New Bacillus subtilis vector, pSSß, as genetic tool for site-specific integration and excision of cloned DNA, and prophage elimination.

Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they have also been used as tools for applications in fields ranging from basic to synthetic biology. SPß is a temperate phage of the Siphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPß encodes a serine-type recombinase, SprA, and recombination directionality factor (RDF), SprB. SprA catalyzes recombination between the attachment site of the phage, attP, and that of the host, attB, to integrate phage genome into the attB site of the host genome and generate attL and attR at both ends of the prophage genome. SprB works in conjunction with SprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage. In the present study, we took advantage of this highly efficient recombination system to develop a site-specific integration and excision plasmid vector, named pSSß. It was constructed using pUC plasmid and the SSR system components, attP, sprA and sprB of SPß. pSSß was integrated into the attB site with a significantly high efficiency, and the resulting pSSß integrated strain also easily eliminated pSSß itself from the host genome by the induction of SprB expression with xylose. This report presents two applications using pSSß that are particularly suitable for gene complementation experiments and for a curing system of SPß prophage, that may serve as a model system for the removal of prophages in other bacteria.

Effects of eicosapentaenoic acid on serum levels of selenoprotein P and organ-specific insulin sensitivity in humans with dyslipidemia and type 2 diabetes.

AIM: Selenoprotein P (SeP, encoded by SELENOP in humans) is a hepatokine that causes insulin resistance in the liver and skeletal muscle. It was found that polyunsaturated fatty acid eicosapentaenoic acid (EPA) downregulates Selenop expression by inactivating SREBP-1c. The present study aimed to examine the effect of EPA for 12 weeks on circulating SeP levels and insulin sensitivity in humans with type 2 diabetes. METHODS: A total of 20 participants with dyslipidemia and type 2 diabetes were randomly assigned to an EPA (900 mg, twice daily) group and a control group. The primary endpoint was a change in serum SeP levels. Organ-specific insulin sensitivity in the liver (HGP and %HGP), skeletal muscle (Rd), and adipose tissue (FFA and %FFA) were assessed using a hyperinsulinemic-euglycemic clamp study with stable isotope-labeled glucose infusion. RESULTS: Serum SeP levels were not changed in either group at the end of the study. In the EPA group, the changes in SeP levels were positively correlated with the change in serum EPA levels (r = 0.709, P = 0.022). Treatment with EPA significantly enhanced %FFA but not %HGP and Rd. The change in serum EPA levels was significantly positively correlated with the change in %HGP, and negatively correlated with changes in Rd. CONCLUSIONS: The change in serum EPA levels was positively correlated with serum SeP levels, hepatic insulin sensitivity, and negatively with skeletal muscle insulin sensitivity in humans with type 2 diabetes. The EPA-induced enhancement of hepatic insulin sensitivity might be associated with a mechanism independent of serum SeP levels.

The clinical course and potential underlying mechanisms of everolimus-induced hyperglycemia.

The mechanistic target of rapamycin (mTOR) inhibitor everolimus is an antitumor agent known to cause hyperglycemia. However, the clinical course of everolimus-induced hyperglycemia, its pathophysiological basis, and the treatment strategy are not clear. In this case series report, we present the clinical course of everolimus-induced hyperglycemia in four patients. Hyperglycemia occurred 3-8 weeks after the administration of everolimus irrespective of the body mass index (range, 21.3-29.1 kg/m2) or pre-existing diabetes. Insulin or insulin secretagogues were required for glycemic control in most of the patients. Of note, the hyperglycemia was reversible in all patients, and none of the patients required anti-diabetic agents to achieve adequate glycemic control after cessation of everolimus therapy. To investigate the underlying mechanism of everolimus-induced hyperglycemia, we assessed insulin secretion and sensitivity by 75 g oral glucose tolerance test, arginine challenge test, and/or hyperinsulinemic-euglycemic clamp study using stable isotope-labeled glucose tracer in two patients. Everolimus did not affect insulin sensitivity in the liver, skeletal muscle, or the adipose tissue. In contrast, everolimus impaired insulin secretion and thereby increased basal hepatic glucose production. These findings further our understanding of the role of mTOR in glucose homeostasis in humans and provide insights for treatment strategies against everolimus-induced hyperglycemia.