Objective:To investigate molecular mechanisms underlying the inflammatory response induced by Cutibacterium acnes ( C. acnes) biofilms in human primary keratinocytes. Methods:A C. acnes biofilm model was established in vitro, and confocal fluorescence microscopy was performed to examine its three-dimensional structure. The cultured human primary keratinocytes were divided into 3 groups: a dimethyl sulfoxide (DMSO) control group (treated with 0.01% DMSO alone), a C. acnes suspension group (co-incubated with C. acnes suspensions), and a C. acnes biofilm group (co-incubated with C. acnes biofilms). Real-time fluorescence-based quantitative PCR (RT-qPCR) was performed to determine the relative mRNA expression of interleukin (IL) -6, IL-8, and tumor necrosis factor (TNF) -α in the groups after 6-hour culture, enzyme-linked immunosorbent assay to detect the free protein levels of IL-6, IL-8, and TNF-α in the groups after 24-hour culture, and Western blot analysis to determine the protein expression of Toll-like receptor 2 (TLR2) in keratinocytes. In addition, some human primary keratinocytes were pretreated with key molecular blockers targeting the TLR2/mitogen-activated protein kinase (MAPK) /nuclear factor (NF) -κB signaling pathway (C29, ST2825, BAY11-7082, SB203580, U0126-EtOH), and then co-incubated with C. acnes biofilms; the DMSO control group and the C. acnes biofilm group receiving no pretreatment were simultaneously set as negative and positive controls, respectively. The mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were then detected in the above groups. One-way analysis of variance was used for comparisons among multiple groups, and the Bonferroni method was used for multiple comparisons. Results:Confocal fluorescence microscopy demonstrated a three-dimensional C. acnes biofilm structure resembling a lawn, and the biofilm grew well. RT-qPCR and ELISA showed significant differences in the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α among the C. acnes biofilm group, C. acnes suspension group and DMSO control group (mRNA: F = 89.70, 312.17, 46.09, respectively, all P < 0.001; free protein: F = 886.12, 634.25, 307.01, respectively, all P < 0.001) ; in detail, the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were significantly higher in the C. acnes biofilm group than in the C. acnes suspension group and DMSO control group (all P < 0.001) ; the C. acnes suspension group showed significantly increased expression levels of IL-6 mRNA and TNF-α free protein compared with the DMSO control group ( P < 0.001, = 0.003, respectively), while there were no significant differences in the expression of IL-6 free protein, TNF-α mRNA, or IL-8 mRNA and free protein between the 2 groups (all P > 0.05). Western blot analysis showed that the TLR2 protein expression was significantly higher in the C. acnes suspension group and C. acnes biofilm group than in the DMSO control group. After the pretreatment with molecular blockers targeting the MAPK/NF-κB signaling pathway and co-incubation with C. acnes biofilms, the mRNA and free protein expression levels of IL-6, IL-8 and TNF-α were all significantly lower in the C29 group, ST2825 group, BAY11-7082 group, SB203580 group, U0126-EtOH group, as well as in the DMSO control group compared with the C. acnes biofilm group (all P < 0.05) . Conclusion:The C. acnes biofilms exhibited a strong ability to induce inflammatory responses in human keratinocytes, possibly through the activation of the TLR2/MAPK/NF-κB signaling pathway.

