AMRViz enables seamless genomics analysis and visualization of antimicrobial resistance.

We have developed AMRViz, a toolkit for analyzing, visualizing, and managing bacterial genomics samples. The toolkit is bundled with the current best practice analysis pipeline allowing researchers to perform comprehensive analysis of a collection of samples directly from raw sequencing data with a single command line. The analysis results in a report showing the genome structure, genome annotations, antibiotic resistance and virulence profile for each sample. The pan-genome of all samples of the collection is analyzed to identify core- and accessory-genes. Phylogenies of the whole genome as well as all gene clusters are also generated. The toolkit provides a web-based visualization dashboard allowing researchers to interactively examine various aspects of the analysis results. Availability: AMRViz is implemented in Python and NodeJS, and is publicly available under open source MIT license at https://github.com/amromics/amrviz .

Mimosa pudica L. extract ameliorates pulmonary fibrosis via modulation of MAPK signaling pathways and FOXO3 stabilization.

ETHNOPHARMACOLOGICAL RELEVANCE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing pulmonary disorder that has a poor prognosis and high mortality. Although there has been extensive effort to introduce several new anti-fibrotic agents in the past decade, IPF remains an incurable disease. Mimosa pudica L., an indigenous Vietnamese plant, has been empirically used to treat respiratory disorders. Nevertheless, the therapeutic effects of M. pudica (MP) on lung fibrosis and the mechanisms underlying those effects remain unclear. AIM OF THE STUDY: This study investigated the protective effect of a crude ethanol extract of the above-ground parts of MP against pulmonary fibrogenesis. MATERIALS AND METHODS: Inflammatory responses triggered by TNFα in structural lung cells were examined in normal human lung fibroblasts and A549 alveolar epithelial cells using Western blot analysis, reverse transcription-quantitative polymerase chain reaction assays, and immunocytochemistry. The epithelial-to-mesenchymal transition (EMT) was examined via cell morphology observations, F-actin fluorescent staining, gene and protein expression measurements, and a wound-healing assay. Anti-fibrotic assays including collagen release, differentiation, and measurements of fibrosis-related gene and protein expression levels were performed on TGFß-stimulated human lung fibroblasts and lung fibroblasts derived from mice with fibrotic lungs. Finally, in vitro anti-fibrotic activities were validated using a mouse model of bleomycin-induced pulmonary fibrosis. RESULTS: MP alleviated the inflammatory responses of A549 alveolar epithelial cells and lung fibroblasts, as revealed by inhibition of TNFα-induced chemotactic cytokine and chemokine expression, along with inactivation of the MAPK and NFκB signalling pathways. MP also partially reversed the TGFß-promoted EMT via downregulation of mesenchymal markers in A549 cells. Importantly, MP decreased the expression levels of fibrosis-related genes/proteins including collagen I, fibronectin, and αSMA; moreover, it suppressed collagen secretion and prevented myofibroblast differentiation in lung fibroblasts. These effects were mediated by FOXO3 stabilization through suppression of TGFß-induced ERK1/2 phosphorylation. MP consistently protected mice from the onset and progression of bleomycin-induced pulmonary fibrosis. CONCLUSION: This study explored the multifaceted roles of MP in counteracting the pathobiological processes of lung fibrosis. The results suggest that further evaluation of MP could yield candidate therapies for IPF.

Prevalence and determinants of constipation in children in Asia: a systematic review and meta-analysis.

