Structural Characterization and Physiological Role of Bombyx mori Fibroinase in the Silk Gland Development.

Silkworm is a highly valuable insect that produces silk through secretion by a silk gland. Within this gland, a type of cathepsin L protease called Fibroinase was identified as an enzyme for hydrolyzing the primary components of silk, including fibroin and sericin. Here, we determined the crystal structure of Fibroinase fromBombyx mori at a resolution of 1.56 Å. Comparative structural analysis revealed that Fibroinase adopted a similar structural pattern with papain-type cathepsin, consisting of an N-terminal domain and a C-terminal domain. The interface between the domains forms a substrate-binding cleft, where the E64 inhibitor noncovalently binds in a novel manner. Additionally, computational simulations combined with biochemical analysis allowed us to define the binding mode and inhibition mechanism of physiological inhibitor Bombyx cysteine protease inhibitor (BCPI) with Fibroinase. Moreover, the expression profiles and RNA interference of Fibroinase indicated its critical role in removing silk proteins in the silk gland lumen and the destruction of silk gland tissue during the larval-pupal metamorphosis. These findings enhance our understanding of the structural and biochemical features of Fibroinase and its inhibitors, while also providing evidence for the physiological role of Fibroinase in silk gland development.

Antimony and arsenic migration in a heterogeneous subsurface at an abandoned antimony smelter under rainfall.

While antimony (Sb) and arsenic (As) co-contamination in subsurface soil systems due to the legacy of Sb smelting wastes has been documented, the role of inherent heterogeneity on pollutant migration is largely overlooked. Herein this study investigated Sb and As migration in a slag impacted, vertically stratified subsurface at an abandoned Sb smelter. A 2-dimensional flume was assembled as a lab-scale analogue of the site and subject to rainfall and stop-rain events. Reactive transport modeling was then performed by matching the experimental observations to verify the key factors and processes controlling pollutant migration. Results showed that rainfall caused Sb and As release from the shallow slag layer and promoted their downward movement. Nevertheless, the less permeable deeper layers limited physical flow and transport, which led to Sb and As accumulation at the interface. The re-adsorption of Sb and As onto iron oxides in the deeper, more acidic layers further retarded their migration. Because of the large difference between Sb and As concentrations, Sb re-adsorption was much less effective, which led to higher mobility. Our findings overall highlight the necessity of understanding the degree and impacts of physicochemical heterogeneity for risk exposure assessment and remediation of abandoned Sb smelting sites.

Toonanoronoids A-E, five new limonoids from Toona ciliate var. yunnanensis.

Five new B-seco-limonoids, namely toonanoronoids A-E (1-5), in conjunction with three previously reported compounds, were isolated from the EtOAc extract of the twigs and leaves of Toona ciliata var. yunnanensis. Their structures were elucidated through comprehensive spectroscopic and X-ray crystallographic analysis. The cytotoxic activities of new compounds against five human tumor cell lines (HL-60, SMMC-7721, A549, MCF-7, and SW480) were screened, Compounds 4 and 5 exerted inhibition toward two tumor cell lines (HL-60, SW-480) with IC50 values between 1.7 and 5.9 µM.

Application of enhanced recovery after surgery during the perioperative period in children with Meckel's diverticulum-a single-center prospective clinical trial.

