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Wang, B.

Publications and source records attributed to Wang, B..

At least 307 records · Page 17Linked to original sources

Robust self-supervised denoising of voltage imaging data using CellMincer

Voltage imaging enables high-throughput investigation of neuronal activity, yet its utility is often constrained by a low signal-to-noise ratio (SNR). Conventional denoising algorithms, such as those based on matrix factorization, impose limiting assumptions about the noise process and the spatiotemporal structure of the signal. While deep learning based denoising techniques offer greater adaptability, existing approaches fail to fully exploit the fast temporal dynamics and unique short- and long-range dependencies within voltage imaging datasets. Here, we introduce CellMincer, a novel self-supervised deep learning method designed specifically for denoising voltage imaging datasets. CellMincer operates on the principle of masking and predicting sparse sets of pixels across short temporal windows and conditions the denoiser on precomputed spatiotemporal auto-correlations to effectively model long-range dependencies without the need for large temporal denoising contexts. We develop and utilize a physics-based simulation framework to generate realistic datasets for rigorous hyperparameter optimization and ablation studies, highlighting the key role of conditioning the denoiser on precomputed spatiotemporal auto-correlations to achieve 3-fold further reduction in noise. Comprehensive benchmarking on both simulated and real voltage imaging datasets, including those with paired patch-clamp electrophysiology (EP) as ground truth, demonstrates CellMincers state-of-the-art performance. It achieves substantial noise reduction across the entire frequency spectrum, enhanced detection of subthreshold events, and superior cross-correlation with ground-truth EP recordings. Finally, we demonstrate how CellMincers addition to a typical voltage imaging data analysis workflow improves neuronal segmentation, peak detection, and ultimately leads to significantly enhanced separation of functional phenotypes.

neuroscience↗

Random adversarial threshold search enables automated DNA screening

Custom DNA synthesis underpins modern biology, but controlled genes in the wrong hands could threaten many lives and public trust in science. In .1992, a virology-trained mass murderer tried and failed to obtain physical samples of Ebola; today, viruses can be assembled from synthetic DNA fragments. Screening orders for controlled sequences is unreliable and expensive because current similarity search algorithms yield false alarms requiring expert human review. Exact-match search can achieve perfect specificity among short known sequences by detecting subsequences unique to controlled genes, but can be trivially evaded by incorporating mutations. Here we rescue exact-match search by additionally screening for predicted functional variants of pseudo-randomly chosen subsequences that arent found in known unregulated genes. To experimentally assess robustness, we protected nine windows from the M13 bacteriophage virus, then invited a "red team" to launch up to 21,000 attacks at each window and measure the fitness of their designed mutants. We identified defensible windows from regulated pathogens, built a test database that our experiments indicate will block 99.999% of functional attacks, and verified its sensitivity against redesigned enzymes. Exact-match search with functional variant prediction offers a promising way to safeguard biotechnology by automating DNA synthesis screening. SummarySearching for exact matches to pre-computed functional variants unique to controlled genes enables sensitive, secure, and automated DNA synthesis screening.

synthetic biology↗

Regulatory Functional Landscape of the HMX1 Gene for Normal Ear Development

Enhancers, through the combinatorial action of transcription factors (TFs), dictate both the spatial specificity and the levels of gene expression, and their aberrations can result in diseases. The association between HMX1 and its downstream enhancer with ear deformities has been previously documented. However, the pathogenic variations and molecular mechanisms underlying the bilateral constricted ear (BCE) malformations remain unclear. This study identifies a copy number variation (CNV) encompassing three enhancers that induces BCE. These enhancers, collectively termed the positional identity hierarchical enhancer cluster (PI-HEC), co-regulate spatiotemporal expression of the HMX1 gene in a coordinated and synergistic mode, each displaying unique activity-location-structure characteristics. A thorough exploration of this regulatory locus reveals that specific motif clusters within the PI-HEC variably modulate its activity and specificity, with the high mobility group (HMG) box combined with Coordinator and homeodomain (HD)-TFs notably influencing them respectively. Our findings based on various types of mouse models, reveal that both aberrant Hmx1 expression in the fibroblasts of the basal pinna, originating from neural crest cells, and ectopic expression in the distal pinna structures contribute to the abnormal development of the outer ear, including the cartilage, muscle, and epidermis tissues. This study deepens our understanding of mammalian ear morphogenesis and sheds light on the complexity of gene expression regulation by enhancers and specific sequence motifs.

