BACKGROUND:Rev-erbα is involved in the regulation of inflammation,but pharmacological activation of Rev-erbα increases the risk for cardiovascular diseases.To reduce the relevant risk,an exploration on SR9009,a Rev-erbα agonist,combined with other drugs to relieve inflammation in skeletal myoblasts was conducted,laying the theoretical foundation for the treatment of inflammation-associated skeletal muscle atrophy. OBJECTIVE:To investigate the relationship of SR9009,indolepropionic acid and nuclear factor-κB signaling pathways in lipopolysaccharide-induced C2C12 myoblasts. METHODS:(1)C2C12 myoblasts were induced to differentiate in the presence of lipopolysaccharide(1 μg/mL).RNA-seq and KEGG pathway analysis were used to study signaling pathways.(2)C2C12 myoblast viability was assessed using the cell counting kit-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,cells were categorized into control group,lipopolysaccharide(1 μg/mL)group,SR9009(10 μmol/L)+lipopolysaccharide group,indolepropionic acid(80μmol/L)+lipopolysaccharide group,and SR9009+indolepropionic acid+lipopolysaccharide group.ELISA was employed to measure protein expression levels of interleukin-6 in the cultured supernatant.Real-time quantitative PCR were employed to measure mRNA expression levels of interleukin-6,tumor necrosis factor α,TLR4 and CD14.Western blot assay were employed to measure protein expression levels of NF-κB p65 and p-NF-κB p65.(3)After Rev-erbα was knocked down by siRNA,knockdown efficiency was assessed by RT-qPCR.And mRNA levels of interleukin-6 and tumor necrosis factor α were also measured. RESULTS AND CONCLUSION:Compared with the blank control group,lipopolysaccharide time-dependently inhibited myofibroblast fusion to form myotubes,the mRNA expression levels of interleukin-6 and tumor necrosis factor α were elevated,and the level of interleukin-6 in the cell supernatant was significantly increased.The results of KEGG pathway showed that the nuclear factor-κB signaling pathway was activated by lipopolysaccharide.Indolepropionic acid exhibited significant suppression of C2C12 myoblasts viability when its concentration exceeded 80 μmol/L.Indolepropionic acid and SR9009 inhibited the activation of NF-κB signaling pathway,thereby played an anti-inflammatory role,and suppressed the mRNA expression levels of interleukin-6,tumor necrosis factor α,TLR4 and CD14.Compared with the lipopolysaccharide group,the ratio of p-NF-κB p65/NF-κB p65 protein expression were downregulated.SR9009 combined with indolepropionic acid notably reduced lipopolysaccharide-induced inflammation,further downregulated the mRNA expression levels of interleukin-6,tumor necrosis factor α,TLR4 and CD14.The ratio of p-NF-κB p65/NF-κB p65 protein expression was significantly lower than that in the SR9009+lipopolysaccharide group or indolepropionic acid+lipopolysaccharide group.Rev-erbα increases time-dependently with lipopolysaccharide induction.The knockdown efficiency of Rev-erbα by siRNA reached over 58%,and lipopolysaccharide was added after Rev-erbα was successfully knocked down.Compared with the lipopolysaccharide group,the mRNA expression levels of interleukin-6 and tumor necrosis factor α were significantly up-regulated.These results conclude that Rev-erbα may act as a promising pharmacological target to reduce inflammation.SR9009 targeted activation of Rev-erbα combined with indolepropionic acid significantly inhibits the nuclear factor-κB signaling pathway and attenuates the inflammatory response of C2C12 myofibroblasts.Moreover,the combined anti-inflammatory effect is superior to that of the intervention alone.

