Average Rheological Quantities of Cells in Monolayers.

The method of cell monolayer rheology enables quantifying average rheological properties of cell in a single experimental run of few millions cells together in a single layer. Here we describe step-by-step procedure as to how to employ a modified commercial rotational rheometer to run rheological measurement and detect average viscoelastic properties of cells while maintaining the necessary precision level at the same time.

Shear rheology of methyl cellulose based solutions for cell mechanical measurements at high shear rates.

Methyl cellulose (MC) is a widely used material in various microfluidic applications in biology. Due to its biocompatibility, it has become a popular crowding agent for microfluidic cell deformability measurements, which usually operate at high shear rates (>10 000 s-1). However, a full rheological characterization of methyl cellulose solutions under these conditions has not yet been reported. With this study, we provide a full shear-rheological description for solutions of up to 1% MC dissolved in phosphate-buffered saline (PBS) that are commonly used in real-time deformability cytometry (RT-DC). We characterized three different MC-PBS solutions used for cell mechanical measurements in RT-DC with three different shear rheometer setups to cover a range of shear rates from 0.1-150 000 s-1. We report viscosities and normal stress differences in this regime. Viscosity functions can be well described using a Carreau-Yasuda model. Furthermore, we present the temperature dependency of shear viscosity and first normal stress difference of these solutions. Our results show that methyl cellulose solutions behave like power-law liquids in viscosity and exhibit first normal stress difference at shear rates between 5000-150 000 s-1. We construct a general viscosity equation for each MC solution at a certain shear rate and temperature. Furthermore, we investigated how MC concentration influences the rheology of the solutions and found the entanglement concentration at around 0.64 w/w%. Our results help to better understand the viscoelastic behavior of MC solutions, which can now be considered when modelling stresses in microfluidic channels.

Measuring the linear viscoelastic regime of MCF-7 cells with a monolayer rheometer in the presence of microtubule-active anti-cancer drugs at high concentrations.

The rheological properties of cells have vital functional implications. Depending, for instance, on the life cycle, cells show large cell-to-cell variations making it cumbersome to quantify average viscoelastic properties of cells by single-cell techniques. Microfluidic devices, typically working in the nonlinear viscoelastic range, allow fast analysis of single-cell deformation. Averaging over a large number of cells can also be achieved by studying them in a monolayer between rheometer discs. This technique allows applying well-established rheological standard procedures to cell rheology. It offers further advantages like studying cells in the linear viscoelastic range while quantifying cell vitality. Here, we study the applicability of the technique to rather adverse conditions, like for microtubule-active anti-cancer drugs and for a cell line with large size variation. We found a strong impact of the gap width and of normal forces on the moduli and obtained high vitality levels during the rheological study. To enable studying the impact of microtubule-active drugs on vital cells at concentrations several orders of magnitude beyond the half maximal effective concentration for cytotoxicity, we arrested the cell cycle with hydroxyurea. Irrespective of the high concentrations, we observed no clear impact of the microtubule-active drugs.

Refractance window drying of food and biological materials: Status on mechanisms, diffusion modelling and hybrid drying approach.

Refractance window (RW) dryer has an immense advantage in terms of final product quality (textural and color attributes, nutrient retention), energy consumption, and drying time over other conventional dryers. RW is a thin film drying system and a technologically evolving drying process. RW drying is an energy-efficient (re-circulation of water) short drying process as the drying of food materials occurs due to a combined mode of heat transfer conduction, radiation, and convection (hot air circulates over film). The high-quality dried product is obtained because the product temperature remains below 80 °C. RW dryer application is not only limited to drying food products, but it can also be further used for improving the gelling and emulsion properties, formation of leather and edible film, and can be used for handling high protein products, drying leafy vegetables or marine foods as this process does not change any functional properties. Due to these advantages over other drying techniques, RW drying has gained academic and industrial interest in recent years. The industrial application of this technology at large scale is becoming difficult due because of large surface area requirement for mass production. Researchers are trying to scale-up by combing this technology with others technology (Infrared, ultrasound, solar energy, and osmotic dehydration). RW dryer is now extending from the food sector to other sectors like pharmaceutical, cosmetic, pigment, edible film formation, and encapsulation. Majority of the reviews on RW drying focuses on the product quality aspects. This review paper aims to comprehend the RW drying system more mechanistically to understand better the principles, diffusion models explaining the transfer processes, and emerging novel hybrid drying approaches.

Narrow-Gap Rheometry: A Novel Method for Measuring Cell Mechanics.

