(+)-Borneol, the main component of "Natural Borneol" in the Chinese Pharmacopoeia, is a high-end spice and precious medicine. Plant extraction cannot meet the increasing demand for (+)-borneol, while microbial biosynthesis offers a sustainable supply route. However, its production was extremely low compared with other monoterpenes, even with extensively optimizing the mevalonate pathway. We found that the key challenge is the complex and unusual dephosphorylation reaction of bornyl diphosphate (BPP), which suffers the side-reaction and the competition from the cellular dephosphorylation process, especially lipid metabolism, thus limiting (+)-borneol synthesis. Here, we systematically optimized the dephosphorylation process by identifying, characterizing phosphatases, and balancing cellular dephosphorylation metabolism. For the first time, we identified two endogenous phosphatases and seven heterologous phosphatases, which significantly increased (+)-borneol production by up to 152%. By engineering BPP dephosphorylation and optimizing the MVA pathway, the production of (+)-borneol was increased by 33.8-fold, which enabled the production of 753 mg/L under fed-batch fermentation in shake flasks, so far the highest reported in the literature. This study showed that rewiring dephosphorylation metabolism was essential for high-level production of (+)-borneol in Saccharomyces cerevisiae, and balancing cellular dephosphorylation is also helpful for efficient biosynthesis of other terpenoids since all whose biosynthesis involves the dephosphorylation procedure.

【Objective】 To objectively evaluate the quality control level of blood testing process in blood banks through quantitative monitoring and trend analysis, and to promote the homogenization level and standardized management of blood testing laboratories in blood banks. 【Methods】 A quality monitoring indicator system covering the whole process of blood collection and supply, including blood donation service, blood component preparation, blood testing, blood supply and quality control was established. The questionnaire Quality Monitoring Indicators for Blood Collection and Supply Process with clear definition of indicators and calculation formulas was distributed to 17 blood banks in Shandong province. Quality monitoring indicators of each blood bank from January to December 2022 were collected, and 31 indicators in terms of blood testing were analyzed using SPSS25.0 software. 【Results】 The proportion of unqualified serological tests in 17 blood bank laboratories was 55.84% for ALT, 13.63% for HBsAg, 5.08% for anti HCV, 5.62% for anti HIV, 18.18% for anti TP, and 1.65% for other factors (mainly sample quality). The detection unqualified rate and median were (1.23±0.57)% and 1.11%, respectively. The ALT unqualified rate and median were (0.74±0.53)% and 0.60%, respectively. The detection unqualified rate was positively correlated with ALT unqualified rate (r=0.974, P<0.05). The unqualified rate of HBsAg, anti HCV, anti HIV and anti TP was (0.15±0.09)%, (0.05±0.04)%, (0.06±0.03)% and (0.20±0.05)% respectively. The average unqualified rate, average hemolysis rate, average insufficient volume rate and the abnormal hematocrit rate of samples in 17 blood bank laboratories was 0.21‰, 0.08‰, 0.01‰ and 0.02‰ respectively. There were differences in the retest concordance rates of four HBsAg, anti HCV and anti HIV reagents, and three anti TP reagents among 17 blood bank laboratories (P<0.05). The usage rate of ELISA reagents was (114.56±3.30)%, the outage rate of ELISA was (10.23±7.05) ‰, and the out of range rate of ELISA was (0.90±1.17) ‰. There was no correlation between the out of range rate, outrage rate and usage rate (all P>0.05), while the outrage rate was positively correlated with the usage rate (r=0.592, P<0.05). A total of 443 HBV DNA positive samples were detected in all blood banks, with an unqualified rate of 3.78/10 000; 15 HCV RNA positive samples were detected, with an unqualified rate of 0.13/10 000; 5 HIV RNA positive samples were detected, with an unqualified rate of 0.04/10 000. The unqualified rate of NAT was (0.72±0.04)‰, the single NAT reaction rate [(0.39±0.02)‰] was positively correlated with the single HBV DNA reaction rate [ (0.36±0.02) ‰] (r=0.886, P<0.05). There was a difference in the discriminated reactive rate by individual NAT among three blood bank laboratories (C, F, H) (P<0.05). The median resolution rate of 17 blood station laboratories by minipool test was 36.36%, the median rate of invalid batch of NAT was 0.67%, and the median rate of invalid result of NAT was 0.07‰. The consistency rate of ELISA dual reagent detection results was (99.63±0.24)%, and the median length of equipment failure was 14 days. The error rate of blood type testing in blood collection department was 0.14‰. 【Conclusion】 The quality monitoring indicator system for blood testing process in Shandong can monitor potential risks before, during and after the experiment, and has good applicability, feasibility, and effectiveness, and can facilitate the continuous improvement of laboratory quality control level. The application of blood testing quality monitoring indicators will promote the homogenization and standardization of blood quality management in Shandong, and lay the foundation for future comprehensive evaluations of blood banks.

