Colorimetric dimethyl sulfide sensor using Rhodovulum sulfidophilum cells based on intrinsic pigment conversion by CrtA

A colorimetric whole-cell sensor for dimethyl sulfide (DMS) was constructed based on the in vivo conversion of intrinsic pigments in response to the analyte. In a marine bacterium, Rhodovulum sulfidophilum, carotenoids are synthesized via the spheroidene pathway. In this pathway, demethylspheroidene, a yellow carotenoid, is converted to spheroidene under catalysis of O-methyltransferase. Spheroidene monooxygenase (CrtA) catalyzes the terminal step of the pathway and converts spheroidene to spheroidenone, a red carotenoid. Here, the CrtA gene in R. sulfidophilum was removed and then reintroduced downstream of the DMS dehydrogenase gene promoter. Using this whole-cell sensor, 3 μM DMS or dimethyl sulfoxide can be detected without adding any color-forming reagent. The ratio of the red spheroidenone to total carotenoids increased, as the DMS concentration was raised to 0.3 mM. Comparison of the signal to the background color indicated a shift in the color coordinate from a yellow to a red hue. An intense signal was obtained with 1-day incubation at a high cell density when sensor cells at the exponential growth phase were used. These results show that the genetically engineered R. sulfidophilum cells can be used to monitor the quality of marine aquacultural environments by the naked eye.     

Modulation of immunogenicity of poly(sarcosine) displayed on various nanoparticle surfaces due to different physical properties

Poly(sarcosine) displayed on polymeric micelle is reported to trigger a T cell‐independent type2 reaction with B1a cells in the mice to produce IgM and IgG3 antibodies. In addition to polymeric micelle, three kinds of vesicles displaying poly(sarcosine) on surface were prepared here to evaluate the amounts and avidities of IgM and IgG3, which were produced in mice, to correlate them with physical properties of the molecular assemblies. The largest amount of IgM was produced after twice administrations of a polymeric micelle of 35 nm diameter ( G1 ). On the other hand, the production amount of IgG3 became the largest after twice administrations of G3 (vesicle of 229 nm diameter) or G4 (vesicle of 85 nm diameter). The augmented avidity of IgG3 after the twice administrations compared with that at the single administration was the highest with G3 . These differences in immune responses are discussed in terms of surface density of poly(sarcosine) chains, nanoparticle size, hydrophobic component of poly(L‐lactic acid) or (Leu‐ or Val‐Aib)_n, and membrane elasticity of the nanoparticles. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Ipragliflozin Improves Hepatic Steatosis in Obese Mice and Liver Dysfunction in Type 2 Diabetic Patients Irrespective of Body Weight Reduction

Type 2 diabetes mellitus (T2DM) is associated with a high incidence of non-alcoholic fatty liver disease (NAFLD) related to obesity and insulin resistance. Currently, medical interventions for NAFLD have focused on diet control and exercise to reduce body weight, and there is a requirement for effective pharmacological therapies. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote the urinary excretion of glucose by blocking its reabsorption in renal proximal tubules. SGLT2 inhibitors lower blood glucose independent of insulin action and are expected to reduce body weight because of urinary calorie loss. Here we show that an SGLT2 inhibitor ipragliflozin improves hepatic steatosis in high-fat diet-induced and leptin-deficient (ob/ob) obese mice irrespective of body weight reduction. In the obese mice, ipragliflozin-induced hyperphagia occurred to increase energy intake, attenuating body weight reduction with increased epididymal fat mass. There is an inverse correlation between weights of liver and epididymal fat in ipragliflozin-treated obese mice, suggesting that ipragliflozin treatment promotes normotopic fat accumulation in the epididymal fat and prevents ectopic fat accumulation in the liver. Despite increased adiposity, ipragliflozin ameliorates obesity-associated inflammation and insulin resistance in epididymal fat. Clinically, ipragliflozin improves liver dysfunction in patients with T2DM irrespective of body weight reduction. These findings provide new insight into the effects of SGLT2 inhibitors on energy homeostasis and fat accumulation and indicate their potential therapeutic efficacy in T2DM-associated hepatic steatosis.     

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.     

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.     

The relationship between muscle deoxygenation and activation in different muscles of the quadriceps during cycle ramp exercise

The relationship between muscle deoxygenation and activation was examined in three different muscles of the quadriceps during cycling ramp exercise. Seven young male adults (24 ± 3 yr; mean ± SD) pedaled at 60 rpm to exhaustion, with a work rate (WR) increase of 20 W/min. Pulmonary oxygen uptake was measured breath-by-breath, while muscle deoxygenation (HHb) and activity were measured by time-resolved near-infrared spectroscopy (NIRS) and surface electromyography (EMG), respectively, at the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM). Muscle deoxygenation was corrected for adipose tissue thickness and normalized to the amplitude of the HHb response, while EMG signals were integrated (iEMG) and normalized to the maximum iEMG determined from maximal voluntary contractions. Muscle deoxygenation and activation were then plotted as a percentage of maximal work rate (%WR(max)). The HHb response for all three muscle groups was fitted by a sigmoid function, which was determined as the best fitting model. The c/d parameter for the sigmoid fit (representing the %WR(max) at 50% of the total amplitude of the HHb response) was similar between VL (47 ± 12% WR(max)) and VM (43 ± 11% WR(max)), yet greater (P < 0.05) for RF (65 ± 13% WR(max)), demonstrating a "right shift" of the HHb response compared with VL and VM. The iEMG also showed that muscle activation of the RF muscle was lower (P < 0.05) compared with VL and VM throughout the majority of the ramp exercise, which may explain the different HHb response in RF. Therefore, these data suggest that the sigmoid function can be used to model the HHb response in different muscles of the quadriceps; however, simultaneous measures of muscle activation are also needed for the HHb response to be properly interpreted during cycle ramp exercise.     

Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.     

Peptide nanotube composed of cyclic tetra-β-peptide having polydiacetylene

A cyclic tetra-β-peptide composed of three β-alanine residues and a L-β-homolysine residue having 10,12-pentacosadiynoic amide at the side chain was synthesized. The cyclic tetra-β-peptide formed peptide nanotubes in CDCl(3) . The diacetylene units along the peptide nanotubes were polymerized by UV irradiation to obtain peptide nanotubes with polydiacetylene along the nanotube. TEM and AFM observations of the polymerized cyclic tetra-β-peptides confirmed that the peptide nanotube structure was preserved after polymerization.     

Near-infrared fluorescent labeled peptosome for application to cancer imaging

Nonionic amphiphilic copolypeptides, which were composed of hydrophilic poly(sarcosine) and hydrophobic poly(gamma-methyl L-glutamate) blocks, were synthesized with varying chain lengths of the blocks. The polypeptides having a suitable hydrophilic and hydrophobic balance were found to form vesicular assemblies of 100 nm size in buffer, which was evidenced by the TEM observation, the DLS analysis, and the encapsulation experiment. The genuine peptide vesicles, peptosomes, were labeled with a near-infrared fluorescence (NIRF) probe. In vivo retention in blood experiment showed long circulation of the peptosome in rat blood as stable as the PEGylated liposome. NIRF imaging of a small cancer on mouse by using the peptosome as a nanocarrier was successful due to the EPR effect of the peptosome. Peptosome is shown here as a novel excellent nanocarrier for molecular imaging.