Cryo-EM structure of a RAS/RAF recruitment complex.

RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.

Comparative effectiveness of different acupuncture therapies for neck pain.

BACKGROUND: Neck pain is a common musculoskeletal symptom that has negative effects on quality of life and work productivity. Acupuncture has been widely used for neck pain, and a number of randomized controlled trials (RCTs) and systematic reviews (SRs) have evaluated its effectiveness. However, previous studies have obtained inconsistent results regarding the effects of acupuncture for neck pain, and there is no SR for the comparative efficacy and safety of various types of acupuncture. Therefore, we herein conducted a SR and network meta-analysis to compare and rank different types of acupuncture with respect to their effectiveness in treating neck pain. METHODS: We searched 9 electronic databases for relevant RCTs published from their inception to July 1, 2021. Pairwise meta-analyses and network meta-analysis were performed with R software using the frequentist framework. Change of pain intensity was assessed as the primary outcome, and change of pain-related disability and efficacy rate were assessed as secondary outcomes. The Cochrane risk of bias tool and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) instrument were used to evaluate the quality of the included RCTs and the certainty of the evidence. RESULTS: A total of 65 RCTs involving 5266 participants and 9 interventions were included. Three network meta-analyses were constructed for the following: pain intensity (42 RCTs, 3158 participants), pain-related disability (21 RCTs, 1581 participants), and efficacy rate (40 RCTs, 3512 participants). The results indicated that fire acupuncture, electroacupuncture, and warm acupuncture were more effective than manual acupuncture in terms of pain intensity reduction and efficacy rate, and that electroacupuncture decreased pain-related disability more effectively than manual acupuncture. Fire acupuncture ranked first among the 9 interventions. The overall q of evidence was very low according to the GRADE assessment. The reported adverse events were not serious. CONCLUSION: Fire acupuncture, warm acupuncture, acupoint catgut embedding, and electroacupuncture ranked higher than other interventions (usual care, sham acupuncture, no treatment) in reducing the pain and disability index scores and the efficacy rate. However, the included trials were evaluated as being of low quality; thus, we recommend additional well-designed RCTs with larger sample sizes to confirm these findings. SYSTEMATIC REVIEW REGISTRATION: PROSPERO, CRD42021235274.

Evaluation of the Privacy Risks of Personal Health Identifiers and Quasi-Identifiers in a Distributed Research Network: Development and Validation Study.

BACKGROUND: Privacy should be protected in medical data that include patient information. A distributed research network (DRN) is one of the challenges in privacy protection and in the encouragement of multi-institutional clinical research. A DRN standardizes multi-institutional data into a common structure and terminology called a common data model (CDM), and it only shares analysis results. It is necessary to measure how a DRN protects patient information privacy even without sharing data in practice. OBJECTIVE: This study aimed to quantify the privacy risk of a DRN by comparing different deidentification levels focusing on personal health identifiers (PHIs) and quasi-identifiers (QIs). METHODS: We detected PHIs and QIs in an Observational Medical Outcomes Partnership (OMOP) CDM as threatening privacy, based on 18 Health Insurance Portability and Accountability Act of 1996 (HIPPA) identifiers and previous studies. To compare the privacy risk according to the different privacy policies, we generated limited and safe harbor data sets based on 16 PHIs and 12 QIs as threatening privacy from the Synthetic Public Use File 5 Percent (SynPUF5PCT) data set, which is a public data set of the OMOP CDM. With minimum cell size and equivalence class methods, we measured the privacy risk reduction with a trust differential gap obtained by comparing the two data sets. We also measured the gap in randomly sampled records from the two data sets to adjust the number of PHI or QI records. RESULTS: The gaps averaged 31.448% and 73.798% for PHIs and QIs, respectively, with a minimum cell size of one, which represents a unique record in a data set. Among PHIs, the national provider identifier had the highest gap of 71.236% (71.244% and 0.007% in the limited and safe harbor data sets, respectively). The maximum size of the equivalence class, which has the largest size of an indistinguishable set of records, averaged 771. In 1000 random samples of PHIs, Device_exposure_start_date had the highest gap of 33.730% (87.705% and 53.975% in the data sets). Among QIs, Death had the highest gap of 99.212% (99.997% and 0.784% in the data sets). In 1000, 10,000, and 100,000 random samples of QIs, Device_treatment had the highest gaps of 12.980% (99.980% and 87.000% in the data sets), 60.118% (99.831% and 39.713%), and 93.597% (98.805% and 5.207%), respectively, and in 1 million random samples, Death had the highest gap of 99.063% (99.998% and 0.934% in the data sets). CONCLUSIONS: In this study, we verified and quantified the privacy risk of PHIs and QIs in the DRN. Although this study used limited PHIs and QIs for verification, the privacy limitations found in this study could be used as a quality measurement index for deidentification of multi-institutional collaboration research, thereby increasing DRN safety.

