Spatial enrichment and genomic analyses reveal the link of NOMO1 with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease with uncertain genetic predisposition in most sporadic cases. The spatial architecture of cell types and gene expression are the basis of cell-cell interactions, biological function and disease pathology, but are not well investigated in the human motor cortex, a key ALS-relevant brain region. Recent studies indicated single nucleus transcriptomic features of motor neuron vulnerability in ALS motor cortex. However, the brain regional vulnerability of ALS-associated genes and the genetic link between region-specific genes and ALS risk remain largely unclear. Here, we developed an entropy-weighted differential gene expression matrix-based tool (SpatialE) to identify the spatial enrichment of gene sets in spatial transcriptomics. We benchmarked SpatialE against another enrichment tool (multimodal intersection analysis) using spatial transcriptomics data from both human and mouse brain tissues. To investigate regional vulnerability, we analysed three human motor cortex and two dorsolateral prefrontal cortex tissues for spatial enrichment of ALS-associated genes. We also used Cell2location to estimate the abundance of cell types in ALS-related cortex layers. To dissect the link of regionally expressed genes and ALS risk, we performed burden analyses of rare loss-of-function variants detected by whole-genome sequencing in ALS patients and controls, then analysed differential gene expression in the TargetALS RNA-sequencing dataset. SpatialE showed more accurate and specific spatial enrichment of regional cell type markers than multimodal intersection analysis in both mouse brain and human dorsolateral prefrontal cortex. Spatial transcriptomic analyses of human motor cortex showed heterogeneous cell types and spatial gene expression profiles. We found that 260 manually curated ALS-associated genes are significantly enriched in layer 5 of the motor cortex, with abundant expression of upper motor neurons and layer 5 excitatory neurons. Burden analyses of rare loss-of-function variants in Layer 5-associated genes nominated NOMO1 as a novel ALS-associated gene in a combined sample set of 6814 ALS patients and 3324 controls (P = 0.029). Gene expression analyses in CNS tissues revealed downregulation of NOMO1 in ALS, which is consistent with a loss-of-function disease mechanism. In conclusion, our integrated spatial transcriptomics and genomic analyses identified regional brain vulnerability in ALS and the association of a layer 5 gene (NOMO1) with ALS risk.

[Effect of Yunkang Oral Solution on pregnant mice with spleen deficiency syndrome].

This study aimed to investigate the therapeutic effect of Yunkang Oral Solution on the improvement of spleen deficiency and pregnancy outcomes in pregnant mice with spleen deficiency syndrome induced by irregular diet and over consumption of cold and bitter foods. To simulate human irregular diet and over consumption of cold and bitter foods leading to spleen deficiency, the pregnant mice with spleen deficiency syndrome were prepared using an alternate-day fasting and high-fat diet combined with oral administration of Sennae Folium. During the experiment, spleen deficiency-related indicators and diarrhea-related parameters were measured. Gastric and intestinal motility(gastric emptying rate and intestinal propulsion rate) were evaluated. The levels of serum ghrelin, growth hormone(GH), gastrin(Gas), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-c), chorionic gonadotropin ß(ß-CG), progesterone(P), and estradiol(E_2) were measured. Intestinal barrier function in pregnant mice with spleen deficiency syndrome was assessed. Conception rate, ovarian coefficient, litter-bearing uterine coefficient, number of live fetuses, average fetal weight, and fetal length were calculated. The results showed that Yunkang Oral Solution significantly improved spleen deficiency-related indicators and diarrhea in pregnant mice with spleen deficiency syndrome, increased gastric emptying rate and intestinal propulsion rate, elevated the levels of gastrointestinal hormones(ghrelin, GH, and Gas) in the serum, and reduced lipid levels(TC and LDL-c), thereby improving lipid metabolism disorders. It also improved colonic tissue morphology, increased the number of goblet cells, and promoted the mRNA and protein expression of occludin and claudin-1 in colonic tissues, thereby alleviating intestinal barrier damage. Yunkang Oral Solution also regulated the levels of pregnancy hormones(ß-CG, P, and E_2) in the serum of pregnant mice with spleen deficiency syndrome. Moreover, it increased the conception rate, ovarian coefficient, litter-bearing uterine coefficient, number of live fetuses, average fetal weight, and fetal length. These findings suggest that Yunkang Oral Solution can improve spleen deficiency-related symptoms in pregnant mice before and during pregnancy, regulate pregnancy-related hormones, and improve pregnancy outcomes.

