The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose-catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16 ℃ in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni-NTA affinity column.Size-exclusion chromatography and SDS-PAGE analysis demonstrated that the puri-fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD+as a coenzyme to NADP+.The optimal temperature and pH of the TeXDH were 40 ℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26％of the initial enzyme activity,while the divalent metal ions Mg2+or Ca2+could recover the enzyme activity of TeXDH,reaching 103.32％and 110.69％of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn2+,Ni2+,Cu2+,Zn2+,Co2+,and Cd2+significantly inhibited the activity of TeXDH.ICP-MS and molecular doc-king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn2+ions and 1 mol/L Mg2+ion.Further-more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.

OBJECTIVE: To assess the efficacy of Qingda Granule (QDG) in ameliorating hypertension-induced cardiac damage and investigate the underlying mechanisms involved. METHODS: Twenty spontaneously hypertensive rats (SHRs) were used to develope a hypertension-induced cardiac damage model. Another 10 Wistar Kyoto (WKY) rats were used as normotension group. Rats were administrated intragastrically QDG [0.9 g/(kg•d)] or an equivalent volume of pure water for 8 weeks. Blood pressure, histopathological changes, cardiac function, levels of oxidative stress and inflammatory response markers were measured. Furthermore, to gain insights into the potential mechanisms underlying the protective effects of QDG against hypertension-induced cardiac injury, a network pharmacology study was conducted. Predicted results were validated by Western blot, radioimmunoassay immunohistochemistry and quantitative polymerase chain reaction, respectively. RESULTS: The administration of QDG resulted in a significant decrease in blood pressure levels in SHRs (P<0.01). Histological examinations, including hematoxylin-eosin staining and Masson trichrome staining revealed that QDG effectively attenuated hypertension-induced cardiac damage. Furthermore, echocardiography demonstrated that QDG improved hypertension-associated cardiac dysfunction. Enzyme-linked immunosorbent assay and colorimetric method indicated that QDG significantly reduced oxidative stress and inflammatory response levels in both myocardial tissue and serum (P<0.01). CONCLUSIONS Both network pharmacology and experimental investigations confirmed that QDG exerted its beneficial effects in decreasing hypertension-induced cardiac damage by regulating the angiotensin converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor type 1 axis and ACE/Ang II/Ang II receptor type 2 axis.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Hypertension/pathology* ; Renin-Angiotensin System/drug effects* ; Rats, Inbred SHR ; Oxidative Stress/drug effects* ; Male ; Rats, Inbred WKY ; Blood Pressure/drug effects* ; Myocardium/pathology* ; Rats ; Inflammation/pathology*

