Phosphorylation-Mediated IFN-γR2 Membrane Translocation Is Required to Activate Macrophage Innate Response.

As a critical step during innate response, the cytoplasmic ß subunit (IFN-γR2) of interferon-γ receptor (IFN-γR) is induced and translocates to plasma membrane to join α subunit to form functional IFN-γR to mediate IFN-γ signaling. However, the mechanism driving membrane translocation and its significance remain largely unknown. We found, unexpectedly, that mice deficient in E-selectin, an endothelial cell-specific adhesion molecule, displayed impaired innate activation of macrophages upon Listeria monocytogenes infection yet had increased circulating IFN-γ. Inflammatory macrophages from E-selectin-deficient mice had less surface IFN-γR2 and impaired IFN-γ signaling. BTK elicited by extrinsic E-selectin engagement phosphorylates cytoplasmic IFN-γR2, facilitating EFhd2 binding and promoting IFN-γR2 trafficking from Golgi to cell membrane. Our findings demonstrate that membrane translocation of cytoplasmic IFN-γR2 is required to activate macrophage innate response against intracellular bacterial infection, identifying the assembly of functional cytokine receptors on cell membrane as an important layer in innate activation and cytokine signaling.

Heparin prevents caspase-11-dependent septic lethality independent of anticoagulant properties.

Heparin, a mammalian polysaccharide, is a widely used anticoagulant medicine to treat thrombotic disorders. It is also known to improve outcomes in sepsis, a leading cause of mortality resulted from infection-induced immune dysfunction. Whereas it is relatively clear how heparin exerts its anticoagulant effect, the immunomodulatory mechanisms enabled by heparin remain enigmatic. Here, we show that heparin prevented caspase-11-dependent immune responses and lethality in sepsis independent of its anticoagulant properties. Heparin or a chemically modified form of heparin without anticoagulant function inhibited the alarmin HMGB1-lipopolysaccharide (LPS) interaction and prevented the macrophage glycocalyx degradation by heparanase. These events blocked the cytosolic delivery of LPS in macrophages and the activation of caspase-11, a cytosolic LPS receptor that mediates lethality in sepsis. Survival was higher in septic patients treated with heparin than those without heparin treatment. The identification of this previously unrecognized heparin function establishes a link between innate immune responses and coagulation.

The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.

The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.

E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.

TBK1 is essential for interferon-ß (IFN-ß) production and innate antiviral immunity. Here we identified the T cell anergy-related E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its protease-associated (PA) domain and catalyzed the K63-linked polyubiquitination of TBK1, which led to TBK1 activation, IRF3 activation and IFN-ß production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN-ß production and innate antiviral immune responses to RNA and DNA viruses, in vitro and in vivo. Our study identified RNF128 as an E3 ligase for K63-linked ubiquitination and activation of TBK1 and delineated a previously unrecognized function for RNF128.

Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate the kinase TBK1 for activation of antiviral innate immunity.

The DNA methyltransferase Dnmt3a has high expression in terminally differentiated macrophages; however, its role in innate immunity remains unknown. Here we report that deficiency in Dnmt3a selectively impaired the production of type I interferons triggered by pattern-recognition receptors (PRRs), but not that of the proinflammatory cytokines TNF and IL-6. Dnmt3a-deficient mice exhibited enhanced susceptibility to viral challenge. Dnmt3a did not directly regulate the transcription of genes encoding type I interferons; instead, it increased the production of type I interferons through an epigenetic mechanism by maintaining high expression of the histone deacetylase HDAC9. In turn, HDAC9 directly maintained the deacetylation status of the key PRR signaling molecule TBK1 and enhanced its kinase activity. Our data add mechanistic insight into the crosstalk between epigenetic modifications and post-translational modifications in the regulation of PRR signaling and activation of antiviral innate immune responses.

INSIDER: Interpretable sparse matrix decomposition for RNA expression data analysis.

