Development and Characterization of 99mTc-timolol Maleate for Evaluating Efficacy of In Situ Ocular Drug Delivery System

In situ gel-forming systems have drawn much attention of current researchers to overcome the poor bioavailability from the conventional eye drops. The present work described formulation and pharmacoscintigraphic evaluation of timolol-maleate-loaded chitosan/hydroxy propyl methyl cellulose (HPMC)-based polymer matrix for enhanced ocular retention. Chitosan and HPMC ratio was optimized and formulation was characterized for various in vitro parameters. The ocular retention was studied on New Zealand rabbits by gamma scintigraphy, which is a very simple and noninvasive technique. For scintigraphy study, the drug timolol maleate was radiolabeled ^99mTc by direct labeling method using SnCl₂·2H₂O as reducing agent. The labeling procedure was optimized to get maximum labeling efficiency (>98%). In vitro stability of the radiolabeled drug (^99mTc-timolol maleate complex) was checked and it was found to be stable for up to 24 h. Plain drug eliminates rapidly as significant activity was recorded in kidney and bladder after 2 h of ocular administration. It was evident from the scintigraphic images and the time–activity curve plotted from the data that the plain drug solution cleared very rapidly from the corneal region and reached into systemic circulation via nasolachrymal drainage system, as significant activity was recorded in kidney and bladder after 2 h of ocular administration. Developed formulation cleared at a slow rate and remained at corneal surface for longer time duration. No radioactivity was observed in systemic circulation after 2 h. Ocular irritation of the developed formulation was also checked by hen’s egg chorioallantoic membrane test and formulation was found to be practically nonirritant. The study signified the potential of gamma scintigraphy in evaluation of novel drug delivery systems in a noninvasive manner.     

A Translatable Predictor of Human Radiation Exposure

Terrorism using radiological dirty bombs or improvised nuclear devices is recognized as a major threat to both public health and national security. In the event of a radiological or nuclear disaster, rapid and accurate biodosimetry of thousands of potentially affected individuals will be essential for effective medical management to occur. Currently, health care providers lack an accurate, high-throughput biodosimetric assay which is suitable for the triage of large numbers of radiation injury victims. Here, we describe the development of a biodosimetric assay based on the analysis of irradiated mice, ex vivo-irradiated human peripheral blood (PB) and humans treated with total body irradiation (TBI). Interestingly, a gene expression profile developed via analysis of murine PB radiation response alone was inaccurate in predicting human radiation injury. In contrast, generation of a gene expression profile which incorporated data from ex vivo irradiated human PB and human TBI patients yielded an 18-gene radiation classifier which was highly accurate at predicting human radiation status and discriminating medically relevant radiation dose levels in human samples. Although the patient population was relatively small, the accuracy of this classifier in discriminating radiation dose levels in human TBI patients was not substantially confounded by gender, diagnosis or prior exposure to chemotherapy. We have further incorporated genes from this human radiation signature into a rapid and high-throughput chemical ligation-dependent probe amplification assay (CLPA) which was able to discriminate radiation dose levels in a pilot study of ex vivo irradiated human blood and samples from human TBI patients. Our results illustrate the potential for translation of a human genetic signature for the diagnosis of human radiation exposure and suggest the basis for further testing of CLPA as a candidate biodosimetric assay.     

Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.     

The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-sensitive Heme and Is Associated with the Small Colony Variant Phenotype

