Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Genetic characteristics of influenza A H3N2 virus influenza season in Xiangyang City in 2022-2023 / 公共卫生与预防医学

Objective To analyze the prevalence and genetic characteristics of influenza A(H3N2) viruses in the city of Xiangyang in 2022-2023, and to provide a scientific basis for predicting the epidemic and mutation of influenza virus. Methods Throat swab specimens of the influenza like cases were collected from national influenza monitoring sentinel hospitals in Xiangyang every week. RNA was extracted from the specimens for influenza diagnosing using real-time RT-PCR．Viruses were isolated from H3N2 positive specimens, and HA and NA genes were amplified and sequenced．3D modeling analyses were conducted. Results The gene phylogenetic tree showed that the H3N2 isolates in 2022-2023 belonged to 3C.2a1b.2a1 and 3C.2a1b.2a2 branches, respectively. The A(H3N2) influenza virus strains all had amino acid point mutation sites on important antigenic determinants of HA protein. The epitope mutations of the 2022 A(H3N2) strain mainly occurred in regions B, C, and D. The epitope mutations of the A(H3N2) strain in 2023 mainly occurred in regions C and D. Different glycosylation sites of HA gene were found in 2022-2023 strains. No variation was found in key amino acid sites associated with neuraminidase inhibitor resistance. The difference of overall structure was not obvious in the three-dimensional simulation structure diagram. Conclusion The A(H3N2) influenza strains isolated in this study have shown antigenic drift, especially the mutation of HA, which may affect the protective effect of the vaccine on the local population and lead to influenza epidemic. The variations of HA and NA suggest that close attention should be paid to the epidemic and genetic variation of H3N2 subtype influenza virus, to provide a scientific basis for the selection of influenza virus vaccine strains and the prevention and control of influenza.

Prokaryotic expression and purification of influenza virus globular head domain HA1 / 中国生物制品学杂志

@#Objective To express the globular head domain HA1 gene of influenza virus(IV)in prokaryotic cells,optimize the induced expression conditions,and purify the protein in order to obtain IV HA1 protein with good immunogenicity.Methods Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)],Influenza A virus[A/Hebei/F076/2018(mixed)]and Influenza A virus[A/USA/LAN＿(P5)＿HA/2018(H1N1)]were truncated,and amino acid sequence of63-286 globular head domain was obtained. The genes were optimized and synthesized according to the codon commonly used in E.coli,and named 17Sa,Hebei and USA. The 17Sa point mutation gene was named as 17Sa change. The four genes were cloned into prokaryotic expression vector pET-28a(+)to construct recombinant plasmids respectively,which were transformed into E.coli BL21(DE3)competent cells and induced by IPTG. The protein expression conditions were optimized,and the protein was purified by His labeled nickel ion protein purification column.Results The 762 bp target gene was successfully inserted,and the recombinant plasmid was confirmed to be constructed correctly by double enzyme digestion(NheⅠ/XhoⅠ). The expressed recombinant proteins USA with a relative molecular mass of about 26 000,17Sa,p17Sa and Hebei with a relative molecular mass of about 28 000,showed specific binding to mouse anti-His antibody. The recombinant proteins were all expressed in the form of inclusion bodies,and the expression was highest after induction at 37 ℃ for 8 h. The purified recombinant proteins 17Sa,17Sa change,Hebei and USA had a purity of 90%,85%,95% and 80%,respectively.Conclusion The target protein was successfully expressed and purified by prokaryotic expression system with good reactivity,which lays a foundation of the detection of influenza antibody and the development of new vaccines.

Two case reports of rare antisynthetase syndrome and literature review / 中华风湿病学杂志

Objective:To study the clinical and immunological features of two case of rare antisynthetase syndrome (ASS), so as to improve the level of diagnosis and treatment.Methods:Two cases with rare antisynthetase syndrome admitted to the First Affiliated Hospital of Zhengzhou University from July 2020 to August 2022 were collected.Results:The two rare ASS were anti-Zo antibody and anti-Ha antibody positive patients, both of which had interstitial lung disease (ILD) as the main clinical manifestation and positive anti-Ro52 antibody. Two rare antisynthetase autoantibodies manifested cytoplasmic ANA indirect immunofluorescence (IIF) staining pattern, but it is different from the cytoplasmic dense speckled pattern of several common ASS antibodies. After treatment with glucocorticoids and immunosuppressants, case 1 died of respiratory failure due to a long course of disease and late diagnosis, the lung lesions of case 2 improved significantly.Conclusion:When encountering the cytoplasmic ANA fluorescent pattern in ILD patients, especially with anti-Ro52 antibody, it is necessary to screen more myositis specific antibodies to rule out the possibility of rare ASS.

