Exosome-Mediated Differentiation of Mouse Embryonic Fibroblasts and Exocrine Cells into ß-Like Cells and the Identification of Key miRNAs for Differentiation.

Diabetes is a concerning health malady worldwide. Islet or pancreas transplantation is the only long-term treatment available; however, the scarcity of transplantable tissues hampers this approach. Therefore, new cell sources and differentiation approaches are required. Apart from the genetic- and small molecule-based approaches, exosomes could induce cellular differentiation by means of their cargo, including miRNA. We developed a chemical-based protocol to differentiate mouse embryonic fibroblasts (MEFs) into ß-like cells and employed mouse insulinoma (MIN6)-derived exosomes in the presence or absence of specific small molecules to encourage their differentiation into ß-like cells. The differentiated ß-like cells were functional and expressed pancreatic genes such as Pdx1, Nkx6.1, and insulin 1 and 2. We found that the exosome plus small molecule combination differentiated the MEFs most efficiently. Using miRNA-sequencing, we identified miR-127 and miR-709, and found that individually and in combination, the miRNAs differentiated MEFs into ß-like cells similar to the exosome treatment. We also confirmed that exocrine cells can be differentiated into ß-like cells by exosomes and the exosome-identified miRNAs. A new differentiation approach based on the use of exosome-identified miRNAs could help people afflicted with diabetes.

Improved differentiation of human adipose stem cells to insulin-producing ß-like cells using PDFGR kinase inhibitor Tyrphostin9.

Diabetes mellitus (DM) is a metabolic syndrome where insulin secretion or the response to insulin produced by the body is compromised. The only available long-term treatment is the transplantation of pancreas or islet for procuring ß-cells. However, due to the shortage of ß-cell sources from the tissues, differentiation of pluripotent stem cells or terminally differentiated cells into ß-cell is proposed as an alternative strategy. Previously, human adipose-derived stem cells (ADSCs) were reported to be converted into ß-like cells by a stepwise treatment of chemicals and growth factors. However, due to the low conversion efficiency, the clinical application was not feasible. In this study, we developed a modified conversion protocol with improved yield and functionality, which is achieved by changing the culture method and addition of Tyrphostin9, a platelet-derived growth factor receptor (PDGFR) kinase inhibitor. Tyrphostin9 was identified from a cell-based chemical screening using the mCherry reporter under the control of the Pdx1 promoter. The ß-like cells differentiated under the new protocol showed a 3.6-fold increase in the expression of Pdx1, a marker for pancreatic differentiation, as compared to the previous protocol. We propose that Tyrphostin9 contributes to the ß-like cell differentiation by playing a dual role; enhancing the definitive endoderm generation by inhibiting the PI3K signaling and suppressing the taurine-mediated proliferation of definitive endoderm. Importantly, these differentiated cells responded well to low and high glucose stimulations compared to cells differentiated by the previous protocol, as confirmed by the 2.0-fold increase in the C-peptide release. As ADSCs are abundant, easily isolated, and autologous in nature, improved differentiation approaches to generate ß-like cells from ADSCs would provide a better opportunity for treating diabetes.

Sensing of Different Human Telomeric G-Quadruplex DNA Topologies by Natural Alkaloid Allocryptopine Using Spectroscopic Techniques.

This article describes how a natural alkaloid allocryptopine (ALL) is able to differentiate two forms of biologically relevant human telomeric (htel22) G-quadruplex DNAs (GQ-DNA) depending on the presence of K+ and Na+ ions by steady-state and time-resolved spectroscopic techniques. For both interactions, predominant involvements of static-type quenching mechanism with the negligible influence of dynamic collision are established by UV-vis absorption and fluorescence emission study, which is further supported by fluorescence lifetime measurements. ALL exhibits appreciable affinity toward both GQ-DNAs. Both the mixed-hybrid (3 + 1) quadruplex structures in K+ ions and the basket-type antiparallel quadruplex structure under Na+ condition are converted to parallel types in the presence of ALL. Fluorescence intercalator displacement assay experiment revealed modest selectivity of ALL to both quadruplexes over duplex DNA along with higher selectivity for antiparallel types among the two quadruplexes via groove and/or loop binding, which is distinct from the conventional π-stacking of the ligands on external G-quartets. ALL stabilized both GQ-DNA topologies moderately. The differences in the dynamics of ALL within both DNA environments have been demonstrated vividly by time-resolved anisotropy measurements using the wobbling-in-cone model. These results suggest groove binding with antiparallel G-quartet with high affinity and moderate loop binding with mixed-hybrid G-quartet accompanied by the partial end stacking additionally in both of the cases. Our conclusions are further supported by steady-state anisotropy measurements and molecular docking. The present investigation can be used in the development of a biocompatible antitumour/anticancer agent targeting particular GQ-DNA conformation.

