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Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms. [ABSTRACT FROM AUTHOR]

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein–lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR–G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid l-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands. Cryo-electron microscopy structures of the human calcium-sensing receptor in complex with Gi and Gq proteins reveal how this receptor activates distinct G protein subtypes and how its function is modulated by a variety of ligands. [ABSTRACT FROM AUTHOR]

Objective To analyze the difference in the use of free fixed dose combination (FDC), and indirect evaluation of the rationality of anti-tuberculosis drug use before and after transformation of new TB prevention and control service mode in Chongqing from 2015 to 2019, it can provide reference for drug demand measurement, procurement, distribution, use and supervision. Methods We used China Information System for Disease Control and Prevention to obtain the number of TB patients, the quarterly reports of anti-tuberculosis drug distribution and other data registered in 2015-2019, and calculated the annual theoretical consumption and actual consumption of FDC-HRZE and FDC-HR respectively, and then utilizing the calculation formula of the difference in the use of drugs, checked the FDC use verification difference of Chongqing city and its four regions. Results From 2015 to 2019, the average differences of FDC-HRZE and FDC-HR in Chongqing were -43.70% and -31.34%, respectively. The main urban area were -52.91% and -45.38%, the metropolitan area were -39.01% and - 24.51%, the town group of the three gorges reservoir area in Northeast Chongqing were -42.00% and -30.75%, the town group of Wuling mountain area in Southeast Chongqing were -47.73% and -29.25%, respectively, all beyond the set range of ±25%. After the transformation of new TB prevention and control service mode, the average differences of FDC-HRZE and FDC-HR were -22.19% and -21.36% in 2018, and -21.65% and -16.43% in 2019, respectively,both in the set range of ±25%. And the average differences of FDC-HRZE, and FDC-HR of the main urban area in 2018 were -29.90%, -36.36% and -29.69%, - 11. 67% in 2019; the metropolitan area -20.34%, -20.54% in 2018 and -8.73%,-0.19% in 2019; the town group of the three gorges reservoir area in Northeast Chongqing were -18.51%, -17.07% in 2018 and -19.72%, -21.36% in 2019; the town group of Wuling mountain area in Southeast Chongqing were -26.54%, -11.47% in 2018 and -54.10%, -56.45% in 2019, respectively. Conclusions In recent years, the administration of free anti-tuberculosis drugs in our city had achieved certain results, the use of free anti-tuberculosis drugs was more reasonable after the transformation, but there were big regional differences. It was suggested to carry out drug use verification every half a year or once a year, once a quarter for regions with large differences, combined with on-the-spot supervision. It was to strengthen the management of patients and ensure the standardized use of drugs in the whole course of treatment. [ABSTRACT FROM AUTHOR]

Positive allosteric modulator (PAM) drugs enhance the activation of the calcium-sensing receptor (CaSR) and suppress parathyroid hormone (PTH) secretion. Unfortunately, these hyperparathyroidism-treating drugs can induce hypocalcemia and arrhythmias. Seeking improved modulators, we docked libraries of 2.7 million and 1.2 billion molecules against the CaSR structure. The billion-molecule docking found PAMs with a 2.7-fold higher hit rate than the million-molecule library, with hits up to 37-fold more potent. Structure-based optimization led to nanomolar leads. In ex vivo organ assays, one of these PAMs was 100-fold more potent than the standard of care, cinacalcet, and reduced serum PTH levels in mice without the hypocalcemia typical of CaSR drugs. As determined from cryo-electron microscopy structures, the PAMs identified here promote CaSR conformations that more closely resemble the activated state than those induced by the established drugs.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D ) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant., Competing Interests: Competing interests statement:The authors declare no competing interest.