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets

Objective To construct a model of knee osteoarthritis(KOA)through the co-culture of dorsal root ganglia(DRG)and fibroblast-like synoviocytes(FLSs).To investigate the effects of transient receptor potential vanilloid type 1(TRPV1)desensitization of sensory neurons induced by mechanical stimulation,including the alleviation of the FLSs inflammatory response.Methods DRG neuronal cells were identified through immunofluorescence.The stress loading of DRG neurons was realized using the FX-6000T cell stress system,and the effect of mechanical strain on the activity of DRG neurons was measured using the CCK-8 method.Ca2+ion flux in DRG neurons was studied through flow cytometry.A Transwell chamber and FLSs were used to establish a co-culture system.The contents of the pro-inflammatory factors IL-1β,TNF-α,and TGF-β in the supernatant were determined by ELISA.Gene and protein expression levels of TRPV1 and its desensitizing negative regulatory proteins PP2B,CaM,IL-1β,TNF-α,TGF-β,and α-SMA in DRG neurons were evaluated using RT-qPCR and Western blot,respectively.Results The Ca2+ion flux in DRG neurons increased under inflammatory conditions,and low intensity(sinusoidal,2％,1 Hz,6 h)thumb-pressing-induced mechanical stimulation did not alter Ca2+ion flux(P＞0.05).However,middle intensity(sinusoidal,4％,1 Hz,6 h)and high intensity(sinusoidal,8％,1 Hz,6 h)stimulation increased Ca2+ion flux significantly(P＜0.05).Notably,high intensity stimulation did not lead to a further increase in Ca2+ion flux over that for middle intensity stimulation(P＞0.05).There was no significant effect of low intensity stimulation on TRPV1,PP2B,or CaM gene or protein expression in DRG neurones,on IL-1β,TNF-α,or TGF-βconcentrations in the supernatant of co-cultured cells,or on IL-1β,TNF-α,TGF-β,or α-SMA gene or protein expression in FLSs(P＞0.05).Middle and high intensity mechanical stimulation up-regulated TRPV1 at the gene and protein expression levels in DRG neurons in the inflammatory group(P＜0.05)but down-regulated PP2B and CaM at the gene and protein expression levels(P＜0.05).Middle and high intensity mechanical stimulation decreased IL-1β,TNF-α,and TGF-β levels in the supernatants of co-cultured cells(P＜0.05)and decreased the gene and protein expression levels of IL-1β,TNF-α,TGF-β,and α-SMA in FLSs(P＜0.05).Conclusions Middle and high intensity thumb-pressing-induced mechanical stimulation induced TRPV1 desensitization of rat sensory neurons,reduced the release of pain mediators,and suppressed the FLSs inflammatory response through downregulating IL-1β,TNF-α,and TGF-β.

The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose-catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16 ℃ in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni-NTA affinity column.Size-exclusion chromatography and SDS-PAGE analysis demonstrated that the puri-fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD+as a coenzyme to NADP+.The optimal temperature and pH of the TeXDH were 40 ℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26％of the initial enzyme activity,while the divalent metal ions Mg2+or Ca2+could recover the enzyme activity of TeXDH,reaching 103.32％and 110.69％of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn2+,Ni2+,Cu2+,Zn2+,Co2+,and Cd2+significantly inhibited the activity of TeXDH.ICP-MS and molecular doc-king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn2+ions and 1 mol/L Mg2+ion.Further-more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.

Objective To construct a model of knee osteoarthritis(KOA)through the co-culture of dorsal root ganglia(DRG)and fibroblast-like synoviocytes(FLSs).To investigate the effects of transient receptor potential vanilloid type 1(TRPV1)desensitization of sensory neurons induced by mechanical stimulation,including the alleviation of the FLSs inflammatory response.Methods DRG neuronal cells were identified through immunofluorescence.The stress loading of DRG neurons was realized using the FX-6000T cell stress system,and the effect of mechanical strain on the activity of DRG neurons was measured using the CCK-8 method.Ca2+ion flux in DRG neurons was studied through flow cytometry.A Transwell chamber and FLSs were used to establish a co-culture system.The contents of the pro-inflammatory factors IL-1β,TNF-α,and TGF-β in the supernatant were determined by ELISA.Gene and protein expression levels of TRPV1 and its desensitizing negative regulatory proteins PP2B,CaM,IL-1β,TNF-α,TGF-β,and α-SMA in DRG neurons were evaluated using RT-qPCR and Western blot,respectively.Results The Ca2+ion flux in DRG neurons increased under inflammatory conditions,and low intensity(sinusoidal,2％,1 Hz,6 h)thumb-pressing-induced mechanical stimulation did not alter Ca2+ion flux(P＞0.05).However,middle intensity(sinusoidal,4％,1 Hz,6 h)and high intensity(sinusoidal,8％,1 Hz,6 h)stimulation increased Ca2+ion flux significantly(P＜0.05).Notably,high intensity stimulation did not lead to a further increase in Ca2+ion flux over that for middle intensity stimulation(P＞0.05).There was no significant effect of low intensity stimulation on TRPV1,PP2B,or CaM gene or protein expression in DRG neurones,on IL-1β,TNF-α,or TGF-βconcentrations in the supernatant of co-cultured cells,or on IL-1β,TNF-α,TGF-β,or α-SMA gene or protein expression in FLSs(P＞0.05).Middle and high intensity mechanical stimulation up-regulated TRPV1 at the gene and protein expression levels in DRG neurons in the inflammatory group(P＜0.05)but down-regulated PP2B and CaM at the gene and protein expression levels(P＜0.05).Middle and high intensity mechanical stimulation decreased IL-1β,TNF-α,and TGF-β levels in the supernatants of co-cultured cells(P＜0.05)and decreased the gene and protein expression levels of IL-1β,TNF-α,TGF-β,and α-SMA in FLSs(P＜0.05).Conclusions Middle and high intensity thumb-pressing-induced mechanical stimulation induced TRPV1 desensitization of rat sensory neurons,reduced the release of pain mediators,and suppressed the FLSs inflammatory response through downregulating IL-1β,TNF-α,and TGF-β.

