Objective: To establish a rapid, accurate, and decentralized workflow for bacterial whole-genome sequencing (WGS) and risk profiling within the shelf-life of platelet concentrates, and to characterize the species, virulence, antimicrobial resistance (AMR), and immune evasion mechanisms of co-existing bacteria in qualified platelet products, thereby providing a scientific basis for transfusion safety assessment. Methods: Three units of platelet concentrates, which tested negative by routine bacterial screening, were collected from the Chengdu Blood Center between May and June 2025. Samples were enriched at 37℃under six aerobic and nine anaerobic conditions for 7 days. Using a culturomics strategy, aliquots were plated for isolation on days 1, 3, 5, and 7 to obtain cultivable isolates, with negative culture controls included to exclude contamination. High-molecular-weight genomic DNA was extracted via mechanical grinding, purified, and size-selected. Sequencing libraries were constructed and sequenced on the G-seq500 single-molecule nanopore sequencing platform. Genomes were assembled using Flye and polished with NextPolish, with quality evaluated by BUSCO and CheckM. Taxonomic identification was performed using GTDB-Tk. Functional annotation and database comparisons were conducted to analyze virulence factors, AMR genes, and genes related to immune evasion and environmental adaptation. Results: Viable bacteria were successfully isolated from all three qualified platelet units within their shelf life. The isolates were identified as Bacillus albus, Niallia taxi, and Staphylococcus warneri. Nanopore sequencing generated 92 227-109 813 reads (totaling 680-758 Mb) with an N50 of 7 625-8 584 bp and Q20/Q30 scores of 97%/93%, respectively. All three genomes were assembled into complete circular chromosomes with 1-3 plasmids, achieving >93% completeness. Functional analysis revealed that B. albus carried multiple hemolysins, metalloproteases, and multidrug resistance genes, indicating the highest potential pathogenicity and AMR risk. S. warneri exhibited a typical multidrug resistance profile and regulatory network characteristic of coagulase-negative staphylococci, suggesting intermediate virulence. N. taxi harbored few canonical virulence factors and lacked functional AMR determinants, presenting a "low-virulence, low-resistance" profile. Notably, all three strains were enriched in genes encoding antimicrobial peptide resistance systems (e.g., dltABCD, mprF, GraRS, BceAB) and antioxidant enzymes, suggesting a strong capacity to withstand immune stress in the blood environment. Conclusion: Viable bacteria can be recovered from qualified platelet concentrates that test negative by routine screening. Nanopore WGS enables rapid strain-level identification and comprehensive risk profiling of virulence, resistance, and immune adaptation traits. The functional repertoires of these "co-existing" isolates range from environmental adaptation to potential pathogenicity, representing an underappreciated risk for transfusion-transmitted infections in susceptible recipients.

【Objective】 To explore the composition of culturable bacteria in platelets through bacterial culturomics and verify the results of culturomics and metagenomics to improve the detection rate of bacteria in platelets. 【Methods】 Platelet samples from 6 healthy people were collected. Eight kinds of culture media were placed in aerobic conditions and 12 kinds of culture media were placed in anaerobic conditions for large-scale culture and isolation of bacteria in platelets. The isolated single colony was identified by 16S rRNA gene sequencing. The bacterial abundance of healthy human platelet microbiome was analyzed by metagenomic sequencing, and the cultivable bacterial species in platelets was confirmed based on metagenomic and culturomics results. 【Results】 A total of 90 strains of bacteria belonging to 3 phylums, 5 classes, 5 orders, 7 families, 9 genus and 23 species were isolated from 6 platelet samples by culturomics. Among them, the strains with more monoclonal clones at the species level were Brevundimonas aurantiaca (16.7%), Bacillus sp. Y1 (15.6%), Cutibacterium acnes (14.4%) and Brevibacillus brevis (13.3%). The platelet samples sequenced by mNGS showed that the abundance values of Proteobacteria, Firmicutes and Actinobacteria were high. The bacteria detected by both culturomics and metagenomic sequencing methods were as follows: Firmicutes: Bacillus sp. Y1, B. thuringiensis, B. cereus, B. mobilis, B. velezensis, Staphylococcus epidermidis, and Brevibacillus brevis; Actinobacteria: Cutibacterium acnes; Proteobacteria: Escherichia coli and Delftia tsuruhatensis. 【Conclusion】 The mutual validation of culturomics and metagenomics has identified some bacteria, proving that bacteria exist in platelets.

