ObjectiveBased on the concept of quality by design（QbD）， the processing process of calcined Pyritum was optimized， and its X-ray diffraction（XRD） fingerprint was established. MethodsThe safety， effectiveness and quality controllability of calcined Pyritum were taken as the quality profile（QTPP）， the color， hardness， metallic luster， phase composition， the contents of heavy metals and hazardous elements were taken as the critical quality attributes（CQAs）， and the calcination temperature， calcination time， paving thickness and particle size were determined as the critical process parameters（CPPs）. Differential thermal analysis， X-ray diffraction（XRD） and inductively coupled plasma mass spectrometry（ICP-MS） were used to analyze the correlation between the calcination temperature and CQAs of calcined Pyritum. Then， based on the criteria importance through intercriteria correlation（CRITIC）-entropy weight method， the optimal processing process of calcined Pyritum was optimized by orthogonal test. Powder XRD was used to analyze the phase of calcined Pyritum samples processed according to the best process， and the mean and median maps of calcined Pyritum were established by the superposition of geometric topological figures， and similarity evaluation and cluster analysis were carried out. ResultsThe results of single factor experiments showed that the physical phase of Pyritum changed from FeS₂ to Fe₇S₈ during the process of temperature increase， the color gradually deepened from dark yellow， and the contents of heavy metals and harmful elements decreased. The optimized processing process of calcined Pyritum was as follows：calcination temperature at 750 ℃， calcination time of 2.5 h， paving thickness of 3 cm， particle size of 0.8-1.2 cm， vinegar quenching 1 time［Pyritum-vinegar（10∶3）］. After calcination， the internal structure of Pyritum was honeycomb-shaped， which was conducive to the dissolution of active ingredients. XRD fingerprints of 13 batches of calcined Pyritum characterized by 10 common peaks were established. The similarities of the relative peak intensities of the XRD fingerprints of the analyzed samples were>0.96， and it could effectively distinguish the raw products and unqualified products. ConclusionTemperature is the main factor affecting the quality of calcined Pyritum. After processing， the dissolution of the effective components in Pyritum increases， and the contents of heavy metals and harmful substances decrease， reflecting the function of processing to increase efficiency and reduce toxicity. The optimized processing process is stable and feasible， and the established XRD fingerprint can be used as one of the quality control standards of calcined Pyritum.

ObjectiveBased on the concept of quality by design（QbD）， the processing process of calcined Pyritum was optimized， and its X-ray diffraction（XRD） fingerprint was established. MethodsThe safety， effectiveness and quality controllability of calcined Pyritum were taken as the quality profile（QTPP）， the color， hardness， metallic luster， phase composition， the contents of heavy metals and hazardous elements were taken as the critical quality attributes（CQAs）， and the calcination temperature， calcination time， paving thickness and particle size were determined as the critical process parameters（CPPs）. Differential thermal analysis， X-ray diffraction（XRD） and inductively coupled plasma mass spectrometry（ICP-MS） were used to analyze the correlation between the calcination temperature and CQAs of calcined Pyritum. Then， based on the criteria importance through intercriteria correlation（CRITIC）-entropy weight method， the optimal processing process of calcined Pyritum was optimized by orthogonal test. Powder XRD was used to analyze the phase of calcined Pyritum samples processed according to the best process， and the mean and median maps of calcined Pyritum were established by the superposition of geometric topological figures， and similarity evaluation and cluster analysis were carried out. ResultsThe results of single factor experiments showed that the physical phase of Pyritum changed from FeS₂ to Fe₇S₈ during the process of temperature increase， the color gradually deepened from dark yellow， and the contents of heavy metals and harmful elements decreased. The optimized processing process of calcined Pyritum was as follows：calcination temperature at 750 ℃， calcination time of 2.5 h， paving thickness of 3 cm， particle size of 0.8-1.2 cm， vinegar quenching 1 time［Pyritum-vinegar（10∶3）］. After calcination， the internal structure of Pyritum was honeycomb-shaped， which was conducive to the dissolution of active ingredients. XRD fingerprints of 13 batches of calcined Pyritum characterized by 10 common peaks were established. The similarities of the relative peak intensities of the XRD fingerprints of the analyzed samples were>0.96， and it could effectively distinguish the raw products and unqualified products. ConclusionTemperature is the main factor affecting the quality of calcined Pyritum. After processing， the dissolution of the effective components in Pyritum increases， and the contents of heavy metals and harmful substances decrease， reflecting the function of processing to increase efficiency and reduce toxicity. The optimized processing process is stable and feasible， and the established XRD fingerprint can be used as one of the quality control standards of calcined Pyritum.

