Efficient mapping of RNA-binding residues in RNA-binding proteins using local sequence features of binding site residues in protein-RNA complexes.

Protein-RNA interactions play vital roles in plethora of biological processes such as regulation of gene expression, protein synthesis, mRNA processing and biogenesis. Identification of RNA-binding residues (RBRs) in proteins is essential to understand RNA-mediated protein functioning, to perform site-directed mutagenesis and to develop novel targeted drug therapies. Moreover, the extensive gap between sequence and structural data restricts the identification of binding sites in unsolved structures. However, efficient use of computational methods demanding only sequence to identify binding residues can bridge this huge sequence-structure gap. In this study, we have extensively studied protein-RNA interface in known RNA-binding proteins (RBPs). We find that the interface is highly enriched in basic and polar residues with Gly being the most common interface neighbor. We investigated several amino acid features and developed a method to predict putative RBRs from amino acid sequence. We have implemented balanced random forest (BRF) classifier with local residue features of protein sequences for prediction. With 5-fold cross-validations, the sequence pattern derived dipeptide composition based BRF model (DCP-BRF) resulted in an accuracy of 87.9%, specificity of 88.8%, sensitivity of 82.2%, Mathew's correlation coefficient of 0.60 and AUC of 0.93, performing better than few existing methods. We further validated our prediction model on known human RBPs through RBR prediction and could map ~54% of them. Further, knowledge of binding site preferences obtained from computational predictions combined with experimental validations of potential RNA binding sites can enhance our understanding of protein-RNA interactions. This may serve to accelerate investigations on functional roles of many novel RBPs.

Involvement of the non-active site Residues in the Catalytic Activity of NDM-4 Metallo beta-lactamase.

The rise of New Delhi metallo beta-lactamase (NDM) producing bacteria imposes a significant threat to the treatment of bacterial infections due to their broad spectrum against beta-lactams. The activity of metallo beta-lactamases is affected by active site residues as well as residues near the active site. Therefore, we aimed to identify the amino acid residues around the active site of NDM-4 which influence its function. To achieve that, seven substitution mutations (S191A, D192A, S213A, K216A, S217A, D223A and D225A) of NDM-4 were generated through site-directed mutagenesis. Out of these, expression of NDM-4_D192A and NDM-4_S217A in Escherichia coli cells increased the beta-lactam susceptibility as compared to NDM-4. Further, proteins were purified to assess the effect of substitution mutations on zinc content, in vitro catalytic efficiency, and stability of NDM-4. The catalytic efficiency was reduced for these mutants (D192A and S217A) towards beta-lactam substrates, while the thermal stability remained insubstantial as compared to NDM-4. However, the purified NDM-4_D192A exhibited altered zinc content. In silico studies reveal that these changes might be the outcomes of alterations in hydrogen bonding networks and substrate interactions. Taken together, we infer that the D192 and the S217 residues play a substantial role in the activity of NDM-4.

Molecular insights into binding dynamics of tandem RNA recognition motifs (tRRMs) of human antigen R (HuR) with mRNA and the effect of point mutations in impaired HuR-mRNA recognition.

Human antigen R (HuR) is a key regulatory protein with prominent roles in RNA metabolism and post-transcriptional gene regulation. Many studies have shown the involvement of HuR in plethora of human diseases, which are often manifestations of impaired HuR-RNA interactions. However, the inherent complexities of highly flexible protein-RNA interactions have limited our understanding of the structural basis of HuR-RNA recognition. In this study, we dissect the underlying molecular mechanism of interaction between N-terminal tandem RNA-recognition motifs (tRRMs) of HuR and mRNA using molecular dynamics simulation. We have also explored the effect of point mutations (T90A, R97A and R136A) of three reported critical residues in HuR-mRNA binding specificity. Our findings show that N-terminal tRRMs exhibit conformational stability upon RNA binding. We further show that R136A and R97A mutants significantly lose their binding affinity owing to the loss of critical interactions with mRNA. This may be attributed to the larger domain rearrangements in the mutant complexes, especially the ß2ß3 loops in both the tRRMs, leading to unfavourable conformations and loss of binding affinity. We have identified critical binding residues in tRRMs of HuR, contributing favourable binding energy in mRNA recognition. This study contributes significantly to understand the molecular mechanism of RNA recognition by tandem RRMs and provides a platform to modulate binding affinities through mutations. This may further guide in future structure-based drug-therapies targeting impaired HuR-RNA interactions.Communicated by Ramaswamy H. Sarma.