Background: Constipation is prevalent worldwide, significantly increasing healthcare costs and diminishing the quality of life in children affected. Current studies have yielded mixed results regarding the factors associated with constipation, and mainly focusing on patients outside of Asia. Moreover, most of these studies lack focus on the paediatric population. This study aimed to identify the prevalence and associated factors of constipation among children in Asia. Methods: In this systematic review and meta-analysis, we systematically searched PubMed, Scopus, and Cochrane for cohort and cross-sectional studies published from database inception up to October 12, 2022, and continued with manual searching until September 2, 2023. Eligible studies were those that included children in Asia aged 0-18 years old suffering from idiopathic constipation, with prevalence value provided in the English abstract. The analysis included clinical and general population. Children with organic constipation, who had undergone gastrointestinal surgery, or with congenital defects were excluded, as these factors affect the incidence of constipation. Data included in the analysis were extracted from published reports only. The extracted data were pooled using random-effects model to analyse the prevalence of constipation in children in Asia. This study is registered with PROSPERO, CRD42022367122. Findings: Out of 4410 systematically searched studies and 36 manually searched ones, a total of 50 studies were included in the final analysis, encompassing data from 311,660 children residing in Asia. The pooled prevalence of constipation was 12.0% (95% CI 9.3-14.6%, I2 = 99.8%). There was no significant difference in constipation prevalence observed by sex and geographical location. Nonetheless, adolescents and children aged 1-9 years exhibited a significantly higher prevalence constipation compared to infants (p < 0.0001) Additionally, significant differences in constipation rates were observed across various diagnostic methods, population sources, and mental health conditions. Interpretation: Despite the high heterogeneity resulting from varying diagnostic tools or definitions used among studies, our review adds to the literature on constipation among children in Asia. It reveals a notably high prevalence of constipation in this demographic. Diagnostic methods, age, and compromised mental health emerged as significant influencers of constipation among children in Asia, highlighting potential strategies to mitigate constipation prevalence in children in Asia. Funding: The National Science and Technology Council, Taiwan.

Antibiotic Resistance, Biofilm Formation, and Persistent Phenotype of Klebsiella pneumoniae in a Vietnamese Tertiary Hospital: A Focus on Amikacin.

Klebsiella pneumoniae stands out as a major opportunistic pathogen responsible for both hospital- and community-acquired bacterial infections. This study comprehensively assesses the antibiotic resistance, amikacin persistent patterns, and biofilm-forming ability of 247 isolates of K. pneumoniae obtained from an intensive care unit of a tertiary hospital in Vietnam. Microdilution assays, conducted on a 96-well plate, determined the minimum inhibitory concentrations (MICs) of amikacin. Susceptibility data for other antibiotics were gathered from the antibiogram profile. Stationary-phase bacteria were exposed to 50 × MIC, and viable bacteria counts were measured to determine amikacin persistence. Biofilm forming capacity on 96-well polystyrene surfaces was assessed by biomass and viable bacteria. The prevalence of resistance was notably high across most antibiotics, with 64.8% classified as carbapenem-resistant K. pneumoniae and 81.4% as multidrug resistant. Amikacin, however, exhibited a relatively low rate of resistance. Of the isolates, 58.2% demonstrated a moderate to strong biofilm formation capacity, and these were found to be poorly responsive to amikacin. K. pneumoniae reveals a significant inclination for amikacin persistence, with ∼45% of isolates displaying an antibiotic antibiotic-survival ratio exceeding 10%. The study sheds light on challenges in treating of K. pneumoniae infection in Vietnam, encompassing a high prevalence of antibiotic resistance, a substantial ability to form biofilm, and a notable rate of antibiotic persistence.

Pasa: leveraging population pangenome graph to scaffold prokaryote genome assemblies.

Whole genome sequencing has increasingly become the essential method for studying the genetic mechanisms of antimicrobial resistance and for surveillance of drug-resistant bacterial pathogens. The majority of bacterial genomes sequenced to date have been sequenced with Illumina sequencing technology, owing to its high-throughput, excellent sequence accuracy, and low cost. However, because of the short-read nature of the technology, these assemblies are fragmented into large numbers of contigs, hindering the obtaining of full information of the genome. We develop Pasa, a graph-based algorithm that utilizes the pangenome graph and the assembly graph information to improve scaffolding quality. By leveraging the population information of the bacteria species, Pasa is able to utilize the linkage information of the gene families of the species to resolve the contig graph of the assembly. We show that our method outperforms the current state of the arts in terms of accuracy, and at the same time, is computationally efficient to be applied to a large number of existing draft assemblies.

Histamine-degrading halophilic bacteria from traditional fish sauce: Characterization of Virgibacillus campisalis TT8.5 for histamine reduction.

