cytokine storm within 48h and thus kill most experimental mice. Rapid, but not late dexamethasone administration remarkably inhibits cytokine storm and rescues LPS-treated mice. Monocytes and macrophages are the major source of cytokine storm. In these cells, pro-inflammatory genes (i.e., Tnf, Il6 and Il1{beta}) have preassembled RNA polymerase II (RNA Pol II), but stay at the pause stage of transcriptional elongation in the absence of stimulation. LPS rapidly activates transcription of these "pre-loaded" genes within 2h. Administration of dexamethasone within this time window inhibits RNA Pol II ser2 binding to the core promoters of pro-inflammatory genes and thus reduces LPS-induced cytokine transcription. Therefore, rapid utilization of dexamethasone might be efficacious to prevent severe bacterium-induced cytokine storm in clinical practice.

molecular biology↗

bioRxiv2023

Emergence of RNA-guided transcription factors via domestication of transposon-encoded TnpB nucleases

Transposon-encoded tnpB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. This widespread gene family was repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas125,6. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRIS-PR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Col-lectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of genes encoded by transposable elements, and reveals that RNA-guided transcription factors emerged long before the development of dCas9-based editors.

genetics↗

bioRxiv2023

Antagonistic conflict between transposon-encoded introns and guide RNAs

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life, presumably due to the critical roles they play in transposon proliferation. IS605-family TnpB homologs function in bacteria as programmable homing endonucleases by exploiting transposon-encoded guide RNAs to cleave vacant genomic sites, thereby driving transposon maintenance through DSB-stimulated homologous recombination. Whether this pathway is conserved in other genetic contexts, and in association with other transposases, is unknown. Here we uncover molecular mechanisms of transposition and RNA-guided DNA cleavage by IS607-family elements that, remarkably, also encode catalytic, self-splicing group I introns. After reconstituting and systematically investigating each of these biochemical activities for a candidate IStron derived from Clostridium botulinum, we discovered sequence and structural features of the transposon-encoded RNA that satisfy molecular requirements of a group I intron and TnpB guide RNA, while still retaining the ability to be faithfully mobilized at the DNA level by the TnpA transposase. Strikingly, intron splicing was strongly repressed not only by TnpB, but also by the secondary structure of {omega}RNA alone, allowing the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work explains how diverse enzymatic activities emerged during the selfish spread of IS607-family elements and highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.

molecular biology↗

bioRxiv2023

Striatum supports fast learning but not memory recall

Animals learn to carry out motor actions in specific sensory contexts to achieve goals. The striatum has been implicated in producing sensory-motor associations, yet its contribution to memory formation or recall is not clear. To investigate the contribution of striatum to these processes, mice were taught to associate a cue, consisting of optogenetic activation of striatum-projecting neurons in visual cortex, with forelimb reaches to access food pellets. As necessary to direct learning, striatal neural activity encoded both the sensory context and outcome of reaching. With training, the rate of cued reaching increased, but brief optogenetic inhibition of striatal activity arrested learning and prevented trial-to-trial improvements in performance. However, the same manipulation did not affect performance improvements already consolidated into short- (within an hour) or long-term (across days) memories. Hence, striatal activity is necessary for trial-to-trial improvements in task performance, leading to plasticity in other brain areas that mediate memory recall.

neuroscience↗

bioRxiv2023

Vina-GPU 2.1: towards further optimizing docking speed and precision of AutoDock Vina and its derivatives

