Modulation of Membrane Fluidity to Control Interfacial Water Structure and Dynamics in Saturated and Unsaturated Phospholipid Vesicles.

The structure and dynamics of interfacial water in biological systems regulate the biochemical reactions. But, it is still enigmatic how the behavior of the interfacial water molecule is controlled. Here, we have investigated the effect of membrane fluidity on the structure and dynamics of interfacial water molecules in biologically relevant phopholipid vesicles. This study delineates that modulation of membrane fluidity through interlipid separation and unsaturation not only mitigate membrane rigidity but also disrupt the strong hydrogen bond (H-bond) network around the lipid bilayer interface. As a result, a disorder in H-bonding between water molecules arises several layers beyond the first hydration shell of the polar headgroup, which essentially modifies the interfacial water structure and dynamics. Furthermore, we have also provided evidence of increasing transportation through these modulated membranes, which enhance the membrane mediated isomerization reaction rate.

Selective Self-Assembly of 5-Fluorouracil through Nonlinear Solvent Response Modulates Membrane Dynamics.

Controllable self-assembly and understanding of the interaction between single metabolite fibrils and live-cell membranes have paramount importance in providing minimal treatment in several neurodegenerative disorders. Here, utilizing the nonlinear nature and peculiar hydrogen bonding behavior of the dimethyl sulfoxide (DMSO)-water mixture, the selective self-assembly of a single metabolite 5-fluorouracil (5-FU) is achieved. A direct correlation between water availability and selective self-assembly of 5-FU is ratified from the excited-state dynamics. The specific fibrillar structures of 5-FU exhibit a great potential to modulate live cell membrane fluidity and model membrane lipid distribution. After 5-FU fibril addition, a disorder of H-bonded water molecules arises several layers beyond the first hydration shell of the polar headgroups, which essentially modifies interfacial water structure and dynamics. Overall, our results shed light on the role of solvent to govern specific self-assembly and also lay the foundation accounting for the earlier stage of several diseases and multidrug resistance.

Hepatic transcriptome signature correlated with HOMA-IR explains early nonalcoholic fatty liver disease pathogenesis.

INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is multistage with heterogeneous outcomes. We studied the influence of insulin resistance (IR) on the hepatic transcriptome of early NAFLD stages, to understand disease development. MATERIALS AND METHODS: In this cross-sectional study, possible clinicopathological risk factors were compared between mild-NAFL (N = 72) and non-alcoholic steatohepatitis (NASH; N = 51) patients. Liver tissue-transcriptome difference was studied between a subset of 25 mild-NAFL and 20 NASH biopsies and validated on another subset of 12 mild-NAFL and 13 NASH biopsies, using RT-PCR. The relationship between IR driven gene expression changes with fibrosis in NASH was investigated. RESULTS: Significantly higher weight (p = 0.005) and elevated levels of HbA1c (p = 0.009), FBG (p = 0.03) and HOMA-IR (p = 0.009) were found in NASH patients. Five differentially expressed genes (DEGs, fold change > 1.5) were identified in NASH-FABP4, FABP5L2, CD24, PRAP1, and SPP1. The DEGs were positively associated with disease severity and HOMA-IR, and were found to be efficient classifiers of mild-NAFL and NASH. Additional 1218 genes identified related to IR (IrCGs), which can classify NASH-with-fibrosis patients separately from mild-NAFL and NASH patients. IrCGs can promote intra-hepatic fat accumulation, dysregulation of the lipid metabolism, lipotoxicity, and activation of cell survival pathways including activation of cell proliferation and differentiation pathways. CONCLUSIONS: Hepatic expression of genes associated with insulin resistance may drive NAFLD development and progression.

Modulation of Membrane Fluidity Performed on Model Phospholipid Membrane and Live Cell Membrane: Revealing through Spatiotemporal Approaches of FLIM, FAIM, and TRFS.

