DNA methylation episignature, extension of the clinical features, and comparative epigenomic profiling of Hao-Fountain syndrome caused by variants in USP7.

PURPOSE: Hao-Fountain syndrome (HAFOUS) is a neurodevelopmental disorder caused by pathogenic variants in USP7. HAFOUS is characterized by developmental delay, intellectual disability, speech delay, behavioral abnormalities, autism spectrum disorder, seizures, hypogonadism, and mild dysmorphic features. We investigated the phenotype of 18 participants with HAFOUS and performed DNA methylation (DNAm) analysis, aiming to generate a diagnostic biomarker. Furthermore, we performed comparative analysis with known episignatures to gain more insight into the molecular pathophysiology of HAFOUS. METHODS: We assessed genomic DNAm profiles of 18 individuals with pathogenic variants and variants of uncertain significance (VUS) in USP7 to map and validate a specific episignature. The comparison between the USP7 cohort and 56 rare genetic disorders with earlier reported DNAm episignatures was performed with statistical and functional correlation. RESULTS: We mapped a sensitive and specific DNAm episignature for pathogenic variants in USP7 and utilized this to reclassify the VUS. Comparative epigenomic analysis showed evidence of HAFOUS similarity to a number of other rare genetic episignature disorders. CONCLUSION: We discovered a sensitive and specific DNAm episignature as a robust diagnostic biomarker for HAFOUS that enables VUS reclassification in USP7. We also expand the phenotypic spectrum of 9 new and 5 previously reported individuals with HAFOUS.

Hao-Fountain syndrome: 32 novel patients reveal new insights into the clinical spectrum.

Hao-Fountain syndrome (HAFOUS, OMIM: #616863) is a neurodevelopmental disorder caused by pathogenic variants in the gene USP7 coding for USP7, a protein involved in several crucial cellular homeostatic mechanisms and the recently described MUST complex. The phenotype of HAFOUS is insufficiently understood, yet there is a great need to better understand the spectrum of disease, genotype-phenotype correlations, and disease trajectories. We now present a larger cohort of 32 additional individuals and provide further clinical information about six previously reported individuals. A questionnaire-based study was performed to characterize the phenotype of Hao-Fountain syndrome more clearly, to highlight new traits, and to better distinguish the disease from related neurodevelopmental disorders. In addition to confirming previously described features, we report hyperphagia and increased body weight in a subset of individuals. HAFOUS patients present an increased rate of birth complications, congenital anomalies, and abnormal pain thresholds. Speech impairment emerges as a potential hallmark of Hao-Fountain syndrome. Cognitive testing reports reveal borderline intellectual functioning on average, although some individuals score in the range of intellectual disability. Finally, we created a syndrome-specific severity score. This score neither indicates a sex- nor age-specific difference of clinical severity, yet highlights a more severe outcome when amino acid changes colocalize to the catalytic domain of the USP7 protein.

Blue- and Red-Shifting C-H⋯O Hydrogen Bonds of Cyclic Ethers with Haloforms: Effect of Ring-Size and Consistency with Bent's Rule.

A DFT-based computational study is carried out to delve into the interplay between hyperconjugation and rehybridization effects underlying the formation of blue- or red-shifting H-bonds (HBs) in 1 : 1 complexes of cyclic ethers (HB acceptor) of varying ring-size with haloforms, CHF3 and CHCl3 (HB donor). The calculations reveal that with decreasing angular strain (increasing ring-size) of the cyclic ethers, the extent of blue-shift increases for 1 : 1 complexes with CHF3, while a reverse sequence is observed with CHCl3, eventually leading to a red-shifting HB in the oxepane : CHCl3 complex. It is noted that the trend in the shift of C-H stretching fundamental is not mirrored by the C-H bond length or interaction energies for both the systems studied, that is, the low sensitivity of the changes on the strain on the O-atom of HB acceptor (cyclic ethers) is to be emphasized.

Experimental study of endurance characteristics of Al-doped HfO2ferroelectric capacitor.