Objective:Based on the specific cleavage and non-specific "trans-cleavage" activities of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein(CRISPR/Cas13), we established a visually amplification-free rapid detection technique of 2019-nCoV nucleic acid. This technique is easily processed with a low detection limit and good specificity.Methods:According to the 2019-nCoV gene sequence, specific CRISPR RNAs were screened and designed by bioinformatics analysis, and then synthesized as universal signal-strained RNA transcription targets in vitro to establish and optimize the reaction system. Moreover, the 2019-nCoV pseudoviral nucleic acid was used as a standard substance to evaluate the detection limit. A total of 65 positive samples were collected from various 2019-nCoV variants, while 48 negative samples included other clinically common respiratory pathogens, such as influenza A virus, influenza B virus, human parainfluenza virus, Klebsiella pneumonia, etc. All samples were tested by quantitative PCR (qPCR), digital PCR, and the method established in this study. The sensitivity and specificity of the newly established method were analyzed and evaluated. Results:With the newly established technique, the detection time for 2019-nCoV nucleic acid could be minimized to 6 minutes. In addition, the detection limit was 14 copies/μl when assisted by the displaying instrument, whereas it increased to 28 copies/μl with the naked eye. This technique had a sensitivity and specificity of 98.5% (66/67) and 100% (46/46) respectively, showing no statistically significant difference compared to the gold standard qPCR( P=1). Conclusions:This study has successfully established a CRISPR/Cas13a-based visually rapid detection technique for 2019-nCoV nucleic acid. This technique offers the advantages of a simple process, convenient operation, low environmental operating requirements, a detection limit close to qPCR, and a strong potential for on-site testing applications.

Objective To observe the value of 3D Res2Net deep learning model for predicting volume doubling time(VDT)of solid pulmonary nodule.Methods Chest CT data of 734 patients with solid pulmonary nodules were retrospectively analyzed.The patients were divided into progressive group(n=218)and non-progressive group(n=516)according to whether lung nodule volume increased by ≥25％during follow-up or not,also assigned into training set(n=515)and validation set(n=219)at a ratio of 7∶3.Then a clinical model was constructed based on clinical factors being significantly different between groups,CT features model was constructed based on features of nodules on 2D CT images using convolutional neural network,and 3D Res2Net model was constructed based on Res2Net network using 3D CT images as input.Receiver operating characteristic curve was drawn,and the area under the curve(AUC)was calculated.Taken actual VDT as gold standard,the efficacy of the above models for predicting solid pulmonary nodule'VDT≤400 days were evaluated.Results No significant difference of predicting efficacy for solid pulmonary nodule'VDT≤400 days was found among clinical model,CT feature model and 3D Res2Net model,the AUC of which was 0.689,0.698 and 0.734 in training set,0.692,0.714 and 0.721 in validation set,respectively.3D Res2Net model needed 5-7 s to predict VDT of solid pulmonary nodules,with an average time of(5.92±1.08)s.Conclusion 3D Res2Net model could be used to predict VDT of solid pulmonary nodules,which might obviously reduce manual interpreting time.

Objective To explore the drug resistance mechanism and characteristics of carbopenem-resistant pseudo-monas aeruginosa(CRPA)in our hospital.Methods BD phoenix 100 automatic bacterial identification and drug sensitivity an-alyzer was used to identify and detect the drug sensitivity of the strains.The minimum inhibitory concentration(MIC)of ceftazi-dime/acibactam was detected by micro broth dilution method.The modified carbapenem inactivation method(mCIM)and colloi-dal gold immunochromatography were used to detect the carbapenemase phenotype of the strains.The whole genome sequencing was used to detect the carbapenemase resistance gene and ST typing of the screened positive strains.Results A total of 22 strains of clinically isolated CRPA were collected,of which the antibacterial drugs with the lowest resistance rate were ceftidine/avibatan(22.7% ),followed by gentamicin and amikacin(27.3% ),pyracillin/tazobactam(59.09% ),cefuroxime(63.6% ).Ceftazide and aminotransferrane(77.27% ),ciprofloxacin(86.36% ),levofloxacin(95.45% ).There are a total of 5 strains(22.7% )of carbapenems in 22 CRPA by phenotypic detection.The whole genome sequencing results show that 4 strains of ST549 CRPA carry metal β-lactamase IMP-45 and serine β-lactamase OXA-1,OXA-50,one strain is ST245 CRPA carries metal β-lactamase NDM-1,that is,all five CRPA strains produce metal β-lactamase.Conclusion The resistance rate of CRPA to ceftazidime/avibactam is low in our hospital.Carbapenemase-producing is not the main mechanism of CRPA resistance to car-bapenems,while metal β-Lactamase-producing is the main mechanism of CRPA resistance to ceftazidime avibactam.