Objective To analyze the influencing factors for intestinal colonization and secondary infection of car-bapenem-resistant Klebsiella pneumoniae(CRKP)in neonates,and provide a basis for formulating prevention and control strategies for CRKP infection.Methods Neonates who were admitted to the neonatal ward of a hospital from January 2021 to October 2022 were selected as the study subjects,and the first screening of CRKP was con-ducted within 48 hours after admission.In addition,active anal swab screening for carbapenem-resistant Ente-robacterales(CRE)was performed weekly during hospitalization,and the infection status of CRKP strains was mo-nitored.Clinical data of neonates in the colonization group,non-colonization group,and infection group were ana-lyzed.Intestinal colonized strains and the non-repetitive CRKP strains isolated from clinical specimens of neonates with secondary infection after colonization were performed carbapenemase gene detection,multilocus sequence ty-ping(MLST)and pulsed-field gel electrophoresis(PFGE)analysis.Results A total of 1 438 neonates were active-ly screened for CRE,174 were CRKP positive,CRKP colonization rate was 12.1％.Among 174 neonates,35 were with secondary infection,with the incidence of 20.1％.The independent risk factors for neonatal CRKP intestinal colonization were cesarean section(OR=2.050,95％CI:1.200-3.504,P=0.009),use of cephalosporins(OR=1.889,95％CI:1.086-3.288,P=0.024),nasogastric tube feeding(OR=2.317,95％CI:1.155-4.647,P=0.018).Protective factors were breast-feeding(OR=0.506,95％CI:0.284-0.901,P=0.021),oral probiotics(OR=0.307,95％CI:0.147-0.643,P=0.002),and enema(OR=0.334,95％CI:0.171-0.656,P=0.001).Independent risk factors for secondary infection after intestinal colonization of neonatal CRKP were carbapenem anti-biotic use(OR=19.869,95％CI:1.778-222.029,P=0.015)and prolonged hospital stay(OR=1.118,95％CI:1.082-1.157,P＜0.001).The detection results of drug resistance genes showed that carbapenemase-producing genes of CRKP strains were all blaKPC-2,all belonged to type ST11.Homologous analysis showed that intestinal CRKP colonization was highly homologous with the secondary infection strains after colonization.Conclusion CRKP intestinal colonization during neonatal hospitalization may increase the risk of CRKP infection.Risk and pro-tective factors of neonatal intestinal colonization and secondary infections after colonization should be paid attention,and corresponding preventive and control measures should be taken,so as to reduce the occurrence and transmission CRKP healthcare-associated infection.

Objective To understand the spectrum and changes of pathogens causing healthcare-associated infec-tion(HAI)in neonatal intensive care unit(NICU).Methods Clinical medical records of neonates with HAI in a hospital from January 2018 to December 2022 were collected,spectrum of pathogens causing HAI were and analyzed retrospectively.Results A total of 7 597 hospitalized neonates were investigated,and 240 of whom had 263 cases of HAI,with an HAI incidence of 3.16％and healthcare-associated case infection incidence of 3.46％.96 cases(36.50％)were bloodstream infection,70(26.62％)were respiratory system infection,and 57(21.67％)were in-fection without clear sites.A total of 170 pathogens were detected from specimens,78(45.88％)of which were Gram-positive bacteria,with Staphylococcus spp.accounting for the highest proportion,78(45.88％)were Gram-negative bacteria,mainly Klebsiella pneumoniae,and 14(8.24％)were fungi.The detection rate of Gram-negative bacteria showed an upward trend from 2018 to 2022(P＜0.01).Conclusion The majority of HAI in NICU is bloodstream infection.In recent years,the detection rate of Gram-negative bacteria has been increasing year by year,and it is necessary to streng-then the prevention and control of HAI in clinical practice.