The effectiveness of different acupuncture therapies for neck pain: A protocol for systematic review and/or network meta-analysis.

BACKGROUND: Neck pain is common musculoskeletal disorders in adult population. Acupuncture treatment has been widely used for treating neck pain. Nevertheless, previous systematic reviews (SRs) on acupuncture for neck pain remain controversial, and there is no SR for the comparative efficacy and safety of various types of acupuncture. Therefore, this study aims to evaluate and rank the effectiveness and safety of different types of acupuncture for neck pain by SR and network meta-analysis. METHODS: Nine databases will be searched, including Ovid-MEDLINE, EMBASE, Cochrane library, China National Knowledge Infrastructure (CNKI), KoreaMed, Korean medical database (KMBASE), Korean Studies Information Service System (KISS), ScienceON, and Oriental Medicine Advanced Searching Integrated System (OASIS) from their inception to July 2021. The primary outcome is the change of pain intensity. A frequentist network meta-analysis will be performed to compare all relative outcomes of different acupuncture methods, using R software. The quality of included randomized controlled trials will be assessed by Cochrane Collaboration "risk of bias" tools and the evidence will be evaluated by the Grading of Recommendations Assessment, Development and Evaluation instrument. RESULTS: The final findings of this network meta-analysis will be published in a recognized journal. CONCLUSIONS: Our study will evaluate and compare the effectiveness of various types of acupuncture for neck pain and provide clinicians with best option for what types of acupuncture treatments are effective. TRIAL REGISTRATION NUMBER: INPLASY202120041.

microRNA-944 overexpression is a biomarker for poor prognosis of advanced cervical cancer.

BACKGROUND: One-third of cervical cancer patients are still diagnosed at advanced stages. The five-year survival rate is decreased in about 50% of advanced stage cervical cancer patients worldwide, and the clinical outcomes are remarkably varied and difficult to predict. One of the miRNAs known to be associated with cancer tumorigenesis is miR-944. However, the prognostic value of miR-944 in cervical cancer has not been fully investigated. The aim of this study was to analyze clinical significance and prognostic value of miR-944 in cervical cancer. METHODS: The expression levels of miR-944 were detected using quantitative reverse transcription polymerase chain reaction in five types of cervical cancer cell lines and 116 formalin-fixed paraffin-embedded (FFPE) cervical tissues. The association between the expression levels of miR-944 and prognostic value was analyzed using the Kaplan-Meier analysis and Cox proportional hazards model. RESULTS: The expression levels of miR-944 in cervical cancer tissues were significantly higher compared with those in normal tissues (P < 0.0001). Moreover, the expression levels of miR-944 in cervical cancer cell lines and FFPE tissues with human papillomavirus (HPV) infection were significantly higher compared to those without HPV infection (P < 0.01 and P = 0.02). High miR-944 expression was also markedly associated with bulky tumor size (P = 0.026), advanced International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.042), and lymph node metastasis (P = 0.030). In particular, high miR-944 expression group showed shorter overall survival than the low miR-944 expression group in the advanced FIGO stage (84.4% vs. 44.4%, HR = 4.0, and P = 0.01). CONCLUSIONS: These results suggest that miR-944 may be used as a novel biomarker for improving prognosis and as a potential therapeutic target.

Prediction of Y haplogroup by polymerase chain reaction-reverse blot hybridization assay.

