The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

Human exhaled breath has great application prospects,e.g.,monitoring pharmacokinetics,disease diagnosis,due to its advantages such as non-invasive and high-frequency sampling.Breath samples can be collected from the oral and nasal cavity.However,the oral and nasal environment affect the chemical composition of breath sample.Therefore,the investigation on the chemical composition of mouth-exhaled breath and nose-exhaled breath is crucial for selection of appropriate sampling strategy for individual studies.In this work,secondary electrospray ionization-high resolution mass spectrometry(SESI-HRMS)was applied to analysis of respiratory metabolomics in real time.A quantitative analysis approach was established for 9 kinds of volatile organic compounds(VOCs)e.g.2-butanone,2-pentanone,ethyl acetate,methyl methacrylate,toluene,styrene,mesitylene,isoprene and limonene.The limit of detection was 2.3?240.8 ng/m3.The intra-day(n=6)and inter-day(n=18)relative standard deviations were 0.6%?4.6%and 4.3%?12.2%,respectively.Nine healthy subjects were recruited to investigate the chemical composition of mouth-exhaled and nose-exhaled breath.The results showed the good performance in quantitative analysis of 9 VOCs in breath air.It was found that the number of unique component(m/z)detected in mouth-exhaled breath(167)was 2.2 times greater than that detected in nose-exhaled breath(76),which might result from the complex environment in oral cavity.The signal intensity of commun component(163)was significantly different between mouth-exhaled breath and nose-exhaled breath.Additionally,the elemental composition analysis showed that the proportion of polar compounds detected in nose-exhaled breath was higher than that in mouth-exhaled breath.This study demonstrated that there was significant differences in the chemical composition between mouth-exhaled and nose-exhaled breath,which provided a theoretical basis for selection of exhalation mode.

Objective:To explore the clinical application and effect of FilmArray ? meningitis/encephalitis (ME) panel in identifying pathogens of central nervous system (CNS) infections in children. Methods:Molecular biology study.Cerebrospinal fluid (CSF) samples were prospectively obtained through lumbar puncture from children with suspected CNS infections admitted to the Department of Infectious Diseases at Beijing Children′s Hospital, Capital Medical University from May to November 2019.These samples were subjected to both routine clinical pathogen testing and FilmArray ME panel testing.Polymerase chain reaction was used to validate all samples.Independent sample t-test or Mann-Whitney U-test was used for comparative analysis of the results and influence factors obtained by the two detection methods. Results:A total of 113 cases of suspected CNS infections were enrolled.Routine clinical testing yielded 17 cases, with a positive rate of 15.0%, including 4 positive CSF cultures, with a positivity rate of 3.5%.FilmArray ME panel detected 23 positive cases, with a positive rate of 20.4%.FilmArray ME panel detected bacteria in 7 cases, viruses in 13 cases, fungi in 1 case, and both viruses and bacteria in 2 cases.Among the common pathogens detected, FilmArray ME panel obtained the results on average 2.7 days in advance.Conclusions:Compared with CSF culture, FilmArray ME panel has the advantages of shorter detection period, higher positive detection rate, and higher virus detection rate.

Redox-sensitive cysteine(RSC)thiol plays an important role in many biological processes such as photosynthesis,cellular metabolism,and transcription.Therefore,it is necessary to identify red-ox-sensitive cysteine accurately.However,traditional redox-sensitive cysteine identification is very ex-pensive and time-consuming.At present,there is an urgent need for a mathematical calculation method to identify sequence information and redox-sensitive cysteines quickly and accurately.Here,we devel-oped an effective predictor called iRSC-PseAAC,which used the dimension reduction algorithm LDA combined with the support vector machine to predict redox-sensitive cysteine sites.In the cross-validation results,the specificity(Sp),sensitivity(Sn),accuracy(Acc)and Matthews correlation coefficient(MCC)were 0.841,0.868,0.859 and 0.692 respectively.In the independent dataset results,the Sp,Sn,Acc and MCC were 0.906,0.882,0.890 and 0.767 respectively.compared with existing prediction methods,iRSC-PseAAC had obvious improvement effect.The method proposed for this study can also be used for many problems in computational proteomics.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

Epigenomic imbalance drives abnormal transcriptional processes,promoting the onset and progression of cancer.Although defective gene regulation generally affects carcinogenesis and tumor suppression networks,tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes,which may have significant implications for the development and application of epigenetic therapy,cancer immunotherapy,and their combinations.Herein,we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes,DNA methylation,histone post-translational modification,and chromatin structure in tumor immunogenicity,and introduce these epigenetic research methods.We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immuno-therapy through the complex interaction between cancer epigenetics and cancer immunology.

