Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

Alpinetin, a composition of Alpinia katsumadai Hayata, has been reported to have a number of biological properties, such as antibacterial, antitumor and other important therapeutic activities. However, the effect of alpinetin on inflammatory bowel disease (IBD) has not yet been reported. The purpose of this study was to investigate the anti-inflammatory effect and mechanism of alpinetin on dextran sulfate sodium (DSS)-induced colitis in mice. In vivo, DSS-induced mice colitis model was established by giving mice drinking water containing 5% (w/v) DSS for 7 days. Alpinetin (25, 50 and 100 mg/kg) were administered once a day by intraperitoneal injection 3 days before DSS treatment. In vitro, phorbol myristate acetate (PMA)-differentiated monocytic THP-1 macrophages were treated with alpinetin and stimulated by lipopolysaccharide (LPS). The results showed that alpinetin significantly attenuated diarrhea, colonic shortening, histological injury, myeloperoxidase (MPO) activity and the expressions of tumor necrosis factor (TNF-α) and interleukin (IL-1β) production in mice. In vitro, alpinetin markedly inhibited LPS-induced TNF-α and IL-1β production, as well as Toll-like receptor 4 (TLR4) mediated nuclear transcription factor-kappaB (NF-κB) and NOD-like receptor protein 3 (NLRP3) inflammasome activation. In conclusion, this study demonstrated that alpinetin had protective effects on DSS-induced colitis and may be a promising therapeutic reagent for colitis treatment.

In plants, high disease resistance often results in a reduction of yield. Therefore, breeding crops with balanced yield and disease resistance has become a major challenge. Recently, microRNA (miRNA)-mediated R gene turnover has been shown to be a protective mechanism used by plants to prevent autoimmunity in the absence of pathogens. However, whether these miRNAs play a role in plant growth and how miRNA-mediated R gene turnover responds to pathogen infection have rarely been explored. Here, we found that a Brassica miRNA, miR1885, targets both an immune receptor gene and a development-related gene for negative regulation through distinct modes of action. MiR1885 directly silences the TIR-NBS-LRR class of R gene BraTNL1 but represses the expression of the photosynthesis-related gene BraCP24 by targeting the Trans-Acting Silencing (TAS) gene BraTIR1 for trans-acting small interfering RNAs (tasiRNAs)mediated silencing. We found that, under natural conditions, miR1885 was kept at low levels to maintain normal development and basal immunity but peaked during the floral transition to promote flowering. Interestingly, upon Turnip mosaic virus (TuMV) infection, miR1885-dependent trans-acting silencing of BraCP24 was enhanced to speed up the floral transition, whereas miR1885-mediated R gene turnover was overcome by TuMV-induced BraTNL1 expression, reflecting precise regulation of the arms race between plants and pathogens. Collectively, our results demonstrate that a single Brassica miRNA dynamically regulates both innate immunity and plant growth and responds to viral infection, revealing that Brassica plants have developed a sophisticated mechanism in modulating the interplay between growth, immunity, and pathogen infection.

No abstract

Morin, a flavonoid isolated from Chinese herbs of the Moraceae family, has been reported to possess antiinflammatory activity. However, the effects of morin on mastitis have not been investigated. The present study was conducted to elucidate the antiinflammatory properties of morin on lipopolysaccharide (LPS)-stimulated primary bovine mammary epithelial cells (bMEC). The viability of bMEC was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay. Subsequently, bMEC were stimulated with LPS in the presence or absence of morin. Gene expression of proinflammatory cytokines was determined by quantitative real-time PCR (qRT-PCR). Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were detected by Western blotting. The results showed that cell viability was not affected by morin. Moreover, morin inhibited the gene expression of tumor necrosis factor-α (TNF-α), IL-6, and IL-1β in LPS-stimulated bMEC in a dose-dependent manner. Western blot analysis showed that morin suppressed the phosphorylation of IκBα, NF-κB unit p65, ERK, p38, and JNK in LPS-stimulated bMEC. In conclusion, the protective effects of morin on LPS-induced inflammatory response in bMEC may be due to its ability to suppress NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. These findings suggest that morin may be used as antiinflammatory drug for mastitis.

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.

Cadillo is an invasive species commonly found in pastures, rangelands, and disturbed areas. It is becoming a significant problem weed in Florida pastures and natural areas. The objectives of this research were to determine effective techniques to break seed dormancy and the effect of light, temperature, pH, water stress, and depth of seed burial on Cadillo germination. Cadillo seeds had significant levels of innate dormancy imposed by a hard seed coat; chemical scarification was the most effective technique for removing dormancy. Seeds germinated from 15 to 40 C, with an optimal temperature of 28 C. Germination was unaffected by pH levels. Water stress below −0.2 MPa reduced seed germination. Cadillo germination was not light-dependent and seeds emerged from depths up to 9 cm, with the greatest occurring emergence near the soil surface. Considering that Cadillo seed can germinate under a wide range of environmental conditions, it is not surprising that it has become a serious invasive weed in Florida.

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.