ObjectiveTo analyze the evolutionary characteristics and genetic variations of the HA (hemagglutinin) and NA (neuraminidase) genes of influenza A(H1N1) viruses in Shanghai during 2024, to investigate their transmission patterns, and to evaluate their potential impact on vaccine effectiveness. MethodsFrom January to October 2024, throat swab specimens were collected from influenza like illness (ILI) patients at 4 hospitals in Shanghai. Real-time fluorescence ploymerase chain reaction (RT-PCR) was used for virus detection and isolation of H1N1 influenza viruses. Forty influenza A(H1N1) virus strains were sequenced using Illumina NovaSeq 6000 platform, followed by phylogenetic analyses, genetic distance analysis, and amino acid variation analyses of HA and NA genes. ResultsPhylogenetic tree of the HA and NA genes revealed that the 40 influenza A(H1N1) virus strains circulating in Shanghai in 2024 exhibited no significant geographic clustering, with a broad origin of strains and complex transmission chains. Genetic distance analyses demonstrated that the average intra-group genetic distances of HA and NA genes among the Shanghai strains were 0.005 1±0.000 6 and 0.004 6±0.000 6, respectively, which were comparable to or higher than those observed in global surveillance strains. Both HA and NA genes displayed frequent mutations. Compared to the 2023‒2024 and 2024‒2025 Northern Hemisphere A(H1N1) vaccine strains (WHO-recommended), the HA proteins of 40 Shanghai strains exhibited amino acid substitutions at positions 120, 137, 142, 169, 216, 223, 260, 277, 356 and 451, with critical mutations at positions 137 and 142 located within the Ca2 antigenic determinant. Furthermore, mutations in the NA protein were observed at positions 13, 50, 200, 257, 264, 339 and 382. ConclusionThe genetic background of the 2024 Shanghai influenza A(H1N1) virus strains is complex and diverse, and antigenic variation may affect vaccine effectiveness. Therefore, it is recommended to enhance genomic surveillance of influenza viruses, evaluate vaccine suitability, and implement more targeted prevention and control strategies against imported influenza viruses.

Objective:To explore the safety and feasibility of optimized pathological evaluation system of donor's kidney and modified surgery during adult dual kidney transplantation(DKT)and evaluate its effectiveness to provide more alternative protocols for kidney transplantation from extended criteria donors.Methods:DKT was performed in 10 recipients using the same protocol from June 2019 to May 2021.And retrospective reviewing was performed for clinical data, including characteristics of donors and recipients, optimized pathological evaluation system, modified surgery, treatment regimens, complications and follow-ups.Results:There were 8 male and 2 female donors with an age of(57.9±12.8)years and BMI(24.1±4.1)kg/m 2.The percentage of DCD was 70% and DBD 30%.The serum creatinine before procurement was 107.6(93.3-163.5)μmol/l.Zero-point puncture biopsy was performed for both kidneys and optimized pathological evaluation system was implemented(Banff criteria & Remuzzi score). The pathological results indicated that glomerular sclerosis for left and right kidneys were 2.0(1.5-2.0)and 1.5(1.0-2.0). And Remuzzi score for left and right kidneys were(4.4±1.2)and(3.6±1.5)points respectively.All recipients were male with an age of(43.1±9.0)years and BMI(22.2±1.9)kg/m 2.All PRAs were negative pre-operation.Modified surgery was performed in all recipients(two kidneys were implanted outside iliac vessels without patch and artery of superior kidney was anastomosed to internal iliac artery). Operative duration was(195±54.3)min and serum creatinine before discharge 125.0(102.0-199.0)μmol/L.Renal dynamic scintigraphy indicated that glomerular filtration rate was(30.0±8.2)ml/min for left kidney and(29.2±13.9)ml/min for right kidney.MRA results indicated that morphologies of renal arteries and veins were regular.The time between operation and discharge was(22.4±4.7)days.Compared with SKT, serum creatinine before discharge of DKT was lower and DGF incidence of DKT was higher without statistical significance.The time between operation and discharge was longer for DKT than that for SKT( P<0.05). The complications consisted of 20% donor derived infection(DDI)and 50% DGF.And there was no surgical complication associated with vessels and ureter.Renal function remained stable during 6-month follow-ups. Conclusions:Optimized pathological evaluation system of donor's kidney and modified surgery during adult dual kidney transplantation are both safe and feasible.The postoperative function of transplanted dual kidney is successfully restored.However, long-term follow-ups are required for evaluating its effectiveness.

