Electric field-mediated adhesive dynamics of cells inside bio-functionalised microchannels offers important cues for active control of cell-substrate adhesion.

Adhesive dynamics of cells plays a critical role in determining different biophysical processes orchestrating health and disease in living systems. While the rolling of cells on functionalised substrates having similarity with biophysical pathways appears to be extensively discussed in the literature, the effect of an external stimulus in the form of an electric field on the same remains underemphasized. Here, we bring out the interplay of fluid shear and electric field on the rolling dynamics of adhesive cells in biofunctionalised micro-confinements. Our experimental results portray that an electric field, even restricted to low strengths within the physiologically relevant regimes, can significantly influence the cell adhesion dynamics. We quantify the electric field-mediated adhesive dynamics of the cells in terms of two key parameters, namely, the voltage-altered rolling velocity and the frequency of adhesion. The effect of the directionality of the electric field with respect to the flow direction is also analysed by studying cellular migration with electrical effects acting both along and against the flow. Our experiment, on one hand, demonstrates the importance of collagen functionalisation in the adhesive dynamics of cells through micro channels, while on the other hand, it reveals how the presence of an axial electric field can lead to significant alteration in the kinetic rate of bond breakage, thereby modifying the degree of cell-substrate adhesion and quantifying in terms of the adhesion frequency of the cells. Proceeding further forward, we offer a simple theoretical explanation towards deriving the kinetics of cellular bonding in the presence of an electric field, which corroborates favourably with our experimental outcome. These findings are likely to offer fundamental insights into the possibilities of local control of cellular adhesion via electric field mediated interactions, bearing critical implications in a wide variety of medical conditions ranging from wound healing to cancer metastasis.

Magnolol induces cytotoxic autophagy in glioma by inhibiting PI3K/AKT/mTOR signaling.

Glioma is difficult-to-treat because of its infiltrative nature and the presence of the blood-brain barrier. Temozolomide is the only FDA-approved drug for its management. Therefore, finding a novel chemotherapeutic agent for glioma is of utmost importance. Magnolol, a neolignan, has been known for its apoptotic role in glioma. In this work, we have explored a novel anti-glioma mechanism of Magnolol associated with its role in autophagy modulation. We found increased expression levels of Beclin-1, Atg5-Atg12, and LC3-II and lower p62 expression in Magnolol-treated glioma cells. PI3K/AKT/mTOR pathway proteins were also downregulated in Magnolol-treated glioma cells. Next, we treated the glioma cells with Insulin, a stimulator of PI3K/AKT/mTOR signaling, to confirm that Magnolol induced autophagy by inhibiting this pathway. Insulin reversed the effect on Magnolol-mediated autophagy induction. We also established the same in in vivo glioma model where Magnolol showed an anti-glioma effect by inducing autophagy. To confirm the cytotoxic effect of Magnolol-induced autophagy, we used Chloroquine, a late-stage autophagy inhibitor. Chloroquine efficiently reversed the anti-glioma effects of Magnolol both in vitro and in vivo. Our study revealed the cytotoxic effect of Magnolol-induced autophagy in glioma, which was not previously reported. Additionally, Magnolol showed no toxicity in non-cancerous cell lines as well as rat organs. Thus, we concluded that Magnolol is an excellent candidate for developing new therapeutic strategies for glioma management.

Oral Delivery of Nanoparticles Carrying Ancestral Uricase Enzyme Protects against Hyperuricemia in Knockout Mice.

The therapeutic value of delivering recombinant uricase to human patients has been appreciated for decades. The development of therapeutic uricases has been hampered by the fact that humans do not encode an endogenous uricase and therefore most recombinant forms of the protein are recognized as foreign by the immune system and are therefore highly immunogenic. In order to both shield and stabilize the active enzyme, we encapsulated a functional ancestral uricase in recombinant, noninfectious Qß capsid nanoparticles and characterized its catalytic activity. Oral delivery of the nanoparticles moderated key symptoms of kidney dysfunction in uricase-knockout mice by lowering uric acid levels. Histological kidney samples of the treated mice suggest that delivery of recombinant uricase had a protective effect against the destructive effects of uric acid that lead to renal failure caused by hyperuricemia.

Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor.

Enzyme activity requires sequential binding and chemical transformation of substrates. While directed evolution and random mutagenesis are common methods for improving catalytic activity, these methods do not allow for independent control of KM and kcat. To achieve such control, we envisioned that the colocalization of aptamers and enzymes that act on the same molecule could increase catalytic efficiency through preconcentration of substrate. We explored this concept with cocaine esterase and anticocaine aptamers having varying KD values, both encapsulated in MS2 virus-like particles. Rate enhancements were observed with magnitudes dependent on both aptamer:enzyme stoichiometry and aptamer KD, peaking when aptamer KD and enzyme KM were roughly equivalent. This beneficial effect was lost when either aptamer binding was too tight or the aptamers were not constrained to be close to the catalyst. This work demonstrates a modular way to enhance catalysis by independently controlling substrate capture and release to the processing enzyme.

Design, characterization and evaluation of a new 99mTc-labeled folate derivative with affinity towards folate receptor.

Folate receptors (FRs) are known to be over-expressed in several human malignancies and therefore serve as an important target for small radiolabeled folate derivatives for non-invasive imaging of tumor, which is an important tool for future treatment recourse. In the present article, we report the synthesis of a new 99mTc-labeled radiotracer for the aforementioned application following the well-established 99mTc-'4+1' chemistry. Formation of the desired [99mTc]Tc-complex with >95% radiochemical purity was confirmed by radio-HPLC and its structure was ascertained by characterizing a natural rhenium analogue of the said complex. Although the ligand exhibited a weaker affinity towards FRs compared to native folic acid (IC50 8.09 µM vs 29.46 nM), the 99mTc-labeled complex was found to bind folate receptor-positive KB cells with high specificity (∼90%). Similar studies in a folate receptor negative cell line viz. A549 further corroborated the receptor-specificity of the synthesized complex. In vivo studies in KB tumor xenograft showed moderate uptake of ∼2.6% upto 3 h post-injection with high specificity (∼80%). The favorable features observed warrant further screening of the current design towards achieving an improved molecular probe for the said application.

CsPbI3/N-GQDs dual layer phosphor-converted white-LEDs with ultrahigh luminous efficiency and color rendering index.

Phosphor-converted LEDs or pc-LEDs, as a solid-state lighting source, are attractive for next-generation display technologies because of their energy savings, and green environmentally friendly nature. Recently, white LEDs are being produced commercially by coating blue LED (440-470 nm) chips with various yellow-emitting phosphors. However, the LEDs produced by this technique often exhibit high correlated color temperature (CCT) and low color rendering index (CRI) values, due to sufficient red spectral components not being present, and thus aren't suitable for commercial grade white illumination. To circumvent this drawback, our work reports for the first time the use of blue and green-emitting nitrogen-functionalized graphene quantum dots (GQDs) coupled with red-emitting CsPbI3NCs for phosphor-based LED applications. We deployed near-UV to visible excitable red-emitting perovskite CsPbI3nanocrystals which contribute toward the red spectral component, thus greatly improving the CRI of the LEDs. CsPbI3nanocrystals are optically excited by nitrogen-functionalized GQD with blue and green emissions in a remote double-layer phosphor stack technique. This double phosphor layer stacking greatly improves both the CRI and luminous efficiency of radiation (LER), which usually has a trade-off in previously reported phosphor stacks. A CCT of ∼5182 K providing daylight white tonality, with superior CRI (∼90%) and ultrahigh LER (∼250 lumens/watt) are reported, which are significantly higher than the established benchmarks.

Transport of Molecular Cargo by Interaction with Virus-Like Particle RNA.

Virus-like particles (VLPs) derived from Leviviridae virions contain substantial amounts of cellular and plasmid-derived RNA. This encapsidated polynucleotide serves as a reservoir for the efficient binding of the intercalating dye thiazole orange (TO). Polyethylene glycol (PEG) molecules and oligopeptides of varying length, end-functionalized with TO, were loaded into VLPs up to approximately 50 % of the mass of the capsid protein (hundreds to thousands of cargo molecules per particle, depending on size). The kinetics of TO-PEG binding included a significant entropic cost for the reptation of long chains through the capsid pores. Cargo molecules were released over periods of 20-120 hours following simple reversible first-order kinetics in most cases. These observations define a simple general method for the noncovalent packaging, and subsequent release, of functional molecules inside nucleoprotein nanocages in a manner independent of modifications to the capsid protein.