Objective:To investigate the related factors and prognosis of invasive Klebsiella pneumoniae liver abscess syndrome (IKLAS). Methods:The in-patients diagnosed with Klebsiella pneumoniae liver abscess in the Department of Infectious Diseases, Huashan Hospital, Fudan University from January 2015 to February 2021 were retrospectively enrolled. The patients were divided into IKLAS group and non-IKLAS group according to whether they had IKLAS or not. The clinical data between the two groups were compared, including the prevalence of diabetes mellitus, the details of liver abscess, clinical symptoms such as fever and abdominal pain, as well as laboratory tests such as glycosylated hemoglobin and hemoglobin. Statistical analysis was performed using chi-square test or independent sample t test. Multivariate logistic regression analysis was used to analyze the factors influencing the occurrence of IKLAS. Results:A total of 75 patients with Klebsiella pneumoniae liver abscess were enrolled, including 55 patients (73.33%) in the IKLAS group and 20 patients (26.67%) in the non-IKLAS group. Fifty-two point seven three percent (29/55) of the patients had diabetes mellitus and 12.73%(7/55) of the patients had abdominal pain in the IKLAS group, which were 20.00%(4/20) and 45.00%(9/20) in the non-IKLAS group, respectively, and the differences were both statistically significant ( χ2=6.38 and 7.28, respectively, both P<0.05). Most of liver abscesses were single (50/75, 66.67%), and more likely to occur in the right liver (50/75, 66.67%). The maximum diameter of liver abscess in the IKLAS group was (4.58±2.04) cm, which was smaller than that in the non-IKLAS group ((6.49±3.11) cm), and the difference was statistically significant ( t=2.82, P=0.011). Compared with those in the non-IKLAS group, patients in the IKLAS group had higher glycosylated hemoglobin (8.69%±2.64% vs 6.18%±1.31%) and hemoglobin ((112.25±22.04) g/L vs (100.05±18.59) g/L), and the differences were both statistically significant ( t=-4.25 and -2.21, respectively, both P<0.05). The proportion of patients using antibiotics combined with abscess drainage in the IKLAS group was 38.18%(21/55), and that in the non-IKLAS group was 85.00%(17/20). The difference between the two groups was statistically significant ( χ2=12.86, P<0.001). A total of 16 patients (21 eyes) were diagnosed as endogenous Klebsiella pneumoniae endophthalmitis (EKPE), and all of them were IKLAS patients, and 14 patients underwent monocular/binocular eyeball injection and/or vitrectomy and silicone oil filling. The visual acuity of 13 patients decreased significantly. Multivariate logistic regression analysis showed that complicated with diabetes mellitus was an independent risk factor for IKLAS (odds ratio ( OR)=5.02, 95% confidence interval (95% CI) 1.01 to 25.03, P=0.049). The large diameter of liver abscess was a protective factor for IKLAS ( OR=0.64, 95% CI 0.47 to 0.86, P=0.003). Conclusions:The patients with IKLAS have less abdominal pain, and most of them complicate with diabetes mellitus. Diabetes mellitus is an independent risk factor for the occurrence of IKLAS, while the large diameter of liver abscess is a protective factor. EKPE is associated with poor visual prognosis.

[Objective]To explore the regulatory mechanism of hypoxia exposure on carbon metabolism pathway in spleen of mice.[Methods]C57BL/6 mice were raised at altitudes of 400 m and 4 200 m,with 5 mice in each group.After 30 days,spleen tissues were aseptically removed for analysis of differentially expressed genes,proteins,and metabolites using transcriptome sequencing,proteomics,and non-targeted metabolomics.GO and KEGG enrichment analysis were conducted to explore key pathways.The key genes and protein in the pathway were validated by RT-qPCR and Western blot.[Results]Transcriptome sequencing revealed a significant difference in the expression of 4 213 genes in hypoxic exposure,of which 1 947 were up-regulated and 2 266 were down-regulated.The analysis of differentially expressed proteins showed that 166 proteins were up-regulated and 39 proteins were down-regulated.The results of non-targeted metabolomics showed that 133 different metabolites were screened under high altitude hypoxia condition,of which 95 were up-regulated and 38 were down-regulated.KEGG enrichment analysis showed that differentially expressed genes,differentially expressed proteins and differentially expressed metabolites were enriched into the carbon metabolic pathway.Therefore,the key genes and proteins in the carbon metabolic pathway were verified.The mRNA and protein expressions of PGAM2、ENO3、PRPS2、PGLS、RPE、IDH3A、SUCLA2 and MDH2 were significantly down-regulated in the carbon metabolism pathway.[Conclusion]Low oxygen environment at high altitude weakens glycolysis,tricarboxylic acid cycle and pentose phosphate pathway by inhibiting the carbon metabolism pathway of the body,resulting in oxidative stress and energy metabolism imbalance.