Objective: To analyze the impact of large hydraulic projects on schistosomiasis transmission and evaluate the transmission risk in potential endemic areas., Methods: During 2008-2010, surveillance on risk factors related to schistosomiasis transmission and risk assessment were carried out in potential endemic sites in counties of Xuyu, Hongze, Jinhu, and Gaoyou in Jiangsu Province, Weishan County in Shangdong, Qianjiang City and Yiling District of Yichang City in Hubei, Juchao District of Chaohu City in Anhui Province, Wanzhou and Kaixian in Chongqing in Three Gorges Dam region or passed by South-to-North Water Diversion Project and Zhangjiagang City in Jiangsu Province located in lower reach of Yangtze River. At least one fixed and three temporary monitoring sites were set in each county (city or district). Local inhabitants aged 6-65 years old were screened by indirect haemagglutination assay (IHA) or ELISA, and the sero-positives were tested by Kato-Katz or miracidium hatching techniques to investigate possible infection in 2008. The endemic status of schistosomiasis in mobile population was surveyed every year during 2008-2010. Infection status in livestocks was surveyed in Juchao, Qianjiang, Gaoyou and Wanzhou Counties in 2008-2010. Oncomelania hupensis distribution was investigated in risky and suspicious areas. Snail spreading investigation was conducted through salvaging floater and snails-inducing by straw curtains in rivers connected with Yangtze River., Results: 8 256 local inhabitants were investigated by serological tests with a positive rate of 0.7% (60/8 256). Among the 60 serologically positive subjects, 55 individuals were examined by stool examination but none of them was egg positive. The antibody prevalence rate of migrating population in 2008-2010 was 2.0%, 1.4%, and 1.7%, respectively, with no significant difference (chi2 = 3.57, P > 0.05). Among the serologically positive subjects, egg-positive cases were found in migrating population in Juchao District each year and one case was found in Jinhu County in 2010. Oncomelania snails were only found in in Jinhu and Gaoyou without infected ones. A lot of aquatic shell-fish and snails were collected in the water floater and straw curtain without Oncomelania snails., Conclusion: Schistosomiasis is not endemic in the original nonendemic areas in Three Gorges Dam region and areas passed by South-to-North Water Diversion Project until now, but potential risk of transmission exists. Long term surveillance scheme on schistosomiasis should be established with varied monitoring factors in different category areas.

Objective: To understand the infection status of Enterobius vermicularis of children aged 3-12 years and its influencing factors in Chongqing City., Methods: One rural county and 1 urban county were selected as investigation areas. The children aged 3-12 years of 5 towns from 5 different directions of each county were investigated. About 150 children were investigated every town. The Enterobius vermicularis eggs of children were detected by the adhesive cellophane anal swab method. The children's parent education levels and personal circumstances of sanitation were investigated by the questionnaire survey., Results: Among 1 592 children investigated, the total infection rate of Enterobius vermicularis was 6.85%. The infection rates of boys and girls were 6.29% and 7.40%, respectively. The infection rates of rural and urban areas were 12.13% and 2.14%, respectively. The infection rate in children aged 5-7 years was the highest (13.56%) among all the child aged groups. There were significant differences for the infection rates of Enterobius vermicularis among children of different aged groups, different areas, different education levels and occupations of parents, washing hand before meal or not, washing hand after WC or not, different classroom grounds, and lodging or not (P < 0.01 or P < 0.05). The multivariate logistic analysis indicated that aged group, different areas, classroom ground and lodging children were independent risk factors for Enterobius vermicularis infection., Conclusions: The infection rate of Enterobius vermicularis in rural area is higher than that in urban area. The control emphases should be the children of low group, rural area, poor classroom and lodging.

Objective: To investigate Enterobius vermicularis infection among primary school students and its influence factors in Chongqing., Methods: Beibei and Changshou were selected as investigated points from October to December 2011. One primary school was randomly chosen from each of the 5 different directions in every investigated point. Adhesive cellophane anal swab was used to examine pinworm eggs for 3 consecutive days. Information on children's family, hygiene habits and school environment was collected by questionnaire., Results: The total infection rate of E. vermicularis was 6.7% (71/1 071). The infection rate in rural schools (7.9%, 60/755) was higher than that of urban schools (3.8%, 12/316) (chi2 = 6.1169, P < 0.05). The rate in males and females was 6.3% (34/536) and 7.1% (38/535), respectively (chi2 = 0.2463, P > 0.05). Among the investigated children aged 6-12 years, the infection rate in 6-year-old children (16.03%) was highest. There was a statistical significance among age groups (chi2 = 29.1492, P < 0.01). With the increase of age, the rate decreased. Multivariate logistic regression analysis showed that location (OR = 0.411), age groups (OR = 0.714), education level of mothers (OR = 0.568), materials of classroom-ground (OR = 0.116) and types of boarding (OR = 0.272) were the influence factors on E. vermicularis infection in primary schools (P < 0.05)., Conclusions: Pinworm control should more focused on rural children, younger group, mothers with lower education, classroom with cement ground and lodging schools in Chongqing City.

