Collateral sensitivity between tetracyclines and aminoglycosides constrains resistance evolution in carbapenem-resistant Klebsiella pneumoniae.

AIMS: The acquisition of resistance to one antibiotic may confer an increased sensitivity to another antibiotic in bacteria, which is an evolutionary trade-off between different resistance mechanisms, defined as collateral sensitivity (CS). Exploiting the role of CS in treatment design could be an effective method to suppress or even reverse resistance evolution. METHODS: Using experimental evolution, we systematically studied the CS between aminoglycosides and tetracyclines in carbapenem-resistant Klebsiella pneumoniae (CRKP) and explored the underlying mechanisms through genomic and transcriptome analyses. The application of CS-based therapies for resistance suppression, including combination therapy and alternating antibiotic therapy, was further evaluated in vitro and in vivo. RESULTS: Reciprocal CS existed between tetracyclines and aminoglycosides in CRKP. The increased sensitivity of aminoglycoside-resistant strains to tetracyclines was associated with the alteration of bacterial membrane potential, whereas the unbalanced oxidation-reduction process of tetracycline-resistant strains may lead to an increased bacterial sensitivity to aminoglycosides. CS-based combination therapy could efficiently constrain the evolution of CRKP resistance in vitro and in vivo. In addition, alternating antibiotic therapy can re-sensitize CRKP to previously resistant drugs, thereby maintaining the trade-off. CONCLUSIONS: These results provide new insights into constraining the evolution of CRKP resistance through CS-based therapies.

Metabolic landscape dysregulation in bronchoalveolar lavage fluid of checkpoint inhibitor pneumonitis.

BACKGROUND: Checkpoint inhibitor pneumonitis (CIP) is a potentially fatal adverse event resulting from immunotherapy in patients with malignant tumors. However, the pathogenesis of CIP remains poorly understood. METHODS: We collected bronchoalveolar lavage fluid (BALF) from cohorts of patients with CIP, new-onset lung cancer (LC), and idiopathic pulmonary fibrosis (IPF). Non-targeted metabolomics analysis was conducted to analyze metabolic signatures. Flow cytometry was used to evaluate immune cell subsets. RESULTS: Lymphocytes were predominant in the BALF of patients with CIP. A total of 903 metabolites were identified, among which lipid compounds were the most abundant. In a comparison between patients with CIP and LC, enrichment analysis of the altered metabolites showed suppressed amino sugar metabolism, and spermidine and spermine biosynthesis in the CIP group. Metabolism of alpha linolenic acid, linoleic acid, and their fatty acid derivatives was enriched in the CIP group relative to the IPF group. The twelve metabolites found to be enriched in the CIP group were positively correlated with the proportion of CD8+ T cells. One cluster of BALF metabolites, 57.14% of which were lipid molecules, was inversely correlated with the proportion of natural killer cells. CONCLUSIONS: In this study, the metabolomic landscape of BALF in patients with CIP was determined. We elucidated suppressed tumor metabolic signatures, enhanced pulmonary inflammatory signaling, and the characteristics of responsible immune cells, which helps to understand the pathogenesis of CIP.

Relationship between clinical features and droplet digital PCR copy number in non-HIV patients with pneumocystis pneumonia.

OBJECTIVE: Droplet digital PCR (ddPCR) is a novel assay to detect pneumocystis jjrovecii (Pj) which has been defined to be more sensitive than qPCR in recent studies. We aimed to explore whether clinical features of pneumocystis pneumonia (PCP) were associated with ddPCR copy numbers of Pj. METHODS: A total of 48 PCP patients were retrospectively included. Pj detection was implemented by ddPCR assay within 4 h. Bronchoalveolar fluid (BALF) samples were collected from 48 patients with molecular diagnosis as PCP via metagenomic next generation sequencing (mNGS) or quantitative PCR detection. Univariate and multivariate logistic regression were performed to screen out possible indicators for the severity of PCP. The patients were divided into two groups according to ddPCR copy numbers, and their clinical features were further analyzed. RESULTS: Pj loading was a pro rata increase with serum (1,3)-beta-D glucan, D-dimmer, neutrophil percentage, procalcitonin and BALF polymorphonuclear leucocyte percentage, while negative correlation with albumin, PaO2/FiO2, BALF cell count, and BALF lymphocyte percentage. D-dimmer and ddPCR copy number of Pj were independent indicators for moderate/severe PCP patients with PaO2/FiO2 lower than 300. We made a ROC analysis of ddPCR copy number of Pj for PaO2/FiO2 index and grouped the patients according to the cut-off value (2.75). The high copy numbers group was characterized by higher level of inflammatory markers. Compared to low copy number group, there was lower level of the total cell count while higher level of polymorphonuclear leucocyte percentage in BALF in the high copy numbers group. Different from patients with high copy numbers, those with high copy numbers had a tendency to develop more severe complications and required advanced respiratory support. CONCLUSION: The scenarios of patients infected with high ddPCR copy numbers of Pj showed more adverse clinical conditions. Pj loading could reflect the severity of PCP to some extent.