Objective:To summarize and analyze the characteristics of delayed hemolytic transfusion reaction in children,in order to provide a scientific basis for clinical prevention,and ensure the safety of children's blood transfusion.Methods:The basic situation,clinical symptoms and signs,diagnosis time and disappearance time of alloantibody of delayed hemolytic transfusion reaction in children were retrospectively analyzed.The serological test,routine blood test,biochemical detection and urine analysis results were compared pre-and post-transfusion.Results:Among 15 164 children with repeated blood transfusion,23 cases occurred delayed hemolytic transfusion reactions,with an incidence rate of 0.15％,and mainly children with thalassemia and acute leukemia.39.13％of delayed hemolytic reactions occurred in children with more than 20 times of blood transfusions.Anemia was the main clinical symptom in 86.96％of children.4.35％of children had hypotension and dyspnea.Serological test results showed that the positive rate of direct antiglobulin test was 91.30％,and that of erythrocyte homologous antibody test was 100％.Erythrocyte alloantibodies were common in Rh and Kidd blood group systems,accounting for 73.91％and 13.04％,respectively.Laboratory test results showed that hemoglobin,reticulocyte,spherocyte,total bilirubin,indirect bilirubin,lactate dehydrogenase,serum ferritin and urine color were significantly different after transfusion compared with those before transfusion(all P＜0.05).The average diagnosis time of delayed hemolytic transfusion reactions was 18.56 days,and the average disappearance time of erythrocyte alloantibodies was 118.43 days.Conclusion:The incidence of delayed hemolytic transfusion reaction is high in children with repeated blood transfusion,and the disappearance time of erythrocyte homologous antibody is long.Blood matched ABO,Rh and Kidd blood group antigens should be transfused prophylactically.Once diagnosed,erythrocyte alloantibody corresponding to antigen-negative blood should be used throughout the whole process.

Objective To analyze and compare the changes in local functional activity of the brain under different recovery times after a 90-day,-6° head down bed rest(HDBR)experiment,clarify the mechanism of brain function remodeling during gravity adaptation,and provide imaging basis and guidance for the model construction and health assessment.Methods 36 healthy male volunteers underwent resting-state functional magnetic resonance imaging(rs-fMRI)scans before HDBR(PRE),3 days after 90-day HDBR(R+3),and 26-28 days after 90-day HDBR(R+26-28),and calculated regional homogeneity(ReHo)and low-frequency amplitude(ALFF)and perform statistical analysis between different time points.Results Compared with PRE-status,ReHo value in the left inferior parietal gyrus and ALFF value in the right posterior cingulate gyrus decreased significantly in R+3 status,while ReHo value in the left calcarine,right lingual gyrus and ALFF value in the precuneus,left paracentral lobule,and right postcentral gyrus increased significantly.At R+26-28 status,more brain areas showed significant changes.Conclusion 90 days of long-term HDBR and subsequent recovery will lead to changes in the neural functional activities of the brain's default network,sensorimotor network and visual network,and obvious adaptive remodeling will occur after a longer period of gravity re-adaptation and recovery.