A comparative analysis of machine learning classifiers for predicting protein-binding nucleotides in RNA sequences.

RNA-protein interactions play vital roles in driving the cellular machineries. Despite significant involvement in several biological processes, the underlying molecular mechanism of RNA-protein interactions is still elusive. This may be due to the experimental difficulties in solving co-crystallized RNA-protein complexes. Inherent flexibility of RNA molecules to adopt different conformations makes them functionally diverse. Their interactions with protein have implications in RNA disease biology. Thus, study of binding interfaces can provide a mechanistic insight of the molecular functioning and aberrations caused due to altered interactions. Moreover, high-throughput sequencing technologies have generated huge sequence data compared to available structural data of RNA-protein complexes. In such a scenario, efficient computational algorithms are required for identification of protein-binding interfaces of RNA in the absence of known structures. We have investigated several machine learning classifiers and various features derived from nucleotide sequences to identify protein-binding nucleotides in RNA. We achieve best performance with nucleotide-triplet and nucleotide-quartet feature-based random forest models. An overall accuracy of 84.8%, sensitivity of 83.2%, specificity of 86.1%, MCC of 0.70 and AUC of 0.93 is achieved. We have further implemented the developed models in a user-friendly webserver "Nucpred", which is freely accessible at "http://www.csb.iitkgp.ac.in/applications/Nucpred/index".

Association of Thr715Pro P-Selectin Gene Polymorphism in Diabetic Retinopathy in North Indian Population: A Prospective Clinical Study.

INTRODUCTION: The aim of this study was to evaluate the role of Thr715Pro P-Selectin gene polymorphism in patients with Diabetic Retinopathy in North Indian population and establish its role in the pathophysiology as an independent factor. MATERIALS AND METHODS: This is a prospective clinical study conducted on 60 patients at a tertiary care centre in North India over a period of eighteen months. Sixty patients satisfying the inclusion criteria were selected from the Vitreoretina clinic in the department. They were categorised equally in three groups namely Diabetics with diabetic retinopathy (DwDR), Diabetics without diabetic retinopathy (DwoDR), and non diabetics. The non-diabetics group was further divided into healthy controls, Hypertensive Retinopathy (HR) and Non-exudative Age Related Macular Degeneration (NEAMD). All the patients underwent complete ophthalmic evaluation and blood samples were drawn for the genetic study with their informed consent. Data was analysed using SPSS software version 16. RESULTS: The genotypic analysis between DwDR, DwoDR and the three subgroup of controls comprising of healthy controls, HR and NEAMD showed that Thr715Pro (A/C) polymorphism prevalence was significantly high in DwDR (p = 0.003) and DwoDR (p = 0.003) compared to healthy controls. No significant difference was noted between DwDR, DwoDR and the HR and NEAMD groups. CONCLUSION: Thr715Pro P-Selectin gene Polymorphism could not be established as an independent factor in the pathogenesis of diabetic retinopathy, as its association is found with other systemic diseases which create a prothrombotic state.

Therapeutic Effectiveness in the Treatment of Experimental Bacterial Keratitis with Ion-activated Mucoadhesive Hydrogel.

PURPOSE: To investigate the therapeutic effectiveness of ion-activated mucoadhesive hydrogel system in the treatment of experimental bacterial keratitis. MATERIALS AND METHODS: Mucoadhesive systems were prepared using gellan or sodium alginate alone and combined with sodium carboxymethylcellulose (NaCMC) to enhance the gel bioadhesion properties. The in vivo antimicrobial efficacy of selected mucoadhesive systems was studied in an experiment on bacterial keratitis in rabbit's eyes and compared with that of the marketed conventional eyedrops. RESULTS: Ocular tolerance was studied in the eye of albino rabbits and tested formulations were non-irritant with no sign of inflammation. Better improvement in experimental bacterial keratitis in rabbit eyes was observed in animals treated with mucoadhesive hydrogel formulation (GG5 and GS5) compared with marketed drug solution. CONCLUSION: The developed system is a viable alternative to conventional eyedrops of GTN due to its ability to enhance bioavailability through its longer precorneal residence time.

