Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4).

Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4-Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

Severe Acute Hepatitis Outbreaks Associated with a Novel Hepacivirus in Rhizomys pruinosus in Hainan, China.

Members of the genus Hepacivirus have a broad range of hosts, with at least 14 species identified. To date, a highly pathogenic hepacivirus causing severe disease in animals has not been found. Here, by using high-throughput sequencing, a new hepacivirus was identified as the dominant and highly pathogenic virus in severe acute hepatitis outbreaks in bamboo rats (Rhizomys pruinosus), with ≈80% mortality; this virus emerged in February 2020 in two bamboo rat farms in China. Hepaciviral genome copies in bamboo rat liver were significantly higher than in other organs. Genomic sequences of hepacivirus strains from 12 sick bamboo rats were found to share 85.3 to 100% nucleotide (nt) identity and 94.9 to 100% amino acid (aa) identity and to share 79.7 to 87.8% nt and 90.4 to 97.8% aa identities with previously reported bamboo rat hepaciviruses of Vietnam and China. Sequence analysis further revealed the simultaneous circulation of genetically divergent hepacivirus variants within the two outbreaks. Phylogenetic analysis showed that hepacivirus strains from the present and previous studies formed an independent clade comprised of at least two genotypes, clearly different from all other known species, suggesting a novel species within the genus Hepacivirus. This is the first report of a non-human-infecting hepacivirus causing potentially fatal infection of bamboo rats, and the associated hepatitis in the animals potentially can be used to develop a surrogate model for the study of hepatitis C virus infection in humans and for the development of therapeutic strategies. IMPORTANCE Members of the genus Hepacivirus have a broad host range, with at least 14 species identified, but none is highly pathogenic to its host except for hepatitis C virus, which causes severe liver diseases in humans. In this study, a new liver-tropic hepacivirus species was identified by high-throughput sequencing as the pathogen associated with two outbreaks of severely acute hepatitis in hoary bamboo rats (Rhizomys pruinosus) on two farms in Hainan Province, China; this is the first reported highly pathogenic animal hepacivirus to our knowledge. Further phylogenetic analysis suggested that the hepaciviruses derived from hoary bamboo rats in either the current or previous studies represent a novel species within the genus Hepacivirus. This finding is a breakthrough that has significantly updated our understanding about the pathogenicity of animal hepaciviruses, and the hepacivirus-associated hepatitis in bamboo rats may have a use as an animal infection model to understand HCV infection and develop therapeutic strategies.

A distinctive ligand recognition mechanism by the human vasoactive intestinal polypeptide receptor 2.

Class B1 of G protein-coupled receptors (GPCRs) comprises 15 members activated by physiologically important peptide hormones. Among them, vasoactive intestinal polypeptide receptor 2 (VIP2R) is expressed in the central and peripheral nervous systems and involved in a number of pathophysiological conditions, including pulmonary arterial hypertension, autoimmune and psychiatric disorders, in which it is thus a valuable drug target. Here, we report the cryo-electron microscopy structure of the human VIP2R bound to its endogenous ligand PACAP27 and the stimulatory G protein. Different from all reported peptide-bound class B1 GPCR structures, the N-terminal α-helix of VIP2R adopts a unique conformation that deeply inserts into a cleft between PACAP27 and the extracellular loop 1, thereby stabilizing the peptide-receptor interface. Its truncation or extension significantly decreased VIP2R-mediated cAMP accumulation. Our results provide additional information on peptide recognition and receptor activation among class B1 GPCRs and may facilitate the design of better therapeutics.

Burden of whooping cough in China (PertussisChina): study protocol of a prospective, population-based case-control study.

INTRODUCTION: Pertussis is one of the top 10 diseases of children under 10 years of age, and the few vaccine-preventable diseases who is on a rise in China in recent years; however, the true burden of pertussis, including age-stratified incidence and risk factors of severe sequelae, are under-recognised. We aim to estimate the health burden of laboratory-confirmed pertussis by age groups, considering the setting of illness onset (ie, in community, outpatient and inpatient), in a Chinese population (~2.23 million in total) at two sites. METHODS AND ANALYSIS: This paper describes the study design of a 1-year, prospective, age-stratified and population-based case-control study, including site selection, study population, case registry, ascertainment and enrolment, control recruitment, follow-up of case, microbiological methods, data collection, quality control activities and statistical methods used to generate incidence estimates. During June 2021 through May 2022, registry of suspected pertussis cases (namely chronic/persistent cough) will be conducted in several participating hospitals (SHs) at the two sites, which are selected based on Healthcare Utilisation and Attitudes Surveys (HUAS) carried out before study initiation. A case-control study will be conducted in the SHs and we aim to enrol a total of 1000 suspected pertussis cases (ie, all hospital admissions and the first 1-3 outpatient visits each week each hospital) and 2000 frequency matched healthy controls in community. Our primary study outcome, the laboratory-confirmed Bordetella pertussis infection, will be determined by a comprehensive laboratory methods and procedures (ie, culture, PCR and serological tests) in both cases and controls at enrolment and during 60-day's follow-up visits. Finally, data from HUAS (ie, population size), case registry (ie, the total number of suspected pertussis cases) and case-control study (ie, the prevalence or population attributable fraction of Bordetella pertussis) will be combined to calculate incidence and its 95% CI through bootstrap method. Epidemiological analyses will be conducted to determine the risk factors associated with severe sequelae of pertussis. ETHICS AND DISSEMINATION: This study has been approved by Chinese Centre for Disease Control and Prevention's Institutional Review Board (no. ICDC-202110). Results will be disseminated via academic presentations and publication in peer-reviewed journals, and will provide valuable scientific data and some new insights into the incidence, aetiology and risk factors for severe sequelae of pertussis to academic societies and the public health authorities who is currently struggling and fighting against this burdensome disease worldwide.

Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and the risk of primary hepatocellular carcinoma (HCC) in a Chinese population.

OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR), which is expressed in the liver, may be involved in both DNA methylation and DNA synthesis. It is also indicated as a potential risk factor of liver cancer in patients with chronic liver disease. To date, no study has been conducted on MTHFR and hepatocellular carcinoma (HCC) using a population-based design. The objective of this study was to evaluate the effects of polymorphisms of the MTHFR gene on the risk of primary liver cancer and their possible effect modifications on various environmental risk factors. METHODS: A population-based case-control study was conducted in Taixing, China. MTHFR C677T and A1298C were assayed by PCR-RFLP techniques. RESULTS: The frequency of MTHFR 677 C/C wild homozygotes genotype was 25.8% in cases, which was lower than that in controls (34.5%). The adjusted odds ratios (ORs) for the MTHFR 677 C/T and T/T genotype were 1.66(95% CI: 1.06-2.61), 1.21(95% CI: 0.65-2.28) respectively when compared with the MTHFR 677 C/C genotype. Subjects carrying any T genotype have the increased risk of 1.55(95% CI: 1.01-2.40) for development of primary hepatocellular carcinoma. A high degree of linkage disequilibrium was observed between the C677T and A1298C polymorphisms, with the D' of 0.887 and p < 0.01. The MTHFR 677 any T genotype was suggested to have potentially more than multiplicative interactions with raw water drinking with p-value for adjusted interaction of 0.03. CONCLUSION: We observed that the MTHFR 677 C/T genotype was associated with an increased risk of primary liver cancer in a Chinese population. The polymorphism of MTHFR 677 might modify the effects of raw water drinking on the risk of primary hepatocellular carcinoma.

[A case-control study on the relationship between methyl-tetra-hydrofolic acid reductase 677 gene polymorphism and the risk of stomach cancer].

OBJECTIVE: To explore the relationship between methyl-tetra-hydrofolic acid (MTHFR) 677 gene polymorphism and the risk of stomach cancer. METHODS: A population based case-control study was conducted and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to detect its genotypes. RESULTS: Among cases with stomach cancer, the frequency of C/C, C/T, T/T genotype were 25.8%, 54.6%, 19.6%, compared with controls as 34.5%, 50.9%, 14.6% respectively. Using C/C genotype as reference, the OR of C/T or T/T genotype was 1.52 (95% CI: 1.04 - 2.23). 53.3% C and 46.7% T allele were distributed in stomach cancer cases, while 60.0% C and 40.0% T in controls. The OR for T allele in relation to C allele was 1.31 (1.02 - 1.69) when C allele was used as reference. In addition, the present study showed that MTHFR677 AnyT genotype might interact with smoking, moldy food intake, wheat porridge intake, eating salty food and Hp CagA infection to increase the risk of stomach cancer. No interaction was observed between MTHFR677 AnyT genotype and alcohol drinking or green tea intake. CONCLUSION: MTHFR677 AnyT genotype, might increase the risk of stomach cancer development and the genotype might also interact with other environmental risk factors to increase the risk of stomach cancer.

[Investigation of concentration of microcystin in raw materials and finished products of spires health food].

In order to investigate the concentration of micromystin in raw materials and finished products of spires health food, we collected 33 water samples from water resources, 160 water samples from culturing pool, 86 samples of Spires slurry and 70 samples of powders of Spires raw materials from 7 Spires manufacturers in Jiangsu, Yunnan, Fujian and Guandong provinces which are the main spires producing areas from July to August 2002. At the same time, we also collected 71 samples of 19 finished products of Spires health foods in markets of above-mentioned 4 provinces. Microcystin were tested for all samples by ELISA. The results show: microcystin were not detected in 12 tap water samples, were detected in 6 of 9 under water samples, 8 of 12 surface water samples; microcystin concentration of water samples from culturing pool was 207.9 pg/ml and Spires slurry was 31.9 ng/g, the former is 153 times as the latter; powder of Spires raw material was 206.4 ng/g. The average concentration of microcystin of all Spires health food samples from market was 317.2 ng/g, there into, microcystin was 142.7 ng/g in a tables and 222.6 ng/g in capsule, respectively. The results suggested: we shouldn't ignore the risk of exposuring microcystin when consuming Spires health food, it is necessary to further study and set a standard of limited microcystin in Spires health food.

