Comorbidity increases the risk of pulmonary tuberculosis: a nested case-control study using multi-source big data.

BACKGROUND: Some medical conditions may increase the risk of developing pulmonary tuberculosis (PTB); however, no systematic study on PTB-associated comorbidities and comorbidity clusters has been undertaken. METHODS: A nested case-control study was conducted from 2013 to 2017 using multi-source big data. We defined cases as patients with incident PTB, and we matched each case with four event-free controls using propensity score matching (PSM). Comorbidities diagnosed prior to PTB were defined with the International Classification of Diseases-10 (ICD-10). The longitudinal relationships between multimorbidity burden and PTB were analyzed using a generalized estimating equation. The associations between PTB and 30 comorbidities were examined using conditional logistic regression, and the comorbidity clusters were identified using network analysis. RESULTS: A total of 4265 cases and 17,060 controls were enrolled during the study period. A total of 849 (19.91%) cases and 1141 (6.69%) controls were multimorbid before the index date. Having 1, 2, and ≥ 3 comorbidities was associated with an increased risk of PTB (aOR 2.85-5.16). Fourteen out of thirty comorbidities were significantly associated with PTB (aOR 1.28-7.27), and the associations differed by sex and age. Network analysis identified three major clusters, mainly in the respiratory, circulatory, and endocrine/metabolic systems, in PTB cases. CONCLUSIONS: Certain comorbidities involving multiple systems may significantly increase the risk of PTB. Enhanced awareness and surveillance of comorbidity are warranted to ensure early prevention and timely control of PTB.

Epidemiological behaviour and interventions of malaria in Niger, 2010-2019: a time-series analysis of national surveillance data.

BACKGROUND: Malaria remains a significant public health concern in Niger, with the number of cases increasing from 592,334 in 2000 to 3,138,696 in 2010. In response, a concerted campaign against the disease has been initiated. However, the implementation of these malaria interventions and their association with epidemiological behaviour remains unclear. METHODS: A time-series study was conducted in Niger from 2010 to 2019. Multiple data sources concerning malaria were integrated, encompassing national surveillance data, Statistic Yearbook, targeted malaria control interventions, and meteorological data. Incidence rate, mortality rate, and case fatality ratio (CFR) by different regions and age groups were analysed. Joinpoint regression models were used to estimate annual changes in malaria. The changes in coverage of malaria interventions were evaluated. RESULTS: Between 2010 to 2019, the incidence rate of malaria decreased from 249.43 to 187.00 cases per 1,000 population in Niger. Niamey had a high annual mean incidence rate and the lowest CFR, while Agadez was on the contrary. Joinpoint regression analysis revealed a declining trend in malaria incidence for all age groups except the 10-24 years group, and the mortality rate and the CFR initially decreased followed by an increase in all age groups. Niger has implemented a series of malaria interventions, with the major ones being scaled up to larger populations during the study period. CONCLUSIONS: The scale-up of multi-interventions in Niger has significantly reduced malaria incidence, but the rise in mortality rate and CFR addresses the challenges in malaria control and elimination. Malaria endemic countries should enhance surveillance of malaria cases and drug resistance in Plasmodium, improve diagnosis and treatment, expand the population coverage of insecticide-treated bed nets and seasonal malaria chemoprevention, and strengthen the management of severe malaria cases.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/ß-catenin signaling.

Despite the diverse roles of tripartite motif (Trim)-containing proteins in the regulation of autophagy, the innate immune response, and cell differentiation, their roles in skeletal diseases are largely unknown. We recently demonstrated that Trim21 plays a crucial role in regulating osteoblast (OB) differentiation in osteosarcoma. However, how Trim21 contributes to skeletal degenerative disorders, including osteoporosis, remains unknown. First, human and mouse bone specimens were evaluated, and the results showed that Trim21 expression was significantly elevated in bone tissues obtained from osteoporosis patients. Next, we found that global knockout of the Trim21 gene (KO, Trim21-/-) resulted in higher bone mass compared to that of the control littermates. We further demonstrated that loss of Trim21 promoted bone formation by enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and elevating the activity of OBs; moreover, Trim21 depletion suppressed osteoclast (OC) formation of RAW264.7 cells. In addition, the differentiation of OCs from bone marrow-derived macrophages (BMMs) isolated from Trim21-/- and Ctsk-cre; Trim21f/f mice was largely compromised compared to that of the littermate control mice. Mechanistically, YAP1/ß-catenin signaling was identified and demonstrated to be required for the Trim21-mediated osteogenic differentiation of BMSCs. More importantly, the loss of Trim21 prevented ovariectomy (OVX)- and lipopolysaccharide (LPS)-induced bone loss in vivo by orchestrating the coupling of OBs and OCs through YAP1 signaling. Our current study demonstrated that Trim21 is crucial for regulating OB-mediated bone formation and OC-mediated bone resorption, thereby providing a basis for exploring Trim21 as a novel dual-targeting approach for treating osteoporosis and pathological bone loss.