Background: Enhanced recovery after surgery (ERAS) has been widely used in adult surgery. However, few studies have reported the efficacy of ERAS in paediatric patients with Meckel's diverticulum (MD), the aim of the study was to prospectively evaluate the safety and efficacy of ERAS in treating MD. Methods: A prospective randomised controlled study of children with MD admitted to our hospital from Jan 1, 2021 to Dec 31, 2023 were conducted, we developed and implemented an ERAS program for this patients. All cases were strictly selected according to the inclusion and exclusion criteria. Among these patients, they were randomly assigned to the ERAS group or the traditional (TRAD) group with random number table row randomization. The main observational indicators were operation time, intraoperative hemorrhage, FLACC pain scale results on 2 h, 6 h, 12 h, 24 h after surgery, length of postoperative stay (LOPS), time to first defecation, time to first eating after surgery, time to discontinuation of intravenous infusion, total treatment cost, incidence of postoperative complications, 30-day readmission rate and parental satisfaction rate. Results: A total of 50 patients underwent Meckel's diverticulectomy during this period, 7 patients were excluded, 23 patients were assigned to the ERAS group and 20 patients were assigned to the TRAD group. There were no significant differences in demographic data and operation time, intraoperative hemorrhage. The FLACC pain scale results on 2 h, 6 h, 12 h, 24 h after surgery were significantly lower in the ERAS group. The LOPS was 6.17 ± 0.89 days in the ERAS group and 8.30 ± 1.26 days in the TRAD group, resulting in a significantly shorter LOPS in ERAS group. ERAS could also reduce the first postoperative defecation time, the time to first eating after surgery and the time to discontinuation of intravenous infusion. The treatment cost was decreased in the ERAS group. The rate of complications and 30-day readmission were not significantly different between the two groups. Conclusions: In this single-center study, the ERAS protocol for patients with MD requiring surgery was safe and effective.

Two-dimensional spin-crossover coordination polymers based on the 1,1,2,2-tetra(pyridin-4-yl)ethene ligand.

A series of two-dimensional (2D) spin-crossover coordination polymers (SCO-CPs) [FeII(TPE)(NCX)2]·solv (1: X = BH3, solv = H2O·2CH3OH·DMF; 2: X = Se, solv = H2O·2CH3OH·0.5DMF; 3: X = S, solv = H2O·2CH3OH·0.5DMF) were synthesized by employing 1,1,2,2-tetra(pyridin-4-yl)ethene (TPE) and pseudohalide (NCX-) coligands. Magnetic measurements indicated that complexes 1-3 exhibited SCO behaviors with diminishing thermal hysteresis (7/4/0 K) upon decreasing the ligand-field strength. The critical temperatures (Tc) during spin transition were found to be inversely proportional to the coordination ability parameters (a™) with a linear correlation. The guest effect was also investigated in the solvent-exchanged phases 1-SE/2-SE/3-SE wherein the DMF molecules were replaced by methanol molecules. Compared with 1-3, 1-SE/2-SE/3-SE displayed more abrupt and complete single-step SCO behaviors but narrower thermal hysteretic loops. The results reported here demonstrate that the Tc values of these two families were dominated by the ligand-field strength of the NCX- anions (NCBH3 > NCSe > NCS), whereas the guest effect only modulated the kinetic factor of the SCO nature in this system.

Insights into the alleviation of cadmium toxicity in rice by nano-zinc and Serendipita indica: Modulation of stress-responsive gene expression and antioxidant defense system activation.

The adversities of cadmium (Cd) contamination are quite distinguished among other heavy metals (HMs), and so is the efficacy of zinc (Zn) nutrition in mitigating Cd toxicity. Rice (Oryza sativa) crop, known for its ability to absorb HMs, inadvertently facilitates the bioaccumulation of Cd, posing a significant risk to both the plant itself and to humans consuming its edible parts, and damaging the environment as well. The use of nanoparticles, such as nano-zinc oxide (nZnO), to improve the nutritional quality of crops and their counteractive implications against HMs, have gained substantial attention among scientists and farmers. While previous studies have explored the individual effects of nZnO or Serendipita indica (referred to as S.i) on Cd toxicity, the synergistic action of these two agents has not been thoroughly investigated. Therefore, the gift of nature, i.e., S. indica, was incorporated alongside nZnO (50 mg L-1) against Cd stress (15 µM L-1) and their alliance manifested as phenotypic level modifications in two rice genotypes (Heizhan43; Hz43 and Yinni801; Yi801). Antioxidant activities were enhanced, specifically peroxidase (61.5 and 122.5% in Yi801 and Hz43 roots, respectively), leading to a significant decrease in oxidative burst; moreover, Cd translocation was reduced (85% for Yi801 and 65.5% for Hz43 compared to Cd alone treatment). Microstructural study showed a decrease in number of vacuoles and starch granules with ameliorative treatments. Overall, plants treated with nZnO displayed gene expression pattern (particularly of ZIP genes), different from the ones with alone or combined S.i and Cd. Inferentially, the integration of nZnO and S.i holds great promise as an effective strategy for alleviating Cd toxicity in rice plants. By immobilizing Cd ions in the soil and promoting their detoxification, this novel approach contributes to environmental restoration and ensures food safety worldwide.