Traditionally produced fish sauce can contain significant amounts of histamine. In some instances, the histamine concentration may be well above the limit recommended by the Codex Alimentarius Commission. The aim of this study was to discover new bacterial strains capable of growing under the stressful environmental conditions of fish sauce fermentation and metabolizing histamine. In this study, 28 bacterial strains were isolated from Vietnamese fish sauce products based on their ability to grow at high salt concentrations (23% NaCl) and tested for their ability to degrade histamine. Strain TT8.5 showed the highest histamine-degradation (45.1 ± 0.2% of initially 5 mM histamine within 7 days) and was identified as Virgibacillus campisalis TT8.5. Its histamine-degrading activity was shown to be localized intracellularly and the enzyme is a putative histamine dehydrogenase. The strain exhibited optimal growth and histamine-degrading activity at 37°C, pH 7%, and 5% NaCl in halophilic archaea (HA) histamine broth. It also showed pronounced histamine-degrading activity in HA histamine broth when cultivated at temperatures of up to 40 °C as well as in the presence of up to 23% NaCl. After treatment with immobilized cells, 17.6-26.9% of the initial histamine in various fish sauce products were reduced within 24 h of incubation, while no significant changes in other parameters of fish sauce quality were observed after this treatment. Our results indicate that V. campisalis TT8.5 is of potential interest to be applied in histamine degradation of traditional fish sauce.

Ginsenoside protopanaxadiol protects adult retinal pigment epithelial-19 cells from chloroquine by modulating autophagy and apoptosis.

Chloroquine often causes serious eye and vision problems, which are mainly mediated by lysosomotropic alteration. In this study, we investigated whether the ginsenoside protopanaxadiol relieves chloroquine-induced retinopathy by restoring lysosomotropic abnormalities in human adult retinal pigment epithelial-19 cells. Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological alterations in autophagosomes of adult retinal pigment epithelial-19 cells was detected using confocal microscopy. Apoptosis was examined using flow cytometry, whereas cellular reactive oxygen species levels were determined by measuring the fluorescence intensity of 5-(and-6)-carboxy-2'-7'-dichlorohydrofluorescein diacetate. Lysosomal function was assessed by measuring lysosomal pH and enzyme activity. Immunoprecipitation and western blotting analyses were performed. Adult retinal pigment epithelial-19 cells accumulated autophagosomes with fusion defects in lysosomes and reactive oxygen species formation following exposure to chloroquine. This effect trapped Beclin-1 and B-cell lymphoma 2 interfering with autophagy initiation and autophagosome development. Protopanaxadiol alleviated chloroquine-induced toxicity by modulating the interaction between Beclin-1 and Bcl-2, which was mediated by the AMP-activated protein kinase-mammalian target of rapamycin signal axis. Furthermore, autophagy and apoptosis were simultaneously controlled by protopanaxadiol via upregulation of autophagy flux and decreased reactive oxygen species formation and apoptotic protein expression. These findings suggest that protopanaxadiol is a promising treatment strategy for chloroquine-mediated retinopathy.

Optimizing Meropenem in Highly Resistant Klebsiella pneumoniae Environments: Population Pharmacokinetics and Dosing Simulations in Critically Ill Patients.

Critically ill patients are characterized by substantial pathophysiological changes that alter the pharmacokinetics (PK) of hydrophilic antibiotics, including carbapenems. Meropenem is a key antibiotic for multidrug-resistant Gram-negative bacilli, and such pathophysiological alterations can worsen treatment outcomes. This study aimed to determine the population PK of meropenem and to propose optimized dosing regimens for the treatment of multidrug-resistant Klebsiella pneumoniae in critically ill patients. Two plasma samples were collected from eligible patients over a dosing interval. Nonparametric population PK modeling was performed using Pmetrics. Monte Carlo simulations were applied to different dosing regimens to determine the probability of target attainment and the cumulative fraction of response, taking into account the local MIC distribution for K. pneumoniae. The targets of 40% and 100% for the fraction of time that free drug concentrations remained above the MIC (ƒT>MIC) were tested, as suggested for critically ill patients. A one-compartment PK model using data from 27 patients showed high interindividual variability. Significant PK covariates were the 8-h creatinine clearance for meropenem and the presence of an indwelling catheter for pleural, abdominal, or cerebrospinal fluid drainage for the meropenem volume of distribution. The target 100% ƒT>MIC for K. pneumoniae, with a MIC of ≤2 mg/liter, could be attained by the use of a continuous infusion of 2.0 g/day. Meropenem therapy in critically ill patients could be optimized for K. pneumoniae isolates with an MIC of ≤2 mg/liter by using a continuous infusion in settings with more than 50% isolates have a MIC of ≥32mg/L.