developmental biology↗

Whole-genome resequencing revealed the Origin and Domestication of Chinese Domestic Rabbits

The evolutionary and genetic origins of Chinese indigenous rabbits (Oryctolagus cuniculus) remain largely unclear, despite being among the most recently domesticated animals. We sequenced the whole genomes of 142 individual rabbits and collected 25 resequencing accessions from the NCBI, representing six Chinese indigenous breeds, three other domesticated breeds (serving as a bridge between European wild-type and Chinese domestic populations), and two wild rabbit populations from the Iberian Peninsula and Southern France. Population and demographic analyses suggest that Chinese domestic rabbits are most likely descendants of O. c. cuniculus, native to France 800-1500 years ago. These rabbits likely first arrived in the southeast coastal areas of China through trade before spreading to inland regions. Additionally, there may be other origins for Chinese domestic rabbits. We observed considerable variation in the genetic makeup of maternal ancestry between Chinese domestic rabbits and European wild populations, with Chinese rabbits possessing unique mitochondrial haplotypes. Our analysis also highlights selective sweeps on genes affecting brain and neuronal development, which may have been under strong positive selection during domestication; genes related to starch digestion and fat metabolism, suggesting an evolutionary adaptation to digest high-starch diets; and the white coat phenotype in rabbits, resulting from selection at the melanogenesis-associated transcription factor locus. Overall, our data provide comprehensive insights into the origin and domestication of rabbits and lay the foundation for genome-based breeding.

evolutionary biology↗

Exploring the Dynamics of Social Interactions During the Juvenile Stage in a Mouse Model of Fragile X Syndrome

IntroductionFragile X Syndrome (FXS), caused by Fmr1 mutations, is linked to cognitive and behavioral differences, including altered social interactions. Most mouse studies focus on adults, despite human research showing critical developmental changes in childhood and adolescence. We examined social behavior in juvenile male and female Fmr1 knockout (KO) mice as well as heterozygous (HET) females. We further assessed cortical activity in KO females to better understand early phenotypes. MethodsJuvenile mice of both sexes and genotypes were paired in same-sex, novel dyads for 10-minute interactions. Key social behaviors such as head, anogenital, and body sniffing, and physical touch, as well as distance travelled, were analyzed with a marker-less tracking software. Frontal-parietal EEG recordings were collected from wild-type (WT) and KO females in home cage and social contexts to analyze power spectra across frequency bands. ResultsHET and KO females engaged in more frequent but shorter interaction events compared to WT females, with HET females showing the highest counts. Males displayed similar trends when comparing KO and WT. Males engaged in overall higher interaction events than females. EEG analyses revealed altered oscillatory activity in KO females compared to WT females, especially within theta, alpha, and beta bands, most prominently during the early interaction phase. Locomotor activity correlated weakly with head/anogenital sniffing but more strongly with body sniffing and touch. DiscussionThese findings suggest that Fmr1-related differences in juvenile social behavior are sex-dependent and associated with cortical oscillatory changes. Characterizing these early phenotypes in both sexes allows us to further understand FXS development and informs potential routes for early intervention. Key PointsO_LIFragile X Syndrome (FXS), the leading inherited cause of autism, is associated with disruptions in social behavior. C_LIO_LIWhile social phenotypes are relatively well described in adult mouse models of FXS, juvenile manifestations remain poorly understood. C_LIO_LISocial behavior was assessed in juvenile male and female Fmr1 knockout (KO), heterozygous (HET, female only), and wildtype (WT) mice, and frontal-parietal EEG recordings were collected from WT and KO females. C_LIO_LIHET and KO females exhibited more frequent but shorter social interactions than WT females, with HET showing the greatest number of events. Males showed similar patterns when comparing KO and WT. Males engaged in higher overall interaction events than females. EEG recordings revealed altered oscillatory activity in KO compared to WT females, most pronounced during the early phase of social encounter. C_LIO_LIThese findings reveal sex- and genotype-dependent differences in juvenile social behavior and cortical activity, highlighting the importance of studying juvenile development in FXS. C_LI

animal behavior and cognition↗

Near telomere-to-telomere genome assemblies of two Chlorella species unveil the composition and evolution of centromeres in green algae

BackgroundCentromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown. ResultsWe constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and Chlorella pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated their centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromeric CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements, although they differed significantly in their composition. Species within the Chlorella genus display a more diverse centromere composition, with major constituents including members of the LTR/Copia, LINE/L1, and LINE/RTEX families. This is in contrast to green algae including Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, in which centromere composition instead has a pronounced single-element composition. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeric sequence evolves more rapidly than sequence in non-centromeric regions. ConclusionsThis study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.

genomics↗

Phylogenomics resolves a 100-year-old debate regarding the evolutionary history of caddisflies (Insecta: Trichoptera)

Trichoptera (caddisfly) phylogeny provides an interesting example of aquatic insect evolution, with rich ecological diversification, especially for underwater architecture. Trichoptera provide numerous critical ecosystem services and are also one of the most important groups of aquatic insects for assessing water quality. The phylogenetic relationships of Trichoptera have been debated for nearly a century. In particular, the phylogenetic position of the "cocoon-makers" within Trichoptera has long been contested. Here, we designed a universal single-copy orthologue and sets of ultraconserved element markers specific for Trichoptera for the first time. Simultaneously, we reconstructed the phylogenetic relationship of Trichoptera based on genome data from 111 species, representing 29 families and 71 genera. Our phylogenetic inference clarifies the probable phylogenetic relationships of "cocoon-makers" within Integripalpia. Hydroptilidae is considered as the basal lineage within Integripalpia, and the families Glossosomatidae, Hydrobiosidae, and Rhyacophilidae formed a monophyletic clade, sister to the integripalpian subterorder Phryganides. The resulting divergence time and ancestral state reconstruction suggest that the most recent common ancestor of Trichoptera appeared in the early Permian and that diversification was strongly correlated with habitat change.

zoology↗

Targeted NAD+ Delivery for Intimal Hyperplasia and Re-endothelialization: A Novel Anti-restenotic Therapy Approach

Endovascular interventions often fail due to restenosis, primarily caused by smooth muscle cell (SMC) proliferation, leading to intimal hyperplasia (IH). Current strategies to prevent restenosis are far from perfect and impose significant collateral damage on the fragile endothelial cell (EC), causing profound thrombotic risks. Nicotinamide adenine dinucleotide (NAD+) is a co-enzyme and signaling substrate implicated in redox and metabolic homeostasis, with a pleiotropic role in protecting against cardiovascular diseases. However, a functional link between NAD+ repletion and the delicate duo of IH and EC regeneration has yet to be established. NAD+ repletion has been historically challenging due to its poor cellular uptake and low bioavailability. We have recently invented the first nanocarrier that enables direct intracellular delivery of NAD+ in vivo. Combining the merits of this prototypic NAD+-loaded calcium phosphate (CaP) nanoparticle (NP) and biomimetic surface functionalization, we created a biomimetic P-NAD+-NP with platelet membrane coating, which enabled an injectable modality that targets IH with excellent biocompatibility. Using human cell primary culture, we demonstrated the benefits of NP-assisted NAD+ repletion in selectively inhibiting the excessive proliferation of aortic SMC, while differentially protecting aortic EC from apoptosis. Moreover, in a rat balloon angioplasty model, a single-dose treatment with intravenously injected P-NAD+-NP immediately post angioplasty not only mitigated IH, but also accelerated the regeneration of EC (re-endothelialization) in vivo in comparison to control groups (i.e., saline, free NAD+ solution, empty CaP-NP). Collectively, our current study provides proof-of-concept evidence supporting the role of targeted NAD+ repletion nanotherapy in managing restenosis and improving re-endothelialization.

pathology↗

Asymmetric Fin Shape changes Swimming Dynamics of Ancient Marine Reptiles' Soft Robophysical Models

