Expanding the Repertoire of Gene Tools for Precise Manipulation of the Clostridium difficile Genome: Allelic Exchange Using pyrE Alleles

Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the ‘correction’ method (5–7 days) makes pyrE ⁻ strains attractive hosts for mutagenesis studies.     

ETHNOBOTANICAL CONSERVATION STATUS OF MARGALLA HILLS NATIONAL PARK, ISLAMABAD

马加拉山国家公园自建立以来,在自然环境保护方面面临着一些问题。园内住民向来使用药用植物治疗各种疾病,长期依赖周围的植物资源提供食品、房舍、饲料、保健品和其他人文生活的需要。然而,工业化的兴起以及随之而来的人们生活方式的改变,使得利用当地植物作医药的情况不断减少。因此,有必要进行民族植物学研究,把本土植物的利用信息在丧失之前记录下来。调查结果表明,ＡｓｐａｒａｇｕｓａｄｅｓｃｅｎｄｅｎｓＲｏｘｂ．,ＢｅｒｂｅｒｉｓｌｙｃｉｕｍＲｏｙｌｅ（柯杞小檗）和ＶｉｏｌａｃａｎｅｓｃｅｎｃｅＷａｌ．ｅｘＲｏｘｂ．只能少量采集,ＡｃａｃｉａｍｏｄｅｓｔａＷａｌ．,Ａ．ｎｉｌｏｔｉｃａ（Ｌ．）Ｄｅｌｉｌｅ（阿拉伯胶金合欢）,ＢｕｘｕｓｐｉｐｉｌｏｓａＣ．Ｋ．Ｓｃｈｎｅｉｄ．和Ｄｏｄｏｎａｅａｖｉｓｃｏｓａ（Ｌ．）Ｊａｃｑ．（车桑子）等作为薪材面临砍伐的压力。而ＧｒｅｗｉａｏｐｔｉｖａＤｒｕｍ．ｅｘＢｕｒｒｅｔ作为持续利用的饲料和制绳用的种类显示良好的前景     

Microtubule-mediated Src tyrosine kinase trafficking in neuronal growth cones

Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they have been implicated in axon guidance; however, the detailed localization, trafficking, and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and we show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry, and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-enhanced green fluorescent protein–positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady-state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones, implicating Src2 in controlling the size of filopodia and lamellipodia.     

Simultaneous determination of rosuvastatin and atorvastatin in human serum using RP-HPLC/UV detection: method development, validation and optimization of various experimental parameters

A novel, precise, accurate and rapid isocratic reversed-phase high performance liquid chromatographic/ultraviolet (RP-HPLC/UV) method was developed, optimized and validated for simultaneous determination of rosuvastatin and atorvastatin in human serum using naproxen sodium as an internal standard. Effect of different experimental parameters and various particulate columns on the analysis of these analytes was evaluated. The method showed adequate separation for rosuvastatin and atorvastatin and best resolution was achieved with Brownlee analytical C18 column (150×4.6 mm, 5 μm) using methanol-water (68:32, v/v; pH adjusted to 3.0 with trifluoroacetic acid) as a mobile phase at a flow rate of 1.5 ml/min and wavelength of 241 nm. The calibration curves were linear over the concentration ranges of 2.0-256 ng/ml for rosuvastatin and 3.0-384 ng/ml for atorvastatin. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for rosuvastatin were 0.6 and 2.0 ng/ml while for atorvastatin were 1.0 and 3.0ng/ml, respectively. All the analytes were separated in less than 7.0 min. The proposed method could be applied for routine laboratory analysis of rosuvastatin and atorvastatin in human serum samples, pharmaceutical formulations, drug-drug interaction studies and pharmacokinetics studies.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

Presowing Seed Treatment with Cytokinins and Its Effect on Growth, Photosynthetic Rate, Ionic Levels and Yield of Two Wheat Cultivars Differing in Salt Tolerance

The effects of presowing seed treatment with different concentrations of cytokinins （kinetin and benzylaminopurine; 100, 150, and 200 mg/L） on growth, photosynthetic capacity, and ion homeostasis were investigated in two spring wheat （Triticum aestivum L.） cultivars, namely MH-97 （salt sensitive） and Inqlab- 91 （salt tolerant）. Primed and non-primed seeds were sown in a field in which NaC1 salinity of 15 dS/m was developed. Of the different concentrations of priming agents tested, the effect of a moderate concentration of kinetin （150 mg/L） was very pronounced, particularly in improving growth and grain yield, in both cultivars. In addition, priming with kinetin alleviated the adverse effect of salt stress on gaseous exchange characteristics （net CO2 assimilation rate and water use efficiency） in both cultivars. Seed priming with a moderate concentration of kinetin also altered the pattern of accumulation of Na^＋ and Clˉ in the shoots, irrespective of the wheat cultivar, under conditions of salt stress. However, all other priming agents at the different concentrations tested did not show any consistent effect on ion levels, except hydropriming, which increased K^＋ levels in the shoots of both cultivars under salt stress. In conclusion, a moderate concentration of kinetin showed a consistent effect in altering the growth and grain yield of both wheat cultivars, which was related to the beneficial effects of kinetin priming on water use efficiency and photosynthetic rate under conditions of salt stress.     