Single-Cell RNA Sequencing Reveals Transcriptional Changes in the Cartilage of Subchondral Insufficiency Fracture of the Knee.

Purpose: Subchondral insufficiency fracture of the knee (SIFK) is a common cause of knee joint pain that mainly afflicts the elderly. Until now, how a sudden insufficiency fracture of subchondral bone affects the transcriptomic profiles of cartilage in SIFK and OA patients are largely unknown. Methods: Single-cell RNA sequencing (scRNA-seq) was used to identify various cell subsets and evaluate transcriptomic differences in cartilage of SIFK and OA patients. In addition, the above findings were confirmed by histological evaluation and immunohistochemical (IHC) staining. Results: We found that the transcriptomic profiles of cartilage in the SIFK patient was completely different from those of normal and OA patients. Accordingly, several novel cell clusters with activation of hypoxia and endochondral ossification signaling were identified in the SIFK cartilage. Chondrocyte trajectories analysis and IHC staining revealed that transcription factors including TCF4 were found to be highly up-regulated during the occurrence of SIFK, which might drive the reactive formation of cartilage and fibrous tissue and the activation of endochondral ossification. Conclusion: This is the first report to elucidate the transcriptomic alterations and distinct cell type subpopulations in the cartilage of SIFK and OA by the use of scRNA-seq, which provides a new insight in the understanding of the initiation and progression of SIFK.

An integrated strategy for comprehensive characterization of metabolites and metabolic profiles of bufadienolides from Venenum Bufonis in rats.

Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable significance in determining the efficacy and safety of traditional Chinese medicine (TCM) in vivo. However, this process is usually hindered by the insufficient characteristic fragments of metabolites, ubiquitous matrix interference, and complicated screening and identification procedures for metabolites. In this study, an effective strategy was established to systematically characterize the metabolites, deduce the metabolic pathways, and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo. The strategy was divided into five steps. First, the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry (MS) system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites. Second, an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites, which reduced approximately 39% of the interfering ions. Third, an acquisition model was developed and used to trigger more tandem MS (MS/MS) fragments of precursor ions based on the targeted ion list. The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode. Fourth, the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction. The main in vivo metabolic pathways of bufadienolides were elucidated. A total of 147 metabolites were characterized, and the main biotransformation reactions of bufadienolides were hydroxylation, dihydroxylation, and isomerization. Finally, the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS, and the metabolic profiles were clearly identified. This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

Profile and dynamics of infectious diseases: a population-based observational study using multi-source big data.

BACKGROUND: The current surveillance system only focuses on notifiable infectious diseases in China. The arrival of the big-data era provides us a chance to elaborate on the full spectrum of infectious diseases. METHODS: In this population-based observational study, we used multiple health-related data extracted from the Shandong Multi-Center Healthcare Big Data Platform from January 2013 to June 2017 to estimate the incidence density and describe the epidemiological characteristics and dynamics of various infectious diseases in a population of 3,987,573 individuals in Shandong province, China. RESULTS: In total, 106,289 cases of 130 infectious diseases were diagnosed among the population, with an incidence density (ID) of 694.86 per 100,000 person-years. Besides 73,801 cases of 35 notifiable infectious diseases, 32,488 cases of 95 non-notifiable infectious diseases were identified. The overall ID continuously increased from 364.81 per 100,000 person-years in 2013 to 1071.80 per 100,000 person-years in 2017 (χ2 test for trend, P < 0.0001). Urban areas had a significantly higher ID than rural areas, with a relative risk of 1.25 (95% CI 1.23-1.27). Adolescents aged 10-19 years had the highest ID of varicella, women aged 20-39 years had significantly higher IDs of syphilis and trichomoniasis, and people aged ≥ 60 years had significantly higher IDs of zoster and viral conjunctivitis (all P < 0.05). CONCLUSIONS: Infectious diseases remain a substantial public health problem, and non-notifiable diseases should not be neglected. Multi-source-based big data are beneficial to better understand the profile and dynamics of infectious diseases.