Distribution of virulence genes in clinical isolates of hospital-associated and community-associated methicillin-susceptible Staphylococcus aureus from Terengganu, Malaysia

@#Staphylococcus aureus is a common bacterial pathogen known to cause various kinds of infections due to its repertoire of virulence factors. This study aimed to investigate the distribution of 19 types of virulence genes among clinical isolates of methicillin-susceptible S. aureus (MSSA) using the polymerase chain reaction. A total of 109 MSSA isolates, i.e., 63 hospital-associated (HA) and 46 community-associated (CA) were collected from Hospital Sultanah Nur Zahirah, the main tertiary hospital in Terengganu, Malaysia, from July 2016 to June 2017. The most frequent virulence genes detected were hla (78.9%, n=86) and hld (78.0%, n=85) encoding hemolysins, lukED (56.9%, n=62) encoding leukotoxin ED, followed by seb (26.6%, n=29) and sea (24.8%, n=27) encoding enterotoxins. Among 34 (31.2%) isolates carrying six or more virulence genes, only five were multidrug resistant (MDR) while the remaining isolates were susceptible. Significant associations were discovered between the hld gene with CA-MSSA (p=0.016) and the seo gene with HA-MSSA (p=0.023). However, there is no significant association between virulence genes among the different types of infection. The clinical MSSA isolates in Terengganu showed high prevalence and high diversity of virulence gene carriage.

Sequence analysis of HA and NA genes of human infected H9N2 avian influenza virus in Yunnan province, 2019 / 中国热带医学

@#Abstract: Objective To investigate the molecular characteristics of the H9N2 avian influenza virus (AIV) causing human infection in Yunnan Province in 2019, and to provide the scientific basis for the prevention and control of avian influenza in Yunnan Province. Methods Influenza virus typing was performed by real-time RT-PCR in two influenza-like illness samples, and the Illumina Miseq high-throughput sequencer was used to determine the viral genome sequence. HA and NA gene sequence alignment and phylogenetic tree construction were performed using Mega7.0 software. Results Real-time RT-PCR results showed that two influenza-like illness samples were positive for H9N2 subtype. The full length of HA and NA were obtained by genomic sequencing. Sequence system evolution analysis showed that the HA and NA of the two AIVs in Yunnan Province were in the same evolutionary clade as A/Chicken/Zhejiang/HJ/2007 and belonged to the G57 type. The HA nucleotide and amino acid homology of the two AIVs were 93.92% and 95.00%, respectively, and the NA nucleotide and amino acid homology was 93.31% and 82.03%, respectively. The nucleotide (amino acid) homology of HA was 92.29%-96.94% (93.77%-98.43%) and 92.84%-94.92% (94.18%-96.23%), respectively, and NA nucleotide homology (amino acid) were 91.81%-97.60% (77.82%-94.83%), 94.38%-97.22% (85.47%-94.55%), respectively, compared with that of human infected H9N2 epidemic strains obtained in China from 2015 to 2020. Both AIVs HA protein cleavage site sequences were PSRSSR↓GLF, which was in line with the characteristics of low pathogenic influenza. The analysis of HA protein receptor binding site showed that amino acids at positions 109, 161, 163, 191, 202, 203 and 234 were consistent with the reference strains, while amino acids at position 198 were mutated to T. N166D and 168N mutations were also found in HA protein, and both AIVs had 7 potential glycosylation sites. Analysis of the erythrocyte binding site of NA gene found that there were amino acid mutations at positions 369, 402, 403, and 432, and amino acid deletion at positions 63-65 was found in the NA genes. There were 4 and 5 potential glycosylation sites in the two AIVs, respectively, and no drug resistance site mutations were found. Conclusions　The receptor binding sites, erythrocyte binding sites and glycosylation sites of HA and NA genes of H9N2 AIV in Yunnan Province have different degrees of variation, and monitoring and prevention and control should be strengthened.

Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae) / 生物工程学报

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

CD44-HA Mediate Adhesion of MDA-MB-231 Cells and HL60 Cells under Fluid Shear Flow / 医用生物力学

Objective To explore how hyaluronic acid ( HA) in extracellular matrix regulates the adhesion ofCD44+tumor cells. Methods MDA-MB-231 cells or HL60 cells were perfused in a parallel plate chamber. Themovement of cells over immobilized HA was observed and analyzed to obtain the characteristics of cell adhesionand rolling. Results The adhesion number of MDA-MB-231 cells on HA substrate was positively regulated by HAconcentration, but not by HA molecular weight. Compared with physically adsorbed HA, immobilized HA byavidin-biotin could significantly improve the cell adhesion ratio. With the increase of shear stress in the range of30-50 mPa, the rolling velocity of cells increased and the adhesion ratio decreased, but the tether lifetime of cellswas not affected. In the same flow field, compared with MDA-MB-231 cells, HL60 cells with low expression ofCD44 rolled more quickly on immobilized HA, with shorter tether lifetime and much lower adhesion ratio(<1. 5% ). Conclusions Fluid shear stress might mediate the rolling velocity of MDA-MB-231 cells by regulatingthe CD44-HA association rate rather than their dissociation rate. The interaction between CD44 and HA is involved in the initial adhesion of HL60 cells, but it does not play a major role. This study will provide references for the design of anti-tumor drugs.

Antitussive Effect of Shegan Zhike Capsule and Its Effect on RARs Receptor,HA and 5-HT / 世界科学技术-中医药现代化

Objective To explore the antitussive action site and potential mechanism of Shegan Zhike Capsule.Methods The mouse cough model induced by concentrated ammonia was used to observe the dose effect relationship of the antitussive effect of Shegan Zhike capsule.The central antitussive effect of Shegan Zhike capsule was observed by using the cough model induced by electrical stimulation of superior laryngeal nerve in guinea pigs.The peripheral antitussive effect of Shegan Zhike capsule was observed by using the cough model of capsaicin desensitized guinea pigs induced by mechanical stimulation.The model of chronic bronchitis in guinea pigs was established by smoking,and the effects of Shegan Zhike Capsule on vasoactive amines histamine(HA)and 5-hydroxytryptamine(5-HT)were observed.Results Shegan Zhike capsule could significantly reduce the number of coughs in mice at the doses of 43.00,86.00 and 172.00 mg extract·kg-1(P<0.05 or P<0.01).The low-dose group,the middle-dose group,and the high-dose group of Shegan Zhike Capsule did not significantly inhibit the cough inducing effect of electrical stimulation of guinea pig superior laryngeal nerve at 30 min and 60 min after administration.Shegan Zhike capsule could significantly inhibit the cough of capsaicin desensitized guinea pigs caused by mechanical stimulation in the low dose group at 60 min,the medium dose group at 30 min and the high dose group at 30 min and 60 min(P<0.05).Compared with the model control group,the content of HA in serum of guinea pigs in low,medium and high dose groups of Shegan Zhike capsule decreased significantly(P<0.05 or P<0.01).The content of serum 5-HT in the high dose group decreased significantly(P<0.05).Conclusion The antitussive effect of Shegan Zhike Capsule was not in the center,and its peripheral antitussive effect was related to the inhibition of RARs receptors,and vasoactive amines such as HA and 5-HT were also involved.

