Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

OBJECTIVE: To investigate the effects of oridonin on platelet function and related mechanisms. METHODS: Washed platelets from healthy adults and mice were incubated with different concentrations of oridonin (2.5, 5 and 10 μmol/L) in vitro . The surface expression level of P-selectin and the activation of integrin αIIbβ3 in platelets were detected by flow cytometry, and the aggregation ability of platelets under the stimulation by various agonists was detected by light transmission aggregometry. The expression of P-AKT (Ser473) was detected by protein immunoblotting. Arterial thrombosis model was established in mice with mesenteric injury induced by ferric chloride, and tail hemorrhage model was established by cutting off the tail of mice. The effect of intraperitoneal injection of oridonin (10 mg/kg) on thrombosis and haemostasis was tested. RESULTS: Oridonin inhibited platelet P-selectin expression and integrin αIIbβ3 activation. In the presence of different stimulants, oridonin inhibited platelet aggregation in a concentration-dependent manner. The phosphorylation level of AKT Ser473 was reduced in the groups treated with different concentrations of oridonin. Oridonin significantly prolonged the time of mesenteric artery thrombosis in mice, but did not affect the tail bleeding time. CONCLUSION Oridonin inhibits platelet activation, aggregation, and thrombosis by inhibiting AKT phosphorylation, and may be used as a potential antiplatelet drug.
Diterpenes, Kaurane/pharmacology* ; Animals ; Mice ; Blood Platelets/drug effects* ; Platelet Aggregation/drug effects* ; P-Selectin/metabolism* ; Thrombosis ; Platelet Glycoprotein GPIIb-IIIa Complex/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Humans ; Phosphorylation ; Platelet Activation/drug effects*

Objective:To report the results of national surveillance on the distribution and antimicrobial resistance profile of clinical Gram-negative bacteria isolates from bloodstream infections in China in 2022.Methods:The clinical isolates of Gram-negative bacteria from blood cultures in member hospitals of national bloodstream infection Bacterial Resistant Investigation Collaborative System（BRICS）were collected during January 2022 to December 2022. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute（CLSI）. WHONET 5.6 and SPSS 25.0 software were used to analyze the data.Results:During the study period，9 035 strains of Gram-negative bacteria were collected from 51 hospitals，of which 7 895（87.4%）were Enterobacteriaceae and 1 140（12.6%）were non-fermenting bacteria. The top 5 bacterial species were Escherichia coli（ n=4 510，49.9%）， Klebsiella pneumoniae（ n=2 340，25.9%）， Pseudomonas aeruginosa（ n=534，5.9%）， Acinetobacter baumannii complex（ n=405，4.5%）and Enterobacter cloacae（ n=327，3.6%）. The ESBLs-producing rates in Escherichia coli， Klebsiella pneumoniae and Proteus spp. were 47.1%（2 095/4 452），21.0%（427/2 033）and 41.1%（58/141），respectively. The prevalence of carbapenem-resistant Escherichia coli（CREC）and carbapenem-resistant Klebsiella pneumoniae（CRKP）were 1.3%（58/4 510）and 13.1%（307/2 340）；62.1%（36/58）and 9.8%（30/307）of CREC and CRKP were resistant to ceftazidime/avibactam combination，respectively. The prevalence of carbapenem-resistant Acinetobacter baumannii（CRAB）complex was 59.5%（241/405），while less than 5% of Acinetobacter baumannii complex was resistant to tigecycline and polymyxin B. The prevalence of carbapenem-resistant Pseudomonas aeruginosa（CRPA）was 18.4%（98/534）. There were differences in the composition ratio of Gram-negative bacteria in bloodstream infections and the prevalence of main Gram-negative bacteria resistance among different regions，with statistically significant differences in the prevalence of CRKP and CRPA（ χ2=20.489 and 20.252， P<0.001）. The prevalence of CREC，CRKP，CRPA，CRAB，ESBLs-producing Escherichia coli and Klebsiella pneumoniae were higher in provinicial hospitals than those in municipal hospitals（ χ2=11.953，81.183，10.404，5.915，12.415 and 6.459， P<0.01 or <0.05），while the prevalence of CRPA was higher in economically developed regions（per capita GDP ≥ 92 059 Yuan）than that in economically less-developed regions（per capita GDP <92 059 Yuan）（ χ2=6.240， P=0.012）. Conclusions:The proportion of Gram-negative bacteria in bloodstream infections shows an increasing trend，and Escherichia coli is ranked in the top，while the trend of CRKP decreases continuously with time. Decreasing trends are noted in ESBLs-producing Escherichia coli and Klebsiella pneumoniae. Low prevalence of carbapenem resistance in Escherichia coli and high prevalence in CRAB complex have been observed. The composition ratio and antibacterial spectrum of bloodstream infections in different regions of China are slightly different，and the proportion of main drug resistant bacteria in provincial hospitals is higher than those in municipal hospitals.