Engagement of AKT and ERK signaling pathways facilitates infection of human neuronal cells with West Nile virus.

West Nile virus (WNV) is an important neurotropic virus that accounts for the emergence of human arboviral encephalitis and meningitis. The interaction of WNV with signaling pathways plays a key role in controlling WNV infection. We have investigated the roles of the AKT and ERK pathways in supporting WNV propagation and modulating the inflammatory response following WNV infection. WNV established a productive infection in neuronal cell lines originated from human and mouse. Expression of IL-11 and TNF-α was markedly up-regulated in the infected human neuronal cells, indicating elicitation of inflammation response upon WNV infection. WNV incubation rapidly activated signaling cascades of AKT (AKT-S6-4E-BP1) and ERK (MEK-ERK-p90RSK) pathways. Treatment with AKT inhibitor MK-2206 or MEK inhibitor U0126 abrogated WNV-induced AKT or ERK activation. Strong activation of AKT and ERK signaling pathways could be detectable at 24 h after WNV infection, while such activation was abolished at 48 h post infection. U0126 treatment or knockdown of ERK expression significantly increased WNV RNA levels and viral titers and efficiently decreased IL-11 production induced by WNV, suggesting the involvement of ERK pathway in WNV propagation and IL-11 induction. MK-2206 treatment enhanced WNV RNA replication accompanied with a moderate decrease in IL-11 production. These results demonstrate that engagement of AKT and ERK signaling pathways facilitates viral infection and may be implicated in WNV pathogenesis.

The proportion of CD161 on CD56+ NK cells in peripheral circulation associates with clinical features and disease activity of primary Sjögren's syndrome.

OBJECTIVES: The purpose of this study was to examine the proportion of CD161 on CD56+ natural killer (NK) cells in peripheral blood of primary Sjögren's syndrome (pSS) and investigate its clinical relevance of pSS. METHODS: The proportion of CD56+ NK cells and CD161 on CD56+ NK cells was detected by flow cytometry in 31 pSS patients and 29 healthy controls (HCs). The correlations between the proportion of CD161+CD56+ NK cells and clinical features and disease activity of pSS were further analyzed. Meanwhile, we drew the receiver operating characteristic curve to evaluate the diagnostic value of CD161+CD56+ NK cells in pSS. In addition, we evaluated the differences in the effects of CD161+ cells and CD161- cells in peripheral blood on the function of CD56+ NK cells in 5 pSS patients. RESULTS: The proportion of CD56+ NK cells and CD161+CD56+ NK cells decreased markedly in pSS patients compared to HCs. The correlation analysis showed that the proportion of CD161+CD56+ NK cells negatively correlated with white blood cells, Immunoglobulin A (IgA), IgM, IgG, European League Against Rheumatism Sjogren's Syndrome Patient Reported Index and European League Against Rheumatism Sjogren's Syndrome Disease Activity Index, and positively correlated with complement C4. The proportion of CD161+CD56+ NK cells in pSS patients with decayed tooth, fatigue, arthralgia, skin involvement, primary biliary cirrhosis, interstitial lung disease, anti-SSA/Ro60 positive, anti-SSB positive and high IgG was lower than that in negative patients. Furthermore, compared with inactive patients, the proportion of CD161+CD56+ NK cells decreased obviously in active patients. The area under the curve was 0.7375 (p = .0016), the results indicated that CD161+CD56+ NK cells had certain diagnostic values for pSS. In addition, the proportion of CD86, HLA-DR, Ki67, FasL, TNF-α, and IFN-γ on CD161+CD56+ NK cells was lower than that on CD161-CD56+ NK cells in the peripheral blood of pSS patients. CONCLUSION: This study suggested that the proportion of CD56+ NK cells and CD161+CD56+ NK cells decreased significantly in pSS patients, and the proportion of CD161+CD56+ NK cells negatively associated with the clinical features and disease activity of pSS patients. CD161 expression inhibited the function of CD56+ NK cells in peripheral blood of pSS patients. The CD161+CD56+ NK cells may present as a potential target for therapy and a biomarker of disease activity in pSS.

