Metagenomic shotgun sequencing reveals the enrichment of Salmonella and Mycobacterium in larynx due to prolonged ethanol exposure.

The exposure of ethanol increases the risk of head and neck inflammation and tumor progression. However, limited studies have investigated the composition and functionality of laryngeal microbiota under ethanol exposure. We established an ethanol-exposed mouse model to investigate the changes in composition and function of laryngeal microbiota using Metagenomic shotgun sequencing. In the middle and late stages of the experiment, the laryngeal microbiota of mice exposed to ethanol exhibited obvious distinguished from that of the control group on principal-coordinate analysis (PCoA) plots. Among the highly abundant species, Salmonella enterica and Mycobacterium marinum were likely to be most impacted. Our findings indicated that the exposure to ethanol significantly increased their abundance in larynxes in mice of the same age, which has been confirmed through FISH experiments. Among the species-related functions and genes, metabolism is most severely affected by ethanol. The difference was most obvious in the second month of the experiment, which may be alleviated later because the animal established tolerance. Notable enrichments concerning energy, amino acid, and carbohydrate metabolic pathways occurred during the second month under ethanol exposure. Finally, based on the correlation between species and functional variations, a network was established to investigate relationships among microbiota, functional pathways, and related genes affected by ethanol. Our data first demonstrated the continuous changes of abundance, function and their interrelationship of laryngeal microbiota under ethanol exposure by Metagenomic shotgun sequencing. Importance: Ethanol may participate in the inflammation and tumor progression by affecting the composition of the laryngeal microbiota. Here, we applied the metagenomic shotgun sequencing instead of 16 S rRNA sequencing method to identify the laryngeal microbiota under ethanol exposure. Salmonella enterica and Mycobacterium marinum are two dominant species that may play a role in the reconstruction of the laryngeal microenvironment, as their local abundance increases following exposure to ethanol. The metabolic function is most evidently impacted, and several potential metabolic pathways could be associated with alterations in microbiota composition. These findings could help us better understand the impact of prolonged ethanol exposure on the microbial composition and functionality in the larynx.

Identifying KRT20 as a Potential Key Gene in Lymphatic Metastasis of Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) was the seventh most common cancer worldwide in 2018. Lymphatic metastasis (LM) is closely related to HNSCC prognosis and recurrence. However, the underlying mechanism of LM remains unclear. Therefore, this study aimed to identify the key genes in the LM of HNSCC. Methods: We used The Cancer Genome Atlas (TCGA) to identify differentially expressed genes (DEGs) between LM and non-LM cases. A random forest model, the Search Tool for the Retrieval of Interacting Genes, Cytoscape, and cytoHubba were used to identify hub genes among DEGs, including KRT20 (Cytokeratins 20). We analyzed the survival of KRT20 in TCGA, and we overexpressed KRT20 in HNSCC cell lines to investigate its effects on migration and invasion. We also correlated the expression of KRT20 in HNSCC tissue microarrays with survival and clinicopathological features. Results: We identified 243 DEGs-143 upregulated genes and 100 downregulated genes. Further analysis revealed that KRT20 is a potential key gene associated with LM and overall survival rates among patients with HNSCC. Overexpression of KRT20 increased the migration and invasion ability of HNSCC cell lines Tu686 and FD-LSC-1. Tissue microarray studies demonstrated an overexpression of KRT20 among N1+ patients (including N1-N3 patients). Survival analysis results and the clinicopathological features of HNSCC tissue microarrays were consistent with our analysis of TCGA. Thus, a high KRT20 expression level might suggest an adverse HNSCC prognosis. Our gene set enrichment analysis showed that KRT20 participates in many metabolic pathways, including those related to tumorigenesis and cancer development. Conclusions: We propose that KRT20 may be a key gene in HNSCC with LM.

Dual inhibition of autophagy and PI3K/mTOR pathway as a potential therapeutic strategy against laryngeal squamous cell carcinoma.