Toll-like receptor 4 polymorphisms and their haplotypes modulate the risk of developing diabetic retinopathy in type 2 diabetes patients.

PURPOSE: Persistent inflammation and impaired neovascularization in type 2 diabetes mellitus (T2DM) patients may lead to development of macro- and microvascular complications. Diabetic retinopathy (DR) is one of the secondary microvascular complications of T2DM. Improper activation of the innate immune system may be an important contributor in the pathophysiology of DR. Toll-like receptor 4 (TLR4) is an important mediator of innate immunity, and genetic alterations in TLR4 support inflammation in the hyperglycemic condition. The present work was designed to investigate whether the TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs10759931, rs1927911, and rs1927914 are associated with DR in a north Indian population. METHODS: The study group of 698 individuals (128 DR, 250 T2DM, 320 controls) was genotyped by PCR-RFLP. Haplotype and linkage disequilibrium between SNPs were determined using Haploview software. RESULTS: Combined risk genotypes of TLR4 SNPs rs10759931 (odds ratio [OR] 1.50, p = 0.05) and rs1927914 (OR 1.48, p = 0.05) were found to be significantly associated with pathogenesis of DR. A total of 14 haplotypes with frequency >1% were obtained using Haploview software. Haplotypes ACATC (37.5%) and ACATT (14.8%) were the two most common haplotypes obtained. CONCLUSIONS: Results of the present case-control study that included 698 north Indian subjects suggested that TLR4 SNPs rs10759931 and rs1927914 modulate the risk of DR in T2DM cases. Association analysis using haplotypes showed none of the haplotypes were associated with either susceptibility or resistance to DR in a north Indian population.

Positively charged microemulsions of dexamethasone: comparative effects of two cosurfactants on ocular drug delivery and bioavailability.

BACKGROUND: The objective of this investigation was to evaluate the potential of mucoadhesive chitosan-coated positively charged microemulsions (CH-MEs) of dexamethasone with respect to the change in nonionic cosurfactants. METHODS: CH-MEs were prepared with different concentrations of surfactant and cosurfactant using the water titration method and coated with low-molecular-weight chitosan. RESULTS: All formulations displayed an average globule size between 85 and 187 nm and a positive surface charge. The optimized CH-MEs showed greater penetration of dexamethasone in the anterior segment of the eye, resulting in twofold and fourfold higher dexamethasone concentration than uncoated ME and drug suspension, respectively. CONCLUSION: The developed CH-MEs shows increases in ocular penetration and bioavailability.

Mucoadhesive chitosan-coated cationic microemulsion of dexamethasone for ocular delivery: in vitro and in vivo evaluation.

PURPOSE: Dexamethasone (DXN) is an effective anti-inflammatory drug in the treatment of acute and chronic eye disease such as uveitis. However, its low aqueous solubility limits its clinical usefulness. The purpose of this study was to investigate the mucoadhesive chitosan-coated cationic microemulsions (CH-MEs) for ophthalmic delivery of DXN to treat uveitis. MATERIALS AND METHODS: The pseudo-ternary phase diagrams were developed and various MEs were prepared using isopropyl myristate as oil, Tween 80 as a surfactant, propylene glycol as a co-surfactant and distilled water. MEs were prepared and coated with chitosan by the dropwise addition of chitosan solution in the ME dispersion. Physicochemical parameters (globule size, zetapotential, drug content, viscosity, refractive index and pH), mucoadhesive properties and the in vitro release of MEs were studied. The in vivo efficacy of prepared formulations and the marketed drug solution were studied by administering them topically to endotoxin-induced uveitis rabbit model. RESULTS: All formulations displayed an average globule size less than 200 nm and a positive surface charge. The developed CH-MEs showed acceptable physico-chemical behavior, good mucoadhesive properties, good stability for three months and exhibited sustained drug release. In vivo studies in rabbit eye showed a marked improvement in the anti-inflammatory activity of mucoadhesive CH-ME-treated eye compared with a marketed suspension formulation in a uveitis-induced rabbit eye model. CONCLUSION: The developed CH-MEs are a viable alternative to conventional eye drops for its ability to enhance bioavailability through its longer precorneal residence time and its ability to sustain the release of the drug.