Safety and effectiveness of all-oral and injectable-containing, bedaquiline-based long treatment regimen for pre-XDR tuberculosis in Vietnam.

Background: The World health organization (WHO) recently recommended standardized all-oral shorter regimens for rifampicin resistant Tuberculosis (RR-TB). For highly resistant Tuberculosis patients such as pre-XDR-TB: RR-TB plus additional resistance to fluoroquinolones (FQ), the 6-9-months bedaquiline (bedaquiline)-based regimens or BDQ-based long regimens are recommended. The role of second-line injectable (SLI) drugs in the treatment of drug resistant TB is restricted because of safety concerns. Nevertheless, it is not well-known how all-oral long regimens (BDQ-long) perform compared to SLI-containing long regimens (BDQ/SLI-long) in terms of safety and effectiveness among patients with highly resistant TB. Method: A prospective observational cohort of patients with RR-TB additionally resistant to fluoroquinolones and/or second-line injectable, treated with either BDQ-long or BDQ/SLI-long regimens according to the guidance of the National Tuberculosis Program of Vietnam, enrolled between December 2015 and June 2017. Results: Of 99 patients enrolled, 42 (42%) patients were treated with BDQ-long and 57 (57%) with BDQ/SLI-long. More than 85% of patients were previously exposed to both FQ and SLI. FQ and SLI resistance were confirmed in 28 (67%) and 41 (98%) in the BDQ-long cohort and 48 (84%) and 17 (30%) in the BDQ/SLI-long cohort, respectively. Treatment success was achieved among 29 (69%) and 46 (81%) patients on the BDQ-long and BDQ/SLI-long regimen, respectively (p = 0.2). For both regimens, median time to first smear/culture sputum conversion was 2 months. All patients experienced at least one adverse event (AE) and 85% of them had at least one severe Adverse events. The median time to a first severe adverse event was 2 months. Among patients treated with BDQ-long a higher proportion of patients had three QT-prolonging drugs in the regimen (26.2% versus 7.0%; p = 0.009). The severe prolonged QTcF was observed in 22 (52.4%) and 22 (38.6%) patients on BDQ-long and BDQ/SLI-long, respectively. Overall, 30 (30%) patients had to either temporary or permanently discontinued or more TB drugs due to AEs. Conclusion: Treatment success was similar for both all-oral and SLI-containing BDQ-based long regimens in highly resistant TB patients. Both regimens had a similar high frequency of AEs. For both BDQ-long and BDQ/SLI-long regimens active AEs monitoring is essential.

Country data on AMR in Vietnam in the context of community-acquired respiratory tract infections: links between antibiotic susceptibility, local and international antibiotic prescribing guidelines, access to medicines and clinical outcome.

