Effects of Neutrophil Extracellular Traps on Bovine Mammary Epithelial Cells in vitro

Bovine mastitis is a common infectious disease which causes huge economic losses in dairy cattle. Bovine mammary epithelial cell (BMEC) damage usually directly causes the decrease of milk production, which is one of the most important causes of economic loss. NETs, novel effector mechanisms, are reported to exacerbate the pathogenesis of several inflammatory diseases. NETs formation has also been observed in the milk and mammary glands of sheep. However, the effects and detailed mechanisms of NETs on BMEC damage remain unclear. Thus, we aim to examine the effects of NETs on BMECs in vitro, and further to investigate the detail mechanism. In this study, the cytotoxicity of NETs on BMECs was determined using lactic dehydrogenase (LDH) levels in culture supernatants. Histone-induced BMEC damage was examined by flow cytometry and immunofluorescence analysis. The activities of caspase 1, caspase 3, caspase 11, and NLRP3 was detected using western blotting and immunohistochemical analysis. The results showed that NETs and their component histone significantly increased cytotoxicity to BMECs, suggesting the critical role of NETs, and their component histone in BMEC damage. In addition, histone could also induce necrosis, pyroptosis, and apoptosis of BMECs, and the mechanisms by which histone leads to BMEC damage occurred via activating caspase 1, caspase 3, and NLRP3. Altogether, NETs formation regulates inflammation and BMEC damage in mastitis. Inhibiting excess NETs formation may be useful to ameliorate mammary gland damage associated with mastitis

Rapid approach for cloning bacterial single-genes directly from soils

Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of cloning strategy in this study. Five GS genes were directly cloned from soils using degenerate primers with two steps of nested polymerase chains reactions. The genes showed 94 to 99% amino acid identities to the homologs in the known database, and encoded proteins affiliated to GS I and GS II families, respectively. All the five genes could rescue the growth of Escherichia coli glutamine auxotroph mutant ET6017 in minimum medium (ammonium chloride was sole nitrogen source in this medium). This study develops one rapid approach for cloning bacterial single-genes directly from soils. Comparing with the conventional strategies for gene cloning from complex environmental samples, this method did not need making genomic library and isolating target genes from large amount of library clones. This approach distinctively demonstrates its advantages of rapidity and effectiveness particularly when it aims at cloning short single-genes that had known homologs in all kinds of nucleic acid databases.Keywords: Gene cloning, soil, glutamine synthetase, nested PCR, single-geneAfrican Journal of Biotechnology Vol. 12(32), pp. 5029-503

Molecular Characterization of Anaplasma , Ehrlichia, Babesia , and Borrelia in Ticks from Guangdong Province of Southern China

Tick-borne pathogens have become emerging global health threats in humans and animals. The objective of the present study was to investigate Anaplasma, Ehrlichia, Babesia , and Borrelia in ticks in southern China. Ticks were collected from animals and identified to the species level. Tick-borne Anaplasma , Ehrlichia , Babesia , and Borrelia were tested by PCR assays, and analyzed by sequencing and phylogenetics. We collected 747 ticks, including Rhipicephalus microplus (n=225) and R. sanguineus (n=522), from pet dogs in Guangdong Province of southern China. Using PCR and phylogenetic analysis we showed that these ticks carried Anaplasma platys , Ehrlichia canis , Borrelia miyamotoi , Babesia vogeli , and an unclassified Ehrlichia species in Rhipicephalus ticks. Of these pathogens, B. miyamotoi was first found in southern China. A. platys , E. canis , and B. miyamotoi were zoonotic pathogens that infected both R. microplus and R. sanguineus , whereas Ba. vogeli only infected R. sanguineus , with a prevalence of 0.3%-1.5%. The results of this study showed diversity and complexity of tick-borne pathogens in Guangdong Province of southern China, implying the importance of tick-borne pathogen surveillance and control

Heating-induced decarboxylative cyclization for the synthesis of 5-trifluoromethyl-1,2,4-triazoles from trifluoroacetimidohydrazides and α-oxocarboxylic acids

A simple metal and additive-free approach for the assembly of 5-trifluoromethyl-1,2,4-triazoles via only heating-induced decarboxylative cyclization of readily available trifluoroacetimidohydrazides and α-oxocarboxylic acids has been developed. In this protocol, the rarely reported tetrahedral carboxylic acids intermediate could be in-situ generated and undergo subsequent decarboxylation to afford the target heterocycle products

Propionate Protects against Lipopolysaccharide-Induced Mastitis in Mice by Restoring Blood–Milk Barrier Disruption and Suppressing Inflammatory Response

Mastitis, an inflammation of the mammary glands, is a major disease affecting dairy animal worldwide. Propionate is one of the main short-chain fatty acid that can exert multiple effects on the inflammatory process. The purpose of this study is to investigate the mechanisms underlying the protective effects of sodium propionate against lipopolysaccharide (LPS)-induced mastitis model in mice. The data mainly confirm that inflammation and blood–milk barrier breakdown contribute to progression of the disease in this model. In mice with LPS, sodium propionate attenuates the LPS-induced histopathological changes, inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) production, myeloperoxidase activity in mammary tissues. Given their importance in the blood–milk barrier, tight junction proteins occludin and claudin-3 are further investigated. Our results show that sodium propionate strikingly increases the expressions of occludin and claudin-3 and reduces the blood–milk barrier permeability in this model. Furthermore, in LPS-stimulated mouse mammary epithelial cells (mMECs), LPS increased the expressions of phosphorylated (p)-p65, p-IκB proteins, which is attenuated by sodium propionate. Finally, we examine the possibility that propionate acts as a histone deacetylase (HDAC) inhibitor, the results show that both sodium propionate and trichostatin A increase the level of histone H3 acetylation and inhibit the increased production of TNF-α, IL-6, and IL-1β in LPS-stimulated mMECs. These data suggest that sodium propionate protects against LPS-induced mastitis mainly by restoring blood–milk barrier disruption and suppressing inflammation via NF-κB signaling pathway and HDAC inhibition

Defect evolution during high temperature tension-tension fatigue of SLM AISi10Mg alloy by synchrotron tomography

Lack of fusion defects and porosity are inevitable characteristics of additive manufacturing and these are expected to play a key role in determining fatigue life and fatigue failure when excluding the influence of inhomogeneous microstructures. This work followed damage accumulation under tension-tension cyclic loading at 250 °C in situ by time-lapse synchrotron radiation X-ray micro-computed tomography (SR-μCT) for AlSi10Mg test-pieces, produced by selective laser melting (SLM) over their complete fatigue lives (ranging from 180 to 38,000 cycles). These samples were found to accumulate widespread plastic strain each cycle in common with ultra-low cycle fatigue (UCLF) at low levels of triaxial constraint. The defects were found to elongate plastically at a rate approximately 10 times larger than their growth rate laterally. This elongation behaviour at room and elevated temperature fatigue is proportional to the accumulated longitudinal strain increment each cycle. Rotation under the influence of shear is also observed for those defects close to the surface of samples. Some defect coalescence was observed, but final failure was found to be associated with the nucleation of a high density of secondary microvoids (occurring at eutectic Si platelets) that form just prior to failure and link up by microvoid coalescence. These steps may take up approximate 90% of the fatigue life. The final stage of cyclic plasticity occurs when the longitudinal strain exceeds ∼0.9. Our results are in line with previous models of strain accumulation and defect growth under ULCF conditions