Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.

COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/µL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.

Acacetin exerts antitumor effects on gastric cancer by targeting EGFR.

Background: Gastric cancer (GC) is a common malignant tumor with a poor prognosis. Combination treatments may prolong the survival of patients with GC. Acacetin, which is a flavonoid, exerts potent inhibitory effects on several types of cancer cells; however, the mechanisms of action remain poorly understood. Methods: Network pharmacology and RNA sequencing were used to predict the targets of acacetin, which were then verified by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking. The biological functions of acacetin in MKN45 and MGC803 cells were investigated using TUNEL assays, crystal staining and colony formation assays. The pathways affected by acacetin were verified through reverse experiments. The in vivo antitumor efficacy of acacetin was assessed in a subcutaneous xenotransplanted tumor model. Results: In this study, we identified EGFR from more than a dozen predicted targets as a protein that directly binds to acacetin. Moreover, acacetin affected the level of phosphorylated EGFR. In vitro, acacetin promoted the apoptosis of GC cells. Importantly, EGFR agonists reversed the inhibitory effects of acacetin on the STAT3 and ERK pathways. In vivo, acacetin decreased the protein levels of pEGFR in tumors, resulting in increased GC xenograft tumor regression without obvious toxicity. Conclusion: Our findings highlight EGFR as one of the direct targets of acacetin in GC cells. Acacetin inhibited the phosphatase activity of EGFR in vitro and in vivo, which played a role in the antitumor effects of acacetin. These studies provide new evidence for the use of acacetin as a potential reagent for the treatment of GC.

[Orthogonal factor analysis on metabolic syndrome].

To elucidate the principal of orthogonal factor analysis, using an example of factor analysis of metabolic syndrome. The basic structures and the fundamental concepts of orthogonal factor analysis were introduced and data involving 1877 women aged of 35-65 years, selected from a cross-sectional study, which was conducted in 1998 - 2001 in Shanghai, were included in this study. Factor analysis was carried out using principle components analysis with Varimax orthogonal rotation of the components of the metabolic syndrome. The different components of the metabolic syndrome were not linked closely with the other components and loaded on the six different factors,which mainly reflected by the variables of obesity, blood pressure, plasma glucose, plasma insulin, triglycerides and HDL-cholesterol respectively. Six major factors of the metabolic syndrome were uncorrelated with each other and explained 86% of the variance in the original data. The factor score and total factor score for the individual could be obtained according to the component score coefficient matrix. Although the components of the metabolic syndrome were related statistically, the finding of six factors suggested that the components of the metabolic syndrome did not show high degrees of intercorrelation. As a linear method of data reduction, the mode reduced a large set of measured intercorrelation variables into a smaller set of uncorrelated factors, which explained the majority of the variance in the original variables. Factor analysis was well suited for revealing underlying patterns or structure among variables showing high degrees of intercorrelation.

[Serum lipid profile changes of the adults in Shanghai communities].

OBJECTIVE: To explore the adult lipid profile of Huayang community from 1998 to 2000 and Caoyang communities in 2001. METHODS: Representative serum samples of 5628 adults (aged 20 - 95 years) were obtained in Huayang and Caoyang communities during 1998.9 and 2001.11. Standard epidemiology questionnaire, physical check-ups and serum lipids data were analyzed. RESULTS: After standardization to Chinese census statistics of 2000, the age-and sex-standardized means of total cholesterol, HDL-C, LDL-C and triglycerides of the two communities (Huayang vs. Caoyang) were 5.01 mmol/L vs. 4.43 mmol/L, 1.28 mmol/L vs. 1.32 mmol/L, 3.37 mmol/L vs. 2.99 mmol/L, 1.97 mmol/L vs. 1.60 mmol/L respectively, and the age- and sex- standardized prevalence of dyslipidemia was 52.9% vs. 25.1%, and the prevalence for borderline dyslipidemia was 76.0% vs. 56.2%, respectively. The decreasing order of dyslipidemia prevalence of the two communities was: elevated TG, decreased HDL-C, elevated LDL-C and TC. The standardized proportions of optimal HDL-C level were only 15.7% and 16.1% in Huayang and Caoyang respectively which was much lower than these of TG, LDL and TC. CONCLUSIONS: The standardized prevalence of adult dyslipidemia and borderline dyslipidemia in the two communities were high. Dyslipidemia of the two communities was TG and decreased.