Objective:To analyze the incidence of co-infection of HIV and HBV and death in HIV/AIDS cases who newly received antiretroviral therapy (ART) from 2005-2020 in Jiangsu Province.Methods:According to the baseline and follow-up data of HIV/AIDS cases on ART enrolled between January 2005 and December 2020, the last follow-up clinical visit was up until December 31, 2022, the national information system was retrospectively collected for HIV/AIDS cases from Chinese System Disease for Control and Prevention. Excel database was established, and statistical analysis was performed using the SPSS 16.0 software. Kaplan-Meier method was used to draw the survival curves, the log rank test was used to compare the survival curves, and Cox proportional hazards modeling was used to assess the mortality and potential risk factors.Results:There were 33 322 HIV/AIDS cases that newly received ART during 2005-2020.The rate of HBsAg test was 57.3%(19 098/33 322). Among HIV/AIDS cases tested HBsAg, the ratio of male to female was 7.1∶1 (16 745∶2 353), the average age was (39.4±14.0) years old, 49.5% (9 446/19 098) of the HIV/AIDS cases were married, 57.8% (11 048/19 098) were infected with HIV through homosexual contact and 36.6% (6 990/19 098) were through heterosexual contact. The M ( Q1, Q3) of CD4 +T lymphocytes (CD4) counts at ART initiation was 297 (166, 445) cells/μl. A total of 8.2% (1 566/19 098, 95% CI:7.8%-8.6%) were HBsAg positive. There were 1 062 HIV/AIDS died by December 31, 2022. The log rank test showed that there were differences in survival curves between HIV/AIDS co-infected with HBV or not ( χ2=28.07, P<0.001). Multivariate analysis of the Cox proportional risk regression model showed that enrollment year, age, marital status, route of HIV infection, baseline CD4 counts before ART, and co-HBV infection were the influencing factors for HIV/AIDS death (all P<0.05), compared with those enrolled in 2015 and before, age ≥45 years, and those who were unmarried. Those enrolled in treatment from 2016 to 2020, those younger than 45 years, and married/cohabitation had a lower risk of death. Compared with baseline CD4 counts ≥201 cells/μl, other routes of infection, and HIV infection alone, baseline CD4 counts ≤200 cells/μl, injecting drug use, and co-HBV infection were associated with a higher risk of death. Conclusion:Effective treatment for coinfection with HBV and HBV vaccination for HBV-negative people with HIV should be integrated into HIV treatment programs to reduce HIV-related mortality in Jiangsu Province, 2005-2020.