AIM: To examine the expression of miRNA-22 in the ovarian tissues and the effect of miRNA-22 over-expression on the proliferation, migration and invasion in SKOV-3 cells.METHODS: The expression levels of miRNA-22 in different ovarian tissues and SKOV-3 cells were determined by qPCR .miRNA-22 was over-expressed by trans-fection of miRNA-22 mimic.The cell viability was examined by CCK-8 assay.The cell migration was measured by wound healing test .The cell invasion was analyzed by Transwell assay .The protein expression levels of VEGF and P 53 were deter-mined by Western blot .RESULTS: Compared with the normal ovarian tissue , the expression level of miRNA-22 was remarkably decreased in the ovarian tumor tissues .After transfection with miRNA-22 mimic, the expression level of miRNA-22 in the SKOV-3 cells was significantly increased , while the cell viability , migration and invasion were obviously decreased .Moreover , the protein expression of VEGF and P 53 was dramatically inhibited after over-expression of miRNA-22.CONCLUSION:The decreased miRNA-22 expression may be correlated with the development of ovarian can-cer.Over-expression of miRNA-22 decreases the cell viability , migration and invasion by reducing the protein expression of VEGF and P53.

OBJECTIVETo establish a culture system for transgenic Salvia miltiorrhiza hairy roots.

METHODInvestigated the success rate of different explants, different infection time and different co-culture time to induce hairy roots of S. miltiorrhiza. Co-cultured explants were sterilizated with 400 g x L(-1) Cef water for 5 min, inoculated on MS solid medium supplied with 400 mg x mL(-1) cef and 2.5 g x L(-1) Hyg, and then transfered to the 67-V liquid medium with 2.5 g x L(-1) Hyg after complete sterilization. GFP fluorescence detection was performed to detect positive hairy root lines. PCR method to detect rolC gene which is the specific gene of hairy root. Biomass was determinated in different growth periods of root lines. HPLC was conducted to measure the content of dihydrotanshinone I of transgenic hairy roots.

RESULTLeaf base of S. miltiorrhiza was used as a perfect explant to Induce hairy roots, the success rate can reach 93.3%. Inducing efficiency was up to 63.3% after Agrobacterium infection for 10 min. Co-culture for 2-3 d can reach the best induced effect. It is a high credibiliy to use PCR method combined with detection of GFP fluorescence to identified positive transformants. There is a close contact between biomass increases and secondary metabolite accumulation of transgenic hairy roots.

CONCLUSIONSuccessfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.

Cells, Cultured ; Culture Media ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; genetics ; growth & development ; metabolism ; Salvia miltiorrhiza ; genetics ; growth & development ; metabolism ; Tissue Culture Techniques ; methods

OBJECTIVEA novel bHLH-like gene, designated SmbHLH1, was isolated from Salvia miltiorrhiza, in order to identify a bHLH gene in related to danshinone biosysnthesis.

METHODSmbHLH1 was isolated by RT-PCR,and Semi-quantitative RT-PCR was used to detect the gene expression level.

RESULTThe full length of SmbHLH1 cDNA has an open reading frame of 999 bp. The deduced amino acid sequence of SmbHLH1 has 332 amino acid residues which forms a 36 kDa polypeptide with a calculated pI of 5.4. SmbHLH1 gene was expressed at high level in root, but low level in stem, leaf and flower of S. miltiorrhiza. The transcripts of SmbHLH1 was suppressed when the plants were treated with exogenous MeJA, Yeast + Ag+. The transcripts of SmbHLH1 constitutively accumulated in response to exogenous ABA and low concentration of salicylic acid.

