Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

In vivo HSC prime editing rescues sickle cell disease in a mouse model.

Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the ß-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of ßS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.

Achieving Remarkable Specific Mechanical Strength and Energy Absorption Capacity in SiC Nanowire Networks through Graded Structural Design.

Lightweight porous ceramics with a unique combination of superior mechanical strength and damage tolerance are in significant demand in many fields such as energy absorption, aerospace vehicles, and chemical engineering; however, it is difficult to meet these mechanical requirements with conventional porous ceramics. Here, we report a graded structure design strategy to fabricate porous ceramic nanowire networks that simultaneously possess excellent mechanical strength and energy absorption capacity. Our optimized graded nanowire networks show a compressive strength of up to 35.6 MPa at a low density of 540 mg·cm-3, giving rise to a high specific compressive strength of 65.7 kN·m·kg-1 and a high energy absorption capacity of 17.1 kJ·kg-1, owing to a homogeneous distribution of stress upon loading. These values are top performance compared to other porous ceramics, giving our materials significant potential in various engineering fields.

Identification of binding partners that facilitate membrane-type 5 matrix metalloproteinase (MT5-MMP) processing of amyloid precursor protein.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular deposits of amyloid beta (Aß) peptide. In addition to Aß as the core component of the amyloid plaque, the amyloid precursor protein (APP) processing fragment Aß was also found accumulated around the plaque. The APPη pathway, mainly mediated by membrane-type 5 matrix metalloproteinase (MT5-MMP), represents an important factor in AD pathogenesis. The proamyloidogenic features of MT5-MMP could result from interactions with APP when trafficking between organelles, so determination of the location within the cell of APPη cleavage and interacting proteins of MT5-MMP affecting this process will be of priority in understanding the role of MT5-MMP in AD. In the present study, MT5-MMP was found to be located in the nucleus, cytosol, and cytosolic subcellular granules of CHO cells that stably expressed wild-type human APP751. MT5-MMP fusion proteins were constructed that could localize enzyme production in the Golgi apparatus, endosome, ER, mitochondria, or plasma membrane. The fusion proteins significantly increased sAPPη when directed to the endosome, Golgi apparatus, plasma membrane, or mitochondria. Since the C-terminal region of MT5-MMP is responsible for its intracellular location and trafficking, this domain was used as the bait in a yeast two-hybrid screen to identify MT5-MMP protein partners in a human brain cDNA library. Identified binding partners included N4BP2L1, TMX3, EIG121, bridging Integrator 1 (BIN1), RUFY4, HTRA1, and TMEM199. The binding of N4BP2L1, EIG121, BIN1, or TMX3 to MT5-MMP resulted in the most significant increase in sAPPη production. Thus, the action of MT5-MMP on APP occurs in multiple locations within the cell and is facilitated by site-specific binding partners.

Huanglian Jiedu Wan intervened with "Shi-Re Shanghuo" syndrome through regulating immune balance mediated by biomarker succinate.

With increasing stress in daily life and work, subhealth conditions induced by "Shi-Re Shanghuo" syndrome was gradually universal. "Huanglian Jiedu Wan" (HLJDW) was the first new syndrome Chinese medicine approved for the treatment of "Shi-Re Shanghuo" with promising clinical efficacy. Preliminary small-sample clinical studies have identified some notable biomarkers (succinate, 4-hydroxynonenal, etc.). However, the correlation and underlying mechanism between these biomarkers of HLJDW intervention on "Shi-Re Shanghuo" syndrome remained ambiguous. Therefore, this study was designed as a randomized, double-blind, multicenter, placebo-controlled Phase II clinical trial, employing integrated analysis techniques such as non-targeted and targeted metabolomics, salivary microbiota, proteomics, parallel peaction monitoring, molecular docking and surface plasmon resonance (SPR). The results of the correlation analysis indicated that HLJDW could mediate the balance between inflammation and immunity through succinate produced via host and microbial source to intervene "Shi-Re Shanghuo" syndrome. Further through the HIF1α/MMP9 pathway, succinate regulated downstream arachidonic acid metabolism, particularly the lipid peroxidation product 4-hydroxynonenal. Finally, an animal model of recurrent oral ulcers induced by "Shi-Re Shang Huo" was established and HLJDW was used for intervention, key essential indicators (succinate, glutamine, 4-hydroxynonenal, arachidonic acid metabolism) essential in the potential pathway HIF1α/MMP9 discovered in clinical practice were validated. The results were found to be consistent with our clinical findings. Taken together, succinate was observed as an important signal that triggered immune responses, which might serve as a key regulatory metabolic switch or marker of "Shi-Re Shanghuo" syndrome treated with HLJDW.