AIM To investigate the effects of Zhuangyao Shuanglu Tongnao Formula on neuronal apoptosis in rats with cerebral ischemia-reperfusion injury based on the study of oxidative stress and inflammatory response.METHODS The rats were randomly divided into the sham operation group,the model group,the edaravone group(3.0 mg/kg),the low,medium and high dose groups(9.0,18.0,36.0 g/kg)of Zhuangyao Shuanglu Tongnao Formula,with 18 rats in each group.The middle cerebral artery occlusion/reperfusion was conducted by thread embolism method to simulate cerebral ischemia reperfusion injury in rats followed by 6 days corresponding drugs administration.Subsequently,the rats had their neurological function deficit scored by Zeal Longa scoring method;their sizes of cerebral infarction areas measured by TTC staining;their pathological damage and apoptosis of neurons in hippocampal CA1 area of ischemic penumbra of the brain tissue detected by HE staining and TUNEL staining;their SOD activity and levels of GSH,MDA,IL-6,IL-1β,TNF-α in brain tissue detected by kits;and their protein expressions of Bax,Bcl-2,caspase-3,cleaved-capase-3,TLR4,NF-κB p65,Nrf2,HO-1 in rat brain tissue determined by Western blot.RESULTS Compared with the model group,the groups intervened with edaravone,medium and high dose of Zhuangyao Shuanglu Tongnao Formula displayed improvements in the scores of nerve function defects,the rate of cerebral infarction,the rate of neuronal apoptosis,the levels of IL-6,IL-1β,TNF-α and MDA in the ischemic penumbra of brain tissues,the protein expressions of Bax and TLR4,the ratio of cleaved-capase-3/caspase-3 and p-NF-κB p65/NF-κB p65(P＜0.05),the levels of GSH,the activity of SOD and the protein expressions of Bcl-2,Nrf2 and HO-1(P＜0.05).CONCLUSION Being an inhibitor of oxidative stress and inflammatory response,Zhuangyao Shuanglu Tongnao Formula can alleviate brain injury in rats with cerebral ischemia reperfusion injury through the inhibition of neuronal apoptosis and improvement of neural function mediated by the inhibition of TLR4/NF-κB signal pathway and activation of Nrf2/HO-1 signal pathway.

Objective To investigate the expression of lymphoid enhancer binding factor 1(LEF1)in B cell chronic lymphoproliferative disorders(B-CLPD)and estimate its value in the differential diagnosis of the subtype of B-CLPD.Methods A retrospective study was conducted on 58 patients diagnosed with B-CLPD by using bone marrow biopsy samples or lymphnode biopsy samples from Hematology Department of The Second People′s Hospital of Wuhu from September 2018 to June 2023,as well as 20 bone marrow biopsy samples which were diagnosis as non-hematologic malignancy in the control group.Immunohistochemical method was used to detect the expression of LEF1 in 78 samples,and statistical analysis was conducted.Results In all 78 cases,16 of 20 chronic lymphocytic leukemia(CLL)patients were LEF1 positive,the positive rate was 80%;mantle cell lymphoma 1/12;Follicular lymphoma 1/5;marginal zone cell lymphoma 0/11;Lymphoplasmacyticlymphom 0/8;hairy cell leukemia 0/2;in Control group no patient was LEF1 positive(P = 0.000).The expression of LEF1 is correlated with CD200 and CLL score(P<0.05).In the LEF1 negative group with 4 CLL patients,2 were detected with +12 chromosomal abnormality,the detective rate was higher than that of the LEF1 positive group(P>0.05).Conclusion LEF1 was a sensitive and specific diagnosis marker in CLL and B-CLPD subtype.

Objective:To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation for the treatment of combined immunodeficiency (CID) and explore prognostic risk factors.Methods:In this retrospective cohort study, clinical characteristics, laboratory tests and prognosis of 73 CID children who underwent allogeneic hematopoietic stem cell transplantation from February 2014 to April 2022 in the Children′s Hospital of Fudan University were analyzed. Based on the subtypes of diseases, all patients were divided into severe combined immunodeficiency disease (SCID) group and other CID group. Based on the types of donors, all patients were divided into matched sibling donor group, matched unrelated donor group, unrelated cord blood group, and haploidentical donor group. Kaplan-Meier method and Log-Rank test were used to analyze the survival data. Cox regression was used to analyze prognostic factors.Results:Among the 73 patients, there were 61 (84%) males and 12 (16%) females. Fifty-five (75%) patients were SCID, and 18 (25%) patients were other CID. Donor source included 2 (3%) matched sibling donors (MSD), 3 (4%) matched unrelated donors (MUD), 64 (88%) unrelated cord blood (UCB), and 4 (5%) haploidentical donors. The age at transplant was 10.7 (5.9, 27.5) months, and the follow-up time was 36.2 (2.5, 62.9) months. The 3-year overall survival rate of 73 patients with CID was (67±6) %. No significant difference was found in the 3-year overall survival rates between patients with SCID (55 cases) and other CID (18 cases) ((64±7) % vs. (78±10) %, χ2=1.31, P=0.252). And no significant difference was found in the 3-year overall survival rates among patients who received MSD or MUD (5 cases), UCB (64 cases), and haploidentical donor (4 cases) transplant (100% vs. (66±6)% vs. (50±25) %, χ2=2.30, P=0.317). Cox regression analysis showed that the medical history of sepsis ( HR=2.55, 95% CI 1.05-6.20, P=0.039) and hypoalbuminemia at transplant ( HR=2.96, 95% CI 1.14-7.68, P=0.026) were independent risk factors for the prognosis of allogeneic hematopoietic stem cell transplantation in pediatric patients with CID. Conclusions:Allogeneic hematopoietic stem cell transplantation is an effective treatment for CID. The medical history of sepsis and hypoalbuminemia at transplant were risk factors for prognosis. Enhancing infection prevention and nutritional intervention before transplant can improve patient prognosis.