BEXCIS: Bayesian methods for estimating the degree of the skewness of X chromosome inactivation.

BACKGROUND: X chromosome inactivation (XCI) is an epigenetic phenomenon that one of two X chromosomes in females is transcriptionally silenced during early embryonic development. Skewed XCI has been reported to be associated with some X-linked diseases. There have been several methods measuring the degree of the skewness of XCI. However, these methods may still have several limitations. RESULTS: We propose a Bayesian method to obtain the point estimate and the credible interval of the degree of XCI skewing by incorporating its prior information of being between 0 and 2. We consider a normal prior and a uniform prior for it (respectively denoted by BN and BU). We also propose a penalized point estimate based on the penalized Fieller's method and derive the corresponding confidence interval. Simulation results demonstrate that the BN and BU methods can solve the problems of extreme point estimates, noninformative intervals, empty sets and discontinuous intervals. The BN method generally outperforms other methods with the lowest mean squared error in the point estimation, and well controls the coverage probability with the smallest median and the least variation of the interval width in the interval estimation. We apply all the methods to the Graves' disease data and the Minnesota Center for Twin and Family Research data, and find that SNP rs3827440 in the Graves' disease data may undergo skewed XCI towards the allele C. CONCLUSIONS: We recommend the BN method for measuring the degree of the skewness of XCI in practice. The R package BEXCIS is publicly available at https://github.com/Wen-YiYu/BEXCIS .

Enhanced DNA and RNA pathogen detection via metagenomic sequencing in patients with pneumonia.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) is an important supplement to conventional tests for pathogen detections of pneumonia. However, mNGS pipelines were limited by irregularities, high proportion of host nucleic acids, and lack of RNA virus detection. Thus, a regulated pipeline based on mNGS for DNA and RNA pathogen detection of pneumonia is essential. METHODS: We performed a retrospective study of 151 patients with pneumonia. Three conventional tests, culture, loop-mediated isothermal amplification (LAMP) and viral quantitative real-time polymerase chain reaction (qPCR) were conducted according to clinical needs, and all samples were detected using our optimized pipeline based on the mNGS (DNA and RNA) method. The performances of mNGS and three other tests were compared. Human DNA depletion was achieved respectively by MolYsis kit and pre-treatment using saponin and Turbo DNase. Three RNA library preparation methods were used to compare the detection performance of RNA viruses. RESULTS: An optimized mNGS workflow was built, which had only 1-working-day turnaround time. The proportion of host DNA in the pre-treated samples decreased from 99 to 90% and microbiome reads achieved an approximately 20-fold enrichment compared with those without host removal. Meanwhile, saponin and Turbo DNase pre-treatment exhibited an advantage for DNA virus detection compared with MolYsis. Besides, our in-house RNA library preparation procedure showed a more robust RNA virus detection ability. Combining three conventional methods, 76 (76/151, 50.3%) cases had no clear causative pathogen, but 24 probable pathogens were successfully detected in 31 (31/76 = 40.8%) unclear cases using mNGS. The agreement of the mNGS with the culture, LAMP, and viral qPCR was 60%, 82%, and 80%, respectively. Compared with all conventional tests, mNGS had a sensitivity of 70.4%, a specificity of 72.7%, and an overall agreement of 71.5%. CONCLUSIONS: A complete and effective mNGS workflow was built to provide timely DNA and RNA pathogen detection for pneumonia, which could effectively remove the host sequence, had a higher microbial detection rate and a broader spectrum of pathogens (especially for viruses and some pathogens that are difficult to culture). Despite the advantages, there are many challenges in the clinical application of mNGS, and the mNGS report should be interpreted with caution.