AutoDock Vina and its derivatives have established themselves as a prevailing pipeline for virtual screening in contemporary drug discovery. Our Vina-GPU method leverages the parallel computing power of GPUs to accelerate AutoDock Vina, and Vina-GPU 2.0 further enhances the speed of AutoDock Vina and its derivatives. Given the prevalence of large virtual screens in modern drug discovery, the improvement of speed and accuracy in virtual screening has become a longstanding challenge. In this study, we propose Vina-GPU 2.1, aimed at enhancing the docking speed and precision of AutoDock Vina and its derivatives through the integration of novel algorithms to facil-itate improved docking and virtual screening outcomes. Building upon the foundations laid by Vina-GPU 2.0, we introduce a novel algorithm, namely Reduced Iteration and Low Complexity BFGS (RILC-BFGS), designed to expedite the most time-consuming operation. Additionally, we implement grid cache optimization to further enhance the docking speed. Furthermore, we employ optimal strategies to individually optimize the structures of ligands, receptors, and binding pockets, thereby enhancing the docking precision. To assess the performance of Vina-GPU 2.1, we conduct extensive virtual screening experiments on three prominent targets, utilizing two fundamental compound libraries and seven docking tools. Our results demonstrate that Vina-GPU 2.1 achieves an average 4.97-fold acceleration in docking speed and an average 342% improvement in EF1% compared to Vina-GPU 2.0. The source code and tools for Vina-GPU 2.1 are freely available at https://github.com/DeltaGroupNJUPT/Vina-GPU-2.1, accompanied by comprehensive instructions and illustrative examples.

developmental biology↗

bioRxiv2023

Ocular dominance-dependent binocular combination of monocular neuronal responses in macaque V1

Primates rely on two eyes to see depth, while keeping a stable vision when one eye is closed. Although psychophysical and modeling studies have investigated how monocular signals are combined to form binocular vision, the corresponding neuronal mechanisms, especially in V1 where most neurons become binocular but with different eye preferences, are not well understood. Here we used two-photon calcium imaging to compare monocular and binocular responses of thousands of V1 superficial-layer neurons in three awake macaques. Under monocular stimulation, neurons preferring the stimulated eye responded substantially stronger than those preferring both eyes. However, under binocular stimulation, the responses of neurons preferring either eye were suppressed, and those preferring both eyes were enhanced, so that neuronal responses became similar regardless of eye preferences. A neuronally realistic model of binocular combination, which includes ocular dominance-dependent divisive interocular inhibition, and binocular summation, is proposed to account for these observations.

neuroscience↗

bioRxiv2023

Integrative Transcriptomic Analysis of Anterior and Posterior IOP-Controlling Tissues in Glaucoma Reveals Enrichment of MHC-II Pathway and T-Cell Infiltration Signatures

BackgroundGlaucoma is a multifactorial neurodegenerative disease indicating by abnormalities in the whole eye, apart from optic neuropathy, and is often associated with abnormal intraocular pressure (IOP). However, the underlying pathological mechanisms of glaucoma and the management of IOP remain largely unaddressed. Emerging evidence suggests the involvement of immune changes in glaucoma. Therefore, characterizations of immune alternations by molecular profiling of IOP-controlling tissues from both anterior and posterior eye segments including trabecular meshwork (TM) and choroid may provide insights to the disease mechanism. MethodsBulk TM microarray data (GSE138125 and GSE27276) from primary open-angle glaucoma (POAG) and choroid single-cell transcriptome data (GSE203499) from glaucoma were downloaded from Gene Expression Omnibus (GEO) database. The two qualified TM datasets were integrated. Analyses of differential expression genes (DEGs), functional and pathway annotations, immune infiltration, and disease-related modules were performed using the POAG bulk data. Similarly, DEGs and pathway annotations, single-cell trajectory and switch gene analysis, and cell-cell communications were conducted using the choroid single-cell RNA-sequencing data. Finally, integrated analyses from the two studies were conducted to determine the glaucomatous changes between anterior and posterior IOP-controlling eye tissues. ResultsA total of 102 DEGs between POAG and healthy tissues were identified from the TM data. Gene set enrichment analysis revealed five glaucoma-enriched pathways: ATP generation and metabolism, cornification and keratinization, metabolism of reactive oxygen species (ROS), humoral immune response, and platelet aggregation. Significant immune infiltrations were observed in POAG tissue, including total T cells (excluding CD8+ T cells), NK cells, monocytic lineage, endothelial cells, and fibroblasts. Furthermore, the POAG enriched pathways are primarily from T-cell infiltrations. On the other hand, fourteen distinct cell clusters were identified from the choroid single-cell data. The most evident findings were fibrosis, represented by extracellular matrix (ECM), the actin-binding pathways enrichment in pericyte-fibroblast transition, reduction in light sensitivity of melanocytes, and complex immune changes among all cell types in the pathological conditions compared to control. Taken together, an integrated analyses between the TM microarray and choroid single-cell data result in 111 significantly dysregulated genes in the disease state. These key genes participated in immune and inflammatory reactions (DUSP1, ADM, CD74, CEBPD, HLA-DPA1, ZNF331), served as potential biomarkers for neurodegenerative and autoimmune diseases (such as NR4A2), or regulated membrane integrity, vasculature calcification, ECM interactions, and cell morphology (PTP4A1, MGP, and RASD1). Overall, our integrated data analyses might provide insight to the understanding of the disease mechanism. ConclusionBy integrating bulk microarray data from TM and single-cell transcriptome data from the choroid, we systematically evaluated the glaucomatous transcriptomic changes between the anterior and posterior IOP-controlling tissue from ocular. Results indicated there was a significantly enrichment of genes belonging to the MHC-II pathway and T-cell infiltration in the disease state, opening new avenues for biomarkers discovery and therapeutic interventions to glaucoma.