The viscoelastic properties of a cell cytoskeleton contain abundant information about the state of a cell. Cells show a response to a specific environment or an administered drug through changes in their viscoelastic properties. Studies of single cells have shown that chemical agents that interact with the cytoskeleton can alter mechanical cell properties and suppress mitosis. This envisions using rheological measurements as a non-specific tool for drug development, the pharmacological screening of new drug agents, and to optimize dosage. Although there exists a number of sophisticated methods for studying mechanical properties of single cells, studying concentration dependencies is difficult and cumbersome with these methods: large cell-to-cell variations demand high repetition rates to obtain statistically significant data. Furthermore, method-induced changes in the cell mechanics cannot be excluded when working in a nonlinear viscoelastic range. To address these issues, we not only compared narrow-gap rheometry with commonly used single cell techniques, such as atomic force microscopy and microfluidic-based approaches, but we also compared existing cell monolayer studies used to estimate cell mechanical properties. This review provides insight for whether and how narrow-gap rheometer could be used as an efficient drug screening tool, which could further improve our current understanding of the mechanical issues present in the treatment of human diseases.

Heat resistant chocolate development for subtropical and tropical climates: a review.

Heat resistant chocolate (HRC) which can retain the desired texture and mouthfeel in tropical and subtropical climatic conditions has become a major research area in the chocolate industry. Liking of the chocolate products keeps on changing with the geographical conditions of the world due to the availability of ingredients from local resources and consumer's taste preferences. The geographical changes also bring about the change in climatic conditions and as such no chocolates have been formulated to withstand the hot tropical or sub-tropical temperature conditions. Textural issues and various storage related problems faced due to meltability of chocolate in different countries has opened up a broad research field of sustainable HRC manufacturing. Over the years, there are broadly three different approaches (fat modification, sugar structure modification and innovative process approach) to develop the HRC and all these scientific approaches have given different scientific insights about improving the heat resistance characteristics and textural stability of chocolate. There is a lack or coordinated fundamental and applied research related to cocoa butter polymorphism, and thermal-textural issues during product development/storage. This review paper is an attempt to describe the different scientific approaches for developing HRC and how they affect the physical/sensory chocolate attributes.

Application and toxicity studies of arabinoxylan and ß-D-glucan stearic acid ester composite coatings in extending postharvest storage of peach.

Peaches are good source of nutrients and known for their taste and aroma. The highly perishable nature of the peaches tends to decay rapidly during transportation and storage is a serious constraint for efficient transportation and storage. Therefore, the effect of arabinoxylan (AX) and ß-D-glucan stearic acid ester (SABG) composite coating material was examined for the postharvest storage quality of peach under storage at 22 ± 2 °C with 85% relative humidity (RH). Both, AX-SABG and shellac (1-2%) coatings significantly reduced the change in the quality attributes like weight loss (1.2-1.4 fold), respiration rate (1.1-1.2 fold), ripening index (1.3-1.5 fold) and firmness (1.3-1.5 fold) during 6 days storage as compared to the uncoated peaches. In addition, AX-SABG (1-2%) coating was more effective in retaining aroma volatiles and reducing disease incidence compared to shellac. Further, acute and chronic toxicological studies have shown no tissue related toxicity and mortality in mice. Our results suggest that AX-SABG as an edible coating has the potential to preserve the fruit quality during 6 days storage at 22 ± 2 °C and extend the postharvest shelf life of peach during storage.

Understanding the effect of milk composition and milking season on quality characteristics of chhana.

The quality characteristics of chhana varied due to the milk composition (cow-, buffalo-, and mixed- milk) which in turn was affected by the milking season (summer and winter). Upon heating and acidification of milk samples water holding phenomena and denatured protein association within and with other components lead to variation in both macroscale properties (color, texture, and rheology) and molecular bonding patterns (FTIR character). Yield, lightness (L* value), textural firmness, and elastic modulus of chhana increased with increasing proportion of buffalo milk in mixed milk due to higher total solids and less moisture content in both the seasons. Total protein, fat, water, and interaction between them and extent of hydrogen bonding significantly affected the rheological and textural properties of chhana samples.

Improved postharvest quality of apple (Rich Red) by composite coating based on arabinoxylan and ß-glucan stearic acid ester.