The chlorite dismutases (C-family proteins) are a widespread family of heme-binding proteins for which chemical and biological roles remain unclear. An association of the gene with heme biosynthesis in Gram-positive bacteria was previously demonstrated by experiments involving introduction of genes from two Gram-positive species into heme biosynthesis mutant strains of Escherichia coli, leading to the gene being renamed hemQ. To assess the gene product''s biological role more directly, a Staphylococcus aureus strain with an inactivated hemQ gene was generated and shown to be a slow growing small colony variant under aerobic but not anaerobic conditions. The small colony variant phenotype is rescued by the addition of exogenous heme despite an otherwise wild type heme biosynthetic pathway. The ΔhemQ mutant accumulates coproporphyrin specifically under aerobic conditions. Although its sequence is highly similar to functional chlorite dismutases, the HemQ protein has no steady state reactivity with chlorite, very modest reactivity with H₂O₂ or peracetic acid, and no observable transient intermediates. HemQ''s equilibrium affinity for heme is in the low micromolar range. Holo-HemQ reconstituted with heme exhibits heme lysis after <50 turnovers with peroxide and <10 turnovers with chlorite. The heme-free apoprotein aggregates or unfolds over time. IsdG-like proteins and antibiotic biosynthesis monooxygenases are close sequence and structural relatives of HemQ that use heme or porphyrin-like organic molecules as substrates. The genetic and biochemical data suggest a similar substrate role for heme or porphyrin, with possible sensor-regulator functions for the protein. HemQ heme could serve as the means by which S. aureus reversibly adopts an SCV phenotype in response to redox stress.     

CHOBIMALT: a cholesterol-based detergent

Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems, a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3?4 μM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210±30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1.     

Dietary fish oil associated with increased apoptosis and modulated expression of Bax and Bcl-2 during 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinogenesis in rats

The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on induction of apoptosis in mammary carcinogenesis model. Mammary carcinogenesis was initiated by a single, tail vein injection of 7,12-dimethylbenz(alpha)anthracene (DMBA) (0.5mg/0.2ml corn oil/100g body weight) at 7 weeks of animal age. Ninety female Sprague-Dawley rats were divided into two parts: part one was used for histology and immunohistochemical study and part two for morphological analysis. Each part consists of three experimental groups having 15 animals, i.e., Group A (DMBA control), Group B (DMBA+fish oil) and Group C (DMBA+corn oil). Rats were fed either fish oil or corn oil (0.5ml/day/rat) by oral gavage, 2 weeks prior to DMBA injection. Treatment was continued 25 weeks, studying histopathology, expression of Bax and Bcl-2 proteins by immunohistochemistry and apoptosis by TUNEL assay and morphological study at 36 weeks. Results showed that the fish oil-treated group exhibited a substantial increase in Bax (p<0.05) immunolabelling and a reduction of Bcl-2 immunopositivity (p<0.05), and increased TUNEL-positive apoptotic cells (p<0.05); however, corn oil treatment did not show these beneficial effects toward mammary preneoplasia. We conclude that fish oil has the potential to play a significant role in limiting mammary tumourigenesis in vivo.     

Response of the chromatophores in relation to the healing of skin wounds in an Indian Major Carp, Labeo rohita (Hamilton)

Chromatophores show significant changes during healing of skin wounds in Labeo rohita (Common Name - Rohu). Wound area can be divided into regions I, II and III. After infliction of wound, skin colour becomes significantly dark by 2 h that is gradually restored by 2 d. In regions II and III at 5 min, epidermal melanophores appear with beaded dendrites. In these regions at 2 h and in region I at 6 h, epidermal melanophores appear small, rounded or irregular shaped having dendritic processes with aggregated melanosomes. Subsequently, melanophores appear having elongated dendrites with dispersed or aggregated melanosomes. At 24 h, clusters of pigmented bodies appear in regions I and II. These bodies increase up to 2 d, and then diminish gradually and disappear by 8 d. Changes in dermal melanophores in region II at 5 min indicate the onset of degeneration. Degenerating melanophores increase up to 12 h, then gradually decline, and disappear by 4 d. Simultaneously, stellate melanophore reappear, gradually increase and appear like control by 8 d. Dermal melanophores in region III at different intervals appear stellate. In region I stellate dermal melanophores appear at 4 d. Stellate melanophores in all regions show different distribution of dispersed or aggregated melanosomes. With the appearance of dermal melanophores, highly refractive, crystalline structures, possibly the refractive platelets of the iridophores, are visualized around them. At subsequent intervals, these are frequently observed. This study provides interesting insights in injury induced changes in chromatophores in fish. The findings could be considered useful in perception of intriguing features in the development of pigment research in future.     