Objective:To report the results of national surveillance on the distribution and antimicrobial resistance profile of clinical Gram-positive bacteria isolates from bloodstream infections in China in 2022.Methods:The clinical isolates of Gram-positive bacteria from blood cultures in member hospitals of National Bloodstream Infection Bacterial Resistant Investigation Collaborative System（BRICS）were collected during January 2022 to December 2022. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute（CLSI）. WHONET 5.6 and SPSS 25.0 software were used to analyze the data.Results:A total of 3 163 strains of Gram-positive pathogens were collected from 51 member units，and the top five bacteria were Staphylococcus aureus（ n=1 147，36.3%），coagulase-negative Staphylococci（ n=928，29.3%）， Enterococcus faecalis（ n=369，11.7%）， Enterococcus faecium（ n=296，9.4%）and alpha-hemolyticus Streptococci（ n=192，6.1%）. The detection rates of methicillin-resistant Staphylococcus aureus（MRSA）and methicillin-resistant coagulase-negative Staphylococci（MRCNS）were 26.4%（303/1 147）and 66.7%（619/928），respectively. No glycopeptide and daptomycin-resistant Staphylococci were detected. The sensitivity rates of Staphylococcus aureus to cefpirome，rifampin，compound sulfamethoxazole，linezolid，minocycline and tigecycline were all >95.0%. Enterococcus faecium was more prevalent than Enterococcus faecalis. The resistance rates of Enterococcus faecium to vancomycin and teicoplanin were both 0.5%（2/369），and no vancomycin-resistant Enterococcus faecium was detected. The detection rate of MRSA in southern China was significantly lower than that in other regions（ χ2=14.578， P=0.002），while the detection rate of MRCNS in northern China was significantly higher than that in other regions（ χ2=15.195， P=0.002）. The detection rates of MRSA and MRCNS in provincial hospitals were higher than those in municipal hospitals（ χ2=13.519 and 12.136， P<0.001）. The detection rates of MRSA and MRCNS in economically more advanced regions（per capita GDP≥92 059 Yuan in 2022）were higher than those in economically less advanced regions（per capita GDP<92 059 Yuan）（ χ2=9.969 and 7.606， P=0.002和0.006）. Conclusions:Among the Gram-positive pathogens causing bloodstream infections in China， Staphylococci is the most common while the MRSA incidence decreases continuously with time；the detection rate of Enterococcus faecium exceeds that of Enterococcus faecalis. The overall prevalence of vancomycin-resistant Enterococci is still at a low level. The composition ratio of Gram-positive pathogens and resistant profiles varies slightly across regions of China，with the prevalence of MRSA and MRCNS being more pronounced in provincial hospitals and areas with a per capita GDP≥92 059 yuan.

The endothelial glycocalyx(EG)is a polyglycoprotein complex present on the internal vascular surface,and its impairment is associated with the progression of multiple diseases,including atherosclerosis,stroke,sepsis,diabetes,kidney disease,hypertension,and lung edema.Therefore,glycocalyx health can be used as a biomarker to evaluate vascular health.Aging leads to dysfunctional changes in the glycocalyx;for example,its thickness decreases,and the genes of enzymes involved in its synthesis and digestion are dysregulated.As a natural barrier to the vascular system,age-related glycocalyx disruption is associated with vascular dysfunction,including impairment of vascular contraction and dilation,enhancement of permeability,dysregulation of inflammatory and immune reactions,and imbalance of anticoagulation and thrombin.From the perspective of'structure determines function'studies on the changing regularity of the thickness,components,microstructure,and mechanical properties of EG with aging and its relationship with vascular dysfunction are of great significance for the prevention,diagnosis,and treatment of atherosclerosis and other age-related cardiovascular diseases.

