Alterations in desmosomal adhesion at protein and ultrastructure levels during the sequential progressive grades of human oral tumorigenesis.

With the aim of developing early diagnostic/prognostic markers for oral cancer, desmosomal adhesion in sequentially progressive grades of tissues from oral normal/disorders (normal, hyperplastic, dysplastic, non-metastatic/metastatic tumours, and metastatic nodes) was investigated at protein and ultrastructural levels using immunohistochemistry and transmission electron microscopy, respectively. The expression of desmosomal proteins was higher in hyperplastic tissues than in normal tissues but was significantly decreased in subsequent progressive stages of the disease. Altered expression of desmosomal proteins was significantly correlated with local recurrence and disease-free survival. Ultrastructural analysis in the corresponding tissues revealed cytoplasmic clustering of desmosomes in hyperplasia; in more advanced disease stages, a significantly lower number of desmosomes and widened intercellular spaces were observed. Altered protein expression resulting in structural changes was confirmed by knocking down desmoplakin expression in non-transformed cells, which failed to form normal desmosome structures and induced a cell-transformation phenotype. Our data suggest that alterations in desmosomal assembly initiate at an early hyperplastic grade and, with more advanced disease stages, the severity of the alterations gradually becomes higher. Alterations in desmosomal adhesion can be useful for early detection of high-risk premalignant lesions, as well as for identification of invasive characteristics of primary non-metastatic tumours. Early detection will help to control further progression of disease by timely intervention.

Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella.

BACKGROUND: Flagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical '9 + 2' cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain. RESULTS: We found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus. CONCLUSION: It appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.

Implications of Volume Exclusion: A Look at Thermodynamic Perspective of DNA-Hemoglobin Complexes and Their Reconstitutes Under Macromolecular Crowding.

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo. Another significant factor which is overlooked is the effects of macromolecular crowding and its consequences in the actual biochemical and physiological environment. Such influences of crowding, its modification and physiological parameters have been reported. The present study investigates the effect of molecular crowding on binding characteristics of Salmon sperm DNA with Bovine hemoglobin and their reconstitutes in presence of molecular crowders viz., Poly ethylene glycol (PEG) and Dextran of different molecular weight by fluorescence, UV visible spectroscopic technique at different temperatures. The results showed that BHb fluorescence was quenched by sDNA through static quenching mechanism which is enhanced in presence of polymers. The number of binding sites 'n' and binding constants 'K' were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely ∆H°, ∆G°, T∆S° were studied at different temperatures and the results indicate that hydrophobic forces are predominant in the sDNA-BHb complex. Negative ∆G° values imply that the binding process is spontaneous.

"Is macromolecular crowding overlooked?"--Effects of volume exclusion on DNA-amino acids complexes and their reconstitutes.

Biological macromolecules evolve and function within intracellular environments that are crowded with other macromolecules. Crowding results in surprisingly large quantitative effects on both the rates and the equilibria of interactions involving macromolecules, but such interactions are commonly studied outside the cell in uncrowded buffers. The addition of high concentrations of natural and synthetic macromolecules to such buffers enables crowding to be mimicked in vitro, and should be encouraged as a routine variable to study. In this study, we propose to understand the changes in DNA character and its modulation in presence of macromolecules such as PEG with reference to binding parameters to amino acids using fluorescence enhancement.

Evaluation of automated image registration algorithm for image-guided radiotherapy (IGRT).

The performance of an image registration (IR) software was evaluated for automatically detecting known errors simulated through the movement of ExactCouch using an onboard imager. Twenty-seven set-up errors (11 translations, 10 rotations, 6 translation and rotation) were simulated by introducing offset up to ± 15 mm in three principal axes and 0° to ± 1° in yaw. For every simulated error, orthogonal kV radiograph and cone beam CT were acquired in half-fan (CBCT_HF) and full-fan (CBCT_FF) mode. The orthogonal radiographs and CBCTs were automatically co-registered to reference digitally reconstructed radiographs (DRRs) and planning CT using 2D-2D and 3D-3D matching software based on mutual information transformation. A total of 79 image sets (ten pairs of kV X-rays and 69 session of CBCT) were analyzed to determine the (a) reproducibility of IR outcome and (b) residual error, defined as the deviation between the known and IR software detected displacement in translation and rotation. The reproducibility of automatic IR of planning CT and repeat CBCTs taken with and without kilovoltage detector and kilovoltage X-ray source arm movement was excellent with mean SD of 0.1 mm in the translation and 0.0° in rotation. The average residual errors in translation and rotation were within ± 0.5 mm and ± 0.2°, ± 0.9 mm and ± 0.3°, and ± 0.4 mm and ± 0.2° for setup simulated only in translation, rotation, and both translation and rotation. The mean (SD) 3D vector was largest when only translational error was simulated and was 1.7 (1.1) mm for 2D-2D match of reference DRR with radiograph, 1.4 (0.6) and 1.3 (0.5) mm for 3D-3D match of reference CT and CBCT with full fan and half fan, respectively. In conclusion, the image-guided radiation therapy (IGRT) system is accurate within 1.8 mm and 0.4° and reproducible under control condition. Inherent error from any IGRT process should be taken into account while setting clinical IGRT protocol.