【Objective】 To explore the influence of common methods of reducing non-viral nucleic acid on the abundance of plasma virus group. 【Methods】 Three kinds of library construction, five kinds of centrifugation conditions, two kinds of filters, four kinds of enzymes and four concentrations of chloroform were used to treat plasma samples added quantitatively 2.16 mL of pseudorabies virus(PRV) and 2.16 mL of porcine parvovirus(PPV). A total of 21.6 mL of plasma samples were processed, including 54 samples. Subsequently, nucleic acid was extracted, mitochondrial DNA(mtDNA) and two viruses were quantitated, the library of the next generation sequencing was constructed, Illumina NovaSeq 6000 was used for the next generation sequencing. The sequencing data were compared with Kraken Py 2.0 software, and the species annotation analysis was conducted. The corresponding species classification information of each segment was obtained to analyze the impact of different reducing non-viral nucleic acid methods on the relative abundance of microorganisms and two indicator viruses. 【Results】 After sequencing by Illumina NovaSeq 6000, 306.27 GB raw data and 193.17 GB clean data were obtained, with Q20>90%, Q30>85%, Error Rate of 0.03%, and average GC Content of 45.02%. The DNA library construction process significantly increased the proportion of microbial sequences and the PRV abundance [(91.8±0.5)%](P<0.05); RNA library construction and combined library construction can increase the abundance of Pestivirus, an RNA virus, and the PRV abundance was(17.7±3.3)% and(8.1±1.5)% respectively. The Ct value of mtDNA was increased and the proportion of human sequence decreased to less than(89.5±1)%, while the proportion of microbial sequence increased to (2.4±0.03)% after treatment of five centrifugation conditions(P<0.05); After centrifugation at 4℃, 100 g, 30 min, the PRV abundance was increased to (40.6±6)%, and centrifugation at 4℃, 4 000 g, 45 min reduced the PRV abundance to (4.1±0.01)%(P<0.05). Both of 0.22-μm filter and 0.45-μm filter increased the Ct value of mtDNA to above 25.56±0.13, decreased the proportion of human sequence to less than (86.1±0.6)%, increased the proportion of microbial sequence to (3.1±0.1)% and (3.4±0.2)%, and decreased the PRV abundance to (1.6±0.3)% and (4.1±0.7)%(P<0.05), while there was no statistical difference in the effect on PPV concentration and abundance. DNase Ⅰ and Benzonase increased the Ct value of PPV to 25.65±0.06 and 25.36±0.45, decreased the proportion of human sequence to (81.7±5.6)% and (72.8±6.7)%, and increased the proportion of microbial sequence and PRV abundance to (11.0±4.1)% and (16.1±4.7)%, (55.8±2.3)% and (39.0±8.9)%, respectively(P<0 05); After treatment with RNase A, the Ct value of PRV increased to 25.20±0.11, and the human sequence proportion decreased to (85.4±5.6)%(P<0 05); Lysozyme had no effect on removing non-viral nucleic acid. The chloroform of 1%, 5%, 10% and 20% increased Ct value of PRV and mtDNA to no less than 27.17±0.21 and 25.68±0.04; Only 10% chloroform increased the proportion of microbial sequences to (3.1±1.2)%(P<0.05); The abundance of PRV with 1% and 5% chloroform treatment was increased to (48.7±13.3)% and (42.1±5.5)%(P<0.05), while 10% and 20% chloroform reduced PRV abundance to (1.0±0.5)% and (3.4±2.8)%(P<0.05). There was no statistical difference in the effect of chloroform with four contents on PPV abundance. 【Conclusion】 Centrifugation at 4℃, 5 000 g, 10 min is suitable for increasing the overall abundance of virus, and centrifugation at 4℃, 100 g, 30 min is suitable for increasing the content of virus similar to PRV. 0.45-μm filter, DNase Ⅰ, Benzonase and low concentration chloroform can effectively reduce the proportion of non-viral nucleic acid sequence in plasma to increase the abundance of the indicated virus group. Thus, the enrichment effect of plasma meta-virome is closely related to the nature of the virus, and the appropriate virus enrichment method should be selected according to the research purpose to establish the corresponding enrichment strategy.

