Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg⁻¹ and 0.96 µg L⁻¹ for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg⁻¹) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L⁻¹). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.     

The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature.

The 5'-end region of cspA mRNA contains a Cold Box sequence conserved among several cold-shock mRNAs. This region forms a stable stem-loop structure followed by an AU-rich sequence. Here we show that the Cold Box region is essential for the normal scale of cspA mRNA induction after cold shock because a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50%. The AU-rich track, however, slightly destabilizes the mRNA. The integrity of the stem is essential for the stabilizing function, whereas that of the loop sequence is less important. Overexpression of a mutant cspA mRNA devoid of both the AUG initiation codon and the coding sequence results in a severe growth inhibition at low temperature along with a derepression of the chromosomal cspA expression. Furthermore, the overexpressed RNA is stably associated with the 30 S and 70 S ribosomes. Our results demonstrate that the AUG initiation codon and the coding region containing the downstream box are not required for cspA mRNA to bind ribosomes and that the 5'-untranslated region by itself has a remarkable affinity to ribosomes at low temperature.     

Computational and Experimental Investigation of the Antimicrobial Peptide Cecropin XJ and its Ligands as the Impact Factors of Antibacterial Activity

Cecropin XJ, as a heat stable antimicrobial peptide (AMP), displayed broad bacteriostatic activities, effectively inhibited proliferation of cancer cells and induced cell apoptosis in vitro. However, it exhibited little hemolytic activity and very low cytotoxicity to erythrocytes and normal cells. Although exerts multiple remarkable bioactivities, the refined molecular conformation of native Cecropin XJ remains unsolved. The aim of the present study is to comprehensively investigate the physicochemical characteristics and structure-function relationship of this antimicrobial peptide by using a series of bioinformatics and experimental approaches. In this study, we revealed that the mature Cecropin XJ consists of 41 amino acids, containing two α-helical structures from Lys⁷ to Lys²⁵ and from Ala²⁹ to Ile³⁹. The phylogenetic tree indicated that Cecropin XJ belongs to the Class I AMPs of cecropin family. Hydrophobic analysis showed Cecropin XJ is a typical amphiphilic molecule. The surface of Cecropin XJ was found to have a much wide range of electrostatic potential from ?83.243 to +83.243. The amphipathicity and surface potential of Cecropin XJ partially supported the AMP pore-forming hypothesis. Scanning electron microscopy experimentally confirmed the damages of Cecropin XJ to microbial membrane. Four predicted docking sites respectively for magnesium ion (Mg²⁺), adenosine diphosphate (ADP), bacteriopheophytin (BPH), and guanosine triphosphate (GTP) were found on the surface of Cecropin XJ. Thereinto, Mg²⁺ was experimentally proved to suppress the antibacterial activity of Cecropin XJ; both GTP and ADP enhanced the bactericidal activities to varying degrees. The present study provides a foundation for further investigation of molecular evolution, structural modification, and functional mechanisms of Cecropin XJ.     

Rapid improvement of rice eating and cooking quality through gene editing toward glutelin as target

Rice eating and cooking quality (ECQ) is a major concern of breeders and consumers, determining market competitiveness worldwide. Rice grain protein content (GPC) is negatively related to ECQ, making it possible to improve ECQ by manipulating GPC. However, GPC is genetically complex and sensitive to environmental conditions; therefore, little progress has been made in traditional breeding for ECQ. Here, we report that CRISPR/Cas9-mediated knockout of genes encoding the grain storage protein glutelin rapidly produced lines with downregulated GPC and improved ECQ. Our finding provides a new strategy for improving rice ECQ.     