The genetic evolution characteristics of HA gene of influenza A(H1N1)pdm09 virus in Changsha City from 2016-2023 / 中国热带医学

@#Abstract: Objective To analysis the genetic evolution characteristics of hemagglutinin (HA) gene of influenza A(H1N1)pdm virus in Changsha City from 2016-2023, to understand the trend of the HA genetic evolution and the mutations of the amino acid. It provides a scientific basis for the prevention and control of influenza epidemics, as well as the screening of vaccines under the new situation. Methods The A(H1N1)pdm09 virus strains from Changsha City from 2016 to 2023 were isolated using SPF chicken embryos, and then the HA genes were sequenced by MiSeq of Illumina Inc. The homology of HA gene was analyzed by MegAlign of the DNASTAR, and the phylogenetic tree was constructed using the Neighbor Joining (NJ) method in the Molecular Evolutionary Genetics Analysis version 11 (MEGA11). Results The homology of the HA gene of A(H1N1)pdm virus in Changsha from 2016 to 2023 was between 94.8%-99.9%, with the HA gene homology decreasing annually. The homology between the isolated strains of A(H1N1)pdm09 in Changsha City from 2016 to 2023 and the WHO recommended vaccine strain ranged from 96.8% to 99.0%, indicating a relatively good match between the flu isolates and the recommended vaccine strain. The phylogenetic tree of the HA gene of the A(H1N1)pdm09 influenza virus in Changsha City showed that the HA gene evolved into several different branches within the 6B branch, and it had currently evolved to 6B.1A.5a.2a branch. Constant mutations had occurred at the amino acid sites of the four antigenic determinant clusters of HA protein. Currently, amino acid mutations had occurred at 15 antigenic sites within the four antigenic determinant clusters, and the newly emerged A186T antigen mutant site in the isolates from 2023 was worth recent notice. The receptor-binding sites are relatively conserved in loop 130, minor amino acid mutations occurred in loop 220, whether the amino acid mutation site in loop 190 is becoming more stable needs to be further monitored. Taking A/California/07/2009 (CY121680) as the reference strain, most of the A(H1N1) pdm09 isolates in Changsha was increased 162 NQTY glycosylation site and was decreased 276 NTTC glycosylation site, and the glycosylation mutations at these two sites have become more stable recently. Conclusions The HA genes of influenza A(H1N1)pdm virus in Changsha are constantly evolving and mutating, suggesting influenza surveillance should be strengthened continuously.

Application progress of high performance liquid chromatography in detection of hemagglutinin content in influenza vaccine / 中国生物制品学杂志

@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.

Effect of activation of RIG-Ⅰ signaling pathway by LRRC23 on replication of influenza virus / 中国生物制品学杂志

@#ObjectiveTo investigate the effect of activation of RIG-Ⅰsignaling pathway by leucine rich repeat containing23(LRRC23)on replication of influenza virus.MethodsOverexpression and knock-down of LRRC23 were performed in A549 cells to investigate its effect on influenza virus replication. A549 cells were transfected with pcLRRC23 plasmid or siLRRC23(small interfering LRRC23)for 24 h and then infected with influenza virus A/jingfang/1/86(H1N1). The virus titer and HA protein expression level in the cell supernatant were determined by plaque assay and ELISA respectively.The expression of LRRC23,RIG-Ⅰ,MAVS,M1 and HA at gene and protein levels were determined by qRT-PCR and Western blot respectively. The interactions between LRRC23 and RIG-Ⅰwere analyzed by co-immunopre-cipitation(Co-IP)and immunofluorescence assay(IFA). IFN-β-luc and NF-κB-luc activities were determined by dual-luciferase reporter assay.ResultsThe LRRC23 overexpression significantly decreased the influenza virus titer and inhibited the expression of HA protein in the supernatant of A549 cells,while enhanced the NF-κB and IFNβ activations by activation of RIG-Ⅰ-MAVS signaling pathway,resulting in the inhibition of expressions of M1 gene and HA protein. Conversely,the knock-down of LRRC23 increased the protein expression level of HA in the supernatant of A549 cells,up-regulated the relative expression level of M1 gene and down-regulated those of RIG-ⅠmRNA and MAVS mRNA.ConclusionLRRC23 plays an essential role in innate antiviral response by inhibiting influenza virus replication through activation of RIG-Ⅰsignaling pathway.