Objective: To explore the effects of long non-coding RNA (LncRNA) MIR22HG on proliferation，apoptosis and inflammatory response of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) and its molecular mechanism． Methods: Synovial tissue samples were collected from 37 RA patients and 30 joint trauma patients in our hospital，and the expression levels of MIR22HG and miR-22-5p in synovial tissue were detected by qRT-PCR. RA-FLSs in human was isolated ，cultured and identified in vitro． MIR22HG siRNA interference plasmid (si-MIR22HG) and its negative control plasmid (si-NC) ，miR-22-5p inhibitor and its negative control (inhibitorNC) were transfected into RA-FLSs respectively or simultaneously．The expression levels of MIR22HG and miR- 22-5p were detected by qRT-PCR. CCK-8 was used to detect the proliferation activity of cells in various groups. Annexin Ⅴ- FITC / PI was used to detect the apoptosis rates of cells in various groups．ELISA was used to detect the levels of TNF-α , IL-1 β and IL-6 in the supernatant of cells in various groups．Western blot was used to detect the protein expression levels of Bcl-2，Bax and Cleaved caspase-3 of cells in various groups．The targeting relationship between MIR22HG and miR-22-5p was verified by dual luciferase reporter gene assay. Results : Compared with joint trauma patients，the expression level of MIR22HG in synovial tissues of RA patients increased (P＜0. 05) ， while the expression level of miR-22-5p decreased (P＜0. 05) ．Interference with MIR22HG inhibited the proliferation activity of RA-FLSs，decreased the levels of TNF-α , IL-1 β and IL-6 in cell supernatant and the protein expression level of Bcl-2 in cells (P＜0. 05) ，and increased the apoptosis rate，the expression level of miR-22-5p and the protein expression levels of Bax and Cleaved casepase-3 (P ＜0. 05 ) ．However，inhibition of miR-22-5p expression reversed the effects of MIR22HG gene silencing on proliferation，apoptosis and inflammation of RA-FLSs (P＜0. 05) ．Dual luciferase reporting assay showed that miR-22-5p was a potential downstream miRNA target of MIR22HG． Conclusion MIR22HG is highly expressed in synovial tissues of RA patients，and it may promote the proliferation and the inflammatory response of RA-FLSs and inhibit cell apoptosis by down regulating the expression of miR-22-5p.

