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.     

Enhancement of hormonal activity with a monoclonal antibody specific to porcine growth hormone

Abstract

The potentiation of the biological effects of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. A monoclonal antibody (mAb), designated PS‐7.6, was raised against pGH and repeatedly shown to enhance the responsiveness of hypophysectomized (hypox) rats to pGH. As a result, animals receiving a combination treatment of pGH and mAb PS‐7.6 together gained significantly more weight than those receiving the same doses of pGH alone. The enhancing action of the mAb was a rapid process and its effective doses ranged from 0.1 to 2 mg/injection. The ability of the antibody to augment the hormonal activity persisted beyond the 5‐day treatment period and the differences in net weight gain between treated and control animals remained significant for 28 days. Results from treatment frequency studies further suggested that pGH when complexed with mAb PS‐7.6 required fewer injections and produced a greater efficacy than being administered alone. Therefore, present findings suggest that mAb PS‐7.6 may prove useful for not only improving the efficiency of pGH, but also developing a novel formulation for sustained pGH release.     

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.     

青藏高原草地承载力空间演变特征及其预警

为研究青藏高原草地承载力的空间演变特征并对其进行预警，以已有的青藏高原净初级生产力数据为基础，核算了该地区的草地理论载畜量及演变趋势，并结合县域实际存栏量，划定了草地承载力的预警等级。结果表明：（1）青藏高原草地承载力整体呈东高西低的格局，其中高寒草原和高寒草甸是该地区草地承载力的主要组成部分；（2）2000-2015年，青藏高原理论载畜量由8614.89万羊单位增至9451.53万羊单位；（3）青藏高原整体处于超载状态，2000-2010年超载状况加剧，至2015年超载状况稍有缓解，草畜平衡指数由67.88%增至79.90%，再降至67.91%。目前亟需优先控制72个红色预警县（超载状态正在加剧）的牲畜存栏量，避免超载状况进一步恶化。未来需要通过控制牲畜存栏量、调整畜牧区发展布局和提高草地生产力等多项措施的结合来改善青藏高原地区的草地承载状况，维持草地生态系统的可持续发展。     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

县域城镇空间形态与居住建筑能耗关联性的连续尺度研究——以浙江长兴、福建连江为例

居住建筑能耗受建筑周边空间形态的直接影响，确定空间形态低碳效应及其最佳尺度将有助于实现县域城镇低碳转型。以浙江省长兴县、福建省连江县为例，共计选取49个具有代表性的居住建筑样本，采用GIS分析与数理统计相结合的方式，在居住建筑1800m半径范围内，以200m为间隔建立缓冲区，开展全年、最冷月份能耗期及最热月份能耗期建筑能耗与周边空间形态关联性的连续尺度研究。结果表明：(1)道路密度、开发密度、容积率均与建筑能耗呈正相关，但各指标对应的能耗时期、尺度范围有较大差异。(2)水面率、土地利用混合度是与建筑能耗相关的共性指标，但在不同地区的正负效应及尺度范围不同；(3)影响浙闽地区县域城镇最热月份能耗期能耗的关键形态因子为1000m-道路密度、800m-水面率、1600m-开放空间率；影响连江最冷月份能耗期能耗的关键形态因子为800m-水面率。(4)就控碳单元设置而言，长兴县可重点关注1000—1200m半径范围，连江县可重点关注800—1000m、1400m半径范围。(5)浙闽地区县域城镇特性指标为开放空间率，与最热月份能耗期建筑能呈负相关；长兴县特性指标为容积率、水岸密度，分别与全年、最冷...     

A cytogenetic characterization comparing a rat 6TG-resistant strain and 6TG-sensitive clones

Summary A 8.3 /ml 6-thioguanine (6TG)-resistant strain was isolated from a rat tetraploid cell line by step-by-step selection in 6TG-medium. In the 6TG-resistant cell population 51% of the cells were tetraploid and 35% of the cells were hypertetraploid, i.e., one chromosome more than a tetraploid. The 6TG-resistant strain grew very well in RPMI 1640 medium with intervals of three days between subcultures. The 6TG-resistant cells all have a homogeneously staining region (HSRs) in one of the X chromosomes which do not stain after chromosome C-banding. They also possess a higher NORs activity and much lower frequency of sister chromatid exchanges (SCE). When the 6TG-resistant RCT cells were subcultured in 6TG-free medium for three days, their SCE frequency did not change. 5-bromodeoxyuridine (BrdU) significantly suppressed the NORs activity for both 6TG-resistant cells and 6TG-sensitive cells (P<0.001).Abbreviations 6TG 6-thioguanine - HSRs homogeneously staining region - NORs nucleolar organizer region - SCE sister chromatid exchange - BrdU 5-bromodeoxyuridine - HPRT Hypoxanthine phosphoribosyl transferase