Incidence trend and disease burden of seven vaccine-preventable diseases in Shandong province, China, 2013-2017: Findings from a population-based observational study.

INTRODUCTION: Although vaccines provide a cost-effective solution to vaccine-preventable diseases (VPDs), the disease burden of VPDs is still very high in most parts of the world. METHODS: A population-based observational study was conducted in Shandong province, China, from 2013 to 2017, giving an insight into the epidemiological characteristics and disease burden of seven VPDs. The incidence trend was estimated using the Poisson regression model. The disease burden was calculated using the disability-adjusted life years (DALYs). RESULTS: Most VPDs included in the China's National Immunization Program had higher incidence density (ID) in inland cities. The ID of mumps decreased significantly, while herpes zoster increased (both P < 0.05). The top three causes of the disease burden as assessed with DALYs included tuberculosis, herpes zoster, and hepatitis B, with the rates of 72.21, 59.99, and 52.10 DALYs/100 000, respectively. The disease burden of influenza and herpes zoster were relatively high in people aged > 50 years, while highest DALYs of hepatitis B were found in young adults. CONCLUSION: Inequalities in the vaccine coverage by geography, socio-economic status, and targeted population contribute to the increasing incidence and high burden of VPDs and call for renewed and sustained immunization strategies in China.

Novel potential functions of amoeboid cells in thraustochytrids revealed by Aurantiochytrium limacinum BL10.

The current study investigated the potential functions of amoeboid cell formation and migration in a thraustochytrid strain, Aurantiochytrium limacinum BL10. Our results showed that: (1) When the surface of an agar plate was inoculated with BL10, amoeboid cells mainly emerged on the periphery of isolated colonies. The amoeboid cells then migrated outwards to form small vegetative cell clusters, which favored rapid colony expansion. In addition, amoeboid cells were capable of self-recognition (i.e. they were able to distinguish BL10 from other thraustochytrid species), and could choose whether to evade (self colonies) or approach (non-self colonies). These observations indicated that amoeboid cells were employed by BL10 to help colonize empty territories and to outcompete other thraustochytrid species in previously colonized territories. (2) When the agar medium was soft, amoeboid cells were able to penetrate the surface and migrate throughout, thereby allowing BL10 to colonize the interior of the solid matrix. This finding suggested that amoeboid cell formation and migration may help Aurantiochytrium colonize the interior of solid matrices to obtain additional nutrients and spatial resources. The mechanisms underlying the regulation of amoeboid cell formation and migration as well as the extraordinary microbial social behaviors of BL10 are also discussed in this article.

Chemical profiling of Huashi Baidu prescription, an effective anti-COVID-19 TCM formula, by UPLC-Q-TOF/MS.

Huashi Baidu prescription (HSBDF), recommended in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Seventh Edition), was clinically used to treat severe corona virus disease 2019 (COVID-19) with cough, blood-stained sputum, inhibited defecation, red tongue etc. symptoms. This study was aimed to elucidate and profile the knowledge on its chemical constituents and the potential anti-inflammatory effect in vitro. In the study, the chemical constituents in extract of HSBDF were characterized by UPLC-Q-TOF/MS in both negative and positive modes, and the pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assays (ELISA) to determine the effects of HSBDF in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that a total of 217 chemical constituents were tentativedly characterized in HSBDF. Moreover, HSBDF could alleviate the expression levels of IL-6 and TNF-α in the cell models, indicating that the antiviral effects of HSBDF might be associated with regulation of the inflammatory cytokines production in RAW264.7 cells. We hope that the results could be served as the basic data for further study of HSBDF on anti-COVID-19 effect.