AIM:To investigate the effects of sodium hydrosulfide(NaHS)on hexokinase 2(HK2)-nucleo-tide-binding oligomerization domain-like receptor protein 3(NLRP3)-gasdermin D(GSDMD)pathway and pyroptosis in-duced by lung ischemia/reperfusion(I/R)in rats.METHODS:Male Sprague-Dawley rats were divided into 6 groups:control group,control+NaHS group,I/R group,low-dose NaHS+I/R(L+I/R)group,medium-dose NaHS+I/R(M+I/R)group,and high-dose NaHS+I/R(H+I/R)group,with 6 rats in each group.The NaHS was administered via intraperi-toneal injection at 1.5 mL,30 min before modeling.The left lung tissues were collected 30 min after ischemia and 1 h af-ter reperfusion,and the wet/dry weight ratio and total lung water content were recorded.Hematoxylin-eosin(HE)staining was used to examine lung tissue morphological changes.The levels of malondialdehyde(MDA),myeloperoxidase(MPO)and lactate in lung tissues were measured with test kits.ELISA was employed to determine the levels of interleukin-1β(IL-1β)and IL-18.The expression of glycolysis-and pyroptosis-related indicators was analyzed by Western blot,qRT-PCR and immunofluorescence staining.RESULTS:Compared with control group,the rats in NaHS group showed no signifi-cant differences in all laboratory tests(P>0.05).The rats in I/R group exhibited significant lung injury,oxidative stress,increased lactate level,and up-regulated glycolysis and pyroptosis(P<0.05 or P<0.01).Compared with I/R group,the indicators in L+I/R group showed a downward trend(P<0.01)or no difference(P>0.05),while those in M+I/R group dis-played a significant reduction(P<0.05 or P<0.01).However,the indexes in H+I/R group exhibited no significant dif-ferences in these tests(all P>0.05).CONCLUSION:A moderate dose(56 μmol·L-1·kg-1)of NaHS mitigated the oc-currence of pyroptosis by inhibiting the HK2-NLRP3-GSDMD pathway,thus contributing to the attenuation of lung I/R in-jury in rats.

Objective:The epidemiological and host animal pathogen data of leptospirosis in the population of Zhejiang Province in 2023 were analyzed, providing scientific basis for formulating prevention and control strategies of leptospirosis.Methods:The data of human leptospirosis in the population were collected from the China Information System for Disease Control and Prevention, and analyzed using descriptive epidemiological methods. The data on isolation, culture, and nucleic acid testing of Leptospira pathogens from mouse kidneys, frog kidneys, pig kidneys, and duck kidneys as well as duck serum antibody data were collected from Zhejiang Provincial Center for Disease Control and Prevention "Leptospirosis Surveillance Project of Zhejiang Province". The carrying and changing status of Leptospira epidemic microbiota in populations and host animals were analyzed. Results:In 2023, a total of 83 cases of leptospirosis were reported in Zhejiang Province, with a incidence rate of 0.126 2/ 100 000, aged (62.66 ± 11.31) years, including 68 males and 15 females. Leptospirosis cases were reported in 11 cities, mainly concentrated in the southern cities of Wenzhou City, Lishui City and Taizhou City(a total of 68 cases), accounting for 81.93% of the total number of cases. August to October were high incidence months for leptospirosis (a total of 70 cases), accounting for 84.34% of the total number cases. The male to female ratio of patients was 4.53 ∶ 1.00, and all were adults ≥20 years old, the middle and old people aged 45 - 79 years were the high-risk population (a total of 77 cases), accounting for 92.77% of the total number of cases. The patient's occupation was mainly farmers, with a total of 54 cases, accounting for 65.06% of the total number cases. The shortest time from onset to initial diagnosis for patients with leptospirosis was 0 day, and the longest was 13 days. The shortest time from initial diagnosis to confirmed diagnosis was 0 day, and the longest was 16 days. The 72.29% of the leptospirosis cases (60 cases) had a history of field labor or suspected contact with contaminated water within one month before the onset of the disease, and 18.07% of the leptospirosis cases (15 cases) had a history of contact with animals such as mice, frogs, pigs, cows, dogs, fish or ducks, or their excreta within one month before the onset of the disease. The average nucleic acid positive rate of host animals with leptospirosis was 5.92% (31/524) in mouse kidney, 6.74% (36/534) in frog kidney, and 0.66% (1/151) in pig kidney. The isolation and culture of leptospirosis from duck kidney, nucleic acid detection, and antibody detection in duck blood were all negative. The leptospirosis bacteria detected in human population were serogroup Icterohaemorrhagiae (3 samples) and Hebdomadis (4 samples), and the bacteria group detected in host animals was serogroup Icterohaemorrhagiae (3 samples). Conclusions:The outbreak of leptospirosis in Zhejiang Province mainly occurs in the summer and autumn, with the affected areas mainly concentrated in the southern region of Zhejiang Province. The affected population is mainly middle-aged and elderly males, and the population carrying Leptospira is still mainly composed of the serogroup Icterohaemorrhagiae and the Hebdomadis, with the host animal being the serogroup Icterohaemorrhagiae.