Hand-drawn electrode based disposable paper chip for artificial sweat analysis using impedance spectroscopy.

Low cost, disposable paper based electrical sensor to examine the analyte concentration in an extremely small volume of sample solution is essential for environmental and healthcare applications. For the development of paper based devices, sophisticated instruments are essential to pattern electrode on the top surface of the paper. In most cases, such fabricated device results in direct contact with the analyte solution on the surface of the electrode during electrical detection and leads to high electrical double layer capacitance. In this work, we have focused to reduce the double layer capacitance by fabricating hand drawn electrode paper sensor utilising the reverse side of the paper. This design acts as a sample storage and facilitate impedimetric sensing of ionic concentration of analyte solution using a few microlitre. Droplet formation at the bottom of the paper in the confined area is visually monitored to reduce sample wastage. The interaction between two different electrode materials (graphite and silver) on the paper substrate with the different volume and concentration of the electrolyte is analysed to improve the robustness and sensitivity of the measurement. Simultaneously, we observed a reduction in the electrical double layer effect on the low sample volumes. The proposed paper based sensor shows the enhanced impedance stability on silver electrode patterned paper chip than graphite electrode paper chip to detect the different ionic concentration of artificial sweat sample. Finally, it demonstrates that paper chip has great potential as a disposable diagnostics sensor in healthcare applications.

Ethyl Cellulose-Assisted Host-Guest Framework for Depositing Solution-Processed Yttria Films on the Inner Surface of a Narrow Quartz Tube.

Straightforward deposition protocols to coat flat surfaces are widely available. However, there are multiple constraints in coating a concave or convex surface, especially on the inner surface of narrow tubes. Coated surface helps in corrosion protection, internal cleanliness, strength, and alloy casting, and it also enhances product aesthetics. In the present work, a solution-based deposition protocol was developed to coat oxide films (Y2O3, Al2O3 among others) of tunable thickness (400 nm to 4 µm) on the inner surface of quartz tubes (inner diameter (ID) ∼ 2, 3, 5, 6, and 10 mm; length (L) ∼ 20, 110, and 500 mm) with the help of a venturimeter-based apparatus. In the course of this study, it was revealed that coating on the curved surface needed substantial optimization of the deposition parameters to minimize mainly the tearing and thinning of the film. Choice of organic solvents, acetic acid, precursor concentrations, and solution containing a binder element, such as ethyl cellulose (EC), was optimized to achieve homogeneous coating. An optimal upward air flow (speed 44 m/min) was maintained during drying the coating to prevent solvent condensation prior to annealing the film at 500-1000 °C in air for 30 min. The coating was studied with X-ray diffractometry (XRD), atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDAX), and Raman spectroscopy. These coated tubes were used as a mold during injection casting of Ni rod at 1450 °C. Surface of the cast Ni was studied for Si as well as yttrium contaminations with EDAX. Raman spectra from a demolded quartz tube (retrieved from casting chamber) revealed characteristic Ag and Fg vibrational modes of cubic Y2O3 phase, indicating good thermal stability and adhesive features of the present coating.

Kinetics of protein aggregation at a temperature gradient condition.

The unconventional multi-sigmoidal kinetic behaviour of protein aggregation at a temperature gradient condition is reported in this study. To establish a feasible theory for protein aggregation kinetics at a temperature gradient condition, the spatial height of the protein solution is divided into hypothetical layers and the kinetic equations in those layers are solved. Furthermore, we endeavour to study numerically the effect of the temperature gradient on the kinetics of oligomer-mediated protein aggregation and protein inhibition.

Impact of long-term doxycycline on lung function & exacerbations: A real-world open, prospective pilot observation on chronic obstructive pulmonary disease.