BACKGROUND: Antimicrobial resistance (AMR) is one of the biggest threats to global public health. Selection of resistant bacteria is driven by inappropriate use of antibiotics, amongst other factors. COVID-19 may have exacerbated AMR due to unnecessary antibiotic prescribing. Country-level knowledge is needed to understand options for action. OBJECTIVES: To review the current situation with respect to AMR in Vietnam and initiatives addressing it. Identifying areas where more information is required will provide a call to action to minimize any further rises in AMR within Vietnam and improve patient outcomes. METHODS: National initiatives to address AMR in Vietnam, antibiotic use and prescribing, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens Streptococcus pneumoniae and Haemophilus influenzae, were identified. National and international antibiotic prescribing guidelines for CA-RTIs (community-acquired pneumonia, acute otitis media and acute bacterial rhinosinusitis) commonly used locally were also reviewed, plus local antibiotic availability. Insights from clinicians in Vietnam were sought to contextualize this information. CONCLUSIONS: In Vietnam there have been some initiatives addressing AMR; Vietnam was the first country in the Western Pacific Region to develop a national action plan to combat AMR, which according to the WHO is being implemented. Vietnam also has one of the highest rates of AMR in Asia due, in part, to the overuse of antimicrobial drugs, both in the animal health sector and in humans in both hospitals and the community. In addition, despite a 2005 law requiring antibiotic prescription, there is unrestricted access to over-the-counter antibiotics. Several global surveillance studies provide antibiotic susceptibility data for CA-RTI pathogens in Vietnam including Survey of Antibiotic Resistance (SOAR) and SENTRY (small isolate numbers only). For management of the common CA-RTIs in Vietnam there are several country-specific local antibiotic prescribing guidelines and in addition, there is a range of international guidelines referred to, but these may have been created based on pathogen resistance patterns that might be very different to those in Vietnam. Expert clinician opinion confirms the high resistance rates among common respiratory pathogens. A more standardized inclusive approach in developing local guidelines, using up-to-date surveillance data of isolates from community-acquired infections in Vietnam, could make management guideline use more locally relevant for clinicians. This would pave the way for a higher level of appropriate antibiotic prescribing and improved adherence. This would, in turn, potentially limit AMR development and improve clinical outcomes for patients.

A National Survey of Dispensing Practice and Customer Knowledge on Antibiotic Use in Vietnam and the Implications.

Misconceptions and pressures have increased the sales of antibiotics without a prescription across countries. There are concerns with such practices in Vietnam given rising antimicrobial resistance rates. A national survey was conducted among 360 private drugstores located in nine provinces in Vietnam. Anonymous interviews were conducted with participants selected by convenience sampling. Subsequently, multivariable logistic regression analyses were undertaken evaluating the relationship between customer characteristics and antibiotic purchases. A total of 480 out of 1626 surveyed participants purchased antibiotics, 81.7% of which did not have a prescription, involving 29 different antibiotics. In 86.4% of these, participants were prescribed antibiotics by drug sellers. Most antibiotics were sold to treat respiratory tract infections (61.4%), with the 'Access' antibiotics (amoxicillin and cephalexin) being the most frequently sold. Only one-fifth of participants understood that they were breaking the law by purchasing antibiotics without a prescription. Participants purchasing antibiotics without a prescription had lower awareness concerning antibiotic laws and treatment duration (p < 0.05). Under 50% agreed to having a doctors' prescription in the future when purchasing antibiotics. Freelancer occupation (OR = 0.52, 95% CI = 0.83−0.96) and a lower educational level (OR = 0.49, 95% CI = 0.25−0.96) were factors related to purchasing antibiotics without a prescription. Overall, we recommend increasing fines and monitoring of drugs stores, greater promotion of the family doctor system as well as increasing media and educational campaigns to limit self-purchasing of antibiotics in Vietnam and reduce resistance.

Harnessing new mHealth technologies to Strengthen the Management of Multidrug-Resistant Tuberculosis in Vietnam (V-SMART trial): a protocol for a randomised controlled trial.