The post-translational modification of proteins is a key mechanism that imparts physiological functions to proteins,among which reversible phosphorylation modifications play a pivotal role in many biological processes.Aberrant changes in phosphorylation are often closely associated with various major disease processes.In recent years,with the aid of proteomic technologies and methods,high-throughput,high-precision qualitative and quantitative approaches for phosphorylated proteins have rapidly advanced.This article reviews the research progress of phosphorylated protein quantification and chemical proteomics analysis methods based on the"bottom-up"strategy,including phosphopeptide enrichment methods,mass spectrometry fragmentation methods,quantification analysis methods and phosphorylation site stoichiometry,and discusses the development trend of quantification and stoichiometric analysis methods for phosphorylated proteins.

Objective To evaluate the biochemical indicators,nutritional status,and immune levels of pa-tients with pulmonary aspergillosis(PA)and other pulmonary diseases,and to construct a predictive model for PA so as to improve the diagnostic efficacy of clinical PA.Methods A total of 40 PA patients and 39 pa-tients with other pulmonary diseases who were hospitalized in the hospital from January 2020 to August 2022 were retrospectively analyzed.The expression trends and differences of serum 1,3-β-D Glucan(G test),galac-tomannan test(GM test),biochemical indexes,blood routine indexes and immune cell subsets were analyzed and compared.The receiver operating characteristic(ROC)curve and binary Logistic regression analysis were used to construct the predictive model for PA by the combination of clinical indicators.Results Serum GM test,G test,albumin,hemoglobin,hematocrit,lymphocytes,B lymphocytes,CD44 T lymphocytes and CD4/CD8 ratio displayed significant differences between PA patients and patients with other lung disease(P＜0.05).The levels of GM test in alveolar lavage fluid of PA patients were significantly higher than that in the serum,and the differences were statistically significant(P＜0.05).The ROC curve analysis showed that the GM test,as an independent predictor of PA,had good predictive accuracy[0.85＜area under the curve(AUC)＜0.95].Besides,albumin,natural killev cells,CD4+T lymphocytes and CD4/CD8 ratio had general predictive efficacy(0.70＜AUC＜0.85).The prediction efficacy of G test and B lymphocytes was poor(AUC＜0.70).The Logistic regression analysis showed that the combination of GM test and serum albumin could construct the optimal prediction model,and the prediction formula of the combined model was as fol-lows:Logit(P)=17.781× GM-0.131×albumin+1.394.The prediction accuracy of the combined model was 0.924(95％CI:0.865-0.982),the sensitivity was 87.5％,the specificity was 81.2％,and the cut off value was 17.781×GM-0.131×albumin-1.735.Conclusion This study retrospectively analyzed the differences in various clinical indicators between patients with PA and patients with other pulmonary diseases,and then screen the key clinical indicators as candidate predictors which displayed significantly different ex-pression between the two groups.The optimal prediction model for the diagnosis of PA is constructed by the combination of GM test and serum albumin through ROC curve and Logistic regression analysis.This model may significantly improve the diagnostic efficiency of PA in clinical,and provide the reference for the early di-agnosis and effective treatment of PA patients.