Objective:To explore the trans-membrane signaling mechanism of interleukin-6 (IL-6)-induced osteogenic differentiation and calcification of human umbilical artery smooth muscle cells (HUASMCs).Methods:HUASMCs were primarily cultured in vitro and were stimulated with IL-6, IL-6+solutable IL-6 receptor (sIL-6R), IL-6+sIL-6R+solutable gp130 (sgp130), or vehicle (blank control). Alizarin red and Von Kossa staining were used for detecting cell calcification, Western blot was used to test the protein expression of tissue-nonspecific alkaline phosphatase (TNAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2) and Runt related transcription factor 2 (Runx2), and immunofluorescence was used to examine the mIL-6R expression of HUASMCs. The comparison of measurement date between the two groups was conducted by t-test. The comparison of measurement date between multiple groups was conducted by one-way analysis of variance (ANOVA). Results:The intensity severity of calcification stain was IL-6+sIL-6R group >IL-6+sIL-6R+sgp130 group>IL-6 group=blank control. After stimulated for 12 hours, the TNAP expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.44±0.08), (0.52±0.14), (0.84±0.16) and (0.55±0.10) respectively ( F=290.96, P<0.001). After stimulated for 3 days, the OPN expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.61±0.84), (0.95±0.16), (1.65±0.24) and (0.99±0.10) respectively ( F=507.72, P<0.001). After stimulated for 12 hours, the BMP-2 expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.77±0.05), (1.69±0.16), (2.81±0.26) and (0.57±0.12) respectively ( F=959.09, P<0.001). After stimulated for 3 days, the Runx2 expression in blank control, IL-6 group, IL-6+sIL-6R group,IL-6+sIL-6R+sgp130 group were (0.57±0.03) , (0.92±0.10), (1.31±0.13) and (0.66±0.06) respectively ( F=1141.27, P<0.001). Comparing with Jurkat cells (positive control) and CEM cells (negative control), HUASMCs limited expressed mIL-6R. Conclusion:IL-6 may induce HUASMCs osteogenic differentiation and calcification mainly via the sIL-6R-mediated trans-signaling pathway.

Objective: To investigate the effect of blocking the B7/CD28 pathway on rheumatoid arthritis mice and its possible mechanism. Methods: 40 DBA/1 mice were randomly divided into normal control group, model group, CTLA4-Ig low-dose group and cytotoxic T lymphocyte antigen-4 immunolobulin(CTLA4-Ig) high-dose group. Except for the normal control group, the other three groups were prepared for rheumatoid arthritis models. At 0, 5 and 10 days after the second immunization, the CTLA4-Ig low-dose group and CTLA4-Ig high-dose group were intraperitoneally injected with 50 mg/kg and 100 mg/kg CTLA4-Ig, respectively, the normal control group and the model group were injected with the same amount of saline. Vernier calipers were used to measure the thickness of the ipsilateral hindfoot of the mouse for arthritis score; the serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) and interleukin-17(IL-17) were detected by ELISA, images were acquired and bone tissue morphometric parameters were measured through Micro-CT scanning, HE staining observed the pathological changes of the knee joint, TRAP staining detected osteoclasts in knee joint tissues, real-time fluorescent quantitative PCR and Western blot detected osteoprotegerin(OPG), receptor activator of NF-κB(RANK), receptor activator of NF-κB ligand(RANKL) mRNA and protein expression, immunohistochemical staining detected nuclear factor κB(NF-κB) expression. Western blot was used to detect the expression of related proteins in the NF-κB signaling pathway. Results: Compared with the model group, after high-dose CTLA4-Ig treatment, RA mouse foot swelling and arthritis scores were reduced, the levels of TNF-α, IL-1β, IL-6 and IL-17 in serum were reduced, the knee joint damage was significantly reduced, bone mineral density(BMD), trabecular thickness(Tb.Th), bonevolume(BV) and bone volume fraction(BV/TV) increased, the number of TRAP positive cells decreased, the relative expression of OPG mRNA and protein in the tissue was up-regulated, while the relative expression of RANK, RANKL mRNA and protein were down-regulated. At the same time, the relative expression of p-p65 and p-IκBα protein decreased, the positive expression area of NF-κB decreased, and the difference were statistically significant(P<0.01); After CTLA4-Ig low-dose treatment, the serum levels of TNF-α, IL-1β, IL-6 and IL-17 in RA mice were significantly lower than those in the model group, and the area of NF-κB positive expression in the tissue was reduced, the difference were statistically significant(P<0.01). Conclusion CTLA4-Ig to block the B7/CD28 pathway has a significant therapeutic effect on mouse rheumatoid arthritis, which can inhibit the inflammatory response, reduce bone destruction and prevent the activation of the NF-κB signaling pathway.