RNA sequencing (RNA-Seq) is widely used to capture transcriptome dynamics across tissues, biological entities, and conditions. Currently, few or no methods can handle multiple biological variables (e.g., tissues/ phenotypes) and their interactions simultaneously, while also achieving dimension reduction (DR). We propose INSIDER, a general and flexible statistical framework based on matrix factorization, which is freely available at https://github.com/kai0511/insider. INSIDER decomposes variation from different biological variables and their interactions into a shared low-rank latent space. Particularly, it introduces the elastic net penalty to induce sparsity while considering the grouping effects of genes. It can achieve DR of high-dimensional data (of > = 3 dimensions), as opposed to conventional methods (e.g., PCA/NMF) which generally only handle 2D data (e.g., sample × expression). Besides, it enables computing 'adjusted' expression profiles for specific biological variables while controlling variation from other variables. INSIDER is computationally efficient and accommodates missing data. INSIDER also performed similarly or outperformed a close competing method, SDA, as shown in simulations and can handle complex missing data in RNA-Seq data. Moreover, unlike SDA, it can be used when the data cannot be structured into a tensor. Lastly, we demonstrate its usefulness via real data analysis, including clustering donors for disease subtyping, revealing neuro-development trajectory using the BrainSpan data, and uncovering biological processes contributing to variables of interest (e.g., disease status and tissue) and their interactions.

Cholinergic macrophages promote the resolution of peritoneal inflammation.

The non-neural cholinergic system plays a critical role in regulating immune equilibrium and tissue homeostasis. While the expression of choline acetyltransferase (ChAT), the enzyme catalyzing acetylcholine biosynthesis, has been well documented in lymphocytes, its role in the myeloid compartment is less understood. Here, we identify a significant population of macrophages (Mϕs) expressing ChAT and synthesizing acetylcholine in the resolution phase of acute peritonitis. Using Chat-GFP reporter mice, we observed marked upregulation of ChAT in monocyte-derived small peritoneal Mϕs (SmPMs) in response to Toll-like receptor agonists and bacterial infections. These SmPMs, phenotypically and transcriptionally distinct from tissue-resident large peritoneal macrophages, up-regulated ChAT expression through a MyD88-dependent pathway involving MAPK signaling. Notably, this process was attenuated by the TRIF-dependent TLR signaling pathway, and our tests with a range of neurotransmitters and cytokines failed to induce a similar response. Functionally, Chat deficiency in Mϕs led to significantly decreased peritoneal acetylcholine levels, reduced efferocytosis of apoptotic neutrophils, and a delayed resolution of peritonitis, which were reversible with exogenous ACh supplementation. Intriguingly, despite B lymphocytes being a notable ChAT-expressing population within the peritoneal cavity, Chat deletion in B cells did not significantly alter the resolution process. Collectively, these findings underscore the crucial role of Mϕ-derived acetylcholine in the resolution of inflammation and highlight the importance of the non-neuronal cholinergic system in immune regulation.

Spin squeezing of 1011 atoms by prediction and retrodiction measurements.

The measurement sensitivity of quantum probes using N uncorrelated particles is restricted by the standard quantum limit1, which is proportional to [Formula: see text]. This limit, however, can be overcome by exploiting quantum entangled states, such as spin-squeezed states2. Here we report the measurement-based generation of a quantum state that exceeds the standard quantum limit for probing the collective spin of 1011 rubidium atoms contained in a macroscopic vapour cell. The state is prepared and verified by sequences of stroboscopic quantum non-demolition (QND) measurements. We then apply the theory of past quantum states3,4 to obtain spin state information from the outcomes of both earlier and later QND measurements. Rather than establishing a physically squeezed state in the laboratory, the past quantum state represents the combined system information from these prediction and retrodiction measurements. This information is equivalent to a noise reduction of 5.6 decibels and a metrologically relevant squeezing of 4.5 decibels relative to the coherent spin state. The past quantum state yields tighter constraints on the spin component than those obtained by conventional QND measurements. Our measurement uses 1,000 times more atoms than previous squeezing experiments5-10, with a corresponding angular variance of the squeezed collective spin of 4.6 × 10-13 radians squared. Although this work is rooted in the foundational theory of quantum measurements, it may find practical use in quantum metrology and quantum parameter estimation, as we demonstrate by applying our protocol to quantum enhanced atomic magnetometry.

A pneumonia outbreak associated with a new coronavirus of probable bat origin.

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.

Assembly of CpcL-phycobilisomes.