AIM To identify the in vivo metabolites of Cynanchum auriculatum Royle ex Wight extract in functional dyspepsia rats by UHPLC Q-Exactive Plus Orbitrap HRMS.METHODS The rat models for functional dyspepsia were established.The analysis was performed on a 40℃ thermostatic Hypersil GOLD C18 column(2.1 mm×100 mm,1.9 μm),with the mobile phase comprising of water(containing 0.1％formic acid)-acetonitrile(containing 0.1％formic acid)flowing at 0.3 mL/min in a gradient elution manner,and electrospray ionization source was adopted in positive and negative ion scanning.RESULTS Total 4 prototypes(baishouwubenzophenone,deacylmetaplexigenin,qingyangshengenin,syringic)and 110 metabolites were identified,12 of which were common metabolites in feces and urine,56 of which were unique metabolites in urine,42 of which were unique metabolites in feces.The metabolic pathway of prototypes contained phase Ⅰ metabolism(reduction,oxidization,etc.),phase Ⅱ metabolism(sulfonation,glucuronidation,etc.)and phase Ⅰ,Ⅱ composite reactions.CONCLUSION This effective and comprehensive method can lay the theoretical foundation for further discovery of potential active metabolites in C.auriculatum.

Intestinal dysbiosis and disrupted bile acid(BA)homeostasis are associated with obesity,but the precise mechanisms remain insufficiently explored.Hepatic protein phosphatase 1 regulatory subunit 3G(PPP1R3G)plays a pivotal role in regulating glycolipid metabolism;nevertheless,its obesity-combatting potency remains unclear.In this study,a substantial reduction was observed in serum PPP1R3G levels in high-body mass index(BMI)and high-fat diet(HFD)-exposed mice,establishing a positive correlation between PPP1R3G and non-12α-hydroxylated(non-12-OH)BA content.Additionally,hepatocyte-specific overexpression of Ppp1r3g(PPP1R3G HOE)mitigated HFD-induced obesity as evidenced by reduced weight,fat mass,and an improved serum lipid profile;hepatic steatosis alleviation was confirmed by normalized liver enzymes and histology.PPP1R3G HOE considerably impacted systemic BA homeostasis,which notably increased the non-12-OH BAs ratio,particularly lithocholic acid(LCA).16S ribosomal DNA(16S rDNA)sequencing assay indicated that PPP1R3G HOE reversed HFD-induced gut dysbiosis by reducing the Firmicutes/Bacteroidetes ratio and Lactobacillus population,and elevating the relative abundance of Blautia,which exhibited a positive correlation with serum LCA levels.A fecal microbiome transplantation test confirmed that the anti-obesity effect of hepatic PPP1R3G was gut microbiota-dependent.Mechanistically,PPP1R3G HOE markedly suppressed hepatic cholesterol 7α-hydroxylase(CYP7A1)and sterol-12α-hydroxylase(CYP8B1),and concurrently upregulated oxysterol 7-α hydroxylase and Takeda G protein-coupled BA receptor 5(TGR5)expression under HFD conditions.Furthermore,LCA administration significantly mitigated the HFD-induced obesity phenotype and elevated non-12-OH BA levels.These findings emphasize the significance of hepatic PPP1R3G in ameliorating diet-induced adiposity and hepatic steatosis through the gut microbiota-BA axis,which may serve as potential ther-apeutic targets for obesity-related disorders.