In this work, the endurance characteristics of Al-doped HfO2(HAO)-based metal-ferroelectric-metal (MFM) capacitors (which were annealed at 1000 °C) with various doping concentrations were investigated. The doping concentration was optimized for the high annealing temperature (1000 °C) process. To investigate the impact of cycling pulses on the endurance characteristics of HAO-based MFM capacitor, the rise/fall time (tr/f) and hold time (th) for the cycling pulses were varied. Moreover, by adopting the recoverable fatigue process, the endurance characteristics under repetitive wake-up/fatigue processes were studied. The HAO capacitors achieved the remnant polarization (2Pr) of 23.767µC cm-2at pristine state under the high annealing temperature. Furthermore, it was demonstrated that the endurance characteristics (∼108cycles) of the HAO capacitors were comparable to them of other HfO2-based ferroelectric capacitors. Lastly but not least, it turned out that the amount of oxygen and oxygen vacancies in the HAO thin film was dependent of doping concentrations for the film. The impact of oxygen and oxygen vacancies was quantitatively analyzed, in detail, with TEM, XPS and GIXRD analysis.

Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link.

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Protective effects of Lacticaseibacillus rhamnosus Hao9 on dextran sulphate sodium-induced ulcerative colitis in mice.

AIMS: Some probiotics used as food additives or food supplements had an anti-inflammatory effect. We tested the potential protective effects of probiotic Lacticaseibacillus rhamnosus Hao9 (Hao9) in mice with dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) and determined whether these effects were related to the modulation of gut microbiota and amelioration of inflammation. METHODS AND RESULTS: Ulcerative colitis mouse model was established by feeding mice with 2.5% (w/v) DSS in drinking water for 7 days. We analysed the disease activity index (DAI), colon length and histological changes in the colon. In addition, we investigated the effects of Hao9 (1 × 109 colony forming unit/day) and curcumin (CUR) (200 mg/kg/day) on gut microbiota and serum inflammatory cytokines. In this study, CUR was used as a positive control. The results showed that both Hao9 and CUR effectively reduced body mass loss and DAI, restored colon length, alleviated colonic pathological variations and reduced histological scores compared with the UC group. Hao9 reduced the serum concentrations of proinflammatory cytokines (tumour necrosis factor alpha, interleukin [IL]-6 and IL-1ß) and increased the concentration of the anti-inflammatory cytokine IL-10. In addition, Hao9 promoted the growth of Faecalibaculum and Romboutsia in the gut and helped to maintain intestinal homeostasis. CONCLUSIONS: Hao9 had a protective effect against DSS-induced colitis, and the mechanisms underlying Hao9 may involve controlling inflammation and maintaining host micro-ecological balance. This study provided experimental evidence for the application of Hao9 in the treatment of ulcerative colitis and suggested that Hao9 may be a promising candidate as a dietary supplement against colitis. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparison of probiotics and prebiotics in terms of therapeutic efficacy in UC helps us to understand their different patterns of regulation of intestinal microbiota.

Studies of Coumarin Derivatives for Constitutive Androstane Receptor (CAR) Activation.

Constitutive androstane receptor (CAR) activation has found to ameliorate diabetes in animal models. However, no CAR agonists are available clinically. Therefore, a safe and effective CAR activator would be an alternative option. In this study, sixty courmarin derivatives either synthesized or purified from Artemisia capillaris were screened for CAR activation activity. Chemical modifications were on position 5,6,7,8 with mono-, di-, tri-, or tetra-substitutions. Among all the compounds subjected for in vitro CAR activation screening, 6,7-diprenoxycoumarin was the most effective and was selected for further preclinical studies. Chemical modification on the 6 position and unsaturated chains were generally beneficial. Electron-withdrawn groups as well as long unsaturated chains were hazardous to the activity. Mechanism of action studies showed that CAR activation of 6,7-diprenoxycoumarin might be through the inhibition of EGFR signaling and upregulating PP2Ac methylation. To sum up, modification mimicking natural occurring coumarins shed light on CAR studies and the established screening system provides a rapid method for the discovery and development of CAR activators. In addition, one CAR activator, scoparone, did showed anti-diabetes effect in db/db mice without elevation of insulin levels.

HAO2 inhibits malignancy of clear cell renal cell carcinoma by promoting lipid catabolic process.