Chemical profiling of Qingfei Paidu Decoction by triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.

Characteristics of oral microbiome of healthcare workers in different clinical scenarios: a cross-sectional analysis.

The environment of healthcare institutes (HCIs) potentially affects the internal microecology of medical workers, which is reflected not only in the well-studied gut microbiome but also in the more susceptible oral microbiome. We conducted a prospective cross-sectional cohort study in four hospital departments in Central China. Oropharyngeal swabs from 65 healthcare workers were collected and analyzed using 16S rRNA gene amplicon sequencing. The oral microbiome of healthcare workers exhibited prominent deviations in diversity, microbial structure, and predicted function. The coronary care unit (CCU) samples exhibited robust features and stability, with significantly higher abundances of genera such as Haemophilus, Fusobacterium, and Streptococcus, and a lower abundance of Prevotella. Functional prediction analysis showed that vitamin, nucleotide, and amino acid metabolisms were significantly different among the four departments. The CCU group was at a potential risk of developing periodontal disease owing to the increased abundance of F. nucleatum. Additionally, oral microbial diversification of healthcare workers was related to seniority. We described the oral microbiome profile of healthcare workers in different clinical scenarios and demonstrated that community diversity, structure, and potential functions differed markedly among departments. Intense modulation of the oral microbiome of healthcare workers occurs because of their original departments, especially in the CCU.

Comparative analysis of oropharyngeal microbiota in healthcare workers post-COVID-19.

Background: To date, more than 770 million individuals have become coronavirus disease 2019 (COVID-19) convalescents worldwide. Emerging evidence highlights the influence of COVID-19 on the oral microbiome during both acute and convalescent disease phases. Front-line healthcare workers are at an elevated risk of exposure to viral infections, and the effects of COVID-19 on their oral microbiome remain relatively unexplored. Methods: Oropharyngeal swab specimens, collected one month after a negative COVID-19 test from a cohort comprising 55 healthcare workers, underwent 16S rRNA sequencing. We conducted a comparative analysis between this post-COVID-19 cohort and the pre-infection dataset from the same participants. Community composition analysis, indicator species analysis, alpha diversity assessment, beta diversity exploration, and functional prediction were evaluated. Results: The Shannon and Simpson indexes of the oral microbial community declined significantly in the post-COVID-19 group when compared with the pre-infection cohort. Moreover, there was clear intergroup clustering between the two groups. In the post-COVID-19 group, the phylum Firmicutes showed a significant increase. Further, there were clear differences in relative abundance of several bacterial genera in contrast with the pre-infection group, including Streptococcus, Gemella, Granulicatella, Capnocytophaga, Leptotrichia, Fusobacterium, and Prevotella. We identified Gemella enrichment in the post-COVID-19 group, potentially serving as a recovery period performance indicator. Functional prediction revealed lipopolysaccharide biosynthesis downregulation in the post-COVID-19 group, an outcome with host inflammatory response modulation and innate defence mechanism implications. Conclusion: During the recovery phase of COVID-19, the oral microbiome diversity of front-line healthcare workers failed to fully return to its pre-infection state. Despite the negative COVID-19 test result one month later, notable disparities persisted in the composition and functional attributes of the oral microbiota.