BACKGROUND: The analysis of Y-SNPs from crime scene samples is helpful for investigators in narrowing down suspects by predicting biogeographical ancestry. OBJECTIVE: In this study, a PCR-reverse blot hybridization assay (REBA) for predicting Y-chromosome haplogroups was employed to determine the major haplogroups worldwide, including AB, DE, C, C3, F, K, NO, O, O2, and O3 and evaluated. METHODS: The REBA detects nine biallelic Y chromosome markers (M9, M89, M122, M145, M175, M214, M217, P31, and RPS4Y711) simultaneously using multiple probes. RESULTS: The REBA for Y-single nucleotide polymorphisms (SNP) genotyping was performed using 40 DNA samples from Asians-14 Koreans, 10 Indonesians, six Chineses, six Thais, and four Mongolians. 40 Asian samples were identified as haplogroup O2 (40%), O3 (32.5%), C3 (17.5%), O (7.5%) and K (2.5%). These cases were confirmed by DNA sequence analysis (κ = 1.00; P < 0.001). CONCLUSION: PCR-REBA is a rapid and reliable method that complements other SNP detection methods. Therefore, implementing REBA for Y-SNP testing may be a useful tool in predicting Y-chromosome haplogroups.

Optical Sensing for Evaluating the Severity of Disease Caused by Cladosporium sp. in Barley under Warmer Conditions.

Crop yield is critically related to the physiological responses and disease resistance of the crop, which could be strongly affected by high temperature conditions. We observed the changes in the growth of barley under higher than ambient air-temperature conditions using a temperature gradient field chamber (TGFC) during winter and spring. Before the stem extension stage of barley growth, Cladosporium sp. spontaneously appeared in the TGFC. The severity of disease became serious under warmer temperature conditions. Further, the stomata closed as the severity of the disease increased; however, stomatal conductance at the initial stage of disease was higher than that of the normal leaves. This was likely due to the Iwanov effect, which explains that stressed plants rapidly and transiently open their stomata before longer-term closure. In this study, we tested three optical methods: soil-plant analysis development (SPAD) chlorophyll index, photochemical reflectance index (PRI), and maximum quantum yield (Fv/Fm). These rapid evaluation methods have not been used in studies focusing on disease stress, although some studies have used these methods to monitor other stresses. These three indicative parameters revealed that diseased barley exhibited lower values of these parameters than normal, and with the increase in disease severity, these values declined further. Our results will be useful in efficient monitoring and evaluation of crop diseases under future warming conditions.

Activation of autophagy rescues amiodarone-induced apoptosis of lung epithelial cells and pulmonary toxicity in rats.

Amiodarone, bi-iodinated benzofuran derivative, is one of the most frequently prescribed and efficacious antiarrhythmic drugs. Despite its low incidence, amiodarone-induced pulmonary toxicity is of great concern and the leading cause of discontinuation. Autophagy is an essential homeostatic process that mediates continuous recycling of intracellular materials when nutrients are scarce. It either leads to a survival advantage or initiates death processes in cells under stress. In the present study, we investigated the role of autophagy in amiodarone-induced pulmonary toxicity. Amiodarone treatment-induced autophagy in H460 human lung epithelial cells and BEAS-2B normal human bronchial epithelial cells was demonstrated by increased LC3-II conversion, Atg7 upregulation, and autophagosome formation. Autophagic flux, as determined by the lysosomal inhibitor bafilomycin A1, was also increased following amiodarone treatment. To determine the role of autophagy in amiodarone toxicity, amiodarone-induced cell death was evaluated in the presence of 3-methyladenine or by knocking down the autophagy-related genes Atg7. Inhibition of autophagy decreased cellular viability and significantly increased apoptosis. Intratracheal instillation of amiodarone in rats increased the number of inflammatory cells recovered from bronchoalveolar lavage fluid, and periodic acid-Schiff-positive staining in bronchiolar epithelial cells. However, induction of autophagy by rapamycin treatment inhibited amiodarone-induced pulmonary toxicity. In conclusion, amiodarone treatment induced autophagy, which is involved in protection against cell death and pulmonary toxicity.

Effects of Medicinal herb Extracts and their Components on Steatogenic Hepatotoxicity in Sk-hep1 Cells.