Mechanochemical Oxidative Coupling of Amine to Azo-based Polymers by Hypervalent Iodine Oxidant.

Among porous organic polymers (POPs), azo-linked POPs represent a crucial class of materials, making them the focus of numerous catalytic systems proposed for their synthesis. However, the synthetic process is limited to metal-catalyzed, high-temperature, and liquid-phase reactions. In this study, we employ mechanochemical oxidative metal-free systems to encompass various syntheses of azo-based polymers. Drawing inspiration from the "rule of six" principle (six or more carbons on an azide group render the organic compound relatively safe), an azo compound featuring significant steric hindrance is obtained using the hypervalent iodine oxidation strategy. Furthermore, during the polymerization process, steric hindrance is enhanced in monomers to effectively prevent explosions resulting from direct contact between hypervalent iodine oxidants and primary amines. Indeed, this approach provides a facile and innovative solid-phase synthesis method for synthesizing azo-based materials.

Rapid static feeding combined with Fe2+ addition for improving the formation and stability of aerobic granular sludge in low-strength wastewater.

Aerobic granular sludge (AGS) needs a long start-up time and always shows unstable performance when it is used to treat low-strength wastewater. In this study, a rapid static feeding combined with Fe2+ addition as a novel strategy was employed to improve the formation and stability of AGS in treating low-strength wastewater. Fe-AGS was formed within only 7 days and showed favorable pollutant removal capability and settling performance. The ammonia nitrogen (NH4+-N) and chemical oxygen demand (COD) concentration in the effluent were lower than 5 mg/L and 50 mg/L after day 23, respectively. The sludge volume index (SVI) and mixed liquid suspended solids (MLSS) was 37 mL/g and 2.15 g/L on day 50, respectively. Rapid static feeding can accelerate granules formation by promoting the growth of heterotrophic bacteria, but the granules are unstable due to filamentous bacteria overgrowth. Fe2+ addition can inhibit the growth of filamentous bacteria and promote the aggregation of functional bacteria (eg. Nitrosomonas, Nitrolancea, Paracoccus, Diaphorobacter) by enhancing the secretion of extracellular polymeric substances (EPS). This study provides a new way for AGS application in low-strength wastewater treatment.

Fate of antibiotic resistance genes during sludge anaerobic fermentation: Roles of different sludge pretreatment.

Excess sludge, the primary by-product of wastewater treatment plants, is the source and sink of antibiotic resistance genes (ARGs). Sludge pretreatments are an indispensable pathway to improve the resource recovery and harmfulness for anaerobic digestion sludge. However, fewer studies have compared the effects of different pretreatment technologies on the distribution of ARGs during anaerobic sludge digestion. Here, this study established seven anaerobic digesters, and four typical ARGs and one integrase gene of class 1 integron (intI1) regarded as the representative mobile genetic elements (MGEs) were examined during the whole anaerobic digestion process. It was found anaerobic digestion could effectively remove ARGs with about 70.86% removal rate of total ARGs. Among these pretreatments, the reduce efficiency of ARGs was the highest in 50 °C pretreatment, followed by oxidant, and the last was acid-alkaline. The microbial community analysis demonstrated the microbial community structure, including ARGs hosts and antibiotic resistant bacteria, was significantly changed and influenced by high temperature pretreatment. In addition, high temperature and K2S2O8 observably decrease the level of ROS production. Macro transcriptome analysis indicated that sludge pretreatment, except for 50 °C pretreatment, up-regulated the genes relevant to lyases and transferase, but down-regulated the genes responsible for peroxidase, antioxidant enzymes and T4SS gene. This study emphasized and compared the different sludge pretreatments on the fate of ARGs in anaerobic sludge, and highlighted concerns regarding the environmental and health risks to our society.

Potential effects of Cu2+ stress on nitrogen removal performance, microbial characteristics, and metabolism pathways of biofilm reactor.

