Intraesophageal chemicals enhance responsiveness of upper thoracic spinal neurons to mechanical stimulation of esophagus in rats

Esophageal hypersensitivity is one of the most common causes of noncardiac chest pain in patients. In this study, we investigated whether exposure of the esophagus to acid and other chemical irritants affected activity of thoracic spinal neurons responding to esophageal distension (ED) in rats. Extracellular potentials of single thoracic (T3) spinal neurons were recorded in pentobarbital sodium-anesthetized, -paralyzed, and -ventilated male rats. ED (0.2 or 0.4 ml, 20 s) was produced by water inflation of a latex balloon placed orally into the middle thoracic region of the esophagus. The chemicals were administered via a tube that was passed through the stomach and placed in the thoracic esophagus. To irritate the esophagus, 0.2 ml of HCl (0.01 N), bradykinin (10 microg/ml), or capsaicin (10 microg/ml) were injected for 1-2 min. Only neurons excited by ED were included in this study. Results showed that intraesophageal instillation of HCl, bradykinin, and capsaicin increased activity in 3/20 (15%), 7/25 (28%), and 9/20 (45%) neurons but enhanced excitatory responses to ED in 9/17 (53%), 8/15 (53%), and 7/11 (64%) of the remaining spinal neurons, respectively. Furthermore, intraesophageal chemicals were more likely to enhance the responsiveness of low-threshold neurons than high-threshold neurons to the esophageal mechanical stimulus. Normal saline (pH 7.4, 0.2 ml) or vehicle instilled in the esophagus did not significantly affect activity or ED responses of neurons. We conclude that enhanced responses of thoracic spinal neurons to ED by the chemically challenged esophagus may provide a possible pathophysiological basis for visceral hypersensitivity in patients with gastroesophageal reflux and/or esophagitis.     

Protective effects of MLIF analogs on cerebral ischemia-reperfusion injury in rats

The monocyte locomotion inhibitory factor (MLIF) is an anti-inflammatory oligopeptide produced by Entamoeba histolytica. Among its different effects, it inhibits locomotion of human monocytes, hence its original name. The carboxyl-terminal end group Cys-Asn-Ser is the pharmacophore of anti-inflammatory peptide Met-Gln-Cys-Asn-Ser. In this study, the N-terminal of Cys-Asn-Ser was modified. With the aim to enhance the antioxidant ability and penetrability of Cys-Asn-Ser, we designed and synthesized two tetrapeptides Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser. The neuroprotective effects of Tyr-Cys-Asn-Ser and His-Cys-Asn-Ser on focal ischemia reperfusion were investigated, and their pharmacological activities compared with Cys-Asn-Ser were studied. In order to study the mechanism of neuroprotective effect of these peptides, the level of oxidative stress markers malondialdehyde (MDA) and superoxide dismutase (SOD) and pro-inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were detected in brain tissue homogenate.     

DNMT3b protects centromere integrity by restricting R-loop-mediated DNA damage

This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.Subject terms: Double-strand DNA breaks, DNA methylation, Primary immunodeficiency disorders     

Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.     

抗原抗体复合物型治疗性疫苗(乙克)临床研究中患者血清乙型肝炎病毒表面抗原下降的分析与启示

近年来全球慢性乙型肝炎(chronic hepatitis B,CHB)防治指南提出了“功能性治愈”(functional cure)的概念,即患者经过治疗达到血清乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)消失,但现有抗病毒治疗很难实现这一目标。本研究对既往临床试验中经抗原抗体复合物型治疗性疫苗(乙克)治疗后的CHB患者HBsAg下降情况进行了归纳分析,结果显示,经乙克治疗随访后达到乙型肝炎e抗原(hepatitis B e antigen,HBeAg)血清学转换者的HBsAg下降高达0.95log₁₀IU/mL,显著高于未达到HBeAg血清学转换者的0.32log₁₀IU/mL(P<0.01),而经氢氧化铝佐剂治疗随访后发生HBeAg血清学转换(0.49log₁₀IU/mL)者与未发生HBeAg血清学转换者(0.36log₁₀IU/mL)之间HBsAg下降无统计学差异。乙克组治疗过程中,丙氨酸氨基转移酶(alanine aminotransferase,ALT)骤升(ALT flare)在HBsAg下降>1.0log₁₀IU/mL者中较多见,氢氧化铝组未观察到此现象。回归分析显示,乙克治疗后HBsAg下降的影响因素有患者出现HBeAg血清学转换、感染的HBV为B基因型、治疗过程中ALT出现10倍增高,以及基线血清HBsAg为高水平。结果提示,乙克诱导的特异性免疫对降低CHB患者血清HBsAg水平有一定效果,采用“抗病毒药物治疗＋针对HBsAg的中和性抗体被动免疫＋乙克主动免疫”的“三明治”治疗策略可能会提高“功能性治愈”率。     

