Epigallocatechin-3-Gallate attenuates lipopolysacharide-induced pneumonia via modification of inflammation, oxidative stress, apoptosis, and autophagy.

BACKGROUND: Pneumonia, the acute inflammation of lung tissue, is multi-factorial in etiology. Hence, continuous studies are conducted to determine the mechanisms involved in the progression of the disease and subsequently suggest effective treatment. The present study attempted to evaluate the effects of Epigallocatechin-3-Gallate (EGCG), an herbal antioxidant, on inflammation, oxidative stress, apoptosis, and autophagy in a rat pneumonia model. METHODS: Forty male Wistar rats, 5 months old and 250-290 g were divided into four groups including control, EGCG, experimental pneumonia (i/p LPS injection, 1 mg/kg), and experimental pneumonia treated with EGCG (i/p, 15 mg/kg, 1 h before and 3 h after LPS instillation). Total cell number in the bronchoalveolar lavage fluid, inflammation (TNF-a, Il-6, IL-1ß, and NO), oxidative stress (Nrf2, HO-1, SOD, CAT, GSH, GPX, MDA, and TAC), apoptosis (BCL-2, BAX, CASP-3 and CASP-9), and autophagy (mTOR, LC3, BECN1) were evaluated. RESULTS: The findings demonstrated that EGCG suppresses the LPS-induced activation of inflammatory pathways by a significant reduction of inflammatory markers (p-value < 0.001). In addition, the upregulation of BCL-2 and downregulation of BAX and caspases revealed that EGCG suppressed LPS-induced apoptosis. Furthermore, ECGC suppressed oxidative injury while promoting autophagy in rats with pneumonia (p-value < 0.05). CONCLUSION: The current study revealed that EGCG could suppress inflammation, oxidative stress, apoptosis, and promote autophagy in experimental pneumonia models of rats suggesting promising therapeutical properties of this compound to be used in pneumonia management.

Clinical characteristics and molecular genetic analysis of ten cases of ornithine carbamoyltransferase deficiency in southeastern China.

BACKGROUND: This study aimed to investigate the clinical and molecular genetic characteristics of ten children with ornithine carbamoyltransferase deficiency (OTCD) in southeastern China, as well as the correlation between the genotype and phenotype of OTCD. METHODS: A retrospective analysis was performed on the clinical manifestations, laboratory testing, and genetic test findings of ten children with OTCD admitted between August 2015 and October 2021 at Quanzhou Maternity and Children's Hospital of Fujian Province in China. RESULTS: Five boys presented with early-onset symptoms, including poor appetite, drowsiness, groaning, seizures, and liver failure. In contrast, five patients (one boy and four girls) had late-onset gastrointestinal symptoms as the primary clinical manifestation, all presenting with hepatic impairment, and four with hepatic failure.Nine distinct variants of the OTC gene were identified, including two novel mutations: c.1033del(p.Y345Tfs*50) and c.167T > A(p.M56K). Of seven patients who died, five had early-onset disease despite active treatment. Three patients survived, and two of them underwent liver transplantation. CONCLUSIONS: The clinical manifestations of OTCD lack specificity. However, elevated blood ammonia levels serve as a crucial diagnostic clue for OTCD. Genetic testing aids in more accurate diagnosis and prognosis assessment by clinicians. In addition, we identified two novel pathogenic variants and expand the mutational spectrum of the gene OTC, which may contribute to a better understanding of the clinical and genetic characteristics of OTCD patients.

Establishment of an at-line nanofractionation-based screening platform by coupling HPLC-MS/MS with high-throughput fluorescence polarization bioassay for natural SARS-CoV-2 fusion inhibitors.