CONCLUSIONSmbHLH is a new member of the S. miltiorrhiza bHLH family, and its possible roles in brassinosteriods signaling responses.

Basic Helix-Loop-Helix Transcription Factors ; genetics ; physiology ; Cloning, Molecular ; Plant Proteins ; genetics ; physiology ; Salvia miltiorrhiza ; genetics

Objective To understand the sensitivity of cytopathogenic effect (CPE) in MadinDarby canine kidney cells(MDCK) that cultured influenza A pharyngeal swab specimens of patients for one,two and three passages. Methods Influenza A pharyngeal swab specimens of patients were inoculated in MDCK for three blind passages. The presence of CPE of every passage was observed by inverted microscope. Results Of the 279 influenza A pharyngeal swab specimens of patients tested by colloidal gold, the presence of CPE in MDCK for one,two and three passages was 65.9%(184/279),91.4%(255/279) and 96.4%(269/279), respectively. Two hundred and seventy-one of 279specimens were identified as influenza A by multiplex reverse transcription-polymerase chain reaction (RT-PCR). Conclusion The positive separation rate can reach more than 95% by inoculating influenza A pharyngeal swab specimens of patients in MDCK for three blind passages.

Objective To understand epidemic characteristics of human influenza A and the genetic and antigenic variations of H1N1 influenza A isolates in Shanghai area in 2009. Methods Throat swabs were collected from patients with influenza-like illness in the sentinel surveillance clinic in Shanghai area in 2009, then inoculated in Madin-Darby canine kidney (MDCK) cell lines. The types of influenza were identified by direct immunofluorescence assay (DIF) and the subtypes were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Segments of hemagglutinin (HA) and neuraminidase (NA) genes of some 2009 H1N1 influenza A isolates were amplified and sequenced. HA and NA gene mutations of 2009 H1N1 influenza A isolates were analyzed. Results Seasonal H1N1 and H3N2 influenza A viruses co-circulated during the spring of 2009 in Shanghai area. Seasonal H3N2 began to co-circulate with 2009 H1N1 in August (the 32nd week) and finally2009 H1N1 became dominate since the 40th week. The phylogenetic tree of 2009 H1N1 HA segment revealed that the isolates from different regions and months were interspersed with each other, but all were clustered into one branch which closed to strains in Spain, Russia, Denmark and other European countries. Mutations were found in some HA amino acid sites, but none of them was in the antigenic determinant region. No change was observed in the 274 NA amino acid residues which were related to the drug resistance to oseltamivir. PB2 protein analysis showed that the 627 and 701 amino acid residues were glutamic acid and aspartic acid respectively, which were the same encoded amino acid with avian flu PB2 protein. Conclusions Seasonal H1N1 and H3N2 co-circulated in the spring of 2009, then both 2009 H1N1 and seasonal H3N2 were prevalent in Summer and Autumn, and 2009 H1N1 finally became dominate in Autumn. Compared to early 2009 H1N1 strains, variations are detected in H1N1 influenza A viruses, but none of them has epidemiological influence, and viruses still show high affinity with human and low-pathogenic characteristics.