Characteristic Malonyl Ginsenosides from the Leaves of Panax notoginseng as Potential Quality Markers for Adulteration Detection.

Due to the high price and limited supply of Panax notoginseng, a large number of samples adulterated with the leaves appear in the market. A group of new malonyl ginsenosides were exclusively detected in the P. notoginseng leaves (PNL). Targeted isolation of the malonyl ginsenosides was guided by UPLC-QDa MS. HRMS, 1D/2D NMR, and chemical methods were used for structural identification. A selected ion monitoring method was developed based on UPLC-QDa MS to detect the adulterations. In addition, the anti-inflammatory activities and the collision-induced dissociation features of the isolated saponins were studied. As a result, eight new 3-OH malonylated dammarane-type triterpene oligoglycosides (notoginsenosides L3-L10) were obtained from PNL. Adulteration with PNL can be easily detected with limit of detection as low as 0.06%. To sum up, the isolated ginsenosides can be used as quality markers for fraud detection, which will promote the quality control of the notoginseng products.

An enhanced strategy integrating offline superimposed two-dimensional separation with mass defect filter and diagnostic ion filter: Comprehensive characterization of steroid alkaloids in Fritillariae Pallidiflorae Bulbus as a case study.

The inherent complexity of traditional Chinese medicines necessitates the application of multi-dimensional information to accomplish comprehensive profiling and confirmative identification of their chemical components. In this study, we display an enhanced strategy by integrating offline superimposed two-dimensional separation (S-2D-LC) with mass defect filter and diagnostic ion filter to comprehensively characterize the alkaloid composition of Fritillariae Pallidiflorae Bulbus (FPB). The superimposed HILIC × RP and UPCC × RP offline two-dimensional liquid chromatography system was constructed with superior orthogonality (R2=0.004 and R2=0.001) for chromatographic separation. In total, 70 fractions were collected after the first-dimensional chromatographic separation (HILIC and UPCC) and then analyzed by the second-dimensional reversed phase (RP) liquid chromatography coupled with Q-TOF/MS/MS in FAST DDA acquisition mode. A four-step interpretation strategy combining mass defect filter with diagnostic ion filter was developed to rapidly characterize alkaloids in Fritillaria species. Ultimately, a sum of 529 Fritillaria alkaloids were characterized from two botanical origins of FPB. The integrated strategy is practical to efficiently expose and comprehensively characterize more trace and isomeric components in complex herbal medicines.

A systematic strategy integrating solid-phase extraction, full scan range splitting, mass defect filter and precursor ion list for comprehensive metabolite profiling of Danqi Tongmai tablet in rats.

In vivo metabolite profiling of herbal medicines remains a challenge due to the complex chemical composition and drastic interference from biological matrix. In this study, a systematic strategy was established for comprehensive metabolite profiling of Danqi Tongmai (DQTM) tablet, a combination of salvianolic acids and notoginsenosides, in rats after oral administration. This strategy was composed of six steps. Firstly, the rat plasma and tissue samples were collected at multiple time points to increase the representativeness of samples. Secondly, different sample preparation methods were systematically investigated including protein precipitation, liquid-liquid extraction and solid-phase extraction to obtain superior extraction efficiency for both salvianolic acids and notoginsenosides. Thirdly, the MS acquisition method was optimized by splitting the full scan range into two separate segments to improve the detection capability for minor components. Fourthly, an extended polygonal mass defect filter (EP-MDF) model was constructed to filter potential metabolites of salvianolic acids and notoginsenosides, and remove large amounts of interference ions. Fifthly, ion intensity-based time point-staggered precursor ion list (IITPS-PIL) was generated to trigger more targeted MS/MS acquisition for potential metabolites at the highest concentration. Finally, the absorbed prototypes and metabolites were comprehensively characterized by reference standards and MS/MS fragmentation. The proposed strategy significantly improved the detection ability for trace prototypes and metabolites in vivo. A total of 370 components, including 94 prototypes (38 confirmed with reference standards) and 276 metabolites, were tentatively characterized in rat plasma and tissue samples after oral administration of DQTM. Collectively, this paper provided an applicable reference for comprehensive metabolite profiling of herbal medicines in complex biological samples.