Sequencing batch biofilm reactors (SBBR) were utilized to investigate the impact of Cu2+ on nitrogen (N) removal and microbial characteristics. The result indicated that the low concentration of Cu2+ (0.5 mg L-1) facilitated the removal of ammonia nitrogen (NH4+-N), total nitrogen (TN), nitrate nitrogen (NO3--N), and chemical oxygen demand (COD). In comparison to the average effluent concentration of the control group, the average effluent concentrations of NH4+-N, NO3--N, COD, and TN were found to decrease by 40.53%, 17.02%, 10.73%, and 15.86%, respectively. Conversely, the high concentration of Cu2+ (5 mg L-1) resulted in an increase of 94.27%, 55.47%, 22.22%, and 14.23% in the aforementioned parameters, compared to the control group. Low concentrations of Cu2+ increased the abundance of nitrifying bacteria (Rhodanobacter, unclassified-o-Sacharimonadales), denitrifying bacteria (Thermomonas, Comamonas), denitrification-associated genes (hao, nosZ, norC, nffA, nirB, nick, and nifD), and heavy-metal-resistant genes related to Cu2+ (pcoB, cutM, cutC, pcoA, copZ) to promote nitrification and denitrification. Conversely, high concentration Cu2+ hindered the interspecies relationship among denitrifying bacteria genera, nitrifying bacteria genera, and other genera, reducing denitrification and nitrification efficiency. Cu2+ involved in the N and tricarboxylic acid (TCA) cycles, as evidenced by changes in the abundance of key enzymes, such as (EC:1.7.99.1), (EC:1.7.2.4), and (EC:1.1.1.42), which initially increased and then decreased with varying concentrations of Cu2+. Conversely, the abundance of EC1.7.2.1, associated with the accumulation of nitrite nitrogen (NO2--N), gradually declined. These findings provided insights into the impact of Cu2+ on biological N removal.

Effect of elemental sulfur on anaerobic ammonia oxidation: Performance and mechanism.

Biological nitrogen removal processes provide effective means to mitigate nitrogen-related issues in wastewater treatment. Previous studies have highlighted the collaborative efficiency between sulfur autotrophic denitrification and Anammox processes. However, the trigger point induced the combination of nitrogen and sulfur metabolism is unclear. In this study, elemental sulfur (S0) was introduced to Anammox system to figure out the performance and mechanism of S0-mediated autotrophic denitrification and Anammox (S0SAD-A) systems. The results showed that the nitrogen removal performance of the Anammox reactor decreased with the increasing concentrations of NH4+-N and NO2--N in influent, denitrification occurred when NH4+-N concentration reached 100 mg/L. At stage ⅳ (150 mg/L NH4+-N), the total nitrogen removal efficiency in S0SAD-A system (95.99%) was significantly higher than that in the Anammox system (77.22%). Throughout a hydraulic retention time, the consumption rate of NH4+-N in S0SAD-A was faster than that in Anammox reactor. And there existed a nitrate-concentration peak in S0SAD-A system. Metagenomic sequencing was performed to reveal functional microbes as well as key genes involved in sulfur and nitrogen metabolism. The results showed that the introduction of S0 elevated the abundance of Ca. Brocadia. Moreover, the relative abundance of Anammox genes, such as hao, hzsA and hzsC were also stimulated by sulfur. Notably, unclassified members in Rhodocyclaceae acted as the primary contributor to key genes involved in the sulfur metabolism. Overall, the interactions between Anammox and denitrification were stimulated by sulfur metabolism. Our study shed light on the potential significance of Rhodocyclaceae members in the S0SAD-A process and disclosed the relationship between anammox and denitrification.

The function and keystone microbiota in typical habitats under the influence of anthropogenic activities in Baiyangdian Lake.

Microbe is an essential driver in regulating the biochemical cycles of carbon, nitrogen, and sulfur. In freshwater lake, microbial communities and functions are influenced by multiple factors, especially anthropogenic activities. Baiyangdian Lake consisted of various habitats, and was frequently interfered with human activities. In this study, 16 S rRNA sequencing and metagenomic sequencing were performed to characterize the microbial communities, determine keystone taxa and reveal dominated metabolic functions in typical habitats in Baiyangdian Lake. The results showed that the diversity of microbial community was significantly higher in sediment compared with corresponding water sample. Microbial community showed strong spatial heterogeneity in sediment, and temporal heterogeneity in water. As for different habitats, significantly higher alpha diversity was observed in ecotone, where the interference of human activities was relatively weak. The shared OTUs were distinguished from the keystone taxa, which indicated the uniqueness of microbiota in different ecological habitat. Moreover, the interactions of microbial in ecological restoration area (abandoned fish pond) were relatively simple, suggesting that this ecosystem was relatively fragile compared with others. Based on the metagenomic sequencing, we recognized that the canal, open water, and abandoned fish pond were beneficial for methanogenic and the ecotone might be a hot zone for the oxidation of methane. Notably, most of the microbes that participated in these predominant metabolisms were unclassified, which indicated the hug potential for exploring functional microorganisms in Baiyangdian Lake. This study provided a comprehensive understanding of the ecology characteristics of microbiota in habitats undergoing various human interference in Baiyangdian Lake.

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Study on chemical profiling of bailing capsule and its potential mechanism against thyroiditis based on network pharmacology with molecular docking strategy.