Complete genotyping of unusual species A rotavirus G12P[11] and G10P[14] isolates and evidence of frequent in vivo reassortment among the rotaviruses detected in children with diarrhea in Kolkata, India, during 2014.

Species A rotaviruses (RVA) are the most important cause of acute gastroenteritis in the young of humans and many animal species globally. G1P[8], G2P[4], G3P[8], G4P[8], G9P[6/8] and G12P[6/8] are the predominantly isolated genotypes throughout the world including India. Unusual genotypes from different host species such as G5, G6, G8, G10 and G11 have also been reported in humans with low frequency. In the present study, among >650 RVA positive stool samples collected from children with diarrhea in Kolkata, India, during 2014, two isolates each of the genotype G12P[11] and G10P[14] were obtained and their genomes completely sequenced. The full genotype constellations were G12-P[11]-I1-R1-C1-M2-A1-N1-T2-E1-H1 and G12-P[11]-I1-R1-C1-M1-A5-N1-T1-E1-H1 for G12P[11] viruses, suggesting several reassortments between Wa- and DS-1-like human RVA strains, including possible reassortment of a simian NSP1 gene. The G10P[14] viruses (G10-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) were found to contain multiple genes closely related to RVAs of artiodactyl origin, highlighting the role of inter-host species transmissions of RVAs. From the G/P constellation of all RVA isolates, it could be concluded that approximately one quarter had likely arisen from reassortment events in vivo among RVAs of 'usual' genotypes.

Hospital based surveillance and genetic characterization of rotavirus strains in children (<5 years) with acute gastroenteritis in Kolkata, India, revealed resurgence of G9 and G2 genotypes during 2011-2013.

INTRODUCTION: India accounts for an estimated 457,000-884,000 hospitalizations and 2 million outpatient visits for diarrhea. In spite of the huge burden of rotavirus (RV) disease, RV vaccines have not been introduced in national immunization programme of India. Therefore, continuous surveillance for prevalence and monitoring of the circulating genotypes is needed to assess the disease burden prior to introduction of vaccines in this region. METHODS: During January 2011 through December 2013, 830 and 1000 stool samples were collected from hospitalized and out-patient department (OPD) patients, respectively, in two hospitals in Kolkata, Eastern India. After primary screening, the G-P typing was done by multiplex semi-nested PCR using type specific primers followed by sequencing. Phylogenetic analysis for the VP7 gene of 25 representative strains was done. RESULTS: Among hospitalized and OPD patients, 53.4% and 47.5% cases were positive for rotaviruses, respectively. Unlike previous studies where G1 was predominant, in hospitalized cases G9 rotavirus strains were most prevalent (40%), followed by G2 (39.6%) whereas G1 and G12 occurred at 16.4% and 5.6% frequency. In OPD cases, the most prevalent strain was G2 (40.3%), followed by G1, G9 and G12 at 25.5%, 22.8%, 9.3%, respectively. Phylogenetically the G1, G2 and G9 strains from Kolkata did not cluster with corresponding genotypes of Rotarix, RotaTeq and Rotavac (116E) vaccine strains. CONCLUSION: The study highlights the high prevalence of RV in children with gastroenteritis in Kolkata. The circulating genotypes have changed over the time with predominance of G9 and G2 strains during 2011-2013. The current G2, G9 and G1 Kolkata strains shared low amino acid homologies with current vaccine strains. Although there is substantial evidence for cross protection of vaccines against a variety of strains, still the strain variation should be monitored post vaccine introduction to determine if it has any impact on vaccine effectiveness.

Formation of oligomers in the early phase of pH-induced aggregation of the Alzheimer Aß(12-28) peptide [corrected].

The early phase in the aggregation process of the Alzheimer's peptide Aß(12-28) with both protected and unprotected ends was studied by time-resolved infrared spectroscopy and circular dichroism spectroscopy. Aggregation in the time-resolved experiments was initiated by a rapid pH drop caused by the photolysis of 1-(2-nitrophenyl)ethyl sulfate (caged sulfate). The infrared spectra indicate aggregates from both versions of the Aß(12-28) peptide. [corrected] They form fast (within 60 ms), presumably from initial aggregates, and their spectral signature is consistent with a ß-barrel structure. The other type arises relatively slowly from unstructured monomers on the seconds-to-minutes time scale and forms at lower pH than the first type. These ß sheets are antiparallel, planar, and large and show an absorption band at 1622 cm(-1) that shifts to 1617 cm(-1) in 12 min with most of the shift occurring in 10 s.

Spectroscopic investigations to reveal the nature of interactions between the haem protein myoglobin and the dye rhodamine 6G.