Predicting recurrent cases of intussusception in children after air enema reduction with machine learning models.

PURPOSE: To develop a model to identify risk factors and predict recurrent cases of intussusception in children. METHODS: Consecutive cases and recurrent cases of intussusception in children from January 2016 to April 2022 were screened. The cohort was divided randomly at a 4:1 ratio to a training dataset and a validation dataset. Three parallel models were developed using extreme gradient boosting (XGBoost), logistic regression (LR), and support vector machine (SVM). Model performance was assessed by the area under the receiver operating characteristic curves (AUC). RESULTS: A total of 2469 cases of intussusception were included, where 225 were recurrent cases. The XGBoost (AUC = 0.718) models showed the best performance in the validation dataset, followed by the LR model (AUC = 0.652), while the SVM model (AUC = 0.613) performed worst among the three models. Based on the Shapley Additive exPlanation values, the most important variables in the XGBoost models were air enema pressure, mass size, age, duration of symptoms, and absence of vomiting. CONCLUSIONS: Machine learning models, especially XGBoost, could be used to predict recurrent cases of intussusception in children. The most important contributing factors to the models are air enema pressure, mass size, age, duration of symptoms and absence of vomiting.

Genome sequencing and heterologous expression of antiporters reveal alkaline response mechanisms of Halomonas alkalicola.

Halomonas alkalicola CICC 11012s is an alkaliphilic and halotolerant bacterium isolated from a soap-making tank (pH > 10) from a household-product plant. This strain can propagate at pH 12.5, which is fatal to most bacteria. Genomic analysis revealed that the genome size was 3,511,738 bp and contained 3295 protein-coding genes, including a complete cell wall and plasma membrane lipid biosynthesis pathway. Furthermore, four putative Na+/H+ and K+/H+ antiporter genes, or gene clusters, designated as HaNhaD, HaNhaP, HaMrp and HaPha, were identified within the genome. Heterologous expression of these genes in antiporter-deficient Escherichia coli indicated that HaNhaD, an Na+/H+ antiporter, played a dominant role in Na+ tolerance and pH homeostasis in acidic, neutral and alkaline environments. In addition, HaMrp exhibited Na+ tolerance; however, it functioned mainly in alkaline conditions. Both HaNhaP and HaPha were identified as K+/H+ antiporters that played an important role in high alkalinity and salinity. In summary, genome analysis and heterologous expression experiments demonstrated that a complete set of adaptive strategies have been developed by the double extremophilic strain CICC 11012s in response to alkalinity and salinity. Specifically, four antiporters exhibiting different physiological roles for different situations worked together to support the strain in harsh surroundings.

Crk-like adapter protein is required for TGF-ß-induced AKT and ERK-signaling pathway in epithelial ovarian carcinomas.

Crk-like adapter protein (CrkL) was identified as an important biomarker in epithelial ovarian carcinomas. At the same time, the transforming growth factor ß (TGF-ß) pathway plays a key role in oncogenesis of advanced cancers. However, more detailed regulation mechanisms are still unclear. So we investigated the role of CrkL in TGF-ß pathways in epithelial ovarian carcinomas. The small interfering RNA (siRNA) was used to suppress CrkL in serous papillary cystic adenocarcinoma (SKOV-3) cell line, TGF-ß downstream signal molecules AKT and ERK phosphorylation status was tested using the Western blot. Wound healing assay was used to evaluate the capacity of cell migration and proliferation. In this study, CrkL can be activated by TGF-ß1 treatment and inhibited by siCrkL. CrkL knockdown markedly suppressed the phosphorylated ERK (p-ERK) as well as the phosphorylated AKT (p-AKT) (p < 0.001) compared with control or TGF-ß1 alone. On the other hand, CrkL knockdown could significantly affect SKOV3 wound closure (p < 0.001) using wound healing assay compared to siControl. In conclusion, CrkL protein is required for TGF-ß signal pathways through AKT and ERK pathway, which can mediate the development of epithelial ovarian carcinomas. CrkL plays a key regulation role in TGF-ß signaling pathway of epithelial ovarian carcinomas, and this study suggested CrkL could be suggested as an efficient target in ovarian cancer treatment.