Objective To know the levels of antibodies against influenza A virus subtypes H1 and H3 of population in Shanghai during 2009, and the detection of antibodies against avian influenza virus subtypes H5 and H9 in population which contacts with avian. Methods The serological survey of the antibodies against influenza A viruses subtypes H1, H3, H5 and H9 in 356 close contacts with avian (professional population) and 332 general subjects (general population) at various age groups were carried out using hemagglutinin inhibit (HI) test. Results The positive rates of antibodies against influenza virus A/Brisbane/59/2007 (H1N1) in general population and professional population were 82.8% (275/332) and 73.9% (263/356), respectively; those of A/Brisbane/10/2007 (H3N2)were 50.6% (168/332) and 54.8% (195/356), respectively. The positive rate of antibodies against influenza virus A/Brisbane/59/2007 (H1N1 )was significantly higher than that of influenza A viruses subtype H3, which was consistent with etiological survey of influenza virus in Shanghai during 2008.The positive rates of antibodies against influenza A virus subtype H5 in professional population and general population were 4.2% (15/356) and 0.3% (1/332), respectively; those of influenza A virus subtype H9 were 34.6% (123/356) and 2.4% (8/332), respectively. The positive rates of antibodies against influenza virus A/Brisbane/59/2007 (H1N1 ) and A/Brisbane/10/2007 (H3N2) in age groups of 6 months-5 years and ≥60 years were lower than other age groups. Conclusions The immune protective response against seasonal influenza A subtype H1 and H3 of population in Shanghai is high,while those of children and the elders were low. The levels of antibodies against influenza A viruses subtype H5 and H9 in professinal population present obviously ascending trend, which indicates that the etiological and serological survey of influenza virus in this population should be enhanced.

Objective To investigate the distribution and genetic characteristic of etiological agents among children with hand,foot and mouth disease(HFMD)in Shanghai and neighbor areas in 2008.Methods Throat swabs were collected from the inpatients with HFMD from May to June 2008 in Pediatrics Hospital affiliated to Fudan University,Shanghai,and Deqing,Zhejiang Province.Cerebral spinal fluid(CSF)from some patients were collected as well.Vero,MRC-5 and RD ceils were used to isolate the possible pathogens by observing cytopathic effect(CPE).Enterovirus genus,Coxsaekie virus group A type 16(CoxA16)and enterovirus type 71(EV71)were detected by reverse transcriptase-polymerase chain reaction(RT-PCR),and finally identified by sequencing.Results A total of 107 swabs and 22 CSF samples were collected from all 100 inpatients.Swabs of 50 children caused CPE observed.Among them,enteroviruses accounted for 74.0%(37/50),which including 26 (52.0%)of EV71,10(20%)of CoxAl6 and 1(2.0%)of CoxB3,and 13(26.0%)of other pathogens.All the 26 EV71 strains were similar with the isolates from Zhejiang Province and Fuyang,Anhui Province in 2008,which belonged tO genotype Cl all the 10 CoxAl6 strains belonged to genetic lineages C.Conclusions The causative agents of HFMD are complicated.CoxA16 and EV71 are predominant among children with HFMD in Shanghai and neighbor areas in 2008,while the pathogens of some patients are still unknown.

Aim To investigate the protective effect of lipoxin A4(LXA4)on ischemic brain injury in a rat model of permanent focal cerebral ischemia.Methods Adult male Sprague-Dawley rats weighing 200～250 g were used and rats were randomly divided into four groups:sham group,ischemia alone group,LXA4 10 ng group and LXA4 100 ng group.Permanent focal cerebral ischemia was induced by improved thread occlusion of right middle cerebral artery.Approximately 10 mm of nylon surgical thread was inserted into the right internal carotid artery in the rats of sham group.After the middle cerebral artery occlusion,the same volume of LXA4(5 ?l)or isotonic Na chloride(5 ?l)was injected respectively into the right lateral ventricle of the rat in 10 minutes.After 24 h of ischemia,the neurological deficit and the infarct volume were assessed by the method of Longa's score and 2,3,5-triphenyltetrazolium chloride(TTC)staining;the levels of malondialdehyde(MDA)and actvities of myeloperoxidase(MPO)in the ischemia cortex were measured by spectrophotometer;the contents of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)were assayed by ELISA method.The histopathological change was observed after HE staining.Results Treatment with LXA4 10 ng or 100 ng significantly improved functional recovery,reduced relative infarction volume,inhibited MPO activity,decreased MDA,TNF-? and IL-1? levels,and improved histopathological injury.Moreover,the effects of neurological recovery and decreasing TNF-? level in LXA4 100 ng group were better than those in 10 ng group.Conclusion Treatment with LXA4 protects against permanent focal cerebral ischemia injury in rats.