We have elucidated the role of unsaturated fatty acid in the in vitro model phospholipid membrane and in vivo live cell membrane. Fluorescence microscopy and time-resolved fluorescence spectroscopy have been employed to uncover how modulation of vesicle bilayer fluidity persuades structural transformation. This unsaturation induced structural transformation due to packing disorder in bilayer has been delineated through spatially resolved fluorescence lifetime imaging microscopy (FLIM) and fluorescence polarization or anisotropy imaging microscopy (FPIM/FAIM). Structure-function relationship of phospholipid vesicle is also investigated by monitoring intervesicular water dynamics behavior, which has been demonstrated by temporally resolved fluorescence spectroscopy (TRFS) techniques. Nevertheless, it has also been manifested from this study that loss of rigidity in bilayer breaks down the strong hydrogen bond (H-bond) network around the charged lipid head groups. The disruption of this H-bond network increases the bilayer elasticity, which helps to evolve various kinds of vesicular structure. Furthermore, the significant influence of unsaturated fatty acid on membrane bilayer has been ratified through in vivo live cell imaging.

The role of viscosity in various dynamical processes of different fluorophores in ionic liquid-cosolvent mixtures: a femtosecond fluorescence upconversion study.

Literature reports provide ample evidence of the dynamical studies of various fluorophores in different room-temperature ionic liquid (RTIL)-cosolvent mixtures. However, most of the experimental and simulation studies reveal that ∼50% of the spectral relaxation dynamics is fast and cannot be resolved using traditional time correlated single photon counting (TCSPC) measurements. Our group has also investigated the dynamics of a solvatochromic probe coumarin 153 (C153) in a RTIL-cosolvent mixture using a TCSPC setup (S. Sarkar, R. Pramanik, C. Ghatak, P. Setua and N. Sarkar, J. Phys. Chem. B, 2010, 114, 2779-2789). Consequently, a major portion of the solvation dynamics remained undetected and moreover we could not monitor the dynamics beyond 0.4 mole fraction of the cosolvents. Thus in this study, we have rekindled our interest to sufficiently capture the rotational anisotropy and solvation dynamics of C153 beyond 0.4 mole fraction of the cosolvents in the RTIL-cosolvent mixture employing femtosecond fluorescence upconversion measurements. Additionally, we have utilized another RTIL with a higher alkyl chain length and viscosity to obtain a comprehensive and quantitative picture of the role of viscosity on the dynamics of the probe molecule. The most interesting observation of the present work is that the viscosities of different RTIL-cosolvent mixtures can efficiently control the cis-trans isomerization kinetics of the anionic fluorophore merocyanine 540 (MC 540) and the translational diffusion of a hydrophobic probe. The optimization of geometrical structures of [EmimOs]- and [EmimOs]-cosolvent mixtures followed by frequency analyses in both gas and solution phases have been carried out using quantum chemical calculations with the aid of density functional theory (DFT) methods. The computation based on the bond distances, electron densities and non-covalent interactions (NCI) has also been used to investigate the existence of the hydrogen-bond (H-bond). Again to comprehend van der Waals interactions and the conventional hydrogen-bond, the evolution of NCI plots are simulated. Therefore, the detailed experimental and theoretical studies presented in this manuscript lead to the inference that addition of the conventional solvents finely tunes the physicochemical properties of RTILs and broadens their scope of applications in the fields of chemistry and biology.

Light-induced morphological transition between unconjugated bilirubin photoisomers.

Morphology switching by an external stimulus creates the possibility to detect and control the activity and functionality of bio-molecules. Unconjugated bilirubin (UCB), a waste product resulting from heme catabolism, is highly sensitive towards blue light induced configurational conversion from (4Z,15Z) to (4Z,15E)-bilirubin. UCB has a distinct elongated nanostructure which is readily photoswitchable to spherical by external blue light (470 nm) irradiation. Herein, the morphology alteration by blue light was nicely correlated with the photoisomerisation of UCB, using different microscopic and spectroscopic techniques. To restrict the other photo-incidents during phototreatment on UCB, a suitable time frame was calibrated by monitoring the absorption, HPLC, lifetime distribution and 1H NMR studies. Furthermore, by the help of this morphological transition as a marker, UCB early stage photoisomerisation has also been triggered by two-photon irradiation (940 nm).