ß-Lactoglobulin-gold nanoparticles interface and its interaction with some anticancer drugs - an approach for targeted drug delivery.

The protein-nanoparticle interface plays a crucial role in drug binding and stability, in turn enhancing efficacy in targeted drug delivery. In the present study, whey protein ß-lactoglobulin (BLG) is conjugated with gold nanoparticles (AuNP) and its interaction with curcumin (CUR) and gemcitabine (GEM) has been explored. Further, AuNP-BLG conjugate interactions with anticancer drugs were characterized using dynamic light scattering (DLS), zeta potential, UV-visible, Raman spectroscopy, fluorescence, circular dichroism along with molecular dynamics simulation. The cytotoxicity studies were performed using breast cancer cell lines (MCF-7). ∼8 µM of BLG resides on AuNP (∼29 nm) surface revealed by DLS. Raman scattering of AuNP-BLG conjugate showed orientation of the central calyx of BLG towards solvent. BLG fluorescence confirmed the interaction between AuNP-BLG conjugate with drugs and indicated strong binding and affinity (for CUR KD = 3.71 x 108 M -1, n = 1.83, and for GEM KD = 3.78 x 103 M -1, n = 0.94), enhanced in the presence of AuNP. CD and Raman analysis exhibited selective hydrophilic and hydrophobic conformations induced by drug binding. Computational studies on BLG-drug complexes revealed that the residues Pro38, Leu39 and Met107 are largely associated with CUR binding, while GEM interaction is via hydrophilic contacts which significantly matches with spectroscopic investigation. IC50 values were calculated for all components of this loading system on MCF-7. The possible mechanisms of interaction between AuNP-BLG with anticancer drugs has been explored at the molecular level. We believe that these conjugates could be considered in the targeted drug delivery studies for cancer research.Communicated by Ramaswamy H. Sarma.

Physical and dosimetric characteristic of high-definition multileaf collimator (HDMLC) for SRS and IMRT.

Physical and dosimetric characteristics of HDMLC were studied for SRS6, 6, and 10 MV X-rays from Novalis Tx. This in-built tertiary collimator consists of 60 pairs (32 × 0.25 cm; 26 × 0.5 cm and 2 × 0.7 cm) of leaves. Properties of HDMLC studied included alignment, readout and radiation field congruence, radiation penumbra, accuracy and reproducibility of leaf position and gap width, static and dynamic leaf shift, tongue-and-groove effect, leaf transmission and leakage, leaf travel speed, and delivery of dynamic conformal arc and IMRT. All tests were performed using a calibrated ionization chamber, film dosimetry and DynaLog file analysis. Alignment of leaves with isocenter plane was better than 0.03 cm at all gantry and collimator positions. The congruence of HDMLC readout and radiation field agreed to within ± 0.03 cm for filed sizes ranging from 1 × 1 to 20 × 20 cm2. Mean 80% to 20% penumbra width parallel (perpendicular) to leaf motion was 0.24 ± 0.05 (0.21 ± 0.02) cm, 0.37 ± 0.12 (0.29 ± 0.07) cm, and 0.51 ± 0.13 (0.43± 0.07) cm for SRS6, 6, and 10 MV X-rays, respectively. Circular field penumbra was comparable to corresponding square field. Average penumbra of 1 × 20 cm2 field was effectively constant over off-axis positions of up to 12 cm with mean value of 0.16 (± 0.01) cm at 1.5 cm depth and 0.38 (± 0.04) cm at 10 cm depth. Minimum and maximum effective penumbra along the straight diagonal edge of irregular fields increased from 0.3 and 0.32 cm at 70° steep angle to 0.35 and 0.56 cm at 20° steep angle. Modified Picket Fence test showed average FWHM of 0.18 cm and peak-to-peak distance of 1.99 cm for 0.1 cm band and 2 cm interband separation. Dynamic multileaf collimation (DMLC) output factor remained within ± 1% for 6 MV and ± 0.5% for 10 MV X-rays at all gantry positions, and was reproducible within ± 0.5% over a period of 14 months. The static leaf shift was 0.03 cm for all energies, while dynamic leaf shift was 0.044 cm for 10 MV and 0.039 cm for both SRS6 and 6 MV X-rays. The dose depression and corresponding tongue-and-groove size were 24% and 0.17 cm for 6 MV and 19% and 0.20 cm for 10 MV X-rays. Average transmission through HDMLC was 1.09%, 1.14% and 1.34% for SRS6, 6 and 10 MV X-rays. Analysis of DynaLog files for leaf speed test in arc dynamic mode, delivery test of dynamic conformal arc, and step-and-shoot and sliding window IMRT showed at least 95% or more of the error counts had misplacements < 0.2 cm, with maximum root mean square (RMS) error value calculated at 0.13cm. Accurate and reproducible leaf position and gap width, and less leakage and small consistent penumbra over the fields demonstrate HDMLC suitable for high-dose resolution SRS and IMRT.

