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.     

内蒙古部分地区骆驼和羊寄生蜱虫病毒组学分析

为了解内蒙古地区蜱虫病毒组学的本底数据,采用病毒宏基因组学方法对在内蒙古阿拉善盟左旗、右旗和四子王旗地区3个采样点采集骆驼和羊体表寄生的1789只蜱虫样品进行病毒宏基因组学分析,并对特定病毒进行巢式PCR扩增和测序,通过Clustal W和MEGA7.0等生物信息学软件对获得的病毒基因序列进行遗传进化分析.数据显示,蜱虫样品携带包括植物、脊椎动物和非脊椎动物等来源的17个病毒科和一些未分类的病毒;其中,2株弹状病毒具有丰富的遗传多样性,与新疆地区和长江地区的弹状病毒的同源性达到98.5％和96.26％,提示蜱虫弹状病毒可能是通过羊和骆驼等动物贸易导致了新疆和内蒙古地区,以及内地的跨区域传播;细小病毒仅在羊来源的蜱虫中检测到,与中国河北地区的山羊血清中的细小病毒形成同一进化分支,我们推测蜱虫细小病毒在国内不同地区间可跨区域传播,在进化分析过程中,发现这种病毒与多种的细小病毒的同源性都不低于50％,提示细小病毒可能具有遗传稳定性;Tamdy病毒与来自阿塞拜疆、乌兹别克斯坦和美国的Tamdy病毒均具有极高的同源性,结果显示该病毒在内蒙古地区已经出现,并存在潜在流行的可能,有必要对Tamdy病毒进行进一步的监测;在本研究中,我们鉴定的白蛉病毒与来自新疆的亚洲璃眼蜱所携带的博乐蜱虱病毒形成同一个进化分支,与新型布尼亚病毒和Heartland virus病毒的同源性达到50％以上,该结果提示,我们发现的蜱虫白蛉病毒可能具有潜在的致病性,需要对其流行情况和致病性进行监测和研究.本研究为完善内蒙古部分地区蜱虫病毒的多样性和本底情况提供了重要的基础数据.     

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.     

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.     

青海省单蚤属两新种记述(蚤目:角叶蚤科)

在鉴定我囱青海省单蚤属(Monopsyllus Kolenati,1857)的跳蚤标本时,除检出别人已记述过的不等单蚤[Monopsyllus anisus(Rotnschild,1907)],新月单蚤(Monopsyllusscaloni Vovchinskaya,1950)和冯氏单蚤(Monopsyllus fengi Liu,Xie et Wang)(新种,待发表)外,尚发现两个在形态上都接近花鼠单蚤[Monopsyllus indages(Rothschild,1908)]的新种。由于两新种阳茎钩突特别,故分别命名为钩状单蚤Monopsyllus hamutus,新种和文状单蚤Monopsyllus forficus,新种。鉴别这三个蚤种的重要特征如表1所示。     

胆碱能神经对人餐后神经降压素释放的影响

本文比较了6名健康成人进食、餐前肌注阿托品以及单纯咀嚼食物后的血浆神经降压素样免疫活性物质(NTLI)水平的变化,以探讨胆碱能神经对神经降压素释放的影响。用放射免疫测定法分别测定NTLI和胰多肽(PP)的含量,以便同时比较两者释放的状态。6人的基础血浆NTLI和PP的水平平均分别为15.7±2.4和16.6±9 7pmol/L。进食后,血浆NTLI和PP水平均显著增高,并呈双相反应。第一个血浆NTLI高峰平均为60.7±13.2pmol/L,出现于餐后的20min。餐后90min,又出现另一个高峰,其平均水平为58.8±8.2pmol/L。在进食前肌注阿托品1mg,餐后的第一个血浆NTLI高峰消失,而第二个高峰仍存在。单纯咀嚼食物后,血浆PP水平明显增高,而对NTLI的释放无刺激作用。本文结果提示,餐后早期的神经降压素释放的调节是由非迷走胆碱能神经参与的,而后期的释放不受胆碱能神经的影响。     

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.