Bailing capsule (BLC), a drug that is clinically administered to modulate the autoimmune system, exhibits promising therapeutic potential in the treatment of thyroiditis. This study elucidates the chemical profile of BLC and its potential therapeutic mechanism in thyroiditis, leveraging network pharmacology and molecular docking techniques. Utilizing ultra-high-performance liquid chromatography coupled with linear trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS), 58 compounds were identified, the majority of which were nucleosides and amino acids. Utilizing the ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC QqQ MS/MS) strategy, 16 representative active components from six batches of BLCs were simultaneously determined. Network pharmacology analysis further revealed that the active components included 5'-adenylate, guanosine, adenosine, cordycepin, inosine, 5'-guanylic acid, and l-lysine. Targets with higher connectivity included AKT1, MAPK3, RAC1, and PIK3CA. The signaling pathways primarily focused on thyroid hormone regulation and the Ras, PI3K/AKT, and MAPK pathways, all of which were intricately linked to inflammatory immunity and hormonal regulation. Molecular docking analysis corroborated the findings from network pharmacology, revealing that adenosine, guanosine, and cordycepin exhibited strong affinity toward AKT1, MAPK3, PIK3CA, and RAC1. Overall, this study successfully elucidated the material basis and preliminary mechanism underlying BLC's intervention in thyroiditis, thus laying a solid basis for further exploration of its in-depth mechanisms.

Sulfur-manganese carbonate composite autotrophic denitrification: nitrogen removal performance and biochemistry mechanism.

A novel composite sulfur-manganese carbonate autotrophic denitrification (SMAD) system was developed to reduce sulfate production and provide pH buffer function while improving denitrification efficiency without external organics. The average removal efficiency of total nitrogen (TN) was 98.09% and 96.29%, and that of NO3--N was 99.53% and 97.77%, respectively, in the SMAD system with a hydraulic retention time (HRT) of 6 h and 3 h. They were significantly higher than that in the controls (quartz sand, manganese carbonate ore, and sulfur systems). The H+ produced by the sulfur autotrophic denitrification (SAD) process promoted the release of Mn2+ in the SMAD system. And this system had a stable pH with no accumulation of NO2--N. The decrease of sulfate and formation of Mn oxides through Mn2+ electron donation confirmed the presence of the manganese autotrophic denitrification (MAD) process in the SMAD system. Dominant functional bacteria in the SMAD system were Thiobacillus, Chlorobium, and Sulfurimonas, which were linked to nitrogen, sulfur, and manganese conversion and promoted denitrification. Meanwhile, Flavobacterium participating in Mn2+ oxidation was found only in the SMAD system. The SMAD system provided a new strategy for advanced tailwater treatment.

Resilient and Antipuncturing Si3N4 Nanofiber Sponge.

Ceramic nanofibrous nanostructure-based sponges have attracted significant attention due to ultrahigh porosity, low thermal conductivity, large specific area, and chemical stability. From the regulation of the fiber itself to the construction method of 3D networks, efforts are being made to improve the mechanical properties of ceramic sponges for practical applications. So far resilient compressibility has been realized in ceramic nanofibrous-based sponges via structural design, but they still show brittle fracture under a more complex stress state. Herein, we introduced a highly aligned and interwoven Si3N4 nanofiber sponge, which exhibits superflexibility, large break elongation (>80%), large-strain reversible stretch (20%), and good resistance to tensile fatigue. The ceramic sponge also displays reversible compressibility up to 60% strain, puncture resistance, high air filtration efficiency (>99.8%), and low pressure drop (38% of cotton fiber), making the ceramic sponge a high-performance wearable respirator to protect us from harm due to PM2.5 pollution and possible microorganisms.

[Synthesis and tissue distribution of bufadienolides in Bufo bufo gargarizans].

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.

[Evaluation of efficacy of Dihuang Baoyuan Granules for diabetes mellitus and composition and in vivo distribution analysis based on UHPLC-LTQ-Orbitrap MS].