[A comparison of the application of two working definitions of metabolic syndrome in Chinese population].

OBJECTIVE: To compare the significance of the application of the working definition for metabolic syndrome (MS) proposed by the World Health Organization (WHO) in 1999 and the one proposed by the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (ATP III) in 2001. METHODS: Data of 2048 subjects, aged 20-74, 896 males and 1152 females, with complete laboratory data, including plasma glucose, lipid profile, blood pressure, serum insulin, urine albumin and creatinine, were gathered from the data of baseline survey on obesity and its co-morbidities conducted in Huayang and Caoyang communities, Shanghai, from 1999 to 2001. MS was diagnosed according to these two definitions respectively. RESULTS: (1) The age-adjusted MS prevalence rates were 17.14% and 10.95% according to the WHO (1999) and ATP III (2001) respectively, the former being significantly higher than the latter. (2) A total of 445 subjects were diagnosed as having MS. The agreement in the diagnosis of MS using two definitions was about 45.17%. 42.02% of the subjects had MS only under the WHO (1999) definition, while 12.81% of the subjects had MS under the ATP III (2001) definition. (3) The prevalence rate of hyperglycemia and central obesity as defined by WHO (1999) definition were 1.74 and 4.38 times higher than those defined by ATP III (2001) definition (16.74% vs 9.57%, 33.04% vs 7.54% respectively). (4) In comparison with non-MS subjects, the body mass index, waist-to-hip ratio, blood pressure, blood glucose, insulin resistant index (HOMA-IR), and albumin-to-creatinine ratio were all significantly increased in MS subjects. CONCLUSION: Both definitions for MS can reflect the clustering degree of risk factors of cerebrovascular and cardiovascular diseases. The WHO definition of MS can better reflect the characteristics of MS in Chinese than the definition proposed by ATP III, however it needs more measurements and costs more. A simple method with high detection ability remains to be worked out.

[Relationship between the prevalence of microalbuminuria and components of metabolic syndrome in Shanghai].

OBJECTIVE: To study the relationship between the prevalence of microalbuminuria and components of metabolic syndrome in Shanghai. METHODS: A total of 3532 Shanghai Chinese (men 1622, women 1910) aged over 20 years were included. Body mass index (BMI), blood pressure, fasting plasma glucose, lipid profile and plasma insulin concentrations were measured in all subjects. Oral glucose tolerance test was performed in those subjects without knowing the diabetic history. Urinary albumin-to-creatinine ratio (ACR) was measured in an early morning spot urine sample. Microalbuminuria was diagnosed when ACR was between 30 and 300 mg/g. RESULTS: (1) The prevalence of microalbuminuria was increasing with aging (P < 0.001). When compared with subjects having normal ACR, the waist-hip ratio, systolic and diastolic pressure, serum triglyceride level, fasting plasma glucose and homeostasis model assessment-insulin resistance (HOMA-IR) were all significantly increased in those subjects with microalbuminuria. (2) Along with the increment of number of items on metabolic disorders, the prevalence of microalbuminuria was significantly increased (P for trend < 0.001). (3) Multiple logistic regression analyses revealed that hypertension and hyperglycemia were independent factors associated with microalbuminuria (OR = 2.15, P < 0.001 and OR = 1.64, P = 0.01 respectively). Those subjects with two or more items on metabolic disorders had higher odd ratio for the development of microalbuminuria (OR = 3.93 and 4.77, both P < 0.001) when compared with the subjects without metabolic disorder. CONCLUSION: The prevalence of microalbuminuria was independently associated with hypertension and hyperglycemia. The subjects with multiple metabolic abnormalities had higher risk for the development of microalbuminuria, especially in metabolic syndrome.