Investigations of anticholinestrase and antioxidant potentials of methanolic extract,subsequent fractions,crude saponins and flavonoids isolated from Isodon rugosus

Background

Based on the ethnomedicinal uses and the effective outcomes of natural products in various diseases, this study was designed to evaluate Isodon rugosus as possible remedy in oxidative stress, alzheimer’s and other neurodegenerative diseases. Acetylecholinestrase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of crude methanolic extract (Ir.Cr), resultant fractions (n-hexane (Ir.Hex), chloroform (Ir.Cf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq)), flavonoids (Ir.Flv) and crude saponins (Ir.Sp) of I. rugosus were investigated using Ellman’s spectrophotometric method. Antioxidant potential of I. rugosus was determined using DPPH, H₂O₂ and ABTS free radicals scavenging assays. Total phenolic and flavonoids contents of plant extracts were determined and expressed in mg GAE/g dry weight and mg RTE/g of dry sample respectively.

Results

Among different fractions Ir.Flv and Ir.Cf exhibited highest inhibitory activity against AChE (87.44 ± 0.51, 83.73 ± 0.64%) and BChE (82.53 ± 0.71, 88.55 ± 0.77%) enzymes at 1 mg/ml with IC₅₀ values of 45, 50 for AChE and 40, 70 μg/ml for BChE respectively. Activity of these fractions were comparable to galanthamine causing 96.00 ± 0.30 and 88.61 ± 0.43% inhibition of AChE and BChE at 1 mg/ml concentration with IC₅₀ values of 20 and 47 μg/ml respectively. In antioxidant assays, Ir.Flv, Ir.Cf, and Ir.EtAc demonstrated highest radicals scavenging activities in DPPH and H₂O₂ assays which were comparable to ascorbic acid. Ir.Flv was found most potent with IC₅₀ of 19 and 24 μg/ml against DPPH and H₂O₂ radicals respectively. Whereas antioxidant activates of plant samples against ABTS free radicals was moderate. Ir.Cf, Ir.EtAc and Ir.Cr showed high phenolic and flavonoid contents and concentrations of these compounds in different fractions correlated well to their antioxidant and anticholinestrase activities.

Conclusion

It may be inferred from the current investigations that the Ir.Sp, Ir.Flv and various fractions of I. rugosus are good sources of anticholinesterase and antioxidant compounds. Different fractions can be subjected to activity guided isolation of bioactive compounds effective in neurological disorders.     

MFG-E8 regulates the immunogenic potential of dendritic cells primed with necrotic cell-mediated inflammatory signals

Dendritic cells (DC) manipulate tissue homeostasis by recognizing dying cells and controlling immune functions. However, the precise mechanisms by which DC recognize different types of dying cells and devise distinct immunologic consequences remain largely obscure. Herein, we demonstrate that Milk-fat globule-EGF VIII (MFG-E8) is a critical mediator controlling DC immunogenicity in inflammatory microenvironments. MFG-E8 restrains DC-mediated uptake and recognition of necrotic cells. The MFG-E8-mediated suppression of necrotic cell uptake by DC resulted in the decreased proinflammatory cytokines production and activated signal components such as STAT3 and A20, which are critical to maintain tolerogenic properties of DC. Furthermore, the DC-derived MFG-E8 negatively regulates the cross-priming and effector functions of antigen-specific T cells upon recognition of necrotic cells. MFG-E8 deficiency enhances an ability of necrotic cell-primed DC to stimulate antitumor immune responses against established tumors. Our findings define what we believe to a novel mechanism whereby MFG-E8 regulates the immunogenicity of DC by modulating the modes of recognition of dying cells. Manipulating MFG-E8 levels in DC may serve as a useful strategy for controlling inflammatory microenvironments caused by various pathological conditions including cancer and autoimmunity.     

Hematologic abnormalities associated with simian immunodeficieny virus (SIV) infection mimic those in HIV infection

Background Studies of hematologic abnormalities in HIV‐infected patients are confounded by a multitude of factors. A retrospective data analysis of simian immunodeficieny virus (SIV)‐infected rhesus macaques (RM) of Indian origin was performed to determine the prevalence of hematologic abnormalities free of these confounds. Methods Hematologic data from RM inoculated with SIV and without antiviral therapy were examined pre‐inoculation, and throughout infection and the development of AIDS. Results Anemia, thrombocytopenia, lymphopenia, eosinophilia, and neutropenia all increased in prevalence with SIV infection. Significant increases in prevalence for both neutropenia and neutrophilia were also detected in SIV‐infected macaques. SIV‐infected macaques also had lower lymphocyte counts and increased prevalence of lymphopenia compared with non‐infected subjects. The prevalence of eosinophilia was significantly increased during SIV infection. Conclusions Concordance of hematologic abnormalities during SIV infection of macaques with similar changes in HIV infection of humans suggests that, like in HIV infection, hematologic abnormalities are major complications of SIV infection.