bioinformatics↗

bioRxiv2023

Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci (Smad1HA/HAand Smad5PA/PA). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers (IGFBP1, PRL, FOXO1) and PR-responsive genes (RORB, KLF15). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.

molecular biology↗

bioRxiv2023

Impaired bone morphogenetic protein signaling pathways disrupt decidualization in endometriosis

It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in individuals with endometriosis. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from individuals with or without endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified differences in key pathways between the two groups, including defective bone morphogenetic protein (BMP)/SMAD4 signaling (ID2, ID3, FST), oxidate stress response (NFE2L2, ALOX15, SLC40A1), and retinoic acid signaling pathways (RARRES, RARB, ALDH1B1). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 and H3K27Ac in the decidualized stromal cells from individuals without endometriosis relative to those with endometriosis, with target genes enriched in pathways related to signaling by transforming growth factor {beta} (TGF{beta}), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation in endometriosis patient-derived assembloids elevated the expression of decidualization markers. In summary, transcriptomic and genome-wide binding analyses of patient-derived endometrial cells and assembloids identified that a functional BMP/SMAD1/5/4 signaling program is crucial for engaging decidualization.

molecular biology↗

bioRxiv2023

Multi-omics resources for the Australian southern stuttering frog (Mixophyes australis) reveal assorted antimicrobial peptides

The number of genome-level resources for non-model species continues to rapidly expand. However, frog species remain underrepresented, with up to 90% of frog genera having no genomic or transcriptomic data. Here, we assemble the first genomic and transcriptomic resources for the recently described southern stuttering frog (Mixophyes australis). The southern stuttering frog is ground-dwelling, inhabiting naturally vegetated riverbanks in south-eastern Australia. Using PacBio HiFi long-read sequencing and Hi-C scaffolding, we generated a high-quality genome assembly, with a scaffold N50 of 369.3 Mb and 95.1% of the genome contained in twelve scaffolds. Using this assembly, we identified the mitochondrial genome, and assembled six tissue-specific transcriptomes. We also bioinformatically characterised novel sequences of two families of antimicrobial peptides (AMPs) in the southern stuttering frog, the cathelicidins and {beta}-defensins. While traditional peptidomic approaches to peptide discovery have typically identified one or two AMPs in a frog species from skin secretions, our bioinformatic approach discovered 12 cathelicidins and two {beta}-defensins that were expressed in a range of tissues. We investigated the novelty of the peptides and found diverse predicted activities. Our bioinformatic approach highlights the benefits of multi-omics resources in peptide discovery and contributes valuable genomic resources in an under-represented taxon.

genomics↗

bioRxiv2023

Bet hedging in a unicellular microalga

AbstractUnderstanding how organisms adapt to unpredictable future environments is a fundamental goal in biology, which becomes even more urgent in an era of rapid climate change. One evolutionary adaptation to randomly fluctuating environments is bet hedging, a strategy that successfully facilitates reproduction and population persistence and has been widely reported from microbes to humans. Empirical evidence for its presence in microalga, one of Earths most important primary producers and carbon sinks, is lacking. Here, we report a bet-hedging strategy in the unicellular microalga Haematococcus pluvialis. In a series of experiments, we show that an isogenic H. pluvialis population reversibly diversifies into hetero-phenotypic mobile and non-mobile subunits, independent of environmental conditions. Mobile cells grow faster but are more susceptible to external stressors, while non-mobile cells hardly grow but are more stress-resistant. This is attributed to dramatic shifts from growth-promoting activities (cell division, photosynthesis) to resilience-promoting cellular metabolic processes, including cell enlargement and aggregation, and accumulation of antioxidant and energy-storaging compounds. Our results provide experimental evidence for bet hedging in microalga, which has implications for their potential to adapt to current and predicted future conditions, and thus for the conservation of ecosystem functions.