Complete genome comparative analysis of Coxsackievirus A6 causing hand,foot and mouth disease and herpangina in Yunnan Province in 2018 / 中国生物制品学杂志

@#ObjectiveTo analyze the genome-wide characteristics of 17 strains of Coxsackievirus A6(CVA6)that cause hand,foot and mouth disease(HFMD)and herpangina(HA)in Yunnan Province in 2018,and understand the genetic differences between different pathogenic CVA6.MethodsA total of 1 909 stool samples clinically diagnosed as HFMD and HA in Kunming Children′s Hospital in 2018 were randomly selected for detection using enterovirus group A universal primers and screening of CVA6 positive samples. The CVA6 whole genome sequence was amplified with CVA6 whole genome primers,spliced by BioEdit splicting software,and analyzed for the whole genome characteristics by BioEdit,MEGA 7.0,Simplot,Heml 1.0 and Phyre2softwares.ResultsA total of 929 CVA6 positive samples were screened,and 17CVA6 complete gene sequences were obtained(9 of which were clinically diagnosed as HFMD and 8 were clinically diagnosed as HA). All 17 CVA6 strains were in type IV clade on the whole phylogenetic tree. No significant recombination occurred in HA and HFMD representative strains,while mutations occurred in non-structural protein 3D region. HFMD and HA representative strains showed differences in VP1 loci S597T,Q705L and Q663L. Online predictive analysis showed that the secondary structure of VP1 was consistent with that of CVA6 with no change.ConclusionThe 17 CVA6 strains causing HFMD and HA had high genomic homology,as well as nucleotide and amino acid differences,which may affect the replication and adaptability of CVA6.

Effects of catgut embedding and PGLA embedding at "Zusanli" (ST 36) on skin mast cells, substance P and histamine in healthy rats / 中国针灸

OBJECTIVE@#To observe the effects of catgut embedding and polyglycolic acid/poly-lactic acid (PGLA) embedding at "Zusanli" (ST 36) on the activation of local skin mast cells (MC), and expression of substance P (SP) and histamine (HA), and to explore the mechanism of the temporal stimulation effect of acupoint catgut embedding and provide a foundation for further research on the initiation mechanism of acupoint catgut embedding.@*METHODS@#One hundred and sixty male SPF-grade SD rats were randomly divided into a blank group (10 rats), a sham-embedding group (50 rats), a catgut group (50 rats), and a PGLA group (50 rats). Each intervention group was further randomly divided into five subgroups according to the time points after intervention: 8 hours, 3 days, 7 days, 14 days, and 21 days, with 10 rats in each subgroup. One-time sham-embedding, catgut embedding and PGLA embedding was given at left "Zusanli" (ST 36) in each intervention group, respectively. The skin and subcutaneous connective tissue of the left "Zusanli" (ST 36) were collected at the corresponding time points after intervention, except for the blank group (only one day before intervention). Toluidine blue staining was used to detect MC count and degranulation, and immunohistochemical staining was used to detect the expression of SP and HA positive cells.@*RESULTS@#There was no significant difference in MC count between the subgroups of each intervention group and the blank group (P>0.05). There was no significant difference in MC count between the subgroups of the catgut group and the PGLA group (P>0.05). The MC count in the 8-hour subgroup of PGLA group was higher than that in the 8-hour subgroup of catgut group (P<0.05), while the MC count in the 21-day subgroup of PGLA group was lower than that in the 21-day subgroup of catgut group (P<0.05). Compared with the blank group, the degranulation rates of MC were increased in the 8-hour and 3-day subgroups of sham-embedding group, 8-hour, 3-day, and 7-day subgroups of catgut group, and 8-hour, 3-day, 7-day, and 14-day subgroups of PGLA group (P<0.01, P<0.05, P<0.001). There was no significant difference in the degranulation rate of MC between the subgroups of the catgut group and the PGLA group (P>0.05), and no significant difference in the degranulation rate of MC between the two embedding groups at the same time point (P>0.05). Compared with the blank group, the expression of SP positive cells was increased in the 8-hour subgroup of sham-embedding group, 8-hour, 3-day, 7-day, and 14-day subgroups of catgut group, and 3-day, 7-day, and 14-day subgroups of PGLA group (P<0.001, P<0.05). The expression of SP positive cells in the 7-day subgroup of catgut group was higher than that in the 8-hour subgroup of catgut group (P<0.05), while the expression of SP positive cells in the 14-day subgroup of catgut group was lower than that in the 7-day subgroup of catgut group (P<0.001). The expression of SP positive cells in the 7-day subgroup of PGLA group was higher than that in the 3-day subgroup of PGLA group (P<0.05), while the expression of SP positive cells in the 14-day subgroup of PGLA group was lower than that in the 7-day subgroup of PGLA group (P<0.01). There was no significant difference in the expression of SP positive cells between the subgroups of the two embedding groups at the same time point (P>0.05). Compared with the blank group, the expression of HA positive cells was increased in the 8-hour, 3-day subgroups of sham-embedding group, 8-hour, 3-day, 7-day, and 14-day subgroups of catgut group, and 8-hour, 3-day, 7-day, 14-day, and 21-day subgroups of PGLA group (P<0.001, P<0.01, P<0.05). The expression of HA positive cells in the 14-day subgroup of catgut group was lower than that in the 7-day subgroup of catgut group (P<0.05), while the expression of HA positive cells in the 3-day subgroup of PGLA group was higher than that in the 8-hour subgroup of PGLA group (P<0.05), and the expression of HA positive cells in the 14-day subgroup of PGLA group was lower than that in the 7-day subgroup of PGLA group (P<0.05). The expression of HA positive cells in the 3-day subgroup of PGLA group was higher than that in the 3-day subgroup of catgut group (P<0.05).@*CONCLUSION@#Catgut and PGLA embedding at "Zusanli" (ST 36) in healthy rats could induce changes in local skin MC, SP, and HA, which may be one of the mechanisms of the temporal stimulation effect after acupoint embedding. There are certain differences between different suture materials. A moderate inflammatory response in the acupoint area, mediated by MC and involving SP and HA, may be one of the initiating factors for the effect of acupoint catgut embedding.