Background: New and effective chemotherapy or targeted therapy strategies are needed against laryngeal squamous cell carcinoma (LSCC). We aimed to explore the antitumor effect of dual PI3K/mTOR inhibitor combined with autophagy suppression on LSCC and its underlying mechanism. Methods: Hep-2 and AMC-HN-8 cell lines were treated with the Akt inhibitor LY294002, mTOR inhibitor rapamycin, and dual inhibitor NVP-BEZ235 separately. The biological characteristics of in vitro proliferation, cell cycle, apoptosis, migration, invasion, and autophagy were analyzed, and the expression levels of PI3K/Akt/mTOR pathway-related proteins were also measured. The in vivo effects of NVP-BEZ235 combined with inhibition of autophagy using pharmacological inhibitor was further assessed. Results: Compared with Akt or mTOR inhibitor, NVP-BEZ235 had the most significant biological effects on LSCC cells. When combined with various autophagy inhibitors, along with siRNA against ATG7, NVP-BEZ235 showed a synergic antitumor effect in LSCC through increasing cell apoptosis and death both in vitro and vivo. Conclusions: NVP-BEZ235 exerted potent antitumor effects on LSCC, especially when combined with the autophagy inhibitor both in vitro and vivo, providing convincing experimental data for new molecular targeted therapy for LSCC.

Genome-wide analysis identified novel susceptible genes of restless legs syndrome in migraineurs.

BACKGROUND: Restless legs syndrome is a highly prevalent comorbidity of migraine; however, its genetic contributions remain unclear. OBJECTIVES: To identify the genetic variants of restless legs syndrome in migraineurs and to investigate their potential pathogenic roles. METHODS: We conducted a two-stage genome-wide association study (GWAS) to identify susceptible genes for restless legs syndrome in 1,647 patients with migraine, including 264 with and 1,383 without restless legs syndrome, and also validated the association of lead variants in normal controls unaffected with restless legs syndrome (n = 1,053). We used morpholino translational knockdown (morphants), CRISPR/dCas9 transcriptional knockdown, transient CRISPR/Cas9 knockout (crispants) and gene rescue in one-cell stage embryos of zebrafish to study the function of the identified genes. RESULTS: We identified two novel susceptibility loci rs6021854 (in VSTM2L) and rs79823654 (in CCDC141) to be associated with restless legs syndrome in migraineurs, which remained significant when compared to normal controls. Two different morpholinos targeting vstm2l and ccdc141 in zebrafish demonstrated behavioural and cytochemical phenotypes relevant to restless legs syndrome, including hyperkinetic movements of pectoral fins and decreased number in dopaminergic amacrine cells. These phenotypes could be partially reversed with gene rescue, suggesting the specificity of translational knockdown. Transcriptional CRISPR/dCas9 knockdown and transient CRISPR/Cas9 knockout of vstm2l and ccdc141 replicated the findings observed in translationally knocked-down morphants. CONCLUSIONS: Our GWAS and functional analysis suggest VSTM2L and CCDC141 are highly relevant to the pathogenesis of restless legs syndrome in migraineurs.

Association Between Alcohol Dehydrogenase Polymorphisms and the Recurrence of Laryngeal Carcinoma.

OBJECTIVES: Alcohol consumption is closely associated with prognosis for laryngeal squamous cell carcinoma (LSCC) patients. As key enzymes in ethanol metabolism, proteins in the alcohol dehydrogenase (ADH) family make for valuable targets to establish a novel predictive nomogram model. This study attempts to do so by focusing on the single nucleotide polymorphisms (SNPs) of ADH1B and ADH1C in LSCC. METHODS: Sixty eight LSCC patients that were followed up for more than 10 years were retrospectively analyzed. Endpoints of the current study included disease-free survival and overall survival. Survival analyses were performed using the Kaplan-Meier method and evaluated by log-rank test. The prognostic value of eight ADH1B SNPs and three ADH1C SNPs were evaluated using univariate and multivariate Cox regression analyses. A nomogram model for disease-free survival was established and evaluated using the receiver operating characteristic curve, the C-index, and a calibration plot. RESULTS: Significant association was exhibited between rs17033 (p < 0.001) and rs1229984 (p = 0.002) with an increase in LSCC recurrence rate on Kaplan-Meier curves. Multivariate logistic regression analysis revealed that the rs17033 polymorphism of ADH1B was independently associated with an increased risk of LSCC recurrence (HR = 3.325, 95% CI = 1.684-6.566, p = 0.001). Based on these findings, a prognostic nomogram of LSCC patients involving ADH1B rs17033 was constructed. CONCLUSION: This study has demonstrated an independent association between ADH1B gene variants and the recurrence of LSCC. A nomogram model based on rs17033 of ADH1B, age, T, and N stages were successfully developed for the first time to predict the probability of recurrence in LSCC patients. LEVEL OF EVIDENCE: 3 Retrospective cohort study Laryngoscope, 132:2169-2176, 2022.