Objective To explore the effect of Kümmell's disease with kyphosis on the sagittal morphology of the spine-pelvis.Methods A retrospective analysis of 34 patients of Kümmell's disease with kyphosis(Kümmell group)admitted from August 2015 to September 2022,including 10 males and 24 females with an average age of(71.1±8.5)years old.A control group of 37 asymptomatic population aged(69.3±6.7)years old was matched.Spinal-pelvic sagittal parameters were measured on the anterior-posterior and lateral X-rays of the whole spine in the standing position,including segmental kyphosis(SK)or thoracolumbar kyphosis(TLK),thoracic kyphosis(TK),lumbar lordosis(LL),pelvic incidence(PI),pelvic tilt(PT),sacral slope(SS),sagittal vertical axis(SVA),T1 pelvic angle(TPA)and PI-LL.Vertebral wedge angle(WA)in Kümmell was mea-sured and differences in parameters among groups were analyzed and the relationship between spino-pelvic parameters and WA,SK were also investigated.Results TK,SK,PT,SVA,TPA and PI-LL in Kümmell group were significantly larger than those in control group(P＜0.05),LL and SS in Kümmell group were significantly decreased than those in control group(P＜0.05),and there was no significant difference in PI between two groups(P＞0.05).In Kümmell group,WA(30.8±5.9)° showed a positive correlation with SK and TK(r=0.366,0.597,P＜0.05),and SK was significantly correlated with LL and SS(r=0.539,-0.591,P＜0.05).Strong positive correlation between LL and PI,SS,SVA,TPA,PI-LL were also confirmed in patients with Kümmell with kyphosis(r=0.559,0.741,-0.273,-0.356,-0.882,P＜0.05).Conclusion Patients with Kümmell with kyphosis not only have segmental kyphosis,but also changes the overall spinal-pelvic sagittal parameters,including loss of lumbar lordosis,pelvic retrorotation,trunk forward tilt.The surgical treatment of Kümmell disease should not only pay attention to the recovery of the height of the collapsed vertebra,but also focus on the overall balance of the spine-pelvic sagittal plane for patients with kyphosis.

In this study,the immunological characteristics of the PhoP protein were explored with a two-component system of Mycobacterium tuberculosis(Mtb).Bioinformatics was used to predict the B and T cell epitopes of the PhoP protein.A re-combinant expression plasmid was constructed by PCR analysis of the phoP sequence and cloning into the prokaryotic expres-sion vector pET-28a(+).Competent Escherichia coli BL21 cells were transformed with the recombinant plasmid and expres-sion was induced with IPTG.The recombinant PhoP protein was purified by affinity chromatography.Serum levels of PhoP-specific antibodies in Mtb-infected mice and tuberculosis(TB)patients were analyzed with an ELISA.BALB/c mice were im-munized with the PhoP recombinant protein by intramuscular injection.Sera of mice were collected and antibody titers were detected with an ELISA and specificity was assessed by West-ern blot analysis.Mouse splenocytes were isolated and the pro-portions of IFN-y-positive cells and cytokine levels were detec-ted with an ELISpot and ELISA,respectively.Bioinformatics i-dentified 24 B cell and 11 T cell epitopes of the PhoP protein.A prokaryotic recombinant vector of PhoP was successfully con-structed and the recombinant PhoP protein was obtained by purification.Specific antibody levels to PhoP in sera of Mtb in-fected mice and TB patients increased significantly,with preci-sion of 99.9％and 82.5％,and specificity of 100％,respectively.PhoP protein immunization successfully induced production of specific antibodies in mice.Stimulated by antigens in vitro,IL-2 and IFN-γ levels were significantly increased in the splenocytes of immunized mice.Immunization with the PhoP protein induce a humoral immune response and Thl-dominated cellular immu-nity,indicating that the PhoP protein was immunogenic with diagnostic efficacy for TB.These results lay a foundation to clari-fy the role of PhoP in Mtb infection and application for diagnosis and prevention of TB.