AIM:To investigate the effect and mechanism of the exogenous hydrogen sulfide donor GYY4137 on hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs).METHODS:Rat PASMCs in optimal growth condition were used.Once the cell density reached 60%～70%,the cells were serum starved for 24 h.The cells were then randomly divided into four groups:normal group,normal+GYY4137 group,hypoxia group,and hypoxia+GYY4137 group.A CCK-8 assay was used to determine the safe concentration range of GYY4137 that exerted no adverse effects on normal cells,and the optimal concentration of GYY4137 to inhibit hypoxia-induced proliferation of PASMCs was identified.EdU staining was employed to assess PASMCs proliferation in each group.Western blot analysis was conducted to evaluate the expression of proliferating cell nuclear antigen(PCNA)and proteins related to glycolysis and pyroptosis in PASMCs.Lactic acid content was quantified using a lactic acid assay kit.Immunofluorescence was used to detect the pro-tein expression of hexokinase 2(HK2)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)in PASMCs,and the levels of interleukin-1β(IL-1β)and IL-18 in PASMCs were measured using ELISA.RESULTS:The effective concentration of GYY4137 in inhibiting hypoxia-induced viability of PAMSCs was 100 μmol/L(P＜0.05).Com-pared with the hypoxia group,the hypoxia+GYY4137 group showed a significant decrease in PCNA protein expression(P＜0.05),reduced PAMSCs proliferation(P＜0.01),decreased HK2 and pyruvate kinase isozyme type M2(PKM2)protein expression(P＜0.05,P＜0.01),increased pyruvate dehydrogenase(PDH)protein expression(P＜0.05),and reduced lac-tic acid content(P＜0.01).Additionally,the expression of pyroptosis-related proteins was significantly decreased(P＜0.015,P＜0.01),and immunofluorescence revealed a significant decrease in HK2 and NLRP3 expression(P＜0.01).ELISA results showed that IL-1β and IL-18 protein levels were significantly decreased(P＜0.05,P＜0.01).CONCLU-SION:GYY4137 inhibits hypoxia-induced proliferation of rat PASMCs by regulating glycolysis and pyroptosis.

AIM:To investigate the effect and mechanism of the exogenous hydrogen sulfide donor GYY4137 on hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs).METHODS:Rat PASMCs in optimal growth condition were used.Once the cell density reached 60%～70%,the cells were serum starved for 24 h.The cells were then randomly divided into four groups:normal group,normal+GYY4137 group,hypoxia group,and hypoxia+GYY4137 group.A CCK-8 assay was used to determine the safe concentration range of GYY4137 that exerted no adverse effects on normal cells,and the optimal concentration of GYY4137 to inhibit hypoxia-induced proliferation of PASMCs was identified.EdU staining was employed to assess PASMCs proliferation in each group.Western blot analysis was conducted to evaluate the expression of proliferating cell nuclear antigen(PCNA)and proteins related to glycolysis and pyroptosis in PASMCs.Lactic acid content was quantified using a lactic acid assay kit.Immunofluorescence was used to detect the pro-tein expression of hexokinase 2(HK2)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)in PASMCs,and the levels of interleukin-1β(IL-1β)and IL-18 in PASMCs were measured using ELISA.RESULTS:The effective concentration of GYY4137 in inhibiting hypoxia-induced viability of PAMSCs was 100 μmol/L(P＜0.05).Com-pared with the hypoxia group,the hypoxia+GYY4137 group showed a significant decrease in PCNA protein expression(P＜0.05),reduced PAMSCs proliferation(P＜0.01),decreased HK2 and pyruvate kinase isozyme type M2(PKM2)protein expression(P＜0.05,P＜0.01),increased pyruvate dehydrogenase(PDH)protein expression(P＜0.05),and reduced lac-tic acid content(P＜0.01).Additionally,the expression of pyroptosis-related proteins was significantly decreased(P＜0.015,P＜0.01),and immunofluorescence revealed a significant decrease in HK2 and NLRP3 expression(P＜0.01).ELISA results showed that IL-1β and IL-18 protein levels were significantly decreased(P＜0.05,P＜0.01).CONCLU-SION:GYY4137 inhibits hypoxia-induced proliferation of rat PASMCs by regulating glycolysis and pyroptosis.