BACKGROUND & OBJECTIVES: Upregulation of matrix metalloproteinases (MMPs) is related to the pathogenesis of chronic obstructive pulmonary disease (COPD). We aimed at assessing the tolerability and impact of long-term use of MMP inhibitor doxycycline in COPD. METHODS: A cohort of COPD patients was randomized to continue a uniform COPD treatment with or without add-on long-term oral doxycycline. The lung exacerbations (spirometry), adverse events and health status (COPD Assessment Test score) were noted at 3, 6, 9 and 12 months of therapy. Measurement of the serum MMP-2, and 9 and high-sensitive C-reactive protein (hs-CRP) levels was done at the start of the study and at three months, whenever possible. RESULTS: There were 27, 19, 13 and 10 patients with add-on doxycycline group and 22, 19, 11 and 7 patients with COPD treatment alone at 3, 6, 9 and 12 months of treatment respectively. The improvement was obvious and mostly (at 6 and 12 months) significant (P >0.05) for lung function parameters [forced expiratory volume in one second (FEV1), FEV1/forced vital capacity (FVC) and forced expiratory flow at 25-75% of FVC (FEF25-75)] and universal for health status at all measurements, with an overall 26.69 per cent reduction in exacerbations. The analysis with the lung function changes in the available population with protocol violation also supported the same trend. The concomitant reduction in serum MMP-9 (P =0.01), MMP-2 (P =0.01) and hs-CRP (P =0.0001) levels (n=21) at three months was also significant. The adverse reactions with add-on doxycycline appeared acceptable. INTERPRETATION & CONCLUSIONS: Long-term doxycycline appears well tolerated and seems to improve lung function, health status and exacerbations in COPD. The claim needs further scientific validations.

Enzyme Stabilization by Virus-Like Particles.

The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such entrainment did not seem to perturb enzyme function, but it did significantly enhance enzyme stability against several denaturing stimuli such as heat, organic solvents, and chaotropic agents. This improvement in performance was found to be general and independent of the number of independent subunits required and of the number of catalytically active enzymes packaged. Packaged enzymes were found by measurements of intrinsic tryptophan fluorescence to retain some of their native folded structure even longer than their catalytic activity, suggesting that protein folding is a significant component of the observed catalytic benefits. While we are unable to distinguish between kinetic and thermodynamic effects - including inhibition of enzyme unfolding, acceleration of refolding, and biasing of folding equilibria - VLP packaging appears to represent a useful general strategy for the stabilization of enzymes that operate on diffusible substrates and products.

Stabilization of Near-Infrared Fluorescent Proteins by Packaging in Virus-like Particles.

Near-IR fluorescent Qß virus-like particles (VLPs) were produced in a high yield by packaging highly red-shifted monomeric and dimeric versions of biliverdin-dependent fluorescent proteins within the capsid shell. The simple addition of biliverdin hydrochloride to the medium during or after Escherichia coli protein expression was enough to produce fully matured encapsidated fluorophores. The packaged near-IR proteins exhibited identical photochemical properties to their nonencapsidated analogues but were far more stable toward heat, chaotrope-induced denaturation, and proteolysis. Noninvasive in vivo imaging showed the VLPs to traffic primarily to the liver after systemic injection in mice, revealing that the particles were easily detected by a standard instrument.

Enzyme-Free Plasmonic Biosensor for Direct Detection of Neurotransmitter Dopamine from Whole Blood.

Complex biological fluids without pretreatment, separation, or purification impose stringent limitations on the practical deployment of label-free plasmonic biosensors for advanced assays needed in point of care applications. In this work, we present an enzyme-free plasmonic neurotransmitter dopamine biosensor integrated with a microfluidic plasma separator. This integrated device allows the in-line separation of plasma directly from the bloodstream and channels it to the active detection area, where inorganic cerium oxide nanoparticles function as local selective dopamine binding sites through strong surface redox reaction. A thorough understanding and engineering of the nanoparticles is carried out to maximize its dopamine sensitivity and selectivity. We obtain detection of dopamine at 100 fM concentration in simulated body fluid and 1 nM directly from blood without any prior sample preparation. The detection selectivity is found to be at least five-times higher compared to the common interfering species. This demonstration shows the feasibility of the practical implementation of the proposed plasmonic system in detection of variety of biomarkers directly from the complex biological fluids.