Oropharynx microbiota transitions in hypopharyngeal carcinoma treatment of induced chemotherapy followed by surgery.

AIMS: To analyze changes in oropharynx microbiota composition after receiving induced chemotherapy followed by surgery for hypopharyngeal squamous cell carcinoma (HPSCC) patients. METHODS: Clinical data and swab samples of 38 HPSCC patients (HPSCC group) and 30 patients with benign disease (control group, CG) were enrolled in the study. HPSCC group was stratified into two groups: induced chemotherapy group (IC) of 10 patients and non-induced chemotherapy group (nIC) of 28 patients. The microbiota from oropharyngeal membrane was analyzed through 16S rRNA sequencing. RESULTS: Alpha-diversity (Shannon and Ace indexes) and weighted UniFrac based beta-diversity severely decreased in the HPSCC group when compared with CG. In pre-operative comparisons, PCoA and NMDS analyses showed microbial structures in the IC group were more similar to CG than nIC. Both IC group and nIC group yielded significantly diverse post-operative communities in contrast to their pre-operative counterparts, evident by the decrease in genera Veillonella and Fusobacterium and increase in genera Streptococcus and Gemella. Given that post-operative oropharynx microbiota showed no difference between IC and nIC groups, the IC group showed less accumulation in anaerobic communities. The abundance of genera Fusobacterium, Parvimonas, Actinomyces were enhanced in the advanced stages (III/IV). CONCLUSIONS: Oropharynx microbiota in the HPSCC group presents dysbiosis with low diversity and abundance. Induced chemotherapy is beneficial in adjusting the oropharynx microbial environment leading to fewer amounts of anaerobe accumulation after operation. Higher amounts of Fusobacterium in advanced stages (III/IV) may influence the progression of HPSCC.

Glycolysis related gene expression signature in predicting prognosis of laryngeal squamous cell carcinoma.

Researches have suggested that aerobic glycolysis can reflect the development and progression of most carcinomas. We aimed to investigate whether glycolysis-related genes (GRGs) are associated with overall survival in laryngeal squamous cell carcinoma (LSCC). Here, we identified differentially expressed GRGs in TCGA dataset and microarray sample of GSE27020 from GEO database. A set of two glycolytic gene signatures, including DDIT4 and PLOD2 was screened through Cox and Lasso regression. The risk score was calculated using the gene expression of the two GRGs. The high-risk group presented a poor prognosis through Kaplan-Meier method. The ROC curve indicated good prediction performance in survival based on the validation of four cohorts. Univariate and multivariate Cox regression analyses suggested that two-gene signature could be an independent risk factor in LSCC. A total of 17 LSCC patients were enrolled to clarify the genetic expression through using reverse transcription-polymerase chain reaction (RT-PCR). A visualized nomogram was then constructed to predict 1-, 3-, and 5-year overall survival. Taken together, two novel glycolytic gene signatures were discovered and validated, providing a potential therapeutic and overall survival (OS)-prediction biomarker for LSCC.

Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (EBA-175) gene in Equatorial Guinea.