Clinical efficacy of combined use of No ha la hu and Ni ru ha in the treatment of incomplete intestinal obstruction / 中国中西医结合急救杂志

Objective To investigate the clinical effect of No ha la hu(Mongolian medicine abdominal massage)combined with Ni ru ha(Mongolian medicine enema)in the treatment of incomplete intestinal obstruction.Methods A total of 80 patients with incomplete intestinal obstruction admitted to the department of emergency and critical care medicine of Inner Mongolia International Mongolian Hospital from April 2020 to April 2022 were selected as the study objects,they were randomly divided into control group and observation group,40 cases in each group.The control group was treated with routine modern medicine,and the observation group was treated with mongolian medicine No ha la hu combined with Ni ru ha on the basis of routine treatment.Specific methods:the patient supine position,both lower limbs flexion,natural calm breathing,the doctor stands on the patient's right side,applies a little butter to the palm of his hand,rub hands,massages the abdomen with the middle finger and ring finger at acupoints such as ileocecal point,small intestine point,small intestine point,small intestine point and lower clockwise direction point.Massage the abdomen with the palm for about 20 minutes,once a day for 3 days.No ha la hu was given after the treatment of Ni ru ha(Rhubarb 250 g,Cold water stone 150 g,Sanai 100 g,Facial alkali 100 g,Hyacinth 50 g,Terminalia chebula 50 g),the patients should lie on the left side,raise the buttocks about 10 cm,insert the enema into the rectum 15-20 cm,take the Mongolian medicine Liuweian powder 12 g and warm water 50-100 mL for enema,once a day for 3 days.The difference of relieving time of abdominal pain and distension,time of exhaustion,time of fasting,and time of hospital stay between the two groups were compared,and the clinical effect was observed.Results Relieving time of abdominal pain,abdominal distension,time of exhaust and time of hospital stay in the observation group were significantly shorter than those in the control group[abdominal pain relief time(hours):5.3±1.8 vs.8.1±1.6,abdominal distension relief time(hours):3.2±1.5 vs.6.5±1.9,time of exhaust(days):2.6±1.7 vs.4.1±2.1,hospital stay(days):8.3±2.9 vs.10.1±2.1,all P<0.05],time of fasting in the observation group were shorter than those in the control group(days:2.9±0.5 vs.3.1±0.6,the difference was not statistically significant(all P>0.05).The effective rate of the observation group was significantly higher than that of the control group[92.5％(37/40)vs.75.0％(30/40),P<0.05].Conclusion On the basis of routine treatment of western medicine,the treatment of incomplete intestinal obstruction by Mongolian medicine No ha la hu combined with Ni ru ha is more effective,which can effectively restore gastrointestinal function,relieve abdominal pain and other symptoms,and shorten the time of exhaust.