In the present investigation, steady-state and time-resolved fluorescence with the combination of circular dichroism (CD) spectroscopic techniques were applied to study the interactions of the well-known dye rhodamine 6 G (R6G) with the haem protein human myoglobin (Mb). From the analysis of the results it appears that the static type of fluorescence quenching mechanism is primarily involved, due to ground-state interactions. Although considerable overlapping of fluorescence emission of the dye R6G with the absorption of Mb in the Q-band region exists, the possibility of occurrences of the excitational singlet-singlet non-radiative energy transfer process from R6G to Mb appears to be unlikely, according to time-resolved fluorescence measurements. From the determinations of the thermodynamic parameters, it was apparent that the combined effect of van der Waals' interactions and hydrogen bonding plays a vital role in Mb-R6G interactions. Induced circular dichroism (ICD) studies demonstrate the possibility of interactions between R6G and Mb. The binding constants, number of binding sites and thermodynamic parameters have been computed. From CD measurements it is apparent that the binding of the dye R6G with the haem protein Mb induces negligible conformational changes in the protein and Mb retains its secondary structure and helicity when it interacts with R6G. The present detailed studies on the interactions with Mb should be helpful in further advancement of medical diagnostics and biotechnology.

Investigations on the photoreactions of phenothiazine and phenoxazine in presence of 9-cyanoanthracene by using steady state and time resolved spectroscopic techniques.

By using electrochemical, steady state and time resolved (fluorescence lifetime and transient absorption) spectroscopic techniques, detailed investigations were made to reveal the mechanisms of charge separation or forward electron transfer reactions within the electron donor phenothiazine (PTZH) or phenoxazine (PXZH) and well known electron acceptor 9-cyanoanthracene (CNA). The transient absorption spectra suggest that the charge separated species formed in the excited singlet state resulted from intermolecular photoinduced electron transfer reactions within the donor PTZH (or PXZH) and CNA acceptor relaxes to the corresponding triplet state. Though alternative mechanisms of via formations of contact neutral radical by H-transfer reaction have been proposed but the observed results obtained from the time resolved measurements indicate that the regeneration of ground state reactants is primarily responsible due to direct recombination of triplet contact ion-pair (CIP) or solvent-separated ion-pair (SSIP).

A detailed spectroscopic study on the interaction of Rhodamine 6G with human hemoglobin.

UV-vis, time-resolved fluorescence and circular dichroism spectroscopic investigations have been made to reveal the nature of the interactions between xanthene dye Rhodamine 6G and the well known protein hemoglobin. From the analysis of the steady-state and time-resolved fluorescence quenching of Rhodamine 6G in aqueous solutions in presence of hemoglobin, it is revealed that the quenching is static in nature. The primary binding pattern between Rhodamine and hemoglobin has been interpreted as combined effect of hydrophobic association and electrostatic interaction. The binding constants, number of binding sites and thermodynamic parameters at various pH of the environment have been computed. The binding average distance between the energy donor Rhodamine and acceptor hemoglobin has been determined from the Forster's theory.

Fluorescence spectroscopic characterization of the interaction of human adult hemoglobin and two isatins, 1-methylisatin and 1-phenylisatin: a comparative study.

In this report, steady state and time-resolved fluorescence along with circular dichroism (CD) spectroscopic, FTIR, and anisotropy investigations were made to reveal the nature of the interactions between human adult hemoglobin (Hb) and the isatins, 1-methylisatin (1-MI) and 1-phenylisatin (1-PI). From the analysis of the steady state and time-resolved fluorescence quenching of Hb in aqueous solution in the presence of an isatin, i.e., 1-MI, it seemed that the nature of the quenching was of static type and a mixture of both static and dynamic nature for 1-PI. The primary binding pattern between isatins and Hb has been interpreted as a combined effect of hydrophobic association and electrostatic interaction for 1-MI. For 1-PI, this was the combined effect of hydrophobic association and ionic interactions and salt bridges or/and proton transfer. The pretwisted structure of 1-PI facilitates ionic interactions with Hb. The binding constants, number of binding sites, and thermodynamic parameters had been computed. The binding average distances between the Hb-1-MI and Hb-1-PI determined from Forster's theory were found to be 4.02 and 5.28 nm, respectively. CD, steady state, and time-resolved anisotropy measurements had been done in support.

Does bimolecular charge recombination in highly exergonic electron transfer afford the triplet excited state or the ground state of a photosensitizer?