A Heparin based dual ratiometric sensor for Thrombin.

Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules, such as, aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T-Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.

An anionic polyelectrolyte induced aggregate assembly of Thioflavin-T: A prospective platform for Protamine sensing.

Protamine, a polycation, is biologically and medically relevant protein. Protamine exhibits a wide array of functions in biological processes like gene transfer, tissue and organogenesis, cell reproduction, etc. Medically, Protamine is the only clinically approved antidote for Heparin and is routinely used in various surgical interventions, and hence controlling Protamine dosing in patients is very crucial. Taking into account the medical significance of Protamine, designing simple, reliable and sensitive fluorescence sensors is highly desirable. In this work, we propose one such sensitive and reliable fluorescent sensor which is based on a template of dye-polyelectrolyte assembly constituting a molecular rotor dye, Thioflavin-T and an anionic synthetic polyelectrolyte, polystyrene sulfonate. The addition of Protamine, prompts drastic modulations in spectral features of dye-polyelectrolyte assembly which enables sensitive detection of Protamine in aqueous solution. Apart from sensitive detection, our sensing platform aids in highly selective sensing of Protamine compared to other proteins. Moreover, our sensor system is constructed on label-free, inexpensive, commercially available molecules posing as an advantage over other sensor systems which involve laborious synthesis protocols. Most importantly, our sensor template is able to sense Protamine in diluted serum sample, indicating the potential practical utility of our sensor system.

Inhibition of crude viper venom action by silver nanoparticles: A biophysical and biochemical study.

This investigation understands the interaction between lyophilized crude Viper snake venom (Doboia russellie) and Silver nanoparticles (SNPs) using biophysical and biochemical approaches. SNPs were synthesized by chemical reduction method and characterized using UV-Visible spectroscopy, Dynamic Light Scattering (DLS) and Transmission electron microscope (TEM). The average hydrodynamic size of SNPs was found to be 52 nm with 0.261 PDI. TEM image revealed the spherical shape of SNP. Interaction of SNPs and viper venom was resulted in the formation of complex which was confirmed by using DLS technique. Spectroscopic results showed an increase in absorbance intensity of venom upon interaction with SNPs which indicated interaction with venom proteins. Fluorescence spectroscopic data revealed the quenching in the fluorescence intensity of viper venom upon incubation with varying concentration of SNPs. The results obtained by biochemical assays (Protease and whole blood clotting test) revealed the inhibition of venom action due to presence of silver nanoparticles. The activity of protease enzyme was found to be decreased (10-13% reduction) in presence of silver nanoparticles. Prolonged clotting time (two fold) of viper venom upon interaction with SNPs compared to native crude viper venom was observed. The overall results confirmed the inhibition action of silver nanoparticles against viper venom.

Albumin corona on nanoparticles - a strategic approach in drug delivery.

Nanomaterials have been used widely for delivery of therapeutic agents. Protein-nanoparticle (NP) complexes have gained importance as vehicles for targeted drug delivery due to increased ease of administration, stability and half-life of drug, and reduced toxic side effects. Designing of phospholipid-bovine serum albumin (BSA) complexes and stealth NPs with BSA has paved the way for drug delivery carriers with prolonged blood circulation times. Preformed albumin corona has shown to decrease non-specific association and thereby reduce the clearance rate. Albumin corona has enabled the localization of drug carriers in specific tissues such as liver and heart, thus regulating biodistribution. Tailored albumin-NP conjugates have also enabled controlled degradation of NP and drug release. However, the binding of albumin with NP is associated with conformational and functional modulations in protein as observed with silver, gold and superparamagnetic iron oxide NPs. In this review, we highlight the various potential albumin-NP hybrids as nano drug carriers.