CpcL-phycobilisomes (CpcL-PBSs) are a reduced type of phycobilisome (PBS) found in several cyanobacteria. They lack the traditional PBS terminal energy emitters, but still show the characteristic red-shifted fluorescence at ~670 nm. We established a method of assembling in vitro a rod-membrane linker protein, CpcL, with phycocyanin, generating complexes with the red-shifted spectral features of CpcL-PBSs. The red-shift arises from the interaction of a conserved key glutamine, Q57 of CpcL in Synechocystis sp. PCC 6803, with a single phycocyanobilin chromophore of trimeric phycocyanin at one of the three ß82-sites. This chromophore is the terminal energy acceptor of CpcL-PBSs and donor to the photosystem(s). This mechanism also operates in PBSs from Acaryochloris marina MBIC11017. We then generated multichromic complexes harvesting light over nearly the complete visible range via the replacement of phycocyanobilin chromophores at sites α84 and ß153 of phycocyanins by phycoerythrobilin and/or phycourobilin. The results demonstrate the rational design of biliprotein-based light-harvesting elements by engineering CpcL and phycocyanins, which broadens the light-harvesting range and accordingly improves the light-harvesting capacity and may be potentially applied in solar energy harvesting.

Multiomics Analyses Provide New Insight into Genetic Variation of Reproductive Adaptability in Tibetan Sheep.

Domestication and artificial selection during production-oriented breeding have greatly shaped the level of genomic variability in sheep. However, the genetic variation associated with increased reproduction remains elusive. Here, two groups of samples from consecutively monotocous and polytocous sheep were collected for genome-wide association, transcriptomic, proteomic, and metabolomic analyses to explore the genetic variation in fecundity in Tibetan sheep. Genome-wide association study revealed strong associations between BMPR1B (p.Q249R) and litter size, as well as between PAPPA and lambing interval; these findings were validated in 1,130 individuals. Furthermore, we constructed the first single-cell atlas of Tibetan sheep ovary tissues and identified a specific mural granulosa cell subtype with PAPPA-specific expression and differential expression of BMPR1B between the two groups. Bulk RNA-seq indicated that BMPR1B and PAPPA expressions were similar between the two groups of sheep. 3D protein structure prediction and coimmunoprecipitation analysis indicated that mutation and mutually exclusive exons of BMPR1B are the main mechanisms for prolific Tibetan sheep. We propose that PAPPA is a key gene for stimulating ovarian follicular growth and development, and steroidogenesis. Our work reveals the genetic variation in reproductive performance in Tibetan sheep, providing insights and valuable genetic resources for the discovery of genes and regulatory mechanisms that improve reproductive success.

Maize RNA 3'-terminal phosphate cyclase-like protein promotes 18S pre-rRNA cleavage and is important for kernel development.

Plant ribosomes contain four specialized ribonucleic acids, the 5S, 5.8S, 18S, and 25S ribosomal RNAs (rRNAs). Maturation of the latter three rRNAs requires cooperative processing of a single transcript by several endonucleases and exonucleases at specific sites. In maize (Zea mays), the exact nucleases and components required for rRNA processing remain poorly understood. Here, we characterized a conserved RNA 3'-terminal phosphate cyclase (RCL)-like protein, RCL1, that functions in 18S rRNA maturation. RCL1 is highly expressed in the embryo and endosperm during early seed development. Loss of RCL1 function resulted in lethality due to aborted embryo cell differentiation. We also observed pleiotropic defects in the rcl1 endosperm, including abnormal basal transfer cell layer growth and aleurone cell identity, and reduced storage reserve accumulation. The rcl1 seeds had lower levels of mature 18S rRNA and the related precursors were altered in abundance compared with wild type. Analysis of transcript levels and protein accumulation in rcl1 revealed that the observed lower levels of zein and starch synthesis enzymes mainly resulted from effects at the transcriptional and translational levels, respectively. These results demonstrate that RCL1-mediated 18S pre-rRNA processing is essential for ribosome function and messenger RNA translation during maize seed development.

Recent development and applications of differential electrochemical mass spectrometry in emerging energy conversion and storage solutions.