Invasive fungal infections play an important factor threatening human health.In recent years, the number of the susceptible population of invasive fungal infections has increased rapidly, leading to a significant increase in the disease burden of human fungi.In 2022, the World Health Organization released a list of key fungal pathogens, aiming to promote global research and investment in fungal infection and antifungal resistance.At present, antifungal drugs are preferred for anti-fungal infection treatment, although the efficacy is limited by the adverse events and drug resistance.Improving the damaged host immune response is a reasonable way to improve the effectiveness of antifungal drugs.This article briefly described the epidemiology of fungal infections, summarized how hosts mediate the clearance of fungi by initiating innate and adaptive immune responses, and discussed the prospects and clinical evidence supporting immunomodulatory therapy for invasive fungal infections.

AIM: To investigate the effect of adalimumab combined with dexamethasone intravitreal implant in the treatment of refractory non-infectious uveitis macular edema(UME).METHODS: A total of 92 cases(131 eyes)of refractory non-infectious UME patients admitted to our hospital from January 2020 to January 2022 were selected and randomly divided into control group, with 46 cases(63 eyes)treated with dexamethasone intravitreal implant and observation group, with 46 cases(68 eyes)treated with adalimumab subcutaneous injection combined with dexamethasone intravitreal implant. The best corrected visual acuity(BCVA), central retinal thickness(CRT), vitreous opacity and Th17/Treg cytokines were measured before and after treatment, and the occurrence of adverse reactions was recorded.RESULTS: Totally 3 cases(4 eyes)were lost to follow-up. After treatment for 1, 3, 6 and 12 mo, BCVA was improved in both groups compared with that before treatment, and CRT, vitreous opacity score, serum interleukin(IL)-17 and IL-22 levels were decreased compared with those before treatment, and serum transforming growth factor-β(TGF-β)and IL-10 levels were increased compared with those before treatment. BCVA in the observation group was better than that in the control group, and CRT, vitreous opacity score, serum IL-17 and IL-22 levels were lower than those in the control group, and serum TGF-β and IL-10 levels were higher than those in the control group(all P<0.05). During treatment and follow-up, no serious adverse reactions occurred in both groups.CONCLUSION: Adalimumab combined with dexamethasone intravitreal implants in the treatment of refractory non-infectious UME can significantly subside the macular edema, reduce vitreous opacity and improve visual acuity.

Objective:To develop a recombinant protein vaccine based on KPC-2, a drug resistance target in Klebsiella pneumoniae, and evaluate its immunogenicity, protective efficacy and mechanism in a mouse model of pneumonia. Methods:KPC-2 was expressed in Escherichia coli and purified using GST affinity chromatography. A recombinant protein vaccine was prepared with KPC-2 and used to immunize New Zealand rabbits through subcutaneous injection. Serum samples were isolated from cardiac blood and Protein G chromatography was used to purify polyclonal antibodies against KPC-2. Opsonophagocytic killing assay was used to assess the bactericidal activity of the polyclonal antibodies in vitro. Female BALB/c mice were immunized three times with the recombinant protein vaccine, and the titers of specific IgG antibodies in serum were measured by indirect ELISA. One week after the last vaccination, the mice were infected with Klebsiella pneumoniae strain SRT through tracheal intubation, and received a single intravenous dose of meropenem (0.1 mg) 1 h later. The protective efficacy of the KPC-2 recombinant protein vaccine was evaluated by comparing the survival rates, bacterial colonization and histopathological changes between vaccine group and adjuvant group as well as the survival rates between meropenem group and normal saline group. Moreover, the protective efficacy of polyclonal antibodies against KPC-2 was evaluated through passive immunization. Results:The level of specific IgG antibodies in serum was significantly higher in the vaccine group than in the adjuvant group ( t=4.325, P<0.05). The survival rate in the vaccine group was also higher than that of the adjuvant group [70% (7/10) vs 10% (1/10), P<0.05]. Furthermore, lung inflammation was less severe and bacterial burden was reduced in the vaccine group as compared with those of the control group ( t=3.127, P<0.05). Both active and passive vaccination strategies demonstrated strong protective efficacy against Klebsiella pneumoniae infection, and had a synergistic effect when used in combination with antibiotic therapy. The polyclonal antibodies against KPC-2 had bactericidal activity in vitro ( t=5.427, P<0.05). Conclusions:The prepared KPC-2 vaccine has better immunogenicity and protective efficacy. It can induce strong humoral immune responses. This study suggest that drug resistance target may be used as a candidate antigen for future vaccine development.