Herbal medicines are widely used in many countries for the treatment of many diseases. Although the use of herb extracts as alternative medicine is growing, their toxicological properties have not been thoroughly investigated. In this study, we have investigated the effects of water and ethanol extracts of 18 herbs on the hepatic lipid metabolism and steatogenic hepatotoxicity. Ethanol extracts of Cirsium japonicum, Carthamus tinctorius, Rehmanniae glutinosa (preparata), Polygala tenuifolia, Foeniculum vulgare, Polygonum multiflorum, and Acorus gramineus and water extracts of Polygonum multiflorum and Rehmanniae glutinosa induced lipid accumulation in Sk-hep1 human hepatoma cells as determined by Nile red staining. These extracts increased the luciferase activity of sterol regulatory element (SRE) and decreased that of peroxisome proliferator response element (PPRE), indicating the possibilities of enhanced fatty acid synthesis and decreased fatty acid oxidation. To identify the components responsible for the fat accumulation, we tested 50 chemicals isolated from the nine herbs. Apigenin, luteolin, pectolinarin and lupeol from Cirsium japonicum, 8-methoxypsoralen and umbelliferone from Foeniculum vulgare and pomonic acid and jiocerebroside from Rehmanniae glutinosa significantly increased the accumulation of lipid droplets. These results suggest that ethanol extracts of Cirsium japonicum, Carthamus tinctorius, Rehmanniae glutinosa (preparata), Polygala tenuifolia, Foeniculum vulgare, Polygonum multiflorum, and Acorus gramineus and water extracts of Polygonum multiflorum and Rehmanniae glutinosa can cause fatty liver disease by decreasing ß-oxidation of fatty acid and increasing lipogenesis.

Association between 24-hour ambulatory blood pressure and erythrocyte n-3 polyunsaturated fatty acids in Korean subjects with hypertension.

N-3 polyunsaturated fatty acids (PUFAs) are known to have antihypertensive properties, but the association between 24-hour ambulatory blood pressure and the tissue content of n-3 PUFA remains controversial. The purpose of the present study was to investigate the hypothesis that the level of erythrocyte n-3 PUFA is inversely related with 24-hour ambulatory blood pressure after adjustment for relevant confounders. Fifty-one male and 49 female Korean patients were included in this study. Twenty-seven of the patients were defined as having hypertension. There were significant differences in age, body mass index, sex, marital status, and family history of hyperlipidemia between hypertensive and nonhypertensive subjects, and these factors were therefore considered to be confounding factors. Multivariate-adjusted regression analysis showed that erythrocyte fatty acids were not significantly associated with the risk of hypertension after adjusting for confounders. However, Pearson correlation analysis showed that 24-hour ambulatory systolic blood pressure (SBP) was significantly and negatively correlated with n-3 PUFA (r = -0.228, P = .027) and eicosapentaenoic acid (r = -0.270, P = .008), but not with docosahexaenoic acid (r = -0.156, P = .131). Multivariate-adjusted regression analysis also showed that intake of protein, vitamin B(2), vitamin E, and cholesterol increased the risk of hypertension after adjusting for confounders. In addition, Pearson correlation analysis showed that fat and cholesterol consumption was positively correlated with SBP, but carbohydrate intake was negatively correlated with SBP. In conclusion, erythrocyte n-3 PUFA did not reduce the risk of hypertension but were negatively correlated with 24-hour ambulatory SBP in the Korean population.

Distributions of total mercury and methylmercury in surface sediments and fishes in Lake Shihwa, Korea.

The concentrations of total mercury (THg) and methylmercury (MeHg) in the sediments of Lake Shihwa, an artificial salt lake in Korea located near two large industrial complexes, were determined to investigate the state of Hg contamination in the lake sediments and the effect of local Hg source. THg and MeHg concentrations in the sediments, monitored for 2 years, ranged from 0.02 to 0.28 microg g(-1) and

Seasonal variation in dissolved gaseous mercury and total mercury concentrations in Juam Reservoir, Korea.

Dissolved gaseous mercury (DGM) and total mercury (TM) concentrations were measured in Juam Reservoir, Korea. DGM concentrations were higher in spring (64+/-13pgL(-1)) and summer (109+/-15pgL(-1)), and lower in fall (20+/-2pgL(-1)) and winter (23+/-6pgL(-1)). In contrast, TM concentrations were higher in fall (3.2+/-0.1ngL(-1)) and winter (3.3+/-0.1ngL(-1)) than in spring (2.3+/-0.1ngL(-1)) and summer (2.2+/-0.4ngL(-1)). DGM concentrations were correlated with water temperature (p<0.0001), ORP (p<0.0001), UV intensity (UV-A: p=0.008; UV-B: p=0.003), and DOC concentration (p=0.0107). DGM concentrations varied diurnally with UV intensity. The average summer DGM (109+/-15pgL(-1)) and TM (2.2+/-0.4ngL(-1)) concentrations in Juam Reservoir were higher than the averages for North American lakes (DGM=38+/-16pgL(-1); TM=1.0+/-1.2ngL(-1)), but lower than levels reported for Baihua Reservoir in China.