Objective To investigate the molecular mechanism of lipid metabolism disorder in mouse spleen tissues due to high-altitude hypoxia.Methods Ten C57BL/6 male mice were randomly divided into normoxia group(maintained at an altitude of 400 m)and high-altitude hypoxia group(maintained at 4200 m)for 30 days(n=5).Lipidomics and metabolomics analyses of the spleen tissue of the mice were conducted using liquid chromatography-mass spectrometry(LC-MS)to identify the differential metabolites,which were further analyzed by KEGG enrichment and pathway analyses,and the differential genes were screened through transcriptome sequencing.Bioinformatics analysis was conducted to identify the upstream target genes of the differential metabolites in specific metabolic pathways.RT-qPCR and Western blotting were used to detect mRNA expressions of 11β-hydroxysteroid dehydrogenase 1(HSD11B1),steroid 5α reductase 1(SRD5A1),prostaglandin-endoperoxide synthase 1(PTGS1),hematopoietic prostaglandin D synthetase(HPGDS),xanthine dehydrogenase(XDH),purine nucleoside phosphorylase(PNP),hypoxanthine guanine-phosphoribosyltransferase(HPRT)and extracellular 5'-nucleotidase(NT5E)and protein expressions of HSD11B1,SRD5A1,XDH,PNP and HPRT in the mouse spleens.Results We identified a total of 41 differential lipid metabolites in the mouse spleens,and these metabolites and the differential genes were enriched in steroid hormone biosynthesis,arachidonic acid metabolism,and purine metabolism pathways.Compared to the mice kept in normoxic conditions,the mice exposed to high-altitude hypoxia showed significantly upregulated expressions of adrenosterone,androsterone,prostaglandin D2,prostaglandin J2,xanthine,xanthosine,and uric acid in the spleen with also changes in the expression levels of HSD11B1,SRD5A1,PTGS1,HPGDS,XDH,PNP,HPRT,and NT5E.Conclusion High-altitude hypoxia can result in lipid metabolism disorder in mouse spleen tissue by affecting steroid hormone biosynthesis,arachidonic acid metabolism,and purine metabolism pathways.

Objective To investigate the molecular mechanism of lipid metabolism disorder in mouse spleen tissues due to high-altitude hypoxia.Methods Ten C57BL/6 male mice were randomly divided into normoxia group(maintained at an altitude of 400 m)and high-altitude hypoxia group(maintained at 4200 m)for 30 days(n=5).Lipidomics and metabolomics analyses of the spleen tissue of the mice were conducted using liquid chromatography-mass spectrometry(LC-MS)to identify the differential metabolites,which were further analyzed by KEGG enrichment and pathway analyses,and the differential genes were screened through transcriptome sequencing.Bioinformatics analysis was conducted to identify the upstream target genes of the differential metabolites in specific metabolic pathways.RT-qPCR and Western blotting were used to detect mRNA expressions of 11β-hydroxysteroid dehydrogenase 1(HSD11B1),steroid 5α reductase 1(SRD5A1),prostaglandin-endoperoxide synthase 1(PTGS1),hematopoietic prostaglandin D synthetase(HPGDS),xanthine dehydrogenase(XDH),purine nucleoside phosphorylase(PNP),hypoxanthine guanine-phosphoribosyltransferase(HPRT)and extracellular 5'-nucleotidase(NT5E)and protein expressions of HSD11B1,SRD5A1,XDH,PNP and HPRT in the mouse spleens.Results We identified a total of 41 differential lipid metabolites in the mouse spleens,and these metabolites and the differential genes were enriched in steroid hormone biosynthesis,arachidonic acid metabolism,and purine metabolism pathways.Compared to the mice kept in normoxic conditions,the mice exposed to high-altitude hypoxia showed significantly upregulated expressions of adrenosterone,androsterone,prostaglandin D2,prostaglandin J2,xanthine,xanthosine,and uric acid in the spleen with also changes in the expression levels of HSD11B1,SRD5A1,PTGS1,HPGDS,XDH,PNP,HPRT,and NT5E.Conclusion High-altitude hypoxia can result in lipid metabolism disorder in mouse spleen tissue by affecting steroid hormone biosynthesis,arachidonic acid metabolism,and purine metabolism pathways.