OBJECTIVE: To explore the genetic basis for a child with mental retardation. METHODS: Whole exome sequencing was carried out for the child. Candidate variant was screened based on his clinical features and verified by Sanger sequencing. RESULTS: The child was found to harbor a c.995_1002delAGACAAAA(p.Asp332AlafsTer84) frameshift variant in the SYNGAP1 gene. Bioinformatic analysis suggested it to be pathogenic. The same variant was not detected in either parent. CONCLUSION The c.995_1002delAGACAAAA(p.Asp332AlafsTer84) frameshift variant of the SYNGAP1 gene probably underlay the mental retardation in this child. Above finding has expanded the spectrum of SYNGAP1 gene variants and provided a basis for the diagnosis and treatment for this child.
Child ; Humans ; Intellectual Disability/genetics* ; Frameshift Mutation ; High-Throughput Nucleotide Sequencing ; Computational Biology ; Heterozygote ; Mutation ; ras GTPase-Activating Proteins/genetics*

Objective:To explore the mechanism of miR-205-5p/E2F1 signal axis in regulating the glioma U251, U87 radiotherapy resistance.Methods:X-ray gradual ascending and intermittent induction method was used to irradiate the glioma U251 cells to establish U251/TR, U87/TR radiation-resistant cell lines. Then, the morphology, migration, invasion and proliferation abilities of cells (U251/TR, U87/TR radiation-resistant cells and U251, U87 radiation-sensitive cells) were analyzed. Luciferase gene detection system and point mutation technique were employed to analyze the mechanism of miR-205-5p and E2F1 gene activity on U251 and U87 radiation-resistant cell lines.Results:Compared with the radiation-sensitive U251 cells, the radiation-resistant cells U251/TR, U87/TR showed increased proliferation activity, enhanced migration and invasion abilities and decreased apoptosis under X-ray irradiation. miR-205-5p mimics transfection could down-regulate the expression of E2F1 factor in U251/TR cells, inhibit cell proliferation, invasion and migration and increase the radiosensitivity of U251/TR cells. miR-205-5p mimics transfection combined with with E2F1 down-regulation exerted anti-tumor effect and decreased cell tolerance by suppressing the Wnt/β-catenin signaling pathway activity.Conclusions:The glioma radiation-resistant cell line U251/TR, U87/TR can be established by X-ray gradual ascending and intermittent induction method. The miR-205-5p/E2F1 signal axis exerts tumor-suppressing effect through the classical Wnt/β-catenin signaling pathway, which can be used as an therapeutic target to increase the radiosensitivity of glioma.

Objective:Analysis of clustering characteristics of coronavirus disease 2019 (COVID-19) in Guangdong Province.Methods:The COVID-19 cases in Guangdong Province onset from January 1 to February 29, 2020 were collected from Chinese information system for disease control and prevention and Emergency Public Reporting System. Obtain the epidemiological survey data of the cluster epidemic situation, and clarify the scale of cluster epidemic situation, the characteristics of the index cases, family and non-family subsequent cases. Calculate serial interval according to the onset time of the index cases and subsequent cases, secondary attack rate based on the close contacts tracking results, the characteristics of different cases in the clustered epidemic were compared.Results:A total of 283 cluster were collected, including 633 index cases, 239 subsequent cases. Families are mainly clustered, the total number involved in each cluster is in the range of 2-27, M( P25, P75) are 2.0 (2.0, 4.0). During January 15 to February 29, the secondary attack rate is 2.86% (239/8 363) in Guangdong Province, the family secondary attack rate was 4.84% (276/3 697), and the non-family secondary attack rate was 1.32% (61/4 632). According to the reporting trend of the number of cases in Guangdong Province, it can be divided into four stages, the rising stage, the high platform stage, the descending stage and the low level fluctuation period. The secondary attack rate of the four stages were 3.5% (140/3 987), 2.3% (55/2 399), 2.6% (37/1 435), 1.3% (7/542), respectively. The difference was statistically significant ( P=0.003). Conclusion:COVID-19 cluster mainly occurs in families in Guangdong Province. The scale of the clustered epidemic was small; the serial interval was short; and the overall secondary attack rate was low.