Animals have evolved highly effective locomotion capabilities in terrestrial, aerial, and aquatic environments. Over lifes history, mass extinctions have wiped out unique animal species with specialized adaptations, leaving paleontologists to reconstruct their locomotion through fossil analysis. Despite advancements, little is known about how extinct megafauna, such as the Ichthyosauria one of the most successful lineages of marine reptiles, utilized their varied morphologies for swimming. Traditional robotics struggle to mimic extinct locomotion effectively, but the emerging soft robotics field offers a promising alternative to overcome this challenge. This paper aims to bridge this gap by studying Mixosaurus locomotion with soft robotics, combining material modeling and biomechanics in physical experimental validation. Combining a soft body with soft pneumatic actuators, the soft robotic platform described in this study investigates the correlation between asymmetrical fins and buoyancy by recreating the pitch torque generated by extinct swimming animals. We performed a comparative analysis of thrust and torque generated by Carthorhyncus, Utatsusaurus, Mixosaurus, Guizhouichthyosaurus, and Ophthalmosaurus tail fins in a flow tank. Experimental results suggest that the pitch torque on the torso generated by hypocercal fin shapes such as found in model systems of Guizhouichthyosaurus, Mixosaurus and Utatsusaurus produce distinct ventral body pitch effects able to mitigate the animals non-neutral buoyancy. This body pitch control effect is particularly pronounced in Guizhouichthyosaurus, which results suggest would have been able to generate high ventral pitch torque on the torso to compensate for its positive buoyancy. By contrast, homocercal fin shapes may not have been conducive for such buoyancy compensation, leaving torso pitch control to pectoral fins, for example. Across the range of the actuation frequencies of the caudal fins tested, resulted in oscillatory modes arising, which in turn can affect the for-aft thrust generated.

biophysics↗

Targeted Dab2 Restoration: A Novel Approach for Enhanced Wound Healing in Diabetic Conditions

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF)-dependent angiogenesis remain uncertain and could reveal new therapeutic targets. In our study, the molecular underpinnings of endothelial dysfunction in diabetes were investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell (EC) function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high concentrations glucose treated primary mouse skin ECs, simulating hyperglycemic conditions in diabetes mellitus. In diabetic mice with a genetic EC deficiency of Dab2 angiogenesis was reduced in vivo and in vitro when compared with wild-type mice. Restoration of Dab2 expression by injected mRNA-containing lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. At the same time, FoxM1 was downregulated in skin ECs subjected to high glucose conditions as determined by RNA-sequencing analysis. FoxM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FoxM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. These findings indicate that restoring Dab2 expression through targeted therapies can enhance angiogenesis and wound repair in diabetes. To explore this therapeutic potential, we tested LyP-1-conjugated lipid nanoparticles (LNPs) containing Dab2 or control mRNAs to target ECs and found the former significantly improved wound healing and angiogenesis in diabetic mice. This study provides evidence of the crucial roles of Dab2 and FoxM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

cell biology↗

Distractor suppression operates exclusively in retinotopic coordinates

Our attention is influenced by past experiences, and recent studies have shown that individuals learn to extract statistical regularities in the environment, resulting in attentional suppression of locations that are likely to contain a distractor (high-probability location). However, little is known as to whether this learned suppression operates in retinotopic (relative to the eyes) or spatiotopic (relative to the world) coordinates. In the current study, two circular search arrays were presented side by side. Participants learned the high-probability location from a learning array presented on one side of the display (e.g., left). After several trials, participants shifted their gaze to the center of the the test array (e.g., located on the right side) in which all locations were equally likely to contain the distractor. Due to the saccade, the test array contained both a spatiotopic and a retinotopic matching location relative to the original high-probability location. The current findings show that, following saccadic eye movements, the learned suppression remained in retinotopic coordinates only, with no measurable transfer to spatiotopic coordinates. Even in a rich environment, attentional suppression still operated exclusively in retinotopic coordinates. We speculate that learned suppression may be resolved by changing synaptic weights in early visual areas. Significance statementIn our daily lives, attention is shaped by past experiences, guiding us to suppress locations that are likely to contain distractions. While this phenomenon has been studied extensively with static search displays, the real world is dynamic - we are constantly moving our eyes. This study addressed this issue by investigating what happens when we learn to suppress a likely distractor location while making eye movements. Do we suppress the same location in space (spatiotopic), or does the learned suppression persist relative to our eyes (retinotopic)? The current findings provide clear evidence of suppression in retinotopic coordinates only.

neuroscience↗

Centromere landscapes resolved from hundreds of human genomes

High-fidelity (HiFi) sequencing has facilitated the assembly and analysis of the most repetitive region of the genome, the centromere. Nevertheless, our current understanding of human centromeres draws from a relatively small number of telomere-to-telomere assemblies, and so has not yet captured its full diversity. In this study, we investigated the genomic diversity of human centromere higher order repeats (HORs) using both HiFi reads and haplotype-resolved assemblies from hundreds of samples drawn from ongoing pangenome-sequencing projects and reprocessed using a novel HOR annotation pipeline, HiCAT-human. We use this wealth of data to provide a global survey of the centromeric HOR landscape, in particular finding that 23 HORs exhibited significant copy number variability between populations. We detected three centromere genotypes with imbalance population frequencies on each of chromosome 5, 8 and 17. An inter-assembly comparison of HOR loci further revealed that while HOR array structures are diverse, they nevertheless tend to form a number of specific landscapes, each exhibiting different levels of HOR subunit expansion and possibly reflecting a cyclical evolutionary transition from homogeneous to nested structures and back.