The essential roles of CCR7 in epithelial-to-mesenchymal transition induced by hypoxia in epithelial ovarian carcinomas.

The chemokine receptor CCR7 and its ligands CCL19/21 mediate the tumor mobility, invasion, and metastasis (Wu et al. Curr Pharm Des. 15:742-57, 2009). Hypoxia induced epithelial-to-mesenchymal transition (EMT) to facilitate the tumor biology. Here, we addressed the roles of CCR7 in epithelial ovarian carcinoma tissues and hypoxia-induced serous papillary cystic adenocarcinoma (SKOV-3) EMT. The expression level of CCR7 protein was analyzed by immunohistochemistry in 30 specimens of epithelial ovarian carcinomas. Western blot was used to investigate the expression of hypoxia-induced CCR7, HIF-1α, and EMT markers (N-cadherin, Snail, MMP-9). In addition, wound healing and Transwell assay were introduced to observe the capacity of migration and invasiveness. Our data showed CCR7 expression was observed in 22 cases of tissues and closely associated with lymph node metastasis and FIGO stage (III + IV). At 6, 12, 24, and 36 h following hypoxia, CCR7 and HIF-1α proteins were both obviously upregulated in a time-dependent method, compared with normal oxygen. In vitro, SKOV-3 expressed N-cadherin, Snail, and MMP-9 once either CCL21 stimulation or hypoxia induction, while hypoxia accompanied with CCL21 induction exhibited strongest upregulation of N-cadherin, Snail, and MMP-9 proteins. Besides, wound healing and Transwell assay further identified that hypoxia with CCL21 stimulation can remarkably promote cell migration and invasiveness. Taken together, CCR7 can constitutively express in epithelial ovarian carcinomas and be induced rapidly in response to hypoxia, which indeed participates in EMT development and prompts the cell migration and invasion. Thus, this study suggested that the epithelial ovarian cancer invasion and metastasis can be inhibited by antagonizing CCR7.

Machine learning for screening and predicting the availability of medications for children: a cross-sectional survey study.

Objective: The aim of the study was to explore the factors influencing the availability of medications for children, and establish a machine learning model to provide an empirical basis for the subsequent formulation and improvement of relevant policies. Methods: Design: Cross-sectional survey. Setting: 12 provinces, China. Medical doctors from 25 public hospitals were enrolled. All data were randomly divided into a training set and a validation set at a ratio of 7:3. Three prediction models, namely random forest (RF), logistic regression (LR), and extreme gradient boosting (XGBoost), were developed and compared. The receiver operating characteristic curve (ROC) and the associated area under the curve (AUC) were used to evaluate the three models. A nomogram and clinical impact curve (CIC) for availability of medication were developed. Results: Fifteen of 29 factors in the database that were most likely to be selected were considered to establish the prediction model. The XGBoost model (AUC = 0.915) demonstrated better performance than the RF model (AUC = 0.902) and the LR model (AUC = 0.890). According to the Shapley additive explanation values, the five factors that most significantly affected the availability of medications for children in the XGboost model were as follows: the relatively small number of specialized dosage forms for children; unaffordable medications for children; public education on the accessibility and safety of medication for children; uneven distribution of medical resources, leading to insufficient access to medication for children; and years of service as a doctor. The CIC was used to assess the practical applicability of the factor prediction nomogram. Conclusions: The XGBoost model can be used to establish a prediction model to screen the factors associated with the availability of medications for children. The most important contributing factors to the models were the following: the relatively small number of specialized dosage forms for children; unaffordable medications for children; public education on the accessibility and safety of medication for children; uneven distribution of medical resources, leading to insufficient access to medication for children; and years of service as a doctor.