Hydroxy acid oxidase 2 (HAO2) has been reported to inhibit tumor progression through metabolic pathway. The current study was designed to evaluate the prognostic significance and probable mechanism of HAO2 in patients with clear cell renal cell carcinoma (ccRCC). The study screened The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) database for patients with ccRCC having complete clinical information and HAO2 expression. Low HAO2 was associated with shorter overall survival (OS) and shorter disease-free survival (DFS). Gene set enrichment analysis (GSEA) showed HAO2 was associated with neutral lipid catabolic process, metabolic process, lipid oxidation, epithelial-mesenchymal transition (EMT), and Kirsten rat sarcoma viral oncogene signaling (KRAS). Western blot analysis and immunohistochemistry analysis checked HAO2 expression in ccRCC cancer tissues, normal tissues, and renal cancer cell lines. HAO2 was downregulated in ccRCC cancer tissues and ccRCC cell lines when compared with their control group. Overexpression of HAO2 by plasmid promoted lipid catabolic process, eliminated lipid accumulation, inhibited KRAS expression, controlled the proliferation, migration, and invasion activity of ccRCC tumor cells. Our results indicated that HAO2 inhibits malignancy ccRCC by promoting lipid catabolic process, HAO2 could be an effective molecular marker and treatment for ccRCC.

Preclinical Pharmacokinetics of Scoparone, Geniposide and Rhein in an Herbal Medicine Using a Validated LC-MS/MS Method.

The herbal formula Yin-Chen-Hao-Tang has been reported to have anti-fibrosis properties. The aim of this study was to reveal the pharmacokinetic characteristics of bioactive compounds in this herbal formula. A new high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of scoparone, geniposide and rhein in rat plasma. A pharmaceutical herbal powder was administered to rats at doses of 1 g/kg and 3 g/kg orally. The method showed excellent linearity (r² > 0.999) and validation was successfully conducted for the pharmacokinetic study. The results show that the Cmax values and areas under the curve of scoparone, geniposide and rhein were higher and not proportional to the dose in rat plasma, while the Tmax and half-life values were consistent in the group that received 1 g/kg. The clearance of the higher dose (3 g/kg) did not decrease proportionally to that of the low dose. The results showed the nonlinear pharmacokinetic properties of scoparone, geniposide and rhein in Yin-Chen-Hao-Tang that suggested possible accumulation of bioactive compounds through oral administration. This pharmacokinetic study reveals that an increased dose of this herbal formula would largely increase the maximum concentration and bioavailability of scoparone, geniposide and rhein.

Identification and screening of chemical constituents with hepatoprotective effects from three traditional Chinese medicines for treating jaundice.

The constituents with hepatoprotective activity were investigated in three traditional Chinese medicine formulae for treating jaundice, namely, Zhi-Zi-Da-Huang-Tang, Yin-Chen-Hao-Tang, and Da-Huang-Xiao-Shi-Tang. By using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry, 79 chemical constituents were identified unambiguously or tentatively in three formulae based on the accurate molecular weight, mass spectrometric fragmentation behavior, and comparison with reference standards. Then the hepatoprotective activities of 27 constituents were evaluated on tert-butylhydroperoxide-injured BRL-3A cells. The results indicated that 11 constituents, including protocatechic acid (19), epijasminoside A (56), rutin (71), tetrahydropalmatine (76), rhaponticin (80), 3,4-dicaffeoylquinic acid (82), 3,5-dicaffeoylquinic acid (85), diosmetin-7-O-glucoside (90), jatrorrhizine (93), berberine (100), and daidzein (101) exerted hepatoprotective activities. Interestingly, most of the crude drugs in three formulae contained hepatoprotective active constituents, and the combinations of constituents from different crude drugs exhibited greater effects. This result provided evidence to the traditional Chinese medicine theory of combining several drugs together to exert synergistic efficacy.

Using Light Microscopy and Liquid Chromatography Tandem Mass Spectrometry for Qualitative and Quantitative Control of a Combined Three-Herb Formulation in Different Preparations.