Root-derived long-distance signals trigger ABA synthesis and enhance drought resistance in Arabidopsis.

Vascular plants have evolved intricate long-distance signaling mechanisms to cope with environmental stress, with reactive oxygen species (ROS) emerging as pivotal systemic signals in plant stress responses. However, the exact role of ROS as root-to-shoot signals in the drought response has not been determined. In this study, we reveal that compared with wild-type plants, ferric reductase defective 3 (frd3) mutants exhibit enhanced drought resistance concomitant with elevated NCED3 transcript levels and ABA contents in leaves as well as increased hydrogen peroxide (H2O2) levels in roots and leaves. Grafting experiments distinctly illustrate that drought resistance can be conferred by the frd3 rootstock regardless of the scion genotype, indicating that long-distance signals originating from frd3 roots promote an increase in ABA levels in leaves. Intriguingly, the drought resistance conferred by the frd3 mutant rootstock is weakened by the CAT2-overexpressing scion, suggesting that H2O2 may be involved in the long-distance signaling. Moreover, the results of comparative transcriptome and proteome analyses support the drought resistance phenotype of the frd3 mutant. Taken together, our findings substantiate the notion that frd3 root-derived long-distance signals trigger ABA synthesis in leaves and enhance drought resistance, providing new evidence for root-to-shoot long-distance signaling in the drought response of plants.

Nutrient and mycoremediation of a global menace 'arsenic': exploring the prospects of phosphorus and Serendipita indica-based mitigation strategies in rice and other crops.

KEY MESSAGE: Serendipita indica induced metabolic reprogramming in colonized plants complements phosphorus-management in improving their tolerance to arsenic stress on multifaceted biological fronts. Restoration of the anthropic damage done to our environment is inextricably linked to devising strategies that are not only economically sound but are self-renewing and ecologically conscious. The dilemma of heavy metal (HM) dietary ingestion, especially arsenic (As), faced by humans and animals alike, necessitates the exploitation of such technologies and the cultivation of healthy and abundant crops. The remarkable symbiotic alliance between plants and 'mycorrhizas' has evolved across eons, benefiting growth/yield aspects as well as imparting abiotic/biotic stress tolerance. The intricate interdependence of Serendipita indica (S. indica) and rice plant reportedly reduce As accumulation, accentuating the interest of microbiologists, agriculturists, and ecotoxicological scientists apropos of the remediation mechanisms of As in the soil-AMF-rice system. Nutrient management, particularly of phosphorus (P), is also praised for mitigating As phytotoxicity by deterring the uptake of As molecules due to the rhizospheric cationic competition. Taking into consideration the reasonable prospects of success in minimizing As acquisition by rice plants, this review focuses on the physiological, metabolic, and transcriptional alterations underlying S. indica symbiosis, recuperation of As stress together with nutritional management of P by gathering case studies and presenting successful paradigms. Weaving together a volume of literature, we assess the chemical forms of As and related transport pathways, discuss As-P-rice interaction and the significance of fungi in As toxicity mitigation, predominantly the role of mycorrhiza, as well as survey of the multifaceted impacts of S. indica on plants. A potential strategy for simultaneous S. indica + P administration in paddy fields is proposed, followed by future research orientation to expand theoretic comprehension and encourage field-based implementation.

UV-modification of Ag nanoparticles on α-MoCx for interface polarization engineering in electromagnetic wave absorption.

The design of electromagnetic wave absorbing materials (EWAMs) has aroused great attention with the express development of electromagnetic devices, which pose a severe EM pollution risk to human health. Herein, an Ag-doped MoCx composite was designed and constructed through a UV-light-induced self-reduction process. The UV-reduction time was controlled on the α-MoC polymer for 0.5-2 hours for modifying different amounts of Ag. As a result, α-MoC@Ag-1.5 exhibited the strongest RLmin of -56.51 dB at 8.8 GHz under a thickness of 3.0 mm and the widest EAB of 4.96 GHz (12.16-17.12 GHz) covering a substantial portion of the Ku-band at a thickness of 2.0 mm due to the synergy of the conductivity loss and abundant interfacial polarization sites. Additionally, a new strategy for computer simulation technology was proposed to simulate substantial radar cross-sectional reduction values with real far-field conditions, whereby absorbing coatings with α-MoC@Ag-1.5 were proved to contribute to a remarkable radar cross-sectional reduction of 37.4 dB m2.