Three approaches for estimating the elastic modulus of the tympanic membrane

The function of the middle ear is to resolve the acoustic impedance mismatch between the air in the ear canal and the fluid of the inner ear. Without this impedance matching, very little acoustic energy would be absorbed into the cochlea. The first step in this process is the tympanic membrane (TM) converting sound in the ear canal into vibrations of the middle ear bones. Understanding how the TM manages its task so successfully over such a broad frequency range should lead to more satisfactory and less variable TM repairs (myringoplasty). In addition, understanding the mechanics of the TM is necessary to improve the coupling between ossicular prostheses and the TM. Mathematical models have played a central role in helping the research community understand the mechanics of the eardrum. However, all models require parameters as inputs. Unfortunately, most of the parameters needed for modeling the TM are not well known. In this work, several approaches for inferring the material properties of the TM are explored. First, constitutive modeling is used to estimate an elastic modulus based on the elastic modulus of collagen and experimentally observed fiber densities. Second, experimental tension and bending test results from the literature are re-interpreted using composite laminate theory. Lastly, dynamic measurements of the cat TM are used in conjunction with a composite shell model to bound the material parameters. Values from the literature, both measurement and modeling efforts, and from the present analysis are brought together to form a coherent picture of the TM's material properties. In the human, the data bound the elastic modulus between 0.1 and 0.3 GPa. In the cat, the data suggest a range of 0.1-0.4 GPa. These values are significantly higher than previous estimates.     

Potential cause of aerobic granular sludge breakdown at high organic loading rates

Aerobic sludge granules are compact, strong microbial aggregates that have excellent settling ability and capability to efficiently treat high-strength and toxic wastewaters. Aerobic granules disintegrate under high organic loading rates (OLR). This study cultivated aerobic granules using acetate as the sole carbon and energy source in three identical sequencing batch reactors operated under OLR of 9–21.3 kg chemical oxygen demand (COD) m⁻³ day⁻¹. The cultivated granules removed 94–96% of fed COD at OLR up to 9–19.5 kg COD m⁻³ day⁻¹, and disintegrated at OLR of 21.3 kg COD m⁻³ day⁻¹. Most tested isolates did not grow in the medium at >3,000 mg COD l⁻¹; additionally, these strains lost capability for auto-aggregation and protein or polysaccharide productivity. This critical COD regime correlates strongly with the OLR range in which granules started disintegrating. Reduced protein quantity secreted by isolates was associated with the noted poor granule integrity under high OLR. This work identified a potential cause of biological nature for aerobic granules breakdown.     

Evidence of salicylic acid pathway with EDS1 and PAD4 proteins by molecular dynamics simulation for grape improvement

Biotic stress is a major cause of heavy loss in grape productivity. In order to develop biotic stress-resistant grape varieties, the key defense genes along with its pathway have to be deciphered. In angiosperm plants, lipase-like protein phytoalexin deficient 4 (PAD4) is well known to be essential for systemic resistance against biotic stress. PAD4 functions together with its interacting partner protein enhanced disease susceptibility 1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defense pathway. Existence and structure of key protein of systemic resistance EDS1 and PAD4 are not known in grapes. Before SA pathway studies are taken in grape, molecular evidence of EDS1: PAD4 complex is to be established. To establish this, EDS1 protein sequence was retrieved from NCBI and homologous PAD4 protein was generated using Arabidopsis thaliana as template and conserved domains were confirmed. In this study, computational methods were used to model EDS1 and PAD4 and simulated the interactions of EDS1 and PAD4. Since no structural details of the proteins were available, homology modeling was employed to construct three-dimensional structures. Further, molecular dynamic simulations were performed to study the dynamic behavior of the EDS1 and PAD4. The modeled proteins were validated and subjected to molecular docking analysis. Molecular evidence of stable complex of EDS1:PAD4 in grape supporting SA defense pathway in response to biotic stress is reported in this study. If SA defense pathway genes are explored, then markers of genes involved can play pivotal role in grape variety development especially against biotic stress leading to higher productivity.