Comparison of extraction processes, characterization and intestinal protection activity of Bletilla striata polysaccharides.

We optimized the extraction process of Bletilla striata polysaccharides using orthogonal design, Box-Behnken design (BBD), and genetic algorithm-back propagation (GA-BP), then compared and evaluated them to confirm that the combination of BBD and GA-BP neural networks was capable of increasing polysaccharide yields and antioxidant activity. The optimal extraction parameters were as follows: liquid-to-solid ratio of 15 mL/g, extraction power of 450 W, and extraction time of 34 min. Under these conditions, the polysaccharide yield and antioxidant activity were 8.29 ± 0.50 % and 26.20 ± 0.28 (mM FE/mg). Subsequently, the polysaccharide was purified to obtain purified Bletilla striata polysaccharides 1 (pBSP1) with a Mw of 255.172 kDa. Scanning electron microscope (SEM), ultraviolet-visible detector (UV), fourier transform infrared spectrometer (FTIR), high performance liquid chromatography (HPLC), X-ray diffraction (XRD), nuclear magnetic resonance (NMR) and periodate oxidation were used to analyze the structure of pBSP1. The results showed pBSP1 had a smooth surface and a rough interior, with a composition of α-D conformation glucose (18.23 %) and ß-D conformation mannose (53.77 %), and an amorphous crystal structure. According to the results of thermogravimetric and rheological tests, pBSP1 exhibits good thermal stability and viscoelastic behavior. Furthermore, pBSP1 protected lipopolysaccharide (LPS)-induced GES - 1 and Caco2 cells, the results showed pBSP1(400 µg/mL) lowered TEER synthesis in Caco2 cells as well as apoptosis and reactive oxygen species (ROS) production in both cells, indicating that pBSP1 may have an intestine protective effect.

Atractylodes macrocephala Koidz polysaccharide improves glycolipid metabolism disorders through activation of aryl hydrocarbon receptor by gut flora-produced tryptophan metabolites.

Polysaccharides are known to confer protection against glycolipid metabolism disorders (GMD) by regulating intestinal flora. In this study, a heterogeneous acidic heteropolysaccharide with high molecular weight mainly composed of fructose was isolated from Atractylodes macrocephala Koidz (AMP). Supplementation with AMP was shown to improve diet-induced GMD in a rat model, including decreasing the levels of serum triglycerides, total cholesterol, and glucose, and improving hepatic lipidosis and islet cells morphologies. AMP-treated rats also exhibited modified intestinal flora with enrichments of intestinal Lactobacillus and Rothia species, which was accompanied by increased tryptophan metabolites such as indole-3-propionic acid, indole, tryptamine, and tryptophol. These metabolites promote the expression of intestinal aryl hydrocarbon receptor (AhR) in nuclear fractions. AhR activation increased the expression levels of IL-22 and GLP-1 proteins and mRNA. IL-22 reduced systemic LPS by upregulating the expression of tight junction proteins, antimicrobial peptides, and mucin to ameliorate intestinal barrier function, and activated the hepatic IL-22R/Stat3/Acox1 signaling pathway to improve lipid metabolism. GLP-1 activated the pancreatic GLP-1R/p-CREB signaling pathway to ameliorate ß-cell injury and improve insulin resistance. Therefore, the intestinal microbial-tryptophan metabolism-AhR pathway was deduced to be a mechanism by which this polysaccharide improves GMD.

[Preparation and physiochemical properties of curcumin-loaded lipid cubic liquid crystalline nanoparticles].

OBJECTIVE: To prepare curcumin-loaded lipid cubic liquid crystalline nanoparticles and evaluate its physiochemical properties. METHODS: The nanoparticles were prepared using hot and high-pressure homogenization. The prescription and preparation process were optimized by uniform design with drug loading and entrapment efficiency as indexes. RESULTS: The nanoparticles were spherical under transmission electron microscope (TEM) with average particle size of 176.1 nm, zeta potential of -25.19 mV, average drug loading of (1.5 +/- 0.2)% and entrapment efficiency of (95 +/- 1.8)%. The release equation: In (1-Q) = -0.0251t-0.0075. The cumulative release percentage was 60% at 36 h in vitro. CONCLUSION: The obtained curcumin-loaded lipid cubic liquid crystalline nanoparticles shows high entrapment efficiency and good sustain release property.