Objective:Analysis of clustering characteristics of coronavirus disease 2019 (COVID-19) in Guangdong Province.Methods:The COVID-19 cases in Guangdong Province onset from January 1 to February 29, 2020 were collected from Chinese information system for disease control and prevention and Emergency Public Reporting System. Obtain the epidemiological survey data of the cluster epidemic situation, and clarify the scale of cluster epidemic situation, the characteristics of the index cases, family and non-family subsequent cases. Calculate serial interval according to the onset time of the index cases and subsequent cases, secondary attack rate based on the close contacts tracking results, the characteristics of different cases in the clustered epidemic were compared.Results:A total of 283 cluster were collected, including 633 index cases, 239 subsequent cases. Families are mainly clustered, the total number involved in each cluster is in the range of 2-27, M( P25, P75) are 2.0 (2.0, 4.0). During January 15 to February 29, the secondary attack rate is 2.86% (239/8 363) in Guangdong Province, the family secondary attack rate was 4.84% (276/3 697), and the non-family secondary attack rate was 1.32% (61/4 632). According to the reporting trend of the number of cases in Guangdong Province, it can be divided into four stages, the rising stage, the high platform stage, the descending stage and the low level fluctuation period. The secondary attack rate of the four stages were 3.5% (140/3 987), 2.3% (55/2 399), 2.6% (37/1 435), 1.3% (7/542), respectively. The difference was statistically significant ( P=0.003). Conclusion:COVID-19 cluster mainly occurs in families in Guangdong Province. The scale of the clustered epidemic was small; the serial interval was short; and the overall secondary attack rate was low.

BACKGROUND: RIPA Buffer exhibits different extraction efficiencies of proteins of cells and tissues, which is not appropriate for all samples. OBJECTIVE: To achieve an optimal lysis buffer for skeletal muscle protein extraction in mice of acidosis, and to provide basis for studies on skeletal muscle atrophy. METHEDS: Twenty male healthy C57BL/6 mice, aged 3 months, weighting 25-30 g, were provided by Laboratory Animal Center of Shanxi Medical University. The mice were sacrificed after anesthesia, and the gastrocnemius muscle of lower extremity was isolated. There were two groups: acidosis group was given 10 g of feed mixed with 0.4 mol/L hydrochloric acid (10 mL) , and control group received 10 g of feed mixed with same volume of water, for 7 consecutive days. The effect of RIPA Buffer, Original Buffer and JP Buffer on the skeletal muscle protein extraction in mice of acidosis was compared. The expression levels of AKT, p-AKT (Thr308) , rpS6 and p-rpS6 (Ser235/236) were detected by western blot assay. GLUT4 mRNA expression was examined by RT-qPCR. RESULTS AND CONCLUSION: (1) Different buffers generated different protein-yields. The protein yield was highest in JP Buffer, but the target protein signal was not high. The protein yield was low in RIPA Buffer. Original Buffer could extract sufficient proteins, and had clear band detected by western blot assay. (2) Western blot assay scores in Original Buffer were higher than those of other two buffers. (3) Western blot assay results showed that the extent of phosphorylation in both groups showed no significant changes. (4) GLUT4 mRNA expression level examined by RT-qPCR showed no significant changes in both groups. (5) These results indicate that Original Buffer is optimal lysate of skeletal muscle protein extraction. Inactivated AKT signaling pathway is seen in the short-term hydrochloric acid-induced acidosis group, so whether lengthening acidosis time can activate the signaling pathway. Selecting the optimal lysis buffer for different samples is premise to ensure western blot assay results.