Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.     

Structural–functional insights of single and multi-domain Capsicum annuum protease inhibitors

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural–functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K_i ～ 57 μM) than -15 (～184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Use of Implantable Loop Recorders to Unravel the Cause of Unexplained Syncope

Syncope is a symptom of many underlying disease states, which range from the relatively benign to the life threatening. There are numerous investigations done for patients with recurrent unexplained syncope which may have very low yield when it comes to making a definitive diagnosis. Recently, the implantable loop recorder (ILR) for continuous monitoring of the cardiac rhythm has been launched in India. This review will briefly discuss these current availabel strategies and focus on the usefulness of an ILR in the definitive diagnosis and treatment of patients with a recurrent unexplained syncope.     

Effect of Different Light Regimes on Pre-Adult Fitness in Drosophila melanogaster Populations Reared in Constant Light for over Six Hundred Generations

Egg to eclosion development time and survivorship were assayed on four laboratory populations of Drosophila melanogaster that had been reared for over 600 generations in continuous light (LL) and constant temperature. The assays were performed in three environments: continuous light (LL), periodically varying light/dark cycles (LD 12:12 hr), and continuous darkness (DD). Development time in LL was significantly less than that in LD, which, in turn, was significantly less than that in DD, whereas survivorship did not differ significantly among the three treatments. The results indicate that individuals from Drosophila populations routinely maintained in LL do not suffer any deleterious effects of LL treatment on pre-adult fitness. Other studies on these populations have shown that free-running period (t) of the eclosion rhythm in DD is greater than that in LD. Our results are, thus, also consistent with the notion that development time may be a function of the free-running period.     

Design Maps for the Hyperthermic Treatment of Tumors with Superparamagnetic Nanoparticles

A plethora of magnetic nanoparticles has been developed and investigated under different alternating magnetic fields (AMF) for the hyperthermic treatment of malignant tissues. Yet, clinical applications of magnetic hyperthermia are sporadic, mostly due to the low energy conversion efficiency of the metallic nanoparticles and the high tissue concentrations required. Here, we study the hyperthermic performance of commercially available formulations of superparamagnetic iron oxide nanoparticles (SPIOs), with core diameter of 5, 7 and 14 nm, in terms of absolute temperature increase ΔT and specific absorption rate (SAR). These nanoparticles are operated under a broad range of AMF conditions, with frequency f varying between 0.2 and 30 MHz; field strength H ranging from 4 to 10 kA m⁻¹; and concentration c_MNP varying from 0.02 to 3.5 mg ml⁻¹. At high frequency field (∼30 MHz), non specific heating dominates and ΔT correlates with the electrical conductivity of the medium. At low frequency field (<1 MHz), non specific heating is negligible and the relaxation of the SPIO within the AMF is the sole energy source. We show that the ΔT of the medium grows linearly with c_MNP, whereas the SAR_MNP of the magnetic nanoparticles is independent of c_MNP and varies linearly with f and H². Using a computational model for heat transport in a biological tissue, the minimum requirements for local hyperthermia (T_tissue >42°C) and thermal ablation (T_tissue >50°C) are derived in terms of c_MNP, operating AMF conditions and blood perfusion. The resulting maps can be used to rationally design hyperthermic treatments and identifying the proper route of administration – systemic versus intratumor injection – depending on the magnetic and biodistribution properties of the nanoparticles.     

Bifunctional recombinant cellulase–xylanase (rBhcell-xyl) from the polyextremophilic bacterium <Emphasis Type="Italic">Bacillus halodurans</Emphasis> TSLV1 and its utility in valorization of renewable agro-residues

The thermostable bifunctional CMCase and xylanase encoding gene (rBhcell-xyl) from Bacillus halodurans TSLV1 has been expressed in Escherichia coli. The recombinant E. coli produced rBhcell-xyl (CMCase 2272 and 910 U L^?1 xylanase). The rBhcell-xyl is a ~62-kDa monomeric protein with temperature and pH optima of 60 °C and 6.0 with T_1/2 of 7.0 and 3.5 h at 80 °C for CMCase and xylanase, respectively. The apparent K _m values (CMC and Birchwood xylan) are 3.8 and 3.2 mg mL^?1. The catalytic efficiency (k _cat/K _m ) values of xylanase and CMCase are 657 and 171 mL mg^?1 min^?1, respectively. End-product analysis confirmed that rBhcell-xyl is a unique endo-acting enzyme with exoglucanase activity. The rBhcell-xyl is a GH5 family enzyme possessing single catalytic module and carbohydrate binding module. The action of rBhcell-xyl on corn cobs and wheat bran liberated reducing sugars, which can be fermented to bioethanol and fine biochemicals.