Objective: To understand the conditions of schistosomiasis laboratories at county level, so as to supply the information for diagnostic capacity building., Methods: A questionnaire survey was conducted at 40 random selected counties which were in charge of national surveillance for schistosomiasis. The information of staffs, areas of laboratories, equipment configuration and diagnostic methods from each laboratory were collected. The serum specimens and Kato-Katz thick smears from the national surveillance sites were randomly selected and retested by provincial staffs to evaluate the testing ability of the personnel., Results: The average age of laboratory staffs was 40.93 +/- 9.56 years old, 69.39% of staffs were older than 35 years, 86.22% of them had education background below bachelor degree. Except missed professional titles of 4 persons, the staffs with primary, middle and high professional titles accounted for 56.63%, 39.29% and 2.04%, respectively. The laboratory areas varied greatly while independent schistosomiasis stations had the lowest areas with 52.81 +/- 40.08 m2, and the equipment configurations of laboratories were in a low level. The consistency rates of primary test and reexamination for serum specimens and thick smear slides were 95.89% and 99.53%, respectively, with the Kappa value over 0.90. Nine immunodiagnostic kits were used in these laboratories, and Kato-Katz technique and miracidium hatching technique were the main parasitological methods., Conclusions: The personnel structure of laboratories at primary prevention and treatment facilities for schistosomiasis is unreasonable, while the basic infrastructure of laboratories is backward and the use of diagnostic assays/methods is disordered. The diagnostic capacity building should be strengthened, and the construction and management of schistosomiasis laboratories should be standardized.

Objective: To construct and express recombinant plasmid pET28a-Sj26GST of Schistosoma japonicum (Sj) in Escherichia coli BL21 (DE3)., Methods: The total RNA was extracted from Sj adult worms by ultrasound-breaking. The Sj26GST antigen gene was amplified by RT-PCR from the total RNA, and then cloned into prokaryotic expression plasmid pET28alpha and transformed into E. coli BL2 (DE3). The BL21(pET28a-Sj26GST) was induced with isopropyl-beta-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified with SDS-PAGE and Western blot., Results: The 676 bp Sj26GST gene was successfully amplified by RT-PCR and cloned into pET28alpha. The recombinant plasmid pET28a-Sj26GST was successfully constructed, with a relative molecular weight of expressed recombinant protein at approximately 36 x 10(3) as determined by SDS-PAGE. The amount of the expressed protein comprised 26% of the total bacterial proteins. The fusion protein could be recognized by the sera of rabbits infected with Sj., Conclusion: The recombinant plasmid pET28alpha-Sj26GST is successfully constructed and highly expressed in E. coli in a fused form with His-tag. The expressed fusion protein shows specific antigenicity.

Objective: To investigate the changes in natural ecology and social environment after the construction of the dam in the Three Gorges Reservoir area, and to understand the social behavior status and education backgrounds of the local residents, and to analyze the potential impact of these factors on schistosomiasis spreading., Methods: Data of nature and social economy after the construction of the dam in the Three Gorge area were collected, and a cross-sectional study with questionnaire survey on information including social demographic characteristics, people's production and life style, knowledge about schistosomiasis was conducted among natives, emigrations., Results: After the construction of the dam, as the stream became slower, the water-level fluctuating zone on the bank of the reservoir was formed. The main source of drinking water and daily life activity are mainly depends on the river supplying (accounting about 68.7% and 75.8%, respectively); due to the needs of farming, washing cloths and vegetables, 45.6% of resident had a close contact with river water. People with away-from-home employment among emigrations were increased from 5.7% to 18.7%. The proportion of using hygienic lavatory was 88.6%. The main source of water for drinking and daily life among emigrations was tap water (85.4% and 87.0%). The residents had no awareness about the health hazard for schistosomiasis, with less health information, Conclusion: The natural environment of reservoir areas and the life style are risk factors for the spread of schistosomiasis. The improved sanitation condition and the economy construction after the construction of the dam is propitious to keeping away from the introduction of schistosomiasis. However, people with away-from-home employment among emigrations are still at risk of schistosomiasis.