These experimental results expand the scope of the protein database, generating targets for further investigation of the pathogenic mechanism of idiopathic asthenozoospermia.
Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six alpha-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host.
The transferability of bacterial resistance to tigecycline, the 'last-resort' antibiotic, is an emerging challenge of global health concern. The plasmidborne tet(X) that encodes a flavin-dependent monooxygenase represents a new mechanism for tigecycline resistance. Natural source for an ongoing family of Tet(X) resistance determinants is poorly understood. Here, we report the discovery of 26 new variants [tet(X18) to tet(X44)] from the poultry pathogen Riemerella anatipestifer, which expands extensively the current Tet(X) family. R. anatipestifer appears as a natural reservoir for tet(X), of which the chromosome harbours varied copies of tet(X) progenitors. Despite that an inactive ancestor rarely occurs, the action and mechanism of Tet(X2/4)-P, a putative Tet(X) progenitor, was comprehensively characterized, giving an intermediate level of tigecycline resistance. The potential pattern of Tet(X) dissemination from ducks to other animals and humans was raised, in the viewpoint of ecological niches. Therefore, this finding defines a large pool of natural sources for Tet(X) tigecycline resistance, heightening the need of efficient approaches to manage the inter-species transmission of tet(X) resistance determinants.
Background Chitinases are enzymes which degrade β-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance. Methods Primers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out. Results The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg−1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0–10.0 and 25–45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and β-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products. Conclusions This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.
Low temperature at the seedling stage is a major damaging factor for rice production in southern China. To better understand the cold response of cultivated and wild rice, cold-sensitive cultivar 93–11 (Oryza sativa L. ssp. Indica) and cold-resistant hybrid wild rice DC907 with a 93–11 genetic background were used for a quantitative proteomic analysis with tandem mass tags (TMT) in parallel. Rice seedlings grown for four weeks at a normal temperature (25°C) were treated at 8–10°C for 24, 72 and 120 h. The number of differentially expressed proteins increased gradually over time in the cold-exposed rice in comparison with the untreated rice. A total of 366 unique proteins involved in ATP synthesis, photosystem, reactive oxygen species, stress response, cell growth and integrity were identified as responding to cold stress in DC907. While both DC907 and 93–11 underwent similar alterations in proteomic profiles in response to cold stress, DC907 responded in a prompter manner in terms of expressing cold-responding proteins, maintained a higher level of photosynthesis to power the cells, and possessed a stable and higher level of DIR proteins to prevent the plant from obtaining irreversible cell structure damage. The observations made in this study may lay a new foundation for further investigation of cold sensitivity or tolerance mechanisms in rice.
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