In this study, a novel at-line nanofractionation platform was established for screening SARS-CoV-2 fusion inhibitors from natural products for the first time by combining HPLC-MS/MS with high-throughput fluorescence polarization (FP) bioassay. A time-course FP bioassay in 384 well-plates was conducted in parallel with MS/MS to simultaneously obtain chemical and biological information of potential fusion inhibitors in Lonicerae Japonicae Flos (LJF) and Lianhua Qingwen capsules (LHQW). Semi-preparative liquid chromatography and orthogonal HPLC separation were employed to enrich and better identify the co-eluted components. After comprehensive evaluation and validation, 28 potential SARS-CoV-2 fusion inhibitors were screened out and identified. Several compounds at low micromolar activity were validated by in vitro inhibitory assay, molecular docking, cytotoxicity test, and pseudovirus assay. Moreover, four potential dual-target inhibitors against influenza and COVID-19 were discovered from LJF using this method, offering novel insights for the development of future pharmaceuticals targeting epidemic respiratory diseases.

Rapid screening of neuraminidase inhibitors using an at-line nanofractionation platform involving parallel oseltamivir-sensitive/resistant neuraminidase bioassays.

In this study, an advanced at-line nanofractionation based screening platform was developed to screen potential neuraminidase inhibitors (NAIs) from Lonicera japonica Thunb by involving two parallel bioassays, for determining both oseltamivir-sensitive neuraminidase (NAS) and oseltamivir-resistant neuraminidase (NAR) inhibitory activities. 20 potential NAIs with both NAS and NAR inhibitory effects were screened from Lonicera japonica Thunb and identified by mass spectrometer, including 11 phenolic acids, 8 flavonoids and one iridoid glycoside. The proposed at-line nanofractionation based screening platform for NAIs was also used to rapidly screen nine batches of water extracts of Lonicera japonica Thunb or its similar species. Clear differences in the number and content of active components were easily observed, demonstrating that the proposed method possesses great potential for the quality control of herb medicines.

In vitro exposure to environmentally relevant concentrations of norgestrel affects sperm physiology and reproductive success of the Pacific oyster Crassostrea gigas.

Progestins in aquatic environments are of increasing concern, as shown by the results of toxicological studies on adult invertebrates with external fertilization. However, their potential effects on the gametes and reproductive success of such animals remain largely unknown. Thus, the current study assessed the effect of in vitro exposure of environmentally relevant concentrations (10 ng/L and 1000 ng/L) of norgestrel (NGT) on the sperm of Pacific oyster Crassostrea gigas, analyzing sperm motility, ultrastructure, mitochondrial function, ATP status, characteristic enzyme activities, and DNA integrity underlying fertilization and hatching success. The results showed that NGT increased the percentage of motile sperm by elevating intracellular Ca2+ levels, Ca2+-ATPase activity, creatine kinase activity, and ATP content. Although superoxide dismutase activity was enhanced to eliminate reactive oxygen species generated by NGT, oxidative stress occurred, as indicated by the increase in malonaldehyde content and damage to plasma membranes and DNA. As a consequence, fertilization rates decreased. However, hatching rates did not alter significantly, possibly as a result of DNA repair processes. This study demonstrates oyster sperm as a useful, sensitive tool for toxicological research of progestins and provides ecologically relevant information on reproductive disturbance in oysters resulting from exposure to NGT.

Antibiotic Prescription Patterns for Acute Respiratory Infections in Rural Primary Healthcare Settings in Guangdong, China: Analysis of 162,742 Outpatient Prescriptions.

