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 investigate the correlation of plasma homocysteine(Hcy)level and the polymorphisms of its key metabolic enzymes methylenetetrahy-drofolate reductase(MTHFR)gene with ischemic stroke(IS).Methods A total of 310 patients with IS were enrolled as the case group and 330 healthy subjects during the same period were selected as the control group.Plasma Hcy concentration was detected by enzyme cycling method,and the real-time fluorescent quantitative PCR(RT-qPCR)was used to detecte the genotypes of MTHFR C677T.Results The frequencies of TT genotype(36.13%),CT genotype(10.00%)and T allele(28.06%)of MTHFR gene C677T locus in stroke patients were significantly higher than that in control group(P＜0.05).The frequency of the TT genotype was significantly higher in IS group compared to control group,indicating a recessive mode of inheritance(P＜0.05);In the dominant mode of inheritance,the frequency of CT+TT genotype in IS group was also significantly higher than that in control group(P＜0.05);The plasma Hay concentration of MTHFR C677T genotype TT,CT and CC patients was statistically different(P＜0.05),which were(20.91±6.78)μmol/L(17.20±5.39)μmol/L,(14.35±4.32)μmol/L,respectively;The area under the ROC curve(AUC)of plas-ma total Hcy level was 0.610(95%CI:0.566～0.653,P＜0.001).It indicated that it might play an impor-tant role in predicting the risk of suffering from IS.Multivariate Logistic regression analysis showed that plasma Hcy level and MTHFR C677T gene polymorphism were important risk factors of IS.Conclusions Elevated plasma Hcy level is associated with IS,and the synergistic effect of elevated Hcy level and MTHFR C677T gene mutation may increase the risk of IS.

Objective To investigate the relationship between methylenetetra-hydrofolatereductase(MTHFR)gene polymorphism and serum homocysteine(Hcy)level and subtypes of ischemic stroke(IS).Methods The study was conducted according to the matched principle of case-control design,310 patients with IS and 330 healthy people during the same period were selected as the case group and the control group.Recycling enzyme method and the real-time fluorescent quantitative PCR(RT-qPCR)method were used to detect the level of serum Hcy and the genotypes of MTHFR C677T,respectively.Results There was a significantly difference in MTHFR C677T genotype and allele frequency between the case and control groups(P＜0.05).The correlation analysis with different subtypes indicated that the frequencies of CT genotype(38.02%),TT genotype(10.74%),and T allele(29.75%)were significantly different in the LAA group(OR=1.662,95%CI:1.058-2.608,P＜0.05;OR=2.373,95%CI:1.110-5.073,P＜0.05;OR=1.663,95%CI:1.190-2.323,P＜0.05);The frequencies of TT genotype(10.53%)and T allele(27.30%)in SAO group were also significantly different(OR=2.130,95%CI:1.046-4.336,P＜0.05;OR=1.474,95%CI:1.075-2.021,P＜0.05).Further analysis of serum Hcy level showed that LAA group(19.55±5.61)μmol/L and SAO group(16.37±5.20)μmol/L were significantly higher than the control group(14.46±4.61)μmol/L(P＜0.001);Among the patients of both subtypes the serum Hcy levels in those with CT genotypes and TT genotypes were significantly higher than those in patients of CC genotypes(P＜0.001).Conclusions The gene polymorphism of MTHFR C677T has a significant effect on Hcy level in pa-tients with LAA and SAO stroke.

Influenza viruses are common pathogens causing respiratory infections in humans. Among the four seasonal influenza viruses, influenza A virus H3N2 has become the leading cause of seasonal influenza illness and death, posing a great threat to public health and the economy. Since it first emerged and caused a pandemic in 1968, H3N2 has been circulating repeatedly in human beings and continually evades host immune attack by antigenic drift, resulting in a decrease in vaccine efficacy. In this paper, the antigenic evolution of influenza A virus H3N2, the impact of antigenic evolution on the selection of vaccine strains and some models for predicting the evolution of influenza viruses were analyzed and reviewed, which paved the road for understanding the antigenic evolution of influenza virus and vaccine development.

Influenza has caused high morbidity and mortality worldwide, seriously endangering human health and life. The continuous mutation of influenza virus has brought new challenges to the prevention and treatment of influenza. Animal models provide convenience for a comprehensive understanding of influenza virus pathogenesis, transmission mechanism, vaccine development, and evaluation of therapeutic effects. The construction and use of animal models of influenza virus infection vary in different studies, and the application of different animal models also has its own characteristics. This article reviewed the current status of the construction and use of various animal models, and summarized the advantages and limitations of animal models in evaluating the efficacy of antibodies, drugs and vaccines, with the aim of providing reference for the selection and optimization of animal models in the future.

Influenza viruses are responsible for a large number of infections and deaths annually, posing a serious threat to public health. Vaccination is the most effective measure to prevent influenza virus infection. However, current seasonal influenza vaccines only protect against closely matched circulating strains. Even with extensive surveillance and annual reformulation, yearly updated vaccines are still a step behind the fast-evolving viruses, often resulting in poor matches or less effective vaccines. Due to the relatively complex evolution of influenza A viruses, it is a new idea and a new means to prevent influenza epidemics by using a series of innovative technologies to develop universal influenza vaccines that can provide extensive and long-lasting protection against influenza viruses. This review summarized the latest progress in the development of universal vaccines targeting HA in the past three years, including design methods for universal vaccines targeting HA, HA stem and other conserved epitopes, compared the advantages and disadvantages of different technologies, explored the impact of immunization programs and strategies, and discussed the potential challenges to be overcome, hoping to provide reference for the successful development of universal vaccines.