genomics↗

Prognostic Significance of preoperative serum CA125, CA19-9, CA72-4, CEA, and AFP in Patients with Endometrial cancer

ObjectiveTo determine preoperative serum CA125, CA19-9, CA72-4, CEA, and AFP with prognostic value, and to establish a risk score based on CA125, CEA, AFP levels for predicting the overall survival (OS) and progression-free survival (PFS) of endometrial cancer (EC) patients. MethodsA retrospective cohort study with 2081 EC patients was conducted at Shengjing Hospital of China Medical University. Patient baseline information, tumor characteristics, and data on five serum biomarkers (CA125, CA19-9, CA72-4, CEA, and AFP) were collected. Hazard ratios (HRs) and 95% confidence intervals (CIs) were determined using univariate or multivariate Cox proportional hazard models. log-rank test and Kaplan-Meier analysis were used to compared survival, Data were randomly divided into a training cohort (50%, N = 1041) and an external validation cohort (50%, n = 1040). the least absolute shrinkage and selection operator (Lasso)-Cox regression model was used to screen the independent factors for establishing risk score. And develop nomograms for survival rate prediction. ResultsMultivariate analysis showed Elevated CA125 (P<0.0001) AFP (P <0.0001) and CEA(P=0.037) were identified as independent biomarkers for PFS. Increased CA125 (P = 0.003) AFP (P <0.0001) and CEA(P=0.014) were independent factors associated with OS. CA125, AFP and CEA were thus incorporated in an innovative Risk score (RS) by Lasso-Cox regression model, The RS was also an independent indicator for PFS (P<0.0001) and OS (P<0.0001). Furthermore, we developed and validated nomogram based on Cox regression models. The discriminative ability and calibration of the nomograms revealed good predictive ability, as indicated by the calibration plots. ConclusionThis study suggests that the risk score based on preoperative serum levels of CA125, CEA, and AFP was prognostic biomarkers for predicting progression-free survival and overall survival for EC patients. Nomograms based on the RS and clinicopathological features accurately predict Prognosis of EC patients.

cancer biology↗

The triose phosphate/phosphate translocator exports photosynthetic glyceraldehyde 3-phosphate from chloroplasts to trigger antimicrobial immunity in plants.

Chloroplasts play a crucial role in plant immunity against invading microbes. However, it remains poorly understood whether photosynthetic metabolites from chloroplasts participate directly in host defenses. Here, we uncovered Arabidopsis thalinana triose phosphate/phosphate translocator (AtTPT), a known translocator for chloroplast inner membrane, plays an indispensable role in suppressing virus infection and evoking defense responses. Interestingly, overexpression of AtTPT impairs virus accumulation in plants, while loss-of-function tpt3 mutants exhibit an increased viral load. The antiviral activity of AtTPT requires its phosphate transport capacity, implying that it actually functions through its metabolite(s). To this end, we found that glyceraldehyde 3-phosphate (GAP), one of AtTPTs translocated metabolites, can drastically enhance expression of defense-related genes and prominently induce defense signaling pathways. More excitingly, AtTPT or GAP robustly restricts the proliferation of multiple types of phytopathogens. Collectively, we propose that AtTPT exports GAP to mediate broad-spectrum resistance to pathogens, which provides new insights into the mechanism underlying the chloroplast-mediated immunity by a photosynthetic metabolite.

plant biology↗

Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation

The spatiotemporal transition of small GTPase Rab5 to Rab7 is crucial for early-to-late endosome maturation, yet the precise mechanism governing Rab5-to-Rab7 switching remains elusive. USP8, a ubiquitin-specific protease, plays a prominent role in the endosomal sorting of a wide range of transmembrane receptors and is a promising target in cancer therapy. Here, we identified that USP8 is recruited to Rab5-positive carriers by Rabex5, a guanine nucleotide exchange factor (GEF) for Rab5. The recruitment of USP8 dissociates Rabex5 from early endosomes (EEs) and meanwhile promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In USP8-deficient cells, the level of active Rab5 is increased, while the Rab7 signal is decreased. As a result, enlarged EEs with abundant intraluminal vesicles accumulate and digestive lysosomes are rudimentary. Together, our results reveal an important and unexpected role of a deubiquitinating enzyme in endosome maturation.