BACKGROUND: Plasmodium falciparum erythrocyte binding antigen-175 (PfEBA-175) is a candidate antigen for a blood-stage malaria vaccine, while various polymorphisms and dimorphism have prevented to development of effective vaccines based on this gene. This study aimed to investigate the dimorphism of PfEBA-175 on both the Bioko Island and continent of Equatorial Guinea, as well as the genetic polymorphism and natural selection of global PfEBA-175. METHODS: The allelic dimorphism of PfEBA-175 region II of 297 bloods samples from Equatorial Guinea in 2018 and 2019 were investigated by nested polymerase chain reaction and sequencing. Polymorphic characteristics and the effect of natural selection were analyzed using MEGA 7.0, DnaSP 6.0 and PopART programs. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 and Foldx program. RESULTS: Both Bioko Island and Bata district populations, the frequency of the F-fragment was higher than that of the C-fragment of PfEBA-175 gene. The PfEBA-175 of Bioko Island and Bata district isolates showed a high degree of genetic variability and heterogeneity, with π values of 0.00407 & 0.00411 and Hd values of 0.958 & 0.976 for nucleotide diversity, respectively. The values of Tajima's D of PfEBA-175 on Bata district and Bioko Island were 0.56395 and - 0.27018, respectively. Globally, PfEBA-175 isolates from Asia were more diverse than those from Africa and South America, and genetic differentiation quantified by the fixation index between Asian and South American countries populations was significant (FST > 0.15, P < 0.05). A total of 310 global isolates clustered in 92 haplotypes, and only one cluster contained isolates from three continents. The mutations A34T, K109E, D278Y, K301N, L305V and D329N were predicted as probably damaging. CONCLUSIONS: This study demonstrated that the dimorphism of F-fragment PfEBA-175 was remarkably predominant in the study area. The distribution patterns and genetic diversity of PfEBA-175 in Equatorial Guinea isolates were similar another region isolates. And the levels of recombination events suggested that natural selection and intragenic recombination might be the main drivers of genetic diversity in global PfEBA-175. These results have important reference value for the development of blood-stage malaria vaccine based on this antigen.

Genetic diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. METHODS: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. RESULTS: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure. CONCLUSIONS: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus.

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/µl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Application of the Ottawa Charter Five Priority Areas of Action for Public Health to an Institution-Wide Diabetes Care Promotion.

This study was the first institution-wide health promotion program in Taiwan to apply the five priority areas for taking action in public health highlighted in the Ottawa Charter for diabetes patients. We aimed to improve the quality of home care received by diabetic patients by training health care professionals in health promotion. This program consisted of developing personal skills, reorienting health services, strengthening community actions, creating supportive environments, and building healthy public policy. It was applied in the Yunlin Christian Hospital located in central Taiwan from August 2011 to November 2011. A health-promoting education course consisting of weight control, diabetes care, and quality management for diabetes was developed and applied to all 323 hospital staff. Then, hospital staff volunteers and diabetes patients were recruited to participate in the program. A total of 61 staff volunteers and 90 diabetes patients were involved in this study. Staff volunteers were trained to participate in communities to provide care and guidance to patients with diabetes. The World Health Organization Quality of Life(WHOQOL)-BREF-Taiwan Version questionnaires were investigated before and after implementation of this program for the patients. A health-promoting lifestyle profile questionnaire was filled by the staff. The investigation data were then analyzed by statistical methods. The diabetes patients experienced a significant increase in their satisfaction with health and health-related quality of life as well as significant improvements in health-promotion and self-management behaviors (p < 0.05). In addition, staff volunteers significantly consumes food from the five major groups than the other staff (p < 0.05). Various improvements in health-promoting behaviors were observed amongst the hospital staff and the diabetic patients. Our project could be a reference for other medical organizations to implement an institution-wide health-promotion program for diabetic patients.

Effects of a single bout of short-duration high-intensity and long-duration low-intensity exercise on insulin resistance and adiponectin/leptin ratio.

OBJECTIVES: Moderate-intensity exercise improves insulin sensitivity, which may depend on the intensity, duration, and frequency of exercise. We examined the effects of a single bout of short-duration high-intensity exercise (HIE) and long-duration lowintensity exercise (LIE) on insulin sensitivity and the adiponectin/leptin ratio in individuals with different body mass indices (BMIs) who do not exercise regularly. METHODS: We enrolled 42 healthy volunteers aged 20-64 years and divided them into two groups based on BMI: BMI <24 kg/m2 and BMI ≥27 kg/m2. They were randomly assigned to either the short-duration (20 min) HIE (70%-80% heart rate reserve, HRR) or long-duration (60 min) LIE training groups (30%-40% HRR). Glucose, insulin, adiponectin, and leptin levels were assessed before training and at 0, 30, 60, and 120 min after training. RESULTS: We finally analyzed 27 normal weight and 9 obese individuals. No significant differences were observed in the baseline information of both BMI groups. Homeostatic model assessment for insulin resistance significantly improved for both exercise patterns in the normal weight group and for the HIE pattern in the obese group (P < 0.01), whereas the adiponectin/leptin ratio increased significantly only among normal weight participants with the LIE intervention. CONCLUSION: Both exercise patterns in BMI <24 kg/m2 and BMI ≥27 kg/m2 benefit on insulin resistance. Therefore, people can choose the way they can fit to improve insulin resistance both short-duration high-intensity exercise and long-duration low-intensity exercise.