Nutritional assessment of servicemen in relation to area of duty and feeding habits: a Pakistani prospective

Abstract The present study was carried out to determine incidence of overweight and obesity in Pakistani servicemen with reference to their area of duty, feeding habits and also to identify risk factors. Accordingly, 2,501 servicemen selected from all over Pakistan using multiple stage stratified sampling protocol. Nutrition assessment performed using body mass index (BMI), waist to hip ratio (WHR) and dietary assessment using food frequency questionnaire. Collected data was analyzed using the SPSS version 25. Regression was used to find risk factors of obesity and WHR. Results indicated that about 1/4th of servicemen were smokers. Approximately, 1/5th of them were overweight and about one quarter were eating fruits and vegetables for 3 days/ week and 4 days/week, respectively. Only 1/3rd of them were physically active for at least 40 minutes per day. Age and fruits intake were significantly predicting BMI with a direct relation and vegetable intake was negatively correlated to BMI of the servicemen. Age and rank were significant predictors of WHR while, physical activity was negatively correlated to WHR. It is concluded and suggested from our study that there is a need to modify eating patterns and habits as well as improving physical activity on daily basis for healthy and long life of the servicemen.

Resumo

Outcome of Single Intra-Articular Injection of High- Molecular-Weight Hyaluronic Acid in Patients with Knee Osteoarthritis

Background: Osteoarthritis (OA) is the most common type of rheumatic disease and a leading cause of disability. Current treatments aim at alleviating these symptoms by several different methods: non-pharmacological and pharmacological treatments and invasive interventions. Among the regenerative methods, hyaluronic acid (HA) is popular now-a-days where differences exist in concentration, molecular weight, dosage, expected duration of effects and added formulations. Based on HA molecular weight, these products are classified in two groups (high and low). Due to cost effectiveness, low molecular weight HA is commonly used. To find out the outcome of single intra-articular injection of high-molecular- weight HA in patients with knee osteoarthritis.Material & Methods:This was a randomized clinical trial conducted among purposively selected 55 patients suffering from Knee OA attending at the Physical Medicine and Rehabilitation outpatient department, DMCH during July 2020 to June 2021. Patients were randomly allocated into two groups; group A received single dose of high molecular weight HA and designed exercise program and group B received same exercise program only. Pre-treatment and 3 weekly post treatment assessment were done up to 12 weeks in each group. In each follow up visit, pain, and functional status were measured in Visual Analog Scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) respectively and compared between group A and group B where was considered as significant (p value <0.050.Results:The mean age of the participants in group A and group B were 56.8 (±9.8) and 53.3 (±7.4) years respectively where majority of the patients in both groups were female patients with overweight. In group A, VAS and WOMAC significantly decreased from base line to each follow up till 12th weeks after treatment with high-molecular-weight HA (P<0.001). In group B, VAS and WOMAC significantly decreased from base line to each follow up till 12th weeks after treatment with conservative management (P<0.001). After 6 weeks of intervention, the mean VAS scores of the patients became 4.9 ±1.1 and 6.0±1.0 in group A and group B respectively which showed significant statistical difference (P<0.001). At the end of 12 weeks, the VAS score was significantly decreased in group A (2.8±1.3) than group B (4.9±1.7) (P<0.001). After 3 weeks of intervention, the mean WOMAC scores of the patients became 71.2±6.6 and 75.0 ±4.9 in group A and group B respectively which showed significant statistical difference (P<0.001). At the end of 12 weeks, the WOMAC score was significantly decreased in group A (61.9±6.6) than group B (68.1±4.6) (P<0.001). Conclusion:Single intra-articular injection of high-molecular-weight Hyaluronic Acid is effective in patients with knee osteoarthritis in reducing pain and improving functional impairment. Long term and large scale research studies are needed to establish the effectiveness and safety of this procedure in patients with knee osteoarthritis.