Overuse and inappropriate use of antibiotics are important contributors to bacterial antimicrobial resistance (AMR), especially in ambulatory primary healthcare (PHC) settings in low- and middle-income countries. This study aimed to investigate antibiotic prescription patterns among patients with acute respiratory infections (ARIs) in rural PHC facilities in the Guangdong Province, China. A total of 444,979 outpatient prescriptions were extracted from the electronic medical record system of 35 township health centers (THCs) and 2 community health centers (CHCs) between November 2017 and October 2018. We used the chi-square test to analyze the antibiotic prescription patterns and binary logistic regression to explore patient-related factors associated with antibiotic prescriptions. Of the 162,742 ARI prescriptions, 85.57% (n = 139,259) included at least one antibiotic. Among the 139,259 prescriptions with antibiotics, 37.82% (n = 52,666) included two or more antibiotics, 55.29% (n = 76,993) included parenteral antibiotics, and 56.62% (n = 78,852) included Watch group antibiotics. The binary logistic regression indicated that (1) female patients were slightly less likely to be prescribed antibiotics than males (adjusted odds ratio (OR) = 0.954, 95% confidence interval [CI] [0.928-0.981]; p = 0.001); and (2) compared to patients aged ≤5 years, those who were 6-15 years old (adjusted OR = 1.907, 95% CI [1.840-1.978]; p < 0.001), 16-60 years old (adjusted OR = 1.849, 95% CI [1.785-1.916]; p < 0.001), and >60 years old (adjusted OR = 1.915, 95% CI [1.810-2.026]; p < 0.001) were more likely to be prescribed antibiotics. The overuse and irrational use of antibiotics in PHC settings remain major healthcare challenges in rural Guangdong. Thus, it is imperative to implement targeted antimicrobial stewardship (AMS) policies to address this problem.

Newborn screening and molecular features of patients with multiple acyl-CoA dehydrogenase deficiency in Quanzhou, China.

OBJECTIVES: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder of fatty acid, amino acid and choline metabolism. Late-onset MADD is caused by ETFDH mutations and is the most common lipid storage myopathy in China. However, few patients with MADD have been identified through newborn screening (NBS). This study assessed the acylcarnitine profiles and molecular features of patients with MADD identified through NBS. METHODS: From January 2014 to June 2020, 479,786 newborns screened via tandem mass spectrometry were recruited for this study. Newborns with elevated levels of multiple acylcarnitines were recalled, those who tested positive in the reassessment were referred for genetic analysis. RESULTS: Of 479,786 newborns screened, six were diagnosed with MADD. The MADD incidence in the Chinese population was estimated to be 1:79,964. Initial NBS revealed five patients with typical elevations in the levels of multiple acylcarnitines; however, in one patient, acylcarnitine levels were in the normal reference range during recall. Notably, one patient only exhibited a mildly increased isovalerylcarnitine (C5) level at NBS. The patient with an atypical acylcarnitine profile was diagnosed with MADD by targeted gene sequencing. Six distinct ETFDH missense variants were identified, with the most common variant being c.250G>A (p.A84T), with an allelic frequency of 58.35 (7/12). CONCLUSIONS: These findings revealed that it is easy for patients with MADD to go unidentified, as they may have atypical acylcarnitine profiles at NBS and the recall stage, indicating the value of genetic analysis for confirming suspected inherited metabolic disorders in the NBS program. Therefore, false-negative (FN) results may be reduced by combining tandem mass spectrometry (MS/MS) with genetic testing in NBS for MADD.

Sequential extraction and enrichment of pesticide residues in Longan fruit by ultrasonic-assisted aqueous two-phase extraction linked to vortex-assisted dispersive liquid-liquid microextraction prior to high performance liquid chromatography analysis.

A simple, green and efficient method for extraction, purification and enrichment of pesticide residues of triazoles and pyrethroids in Longan fruit was developed by ultrasonic-assisted aqueous two-phase extraction (UAATPE) coupled to vortex-assisted dispersive liquid-liquid microextraction (VADLLME). Using an aqueous two-phase system (ATPS) of ethanol/K2HPO4 as extraction solvent, the composition of the ATPS, extraction temperature and time were investigated, respectively. Then VADLLME process also was optimized by investigating type and volume of extracting and dispersive solvents, vortex-assisted time and salt addition. The optimum conditions were as follows: the ATPS composition of ethanol concentration 30.0% (w/w) and K2HPO4 concentration 25% (w/w), extraction temperature 70 °C and extraction time 15 min for UAATPE; 1-dodecanol 200 µL as extraction solvent, ethanol 1.25 mL as dispersive solvent, vortex-assisted time 1.5 min and addition of NaCl 4% (w/v) for VADLLME. Ethanol as extraction solvent and dispersive solvent could directly connect UAATPE with VADLLME without extra steps. By means of HPLC-DAD detection, nine pesticides had good linearity ranged from 0.0200 to 13.59 µg/mL (R2 ≥ 0.9957). LODs and LOQs were in the range of 0.005576-0.01740 µg/mL and 0.01859-0.05010 µg/mL, respectively. UAATPE-VADLLME coupled to HPLC was successfully applied to simultaneous determination of multiple pesticides in Longan fruit, and mean recoveries and RSDs were between 76.95% and 98.63%, 1.2% and 9.8%, respectively. Furthermore, myclobutanil, fenpropathrin and deltamethrin were detected in pericarp and pulp of Longan samples from different districts, respectively.