Apples (Rich Red) were coated with wheat straw arabinoxylan (AX) and ß-glucan stearic acid ester (SABG) composite coating material in the concentration range of 1-2%. The postharvest storage life of coated apples was studied at 22 °C (±2) with 65% and 85% relative humidity for 45 days. Application of both AX-SABG (1-2%) and shellac (1-2%) coatings material significantly reduced the weight loss, respiration process, color degradation, process of fruit softening and ripening index as compared to the uncoated apples up to 30 days storage. However, the AX-SABG coatings were found more effective in preventing the aroma loss, reducing microbial spoilage and maintaining sensorial attributes as compared to shellac and uncoated apples during storage.

Effect of arabinoxylan and ß-glucan stearic acid ester coatings on post-harvest quality of apple (Royal Delicious).

The effect of wheat straw arabinoxylan (AX) and ß-glucan stearic acid ester (SABG) composite coating on the quality and storage life of apple (Royal Delicious) was studied at 22 °C (±2) with relative humidity of 65% and 85% for 60 days. Fresh fruits were coated with surface coatings of AX-SABG, shellac in the concentration range of 1-4%. Application of both AX-SABG (1-4%) and shellac (1-4%) coatings was found to significantly reduce weight loss, respiration rate, fruit softening process, ripening index, color degradation and polyphenol oxidase activity compared to control during the storage period of more than 30 days. However, an AX-SABG coating was more effective in reducing fruit decay and loss of aroma volatiles followed by shellac coated apples; the un-coated apples being showing maximum quality deterioration. These findings confirmed the potential benefits of applying AX-SABG coating to extend the shelf life and quality of apples especially during transportation and storage.

A Novel Therapeutic Strategy for Cancer Using Phosphatidylserine Targeting Stearylamine-Bearing Cationic Liposomes.

There is a pressing need for a ubiquitously expressed antigen or receptor on the tumor surface for successful mitigation of the deleterious side effects of chemotherapy. Phosphatidylserine (PS), normally constrained to the intracellular surface, is exposed on the external surface of tumors and most tumorigenic cell lines. Here we report that a novel PS-targeting liposome, phosphatidylcholine-stearylamine (PC-SA), induced apoptosis and showed potent anticancer effects as a single agent against a majority of cancer cell lines. We experimentally proved that this was due to a strong affinity for and direct interaction of these liposomes with PS. Complexation of the chemotherapeutic drugs doxorubicin and camptothecin in these vesicles demonstrated a manyfold enhancement in the efficacies of the drugs both in vitro and across three advanced tumor models without any signs of toxicity. Both free and drug-loaded liposomes were maximally confined to the tumor site with low tissue concentration. These data indicate that PC-SA is a unique and promising liposome that, alone and as a combination therapy, has anticancer potential across a wide range of cancer types.

Role of microRNAs in senescence and its contribution to peripheral neuropathy in the arsenic exposed population of West Bengal, India.

Arsenic induced senescence (AIS) has been identified in the population of West Bengal, India very recently. Also there is a high incidence of arsenic induced peripheral neuropathy (PN) throughout India. However, the epigenetic regulation of AIS and its contribution in arsenic induced PN remains unexplored. We recruited seventy two arsenic exposed and forty unexposed individuals from West Bengal to evaluate the role of senescence associated miRNAs (SA-miRs) in AIS and their involvement if any, in PN. The downstream molecules of the miRNA associated with the disease outcome, was also checked by immuoblotting. In vitro studies were conducted with HEK 293 cells and sodium arsenite exposure. Our results show that all the SA-miRs were upregulated in comparison to unexposed controls. miR-29a was the most significantly altered, highest expression being in the arsenic exposed group with PN, suggesting its association with the occurrence of PN. We looked for the expression of peripheral myelin protein 22 (PMP22), a specific target of miR-29a associated with myelination and found that both in vitro and in vivo results showed over-expression of the protein. Since this was quite contrary to miRNA regulation, we checked for intermediate players ß-catenin and GSK-3ß upon arsenic exposure which affects PMP22 expression. We found that ß-catenin was upregulated in vitro and was also highest in the arsenic exposed group with PN while GSK-3ß followed the reverse pattern. Our findings suggest that arsenic exposure alters the expression of SA-miRs and the mir-29a/beta catenin/PMP22 axis might be responsible for arsenic induced PN.

Effect of ß-glucan-fatty acid esters on microstructure and physical properties of wheat straw arabinoxylan films.