Artemisia capillaries Thunb, Gardenia jasminoides Ellis, and Rheum officinale Baill have been combined to treat jaundice for thousands of years. Studies have revealed that these herbs induce anti-hepatic fibrosis and anti-hepatic apoptosis and alleviate hepatic oxidative stress. This study aims to determine the quality and quantity of an herbal formulation (Chinese name: Yin-Chen-Hao-Tang) using physical and chemical examinations. Physical examination of Yin-Chen-Hao-Tang in pharmaceutical herbal products, raw fiber powders, and decoction preparations was performed using Congo red and iodine-potassium staining. A sensitive and validated method employing ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed to simultaneously quantify the bioactive compounds scoparone, geniposide, and rhein in the Yin-Chen-Hao-Tang formulation in different preparations. Physical examination indicated that cellulose fibers with irregular round shapes were present in the pharmaceutical herbal products. The developed UHPLC-MS/MS method showed good linearity and was well validated. The quantification results revealed that the decoction preparations had the highest amounts of geniposide and rhein. Scoparone appeared in pharmaceutical herbal products from two manufacturers. This experiment provides a qualitative and quantitative method using physical and chemical examinations to test different preparations of herbal products. The results provide a reference for clinical herbal product preparations and further pharmacokinetic research.

Serum Metabolomic Characterization of Liver Fibrosis in Rats and Anti-Fibrotic Effects of Yin-Chen-Hao-Tang.

Yin-Chen-Hao-Tang (YCHT) is a famous Chinese medicine formula which has long been used in clinical practice for treating various liver diseases, such as liver fibrosis. However, to date, the mechanism for its anti-fibrotic effects remains unclear. In this paper, an ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS)-based metabolomic study was performed to characterize dimethylnitrosamine (DMN)-induced liver fibrosis in rats and evaluate the therapeutic effects of YCHT. Partial least squares-discriminant analysis (PLS-DA) showed that the model group was well separated from the control group, whereas the YCHT-treated group exhibited a tendency to restore to the controls. Seven significantly changed fibrosis-related metabolites, including unsaturated fatty acids and lysophosphatidylcholines (Lyso-PCs), were identified. Moreover, statistical analysis demonstrated that YCHT treatment could reverse the levels of most metabolites close to the normal levels. These results, along with histological and biochemical examinations, indicate that YCHT has anti-fibrotic effects, which may be due to the suppression of oxidative stress and resulting lipid peroxidation involved in hepatic fibrogenesis. This study offers new opportunities to improve our understanding of liver fibrosis and the anti-fibrotic mechanisms of YCHT.

Flower morphology and floral sequence in Artemisia annua (Asteraceae)1.

UNLABELLED: • PREMISE OF THE STUDY: Artemisia annua produces phytochemicals possessing antimalarial, antitumor, anti-inflammatory, and anthelmintic activities. The main active ingredient, artemisinin, is extremely effective against malaria. Breeding to develop cultivars producing high levels of artemisinin can help meet worldwide demand for artemisinin and its derivatives. However, fundamental reproductive processes, such as the sequence of flowering and fertility, are not well understood and impair breeding and seed propagation programs.• METHODS: Capitulum structure and floral sequence were studied using light and scanning electron microscopy to describe inflorescence architecture, floret opening, and seed set.• KEY RESULTS: Florets are minute and born in capitula containing pistillate ray florets and hermaphroditic disk florets. Ray florets have elongated stigmatic arms that extend prior to disk floret opening. Disk florets exhibit protandry. During the staminate phase, pollen is released within a staminate tube and actively presented with projections at the tip of stigmas as the pistil elongates. During the pistillate phase, stigmatic arms bifurcate and reflex. Stigmas are of the dry type and stain positively for polysaccharides, lipids, and an intact cuticle. Floret numbers vary with genotype, and capitula are predominantly composed of disk florets. Both ray and disk florets produce filled seed.• CONCLUSIONS: Gynomonoecy, early opening of ray florets, and dichogamy of disk florets promote outcrossing in A. annua For breeding and seed development, flowering in genotypes can be synchronized under short days according to the floral developmental stages defined. Floret number and percentage seed fill vary with genotype and may be a beneficial selection criterion.

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors.