Role of P4HA2 in promoting cancer progression / 国际肿瘤学杂志

P4HA2, one of the important genes encoding the α subunit of collagen prolyl 4-hydroxylase (C-P4H) , is overexpressed in a variety of tumors and promotes tumor progression, and its high expression correlates with the poor prognosis of patients. In different tumors, P4HA2 can promote the proliferation, migration and invasion of tumor cells, increase the proportion of cancer stem cell population, and promote tumor cells to escape from immune system. An in-depth understanding of the role of P4HA2 in tumor progression may provide new ideas and insights for targeting P4HA2 to prevent or reverse tumor progression.

Genetic evolution of influenza B virus in Guizhou Province during 2017 to 2021 / 中华微生物学和免疫学杂志

Objective:To analyze the molecular evolution characteristics of HA and NA genes of influenza B/Yamagata (BY) and influenza B/Victoria (BV) lineage viruses in Guizhou Province, aiming to provide reference for scientific prevention and control of influenza. Methods:The prevalence of various types of influenza viruses in Guizhou Province from 2017 to 2021 was analyzed. The nucleic acid of influenza B viruses was extracted, and then the HA and NA genes were amplified by RT-PCR. Fourteen strains were sequenced and the sequences of 83 strains were obtained from GISAID. Homologies between the 97 influenza B viruses as well as the phylogenetic characteristics and amino acid site variations were analyzed. Results:Influenza A, BY and BV lineage viruses co-circulated in Guizhou Province and BV lineage was the predominant type. The homologies of HA and NA genes were 98.7%-99.4% and 98.4%-99.6% between BY lineage viruses and the reference vaccine strain B/PHUKET/3073/2013. BV lineage viruses shared 98.3%-99.3% and 98.9%-99.6% homologies with the reference vaccine strain B/Colorado/06/2017. The BY lineage strains in Guizhou Province mainly belonged to Y3 genetic group with HA gene in two branches of Y3-H1-2 and NA gene in three branches of Y3-N1-3. Three reassortant strains were found in Y3 clade. The isolated BV lineage strains mainly belonged to V1A-2 genetic group with HA gene in four branches of V1A-2 H1-4 and NA gene in five branches of V1A-2 N1-5. Twenty reassortant strains were found in V1A-2 clade and no inter-lineage reassortants were found. Analysis of variations at key amino acid sites showed that there was no mutation at epitopes in Y3 genetic group. However, there were point mutations at four main epitopes and a shift mutation in 190 helix in V1A-2 genetic group. There was no mutation in drug resistance sites. Conclusions:Various types of influenza viruses circulated in Guizhou Province. The homology between influenza B viruses and vaccine strains was decreasing. Different branches of HA and NA genes had been evolved and various forms of mutations were detected in the sequences. Intra-lineage reassortant strains and new varieties emerged. Surveillance of influenza B viruses should be strengthened.

High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes

The aims of the present study were to evaluate the expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) in adipocytes and adipose tissue and to explore its effect on obesity and type 2 diabetes mellitus (T2DM). We initially demonstrated that P4HA3 was significantly upregulated in the subcutaneous adipose tissue of obesity and T2DM patients, and its functional roles in adipocyte differentiation and insulin resistance were investigated using in vitro and in vivo models. The knockdown of P4HA3 inhibited adipocyte differentiation and improved insulin resistance in 3T3-L1 cells. In C57BL/6J db/db mice fed with a high fat diet (HFD), silencing P4HA3 significantly decreased fasting blood glucose and triglycerides (TG) levels, with concomitant decrease of body weight and adipose tissue weight. Further analysis showed that P4HA3 knockdown was correlated with the augmented IRS-1/PI3K/Akt/FoxO1 signaling pathway in the adipose and hepatic tissues of obese mice, which could improve hepatic glucose homeostasis and steatosis of mice. Together, our study suggested that the dysregulation of P4HA3 may contribute to the development of obesity and T2DM.