聚乙二醇定点修饰集成干扰素突变体Ⅱ

目的：用聚乙二醇（PEG）修饰集成干扰素突变体Ⅱ（IFN-Con-m2,IIFNm2）,通过纯化获得新型修饰分子并对该分子进行抗胰蛋白酶水解稳定性及初步药代动力学研究。 方法：将mPEG20000定点偶联到IIFNm2的第86位Cys残基上,修饰后的产物经CM层析后,以SDS-PAGE考察其纯度,用WISH-VSV系统进行生物活性测定;在0.1%胰蛋白酶条件下考察体外抗酶解稳定性;并以SD大鼠进行初步药代动力学研究,绘制血药浓度-时间曲线。采用3P87软件进行数据拟合,分析药物动力学参数。 结果：干扰素修饰率约为50%,且绝大多数以单修饰体（mono-PEG- IIFNm2）形式存在;提纯后mono-PEG-IIFNm2 的纯度大于98%,比活性约为修饰前IIFNm2的1%。抗胰蛋白酶水解试验表明：30min后,IIFNm2抗病毒活性残留为8%,mono-PEG-IIFNm2为41%。初步药代动力学研究显示：IIFNm的消除半衰期为(1.57±0.34)h,mono-PEG-IIFNm2为(18.0±4.0)h。 结论：成功地偶联了PEG和IIFNm2,建立了mono-PEG-IIFNm2的纯化工艺,PEG修饰能增加IIFNm2的体外抗胰蛋白酶水解稳定性,并显著延长体内半衰期。     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.     

Comparison and Effects of Acute Lamotrigine Treatment on Extracellular Excitatory Amino Acids in the Hippocampus of PTZ-Kindled Epileptic and PTZ-Induced Status Epilepticus Rats

In this communication, the effect of acute treatment with lamotrigine (LTG) was investigated on release of main excitatory amino acids (EAA) such as glutamate (Glu) and aspartate (Asp) in the hippocampus of pentylenetetrazol (PTZ)-induced and PTZ-kindled freely moving rats using micro dialysis. The results show that, levels of Glu and Asp significantly increased in the rat hippocampus during the seizure/interical periods for PTZ-status epilepticus (SE) and PTZ-kindled epileptic (EP) rats. The levels of Glu and Asp increased more in EP rat hippocampus than in SE rat hippocampus. After administration of 20 mg/kg LTG, the levels of Glu and Asp significantly decreased in the SE and EP rat hippocampus. The results indicate that: (a) excitability of the PTZ-kindled epileptogenic model is higher than that of the status epilepticus model; (b) the modulation of LTG on the EAA neurotransmitters certainly plays an important role in antiepileptic efficacy, especially in PTZ-kindled epileptic model where the release of EAA was influenced more markedly by acute application of 20 mg/kg LTG.     

Genetic Variants in Meiotic Program Initiation Pathway Genes Are Associated with Spermatogenic Impairment in a Han Chinese Population

Background

The meiotic program initiation pathway genes (CYP26B1, NANOS1 and STRA8) have been proposed to play key roles in spermatogenesis.

Objective

To elucidate the exact role of the genetic variants of the meiosis initiation genes in spermatogenesis, we genotyped the potential functional genetic variants of CYP26B1, NANOS1 and STRA8 genes, and evaluated their effects on spermatogenesis in our study population.

Design, Setting, and Participants

In this study, all subjects were volunteers from the affiliated hospitals of Nanjing Medical University between March 2004 and July 2009 (NJMU Infertile Study). Total 719 idiopathic infertile cases were recruited and divided into three groups according to WHO semen parameters: 201 azoospermia patients (no sperm in the ejaculate even after centrifugation), 155 oligozoospermia patients (sperm counts <20×10⁶/ml) and 363 infertility/normozoospermia subjects (sperm counts >20×10⁶/ml). The control group consisted of 383 subjects with normal semen parameters, all of which had fathered at least one child without assisted reproductive technologies.

Measurements

Eight single nucleotide polymorphisms (SNPs) in CYP26B1, NANOS1 and STRA8 genes were determined by TaqMan allelic discrimination assay in 719 idiopathic infertile men and 383 healthy controls.

Results and Limitations

The genetic variant rs10269148 of STRA8 gene showed higher risk of spermatogenic impairment in the groups of abnormospermia (including azoospermia subgroup and oligozoospermia subgroup) and azoospermia than the controls with odds ratios and 95% confidence intervals of 2.52 (1.29–4.94) and 2.92 (1.41–6.06), respectively (P = 0.006, 0.002 respective). Notably, larger sample size studies and in vivo or in vitro functional studies are needed to substantiate the biological roles of these variants.

Conclusions

Our results provided epidemiological evidence supporting the involvement of genetic polymorphisms of the meiotic program initiation genes in modifying the risk of azoospermia and oligozoospermia in a Han-Chinese population.