Isolation, molecular characterization, immunological and anticoagulatant activities of polysaccharides from frankincense and its vinegar processed product.

Frankincense (FRA), the oily resin consisting of essential oils, boswellic acids (BAs) and polysaccharides, has been used to improve the blood circulation and relieve pain against carbuncles. According to the theory of traditional Chinese medicine, vinegar processed frankincense (VPF) can increase the effects of promoting blood circulation and relieving pain. Existing studies have carried out much on BAs and essential oils. However, the comparative analysis of polysaccharides from FRA and VPF has not been reported. In this paper, two polysaccharides were isolated and purified from FRA and the other two were from VPF, and their structures and physicochemical properties were analyzed. The immunological and anticoagulatant activities of the four polysaccharides were tested in RAW 264.7 cell and Sprague-Dawley rats, respectively. The polysaccharides purified from VPF showed better immunological and anticoagulatant activities than those in FRA. Therefore, polysaccharides may be one of the active substances for the synergistic effect of VPF.

Cytotoxicity evaluation and metabolism of hepatotoxicity components of Euodiae Fructus in L02 cells.

Euodiae Fructus (EF), the dried unripe scented fruit of Euodia rutaecarpa (Juss.) Benth., was reported to show anti-hypertensive, antitumor, and anti-obesity effects. The main alkaloids of EF were reported as the reason for toxicity of EF by metabolic activation majority through CYP3A. Up till the present moment, the cytotoxicity mechanisms of EF have not yet to be fully clarified. For the purposes of this article, the influence of CYP3A inducer and inhibitor on cytotoxicity of EF and metabolism in L02 cells of five alkaloids related to toxicity of EF were evaluated. The results indicated that CYP3A inducer aggravated the toxicity and CYP3A inhibitor alleviated the toxicity. UPLC-Q-Exactive-MS was used for the identification of five alkaloids of EF in L02 cells. A total of 13 metabolites were detected in L02 cells. In general, five alkaloids were widely metabolized in L02 cells such as oxygenation, demethylation, dehydrogenation, and etc. In addition, oxygenation was the main metabolic pathway. It was inferred that the toxicity of EF was closely related to the CYP3A and the metabolic intermediate might be one of the reasons for the toxicity of EF. Hence, the choice of optimal dose might be critical to avoid the adverse reactions owing to combination of EF and CYP3A inducer.

Effect of CYP3A inducer/inhibitor on pharmacokinetics of five alkaloids in Evodiae Fructus.

Evodiae Fructus (EF), the dried nearly mature scented fruit of Tetradium ruticarpum (A.Juss.) T.G.Hartley, was typically used to treat headache, abdominal pain, hernias, and menorrhagia for thousands of years. It had been reported to be a mild toxic herb through metabolic activation mainly by CYP3A but was barely explained from pharmacokinetic interaction. The aim of the study was to investigate the role of CYP3A inducer/inhibitor in pharmacokinetics of five alkaloids (evodiamine (EVOD), rutaecarpine (RUTA), 1-methyl-2-undecyl-4(1H)-quinolone (MUDQ), 1-methyl-2-nonyl-4(1H)-quinolone (MNNQ) and evocarpine (EVOC)) associated with hepatotoxicity of EF in Sprague Dawley (SD) rats. The results demonstrated that the metabolism of the five alkaloids of EF were inhibited in presence of CYP3A inhibitor whereas the metabolism of the five alkaloids of EF were promoted in presence of CYP3A inducer. Therefore, the dose is required attention when EF is taken in conjunction with CYP3A inducer as there is an enhancement in drug metabolism, which might lead to toxicity.

Morning glory seed keeps laxative effect while retains less subchronic toxicity after being fried.