INTRODUCTION: Multidrug-resistant tuberculosis (MDR-TB) remains a major public health problem globally. Long, complex treatment regimens coupled with frequent adverse events have resulted in poor treatment adherence and patient outcomes. Smartphone-based mobile health (mHealth) technologies offer national TB programmes an appealing platform to improve patient care and management; however, clinical trial evidence to support their use is lacking. This trial will test the hypothesis that an mHealth intervention can improve treatment success among patients with MDR-TB and is cost-effective compared with standard practice. METHODS AND ANALYSIS: A community-based, open-label, parallel-group randomised controlled trial will be conducted among patients treated for MDR-TB in seven provinces of Vietnam. Patients commencing therapy for microbiologically confirmed rifampicin-resistant or multidrug-resistant tuberculosis within the past 30 days will be recruited to the study. Participants will be individually randomised to an intervention arm, comprising use of an mHealth application for treatment support, or a 'standard care' arm. In both arms, patients will be managed by the national TB programme according to current national treatment guidelines. The primary outcome measure of effectiveness will be the proportion of patients with treatment success (defined as treatment completion and/or bacteriological cure) after 24 months. A marginal Poisson regression model estimated via a generalised estimating equation will be used to test the effect of the intervention on treatment success. A prospective microcosting of the intervention and within-trial cost-effectiveness analysis will also be undertaken from a societal perspective. Cost-effectiveness will be presented as an incremental cost per patient successfully treated and an incremental cost per quality-adjusted life-year gained. ETHICS: Ethical approval for the study was granted by The University of Sydney Human Research Ethics Committee (2019/676). DISSEMINATION: Study findings will be disseminated to participants and published in peer-reviewed journals and conference proceedings. TRIAL REGISTRATION NUMBER: ACTRN12620000681954.

Measuring Orthographic Knowledge of L2 Chinese Learners in Vietnam Using a Handwriting Task - A Preliminary Report.

In the current study, the orthographic knowledge required for writing Chinese characters was assessed among participants with L1 Vietnamese background who learn Chinese as a foreign language. A total of 42 undergraduates were recruited. They were invited to participate in a delayed Chinese character copying task consisting of 32 characters. Their Chinese character reading abilities were also obtained using a character naming task. All the tests were conducted online during the pandemic in 2021. Results indicated that the participants' accuracy in the copying task was affected by the familiarity of the characters and the number of strokes of the characters. These effects minimized as reading performance increased. In the inter-stroke interval (ISI) analysis, results indicated a significant boundary effect where ISIs between orthographic units were longer than ISIs within orthographic units, showing the participants' tendency to chunk Chinese characters into functional units when they write. Only high achievers in the reading task demonstrated the use of both large and small grain-size units in writing (i.e., radical-boundary ISI > logographeme-boundary ISI > non-boundary ISI), while the low achievers only used small grain-size units in their writing. We suggest that the delayed copying task incorporated with handwriting measures is an effective method to assess orthographic knowledge of L2 Chinese learners.

Anti-inflammatory effect of a triterpenoid from Balanophora laxiflora: results of bioactivity-guided isolation.

Balanophora laxiflora, a medicinal plant traditionally used to treat fever, pain, and inflammation in Vietnam, has been reported to possess prominent anti-inflammatory activity. This study examined the active constituents and molecular mechanisms underlying these anti-inflammatory effects using bioactivity-guided isolation in combination with cell-based assays and animal models of inflammation. Among the isolated compounds, the triterpenoid (21α)-22-hydroxyhopan-3-one (1) showed the most potent inhibitory effect on COX-2 expression in LPS-stimulated Raw 264.7 macrophages. Furthermore, 1 suppressed the expression of the inflammatory mediators iNOS, IL-1ß, INFß, and TNFα in activated Raw 264.7 macrophages and alleviated the inflammatory response in carrageenan-induced paw oedema and a cotton pellet-induced granuloma model. Mechanistically, the anti-inflammatory effects of 1 were mediated via decreasing cellular reactive oxygen species (ROS) levels by inhibiting NADPH oxidases (NOXs) and free radical scavenging activities. By downregulating ROS signalling, 1 reduced the activation of MAPK signalling pathways, leading to decreased AP-1-dependent transcription of inflammatory mediators. These findings shed light on the chemical constituents that contribute to the anti-inflammatory actions of B. laxiflora and suggest that 1 is a promising candidate for treating inflammation-related diseases.

Allopurinol-induced severe cutaneous adverse reactions in Vietnamese: the role of HLA alleles and other risk factors.