气候变化和人类活动对中国北方旱区植被变绿的定量贡献

气候变化和大规模的生态恢复使中国北方旱区植被发生了显著变化，量化气候变化和人类活动对植被动态的相对贡献，对于旱区生态系统管理和应对未来气候变化具有重要意义。目前，中国北方旱区植被变化影响因素的时间动态（2000年大规模生态恢复工程实施前后）和空间异质性（沿干旱梯度）仍需进一步的定量研究。基于多源数据，采用趋势分析、偏相关分析和随机森林模型等方法，分析了1981-2018年中国北方旱区气候和植被的时空变化规律，量化了2000年前后气候变化和人类活动对植被动态的相对贡献并分析其在干旱梯度上的空间差异性。结果表明：（1）1981-2018年期间，中国北方旱区的叶面积指数（LAI）平均增加速率为（0.0037±0.0443） a^-1，且增加速率沿干旱梯度增大。2000年前仅10.46%（P<0.05）的地区显著变绿，而2000年后达到36.84%，且植被变绿主要归因于非树木植被。（2）2000年后降水对植被变绿的正效应在不同干旱梯度均增加，而在半干旱区和亚湿润干旱区，温度对植被变绿由正向促进转为负向抑制，而辐射在干旱区由负效应转向正效应。（3）2000年前后，气候变化均主导着植被的动态，贡献率分别为96.07%和73.72%，人类活动的贡献在2000年后进一步增强（从3.93%增加到26.28%），且沿着干旱梯度而增加，其中人类活动对植被变绿的贡献在半干旱地区增加最显著（+0.0289 m² m^-2 a^-1，P<0.05）。研究结果可为未来气候变化下中国北方旱区的植被恢复和可持续发展提供科学依据。     

Iron plaque enhances phosphorus uptake by rice (Oryza sativa) growing under varying phosphorus and iron concentrations

Both solution culture and pot experiments were performed to investigate (a) the effects of external Fe (II) concentrations and forms on the formation of iron plaque on the roots of rice (Oryza sativa) and subsequent P adsorption on iron plaque and shoot P concentrations and (b) the effects of soil moisture regimes on the formation of iron plaque and P adsorption on root surfaces and P accumulation in shoots. The results showed that iron plaque was significantly increased with increasing Fe²⁺ concentrations in the solution culture. The amounts of P adsorbed on the iron plaque were increased significantly with external Fe²⁺ concentrations. Although shoot P concentration was not significantly affected by Fe²⁺ treatment after incubation for 2 days, it was significantly increased in the Fe‐treated plants compared with Fe‐deprived ones after incubation for 4 days. Soil culture experiment showed that the formation of iron plaque on root surfaces was promoted by exogenous iron, with greater amount of iron plaque being formed by addition of ferric hydroxide than of ferric oxide. Phosphorus adsorption on iron plaque also increased with the addition of iron oxides, and increasing soil P increased the amounts of P associated with the iron plaque and shoot P concentration. The amounts of iron plaque were almost sixfold higher under flooding condition than under field capacity condition. Plants pretreated under flooding condition generally had higher shoot P concentrations when they were transplanted to solutions with varying P levels, and this was most pronounced in the treatment with highest solution P concentration. The results suggest that iron plaque acts as a nutrient reservoir for phosphorus in the rhizosphere and helps enhance P acquisition by rice.     

Microrheological aspects of adhesion of Escherichia coli on glass

The adhesion of both live and fixed bacteria (Escherichia coli) on glass has been studied under well-defined hydrodynamic conditions, created in an impinging jet apparatus. With this technique one can accurately measure the initial deposition rate jo on the surface, the average lifetime of a bacterium on the surface, tau esc, and the surface area blocked per deposited bacterium, normalized by its projected area, gamma. The experimental results are compared to theoretical results for equivalent spheres. It is found that near the stagnation point the deposition rate jo is mainly controlled by convective diffusive transport which, for rod-shaped Eschericia coli, with an axis ratio of about 2, is found to be equal to that for spheres. No differences in jo and tau esc were found between live and fixed bacteria at low flow rates. At high flow rates fixed bacteria adhered to the surface at a slower rate. In both systems jo was found to decrease suddenly at a distance of about 150 microns from the stagnation point, in contrast to systems of spherical particles for which jo is uniform over the surface. Most likely this is due to the rotation of the rod-shaped particles, which vary their distance to the surface periodically with time. The main difference between live and fixed bacteria, besides different deposition rates in strong flows, is that gamma is about 30% larger for fixed bacteria than for live ones, resulting in a much lower final coverage for fixed bacteria. These results imply a larger repulsion between fixed bacteria than between living ones. From detachment experiments we can conclude that not all bacteria stick to the surface with the same bond strength. The variation in the bond strength is due to the aging of the bonds between the bacteria and the surface. The average bond strength corresponds to an energy of about 13-15 kT.