Objective Comprehensive mortality risk analyses and therapeutic assessment in real-world practice are beneficial to guide individual treatment in patients with Carbapenem-resistant Klebsiella pneumoniae bloodstream infections(CRKP-BSI).Methods Retrospective analysis of the clinical characteristics of 81 CRKP-BSI patients in our intensive care unit from July 2016 to June 2020,to indentify the risk factors of death and treatment effects of different antibiotic regimens.Results In 81 CRKP-BSI cases,the majority source were from abdominal and respiratory,accounting for 56.79%(46 cases)and 22.22%(18 cases),respectively.The 28-day mortality and hospitalization mortality of CRKP-BSI were 54.32%(44 cases)and 65.43%(53 cases).Multivariate regression analysis suggested that biliary tract disease before admission(P=0.026)and increased SOFA score at the onset of BSI(P=0.006)were independent risk factors for 28-day mortality.There was no statistically significant difference in 28-day mortality between the groups of antibiotic treatment based on tigecycline(44 cases)and polymyxin B(26 cases)[56.82%(25/44)vs.57.69%(15/26),P=0.943].Patients were evaluated based on their age(≤65 years vs.>65 years),gender,body mass index(≤25 kg/m2 vs.>25 kg/m2),and APACHEⅡ score(≤20 vs.>20),the use of renal replacement therapy and mechanical ventilation,there was no difference in the mortality among each subgroup.Conclusions Biliary tract disease before admission and SOFA score were independent risk factors for 28-day mortality.There was no significant difference outcomes between tigecycline-and polymyxin B-based therapy.

Objective: The heart contains a pool of c-kit+ progenitor cells which is believed to be able to regenerate. The differentiation of these progenitor cells is reliant on different physiological cues. Unraveling the underlying signals to direct differentiation of progenitor cells will be beneficial in controlling progenitor cell fate. In this regard, the role of the mitochondria in mediating cardiac progenitor cell fate remains unclear. Specifically, the association between changes in mitochondrial morphology with the differentiation status of c-kit+ CPCs remains elusive. In this study, we investigated the relationship between mitochondrial morphology and the differentiation status of c-kit+ progenitor cells. Methods: and Results: c-kit+ CPCs were isolated from 2-month-old male wild-type FVB mice. To activate differentiation, CPCs were incubated in α-minimal essential medium containing 10 nM dexamethasone for up to 7 days. To inhibit Drp1-mediated mitochondrial fragmentation, either 10 μM or 50 μM mdivi-1 was administered once at Day 0 and again at Day 2 of differentiation. To inhibit calcineurin, either 1 μM or 5 μM ciclosporin-A (CsA) was administered once at Day 0 and again at Day 2 of differentiation. Dexamethasone-induced differentiation of c-kit+ progenitor cells is aligned with fragmentation of the mitochondria via a calcineurin-Drp1 pathway. Pharmacologically inhibiting mitochondrial fragmentation retains the undifferentiated state of the c-kit+ progenitor cells. Conclusions The findings from this study provide an alternative view of the role of mitochondrial fusion-fission in the differentiation of cardiac progenitor cells and the potential of pharmacologically manipulating the mitochondria to direct progenitor cell fate.