Elucidation of active components and target mechanism in Jinqiancao granules for the treatment of prostatitis and benign prostatic hyperplasia.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostatitis and benign prostatic hyperplasia (BPH) are inflammations of the prostate gland, which surrounds the urethra in males. Jinqiancao granules are a traditional Chinese medicine used to treat kidney stones and this medicine consists of four herbs: Desmodium styracifolium (Osbeck) Merr., Pyrrosia calvata (Baker) Ching, Plantago asiatica L. and stigma of Zea mays L. AIM OF THE STUDY: We hypothesized that Jinqiancao granules could be a potential therapy for prostatitis and BPH, and this work aimed to elucidate active compounds in Jinqiancao granules and their target mechanisms for the potential treatment of the two diseases. MATERIALS AND METHODS: Jinqiancao granules were commercially available and purchased. Database-driven data mining and networking were utilized to establish a general correlation between Jinqiancao granules and the two diseases above. Ultra-performance liquid chromatography-mass spectrometry was used for compound separation and characterization. The characterized compounds were evaluated on four G-protein coupled receptors (GPCRs: GPR35, muscarinic acetylcholine receptor M3, alpha-1A adrenergic receptor α1A and cannabinoid receptor CB2). A dynamic mass redistribution technique was applied to evaluate compounds on four GPCRs. Nitric acid (NO) inhibition was tested on the macrophage cell line RAW264.7. Molecular docking was conducted on GPR35-active compounds and GPR35 crystal structure. Statistical analysis using GEO datasets was conducted. RESULTS: Seventy compounds were isolated and twelve showed GPCR activity. Three compounds showed potent GPR35 agonistic activity (EC50 < 10 µM) and the GPR35 agonism action of PAL-21 (Scutellarein) was reported for the first time. Docking results revealed that the GPR35-targeting compounds interacted at the key residues for the agonist-initiated activation of GPR35. Five compounds showed weak antagonistic activity on M3, which was confirmed to be a disease target by statistical analysis. Seventeen compounds showed NO inhibitory activity. Several compounds showed multi-target properties. An experiment-based network reflected a pharmacological relationship between Jinqiancao granules and the two diseases. CONCLUSIONS: This study identified active compounds in Jinqiancao granules that have synergistic mechanisms, contributing to anti-inflammatory effects. The findings provide scientific evidence for the potential use of Jinqiancao granules as a treatment for prostatitis and BPH.

Plasma proteome analysis and validation of patients with community-acquired pneumonia: A cohort study.

PURPOSE: This study aimed to investigate the diagnostic potential of plasma biomarkers of community-acquired pneumonia (CAP) and their severity grading. EXPERIMENTAL DESIGN: Plasma proteomes from cohort I (n = 32) with CAP were analyzed by data-independent acquisition mass spectrometry (MS). MetaboAnalyst 5.0 was used to statistically evaluate significant differences in proteins from different samples, and demographic and clinical data were recorded for all enrolled patients. Cohort II (n = 80) was used to validate candidate biomarkers. Plasma protein levels were determined using quantitative enzyme-linked immunosorbent assay (ELISA). Correlations were assessed using Pearson's correlation coefficient. A receiver operating characteristic curve was used to verify the association between the variables, CAP diagnosis, and prognosis. RESULTS: 121 differentially expressed proteins (DEPs) were obtained between CAP and controls. These DEPs were mainly aggregated in pathways of phagosome(hsa04145) and complement and coagulation cascades (hsa04610). No significant differential proteins were detected in bacterial, viral, and mixed infection groups. The plasma levels of fetuin-A, alpha-1-antichymotrypsin (AACT), α1-acid glycoprotein (A1AG), and S100A8/S100A9 heterodimers detected by ELISA were consistent with those of MS. AACT, A1AG, S100A8/S100A9 heterodimer, and fetuin-A can potentially be used as diagnostic predictors, and fetuin-A and AACT are potential predictors of SCAP. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma protein profiling can successfully identify potential biomarkers for CAP diagnosis and disease severity assessment. These biomarkers should be further studied for their clinical application.

Clinical performance of BALF droplet digital PCR for differential diagnosis of Pneumocystis jirovecii pneumonia and Pneumocystis jirovecii colonization.

BACKGROUND: Accurate differentiation between Pneumocystis jirovecii (Pj) infection and colonization is crucial for effective treatment. METHODS: From September 2016 to June 2022, 89 immunocompromised patients with unexplained lung infiltrates and clinical suspicion of Pj pneumonia were enrolled at Peking University People's Hospital. Bronchoalveolar lavage fluid (BALF) of these patients were detected by quantitative PCR (qPCR) and droplet digital PCR (ddPCR). RESULTS: The performance of ddPCR was superior to qPCR in detecting Pj infection. Area under the curve was 0.97 (95 %CI: 0.94-1) for ddPCR of the BALF in all patients. The optimal threshold value for discriminating Pj infection from colonization by ddPCR was 13.98 copies/test, with a sensitivity of 97.96 %, specificity of 85.71 %. No obvious correlation between ddPCR copy number and disease severity was observed. CONCLUSION: BALF ddPCR exhibits robust potential in detecting Pj and effectively discriminating colonization and infection.