Electrochemical energy conversion and storage are playing an increasingly important role in shaping the sustainable future. Differential electrochemical mass spectrometry (DEMS) offers an operando and cost-effective tool to monitor the evolution of gaseous/volatile intermediates and products during these processes. It can deliver potential-, time-, mass- and space-resolved signals which facilitate the understanding of reaction kinetics. In this review, we show the latest developments and applications of DEMS in various energy-related electrochemical reactions from three distinct perspectives. (I) What is DEMS addresses the working principles and key components of DEMS, highlighting the new and distinct instrumental configurations for different applications. (II) How to use DEMS tackles practical matters including the electrochemical test protocols, quantification of both potential and mass signals, and error analysis. (III) Where to apply DEMS is the focus of this review, dealing with concrete examples and unique values of DEMS studies in both energy conversion applications (CO2 reduction, water electrolysis, carbon corrosion, N-related catalysis, electrosynthesis, fuel cells, photo-electrocatalysis and beyond) and energy storage applications (Li-ion batteries and beyond, metal-air batteries, supercapacitors and flow batteries). The recent development of DEMS-hyphenated techniques and the outlook of the DEMS technique are discussed at the end. As DEMS celebrates its 40th anniversary in 2024, we hope this review can offer electrochemistry researchers a comprehensive understanding of the latest developments of DEMS and will inspire them to tackle emerging scientific questions using DEMS.

Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

Identification of SARS-CoV-2 PLpro and 3CLpro human proteome substrates using substrate phage display coupled with protein network analysis.

Viral proteases play key roles in viral replication, and they also facilitate immune escape by proteolyzing diverse target proteins. Deep profiling of viral protease substrates in host cells is beneficial for understanding viral pathogenesis and for antiviral drug discovery. Here, we utilized substrate phage display coupled with protein network analysis to identify human proteome substrates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteases, including papain-like protease (PLpro) and 3C-like protease (3CLpro). We first performed peptide substrates selection of PLpro and 3CLpro, and we then used the top 24 preferred substrate sequences to identify a total of 290 putative protein substrates. Protein network analysis revealed that the top clusters of PLpro and 3CLpro substrate proteins contain ubiquitin-related proteins and cadherin-related proteins, respectively. We verified that cadherin-6 and cadherin-12 are novel substrates of 3CLpro, and CD177 is a novel substrate of PLpro using in vitro cleavage assays. We thus demonstrated that substrate phage display coupled with protein network analysis is a simple and high throughput method to identify human proteome substrates of SARS-CoV-2 viral proteases for further understanding of virus-host interactions.

Comprehensive analysis of the expression profiles of mRNA, lncRNA, circRNA, and miRNA in primary hair follicles of coarse sheep fetal skin.

BACKGROUND: The Qinghai Tibetan sheep, a local breed renowned for its long hair, has experienced significant deterioration in wool characteristics due to the absence of systematic breeding practices. Therefore, it is imperative to investigate the molecular mechanisms underlying follicle development in order to genetically enhance wool-related traits and safeguard the sustainable utilization of valuable germplasm resources. However, our understanding of the regulatory roles played by coding and non-coding RNAs in hair follicle development remains largely elusive. RESULTS: A total of 20,874 mRNAs, 25,831 circRNAs, 4087 lncRNAs, and 794 miRNAs were annotated. Among them, we identified 58 DE lncRNAs, 325 DE circRNAs, 924 DE mRNAs, and 228 DE miRNAs during the development of medullary primary hair follicle development. GO and KEGG functional enrichment analyses revealed that the JAK-STAT, TGF-ß, Hedgehog, PPAR, cGMP-PKG signaling pathway play crucial roles in regulating fibroblast and epithelial development during skin and hair follicle induction. Furthermore, the interactive network analysis additionally identified several crucial mRNA, circRNA, and lncRNA molecules associated with the process of primary hair follicle development. Ultimately, by investigating DEmir's role in the ceRNA regulatory network mechanism, we identified 113 circRNA-miRNA pairs and 14 miRNA-mRNA pairs, including IGF2BP1-miR-23-x-novel-circ-01998-MSTRG.7111.3, DPT-miR-370-y-novel-circ-005802-MSTRG.14857.1 and TSPEAR-oar-miR-370-3p-novel-circ-005802- MSTRG.10527.1. CONCLUSIONS: Our study offers novel insights into the distinct expression patterns of various transcription types during hair follicle morphogenesis, establishing a solid foundation for unraveling the molecular mechanisms that drive hair development and providing a scientific basis for selectively breeding desirable wool-related traits in this specific breed.