cell biology↗

Epsins promote atherosclerosis through VSMC phenotypic modulation

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA- seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

cell biology↗

Synthetase and Hydrolase Specificity Collectively Excludes Deoxyguanosine from Bacterial Alarmone

In response to starvation, virtually all bacteria pyrophosphorylate the 3-hydroxy group of GTP or GDP to produce two messenger nucleotides collectively denoted as (p)ppGpp. Also known as alarmones, (p)ppGpp reprograms bacterial physiology to arrest growth and promote survival. Intriguingly, although cellular concentration of dGTP is two orders of magnitude lower than that of GTP, alarmone synthetases are highly selective against using 2-deoxyguanosine (2dG) nucleotides as substrates. We thus hypothesize that production of 2dG alarmone, (p)pp(dG)pp, is highly deleterious, which drives a strong negative selection to exclude 2dG nucleotides from alarmone signaling. In this work, we show that the B. subtilis SasB synthetase prefers GDP over dGDP with 65,000-fold higher kcat/Km, a specificity stricter than RNA polymerase selecting against 2-deoxynucleotides. Using comparative chemical proteomics, we found that although most known alarmone-binding proteins in Escherichia coli cannot distinguish ppGpp from pp(dG)pp, hydrolysis of pp(dG)pp by the essential hydrolase, SpoT, is 1,000-fold slower. This inability to degrade 2-deoxy-3-pyrophosphorylated substrate is a common feature of the alarmone hydrolase family. We further show that SpoT is a binuclear metallopyrophoshohydrolase and that hydrolysis of ppGpp and pp(dG)pp shares the same metal dependence. Our results support a model in which 2-OH directly coordinates the Mn2+ at SpoT active center to stabilize the hydrolysis-productive conformation of ppGpp. Taken together, our study reveals a vital role of 2-OH in alarmone degradation, provides new insight on the catalytic mechanism of alarmone hydrolases, and leads to the conclusion that 2dG nucleotides must be strictly excluded from alarmone synthesis because bacteria lack the key machinery to down-regulate such products.

microbiology↗

Capsaicin acts as a novel NRF2 agonist to suppress ethanol induced gastric mucosa oxidative damage by directly disrupting the KEAP1-NRF2 interaction

Excessive alcohol consumption poses significant health risks and is closely associated with oxidative damage. The KEAP1-NRF2-ARE signaling pathway serves as the primary antioxidant system. However, current small molecule inhibitors are all covalently bound to KEAP1, meaning that once bound, they are not easily dissociated, while continuous inhibition of KEAP1 exhibits severe side effects. In this study, BLI, CETSA, Pull-down, Co-IP and HDX-MS assay analysis were conducted to detect the KEAP1 binding behavior of natural product, capsaicin (CAP), both in vitro and in cells. The ethanol-induced acute gastric mucosal damage rat model was also established to evaluate the therapeutic effect of CAP. Our findings demonstrated that CAP mitigated mitochondrial damage, facilitated the nuclear translocation of NRF2, leading to the up-regulation of downstream antioxidant response elements, HMOX1, TXN, GSS and NQO1 in GES-1 cells. Furthermore, CAP directly bind to KEAP1 and inhibit the interaction between KEAP1 and NRF2. In the KEAP1-knockout 293T cells, CAP failed to activate NRF2 expression. We identified that CAP non-covalently bound to Kelch domain and allosterically regulated three specific regions of KEAP1 : L342-L355, D394-G423 and N482-N495. To improve drug solubility and delivery efficiency, we developed IR-Dye800 modified albumin coated CAP nanoparticles. The nanoparticles significantly reduced the gastric mucosal inflammation and activated NRF2 downstream genes in vivo. Our hypothesis was further verified our hypothesis in Nfe2l2-knockout mice. This study provides new insights that CAP is a safe and novel NRF2 agonist by allosterically regulating KEAP1, which may contribute to the development of lead drugs for oxidative stress-related illness, e.g. aging, cancer, neurodegenerative and cardiovascular diseases.

cell biology↗