Arabinoxylans (AX) was isolated from wheat straw, whereas ß-glucan (BG) was extracted from oat flour. The compositional analysis indicated wheat straw AX contained arabinose and xylose as major constituent sugars whereas higher ß-glucan content (77%) was found in the extracted material from oat flour. The BG was conjugated with lauric (LA), myristic (MA), palmitic (PA), stearic (SA) and oleic (OA) acid to prepare corresponding ß-glucan-fatty acid esters (BGFAs) with nearly similar degree of substitution. The effect of BGFAs to AX films on the water barrier, optical and mechanical properties were investigated. The addition of LABG and MABG to AX formed laminar structures in the composite films which limited water vapor permeability, giving rise to more opacity. Films prepared by blending AX with SABG and OABG were less effective as water vapor barrier due to their non-layer film microstructures; however they were less opaque. The laminar structures also imparted less mechanical strength and flexibility in the composite films. Furthermore, thermogravimetric analysis (TGA) revealed that all AX-BGFAs composite films were thermally more stable than pure AX and AX-BG films.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy.

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels.

Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation.

Amyloids are highly ordered protein/peptide aggregates associated with human diseases as well as various native biological functions. Given the diverse range of physiochemical properties of amyloids, we hypothesized that higher order amyloid self-assembly could be used for fabricating novel hydrogels for biomaterial applications. For proof of concept, we designed a series of peptides based on the high aggregation prone C-terminus of Aß42, which is associated with Alzheimer's disease. These Fmoc protected peptides self assemble to ß sheet rich nanofibrils, forming hydrogels that are thermoreversible, non-toxic and thixotropic. Mechanistic studies indicate that while hydrophobic, π-π interactions and hydrogen bonding drive amyloid network formation to form supramolecular gel structure, the exposed hydrophobic surface of amyloid fibrils may render thixotropicity to these gels. We have demonstrated the utility of these hydrogels in supporting cell attachment and spreading across a diverse range of cell types. Finally, by tuning the stiffness of these gels through modulation of peptide concentration and salt concentration these hydrogels could be used as scaffolds that can drive differentiation of mesenchymal stem cells. Taken together, our results indicate that small size, ease of custom synthesis, thixotropic nature makes these amyloid-based hydrogels ideally suited for biomaterial/nanotechnology applications.

Fluorescent probes for the detection of cyanide ions in aqueous medium: cellular uptake and assay for ß-glucosidase and hydroxynitrile lyase.

A chemodosimteric reagent (1) for the efficient detection of cyanide species (CN- and/or HCN) in aq. medium as well as under physiological conditions has been described. Selective reaction of the cyanide species with this reagent in the presence of all common interfering anions, amino acids and glutathione (GSH) led to the generation of the corresponding cyanohydrin derivative. The formation of the cyanohydrin derivative of the probe is associated with a visually detectable change in solution fluorescence in aq. buffer medium with 1.9 µM NaCN, the threshold limit set by WHO for the safe drinking water and this makes this fluorogenic sensor an ideal candidate for in-field applications. An apparent switch on the luminescence response, ultralow detection limit, low response time, cell membrane permeability and insignificant toxicity are key features of a probe molecule, which gives it a distinct edge over previously reported chemodosimetric reagents for the detection of cyanide species (CN- or HCN) in an aqueous environment. This methodology could be used for developing a generalized and efficient fluorescence-based assay for crucial enzymes like ß-glucosidase and hydroxynitrile lyase. Furthermore, spectrally-resolved fluorescence microscopy measurements on single-cells revealed that this sensor molecule could also be used for imaging the cellular uptake of cyanide species from aq. solution contaminated with NaCN. Our results confirmed that statistical analysis of integrated intensity and transition energy obtained from the emission spectra collected over various microscopic sub-cellular regions can potentially be used to discriminate the effects of local cellular environments and that due to cyanide detection.

Sensing Hg(II) in vitro and in vivo using a benzimidazole substituted BODIPY.

A multisignaling Hg(II) sensor based on a benzimidazole substituted BODIPY framework was designed, which displays excellent selectively toward Hg(II) in vitro and in vivo. Optical and fluorogenic measurements in solution reveal that the sensor can detect mercury ions at submicromolar concentrations, with high specificity. The detection of Hg(II) is associated with a blue-shift in optical spectra and a simultaneous increase in the fluorescence quantum yield of the sensor, which is attributed to a decrease in charge delocalization and inhibition of photoinduced electron transfer upon binding to Hg(II). Using several spectroscopic measurements, it is shown that the binding mechanism involves two sensor molecules, where lone pairs of the benzimidazole nitrogen coordinate to a single mercury ion. The utility of this BODIPY sensor to detect Hg(II) in vivo was demonstrated by fluorescence imaging and spectroscopy of labeled human breast adenocarcinoma cells. While average emission intensity of the sensor over a large number of cells increases with incubated mercury concentrations, spatially resolved fluorescence spectroscopy performed on individual cells reveals clear spectral blue-shifts from a subensemble of sensors, corroborating the detection of Hg(II). Interestingly, the emission spectra at various submicrometer locations within cells exhibited considerable inhomogeneity in the extent of blue-shift, which demonstrates the potential of this sensor to monitor the local (effective) concentration of mercury ions within various subcellular environments.