The Dionex CaboPac^TM PA10 BioLC^TM Analyical 2 mm × 250 mm column was used with a protective column (Dionex CaboPac^TM PA10 BioLC^TM Guard 2 mm × 50 mm). 100 mmol·L^-1 sodium hydroxide solution was used as eluent; the flow rate was 0.25 mL·min^-1. Sample tray temperature: 35 ℃. The pulse amperometric detector was adopted, and the waveform was Gold CWE, Ag-AgCl RE, Carbo, Quad. The samples were cultured with 8 concentrations of glycogen substrates (0.31, 1.25, 2.5, 5, 10, 20, 30, and 40 mg·mL^-1). D-Glucose concentrations were measured at 5 different time points (T0, T1, T2, T3 and T4). The glucose concentration from T1 to T4 minus the glucose concentration at T0. The reaction rate was calculated at different glycogen substrate concentrations. These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation. The kinetic parameters were expressed as V_max (nmol·mg^-1·min^-1) and K_m (mg·mL^-1). The RSD of glucose standard curve R² (n = 6, linear range: 1.25-500 μmol·L^-1) was 0.1% and the RSD (n = 6) of the slope of the standard curve was 2.2%. The mean limit of quantitation was 0.14 μmol·L^-1, and the mean limit of detection was 0.05 μmol·L^-1. The RSD of K_m and V_max were 4.4% and 4.6% respectively in three separate experiments. The durability of the method was good. The method was developed for the on-line automatic determination of the hydrolysis kinetics of acid α-glucosidase (GAA) for injection by ion chromatography. The method has good precision, repeatability and durability, and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection, and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.

According to the requirements of the regulatory authorities, degree of modification (DP) should be included in the characterisation of the PEGylated protein drug substance, and is one of the critical quality attributes for quality control. In this study, based on the fundamental assumption that the refractive index (RI) signal and the ultraviolet (UV) signal of PEGylated protein are equal to the sum of the corresponding signal produced by the polyethylene glycol (PEG) and protein parts of the conjugates in their uncoupled state, we developed a method to determine the DP of PEGylated recombinant human growth hormone (inpegsomatropin). In this method, 20 μL of 1 mg·mL^-1 human growth hormone (hGH) standard, 2 mg·mL^-1 PEG reference substance and 1 mg·mL^-1 drug substance solution were each injected to size exclusion chromatographic (SEC) column for separation, detected with ultraviolet and refractive index (UV-RI) detectors in series. Finally, the DP was calculated as the formula derived from the fundamental assumption. The developed SEC-UV-RI method showed good specificity, repeatability (RSD = 0.63%, n = 9) and accuracy, with a recovery of 100.0%, compared with the result obtained from the simultaneously established classical acid hydrolysis method, demonstrating pretty handleability and accessibility, and is appropriate for quality control test of DP for this drug substance.

OBJECTIVE: To derive the Chinese medicine (CM) syndrome classification and subgroup syndrome characteristics of ischemic stroke patients. METHODS: By extracting the CM clinical electronic medical records (EMRs) of 7,170 hospitalized patients with ischemic stroke from 2016 to 2018 at Weifang Hospital of Traditional Chinese Medicine, Shandong Province, China, a patient similarity network (PSN) was constructed based on the symptomatic phenotype of the patients. Thereafter the efficient community detection method BGLL was used to identify subgroups of patients. Finally, subgroups with a large number of cases were selected to analyze the specific manifestations of clinical symptoms and CM syndromes in each subgroup. RESULTS: Seven main subgroups of patients with specific symptom characteristics were identified, including M3, M2, M1, M5, M0, M29 and M4. M3 and M0 subgroups had prominent posterior circulatory symptoms, while M3 was associated with autonomic disorders, and M4 manifested as anxiety; M2 and M4 had motor and motor coordination disorders; M1 had sensory disorders; M5 had more obvious lung infections; M29 had a disorder of consciousness. The specificity of CM syndromes of each subgroup was as follows. M3, M2, M1, M0, M29 and M4 all had the same syndrome as wind phlegm pattern; M3 and M0 both showed hyperactivity of Gan (Liver) yang pattern; M2 and M29 had similar syndromes, which corresponded to intertwined phlegm and blood stasis pattern and phlegm-stasis obstructing meridians pattern, respectively. The manifestations of CM syndromes often appeared in a combination of 2 or more syndrome elements. The most common combination of these 7 subgroups was wind-phlegm. The 7 subgroups of CM syndrome elements were specifically manifested as pathogenic wind, pathogenic phlegm, and deficiency pathogens. CONCLUSIONS There were 7 main symptom similarity-based subgroups in ischemic stroke patients, and their specific characteristics were obvious. The main syndromes were wind phlegm pattern and hyperactivity of Gan yang pattern.
Humans ; Syndrome ; Ischemic Stroke ; Medicine, Chinese Traditional ; Liver ; Phenotype

Itch is an unpleasant sensation that provokes the desire to scratch. While acute itch serves as a protective system to warn the body of external irritating agents, chronic itch is a debilitating but poorly-treated clinical disease leading to repetitive scratching and skin lesions. However, the neural mechanisms underlying the pathophysiology of chronic itch remain mysterious. Here, we identified a cell type-dependent role of the anterior cingulate cortex (ACC) in controlling chronic itch-related excessive scratching behaviors in mice. Moreover, we delineated a neural circuit originating from excitatory neurons of the ACC to the ventral tegmental area (VTA) that was critically involved in chronic itch. Furthermore, we demonstrate that the ACC→VTA circuit also selectively modulated histaminergic acute itch. Finally, the ACC neurons were shown to predominantly innervate the non-dopaminergic neurons of the VTA. Taken together, our findings uncover a cortex-midbrain circuit for chronic itch-evoked scratching behaviors and shed novel insights on therapeutic intervention.
Mice ; Animals ; Gyrus Cinguli/physiology* ; Pruritus/pathology* ; Mesencephalon ; Cerebral Cortex/pathology* ; Neurons/pathology*