ecology↗

bioRxiv2023

Label-Free Composition Analysis of Supramolecular Polymer - Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties following applied stresses, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel compositions is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, Bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e. proteins), and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with high resolution mass spectrometry and a Fourier-transform based deconvolution algorithm. To our knowledge, this is the first RPLC-CAD method for characterizing the critical quality attributes of supramolecular hydrogels. We envision this analytical strategy could be generalized to characterize other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

biochemistry↗

bioRxiv2023

A simplified femtosecond laser repetition frequency divider for two-photon imaging

Commercial Ti:Sapphire femtosecond lasers used for conventional two-photon microscopy typically operate at a [~]80 MHz repetition rate. However, this frequency is often suboptimal for cortical tissue imaging, where lower rates of 20-40 MHz are considered ideal. However, achieving these lower frequencies has remained a significant technical and financial challenge. Here, we present a compact, cost-effective resonant electro-optic modulator that halves the laser repetition rate to 40 MHz. When imaging neurons in the macaque visual cortex in vivo, this 40 MHz configuration yielded a >2-fold increase in fluorescence intensity and a [~]3 dB improvement in signal-to-noise ratio (SNR) for both green (GCaMP5G) and red (mScarlet) indicators. This enhancement proved particularly pronounced in deep-tissue imaging. Furthermore, the system demonstrated excellent long-term stability and induced no detectable phototoxicity. This simple and robust device represents a powerful upgrade for conventional two-photon microscopes, significantly enhancing imaging quality for the investigation of neural circuits within scattering brain tissue.

neuroscience↗

bioRxiv2023

Up-regulation of cholesterol synthesis pathways and limited neurodegeneration in a knock-in Sod1 mutant mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene (SOD1) are associated with [~]20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. To generate a model that more closely represents patient gene expression, we created a knock-in point mutation (G85R, a human ALS-causing mutation) in the endogenous mouse Sod1 gene, leading to mutant SOD1G85R protein expression. Heterozygous Sod1G85R mutant mice resemble wild type, whereas homozygous mutants have reduced body weight and lifespan, a mild neurodegenerative phenotype, and express very low mutant SOD1 protein levels with no detectable SOD1 activity. Homozygous mutants exhibit partial neuromuscular junction denervation at 3-4 months of age. Spinal cord motor neuron transcriptome analyses of homozygous Sod1G85R mice revealed up-regulation of cholesterol synthesis pathway genes compared to wild type. Transcriptome and phenotypic features of these mice are similar to Sod1 knock-out mice, suggesting the Sod1G85R phenotype is largely driven by loss of SOD1 function. By contrast, cholesterol synthesis genes are down-regulated in severely affected human TgSOD1G93A transgenic mice at 4 months. Our analyses implicate dysregulation of cholesterol or related lipid pathway genes in ALS pathogenesis. The Sod1G85R knock-in mouse is a useful ALS model to examine the importance of SOD1 activity in control of cholesterol homeostasis and motor neuron survival. SIGNIFICANCE STATEMENTAmyotrophic lateral sclerosis is a devastating disease involving the progressive loss of motor neurons and motor function for which there is currently no cure. Understanding biological mechanisms leading to motor neuron death is critical for developing new treatments. Using a new knock-in mutant mouse model carrying a Sod1 mutation that causes ALS in patients, and in the mouse, causes a limited neurodegenerative phenotype similar to Sod1 loss-of-function, we show that cholesterol synthesis pathway genes are up-regulated in mutant motor neurons, whereas the same genes are down-regulated in transgenic SOD1 mice with a severe phenotype. Our data implicate dysregulation of cholesterol or other related lipid genes in ALS pathogenesis and provide new insights that could contribute to strategies for disease intervention.

neuroscience↗