Infectious diseases among elderly persons: Results from a population-based observational study in Shandong province, China, 2013-2017.

BACKGROUND: The health of the elderly is one of the major challenges in today's ageing society. However, research on infectious diseases among the elderly is limited. This study aimed to describe the epidemiological characteristics and dynamics of infectious diseases among the elderly population aged ≥60 years in Shandong province, China. METHODS: Incidence data for infectious diseases were collected from the Shandong Multi-Center Healthcare Big Data Platform from January 2013 to June 2017, which involved 550 432 elderly persons. We compared the incidence of each infectious disease and disease category, stratified by age, gender, and region. Annual percentage change (APC) was estimated using logarithmic linear regression to examine the incidence trends. Poisson regression was conducted to identify the effect of demographic factors on incidence, with incidence rate ratio (IRR) and their 95% confidence intervals (CIs) estimated. RESULTS: A total of 27 595 cases of 102 infectious diseases were reported during the study period, with an overall incidence of 1425.51/100 000 person-years. The most common infectious diseases were respiratory and mucocutaneous diseases among the elderly persons, with annual increases of 17.45% and 20.44%, respectively (both P<0.05). In rural areas, the incidence of respiratory, gastrointestinal, blood- and sex-transmitted, and mucocutaneous infections increased significantly, with APCs of 178.52%, 204.66%, 28.24%, 63.01%, respectively (all P<0.05). Elderly males had a higher risk of infections than that of females, with the highest IRRa of 2.94 (95% confidence interval (CI) = 2.89, 3.00) in respiratory diseases. The elderly aged 85-89 years had a much higher risk of respiratory diseases than those aged 60-64 years (IRRa = 9.85, 95%CI: 9.39, 10.33); however, the risk of blood- and sex-transmitted diseases was highest among the elderly aged 65-69 years (IRRa = 1.24, 95% CI = 1.06, 1.45). CONCLUSIONS: Ageing population are facing a substantial challenge on infectious diseases. More attention should be paid to infections with significant growth. Targeted strategies and measures on elderly persons in different regions and subgroups are urgently needed.

Epidemic and control of COVID-19 in Niger: quantitative analyses in a least developed country.

BACKGROUND: The COVID-19 pandemic is challenging the public health response system worldwide, especially in poverty-stricken, war-torn, and least developed countries (LDCs). METHODS: We characterized the epidemiological features and spread dynamics of COVID-19 in Niger, quantified the effective reproduction number (Rt ), evaluated the impact of public health control measures, and estimated the disease burden. RESULTS: As of 4 July 2020, COVID-19 has affected 29 communes of Niger with 1093 confirmed cases, among whom 741 (67.8%) were males. Of them 89 cases died, resulting in a case fatality rate (CFR) of 8.1%. Both attack rates and CFRs were increased with age (P < 0.0001). Health care workers accounted for 12.8% cases. Among the reported cases, 39.3% were isolated and treated at home, and 42.3% were asymptomatic. 74.6% cases were clustered in Niamey, the capital of Niger. The Rt fluctuated in correlation to control measures at different outbreak stages. After the authorities initiated the national response and implemented the strictest control measures, Rt quickly dropped to below the epidemic threshold (<1), and maintained low level afterward. The national disability-adjusted life years attributable to COVID-19 was 1267.38 years in total, of which years of life lost accounted for over 99.1%. CONCLUSIONS: Classic public health control measures such as prohibition of public gatherings, travelling ban, contact tracing, and isolation and quarantine at home, are proved to be effective to contain the outbreak in Niger, and provide guidance for controlling the ongoing COVID-19 pandemic in LDCs.

Gastric H+/K+-ATPase Expression in Normal Laryngeal Tissue and Laryngeal Carcinoma.