Two-Photon Fluorescence Tracking of Colloidal Clusters.

In situ dynamics of colloidal cluster formation from nanoparticles is yet to be addressed. Using two-photon fluorescence (TPF) that has been amply used for single particle tracking, we demonstrate in situ measurement of effective three-dimensional optical trap stiffness of nanoparticles and their aggregates without using any position sensitive detector. Optical trap stiffness is an essential measure of the strength of an optical trap. TPF is a zero-background detection scheme and has excellent signal-to-noise-ratio, which can be easily extended to study the formation of colloidal cluster of nanospheres in the optical trapping regime. TPF tracking can successfully distinguish colloidal cluster from its monomer.

Precise control and measurement of solid-liquid interfacial temperature and viscosity using dual-beam femtosecond optical tweezers in the condensed phase.

We present a novel method of microrheology based on femtosecond optical tweezers, which in turn enables us to directly measure and control in situ temperature at microscale volumes at the solid-liquid interface. A noninvasive pulsed 780 nm trapped bead spontaneously responds to changes in its environment induced by a co-propagating 1560 nm pulsed laser due to mutual energy transfer between the solvent molecules and the trapped bead. Strong absorption of the hydroxyl group by the 1560 nm laser creates local heating in individual and binary mixtures of water and alcohols. "Hot Brownian motion" of the trapped polystyrene bead is reflected in the corner frequency deduced from the power spectrum. Changes in corner frequency values enable us to calculate the viscosity as well as temperature at the solid-liquid interface. We show that these experimental results can also be theoretically ratified.

Epidemiology of Liver Diseases in India.

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Endoscopic ultrasonographic diagnosis of pancreatic tuberculosis in immunocompetent patients-A case series from eastern India.

Tubercular involvement of pancreas is rare. It presents as a focal lesion of pancreas on cross sectional imaging. Endosonography with fine-needle aspiration (EUS-FNA) is crucial for a timely presurgical diagnosis. A retrospective review was conducted on 117 cases of pancreatic focal mass undergoing EUS-FNA at our institution over a period of 3 years, and 5 cases with pancreatic tuberculosis (TB) were detected. The clinical presentation varied from obstructive jaundice, recurrent acute pancreatitis to incidentaloma of pancreas. All patients received antitubercular therapy and were followed up for at least 6 months. In conclusion, pancreatic tuberculosis is a differential of a pancreatic focal lesion and EUS-FNA is the method for diagnosis of this condition that may obviate surgical exploration.

Dynamics of the vesicles composed of fatty acids and other amphiphile mixtures: unveiling the role of fatty acids as a model protocell membrane.

Fundamental research at the interface of chemistry and biology has the potential to shine light on the question of how living cells can be synthesized from inanimate matter thereby providing plausible pathways for the emergence of cellular life. Compartmentalization of different biochemical reactions within a membrane bound water environment is considered an essential first step in any origin of life pathway. It has been suggested that fatty acid-based vesicles can be considered a model protocell having the potential for change via Darwinian evolution. As such, protocell models have the potential to assist in furthering our understanding of the origin of life in the laboratory. Fatty acids, both by themselves and in mixtures with other amphiphiles, can form different self-assembled structures depending on their surroundings. Recent studies of fatty acid-based membranes have suggested likely pathways of protocell growth, division and membrane permeabilisation for the transport of different nutrients, such as nucleotides across the membrane. In this review, different dynamic processes related to the growth and division of the protocell membrane are discussed and possible pathways for transition of the protocell to the modern cell are explored. These areas of research may lead to a better understanding of the synthesis of artificial cell-like entities and thus herald the possibility of creating new form of life distinct from existing biology. Graphical Abstract Table of Content (TOC) only.

