Construction of expression vectors carrying mouse peroxisomal protein gene (PeP) with GST and Flag labels

The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in chimeric cDNA types, encompassingGST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression vector in order to label this gene with GST to purify PEP protein for further biochemical analysis and identifying related proteins thereafter. FLAG-PEP recombinant DNA was produced and sub-cloned inpUcD3 eukaryotic expression vector to express tagged-PEP protein for transient transfection analysis and identifying intracellular localization of PEP protein in future experiments. PEP-cDNA was amplifiedin different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ends of PEP. PCR products with BamHI/SalI restriction sites were treated by restriction enzymes and inserted into the pGEX6p2, downstream of GST tag. PEP-cDNA containingBamHI/ApaI restriction sites and FLAG gene (which amplified using pUcD3-FLAG-PEX3 vector) were used as templates in secondary PCR for amplifying FLAG-PEP recombinant DNA. FLAG-PEP fragment was treated by enzymatic digestion and inserted into the pUcD3 eukaryotic expression vector.pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively. Positive colonies were selected for plasmid preparation. Results confirmed correct amplification of the expected products. PEP-cDNA in both PCRreactions encompasses 630 bp. FLAG fragment containing designed sites was 77 bp and FLAG-PEP fragment was 700 bp. Sequencing of constructed vectors confirmed that PEP-cDNA was tagged appropriately and inserted free of mutation and in frame with GST and FLAG

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Species and tissue-specificity of prokinetic, laxative and spasmodic effects of Fumaria parviflora

<p>Abstract</p> <p>Background</p> <p><it>Fumaria parviflora </it>Linn. (<it>Fumariaceae</it>), is a small branched annual herb found in many parts of the world including Saudi Arabia and Pakistan. This study was designed to provide pharmacological basis for the medicinal use of <it>Fumaria parviflora </it>in gut motility disorders.</p> <p>Methods</p> <p>The <it>in-vivo </it>prokinetic and laxative assays were conducted in mice. Isolated intestinal preparations (ileum and jejunum) from different animal species (mouse, guinea-pig and rabbit) were separately suspended in tissue baths containing Tyrode's solution bubbled with carbogen and maintained at 37°C. The spasmogenic responses were recorded using isotonic transducers coupled with PowerLab data acquisition system.</p> <p>Results</p> <p>The aqueous-methanol extract of <it>Fumaria parviflora </it>(Fp.Cr), which tested positive for the presence of alkaloids, saponins, tannins and anthraquinones showed partially atropine-sensitive prokinetic and laxative activities in the <it>in-vivo </it>in mice at 30 and 100 mg/kg. In the <it>in-vitro </it>studies, Fp.Cr (0.01-1 mg/ml) caused a concentration-dependent atropine-sensitive stimulatory effect both in mouse tissues (jejunum and ileum), and rabbit jejunum but had no effect in rabbit ileum. In guinea-pig tissues (ileum and jejunum), the crude extract showed a concentration-dependent stimulatory effect with higher efficacy in ileum and the effect was partially blocked by atropine, indicating the involvement of more than one types of gut-stimulant components (atropine-sensitive and insensitive). This could be a plausible reason for the greater efficacy of Fp.Cr in gut preparations of guinea-pig than in rabbit or mouse.</p> <p>Conclusions</p> <p>This study shows the prokinetic, laxative and spasmodic effects of the plant extract partially mediated through cholinergic pathways with species and tissue-selectivity, and provides a sound rationale for the medicinal use of <it>Fumaria parviflora </it>in gut motility disorders such as, indigestion and constipation. This study also suggests using different species to know better picture of pharmacological profile of the test material.</p

Habitat use at fine spatial scale: how does patch clustering criteria explain the use of meadows by red deer ?

Large mammalian herbivores are keystone species in different ecosystems. To mediate the effects of large mammalian herbivores on ecosystems, it is crucial to understand their habitat selection pattern. At finer scales, herbivore patch selection depends strongly on plant community traits and therefore its understanding is constrained by patch definition criteria. Our aim was to assess which criteria for patch definition best explained use of meadows by wild, free-ranging, red deer (Cervus elaphus) in a study area in Northeast Portugal. We used two clustering criteria types based on floristic composition and gross forage classes, respectively. For the floristic criteria, phytosociological approach was used to classify plant communities, and its objectivity evaluated with a mathematical clustering of the floristic relevés. Cover of dominant plant species was tested as a proxy for the phytosociological method. For the gross forage classes, the graminoids/forbs ratio and the percentage cover of legumes were used. For assessing deer relative use of meadows we used faecal accumulation rates. Patches clustered according to floristic classification better explained selection of patches by deer. Plant community classifications based on phytosociology, or proxies of this, used for characterizing meadow patches resulted useful to understand herbivore selection pattern at fine scales and thus potentially suitable to assist wildlife management decisions

Gene expression profiling associated with the progression to poorly differentiated thyroid carcinomas

Poorly differentiated thyroid carcinomas (PDTC) represent a heterogeneous, aggressive entity, presenting features that suggest a progression from well-differentiated carcinomas. To elucidate the mechanisms underlying such progression and identify novel therapeutic targets, we assessed the genome-wide expression in normal and tumour thyroid tissues.info:eu-repo/semantics/publishe

Type I IFN and TNFα cross-regulation in immune-mediated inflammatory disease: basic concepts and clinical relevance

A cross-regulation between type I IFN and TNFα has been proposed recently, where both cytokines are hypothesized to counteract each other. According to this model, different autoimmune diseases can be viewed as disequilibrium between both cytokines. As this model may have important clinical implications, the present review summarizes and discusses the currently available clinical evidence arguing for or against the proposed cross-regulation between TNFα and type I IFN. In addition, we review how this cross-regulation works at the cellular and molecular levels. Finally, we discuss the clinical relevance of this proposed cross-regulation for biological therapies such as type I IFN or anti-TNFα treatment

Somatostatin analogues in the treatment of gastroenteropancreatic neuroendocrine tumours, current aspects and new perspectives

Gastroenteropancreatic neuroendocrine tumours (GEP NETs) are rare tumours that present many clinical features