Erratum to upregulation of long noncoding RNA RAB11B-AS1 promotes tumor metastasis and predicts poor prognosis in lung cancer.

[This corrects the article DOI: 10.21037/atm.2020.04.52.].

Upregulation of long noncoding RNA RAB11B-AS1 promotes tumor metastasis and predicts poor prognosis in lung cancer.

BACKGROUND: Lung cancer (LC) is one of the leading causes of cancer-related mortality in China and worldwide. Despite the progress in diagnosis and treatment of LC, the prognosis of LC remains poor. Studies have demonstrated that long non-coding RNAs (lncRNAs) play a critical role in carcinogenesis and cancer development. METHODS: Here we examined the expression and potential function of lnc-RAB11B-AS1 in LC both in vitro and in vivo. All experiments in this study were conducted using A549 and PC-9 cell lines according to protocols described in this paper. The clinic characteristics were analyzed using logistic regression, cox model, log rank test, biochemical analysis using qRT-PCR, transfections, nude mice model, and cell biological analysis using Transwell assay, CCK-8 assay, flow cytometry, and rescue experiments, and immunohistochemistry. RESULTS: The results showed that lnc-RAB11B-AS1 was significantly overexpressed in LC tissues compared to the corresponding non-tumor tissues. Patients with a higher level of lnc-RAB11B-AS1 expression showed a poorer overall survival rate. Functionally, overexpression of lnc-RAB11B-AS1 promotes cell proliferation, migration and invasion abilities of LC cell lines, which suggests lnc-RAB11B-AS1 may play an oncogenic role in LC. lnc-RAB11B-AS1 was located in physical contiguity with RAB11B gene and found positively regulates the RAB11B expression, and the protein levels of RAB11B in LC tissues also found to positively correlated with the level of lnc-RAB11B-AS1 expression. RAB11B silencing partially abrogated lnc-RAB11B-AS1-induced proliferation of the LC cell lines used in this study. CONCLUSIONS: This study provided a novel evidence into the function of lncRNA-driven carcinogenesis. Our findings highlighted the importance of lnc-RAB11B-AS1 and RAB11B in LC progression and indicated that lnc-RAB11B-AS1 may serve as a novel and valuable prognostic biomarker for LC.

An antibiotic stewardship programme to reduce inappropriate antibiotic prescribing for acute respiratory infections in rural Chinese primary care facilities: study protocol for a clustered randomised controlled trial.