Review of the Genus Sycanus Amyot & Serville, 1843 (Heteroptera: Reduviidae: Harpactorinae), from China Based on DNA Barcoding and Morphological Evidence.

Due to the variability of body coloration and morphological similarity among closely related species, unresolved issues and debates still persist in the taxonomic study of the genus Sycanus from China. In this study, we conducted phylogenetic analyses and species delimitation for Sycanus in China based on a COI DNA barcoding dataset comprising 81 samples. The results revealed that all the samples could be classified into 12 species by integrating molecular analyses with morphological comparison. This paper provides a comprehensive systematic review of the Sycanus species found in China, including descriptions of three new species: S. taiwanensis Zhao & Cai sp. nov., S. flavicorius Li & Cai sp. nov., and S. hainanensis Wang & Cai sp. nov. Furthermore, it is proposed that S. croceovittatus Dohrn, 1859, S. leucomesus Walker, 1873, and S. villicus Stål, 1863, are three synonyms of S. bifidus (Fabricius, 1787); S. bicolor Hsiao, 1979, is a synonym of S. versicolor Dohrn, 1859; and S. hsiaoi Maldonado-Capriles, 1990, is a synonym of S. marginellus Putshkov, 1987. Additionally, brief biological information is provided for two species, S. falleni Stål, 1863, and S. croceus Hsiao, 1979.

Domestication Gene Mlx and Its Partner Mondo Are Involved in Controlling the Larval Body Size and Cocoon Shell Weight of Bombyx mori.

Bombyx mori was domesticated from Bombyx mandarina. The long-term domestication of the silkworm has brought about many remarkable changes to its body size and cocoon shell weight. However, the molecular mechanism underlying the improvement in the economic characteristics of this species during domestication remains unclear. In this study, we found that a transposable element (TE)-Bm1-was present in the upstream regulatory region of the Mlx (Max-like protein X) gene in wild silkworms but not in all domesticated silkworms. The absence of Bm1 caused an increase in the promoter activity and mRNA content of Mlx. Mlx and its partner Mondo belong to the bHLHZ transcription factors family and regulate nutrient metabolism. RNAi of Mlx and Mondo decreased the expression and promoter activity of glucose metabolism-related genes (trehalose transport (Tret), phosphofructokinase (PFK), and pyruvate kinase (PK)), lipogenic genes (Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)), and glutamine synthesis gene (Glutamine synthase 2, (GS2)). Furthermore, the transgenic overexpression of Mlx and Mondo in the fat body of silkworms increased the larval body size, cocoon shell weight, and egg number, but the silencing of the two genes resulted in the opposite phenotypes. Our results reveal the molecular mechanism of Mlx selection during domestication and its successful use in the molecular breeding of Bombyx mori.

Kisspeptin prevents pregnancy loss by modulating the immune microenvironment at the maternal-fetal interface in recurrent spontaneous abortion.

PROBLEM: Immune factors are crucial in the development of recurrent spontaneous abortion (RSA). This study aimed to investigate whether kisspeptin regulates immune cells at the maternal-fetal interface and whether G protein-coupled receptor 54 (GPR54) is involved in this process, through which it contributes to the pathogenesis of RSA. METHOD OF STUDY: Normal pregnancy (NP) (CBA/J × BALB/c) and RSA (CBA/J × DBA/2) mouse models were established. NP mice received tail vein injections of PBS and KP234 (blocker of kisspeptin receptor), whereas RSA mice received PBS and KP10 (active fragment of kisspeptin). The changes in immune cells in mouse spleen and uterus were assessed using flow cytometry and immunofluorescence. The expression of critical cytokines was examined by flow cytometry, ELISA, Western blotting, and qPCR. Immunofluorescence was employed to detect the coexpression of FOXP3 and GPR54. RESULTS: The findings revealed that the proportion of Treg cells, MDSCs, and M2 macrophages in RSA mice was lower than that in NP mice, but it increased following the tail vein injection of KP10. Conversely, the proportion of these cells was reduced in NP mice after the injection of KP234. However, the trend of γδT cell proportion change is contrary to these cells. Furthermore, FOXP3 and GPR54 were coexpressed in mouse spleen and uterus Treg cells as well as in the human decidua samples. CONCLUSION: Our results suggest that kisspeptin potentially participates in the pathogenesis of RSA by influencing immune cell subsets at the maternal-fetal interface, including Treg cells, MDSC cells, γδT cells, and M2 macrophages.

