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.     

Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH doubled haploid - LS Linsmaier and Skoog basal medium - BAP benzylaminopurine - GLM Generalized Linear Model - SAS Statistical Analysis System     

大壁虎的染色体及减数分裂联会复合体的研究

大壁虎(Gekko gecko)的染色体数目为2n=38,核型由2对中着丝粒(Nos.1.4.)、3对亚中着丝粒(Nos.2.3.5)及14对端着丝粒和亚端着丝粒(Nos.6—19)染色体组成。一对核仁组织者(NOR_s),位于第7对端着丝粒染色体的末端。同时,本文还对大壁虎的减数分裂以及联会复合体(S.C)的结构和组型,进行了详细的观察和分析。     

白腹锦鸡,红腹锦鸡,中国雉鸡SC组型的比较研究

以微铺展—硝酸银染色技术制备三种鸡的SC标本,进行电镜观察。结果表明:三种鸡的SC组非常相似,即2n=82,ZZ/ZW型性别决定,雄性为ZZ。除1号SC和Z-SC为中着丝粒外,其余均为端着丝粒。Z-SC的相对长度有明显的细胞间差异;平均相对长度度介于第3和第4号SC之间。三者SC组型上的差异主要表现在相应SC长度上的不同。并对其亲缘关系及在鸟类进化中的可能地位进行了讨论。此外,在微铺展法制备的锦鸡精母细胞SC标本中还发现了巨大中心粒,这在高等动物尚属首次。