BACKGROUND: Several studies have suggested that laryngopharyngeal reflux disease (LPRD) or gastroesophageal reflux disease (GERD) is an independent risk factor for laryngeal carcinoma. However, it remains unclear whether either condition affects the level of H+/K+-ATPase expression in laryngeal carcinoma. MATERIALS AND METHODS: Immunohistochemistry, real-time RT-PCR, and Western blotting were used to explore the distributions of proton pump (H+/K+-ATPase) α- and ß-subunits in normal laryngeal tissue and laryngeal carcinoma. RESULTS: Messenger RNAs encoding both the α- and ß-subunits were found in the normal epiglottic, ventricular fold, vocal fold, and arytenoid mucosae, as well as epiglottic cartilage. The distributions and expression levels of H+/K+-ATPase α-subunits in various laryngeal subregions did not significantly differ in IHC, RT-PCR, or Western blotting. However, Western blotting revealed a significant difference between the expression level of the ß-subunit protein in the epiglottic cartilage and the levels in other sites. The expression levels of both subunits were significantly higher in carcinomatous than in paracarcinomatous tissue and normal laryngeal tissue. The mean follow-up duration was 66.2 months (range, 17-162 months). In all, 4 patients died during follow-up, 4 were lost to follow-up, and 22 were alive and free of disease at the end of follow-up. Two patients developed lung metastases and six developed disease recurrences (at 2, 8, 14, 16, 36, and 41 months). The 3- and 5-year overall survival (OS) rates were 93.0% and 77.0%, respectively. Univariate analyses showed that the 5-year OSs were significantly associated with the T, N, and clinical stages but not with age, alcohol use, pathological differentiation, or the expression levels of the α- or ß-subunits (as revealed by IHC, RT-PCR, or Western blotting). However, in multivariate regression analyses, the 5-year OSs were not significantly associated with any clinicopathological factor or the expression levels of either subunit. CONCLUSION: H+/K+-ATPase is expressed in the normal larynx, including in the epiglottic cartilage and the mucosae of the epiglottis, ventricular fold, and arytenoid vocal fold. The expression levels of the H+/K+-ATPase α- and ß-subunits in laryngeal carcinomas were higher than in normal laryngeal tissues.

Systematic comparison of metabolic differences of Uncaria rhynchophylla in rat, mouse, dog, pig, monkey and human liver microsomes.

Metabolites have a close relationship with the efficacy and safety of herbal medicines. However, ubiquitous matrix interferences, complex co-elution, and minor or trace amounts in plasma restrict the comprehensive identification of metabolites. In this study, an efficient strategy comprising a mass defect filter and time-staggered targeted ion lists was established to characterize the metabolites of alkaloids of Uncaria rhynchophylla (UR) for the systematic comparison of metabolic differences in rat, mouse, dog, pig, monkey and human liver microsomes. The mass defect filter model effectively decreased interfering ions by 63-68%, and time-staggered precursor ion lists significantly increased the number of triggered MS/MS fragmentation by 65-120% in liver microsomes of six species. Ultimately, a total of 165 metabolites in the liver microsomes of six species were tentatively characterized, and the main metabolic pathways were demethylation, isomerization, hydrolysis, oxygenation and dehydrogenation. The results showed that the mouse liver microsomes exhibited metabolic behavior most similar to human metabolism of UR alkaloids. We hope that these results provide basic data for further investigation of UR metabolism in different species, and that the strategy can provide a reference for metabolite characterization of herbal medicines in complex biological matrix. Graphical abstract.

Low-dose phloretin alleviates diabetic atherosclerosis through endothelial KLF2 restoration.

We investigated whether low-dose phloretin served as daily dietary supplements could ameliorate diabetic atherosclerosis and the role of kruppel-like factor 2 (KLF2). HUVECs cultured in high glucose medium were treated with different concentrations of phloretin and KLF2 mRNA, and protein level was detected. Diabetes was induced using streptozotocin in Apoe-/- mice after which they were fed a high-cholesterol diet for 8 weeks. Diabetic mice injected with KLF2 shRNA-lentivirus or control virus were treated with 20 mg/kg phloretin. Glucose, lipid profile, aortic atheroma, and endothelial nitric oxide synthase (eNOS) expression were detected. Phloretin retained endothelial function by KLF2-eNOS activation under hyperglycemia. Low-dose phloretin helped with lipid metabolism, and blocked the acceleration of atherosclerosis in STZ-induced diabetic mice since the early stage, which was diminished by KLF2 knockdown. Low-dose phloretin exhibited athero-protective effect in diabetic Apoe-/- mice dependent on KLF2 activation. This finding makes phloretin for diabetic atherosclerosis.