Elucidating optical field directed hierarchical self-assembly of homogenous versus heterogeneous nanoclusters with femtosecond optical tweezers.

Insights into the morphology of nanoclusters would facilitate the design of nano-devices with improved optical, electrical, and magnetic responses. We have utilized optical gradient forces for the directed self-assembly of colloidal clusters using high-repetition-rate femtosecond laser pulses to delineate their structure and dynamics. We have ratified our experiments with theoretical models derived from the Langevin equation and defined the valid ranges of applicability. Our femtosecond optical tweezer-based technique characterizes the in-situ formation of hierarchical self-assembled clusters of homomers as well as heteromers by analyzing the back focal plane displacement signal. This technique is able to efficiently distinguish between nano-particles in heterogeneous clusters and is in accordance with our theory. Herein, we report results from our technique, and also develop a model to describe the mechanism of such processes where corner frequency changes. We show how the corner frequency changes enables us to recognize the structure and dynamics of the coagulation of colloidal homogeneous and heterogeneous clusters in condensed media over a broad range of nanoparticle sizes. The methods described here are advantageous, as the backscatter position-sensitive detection probes the in-situ self-assembly process while other light scattering approaches are leveraged for the characterization of isolated clusters.

Polarization induced control of optical trap potentials in binary liquids.

We illustrate control of a polarized laser optical trapping potential landscape through the nonideal mixing of binary liquids. The inherent trapping potential asymmetry (ITPA) present in the trapping region results from the asymmetric intensity distribution in focal volume due to the high numerical aperture objective lens. Experimentally, we show that this ITPA effect can be modified and/or removed by the use of binary liquid mixtures. From our femtosecond optical tweezers experiments, we determine the topograph of the trapping potential base on the fluctuation-dissipation theorem. Additionally, the Brownian motion of the trapped bead is sensitive to the frictional force (FF) of the surroundings that is exerted by clusters of water and alcohol binary mixture through extended hydrogen bonding. Thus, using these two effects, ITPA and FF of the medium, we have shown that one can indeed modify the effective trapping potential landscape. Water-alcohol binary mixtures display a nonlinear dependence on the microrheological properties of the solvent composition as a result of rigid cluster formation. Volumetrically, at about 30% methanol in water binary mixture, the trapping asymmetry is minimal. In this particular binary mixture composition, the hydrophobic part of the methanol molecule is surrounded by 'cages' of water molecules. Enhanced H-bonding network of water molecules results in higher viscosity, which contributes to the higher frictional force. Increased viscosity decreases the degree of anisotropy due to hindered dipolar rotation. However, at higher methanol concentrations, the methanol molecules are no longer contained within the water cages and are free to move, which decrease their overall bulk viscosity. Thus, for pure solvents, experimentally measured anisotropy matches quite well with the theoretical prediction, but this fails in case of the binary mixtures due to the increased frictional force exerted by binary mixtures that result from the formation of cage-like structures.

Devulcanization of Waste Rubber and Generation of Active Sites for Silica Reinforcement.

Each year, hundreds of millions of tires are produced and ultimately disposed into nature. To address this serious environmental issue, devulcanization could be one of the sustainable solutions that still remains as one of the biggest challenges across the globe. In this work, sulfur-vulcanized natural rubber (NR) is mechanochemically devulcanized utilizing a silane-based tetrasulfide as a devulcanizing agent, and subsequently, silica (SiO2)-based rubber composites are prepared. This method not only breaks the sulfur-sulfur cross-links but also produces reactive poly(isoprene) chains to interact with silica. The silica natural rubber composites are prepared by replacing 30% fresh NR by devulcanized NR with varying contents of silica. The composites exhibit excellent mechanical properties, tear strength, abrasion resistance, and dynamic mechanical properties as compared with the fresh natural rubber silica composites. The tensile strength of devulcanized rubber-based silica composites is ∼20 MPa, and the maximum elongation strain is ∼921%. The devulcanized composites are studied in detail by chemical, mechanical, and morphological analyses. Thus, the value added by the devulcanized rubber could attract the attention of recycling community for its sustainable applications.

