Transplantation of betacellulin-transduced islets improves glucose intolerance in diabetic mice

Type 1 diabetes is an autoimmune disease caused by permanent destruction of insulin-producing pancreatic β cells and requires lifelong exogenous insulin therapy. Recently, islet transplantation has been developed, and although there have been significant advances, this approach is not widely used clinically due to the poor survival rate of the engrafted islets. We hypothesized that improving survival of engrafted islets through ex vivo genetic engineering could be a novel strategy for successful islet transplantation. We transduced islets with adenoviruses expressing betacellulin, an epidermal growth factor receptor ligand, which promotes β-cell growth and differentiation, and transplanted these islets under the renal capsule of streptozotocin-induced diabetic mice. Transplantation with betacellulin-transduced islets resulted in prolonged normoglycemia and improved glucose tolerance compared with those of control virus-transduced islets. In addition, increased microvascular density was evident in the implanted islets, concomitant with increased endothelial von Willebrand factor immunoreactivity. Finally, cultured islets transduced with betacellulin displayed increased proliferation, reduced apoptosis and enhanced glucose-stimulated insulin secretion in the presence of cytokines. These experiments suggest that transplantation with betacellulin-transduced islets extends islet survival and preserves functional islet mass, leading to a therapeutic benefit in type 1 diabetes.     

Improving Site‐Directed RNA Editing In Vitro and in Cell Culture by Chemical Modification of the GuideRNA

Adenosine‐to‐inosine deamination can be re‐addressed to user‐defined mRNAs by applying phosphothioate/2′‐methoxy‐modified guideRNAs. Dense chemical modification of the guideRNA clearly improves performance of the covalent conjugates inside the living cell. Furthermore, careful positioning of a few modifications controls editing selectivity in vitro and was exploited for the challenging repair of the Factor 5 Leiden missense mutation.     

Non‐Heme Dioxygenase Catalyzes Atypical Oxidations of 6,7‐Bicyclic Systems To Form the 6,6‐Quinolone Core of Viridicatin‐Type Fungal Alkaloids

The 6,6‐quinolone scaffold of the viridicatin‐type of fungal alkaloids are found in various quinolone alkaloids which often exhibit useful biological activities. Thus, it is of interest to identify viridicatin‐forming enzymes and understand how such alkaloids are biosynthesized. Here an Aspergillal gene cluster responsible for the biosynthesis of 4′‐methoxyviridicatin was identified. Detailed in vitro studies led to the discovery of the dioxygenase AsqJ which performs two distinct oxidations: first desaturation to form a double bond and then monooxygenation of the double bond to install an epoxide. Interestingly, the epoxidation promotes non‐enzymatic rearrangement of the 6,7‐bicyclic core of 4′‐methoxycyclopenin into the 6,6‐quinolone viridicatin scaffold to yield 4′‐methoxyviridicatin. The finding provides new insight into the biosynthesis of the viridicatin scaffold and suggests dioxygenase as a potential tool for 6,6‐quinolone synthesis by epoxidation of benzodiazepinediones.     

Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement

The widespread application of CRISPR-Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold-switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold-switch-mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering.     

Tetramethylguanidinium‐polyallylamine (Tmg‐PA): A new class of nonviral vector for efficient gene transfection

Highly toxic polyallylamine (PA) was reacted with a varying amount of a novel linker, 6‐(N,N,N′,N′‐tetramethylguanidinium chloride) hexanoic acid (Tmg‐HA), to prepare a series of tetramethylguanidinium‐PA (Tmg‐PA) polymers, which were used as vectors for gene transfection. The extent of attachment of the linker, Tmg‐HA, to the PA backbone was determined by 2,4,6‐trinitrobenzene sulfonic acid assay. The modified polymers (Tmg‐PAs), when complexed with pDNA, exhibited good condensation ability. The nanoparticles, so formed, were characterized by their size and zeta potential and were subsequently evaluated for their toxicity and transfection ability on various mammalian cells, viz., HeLa, CHO, and HEK 293 cells. Mobility shift assay revealed that on increasing the percent substitution of Tmg‐HA onto PA (from Tmg‐PA1 to Tmg‐PA6), relatively higher amounts of modified polymers were required to retard the mobility of a fixed amount of DNA. Besides, Tmg‐PA polymers provided sufficient protection (ca. 84–88%) to bound DNA against nucleases and one of the formulations, Tmg‐PA2 (ca. 15% substitution) displayed the highest transfection efficiency outcompeting the commercial transfection reagent, Lipofectamine? with minimal cytotoxicity. More impressively, the transfection efficiency increased despite recording a decrease in the buffering capacity of the grafted polymers suggesting that buffering capacity is not the sole parameter in determining the gene delivery efficiency of a vector system. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E

The bifunctional activities of α-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E. An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system. The optimum pH of the recombinant amylopullulanase was 6.0. The most stable pH for the α-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the α-amylase activity was 90°C, while its most stable temperature was 80°C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75°C, respectively. Pullulan was found to be the best substrate for the enzyme. The enzyme was activated and stabilized by the presence of Ca²⁺, whereas EDTA, N-bromosuccinimide, and α-cyclodextrin inhibited its bifunctional activities. A malto-2–4-oligosac-charide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen. A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase. Kinetic analysis of the enzyme indicated that the K _m values of α-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the V _max values were 39 and 98 μmol/(min · mg of protein), respectively.     

Analysis of longitudinal multispin orders in strongly coupled spin systems

Longitudinal multispin orders provide an effective way for measurement of scalar couplings and also to probe molecular interactions and dynamics. Analysis of longitudinal orders has been made in strongly coupled AB and ABX spin systems to determine the dependence of strong coupling parameter on these orders. Experimental and simulated spectra of various longitudinal orders are illustrated for these spin systems. This general procedure can be extended to broad range of spin systems to understand the influence of strong coupling on longitudinal orders.