Glycolate oxidase (HAO1) catalyses the synthesis of glyoxylate, a common metabolic intermediate that causes renal failure if accumulated. HAO1 inhibition is an emerging treatment for primary hyperoxaluria, a rare disorder of glyoxylate metabolism. Here we report the first cell-based measurement of inhibitor uptake and engagement with HAO1, by adapting the cellular thermal shift assay (CETSA) based on Nano luciferase complementation and luminescence readout. By profiling the interaction between HAO1 and four well-characterised inhibitors in intact and lysed HEK293T cells, we showed that our CETSA method differentiates between low-permeability/high-engagement and high-permeability/low-engagement ligands and is able to rank HAO1 inhibitors in line with both recombinant protein methods and previously reported indirect cellular assays. Our methodology addresses the unmet need for a robust, sensitive, and scalable cellular assay to guide HAO1 inhibitor development and, in broader terms, can be rapidly adapted for other targets to simultaneously monitor compound affinity and cellular permeability.

Identification of Two Variants c.2697A > C and c.3305A > C in USP7 by Analysis of Whole-Exome Sequencing in Chinese Patients with Hao-Fountain Syndrome.

Background Variants of ubiquitin-specific protease 7 ( USP7 ) gene in humans are associated with a neurodevelopmental disorder-Hao-Fountain syndrome, its core symptoms including developmental delay, intellectual disability, and speech delay. Other variable symptoms can affect multiple systems. In present study, we report two patients with core features from two unrelated consanguineous families originating from the Tianjin Children's Hospital. Methods and Results Genomic DNA was extracted from the peripheral blood samples collected from the probands with their family members and whole-exome sequencing (WES) was used to detect the pathogenic genes in the probands. Suspected variants were subsequently validated by Sanger sequencing. In family 1, WES revealed that the proband carried the de novo variant c.2697A > C (p.Leu899Phe) in USP7 (NM_003470.3). In family 2, WES identified the variant c.3305A > C (p.Asn1102Thr) in USP7 (NM_003470.3) from the proband. Conclusion We reported two cases of Hao-Fountain syndrome caused by novel USP7 variants. In addition, we report the first case of mosaicism with a USP7 variant in Chinese family. Our findings demonstrate the importance of WES in diagnosis of genetic diseases and expands the USP7 variants spectrum in Hao-Fountain syndrome. Moreover, we summarize the cases caused by USP7 variants in the literature. Our study can provide a vital reference for the diagnosis of future cases.

Comparison of Ba-Hao burn ointment gauze and petrolatum gauze in split graft donor site healing: A randomized, prospective, and self-control study.

Background and Aims: To assess patient comfort, wound healing, and scarring at the 6-month follow-up of split-skin graft donor sites treated with Ba-Hao burn ointment (BHBO) gauze, a compound preparation of traditional Chinese medicine since 1970s, compared with petrolatum gauze. Methods: Thirty patients admitted to the Department of Burns of the First Affiliated Hospital of Anhui Medical University between September 2021 and September 2022 participated in this randomized, prospective, self-control clinical study. After harvesting the split skin, donor sites were divided into two parts along the midline. BHBO gauze was applied to half of the donor wounds, and petrolatum gauze was applied to the other half. The wound healing time, pain scores on the postoperative Days 3, 6, and 9, and Vancouver Scar Scale (VSS) score at the 6-month follow-up were assessed. Results: The wound healing time was significantly shorter in the BHBO group than in the control group (10.07 ± 1.48 days vs. 11.50 ± 1.74 days, p < 0.001). On postoperative Days 3 and 6, the pain scores quantified by visual analog scores were significantly lower in the BHBO group than in the control group (5.33 ± 1.54 and 4.17 ± 1.51, respectively vs. 7.57 ± 1.41 and 5.20 ± 1.47, respectively). The difference in the visual analog scale score on postoperative Day 9 between the groups was not significant (p > 0.05). Microbiological assessment revealed the absence of bacterial contamination in both groups. At the 6-month follow up, the VSS score was significantly lower in the BHBO group (6.67 ± 1.92) than in the control group (9.57 ± 1.55). Conclusion: BHBO resulted in faster donor-site healing, reduced postoperative pain, and improved scar quality at the 6-month follow-up than petrolatum gauze alone.

Jiang Tang San Hao Formula exerts its anti-diabetic effect by affecting the gut-microbiota-brain axis.