Antibacterial-Based Hydrogel Coatings and Their Application in the Biomedical Field-A Review.

Hydrogels exhibit excellent moldability, biodegradability, biocompatibility, and extracellular matrix-like properties, which make them widely used in biomedical fields. Because of their unique three-dimensional crosslinked hydrophilic networks, hydrogels can encapsulate various materials, such as small molecules, polymers, and particles; this has become a hot research topic in the antibacterial field. The surface modification of biomaterials by using antibacterial hydrogels as coatings contributes to the biomaterial activity and offers wide prospects for development. A variety of surface chemical strategies have been developed to bind hydrogels to the substrate surface stably. We first introduce the preparation method for antibacterial coatings in this review, which includes surface-initiated graft crosslinking polymerization, anchoring the hydrogel coating to the substrate surface, and the LbL self-assembly technique to coat crosslinked hydrogels. Then, we summarize the applications of hydrogel coating in the biomedical antibacterial field. Hydrogel itself has certain antibacterial properties, but the antibacterial effect is not sufficient. In recent research, in order to optimize its antibacterial performance, the following three antibacterial strategies are mainly adopted: bacterial repellent and inhibition, contact surface killing of bacteria, and release of antibacterial agents. We systematically introduce the antibacterial mechanism of each strategy. The review aims to provide reference for the further development and application of hydrogel coatings.

Lysophosphatidylcholine acyltransferase level predicts the severity and prognosis of patients with community-acquired pneumonia: a prospective multicenter study.

Background: Identifying the diagnosis as well as prognosis for patients presented with community-acquired pneumonia (CAP) remains challenging. We aimed to identify the role of lysophosphatidylcholine acyl-transferase (LPCAT) for CAP along with assessing this protein's effectiveness as a biomarker for severity of disease and mortality. Methods: Prospective multicenter research study was carried out among hospitalized patients. A total of 299 CAP patients (including 97 severe CAP patients [SCAP]) and 20 healthy controls (HC) were included. A quantitative enzyme-linked immunosorbent test kit was employed for detecting the LPCAT level in plasma. We developed a deep-learning-based binary classification (SCAP or non-severe CAP [NSCAP]) model to process LPCAT levels and other laboratory test results. Results: The level of LPCAT in patients with SCAP and death outcome was significantly higher than that in other patients. LPCAT showed the highest predictive value for SCAP. LPCAT was able to predict 30-day mortality among CAP patients, combining LPCAT values with PSI scores or CURB-65 further enhance mortality prediction accuracy. Conclusion: The on admission level of LPCAT found significantly raised among SCAP patients and strongly predicted SCAP patients but with no correlation to etiology. Combining the LPCAT value with CURB-65 or PSI improved the 30-day mortality forecast significantly. Trial registration: NCT03093220 Registered on March 28th, 2017.

Oral microbial dysbiosis in patients with periodontitis and chronic obstructive pulmonary disease.

Background: Oral microbiota is closely related to the homeostasis of the oral cavity and lungs. To provide potential information for the prediction, screening, and treatment strategies of individuals, this study compared and investigated the bacterial signatures in periodontitis and chronic obstructive pulmonary disease (COPD). Materials and methods: We collected subgingival plaque and gingival crevicular fluid samples from 112 individuals (31 healthy controls, 24 patients with periodontitis, 28 patients with COPD, and 29 patients with both periodontitis and COPD). The oral microbiota was analyzed using 16S rRNA gene sequencing and diversity and functional prediction analysis were performed. Results: We observed higher bacterial richness in individuals with periodontitis in both types of oral samples. Using LEfSe and DESeq2 analyses, we found differentially abundant genera that may be potential biomarkers for each group. Mogibacterium is the predominant genus in COPD. Ten genera, including Desulfovibrio, Filifactor, Fretibacterium, Moraxella, Odoribacter, Pseudoramibacter Pyramidobacter, Scardovia, Shuttleworthia and Treponema were predominant in periodontitis. Bergeyella, Lautropia, Rothia, Propionibacterium and Cardiobacterium were the signature of the healthy controls. The significantly different pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) between healthy controls and other groups were concentrated in genetic information processing, translation, replication and repair, and metabolism of cofactors and vitamins. Conclusions: We found the significant differences in the bacterial community and functional characterization of oral microbiota in periodontitis, COPD and comorbid diseases. Compared to gingival crevicular fluid, subgingival plaque may be more appropriate for reflecting the difference of subgingival microbiota in periodontitis patients with COPD. These results may provide potentials for predicting, screening, and treatment strategies for individuals with periodontitis and COPD.