[A case-control study on drinking green tea and decreasing risk of cancers in the alimentary canal among cigarette smokers and alcohol drinkers].

OBJECTIVE: To explore the role of green tea in decreasing the risks of gastric cancer, liver cancer, esophageal cancer among alcohol drinkers or cigarette smokers. METHODS: A population based case-control study was conducted in Taixing, Jiangsu province. RESULTS: In Taixing city, identified cases of stomach, liver and esophageal cancers were chosen with informed consent. The numbers were 206, 204, 218 respectively. Controls were chosen from normal population having lived in the area for longer than 10 years, also with informed consent. Green tea drinking seemed to have decreased 81%, 78%, 39% risk for the development of gastric cancer, liver cancer and esophageal cancer among alcohol drinkers. It might also have decreased 16%, 43%, 31% on the risks of developing the three kinds of cancers among cigarette smokers. Interaction assessment showed that drinking green tea could significantly decrease the risk of gastric cancer and liver cancer among alcohol drinkers, with ORs of interaction item 0.23 (95% CI: 0.10 - 0.55) and 0.25 (95% CI: 0.11 - 0.57) respectively. CONCLUSION: Habit of drinking green tea seemed to have significant protective effects on the development of both gastric and liver cancer among alcohol drinkers while, green tea also having some protective effect on esophageal cancer among alcohol drinkers and on three kinds of cancers among cigarette smokers.

Study on the distribution of nodularin in tissues and cell level in mice.

OBJECTIVES: To study the tissue distribution of nodularin in mice and the cellular location of nodularin in the target organs. METHODS: The nodularin was labeled with radioactive isotope (125)I and then was given to mice via oral, intraperitoneal and intravenous administration. The distribution of nodularin in target organs and the cellular location of nodularin were studied by radioisotope and autoradiography techniques. RESULTS: The radioisotope study results showed that nodularin was mainly distributed in the kidney and liver in mice. Further autoradiography study indicated that nodularin was distributed in the renal cell nuclei and liver cell nuclei. CONCLUSION: The kidney and liver are the two main target organs for nodularin in mice.

[The acute toxic effects of microcystin LR in SD rats].

OBJECTIVE: To assess Microcystin LR (MCLR)-induced acute toxic effects in male Sprague-Dawley rats. METHODS: The rats were injected with MCLR intraperitoneally in different doses for different days. The organs and serum with rats were collected at 1 and 7 days after injection, and 7 days after the final injection (total 14 days). Pathological and enzymatic changes were observed. RESULTS: The rats injected with 122 microg/kg MCLR showed myocardial cells damage including pyknosis, plasma dissolve and myofibrilla (pls check with dictionary) necrosis in the heart muscles after 24 hours. At the same time, the activities of serum glutamate-oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH) and creatine phosphonase (CPK) were higher than these in the other groups (P < 0.01). The kidney was also damaged, kidney cell degeneration, and the increase of blood creatine (BCr) and blood urea nitrogen (BUN) were also seen. In liver pathological study, liver cell hemorrhage, degeneration and/or necrosis was observed. In serum the activities of glutamate-pyruvate transaminase (GPT), alkaline phosphatase (LDH) and GOT were higher than these in the other groups (P < 0.01). CONCLUSION: These results suggested that MCLR can injure the heart, kidney and the liver in SD rats, and there is a dose-response relationship between MCLR and the toxic effect.

[Teratosis and damage of viscera induced by microcystin in SD rat fetuses].

OBJECTIVE: To study the teratogenicity and traumatic effect of microcystin LR (MCLR) on pregnant SD rats and their fetuses. METHODS: Sixty pregnant SD rats were divided into four groups, 16 in each group: three experimental groups in which MCLR was injected into the abdominal cavity at the doses of 4 microgram/kg, 16 microgram/kg, and 62 microgram/kg respectively for 10 days, and control group injected with normal saline. Twenty days after, the pregnant rats were killed. The development, teratosis, and histology of viscera of the fetuses were examined. RESULTS: Deformities were found in the fetuses of the 62 microgram/kg group and 16 microgram/kg group with the teratogenic rate of 11.70 per thousand (2/172) and 6.76 per thousand (1/155). Petechial hemorrhage and severe hydropic degeneration in liver and maldevelopment of glomeruli and renal medulla were found in 62 microgram/kg group. Mild granular degeneration was found in the liver of fetuses in 4 microgram/kg group. CONCLUSION: MCLR passes through the placental barrier and causes damage in kidney and liver, which may be the basis of high incidence of liver cancer microgram in the fetus phase.