Genetic polymorphism of Plasmodium falciparum circumsporozoite protein on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Plasmodium falciparum circumsporozoite protein (PfCSP) is a potential malaria vaccine candidate, but various polymorphisms of the pfcsp gene among global P. falciparum population become the major barrier to the effectiveness of vaccines. This study aimed to investigate the genetic polymorphisms and natural selection of pfcsp in Bioko and the comparison among global P. falciparum population. METHODS: From January 2011 to December 2018, 148 blood samples were collected from P. falciparum infected Bioko patients and 96 monoclonal sequences of them were successfully acquired and analysed with 2200 global pfcsp sequences mined from MalariaGEN Pf3k Database and NCBI. RESULTS: In Bioko, the N-terminus of pfcsp showed limited genetic variations and the numbers of repetitive sequences (NANP/NVDP) were mainly found as 40 (35%) and 41 (34%) in central region. Most polymorphic characters were found in Th2R/Th3R region, where natural selection (p > 0.05) and recombination occurred. The overall pattern of Bioko pfcsp gene had no obvious deviation from African mainland pfcsp (Fst = 0.00878, p < 0.05). The comparative analysis of Bioko and global pfcsp displayed the various mutation patterns and obvious geographic differentiation among populations from four continents (p < 0.05). The global pfcsp C-terminal sequences were clustered into 138 different haplotypes (H_1 to H_138). Only 3.35% of sequences matched 3D7 strain haplotype (H_1). CONCLUSIONS: The genetic polymorphism phenomena of pfcsp were found universal in Bioko and global isolates and the majority mutations located at T cell epitopes. Global genetic polymorphism and geographical characteristics were recommended to be considered for future improvement of malaria vaccine design.

[Clinical Application of Preoperative Autologous Blood Donation in Selective General Surgery].

OBJECTIVE: To study the clinical effects of preoperative autologous blood donation (PABD) in selective general surgery. METHODS: Paired study was performed in PABD group with 70 PABD cases screened from selective general surgery during the period from November 2017 to August 2018 in our hospital, and the control group included 70 cases without preoperative autologous blood donation, the baseline data before surgery were not significantly different. The transfusion quantities of allogeneic RBC and plasma, the levels of perioperative hemoglobin and platelets, the time and expense of hospitalization were compared between two groups. RESULTS: The levels of Hb and Plt in PABD group before and after blood collection were determined as follows: 138.26±14.73 g/L vs 127.52±13.36 g/L (P＜0.05) and (221.67±52.86)×109/L vs (198.35±52.65)×109/L (P＞0.05) respectively. The analysis of allo-RBC and allo-plasma transfusion in PABD group and control group showed that: the quantity of allogeneic RBC transfusion was 0.20±0.71 U and 0.89±0.97 U, and the quantity of allogeneic plasma transfusion was 30.43±100.81 ml and 106.52±152.61 ml (P＜0.05) respectirdy during perioperation. The comparison results of preoperative Hb and plt in PABD group and control group were 135.65±14.16 g/L vs 134.15±11.98 g/L and (270.36±58.28)×109/L vs (271.67±65.02) ×109/L respectively. The levels of postoperative Hb and plt in PABD group and control group were 120.24±14.40 g/L vs 121.20±14.30 g/L at 1 d after operation, and (241.80±63.58)×109/L vs (241.30±69.11)×109/L at 1 d after operation respectively; 123.15±13.80 g/L vs 121.65±14.33 g/L at 3 d after operation and (251.26±72.94)×109/L vs (255.54±73.85)×109/L at 3 d after operation; 122.78±13.92 g/L and 122.00±13.82 g/L (before discharge) and (262.50±80.96)×109/L and (264.56±71.08)×109/L (before discharge, platelet). These data were not statistically different (P＞0.05). The hospitalization time was 14.84±3.37 days and 14.84±2.24 days, respectively, without statistical difference (P＞0.05) in two groups. The expenses of hospitalization and the blood transfusion in two groups were 50627.27±9889.45 RMB and 50979.43±8195.00 RMB; 354.39±362.57 RMB and 684.02±425.53 RMB (P＜0.05). CONCLUSION: The application of PABD reduces the use of allogeneic blood and costs for patients undergoing selective surgery with blood losts of 1000 ml.