Objective To study the influence of long term simulated weightlessness on occurrence of laryngopharyngeal reflux(LPR)in healthy volunteers by detection of pepsin in saliva.Methods During a 90-day head-down tilt bed rest(HDTBR)experiment,11 volunteers(4 from the control group and 7 from the countermeasure group)were recruited in the study in the following seven sessions:before bed rest session(Pre),during bed rest sessions for 3 days(BR3),31 days(BR31),61 days(BR61),85 days(BR85)and post bed rest sessions for 7 days(R7)and 25 days(R25).During each testing session,saliva samples were collected for two consecutive days both in the morning and at night before sleeping.Then pepsin in the saliva was measured by enzyme-linked immunosorbent assay.Scale of otorhinolaryngological symptoms for the duration of"Pre-,During-and Post-"were retrospectively performed.Results Among 288 saliva samples of 11 volunteers,17 saliva samples of 8 volunteers were positive for pepsin.The incidence of LPR was 9.1%(1/11)in the Pre session,and it increased to 36.4%in the BR31 session which was higher than the following consequential sessions(0%in BR31,27.3%in BR61 and 27.3%in BR85).Intriguingly,it rose again to 45.5%in R7 session and decreased to 9.1%until in R25 session.There was no significant difference between the control group and the countermeasure group.Total scores for the scale of otorhinolaryngological symptoms showed that a slight elevation tendency emerged during HDTBR session,compared with the pre-HDTBR session.It recovered during the post bed rest sessions.Conclusion The laryngopharyngeal reflux was found in the Pre,During and Post HDTBR experiment.The incidence was significantly increased both in the BR3 and R7 sessions,which indicates that the change of the posture with the stimulation of gastrointestinal dynamic conditions,despite the changes from vertical to horizontal or from horizontal to vertical,might be the major impact factor contributing to the elevation tendency of the incidence of laryngopharyngeal reflux.

Objective:Melanoma is highly malignant and heterogeneous.It is essential to develop a specific prognostic model for improving the patients'survival and treatment strategies.Recent studies have shown that ferroptosis results from the overproduction of lipid peroxidation and is an iron-dependent form of programmed cell death.Despite this,ferroptosis-related genes(FRGs)and their clinical significances remain unknown in malignant melanoma.This study aims to assess the role of FRGs in melanoma,with the goal of developing a novel prognostic model that provides new insights into personalized treatment and improvement of therapeutic outcomes for melanoma. Methods:We systematically characterized the genetic alterations and mRNA expression of 73 FRGs in The Cancer Genome Atlas(TCGA)-skin cutaneous melanoma(SKCM)dataset in this study.The results were validated with real-time RT-PCR and Western blotting.Subsequently,a multi-gene feature model was constructed using the TCGA-SKCM cohort.Melanoma patients were classified into a high-risk group and a low-risk group based on the feature model.As a final step,correlations between ferroptosis-related signatures and immune features,immunotherapy efficacy,or drug response were analyzed. Results:By analyzing melanoma samples from TCGA-SKCM dataset,FRGs exhibited a high frequency of genetic mutations and copy number variations(CNVs),significantly impacting gene expression.Additionally,compared with normal skin tissue,30 genes with significantly differential expression were identified in melanoma tissues.A prognostic model related to FRGs,constructed using the LASSO Cox regression method,identified 13 FRGs associated with overall survival prognosis in patients and was validated with external datasets.Finally,functional enrichment and immune response analysis further indicated significant differences in immune cell infiltration,mutation burden,and hypoxia status between the high-risk group and the low-risk group,and the model was effective in predicting responses to immunotherapy and drug sensitivity. Conclusion:This study develops a strong ferroptosis-related prognostic signature model which could put forward new insights into target therapy and immunotherapy for patients with melanoma.