Effect of Microheterogeneity of Different Aqueous Binary Mixtures on the Proton Transfer Dynamics of [2,2'-Bipyridyl]-3,3'-diol: A Femtosecond Fluorescence Upconversion Study.

In this article, we have investigated the excited-state intramolecular double proton transfer dynamics of [2,2'-bipyridyl]-3,3'-diol, BP(OH)2, in three alcohol-water binary mixtures, namely, ethanol (EtOH)-water, n-propanol (PrOH)-water, tert-butyl alcohol (TBA)-water, and dimethyl sulfoxide (DMSO)-water utilizing the femtosecond fluorescence upconversion technique. We have found that in alcohol-water binary mixtures the proton transfer (PT) pathway of BP(OH)2 is sequential and the anomalous slowdown in PT dynamics is observed in mole fraction (χ) ranges χEtOH = 0.04-0.07, χEtOH = 0.23-0.28, χPrOH = 0.17-0.30, χTBA = 0.12-0.21, and χTBA = 0.40-0.46. Our study sheds light on the involvement of water network in the PT dynamics. Reduction in water accessibility due to the involvement of water molecules in cluster formation results in hindered PT dynamics, and this retardation is more for the TBA-water binary mixture compared to that for the other two mixtures. Additionally, we have found two anomalous regions for the DMSO-water binary mixture in ranges χDMSO = 0.12-0.16 and χDMSO = 0.26-0.34. However, most interestingly, beyond χDMSO = 0.40, we do not find any growth component in the femtosecond fluorescence upconversion trace, which may be due to the change in the PT mechanism from a sequential water-mediated pathway to a concerted intramolecular pathway.

On-the-Fly Calibrated Measure and Remote Control of Temperature and Viscosity at Nanoscale.

A novel on-the-fly calibration method of optical tweezers is presented, which enables in situ control and measure of absolute temperature and viscosity at nanoscale dimensions. Such noncontact measurement and control at the nanoscale are challenging as the present techniques only provide off-line measurements that do not provide absolute values. Additionally, some of the present methods have a low spatial resolution. We simultaneously apply the high temporal sensitivity of position autocorrelation and equipartition theorem to precisely measure and control in situ temperature and the corresponding microrheological property around the focal volume of the trap at high spatial resolution. The femtosecond optical tweezers (FOTs) use a single-beam high repetition rate laser for optical trapping to result in finer temperature gradients in comparison to the continuous-wave laser tweezers. Such finer temperature gradients are due to the additional nonlinear optical (NLO) phenomena occurring only at the nanoscale focal plane of the FOTs. Because NLO processes are laser peak power-dependent, they promote an effective study of physical properties occurring only at the focal plane. Using FOTs at optically benign near-infrared wavelengths, we demonstrate microrheological control and measurement in water by adding a highly absorbing yet low fluorescent dye (IR780).

A cell-penetrating peptide induces the self-reproduction of phospholipid vesicles: understanding the role of the bilayer rigidity.

Model lipid vesicles self-reproduce to generate unilamellar daughter vesicles in the presence of a cell-penetrating peptide. Fluorescence lifetime and anisotropy image analysis exquisitely capture the change in the lipid bilayer rigidity throughout the process, whereas a solvation dynamic study delineates the change in the dynamics of the encapsulated water inside the vesicles. Overall, our results provide an experimental underpinning of the role of the lipid bilayer structural changes in self-reproduction, which can exhibit great potential for interpreting the protein-membrane interaction in the emergence of life and for developing new therapeutic strategies.