Assembly and substrate recognition of curli biogenesis system.

A major component of bacterial biofilms is curli amyloid fibrils secreted by the curli biogenesis system. Understanding the curli biogenesis mechanism is critical for developing therapeutic agents for biofilm-related infections. Here we report a systematic study of the curli biogenesis system, highlighted by structural, biochemical and functional analysis of the secretion channel complexes (CsgF-CsgG) with and without the curli substrate. The dual-pore architecture of the CsgF-CsgG complex was observed and used to develop an approach to inhibit the curli secretion by physically reducing the size of the CsgF pore. We further elucidated the assembly of the CsgFG complex with curli components (CsgA and CsgB) and curli-cell association through CsgF. Importantly, the recognition of the CsgA substrate by CsgG was uncovered. Nine crevices outside of the CsgG channel provide specific and highly-conserved recognition sites for CsgA N-terminus. Together with analysis of CsgE, our study provides comprehensive insights into curli biogenesis.

Pharmacokinetics and bioequivalence of low-dose clopidogrel in healthy Chinese volunteers under fasted and fed conditions.

Clopidogrel is an antiplatelet drug whose performance at a low dose (25 mg) have not been evaluated in Chinese patients, who may be subject to different effects from Caucasians. We carried out this evaluation and compared a generic (Taijia) and a reference drug (Plavix®). We evaluated Taijia and Plavix® in 128 subjects, with 64 in a fasted state and 64 receiving a high-fat diet, and computed Cmax, AUC0-∞, and AUC0-t. Reference-scaled average bioequivalence (RSABE) methods and average bioequivalence (ABE) methods were used, and adverse events were assessed. Average maximum plasma concentrations (Cmax) of clopidogrel were significantly greater after 25 mg dose under fed conditions compared to fasted. Reference-scaled Cmax, AUC0-t, and AUC0-∞ were higher than the 0.294 cutoff during fasted, meeting RSABE criteria. Under fed conditions, SWR for AUC0-t and AUC0-∞ were lower than 0.294, permitting use of ABE. The 90% confidence intervals for AUC0-t and AUC0-∞ indicated bioequivalence. Pharmacokinetic parameters differed between fasted and fed states. The generic product was bioequivalent to the reference drug, and was safe and well tolerated. This suggests that they can be used interchangeably in a clinical setting.

Structural insight into the assembly of the type II secretion system pilotin-secretin complex from enterotoxigenic Escherichia coli.

Secretin is a large outer-membrane channel found in secretion systems of Gram-negative bacteria, facilitating the last step for transfer of proteins into the extracellular environment. In the type II secretion system, a lipoprotein called pilotin is essential to bind and target its corresponding secretin to the outer membrane. However, there is only limited structural information available about the interaction and assembly of the pilotin-secretin complex. Here we report the first near-atomic-resolution structure of a full-length Vibrio-type pilotin-secretin (AspS-GspD) complex from enterotoxigenic Escherichia coli by cryo-electron microscopy, which reveals the detailed assembly mode of the full-length pilotin-secretin complex. The AspS subunits attach to the secretin channel surface with a 15:15 stoichiometric ratio to GspD subunits, and insert their amino terminus into the outer membrane. The AspS subunits interact with all three secondary structural elements of the S domain of GspD, including strong interaction with the carboxy-terminal α-helix and weak interactions with another two elements, an α-helix and a loop. These structural and biochemical details provide a deeper insight to pilotin-secretin interaction and their assembly mode.

Structural insights into the secretin translocation channel in the type II secretion system.

The secretin GspD of the type II secretion system (T2SS) forms a channel across the outer membrane in Gram-negative bacteria to transport substrates from the periplasm to the extracellular milieu. The lack of an atomic-resolution structure of the GspD channel hinders the investigation of substrate translocation mechanism of T2SS. Here we report cryo-EM structures of two GspD channels (∼1 MDa), from Escherichia coli K12 and Vibrio cholerae, at ∼3 Å resolution. The structures reveal a pentadecameric channel architecture, wherein three rings of GspD N domains form the periplasmic channel. The secretin domain constitutes a novel double ß-barrel channel, with at least 60 ß-strands in each barrel, and is stabilized by S domains. The outer membrane channel is sealed by ß-strand-enriched gates. On the basis of the partially open state captured, we proposed a detailed gate-opening mechanism. Our structures provide a structural basis for understanding the secretin superfamily and the mechanism of substrate translocation in T2SS.

miR-214-mediated downregulation of RNF8 induces chromosomal instability in ovarian cancer cells.

Defective DNA damage response (DDR) is frequently associated with carcinogenesis. Abrogation of DDR leads to chromosomal instability, a most common characteristic of tumors. However, the molecular mechanisms underlying regulation of DDR are still elusive. The ubiquitin ligase RNF8 mediates the ubiquitination of γH2AX and recruits 53BP1 and BRCA1 to DNA damage sites which promotes DDR and inhibits chromosomal instability. Though RNF8 is a key player involved in DDR, regulation of its expression is still poorly understood. Here, we show that miR-214 could abrogate DDR by repressing RNF8 expression through direct binding to 3'-untranslated region (3' UTR) of RNF8 mRNA in human ovarian cancer cells. Antagonizing miR-214 by expressing its inhibitors in A2780 cells significantly increased RNF8 expression and thus promoted DNA damage repair. Consistent with the role of miR-214 in regulating RNF8 expression, the impaired DNA repair induced by miR-214 overexpression can be rescued by overexpressing RNF8 mRNA lacking the 3' UTR. Together, our results indicate that down-regulation of RNF8 mediated by miR-214 impedes DNA damage response to induce chromosomal instability in ovarian cancers, which may facilitate the understanding of mechanisms underlying chromosomal instability.

[Relationship between polymorphism of interleukin-1 and pneumoconiosis susceptibility].

OBJECTIVE: To explore the relationship between the polymorphism of Interleukin-1 and the pneumoconiosis susceptibility. METHODS: Eighty patients with silicosis and 45 with coal workers' pneumoconiosis (CWP) were selected while 125 male workers, Han nationality in the same workplace as the patients were selected as the controls. Between the patients and the control, the differences of age and cumulative length of service were less than five years and two years, respectively. The controls were exposed to dusts but did not suffer from pneumoconiosis. Moreover, the patients and the controls were paired by 1:1. DNA was extracted from leucocytes by the hydroxybenzene chloroform method. The polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) techniques and PCR were used to examine polymorphism of IL-1alpha (-889), IL-1beta (-511) and IL-1Ra (+2018) and variable number of tandem repeats (VNTR) of IL-1Ra. After the preliminary experiment, the most adaptive PCR reaction, the restriction enzyme digest and electrophoresis system were used. RESULTS: The difference in IL-1alpha (-889) 1/2 + 2/2 between the pneumoconiosis patients and the controls was significant (P < 0.01). The result of conditional Logistic regression showed that heterozygote and allele 2 of IL-1a (-889) were risk factors of pneumoconiosis. The difference in the genotype frequencies of IL-1beta (-511) 1/2 + 2/2, IL-1Ra (+2018) 1/2 + 2/2 and IL-Ra VNTR1/2 + 2/2 between the patients and the controls were not statistically significant (P > 0.05). CONCLUSION: IL-1alpha (-889) gene polymorphism is related to pneumoconiosis. Workers with IL-1alpha (-889) allele 2 are susceptible to the pneumoconiosis. The relationship between IL-1beta (-511), IL-1Ra (+2018), IL-1Ra VNTR genes polymorphisms and pneumoconiosis are not found.