Age-related decline in melatonin contributes to enhanced osteoclastogenesis via disruption of redox homeostasis.

BACKGROUND: Increased oxidative stress contributes to enhanced osteoclastogenesis and age-related bone loss. Melatonin (MT) is an endogenous antioxidant and declines with aging. However, it was unclear whether the decline of MT was involved in the enhanced osteoclastogenesis during the aging process. METHODS: The plasma level of MT, oxidative stress status, bone mass, the number of bone marrow-derived monocytes (BMMs) and its osteoclastogenesis were analyzed in young (3-month old) and old (18-month old) mice (n = 6 per group). In vitro, BMMs isolated from aged mice were treated with or without MT, followed by detecting the change of osteoclastogenesis and intracellular reactive oxygen species (ROS) level. Furthermore, old mice were treated with MT for 2 months to investigate the therapeutic effect. RESULTS: The plasma level of MT was markedly lower in aged mice compared with young mice. Age-related decline in MT was accompanied by enhanced oxidative stress, osteoclastogenic potential and bone loss. MT intervention significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, decreased intracellular ROS and enhanced antioxidant capacity of BMMs from aged mice. MT supplementation significantly attenuated oxidative stress, osteoclastogenesis, bone loss and deterioration of bone microstructure in aged mice. CONCLUSIONS: These results suggest that age-related decline of MT enhanced osteoclastogenesis via disruption of redox homeostasis. MT may serve as a key regulator in osteoclastogenesis and bone homeostasis, thereby highlighting its potential as a preventive agent for age-related bone loss.

In Situ Construction of NiCoMn-LDH Derived from Zeolitic Imidazolate Framework on Eggshell-Like Carbon Skeleton for High-Performance Flexible Supercapacitors.

Active compounds based on LDH (ternary layered double hydroxide) are considered the perfect supercapacitor electrode materials on account of their superior electrochemical qualities and distinct structural characteristics, and flexible supercapacitors are an ideal option as an energy source for wearable electronics. However, the prevalent aggregation effect of LDH materials results in significantly compromised actual specific capacitance, which limits its broad practical applications. In this research, a 3D eggshell-like interconnected porous carbon (IPC) framework with confinement and isolation capability is designed and synthesized by using glucose as the carbon source to disperse the LDH active material and enhance the conductivity of the composite material. Second, by constructing NiCoMn-LDH nanocage structure based on ZIF-67 (zeolitic imidazolate framework-67) at the nanometer scale the obtained IPC/NiCoMn-LDH electrode material can expose more active sites, which allows to achieve excellent specific capacitance (2236 F g-1/ 310.6 mAh g-1 at 1 A g-1), good rate as well as the desired cycle stability (85.9% of the initial capacitance upon 5000 cycles test). The constructed IPC/NiCoMn-LDH//IPC ASC (asymmetric supercapacitor) exhibits superior capacitive property (135 F g-1/60.1 mAh g-1 at 0.5 A g-1) as well as desired energy density (40 Wh kg-1 at 800 W kg-1).

Monomeric Far-red and Near-infrared Fluorescent Biliproteins of Ultrahigh Brightness.

Far-red and near-infrared fluorescent proteins have regions of maximum transmission in most tissues and can be widely used as fluorescent biomarkers. We report that fluorescent phycobiliproteins originating from the phycobilisome core subunit ApcF2 can covalently bind biliverdin, named BDFPs. To further improve BDFPs, we conducted a series of studies. Firstly, we mutated K53Q and T144A of BDFPs to increase their effective brightness up to 190 % in vivo. Secondly, by homochromatic tandem fusion of high-brightness BDFPs to achieve monomerization, which increases the effective brightness by up to 180 % in vivo, and can effectively improve the labeling effect. By combining the above two approaches, the brightness of the tandem BDFPs was much improved compared with that of the previously reported fluorescent proteins in a similar spectral range. The tandem BDFPs were expressed stably while maintaining fluorescence in mammalian cells and Caenorhabditis elegans. They were also photostable and resistant to high temperature, low pH, and chemical denaturation. The tandem BDFPs advantages were proved in applications as biomarkers for imaging in super-resolution microscopy.