The Effect and Mechanism of Fructus lycii on Improvement of Exercise Fatigue Using a Network Pharmacological Approach with in vitro Experimental Verification.

Objective: This study aimed to investigate the effect and underlying mechanism of Fructus lycii in improving exercise fatigue. Methods: A network pharmacological approach was used to explore potential mechanisms of action of Fructus lycii. Skeletal muscle C2C12 cells and immunofluorescence were employed to verify the effect and mechanism of the representative components in Fructus lycii predicted by network pharmacological analysis. Results: Six potential active components, namely quercetin, ß-sitosterol, stigmasterol, 7-O-methylluteolin-6-C-beta-glucoside_qt, atropine, and glycitein, were identified to have potency in improving exercise fatigue via multiple pathways, such as the PI3K-Akt, neuroactive ligand-receptor interaction, IL-17, TNF, and MAPK signaling pathways. The immunofluorescence results indicated that quercetin, a significant active component in Fructus lycii, increased the mean staining area of 2-NBDG, TMRM, and MitoTracker, and decreased the area of CellRox compared to the control. Furthermore, the protein expression levels of p-38 MAPK, p-MAPK, p-JNK, p-PI3K, and p-AKT markedly increased after quercetin treatment. Conclusion: Fructus lycii might alleviate exercise fatigue through multiple components and pathways. Among these, quercetin appears to improve exercise fatigue by enhancing energy metabolism and reducing oxidative stress. The PI3K-AKT and MAPK signaling pathways also appear to play a role in this process.

Protective role of arachidonic acid against diabetic myocardial ischemic injury: a translational study of pigs, rats, and humans.

AIM: Patients with diabetes mellitus have poor prognosis after myocardial ischemic injury. However, the mechanism is unclear and there are no related therapies. We aimed to identify regulators of diabetic myocardial ischemic injury. METHODS AND RESULTS: Mass spectrometry-based, non-targeted metabolomic approach was used to profile coronary sinus blood from diabetic and non-diabetic Bama-mini pigs at 0.5-h post coronary artery ligation. Six metabolites had a |log2 (Fold Change)|> 1.3. Among them, the most changed is arachidonic acid (AA), levels of which were 32 times lower in diabetic pigs than in non-diabetic pigs. The AA-derived products, PGI2 and 6-keto-PGF1α, were also significantly reduced. AA treatment of cultured cardiomyocytes protected against cell death by 30% at 48 h of high glucose and oxygen deprivation, which coincided with increased mitophagic activity (as indicated by increased LC3II/LC3I, decreased p62 and increased parkin & PINK1), improved mitochondrial renewal (upregulation of Drp1 and FIS1), reduced ROS generation and increased ATP production. These cardioprotective effects were abolished by PINK1(a crucial mitophagy protein) knockdown or the autophagy inhibitor 3-Methyladenine. The protective effect of AA was also inhibited by indomethacin and Cay10441, a prostacyclin receptor antagonist. Furthermore, diabetic Sprague Dawley rats were subjected to coronary ligation for 40 min and AA treatment (10 mg/day per animal gavaged) decreased myocardial infarct size, cell apoptosis index, inflammatory cytokines and improved heart function. Scanning electron microscopy showed more intact mitochondria in the border zone of infarcted myocardium in AA treated rats. Lastly, diabetic patients after myocardial infarction had lower plasma levels of AA and 6-keto-PGF1α and reduced cardiac ejection fraction, compared with non-diabetic patients after myocardial infarction. Plasma AA level was inversely correlated with fasting blood glucose. CONCLUSIONS: AA protects against diabetic ischemic myocardial damage by promoting mitochondrial autophagy and renewal, which is related to AA derived PGI2 signaling. AA may represent a new strategy to treat diabetic myocardial ischemic injury.