An improved kit formulation for one-pot synthesis of [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 for routine clinical use in cancer imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide derivatives have immense potential for non-invasive monitoring of malignancies overexpressing integrin αv ß3 receptors. Easy availability of suitable radiotracers would augment the utility of this class of molecular imaging agents. Towards this, the present article describes the development of an improved lyophilized kit for the routine clinical formulation of [99m Tc]Tc complex of HYNIC-conjugated dimeric cyclic RGD peptide derivative E-[c(RGDfK)]2 (E = glutamic acid, f = phenyl alanine, K = lysine) without using Sn2+ and systematic evaluation of its efficacy. Five batches of the kits were prepared, and [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 radiotracer was synthesized with high radiochemical purity (98.6 ± 0.5%) and specific activity (124.8 GBq/µmol) using the kits. Biodistribution studies in C57BL/6 mice bearing melanoma tumor exhibited significant accumulation of the radiotracer in tumor (5.32 ± 0.56 %ID/g at 60 min p.i.), and this uptake was also found to be receptor-specific by blocking studies. Preliminary human clinical investigations carried out in 10 breast cancer patients revealed high radiotracer uptake in the tumor along with good tumor-to-background contrast. The developed kit formulation showed an exceptionally high shelf-life of at least 18 months. These results demonstrated promising attributes of the developed kit formulation and warrant more extensive clinical investigations.

Anisotropic Electrowetting on Wrinkled Surfaces: Enhanced Wetting and Dependency on Initial Wetting State.

Electrowetting on dielectric (EWOD) on unidirectional microstructured surfaces has recently evoked significant interest as they can modulate the effect of electrowetting, and can thus find applications in directional wetting in microfluidic systems. However, the dependency of such EW phenomenon on their initial state of wetting and anisotropy is far from being well understood. The current study addresses the initial wetting states and their implication on the anisotropic electrowetting using a wrinkled EWOD platform. Herein we demonstrate a facile stampless and maskless structure generation technique to fabricate wrinkles of varying topography. Further, we have demonstrated alteration in the interfacial wetting conditions by modulating the wrinkle topography, and its effect on the droplet behavior during electrowetting. The capillary wicking-assisted electrowetting on these wrinkled surfaces is in specific direction dictated by the ordered wrinkles and prompts enhanced spreading of the droplet. We also demonstrate that while the enhancement of unidirectional electrowetting is stronger in conformal wetting state surfaces, composite wetting state surfaces depict a reversal in anisotropy.

Tailored topography: a novel fabrication technique using an elasticity gradient.

A facile methodology to create a wrinkled surface with a tailored topography is presented herein. The dependency of the elasticity of poly(dimethyl)siloxane (PDMS) on the curing temperature has been exploited to obtain a substrate with an elasticity gradient. The temperature gradient across the length of PDMS is created by a novel set-up consisting of a metal and insulator connected to a heater and the highest usable (no degradation of PDMS) temperature gradient is used. The time-dependent temperature distributions along the substrate are measured and the underlying physics of the dependence of the PDMS elasticity on the curing temperature is addressed. The PDMS substrate with the elasticity gradient is first stretched and subsequently oxidized by oxygen plasma. Upon relaxation, an ordered wrinkled surface with continuously varying wavelength and amplitude along the length of PDMS is obtained. The extent of hydrophobicity recovery of this plasma oxidized PDMS with varying elasticity has been studied. The change in the wavelength and amplitude of the regular patterns on the substrate can be controlled by varying operational parameters like applied pre-strain, plasma power and the heater temperature. It has been found that the spatial distributions of the topography and the hydrophobicity collectively decide the resultant wettability of the substrate. Such surfaces with gradients in the substructure dimensions demonstrate different wetting characteristics that may lead to a wide gamut of applications including droplet movement, cell adhesion and proliferation, diffraction grating etc.

Synthesis and biodistribution studies of 99m Tc labeled fatty acid derivatives prepared via "Click approach" for potential use in cardiac imaging.