The investigations were made on photoinduced electron transfer (ET) from the singlet excited state of rubrene (1RU*) to p-benzoquinone derivatives (duroquinone, 2,5-dimethyl-p-benzoquinone, p-benzoquinone, 2,5-dichloro-p-benzoquinone, and p-chloranil) in benzonitrile (PhCN) by using the steady state and time-resolved spectroscopies. The photoinduced ET produces solvent-separated type charge-separated (CS) species and the charge-recombination (CR) process between RU radical cation and semiquinone radical anions obeys second-order kinetics. Not only the CS species but also the triplet excited state of RU (3RU*) is seen in the transient absorption spectra upon laser excitation of a PhCN solution of RU and p-benzoquinone derivatives. The comparison of their time profiles clearly suggests that the CR process between RU radical cation and semiquinone radical anions to the ground state is independent from the deactivation of 3RU*. This indicates that the CR in a highly exergonic ET occurs at a longer distance with a large solvent reorganization energy, which results in faster ET to the ground state than to the triplet excited state that is lower in energy than the CS state. Photoinduced ET from 3RU* in addition from 1RU* also occurs when p-benzoquinone derivatives with electron-withdrawing substituents were employed as electron acceptors.

Effects of liquid crystal environment on the spectroscopic and photophysical properties of well-known reacting systems 2,3-dimethylindole (DMI) and 9-cyanoanthracene (9CNA).

Electrochemical and steady-state and time-resolved spectroscopic studies on a disubstituted indole, 2,3-dimethylindole (DMI), and well-known electron acceptor 9-cyanoanthracene (9CNA) in liquid crystal (LC) 4-(n- pentyl)-4'-cyanobiphenyl (5CB) environment demonstrate entirely different spectroscopic and photophysical behaviors from those observed earlier by our research group with the same reacting systems in isotropic media n-heptane and acetonitrile (ACN). From the UV-vis absorption spectral measurements of the donor DMI in the presence of the acceptor 9CNA in liquid crystal medium (in 5CB) in various temperatures above the nematic-isotropic phase transition from 308 to 313 K (pseudo-ordered domain), it was observed that the lower energy lying absorption band of DMI situates in a longer wavelength region than the corresponding band observed in isotropic medium n-heptane or ACN. The possibility of the photochromic effect is discussed. In this band, the degree of mixing of the two closely spaced electronic states (1)L(a) (S(2)) and (1)L(b) (S(1)) of DMI was very prominent in the ordered LC environment (5CB) whereas in isotropic medium the dominant contribution for the formation of the lower energy band system primarily originates from the (1)Lb (S(1)) state, as evidenced from the steady-state polarization measurements. Both steady-state fluorescence quenching and time-resolved fluorescence studies clearly demonstrate in favor of the presence of only the static mode in LC environment. The situation differs in isotropic media where the dynamic process possesses the key role in the quenching mechanism. Expectedly, the transient absorption measurements by the nanosecond laser flash photolysis technique show a lack of formation of transient ionic species in the pseudo-ordered domain of 5CB. On the contrary, in isotropic solvents n-heptane and ACN, the transient absorption spectra measured by the same nanosecond laser flash photolysis technique exhibit the broad band of 9CNA radical anion at around 560 nm (9CNA-) and the band of neutral radical of DMI at 540 nm. It is inferred that the charge-separation reactions occurring within the present intermolecular systems could be stopped significantly by changing the nature of the environment from the isotropic to the LC's pseudo-ordered domain which situates closely above the nematic (N)-isotropic (I) phase transition temperature. From the steady-state and time-resolved investigations, it is revealed that, due to the hysteresis phenomenon, the nematic phase properties persist over a wide temperature range well within pseudo-ordered domain to some extent into the isotropic phases. The investigations with the different donor-acceptor inter- and intramolecular systems in 5CB and some other LC's environment are underway.

Photophysical processes involved within the bichromophoric system 9-benzotriazole-1-ylmethyl-9H-carbazole and its role as an artificial photosynthetic device.

From both steady state and fluorescence lifetime measurements it reveals that due to photoexcitation of benzotriazole (BZ) part of the bichromophore, 9(1-H-benzotriazole-lylmethyl)-9H-carbazole (BHC), singlet-singlet energy transfer takes place to populate the lowest excited singlet of carbazole (CZ). CZ, thus being excited indirectly via energy transfer process, undergoes strong charge transfer (CT) reaction with the surrounding polar medium acetonitrile (ACN). On the other hand, very weak CT band was apparent when CZ part, within BHC, was directly excited. In less polar tetrahydrofuran (THF) and polar benzonitrile (BN) environment, lack of formation of CT band strongly suggests in favor of the electron-accepting behavior of ACN. Moreover, by measuring the emission spectra of BHC in microcrystals and of 30 bilayers mixed LB film at high mole fraction of BHC molecules, the possibility of excimer formation or aggregation has been ruled out. Thus, BHC, when dissolved in ACN, acts as a triad system of BZ-CZ-ACN where BZ acts as an antenna molecule and CZ as a reaction center. The possible role of the bichromophoric system BHC as an artificial photosynthetic or solar energy conversion device has been hinted.