Effect of hydroxypropyl-ß-cyclodextrin on the ocular bioavailability of dexamethasone from a pH-induced mucoadhesive hydrogel.

PURPOSE: Dexamethasone (DXN) is an effective anti-inflammatory drug in the treatment of acute and chronic eye disease such as uveitis. It is relatively lipophilic and permeates biological membranes quite easily. However, its low aqueous solubility limits its clinical usefulness. To circumvent this problem Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was used as solubilizer and penetration enhancer for DXN. The purpose of this study was to develop HP-ß-CD based pH-induced mucoadhesive hydrogel for ophthalmic delivery of DXN to treat uveitis. MATERIALS AND METHODS: The formation of inclusion complex of DXN with HP-ß-CD was characterized in solution and solid states by phase solubility, X-ray diffractometry and IR spectrum analyses. To improve ocular retention and sustained action Carbopol 980 NF and sodium carboxymethylcellulose (NaCMC) were added to the formulations as phase transition and mucoadhesive agents, respectively. RESULTS: The HP-ß-CD-based hydrogel system enhanced the solubility of DXN and the apparent stability constant (k') of the DXN-HP-ß-CD inclusion complex was found to be 258.62 M(-1). The optimum concentrations of Carbopol 980NF and NaCMC for the mucoadhesive hydrogel were 0.2% (w/v) and 0.4% (w/v), respectively. This mucoadhesive hydrogel could flow freely under non-physiological condition and showed the character of pseudoplastic fluid under both physiological and non-physiological conditions. In vitro release of DXN from the HP-ß-CD complex in simulated tear fluid (STF, pH- 7.4), was influenced significantly by the properties and concentration of Carbopol and NaCMC. In vivo studies in rabbit eye showed a marked improvement in anti-inflammatory activity of mucoadhesive hydrogel-treated eye compared with a marketed solution formulation in a uveitis-induced rabbit eye model. CONCLUSION: The developed HP-ß-CD-based mucoadhesive system is a viable alternative to conventional eye drops of DXN due to its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain the release of the drug.

Chitosan nanoparticles of 5-fluorouracil for ophthalmic delivery: characterization, in-vitro and in-vivo study.

The aim of this investigation was to develop 5-fluorouracil (5-FU) loaded chitosan nanoparticles (CH-DNPs) for ophthalmic delivery. CH-DNPs were fabricated by ionotropic gelation mechanism using chitosan (CH) and a polyanion (TPP). The nanoparticles were smooth and spherical, confirmed by scanning electron microscopy (SEM) and atomic force microscope (AFM). CH/TPP mass ratio and TPP significantly changed the particles size morphology and encapsulation efficiency. The nanoparticles size ranged from approximately 114 to 192 nm and had a positive zeta potential (30±4 mV). The encapsulation efficiency, loading capacity and recovery of DNPs were 8.12-34.32%, 3.14-15.24% and 24.22 to 67% respectively. Physical characterization was done by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD). No interaction was observed in between drug and polymer and crystallinity of drug was not changed in drug loaded nanoparticles. In-vitro release study of DNPs showed diffusion controlled release. Bioavailability study of batch CS9 was studied in rabbit eye and compare to 5-FU solution. 5-FU level was significantly higher in aqueous humor of rabbit eye. Ocular tolerance was studied in the eye of New Zealand rabbits and tested formulation was non-irritant with no sign of inflammation.

Polymeric nanoparticulate system: a potential approach for ocular drug delivery.