BACKGROUND: Type 2 diabetes is a complex metabolic disorder characterized by insulin resistance and impaired insulin secretion, with growing evidence highlighting the critical role of the gut-microbiota-brain axis in modulating glucose and lipid metabolism. OBJECTIVE: To evaluate the effects of Jiang Tang San Hao Formula (JTSHF) on blood glucose control in type 2 diabetic mouse model and to explore its mechanism through the gut- microbiota-brain axis. METHODS: A type 2 diabetes model was established using six-week-old male C57BL6/J mice, induced by a high-fat diet combined with streptozotocin injection. The diabetic mice then randomly assigned to the model group, metformin (Glucophage) group and JTSHF group, receiving 11 weeks of treatment by gavage. Body weight and fasting blood glucose were monitored biweekly. The oral glucose tolerance test was performed during the fifth and 10th weeks of the intervention. The measurements of body composition were conducted pre- and post-treatment. After the intervention, serum insulin, lipid levels, glucagon like peptide-1 (GLP-1), peptide YY, ghrelin, and leptin were detected. The fresh feces of mice were collected before sacrifice for gut microbiota analysis and short chain fatty acids quantification. The colon tissues of mice in each group were collected to observe the morphological structure and to measure the expression levels of GPR41 and GPR43. The hypothalamus was collected to assess the expression of POMC, AgRP and NPY. RESULTS: JTSHF significantly boosted sugar and lipid metabolism and contributed to weight reduction in diabetic mice (p < 0.05). At the genus level, JTSHF increased the relative abundance of Bacteroides, Prevotella, and Parabacteroides, and decreased Clostridium, Lactobacillus, and Oscillibacter in the gut microbiota. JTSHF enhanced the content of short chain fatty acids, improved the expression level of GPR43/41 in colonic tissue (p < 0.05), and increased POMC expression while decreasing AgRP and NPY expression in the hypothalamus (p < 0.05). Serum GLP-1 was increased, and ghrelin was decreased significantly after JTSHF intervention (p < 0.05). CONCLUSION: By affecting the composition, relative abundance, and metabolites of gut microbiota, JTSHF regulates various gut brain peptides, affects the hypothalamic feeding center, improves glucose and lipid metabolism, and thus plays the anti-diabetic role. The study provides novel insights into how traditional Chinese medicine modulates the gut-brain connection to exert anti-diabetic effects, highlighting the innovative potential of JTSHF in metabolic disease management.

HAO2 protects from proximal tubular cells injured in rats with chronic kidney disease by promoting fatty acid metabolic processes.

The complex pathogenesis of kidney disease is closely related to the diversity of kidney intrinsic cells. In this study, single-cell transcriptome sequencing technology was used to sequence and analyze blood and kidney tissue cells in normal control rats and rats with chronic kidney disease (CKD), focusing on key cell populations and functional enrichment to explore the pathogenesis of CKD. Oil red O staining and enzyme-linked immunosorbent assay (ELISA) were used to detect lipid droplets and free fatty acid (FFA). Quantitative real-time polymerase chain reaction (RT-PCR), western blot (WB) were used to verify the differential gene hydroxyacid oxidase 2 (HAO2) and fatty acid metabolic process in tissue to ensure the reliability of single-cell sequencing results. We successfully established a single-cell transcriptome atlas of blood and kidney tissue in rats with CKD, which were annotated into 14 cell subsets (MPCs, PT, Tc, DCT, B-IC, A-IC, CNT, ALOH, BC, Neu, Endo, Pla, NKT, Baso) according to marker gene, and the integrated single-cell atlas of rats showed a significant increase and decrease of MPCs and PTs in the CKD group, respectively. Functional analysis found extensive enrichment of metabolic-related pathways in PT cells, includes fatty acid metabolic process, cellular amino acid metabolic process and generation of precursor metabolites and energy. Immunohistochemical experiments determined that the differential gene HAO2 was localized in the renal tubules, and its expression was significantly reduced in CKD group compared with control, and oil red O staining showed that lipid droplets increased in the CKD group, after overexpression of HAO2 the lipid droplets was inhibited. ELISA assay showed that ATP content decreased in the CKD group and FFA increased in the CKD group. Moreover, the mitochondrial membrane potential of the cells in the OE-HAO2 group was significantly increased compared with OE-NC. The acyl-CoA oxidase 1(ACOX1), peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were decreased in the CKD group, while genes and proteins were increased after overexpression of HAO2, and the AMP-activated protein kinase (AMPK) phosphorylated proteins were increased, the acetyl-CoA carboxylase (ACC) phosphorylated proteins were decreased, reversely. Therefore, HAO2 may be an important regulator of fatty acid metabolic processes in CKD, and overexpression of HAO2 can enhance fatty acid metabolism by promoting fatty acid oxidation (FAO) pathway.