Lung microbiome and cytokine profiles in different disease states of COPD: a cohort study.

Increasing evidence indicates that respiratory tract microecological disorders may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Understanding the composition of the respiratory microbiome in COPD and its relevance to respiratory immunity will help develop microbiome-based diagnostic and therapeutic approaches. One hundred longitudinal sputum samples from 35 subjects with acute exacerbation of COPD (AECOPD) were analysed for respiratory bacterial microbiome using 16S ribosomal RNA amplicon sequencing technology, and the sputum supernatant was analysed for 12 cytokines using a Luminex liquid suspension chip. Unsupervised hierarchical clustering was employed to evaluate the existence of distinct microbial clusters. In AECOPD, the respiratory microbial diversity decreased, and the community composition changed significantly. The abundances of Haemophilus, Moraxella, Klebsiella, and Pseudomonas increased significantly. Significant positive correlations between the abundance of Pseudomonas and TNF-α, abundance of Klebsiella and the percentage of eosinophils were observed. Furthermore, COPD can be divided into four clusters based on the respiratory microbiome. AECOPD-related cluster was characterized by the enrichment of Pseudomonas and Haemophilus and a high level of TNF-α. Lactobacillus and Veillonella are enriched in therapy-related phenotypes and may play potential probiotic roles. There are two inflammatory endotypes in the stable state: Gemella is associated with the Th2 inflammatory endotypes, whereas Prevotella is associated with the Th17 inflammatory endotypes. Nevertheless, no differences in clinical manifestations were found between these two endotypes. The sputum microbiome is associated with the disease status of COPD, allowing us to distinguish different inflammatory endotypes. Targeted anti-inflammatory and anti-infective therapies may improve the long-term prognosis of COPD.

[Investigation of the chemical components of Ciwujia injection using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry].

Ciwujia injection is commonly used to treat cerebrovascular and central nervous system diseases in clinical practice. It can significantly improve blood lipid levels and endothelial cell function in patients with acute cerebral infarction and promote the proliferation of neural stem cells in cerebral ischemic brain tissues. The injection has also been reported to have good curative effects on cerebrovascular diseases, such as hypertension and cerebral infarction. At present, the material basis of Ciwujia injection remains incompletely understood, and only two studies have reported dozens of components, which were determined using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS). Unfortunately, the lack of research on this injection restricts the in-depth study of its therapeutic mechanism.In the present study, a qualitative method based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS) was developed to analyze the chemical components of Ciwujia injection. Separation was performed on a BEH Shield RP18 column (100 mm×2.1 mm, 1.7 µm) using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phases, and gradient elution was performed as follows: 0-2 min, 0%B; 2-4 min, 0%B-5%B; 4-15 min, 5%B-20%B; 15-15.1 min, 20%B-90%B; 15.1-17 min, 90%B. The flow rate and column temperature were set to 0.4 mL/min and 30 ℃ respectively. MS1 and MS2 data were acquired in both positive- and negative-ion modes using a mass spectrometer equipped with an HESI source. For data post-processing, a self-built library including component names, molecular formulas, and chemical structures was established by collecting information on the isolated chemical compounds of Acanthopanax senticosus. The chemical components of the injection were identified by comparison with standard compounds or MS2 data in commercial databases or literature based on precise relative molecular mass and fragment ion information. The fragmentation patterns were also considered. For example, the MS2 data of 3-caffeoylquinic acid (chlorogenic acid), 4-caffeoylquinic acid (cryptochlorogenic acid), and 5-caffeoylquinic acid (neochlorogenic acid) were first analyzed. The results indicated that these compounds possessed similar fragmentation behaviors, yielding product ions at m/z 173 and m/z 179 simultaneously. However, the abundance of the product ion at m/z 173 was much higher in 4-caffeoylquinic acid than in 5-caffeoylquinic acid or 3-caffeoylquinic acid, and the fragment signal at m/z 179 was much stronger for 5-caffeoylquinic acid than for 3-caffeoylquinic acid. Four caffeoylquinic acids were identified using a combination of abundance information and retention times. MS2 data in commercial database and literature were also used to identify unknown constituents. For example, compound 88 was successfully identified as possessing a relative molecular mass and neutral losses similar to those of sinapaldehyde using the database, and compound 80 was identified as salvadoraside because its molecular and fragmentation behaviors were consistent with those reported in the literature. A total of 102 constituents, including 62 phenylpropanoids, 23 organic acids, 7 nucleosides, 1 iridoid, and 9 other compounds, were identified. The phenylpropanoids can be further classified as phenylpropionic acids, phenylpropanols, benzenepropanals, coumarins, and lignans. Among the detected compounds, 16 compounds were confirmed using reference compounds and 65 compounds were identified in Ciwujia injection for the first time. This study is the first to report the feasibility of using the UHPLC-Q/Orbitrap HRMS method to quickly and comprehensively analyze the chemical components of Ciwujia injection. The 27 newly discovered phenylpropanoids provide further material basis for the clinical treatment of neurological diseases and new research targets for the in-depth elucidation of the pharmacodynamic mechanism of Ciwujia injection and its related preparations.