Dihuang Baoyuan Granules is a prescription endorsed by HU Tianbao, a renowned and elderly Chinese medicine practitioner from Beijing, and has demonstrated definite clinical efficacy. The composition of this prescription is intricate as it includes 7 distinct herbal medicines. This study aims to analyze the chemical composition of Dihuang Baoyuan Granules, evaluate its efficacy in the treatment of diabetes and analyze the distribution of the drug components in the plasma, liver, and kidney after administration. The findings will serve as a reference for future research on pharmacodynamic substances of this prescription. UHPLC-LTQ-Orbitrap MS was employed to analyze the main chemical components of Dihuang Baoyuan Granules. A Waters ACQUITY Premier HSS T3 column(2.1 mm×100 mm, 1.8 µm) was used for chromatographic separation with 0.1% formic acid(A)-acetonitrile(B) as the mobile phases in a gradient elution at a flow rate of 0.3 mL·min~(-1). Electrospray ionization(ESI) source was used to acquire data in positive and negative ion modes. Furthermore, a rat model of diabetes mellitus was established by feeding with a high-sugar high-fat diet, and injection with streptozocin at a dose of 35 mg·kg~(-1), and the modeled rats were then administrated with Dihuang Baoyuan Granules. The fasting blood glucose, hemoglobin A1c, and other relevant indicators were measured, and the substances present in the plasma, liver, and kidney were identified. By reference to quasi-molecular ions, MS/MS fragment ions, MS spectra of reference substances, and compound information in available reports, 191 components were identified in Dihuang Baoyuan Granules, including 29 alkaloids, 24 flavonoids, 22 organic acids, 16 amino acids, 12 terpenes, 11 steroid saponins, 9 sugars, 8 phenylethanoid glycosides, 8 nucleosides, 2 phenylpropanoids, and 49 others compounds. Eighty-three chemical components were identified in rat plasma, 109 in the liver, and 98 in the kidney. Component identification and characterization of Dihuang Baoyuan Granules in vitro and in vivo provide efficacy information and guidance for the basic research on the pharmacodynamic substances and further clinical application of this prescription.

ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.

High-quality chromosome-level de novo assembly of the Trifolium repens.

BACKGROUND: White clover (Trifolium repens L.), an excellent perennial legume forage, is an allotetraploid native to southeastern Europe and southern Asia. It has high nutritional, ecological, genetic breeding, and medicinal values and exhibits excellent resistance to cold, drought, trample, and weed infestation. Thus, white clover is widely planted in Europe, America, and China; however, the lack of reference genome limits its breeding and cultivation. This study generated a white clover de novo genome assembly at the chromosomal level and annotated its components. RESULTS: The PacBio third-generation Hi-Fi assembly and sequencing methods generated a 1096 Mb genome size of T. repens, with contigs of N50 = 14 Mb, scaffolds of N50 = 65 Mb, and BUSCO value of 98.5%. The newly assembled genome has better continuity and integrity than the previously reported white clover reference genome; thus provides important resources for the molecular breeding and evolution of white clover and other forage. Additionally, we annotated 90,128 high-confidence gene models from the genome. White clover was closely related to Trifolium pratense and Trifolium medium but distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum. The expansion, contraction, and GO functional enrichment analysis of the gene families showed that T. repens gene families were associated with biological processes, molecular function, cellular components, and environmental resistance, which explained its excellent agronomic traits. CONCLUSIONS: This study reports a high-quality de novo assembly of white clover genome obtained at the chromosomal level using PacBio Hi-Fi sequencing, a third-generation sequencing. The generated high-quality genome assembly of white clover provides a key basis for accelerating the research and molecular breeding of this important forage crop. The genome is also valuable for future studies on legume forage biology, evolution, and genome-wide mapping of quantitative trait loci associated with the relevant agronomic traits.

CD248 induces a maladaptive unfolded protein response in diabetic kidney disease.

Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of kidney failure. However, the underlying molecular mechanisms are incompletely understood. By unbiased gene expression analysis of glucose-exposed mesangial cells, we identified the transmembrane receptor CD248 as the most upregulated gene, and the maladaptive unfolded protein response (UPR) as one of the most stimulated pathways. Upregulation of CD248 was further confirmed in glucose-stressed mesangial cells in vitro, in kidney glomeruli isolated from diabetic mice (streptozotocin; STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) in vivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by transcription factor C/EBP homologous protein (CHOP) induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (XBP1). Mechanistically, CD248 promoted maladaptive UPR signaling via inhibition of the inositol requiring enzyme 1α (IRE1α)-mediated transcription factor XBP1 splicing in vivo and in vitro. CD248 induced a multiprotein complex comprising heat shock protein 90, BH3 interacting domain death agonist (BID) and IRE1α, in which BID impedes IRE1α-mediated XBP1 splicing and induced CHOP mediated maladaptive UPR signaling. While CD248 knockout ameliorated DKD-associated glomerular dysfunction and reverses maladaptive unfolded protein response signaling, concomitant XBP1 deficiency abolished the protective effect in diabetic CD248 knockout mice, supporting a functional interaction of CD248 and XBP1 in vivo. Hence, CD248 is a novel mesangial cell receptor inducing maladaptive UPR signaling in DKD.