Association of NALP2 polymorphism with arsenic induced skin lesions and other health effects.

Prolonged consumption of arsenic-laden water above the threshold limit of 10µg/L causes a plethora of dermatological and non-dermatological multi-organ health problems, including cancer and death. Among several mechanisms of arsenic-induced toxicity and carcinogenicity studied so far, role of arsenic in impairment of immune system is less understood. Epidemiological data, animal model as well as cell line based studies have indicated that arsenic targets immune system and is associated with characteristic immunosupression, which may further adversely affect respiratory function. However, to the best of our knowledge, there is no study with respect to arsenic susceptibility investigating the role of genetic variation having immunological function. Hence, we have recruited a total of 432 arsenic-exposed individuals, of which 219 individuals with characteristic arsenic-induced skin lesions (cases) and 213 individuals without arsenic-induced skin lesion(controls), from arsenic-exposed districts of West Bengal, India. To find any probable association between arsenicism and the exonic single nucleotide polymorphisms (SNPs) in NALP2 gene, an important component of inflammasome complex, we screened the entire coding region (exon) in all the study participants. Among 9 SNPs found in NALP2 gene, the A1052E polymorphism (at least with one minor allele), was significantly overrepresented in controls and hence implies decreased risk toward the development of skin lesions [OR=0.67, 95% CI: 0.46-0.97]. Since, development of non-dermatological health effects are also important factor to properly look into, we have attempted to correlate the genetic variation of NALP2 with the extent of cytogenetic damage as measured by chromosomal aberration assay and adverse health effects including peripheral neuropathy, eye problem and respiratory diseases in the study population. We observed individuals with the protective genotype had less chromosomal aberration (p<0.05), and were also less susceptible toward arsenic-related respiratory diseases [OR=0.47; 95%CI: 0.23-0.89]. These findings suggest that NALP2 A1052E SNP plays an important role toward development of arsenic-induced skin lesions, chromosomal damage and respiratory diseases.

Quinoline driven fluorescence turn on 1,3-bis-calix[4]arene conjugate-based receptor to discriminate Fe3+ from Fe2+.

The synthesis and characterization of a triazole linked quinoline appended calix[4]arene conjugate, L, and its fluorescence turn on receptor property for Fe(3+) have been demonstrated. The selective and sensitive discrimination of Fe(3+) has been shown using fluorescence and absorption titration experiments. The Fe(3+) binding to L has been further shown by ITC and ESI MS. The mode of binding of Fe(3+) by calix[4]arene conjugate has been shown by absorption, (1)H NMR and visual color change and the species were modeled based on DFT computations. The {L + Fe(3+)} has been shown to label cells with fluorescence imaging. Moreover the utility of this conjugate has been demonstrated by the combination logic gate system.

Arsenic-induced toxicity and carcinogenicity: a two-wave cross-sectional study in arsenicosis individuals in West Bengal, India.

In the state of West Bengal in India, over 26 million individuals are exposed to arsenic via drinking water. Dermatological, non-dermatological disorders and cancers are associated with arsenic toxicity. Of late, there has been a decrease in the arsenic concentration in drinking water owing to governmental efforts, raising the possibility of remediation. A cross-sectional study was conducted, where 189 arsenicosis and 171 unexposed individuals were recruited at two time points, (2005-06 and 2010-11) with concomitant decrease in the level of arsenic exposure via drinking water in the arsenicosis group in 2010-11. Parameters studied included dermatological, non-dermatological health status and cytogenetic damage. Decrease of arsenic exposure (190.1 µg/l to 37.94 µg/l) resulted in significant decline in the number of individuals having dermatological disorders (P<0.01) and in the severity of each dermatological outcome (P<0.0001). Micronucleus formation in urothelial cells and lymphocytes decreased significantly (P<0.001). However, there was a significant (P<0.001) rise in the incidence of each of the non-dermatological diseases, that is, peripheral neuropathy, conjunctivitis and respiratory distress over the period. Thirteen (6.87%) of the initially recruited arsenicosis individuals died of cancer, in this period. Remediation by arsenic-safe drinking water can reduce dermatological manifestations and cytogenetic insult; but is unable to counter the non-dermatological symptoms.