Objective To observe the clinical effect of neuroendoscopic minimal invasive surgery in treating the patients with intraventricular hemorrhage cast.Methods The prospective non-randomized con-trolled study was adopted.Sixty-eight inpatients with intraventricular hematoma cast receiving surgical treat-ment in the neurosurgery department of this hospital from January 2020 to January 2023 were selected as the study subjects;thirty-four cases adopting neuroendoscopic minimal invasive surgery served as the observation group and 34 cases adopting lateral ventricle drilling drainage served as the control group;the surgical time,in-traoperative bleeding volume,hospitalization duration,ICU duration,clearance time of postoperative ventricle hematoma,postoperative hydrocephalus occurrence,occurrence rate of recurrent bleeding in operating area and postoperative complications occurrence rate were observed in the two groups.The levels of serum TNF-α,L-6,CRP,GFAP,S100-β and NSE before operation and on postoperative 7 d were detected;the GCS scores,BI,NIHSS scores before operation and on postoperative 14 d were observed;the GOS scores in postoperative 6 months were observed.Results The surgical time and intraoperative bleeding amount in the control group were significantly less than those in the observation group(P＜0.05);the hospitalization duration,ICU dura-tion,clearance time of postoperative ventricular hematoma and incidence rate of hydrocephalus in the observa-tion group were significantly short or less than those in the control group(P＜0.05);there was no statistical-ly significant difference in postoperative rebleeding incidence rate between the two groups(P＞0.05);the in-cidence rates of pulmonary infection,urinary tract infection,deep venous thrombosis and surgical site infection in the observation group were significantly less than those in the control group(P＜0.05);there was no sta-tistically significant difference in organ dysfunction incidence rate between the two groups(P＞0.05);the lev-els of postoperative TNF-α,L-6,CRP,GFAP,S100-β and NSE in the both groups were significantly decreased compared with those before operation(P＜0.05);the observation group was significantly lower than the con-trol group(P＜0.05);the GCS,BI and NIHSS scores on postoperative 14 d in the two groups were signifi-cantly improved compared with before operation(P＜0.05);the observation group was significantly better than the control group(P＜0.05);the GOS score at postoperative 6 months in the observation group was bet-ter than that in the control group(P＜0.05).Conclusion Neuroendoscopic minimally invasive surgery is ef-fective in treating intraventricular hemorrhage cast with low incidence rate of postoperative complications,which is worthy of clinical promotion.

OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Humans ; Autophagy ; Cell Death ; Cell Nucleus ; Human Umbilical Vein Endothelial Cells/virology* ; Dengue ; Proteome

Human gut is a huge microbial habitat.Gut microbiota provides nutrition for human body, regulates metabolism and intestinal epithelial development, and induces innate immunity, and has significant impacts on growth, development and aging.Gut microbiota is influenced by genetics, living environment or life pattern, diseases and other factors, and also interacts with organs of the whole body through various of ways.The lung and the large intestine have embryological homology, common mucosal immune system, secretory function and other modern biological basis.Gut microbiota not only regulates the function of the gastrointestinal tract, but also affects the health and disease of the respiratory system, forming the " gut-lung axis". Intestinal microecological mediation and regulation based on the theory of gut-lung axis has achieved beneficial effects on the prevention and treatment of respiratory tract infection, asthma, and other respiratory diseases.