Supplementation of Clostridium butyricum Alleviates Vascular Inflammation in Diabetic Mice.

Background: Gut microbiota is closely related to the occurrence and development of diabetes and affects the prognosis of diabetic complications, and the underlying mechanisms are only partially understood. We aimed to explore the possible link between the gut microbiota and vascular inflammation of diabetic mice. Methods: The db/db diabetic and wild-type (WT) mice were used in this study. We profiled gut microbiota and examined the and vascular function in both db/db group and WT group. Gut microbiota was analyzed by 16s rRNA sequencing. Vascular function was examined by ultrasonographic hemodynamics and histological staining. Clostridium butyricum (CB) was orally administered to diabetic mice by intragastric gavage every 2 days for 2 consecutive months. Reactive oxygen species (ROS) and expression of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were detected by fluorescence microscopy. The mRNA expression of inflammatory cytokines was tested by quantitative polymerase chain reaction. Results: Compared with WT mice, CB abundance was significantly decreased in the gut of db/db mice, together with compromised vascular function and activated inflammation in the arterial tissue. Meanwhile, ROS in the vascular tissue of db/db mice was also significantly increased. Oral administration of CB restored the protective microbiota, and protected the vascular function in the db/db mice via activating the Nrf2/HO-1 pathway. Conclusion: This study identified the potential link between decreased CB abundance in gut microbiota and vascular inflammation in diabetes. Therapeutic delivery of CB by gut transplantation alleviates the vascular lesions of diabetes mellitus by activating the Nrf2/HO-1 pathway.

Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes multi-organ damage, which includes hepatic dysfunction, as observed in over 50% of COVID-19 patients. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (ACE2) is the primary receptor for SARS-CoV-2 entry into host cells, and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2, but at extremely low levels. Consequently, we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells. To address this question, we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1 (ASGR1) promoted SARS-CoV-2 pseudovirus infection of HeLa cells. In Huh-7 cells, simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells, both of which barely expressed ACE2, SARS-CoV-2 pseudovirus could successfully establish an infection. However, after treatment with ASGR1 antibody or siRNA targeting ASGR1, the infection rate significantly dropped, suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism. We confirmed that ASGR1 could interact with Spike protein, which depends on receptor binding domain (RBD) and N-terminal domain (NTD). Finally, we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells. After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA, the infection efficiency of the live virus decreased significantly. Collectively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells.

Design, synthesis, and inhibitory activity of hydroquinone ester derivatives against mushroom tyrosinase.

Tyrosinase is a widely distributed copper-containing enzyme found in various organisms, playing a crucial role in the process of melanin production. Inhibiting its activity can reduce skin pigmentation. Hydroquinone is an efficient inhibitor of tyrosinase, but its safety has been a subject of debate. In this research, a scaffold hybridization strategy was employed to synthesize a series of hydroquinone-benzoyl ester analogs (3a-3g). The synthesized compounds were evaluated for their inhibitory activity against mushroom tyrosinase (mTyr). The results revealed that these hydroquinone-benzoyl ester analogs exhibited inhibitory activity against mTyr, with compounds 3a-3e displaying higher activity, with compound 3b demonstrating the highest potency (IC50 = 0.18 ± 0.06 µM). Kinetic studies demonstrated that the inhibition of mTyr by compounds 3a-3e was reversible, although their inhibition mechanisms varied. Compounds 3a and 3c exhibited non-competitive inhibition, while 3b displayed mixed inhibition, and 3d and 3e showed competitive inhibition. UV spectroscopy analysis indicated that none of these compounds chelated with copper ions in the active center of the enzyme. Molecular docking simulations and molecular dynamics studies revealed that compounds 3a-3e could access the active pocket of mTyr and interact with amino acid residues in the active site. These interactions influenced the conformational flexibility of the receptor protein, subsequently affecting substrate-enzyme binding and reducing enzyme catalytic activity, in line with experimental findings. Furthermore, in vitro melanoma cytotoxicity assay of compound 3b demonstrated its higher toxicity to A375 cells, while displaying low toxicity to HaCaT cells, with a dose-dependent effect. These results provide a theoretical foundation and practical basis for the development of novel tyrosinase inhibitors.