Respiratory infection risk in primary Sjögren's syndrome complicated with interstitial lung disease: a retrospective study.

OBJECTIVES: To explore clinical and laboratory characteristics of primary Sjögren's syndrome (pSS) complicated with interstitial lung disease (ILD) and investigate the risk factors for respiratory infections in pSS-ILD. METHODS: A cohort of 162 pSS-ILD patients in Peking University People's Hospital from 2015 to 2020 were included, and all medical records were completely collected. We screened 53 patients suffering from respiratory infections as study cases, compared with 109 age- and sex-matched controls. Differences between infection group and control group were compared. Univariate and multivariate binary logistic regression tests were conducted to identify potential risk factors for respiratory infections in pSS-ILD patients. RESULTS: Among 162 pSS-ILD patients, 32.72% (53/162) suffered from respiratory infections. The most frequent type of ILD was nonspecific interstitial pneumonia (32.08%, 51/159), and the most common type of pathogen was bacteria (64.25%, 34/53). Infection group showed higher levels of ESSDAI (P < 0.001), CRP (P < 0.001), ESR (P = 0.003), and C3 (P = 0.020) but lower level of DLCO-SB (P = 0.015). Univariate logistic model revealed that PAH and the use of glucocorticoid increased infection risk in pSS-ILD patients. On multivariate logistic regression analysis, PAH (OR = 3.993, 95% CI = 1.192-13.373, P = 0.025) and severe reduction of DLCO (DLCO-SB < 40%, OR = 4.625, 95% CI = 1.281-16.702, P = 0.019) were significantly associated with increased risk of respiratory infections in pSS-ILD patients. CONCLUSION: Among pSS-ILD patients, the most frequent type of ILD was nonspecific interstitial pneumonia. In patients with infection, bacteria were the most common pathogen. Higher levels of ESSDAI, CRP, ESR, and C3 may be correlated with increased infection risk. PAH and reduction of DLCO were identified as independent risk factors. Key Points • ILD and infectious diseases severely affect pSS patient conditions. • Higher levels of ESSDAI, CRP, ESR, and C3 may be correlated with increased infection risks in pSS-ILD. • PAH and reduction of DLCO were identified as independent risk factors for lower respiratory infection.

Shen-yan-yi-hao oral solution ameliorates IgA nephropathy via intestinal IL-17/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world, it is one of the most common causes of kidney disease and can lead to end-stage kidney disease, however, its pathogenesis is still complicated. The Shen-yan-yi-hao oral solution (SOLI) is an effective prescription for the clinical treatment of IgAN while its specific mechanism remains to be further elucidated. AIM OF THE STUDY: This study investigates SOLI's effects on IgAN in rats, particularly on the intestinal mucosal barrier, and identifies potential therapeutic targets through network pharmacology and molecular docking, validated experimentally. MATERIALS AND METHODS: Target genes for SOLI in IgAN were identified and analysed through molecular docking and KEGG pathway enrichment. An IgAN rat model examined SOLI's effect on renal biomarkers and cytokines involved in specific pathways, ileum mucosal lesions, and the intestinal immune system. The IL-17 pathway's role was studied in IEC-6 cells with SOLI in vitro. RESULT: Rats developed increased proteinuria and kidney damage marked by IgA deposition and inflammation. SOLI treatment significantly ameliorated these symptoms, reduced galactose-deficient Ig A1 (Gd-IgA1), and decreased cytokines like IL-17, TNF-α, IL-6 and IL-1ß etc. SOLI also normalized intestinal tight junction protein expression, ameliorated intestinal damage, and regulated intestinal immune response (focused on IL-17/NF-κB signal pathway). SOLI moderated the abnormally activated IL-17 pathway, which damages intestinal epithelial cells, suggesting IgAN treatment potential. CONCLUSION: SOLI reduces proteinuria and enhances intestinal mucosal function in IgAN rats, kidney protection in the IgAN rat model may initiate from modulating the intestinal IL-17/NF-κB pathway and subsequent Gd-IgA1 accumulation.