ETHNOPHARMACOLOGICAL RELEVANCE: Morning glory seed (MGS), has been widely used in treating constipation especially towards children. Clinically, people usually take fried MGS (MGSF) in formulas to reduce its side effect. However, the safety of MGSF other than MGS has yet to be explored. OBJECTIVE: The study aimed to reveal the potential mechanisms of using MGSF instead of MGS basing on chemistry, pharmacodynamics and toxicology. METHODS: The chemical compositions of the extracts of MGS and MGSF were compared using UPLC-Q-TOF/MS method. Simultaneously, to prove the availability and safety of MGSF, we investigated the laxative effect and subchronic toxicity of MGS and MGSF and addressed the mechanism of laxative effect of them. RESULTS: In this study, less phenolic acids and more fatty acids were detected in MGSF compared with the compounds in MGS. Moreover, we found that MGS group had stronger laxative effect than MGSF group via downregulating the expression of AQP3 protein. As for subchronic toxicity test, the body weights of MGS group were lower than MGSF group. In serum biochemistry and histopathological examinations, MGS group could cause more serious toxicity in liver, kidney and colon than MGSF group with higher values of BUN, Cr, AST and ALP. CONCLUSION: Based on the findings in this study, MGSF with varied compounds contents could still keep the laxative effect while retain less subchronic toxicity, which emphasized the necessity of processing and provided an insight into the rational use of MGSF in clinical practice.

Systematically characterize the substance basis of Jinzhen oral liquid and their pharmacological mechanism using UPLC-Q-TOF/MS combined with network pharmacology analysis.

Jinzhen oral liquid (JZ) is a classical traditional Chinese medicine formula used for the treatment of children lung disease. However, the effective substance of JZ is still unclear. In this study, we used lung injury rat model to study the protective effect of JZ, through UPLC-Q-TOF/MS detection coupled with metabolic research and network pharmacology analysis. Fortunately, 31 absorbed prototype constituents and 41 metabolites were identified or tentatively characterized based on UPLC-Q-TOF/MS analysis, and the possible metabolic pathways were hydroxylation, sulfation and glucuronidation. We optimized the data screening in the early stage of network pharmacology by collecting targets based on adsorbed constituents, and further analyzed the main biological processes and pathways. 24 selected core targets were frequently involved in reactive oxygen species metabolic process, dopaminergic synapse pathway and so on, which might play important roles in the mechanisms of JZ for the treatment of lung injury. Overall, the absorbed constituents and their possible metabolic pathways, as well as the absorbed constituent-target-disease network provided insights into the mechanisms of JZ for the treatment of lung injury. Further studies are needed to validate the biological processes and effect pathways of JZ.

Crk-like adapter protein regulates CCL19/CCR7-mediated epithelial-to-mesenchymal transition via ERK signaling pathway in epithelial ovarian carcinomas.

Recent studies have suggested that Crk-like adapter protein (CrkL) and epithelial-to-mesenchymal transition (EMT) induced by CCL19/CCR7 play an important role in ovarian epithelial carcinogenesis. However, the regulatory mechanisms of CrkL on the CCL19/CCR7 signaling pathways in epithelial ovarian carcinomas (EOC) are not well characterized. Here, CCR7 and CrkL proteins were tested in 30 EOC tissues and cell lines. In vitro, the roles of CrkL in CCL19-stimulated SKOV-3 cell invasion and migration were investigated. In this work, CCR7 and CrkL over-expressed in EOC tissues and cell lines and correlated with FIGO stage and lymph node metastasis. Moreover, CCR7 and CrkL serve as an independent prognostic factor. In SKOV-3 cells, CrkL knockdown markedly suppressed the CCL19-stimulated expression of p-ERK and EMT biomarkers (N-cadherin, Snail and MMP9), compared with control. In contrast, p-AKT expression level did not change. On the other hand, functional analysis revealed CrkL knockdown could significantly decrease SKOV-3 cell invasion number of transwell invasion assay, and wound closure area of wound healing assay, compared to control. In conclusion, CrkL regulates CCL19/CCR7-induced EMT via ERK signaling pathway in EOC patients, which further suggested CrkL could be suggested as an efficient target in ovarian cancer treatment.