Trends in Molecular Markers Associated with Resistance to Sulfadoxine-Pyrimethamine (SP) Among Plasmodium falciparum Isolates on Bioko Island, Equatorial Guinea: 2011-2017.

PURPOSE: Antimalarial drug resistance is one of the major challenges in global efforts to control and eliminate malaria. In 2006, sulfadoxine-pyrimethamine (SP) replaced with artemisinin-based combination therapy (ACT) on Bioko Island, Equatorial Guinea, in response to increasing SP resistance, which is associated with mutations in the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes. PATIENTS AND METHODS: To evaluate the trend of molecular markers associated with SP resistance on Bioko Island from 2011 to 2017, 179 samples collected during active case detection were analysed by PCR and DNA sequencing. RESULTS: Pfdhfr and Pfdhps gene sequences were obtained for 90.5% (162/179) and 77.1% (138/179) of the samples, respectively. For Pfdhfr, 97.5% (158/162), 95.7% (155/162) and 98.1% (159/162) of the samples contained N51I, C59R and S108N mutant alleles, respectively. And Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 25.4% (35/138), 88.4% (122/138), 5.1% (7/138), 1.4% (2/138), and 7.2% (10/138) of the samples, respectively. Two classes of previously described Pfdhfr-Pfdhps haplotypes associated with SP resistance and their frequencies were identified: partial (IRNI-SGKAA, 59.4%) and full (IRNI-SGEAA, 5.5%) resistance. Although no significant difference was observed in different time periods (p>0.05), our study confirmed a slowly increasing trend of the frequencies of these SP-resistance markers in Bioko parasites over the 7 years investigated. CONCLUSION: The findings reveal the general existence of SP-resistance markers on Bioko Island even after the replacement of SP as a first-line treatment for uncomplicated malaria. Continuous molecular monitoring and additional control efforts in the region are urgently needed.

DFSG, a novel herbal cocktail with anti-asthma activity, suppressed MUC5AC in A549 cells and alleviated allergic airway hypersensitivity and inflammatory cell infiltration in a chronic asthma mouse model.

AIM OF THE STUDY: To develop a novel anti-asthma drug. DFSG is a novel herbal cocktail composed of 4 types of herbal medicines. This study explored whether DFSG has the potential to attenuate asthma symptom severity and aimed to determine the immunomodulatory mechanism of DFSG using a chronic asthmatic mouse model induced by repeated challenges with Dermatogoides pteronyssinus (Der p). MATERIALS AND METHODS: BALB/c mice were intratracheally inoculated with Der p (50 µl, 1 mg/ml) once a week for 5 weeks. In addition, 30 min before Der p challenge, the mice were orally administered 1x DFSG (1 g/kg) or 1/2x DFSG (0.5 g/kg). Three days after the final challenge, the mice were sacrificed to evaluate inflammatory cell infiltration, lung histological features, blood total IgE, and cytokine levels in pulmonary alveolar lavage fluid. Furthermore, 30 min after the addition of DFSG, caffeic acid, p-coumaric acid or chlorogenic acid to A549 cells, 10 ng/ml IL-1ß was added to evaluate the effect of the drug on mucin 5AC (MUC5AC) gene expression after stimulation of A549 cells by IL-1ß. RESULTS: DFSG significantly reduced Der p-induced airway hyperresponsiveness, bronchial inflammatory cell infiltration, and total IgE and IgG1 serum levels. Furthermore, DFSG significantly inhibited TH2 cytokines and increased the expression of TH1 cytokines. In addition, immunohistochemical staining demonstrated that DFSG inhibited MUC5AC expression in the bronchial epithelial cells. DFSG and a mixture of caffeic acid, p-coumaric acid, and chlorogenic acid inhibited MUC5AC gene expression in A549 cells after stimulation with IL-1ß. CONCLUSION: These results suggest that by regulating TH1 and TH2 cytokines and MUC5AC expression, DFSG exhibits anti-airway inflammatory cell infiltration and anti-hyperresponsiveness activity and inhibits specific immunity in a chronic asthmatic mouse model. Therefore, DFSG has potential for development into a drug for chronic asthma treatment.