Various efforts in ocular drug delivery have been made to improve the bioavailability and to prolong the residence time of drugs applied topically onto the eye. The potential use of polymeric nanoparticles as drug carriers has led to the development of many different colloidal delivery vehicles. Drug loaded polymeric nanoparticles (DNPs) offer several favorable biological properties, such as biodegradability, nontoxicity, biocompatibility and mucoadhesiveness. These submicron particles are better than conventional ophthalmic dosage forms to enhance bioavailability without blurring the vision. DNPs have been shown to be amenable to targeting of the drug to the site of action, leading to a decrease in the dose required and a decrease in side effects. Additionally, DNPs can be fabricated by simple techniques with better physical stability than liposomes. This unique combination of properties makes DNPs a novel polymeric drug delivery device, which fulfils the requirements for ophthalmic application. This review discusses the polymeric nanoparticles, physiochemical characterization, fabrication techniques, therapeutic significances, patented technology of nanoparticles and future possibility in the field of ocular drug delivery.

Pattern of retinopathy of prematurity and its correlation with insulin-like growth factor-1 (IGF-1).

We estimated serum insulin-like growth factor-1 (IGF-1) level to correlate for development of retinopathy of prematurity (ROP) in serum from 15 premature infants. 73% of the infant developed Stage 1 ROP and the rest develop Stage 2. Zone III involvement never progress beyond Stage 1, and Zone II beyond Stage 2. Severity of ROP could not be related to the level of IGF-1. All cases develop ROP of Stage 1 and 2 irrespective of serum IGF-1.

Vascular endothelial growth factor-A (VEGF-A) in vitreous fluid of patients with proliferative diabetic retinopathy.

VEGA-A concentration was measured in undiluted vitreous fluid samples were taken at the start of vitrectomy procedure from 9 new cases of proliferative diabetic retinopathy and 8 non-diabetic patients. A positive correlation was found between VEGF-A levels and blood sugar and HgbA1c levels of patients with proliferative diabetic retinopathy.

Gellan-based scleral implants of indomethacin: in vitro and in vivo evaluation.

Film-type scleral implants of indomethacin with gellan gum were prepared by solvent casting and evaluated for uniformities of thickness, weight, drug content, and surface pH. The effect of plasticizers like glycerol, propylene glycol (PG), and polyethylene glycol 200, and 400 on the void volume of free gellan films (placebo) was calculated from the water content of the films. The drug release from the prepared implants was determined using a static dissolution set-up developed and optimized in our laboratory. Based on the results of the void volume and initial drug release studies, glycerol and PG were selected as the plasticizers for the gellan-based implants. The morphology of the drug-free films (containing 10% and 40% of PG) and the drug-loaded films (before and after dissolution and crosslinked) was studied using scanning electron microscopy. Further, the effect of plasticizer concentration, gellan concentration, effect of crosslinking technique, and duration of crosslinking using calcium chloride on in vitro drug release characteristics were evaluated. Selected batches of the implants were subjected to pharmacodynamic studies, after scleral placement, in uveitis-induced (intravitreal injection of bovine serum albumin 50 microg/ml) rabbit eyes. The release of indomethacin from the prepared implants followed matrix diffusion kinetics with diffusion co-efficient (n) values ranging between 0.358 to 0.708 and seemed to depend on both gellan and plasticizer concentration. Surface crosslinking with 10% calcium chloride for 8 hr retarded drug release (1.42 times less than noncrosslinked implant) and was optimum. The pharmacodynamic studies showed a marked improvement in the various clinical parameters (congestion, keratitis, flare, clot, aqueous cells, and synechias) in the implanted eye compared with the control eye in the rabbits. The scleral implants survived up to 3 weeks in vivo.

In vitro and in vivo evaluation of the Gelrite gellan gum-based ocular delivery system for indomethacin.

The poor bioavailability and therapeutic response exhibited by the conventional ophthalmic solutions due to pre-corneal elimination of the drug may be overcome by the use of in situ gel forming systems, which upon instillation as drops into the eye undergo a sol-gel transition in the cul-de-sac. This may result in better ocular availability of the drug. The purpose of this work was to develop an ophthalmic delivery system of the NSAID indomethacin, based on the concept of ion activated in situ gelation. Gelrite gellan gum, a novel ophthalmic vehicle, which gels in the presence of mono or divalent cations present in the lacrimal fluid, was used as the gelling agent. The developed formulations were therapeutically efficacious (in a uveitis induced rabbit eye model) and provided sustained release of the drug over an 8-hour period in vitro.