123 I-Iodophenylpentadecanoic acid (IPPA) is a metabolic agent used in nuclear medicine for diagnosis of myocardial defects. Efforts are underway worldwide to develop a 99m Tc substitute of the above radiopharmaceutical for the aforementioned application. Herein, we report synthesis and biodistribution studies of 99m Tc labeled fatty acids (8, 11, and 15 carbons) obtained via "click chemistry" for its potential use in myocardial imaging. ω-Bromo fatty acids (8C/11C/15C) were synthetically modified at bromo terminal to introduce a heterocyclic triazole with glycine sidearm in a five step procedure. Modified fatty acids were subsequently radiolabeled with preformed [99m Tc(CO)3 ]+ synthon to yield the desired fatty acid complexes which were evaluated in Swiss mice. All the radiolabeled complexes were obtained with radiochemical purities >80%, as characterized by HPLC. Biodistribution studies of all three complexes in Swiss mice showed myocardial uptake of ~6-9% ID/g at 2 minutes post-injection, close to* I-IPPA (~9% ID/g). Complexes exhibited significant retention in the myocardium up to 30 minutes (~1% ID/g) but were lower to the standard agent (~7% ID/g). Similar uptake of activity in myocardium for the newly synthesized complexes in comparison to 125 I-IPPA along with favorable in vivo pharmacokinetics merits potential for the present "click" design of complexes for myocardial imaging.

High-Throughput, Protein-Targeted Biomolecular Detection Using Frequency-Domain Faraday Rotation Spectroscopy.

A clinically relevant magneto-optical technique (fd-FRS, frequency-domain Faraday rotation spectroscopy) for characterizing proteins using antibody-functionalized magnetic nanoparticles (MNPs) is demonstrated. This technique distinguishes between the Faraday rotation of the solvent, iron oxide core, and functionalization layers of polyethylene glycol polymers (spacer) and model antibody-antigen complexes (anti-BSA/BSA, bovine serum albumin). A detection sensitivity of ≈10 pg mL-1 and broad detection range of 10 pg mL-1 ≲ cBSA ≲ 100 µg mL-1 are observed. Combining this technique with predictive analyte binding models quantifies (within an order of magnitude) the number of active binding sites on functionalized MNPs. Comparative enzyme-linked immunosorbent assay (ELISA) studies are conducted, reproducing the manufacturer advertised BSA ELISA detection limits from 1 ng mL-1 ≲ cBSA ≲ 500 ng mL-1 . In addition to the increased sensitivity, broader detection range, and similar specificity, fd-FRS can be conducted in less than ≈30 min, compared to ≈4 h with ELISA. Thus, fd-FRS is shown to be a sensitive optical technique with potential to become an efficient diagnostic in the chemical and biomolecular sciences.

PDMS based multielectrode arrays for superior in-vitro retinal stimulation and recording.

Understanding of the neural response to electrical stimulation requires simultaneous recording from the various neurons of retina. Electrodes form the physical interface with the neural or retinal tissue. Successful retinal stimulation and recording demands conformal integration of these electrodes with the soft tissue to ensure establishment of proper electrical connection with the excitable tissue. Mechanical impedance of polydimethylsiloxane (PDMS) being compliant with that of retinal tissue, offers excellent potential as a substrate for metal electrodes. In this paper, Cr/Au micro electrodes with 200 µm diameter were fabricated on rigid and flexible PDMS substrates under crack free condition. Spontaneous buckling of thin films over PDMS substrates improved electrode performance circumventing the fabrication issues faced over a buckled surface. Individual electrodes from the multielectrode arrays (MEAs) were examined with electrochemical impedance spectroscopy and cyclic voltammetry. Controlled fabrication process as described here generates buckles in the metal films leading to increased electrode surface area that increases the charge storage capacity and decreases the interface impedance of the metal electrodes. At 1 kHz, impedance was reduced from 490 ± 27 kΩ to 246 ± 19 kΩ and charge storage capacity was increased from 0.40 ± 0.87 mC/cm2 to 2.1 ± 0.87 mC/cm2. Neural spikes recorded with PDMS based electrodes from isolated retina also contained less noise as indicated by signal to noise ratio analysis. The present study established that the use of PDMS as a substrate for MEAs can enhance the performance of any thin film metal electrodes without incorporation of any coating layers or nanomaterials.