Evidence of positively selected G6PD A- allele reduces risk of Plasmodium falciparum infection in African population on Bioko Island.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Although G6PD-deficient alleles appear to confer a protective effect of malaria, the link with clinical protection against Plasmodium infection is conflicting. METHODS: A case-control study was conducted on Bioko Island, Equatorial Guinea and further genotyping analysis used to detect natural selection of the G6PD A- allele. RESULTS: Our results showed G6PD A- allele could significantly reduce the risk of Plasmodium falciparum infection in male individuals (adjusted odds ratio [AOR], 0.43; 95% confidence interval [CI], 0.20-0.93; p < .05) and homozygous female individuals (AOR, 0.11; 95% CI, 0.01-0.84; p < .05). Additionally, the parasite densities were significantly different in the individuals with different G6PD A- alleles and individual levels of G6PD enzyme activity. The pattern of linkage disequilibrium and results of the long-range haplotype test revealed a strong selective signature in the region encompassing the G6PD A- allele over the past 6,250 years. The network of inferred haplotypes suggested a single origin of the G6PD A- allele in Africans. CONCLUSION: Our findings demonstrate that glucose-6-phosphate dehydrogenase (G6PD) A- allele could reduce the risk of P. falciparum infection in the African population and indicate that malaria has a recent positive selection on G6PD A- allele.

The Integration of Recipes with a Standardizable Food Description FACET for Cadmium Exposure Risk Assessment.

Existing food classification and description systems provide users with limited information related to exposure assessment. Our aim in this work is to propose a standardized food description facet called the Taiwan Food Recipe (TFR) system as an emerging tool for food composition, with detailed food ingredient information, including names, proportions, weights of uncooked and cooked foods, etc. The composite foods listed in the Taiwan Nutrition and Health Survey were collected into a list and as consumption data. The TFR system is intended to help analysts reduce potential estimation bias, where, for example, risk assessment results may be overestimated or underestimated due to the complexity of the composition in the composite foods. Based on a Taiwanese food database, we further illustrate and demonstrate how the TFR system can be applied to the assessment of risk of cadmium (Cd) exposure in rice ingredients in the composite food products. In the original system (HFDFC system), the composite food intakes used total weight to estimate the hazard index (HI) of cadmium in the exposure risk assessment, but the percentage of rice was not 100%. The proposed TFR system estimates the percentage of rice and actual intakes in composite foods. Fried rice, sushi, and rice balls in the study were the most common foods containing rice and had higher consumption rates among Taiwan's rice-based composite foods. The HIs of fried rice, sushi, and rice balls were 0.09, 0.10, and 0.13, respectively, in the HFDFC system. In the TFR system, the HIs of rice in fried rice, sushi, and rice balls were 0.06, 0.04 and 0.05, respectively. The HI of other components in fried rice, sushi, and rice balls were 0.03, 0.06 and 0.08, respectively. More precise HIs were thus shown. The TFR system contributes to global food classification and description systems by providing an appropriate, standardized, and generalized framework for exposure assessments.

Natural selection and genetic diversity of domain I of Plasmodium falciparum apical membrane antigen-1 on Bioko Island.

BACKGROUND: Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity of PfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of the PfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among Bioko P. falciparum isolates were analysed. METHODS: 214 blood samples were collected from Bioko Island patients with P. falciparum malaria between 2011 and 2017. A fragment spanning DI of PfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 global PfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program. RESULTS: 131 different haplotypes of PfAMA-1 were identified in 214 Bioko Island P. falciparum isolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified in PfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of Bioko PfAMA-1 DIs were similar to those in global PfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates. CONCLUSIONS: Patterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of global PfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure of PfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.