β-thalassemia is a single-gene genetic disease caused by β globin gene mutations leading to the fact that red blood cells are unable to form normal adult hemoglobin, and then patients develop hemolytic anemia. Current treatment regimens mainly include allogenetic hematologic stem cell transplantation, symptomatic regular blood transfusions and the use of iron removers to reduce iron load. Some severe patients have quite poor prognoses and deadly consequences if not treated timely. Genetically modified autohematopoietic stem cells can provide a new treatment option for patients with β thalassemia, which may achieve a long-term and stable increase in hemoglobin level through a single dose, making one-time cure β-thalassemia possible. This paper reviews the key elements of clinical trial design for β-thalassemia gene therapy from the aspects of efficacy evaluation endpoints, clinical trial design, enrollment population, and subject monitoring in order to provide a reference for pharma-therapeutic research and development enterprises.

Objective To fabricate a foldable microplate for single cell culture and establish finite element model of the folding microplate, so as to calculate traction force of single cells during contraction in three-dimensional (3D) state.Methods The folding angle of the microplate casued by cell traction force was calculated. Then the relation between bending moment and folding angle as well as the relation between traction force and bending moment were derived by using finite element simulation, so as to realize the characterization of traction force for singel cell in 3D state.Results The folding angles of the microplate with HSF and MC3T3-E1 cells in 3D state were 73°-173° and 49°-138°, respectively. The single cell traction forces of HSF and MC3T3-E1 cells were 55-210 nN and 52-161 nN, respectively.Conclusions The proposed method for measuring traction force of single cells in 3D state by fabricating the foldable microplate for single cell culture will provide some references for further development of calculating traction forces in 3D cell adhesion, spreading and migration.

Protein self-assemblies at the micro- and nano-scale are of great interest because of their morphological diversity and good biocompatibility. High-throughput screening of protein self-assembly at different scales and morphologies using protein crystallization screening conditions is an emerging method. When using this method to screen protein self-assembly conditions, some apparently transparent droplets are often observed, in which it is not clear whether self-assembly occurs. We explored the interaction between β-lactoglobulin and the protein crystallization kit Index™ C10 and observed the presence of micro- and nano-scale protein self-assemblies in the transparent droplets. The diverse morphology of the micro- and nano-scale self-assemblies in the transparent droplets formed by mixing different initial concentrations of β-lactoglobulin and Index™ C10 was further investigated by scanning electron microscope. Self-assembly process of fluorescence-labelled β-lactoglobulin was monitored continuously by laser confocal microscope, allowing real-time observation of the liquid-liquid phase separation phenomenon and the morphology of the final self-assemblies. The internal structure of the self-assemblies was gradually ordered over time by in-situ X-ray diffraction. This indicates that the self-assembly phenomenon within transparent droplets, observed in protein self-assembly condition screening experiments, is worthy of further in-depth exploration.
Crystallization ; Lactoglobulins

Objective:To evaluate the value of combined measurement of urinary insulin-like growth factor-binding protein 7 (IGFBP7) and urinary metalloproteinase inhibitor-2 (TIMP-2) in the early diagnosis and prognosis of cardiac surgery-associated acute kidney injury (CSA-AKI).Methods:From March 2018 to June 2018, cardiac surgery patients admitted to the cardiac macrovascular surgery department of the First Affiliated Hospital of Nanjing Medical University were prospectively included, and the blood creatinine was monitored to observe the presence of acute kidney injury (AKI). The prognostic information of the patients was collected, including in-hospital dialysis, in-hospital death, complete recovery of kidney function at discharge, death in one year after surgery, and progression to chronic kidney disease. The levels of urine IGFBP7 and TIMP-2 at 6 h, 24 h and 48 h after cardiac surgery were detected by enzyme linked immunosorbent assay (ELISA), and the urine creatinine (Cr) was also measured. Moreover, receiver operating characteristic curves (ROC) were plotted and the areas under the curves ( AUC) were calculated to evaluate the predictive value and prognostic value of urinary [TIMP-2]·[IGFBP7] (T*I for short) and urine T*I/urine Cr 2 in CSA-AKI. Results:A total of 74 patients with age of (58.43±10.91) years old and 47 males, were enrolled in this study, of which 24 cases (32.4%) had AKI and 10 cases (13.5%) had stage 2-3 AKI. Compared with the non-AKI group, the AKI group had significantly higher levels of urine T*I levels at 6 h and 24 h (both P<0.05). The AUC of T*I at 24 h predicting for AKI was 0.71(95% CI 0.59-0.81, P=0.001, cutoff value 0.020, sensitivity 79.2%, specificity 56.0%), while the AUC for stage 2-3 AKI was 0.85 (95% CI 0.75-0.92, P<0.001, cutoff value 0.083, sensitivity 70.0%, specificity 90.6%). Urinary T*I normalized for urinary creatinine excretion did not show better predictive value. In addition, of T*I at 24 h predicting for poor hospitalization outcome, renal recovery, and one year postoperative death, the AUC was 0.82(95% CI 0.71-0.90, P=0.001), 0.80(95% CI 0.66-0.86, P<0.001), and 0.81(95% CI 0.70-0.89, P=0.047), respectively. Conclusion:The combined detection of TIMP-2 and IGFBP7 in urine is expected to be a biomarker for early diagnosis of CSA-AKI and has certain clinical value in predicting the prognosis of CSA-AKI.