T2T-YAO: A Telomere-to-telomere Assembled Diploid Reference Genome for Han Chinese.

Since its initial release in 2001, the human reference genome has undergone continuous improvement in quality, and the recently released telomere-to-telomere (T2T) version - T2T-CHM13 - reaches its highest level of continuity and accuracy after 20 years of effort by working on a simplified, nearly homozygous genome of a hydatidiform mole cell line. Here, to provide an authentic complete diploid human genome reference for the Han Chinese, the largest population in the world, we assembled the genome of a male Han Chinese individual, T2T-YAO, which includes T2T assemblies of all the 22 + X + M and 22 + Y chromosomes in both haploid. The quality of T2T-YAO is much better than all currently available diploid assemblies, and its haploid version, T2T-YAO-hp, generated by selecting the better assembly for each autosome, reaches the top quality of fewer than one error per 29.5 Mb, even higher than that of T2T-CHM13. Derived from an individual living in the aboriginal region of the Han population, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors. Each haplotype of T2T-YAO possesses ∼ 330-Mb exclusive sequences, ∼ 3100 unique genes, and tens of thousands of nucleotide and structural variations as compared with CHM13, highlighting the necessity of a population-stratified reference genome. The construction of T2T-YAO, a truly accurate and authentic representative of the Chinese population, would enable precise delineation of genomic variations and advance our understandings in the hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population.

Heteroresistance Is Associated With in vitro Regrowth During Colistin Treatment in Carbapenem-Resistant Klebsiella pneumoniae.

Polymyxins including polymyxin B and colistin (polymyxin E) are considered the last resort for treating infections caused by carbapenem-resistant gram-negative bacteria. However, in vitro regrowth with the emergence of resistance during treatment is common. Polymyxin heteroresistance, particularly in Acinetobacter baumannii and Klebsiella pneumoniae, has been widely reported. This study was primarily performed to evaluate the prevalence of colistin heteroresistance in carbapenem-resistant K. pneumoniae (CR-KP) and the association between in vitro regrowth and heteroresistance. The mechanisms of colistin resistance and the ability of combination therapies to suppress resistance selection were further investigated. A population analysis profile (PAP) analysis showed that 69 (71.9%) of 96 CR-KP strains had colistin heteroresistance. Time-kill assays revealed that the colistin monotherapy could quickly eliminate the bacterial cells in strains without heteroresistance within the first 6 h. Conversely, it could initially reduce the number of cells in heteroresistant strains, but then regrowth occurred rapidly. Resistance screening at 12 and 24 h in the time-kill assays indicated that susceptible populations were killed, and regrowth was the exact result of the continued growth of resistant subpopulations. Colistin resistance in the regrowth subpopulations was mainly due to the overexpression of phoPQ and pmrD. Colistin combined with tetracyclines (tigecycline or minocycline) or aminoglycosides (amikacin or gentamicin) could effectively suppress the resistance selection and significantly elicit in vitro synergistic effects. These findings suggested that the combination therapy can be used to treat infections caused by CR-KP with colistin heteroresistance. Nevertheless, further in vivo studies considering drugs pharmacokinetics/pharmacodynamics are needed to confirm these findings.