BACKGROUND: Inappropriate prescribing of antibiotics for acute respiratory infections at the primary care level represents the major source of antibiotic misuse in healthcare, and is a major driver for antimicrobial resistance worldwide. In this study we will develop, pilot and evaluate the effectiveness of a comprehensive antibiotic stewardship programme in China's primary care hospitals to reduce inappropriate prescribing of antibiotics for acute respiratory infections among all ages. METHODS: We will use a parallel-group, cluster-randomised, controlled, superiority trial with blinded outcome evaluation but unblinded treatment (providers and patients). We will randomise 34 primary care hospitals from two counties within Guangdong province into the intervention and control arm (1:1 overall ratio) stratified by county (8:9 within-county ratio). In the control arm, antibiotic prescribing and management will continue through usual care. In the intervention arm, we will implement an antibiotic stewardship programme targeting family physicians and patients/caregivers. The family physician components include: (1) training using new operational guidelines, (2) improved management and peer-review of antibiotic prescribing, (3) improved electronic medical records and smart phone app facilitation. The patient/caregiver component involves patient education via family physicians, leaflets and videos. The primary outcome is the proportion of prescriptions for acute respiratory infections (excluding pneumonia) that contain any antibiotic(s). Secondary outcomes will address how frequently specific classes of antibiotics are prescribed, how frequently key non-antibiotic alternatives are prescribed and the costs of consultations. We will conduct a qualitative process evaluation to explore operational questions regarding acceptability, cultural appropriateness and burden of technology use, as well as a cost-effectiveness analysis and a long-term benefit evaluation. The duration of the intervention will be 12 months, with another 24 months' post-trial long-term follow-up. DISCUSSION: Our study is one of the first trials to evaluate the effect of an antibiotic stewardship programme in primary care settings in a low- or middle-income country (LMIC). All interventional activities will be designed to be embedded into routine primary care with strong local ownership. Through the trial we intend to impact on clinical practice and national policy in antibiotic prescription for primary care facilities in rural China and other LMICs. TRIAL REGISTRATION: ISRCTN, ID: ISRCTN96892547. Registered on 18 August 2019.

Long noncoding RNA DNAJC3-AS1 promotes osteosarcoma progression via its sense-cognate gene DNAJC3.

Long noncoding RNAs have been proved to play essential roles in tumor development and progression. In this study, we focused on DNAJC3-AS1 and investigated its biological function and clinical significance in osteosarcoma. We detected the expression of DNAJC3-AS1 in 30 pairs of matched osteosarcoma and adjacent nontumorous specimens and osteosarcoma cell lines and analyzed association between DNAJC3-AS1 levels and clinicopathological factors. We found that DNAJC3-AS1 expression was up-regulated in osteosarcoma. High level of DNAJC3-AS1 was correlated with high differentiated degree (P = 0.018) and advanced Enneking stage (P = 0.016). Mechanistically, DNAJC3-AS1 enhanced cell proliferation, migration, and invasion in vitro and in vivo and reduced sensitivity of osteosarcoma to cisplatin. These effects of DNAJC3-AS1 were reversed by down-regulation of its sense-cognate gene DNAJC3. Thus, DNAJC3-AS1 promotes osteosarcoma development and progression by regulating DNAJC3 and might be a biomarker and therapeutic target for osteosarcoma.

Long non-coding RNA RAB11B-AS1 prevents osteosarcoma development and progression via its natural antisense transcript RAB11B.

Long non-coding RNAs (lncRNAs) have been shown to exert essential roles in development and progression of tumors. Here we discovered a novel lncRNA, RAB11B antisense RNA (RAB11B-AS1), which is markedly down-regulated in human osteosarcoma (OS) and associated with OS metastasis and poor prognosis. We find that reduction of RAB11B-AS1 significantly facilitates proliferation, migration and invasiveness and prevents apoptosis of OS cells and results in lower sensitivity to cisplatin in these cells. In contrast, up-regulation of RAB11B-AS1 suppresses the aggressive behaviors of OS cells. Mechanistically, down-regulation of RAB11B-AS1 elevates its sense-cognate gene RAB11B expression at both mRNA and protein levels. RAB11B-AS1 expression correlates negatively with RAB11B expression in OS tissues. Luciferase reporter assay illuminated that RAB11B-AS1 regulates RAB11B expression through antisense pairing. Most importantly, all the effects of RAB11B-AS1 were abrogated by RAB11B down-regulation. Thus our findings revealed that lnc-RAB11B-AS1 prevents osteosarcoma development and progression via inhibiting RAB11B expression, indicating lnc-RAB11B-AS1 as a potential therapeutic target for osteosarcoma.