Objective To explore the effects of heating intravenous fluid infusion and blood transfusion based on guidelines in severe trauma patients with hypothermia. Methods A total of 40 severe trauma patients with hypothermia admitted from July 2014 to December 2015 were enrolled as the control group treated with routine measures to maintain the body temperature at normothermia by such as electrical heating blanket; other 40 severe casualties with hypothermia admitted from January 2016 to July 2017 were recruited as the warming up group treated with heating intravenous fluid infusion and blood transfusion by hot water bath in addition to the routine measures for keeping body temperature at normothermia. The differences in core body temperature, prothrombin time, activated partial thromboplastin time, incidence of shivering and mortality rate were compared between the two groups. Results There was statistically signifi cant difference in core body temperature at 0.5 h, 1.0 h, 1.5 h, 3.0 h between the two groups (P<0.05). Though the prothrombin time and shivering were improved after warming up in both groups, and there were significant differences in prothrombin time at 3.0 h after warming up and the incidence of shivering between two groups(P<0.05).There was no signifi cant difference in mean arterial pressure at all seven intervals between two groups. Conclusion The heating intravenous fl uid infusion and blood transfusion had remarkable effects to prevent hypothermia, improves blood coagulation and reduced the incidence of shivering to provide more simple and convenient warming up intervention for clinical practice.

Objective To investigate the serum concentration of soluble klotho (s-kl) in systemic lupus erythematosus (SLE) and lupus nephritis (LN) ,and elucidate its role in SLE and LN .Methods A total of 34 patients ,definitely diagnosis as SLE with un-treatment firstly ,were enrolled in this study .The patient were divided into two groups ,those who complicated with LN were assigned to LN group (15 cases) ,the others were distributed to SLE group (19 cases) .At the same time ,17 cases of routine physical examination people were take as control group .24 hours urine of all the cases was collected for examining urinary protein (Upro) .Routine hemocyte analysis ,serum biochemical parameters and ANA and dsDNA were measured by routine method .ELISA was used to detect s-kl ,25-hydroxy vitamin D (25-OH-D) and fibroblast grow th factor-23 (FGF-23) .Systemic lupus erythematosus disease activity index (SLEDAI) was performed in SLE and LN group ,and the creatinine clearance rate(CCr)was calculated accord-ing to the Cockcroft-Gault formula .Pearson′s and linear regression were applied to analyisis the correlation of relevant parameters . Results As compared with the control group ,there were statistically significant differences in mean arterial pressure ,blood rou-tine ,creatine kinase (CK) and lactate dehydrogenase (LDH ) ,complement (C3 and C4 ) and dsDNA in SLE and LN group (P <0 .05) .The level of Upro/24 h ,lipids (CHOL and TG) and creatinine (Scr) in LN group was significantly higher ,while serum al-bumin (ALB) and CCr were obviously lower than those in SLE group and control goup(P< 0 .05) .Difference of s-kl ,25-OH-D and FGF-23 in serum were not observed between SLE and control gropu(P> 0 .05) ,but the serum level of s-kl ,25-OH-D and FGF-23 in LN group were showed a statistical significance when compared with SLE or control group(P< 0 .05) .Meanwhile ,the SLEDAI score was higher in LN than in SLE group(P< 0 .05) .Correlation analysis indicted that s-kl exhibited a positive relationship with 25-OH-D ,C3 and C4 ,while showed a negative correlation with FGF-23 ,SLEDAI and dsDNA(all P< 0 .05) .However ,no any corre-lationt was revealed in regression analysis between the s-kl ,25-OH-D ,FGF-23 and the lupus activity .Conclusion The decrease of s-kl maybe one of the pivotal factors that up-regulated the level of FGF-23 in SLE and LN patients ,thus lead to the deficiency of vi-tamin D and lupus activity .