Objective:To investigate the clinical characteristics and short-term follow-up outcomes of primary nephrotic syndrome (PNS) children infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the Omicron variant outbreak in Shanghai, and to provide a reliable reference for clinicians in the diagnosis and treatment.Methods:It was a case-control study. The clinical data of children with PNS (PNS group) who were diagnosed and followed-up up to 1 year in the nephrology department of four children's medical centers in Shanghai, and the children (control group) who had no underlying diseases and were infected with SARS-CoV-2 in Shanghai Jinshan Public Health Center, including the data when they were infected with SARS-CoV-2, were retrospectively analyzed.Results:(1) From March 30th to April 13th, 2022, 6 PNS children in Shanghai were infected with SARS-CoV-2, including 5 boys and 1 girl. The median age was 4.5 (2.0, 11.0) years old. And 30 children were matched by sex, age and disease type as control group, including 20 males and 10 females. The median age was 4.5 (2.0, 9.0) years. There were no significant differences between the PNS group and the control group in clinical symptoms (including fever duration), treatment regimens, vaccine doses and virus clearance time (all P>0.05). (2) The 6 children with PNS included 3 cases of steroid-sensitive type, 3 cases of steroid-resistant type, 2 cases of minimal change disease, 2 cases of focal segmental glomerulosclerosis and 2 cases with no renal biopsy. Before SARS-CoV-2 infection, their primary disease-PNS were stable, and urine protein was negative, four of them were under maintenance treatment with oral steroids or immunosuppressive drugs. At the time of SARS-CoV-2 infection, the symptoms of all of the 6 cases were mild, no severe, critical or fatal cases, and they were all cured and discharged from hospital through medical isolation observation or symptomatic treatment of infections. (3) Five cases of them still had discomfort symptoms such as cough, anorexia, and fatigue after being discharged from the hospital, which lasted for about 1 week. Within 1 year of follow-up, none of the children have suffered from "recurrent positive PCR results" or "secondary infection" of the SARS-CoV-2. (4) Among them, 4 cases of PNS relapsed after SARS-CoV-2 infection, timely addition of steroids was effective, their urine protein quickly turned negative, and there was no recurrence after 1 year of follow-up. (5) Before infection with SARS-CoV-2, the levels of immunoglobulin IgG were lower than the normal reference value in the 4 cases with PNS recurrence. Conclusions:During the Omicron variant outbreak in Shanghai, the infection of SARS-CoV-2 in children with PNS are resulted in high transmission among household contacts. Most of them have mild symptoms and good prognosis. PNS is prone to relapse after SARS-CoV-2 infection, and steroid therapy is effective and safe for these relapse. IgG may be a potential marker for the prognosis of PNS children infected with SARS-CoV-2.

Bibenzyls, a kind of important plant polyphenols, have attracted growing attention for their broad and remarkable pharmacological activities. However, due to the low abundance in nature, uncontrollable and environmentally unfriendly chemical synthesis processes, these compounds are not readily accessible. Herein, one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes. Three types of efficiently post-modifying modular strains were engineered by employing methyltransferases, prenyltransferase, and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules. Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes. Especially, a prenylated bibenzyl derivative ( 12) was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke. RNA-seq, quantitative RT-PCR, and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor, mitochondria associated 3 (Aifm3), suggesting that Aifm3 might be a new target in ischemic stroke therapy. This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.

Objective:To analyze the clinical features of 6 patients with spontaneous intracranial hypotension caused by high-flow spinal cerebrospinal fluid leaks.Methods:The clinical characteristics, auxiliary examinations, treatment, and outcomes in 6 patients of spontaneous intracranial hypotension caused by high-flow spinal cerebrospinal fluid leaks enrolled in the Xuanwu Hospital, Capital Medical University from February 2021 to April 2022 were retrospectively reviewed.Results:All the 6 patients had orthostatic headaches. Brain magnetic resonance imaging showed dural enhancement and brain sagging and magnetic resonance myelography showed longitudinal extradural collection in all the patients. The high-flow spinal cerebrospinal fluid leaks were demonstrated in upper thoracic segments by the dynamic myelography. The headache disappeared after conservative treatment in 2 patients and treatment with targeted epidural blood patch in 4 patients.Conclusions:The diagnosis of spontaneous intracranial hypotension caused by high-flow spinal cerebrospinal fluid leaks with typical orthostatic headache and brain magnetic resonance imaging and myelography findings is not difficult. However, the localization of the site of high-flow spinal cerebrospinal fluid leaks in spontaneous intracranial hypotension depends on the dynamic myelography. Targeted epidural blood patch is effective, but conservative treatment does not always work.