Quantitative analysis of fourteen bufadienolides in Venenum Bufonis crude drug and its Chinese patent medicines by ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis, a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, possessed an array of pharmacological activities, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. However, there were few efficient methods for quality evaluation of Venenum Bufonis medicinal materials and its related Chinese patent medicines. AIM OF THE STUDY: To establish an effective method for quality assessment of crude drugs and Chinese proprietary medicines of Venenum Bufonis, and explore the relationship of primary compounds - target - pathway - disease through a series of network databases. MATERIALS AND METHODS: An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was developed and validated to simultaneously determine 14 bufadienolides for quantitative analysis of 71 batches of crude drugs and 20 kinds of Chinese patent medicines of Venenum Bufonis. Multiple reaction monitoring with good specificity and accuracy was applied to monitor the 14 bufadienolides in positive mode. RESULTS: The methodology was validated with good specificity, precision, stability, repeatability and recovery. The low limits of quantification were in the range of 0.1-2.7 ng/mL. The relative standard deviation values for intra- and inter-day precisions ranged from 0.98% to 6.3% and from 2.39% to 6.76%, respectively. The recovery was varied from 87.78% to 110.57% for crude drugs and 88.32%-100.96% for Chinese proprietary medicine (Shexiang Baoxin Pill). The contents of 14 analytes in 71 batches of crude drugs and 20 sorts of Chinese proprietary medicines were procured, the results showed that the contents of crude drugs collected from the market exhibited great variations. Furthermore, 13 batches of crude drugs were identified as counterfeit with no bufadienolides detected. In addition, the total contents of bufadienolides in the same drug showed great difference among products from various manufacturers or brands. Subsequently, 9 bufadienolides with the higher contents were applied to screen the anti-tumor effect by network pharmacology, and 8 pathways which had prior correlation with bufadienolides were disclosed. CONCLUSION: This method could be used for quality assessment of crude drugs and Chinese patent medicines of Venenum Bufonis, and the data could be served as the fundamental basis for drug research and development of Venenum Bufonis.

Simultaneous determination of resibufogenin and its eight metabolites in rat plasma by LC-MS/MS for metabolic profiles and pharmacokinetic study.

BACKGROUND: Resibufogenin is one of the main active compounds of Venenum Bufonis and exhibits diverse pharmacological activities. It is brought into focus for its potency in heart failure and cancer therapy. PURPOSE: The purpose of this study was to establish a convenient and effective method which was used to simultaneously determine the resibufogenin and its metabolites in rat plasma for further understanding the metabolic profiles of resibufogenin in vivo and pharmacokinetic study by LC-MS/MS. METHODS: The analytes were separated on a BEH C18 column with a mobile phase of water containing 0.05% formic acid and acetonitrile under gradient elution at a flow rate of 0.4 ml/min. Resibufogenin and its eight metabolites were quantified in positive electrospray ionization and MRM mode with transitions of m/z 385.5→349.2 for resibufogenin; m/z 513.7→145.3 for IS (internal standard); m/z 401.23→365.21, m/z 417.23→285.21 and m/z 385.24→349.21 for three main metabolites (hydroxylated-resibufogenin; dihydroxylated-resibufogenin and 3-epi-resibufogenin, respectively). RESULTS: This method was successfully validated with a good linearity over the concentration ranges of 1-200 ng/ml for resibufogenin and the correlation coefficients was more than 0.990. The lower limit of quantification was 1 ng/ml and the precision and accuracy values were less than 15%. The method was applied to study the metabolic profiles of resibufogenin in rat plasma after oral administration of 20 mg/kg. The results indicated that the metabolic reactions of resibufogenin were mainly hydroxylation, dihydroxylation, dehydrogenation and isomerization. Totally eleven metabolites were identified, among which eight were successfully quantified. CONCLUSION: The results could provide further research foundation for the mechanisms study of activity and toxicity in vivo and facilitate the appropriate clinical application of resibufogenin.