Aim: To reveal the association of three class I HLA alleles, including HLA-A*33:03, HLA-B*58:01 and HLA-C*03:02, and allopurinol-induced severe cutaneous adverse reactions (SCARs) in Vietnamese patients. Methods: A case-control study on 100 allopurinol-induced SCARs patients, 183 tolerant controls and 810 population controls was performed. The HLA-A*33:03 and HLA-C*03:02 alleles were detected with the nested allele-specific PCR method; the HLA-B*58:01 allele was detected with the sequence-specific primer PCR method. Results: There were strong associations between HLA-B*58:01 and HLA-C*03:02 and allopurinol-induced SCARs. Specific associations were found between HLA-B*58:01 and Stevens-Johnson syndrome/toxic epidermal necrolysis and between HLA-C*03:02 and drug reaction with eosinophilia and systemic symptoms, with a gene dosage effect. The multivariate regression analysis indicated two significant independent risk factors: HLA-B*58:01/HLA-C*03:02 and estimated glomerular filtration rate <60 ml/min/1.73 m2. The specificity, positive predictive value and negative predictive value of HLA-B*58:01 testing were higher than the HLA-C*03:02 or the multiplex testing, especially in patients with impaired renal function. Conclusion: The results supported pre-treatment HLA-B*58:01 testing in Vietnamese patients with declined renal function to prevent SCARs.

Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2'-7'-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy.

Predicting mortality in intensive care unit patients infected with Klebsiella pneumoniae: A retrospective cohort study.

INTRODUCTION: Although several models to predict intensive care unit (ICU) mortality are available, their performance decreases in certain subpopulations because specific factors are not included. Moreover, these models often involve complex techniques and are not applicable in low-resource settings. We developed a prediction model and simplified risk score to predict 14-day mortality in ICU patients infected with Klebsiella pneumoniae. METHODOLOGY: A retrospective cohort study was conducted using data of ICU patients infected with Klebsiella pneumoniae at the largest tertiary hospital in Northern Vietnam during 2016-2018. Logistic regression was used to develop our prediction model. Model performance was assessed by calibration (area under the receiver operating characteristic curve-AUC) and discrimination (Hosmer-Lemeshow goodness-of-fit test). A simplified risk score was also constructed. RESULTS: Two hundred forty-nine patients were included, with an overall 14-day mortality of 28.9%. The final prediction model comprised six predictors: age, referral route, SOFA score, central venous catheter, intracerebral haemorrhage surgery and absence of adjunctive therapy. The model showed high predictive accuracy (AUC = 0.83; p-value Hosmer-Lemeshow test = 0.92). The risk score has a range of 0-12 corresponding to mortality risk 0-100%, which produced similar predictive performance as the original model. CONCLUSIONS: The developed prediction model and risk score provide an objective quantitative estimation of individual 14-day mortality in ICU patients infected with Klebsiella pneumoniae. The tool is highly applicable in practice to help facilitate patient stratification and management, evaluation of further interventions and allocation of resources and care, especially in low-resource settings where electronic systems to support complex models are missing.

Voltammetric Determination of Amoxicillin Using a Reduced Graphite Oxide Nanosheet Electrode.

A reduced graphite oxide nanosheet electrode (RGOnS) was prepared as a sensor for amoxicillin (AMX) detection, an antibiotic commonly used in the livestock farm, by the square-wave adsorptive stripping voltammetry technique. Graphite oxide with nanosheet shape was produced from a graphite electrode by a chronoamperometry process at 5 V and then an electrochemical reduction process was carried out to form RGOnS with restored long-range conjugated networks and better conductivity. The electrodes were characterized by SEM, EDX, and FTIR spectroscopy. The RGOnS electrode prepared at an optimal reduction potential of -1 V for 120 s exhibits a larger electrochemical active surface area, and the obtained oxidation signal of AMX is approximately ten times higher than that of the pristine graphite electrode. The analytical conditions such as the pH of electrolyte and accumulation time were optimized. The calibration curve built under the optimal conditions provided a good linear relationship in the range of AMX concentration from 0.5-80 µM with the correlation coefficient of 0.9992. The limit of detection was calculated as 0.193 µM. Satisfactory results are obtained from the detection of the AMX in different samples using the prepared electrode.

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.