Human hnRNPA1 reorganizes telomere-bound Replication Protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence Summary: At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 µM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 µM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

A moving target for drug discovery: Structure activity relationship and many genome (de)stabilizing functions of the RAD52 protein.

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Interaction map of Arabidopsis Mediator complex expounding its topology.

Understanding of mechanistic details of Mediator functioning in plants is impeded as the knowledge of subunit organization and structure is lacking. In this study, an interaction map of Arabidopsis Mediator complex was analyzed to understand the arrangement of the subunits in the core part of the complex. Combining this interaction map with homology-based modeling, probable structural topology of core part of the Arabidopsis Mediator complex was deduced. Though the overall topology of the complex was similar to that of yeast, several differences were observed. Many interactions discovered in this study are not yet reported in other systems. AtMed14 and AtMed17 emerged as the key component providing important scaffold for the whole complex. AtMed6 and AtMed10 were found to be important for linking head with middle and middle with tail, respectively. Some Mediator subunits were found to form homodimers and some were found to possess transactivation property. Subcellular localization suggested that many of the Mediator subunits might have functions beyond the process of transcription. Overall, this study reveals role of individual subunits in the organization of the core complex, which can be an important resource for understanding the molecular mechanism of functioning of Mediator complex and its subunits in plants.

A Clinical and Radiographic Evaluation of the Efficacy of Nanohydroxyapatite (Sybograf™) versus Bioactive Calcium Phosphosilicate Putty (Novabone®) in the Treatment of Human Periodontal Infrabony Defects: A Randomized Clinical Trial.

AIM: The aim of this study is to compare and to evaluate clinically and radiographically the bone regeneration and the amount of bone fill (BL) between nanocrystalline hydroxyapatite (Nc-HA) (Sybograf™) and bioactive synthetic NovaBone Putty in the treatment of intrabony component of periodontal osseous defects. MATERIALS AND METHODS: Twenty sites in 20 patients, within the age range of 25-55 years, showing intrabony defects were selected and divided into Group I (Nc-HA) and Group II (Bioactive synthetic NovaBone Putty). All the selected sites were assessed with the clinical and radiographic parameters such as plaque index, gingival index, sulcus bleeding index, probing pocket depth, clinical attachment level, gingival recession, and radiographic BL. All the clinical and radiographic parameter values obtained at different intervals (baseline, 3, and 6 and 9 months) were subjected to statistical analysis. RESULTS: A statistically significant reduction in pocket depth of 4.400 ± 0.843 mm (Group I), 3.800 ± 0.789 mm (Group II) and gain in clinical attachment level of 6.2 mm (Group I), 5.9 mm (Group II) were recorded at the end of the study. A slight increase in gingival recession was observed. The mean percentage changes in the amount of radiographic BL of Group II and Group I were significant, However, when compared between the groups, there is no significant difference in BL observed. CONCLUSION: Both the graft materials appear to have nearly comparable effects, with nanocrystalline hydroxyapatite (Sybograf™), displaying slightly superior effect over bioactive glass especially in relation to clinical parameters. However, long-term, controlled clinical trials are required to confirm these findings.

Expression of AtMed15 of Arabidopsis in yeast causes flocculation and increases ethanol production in yeast culture.

Mediator, a multiprotein complex involved in transcription of class II genes, was first discovered in yeast and then characterized in many metazoans revealing a striking structural conservation of the complex. However, sequences of Mediator subunits are not well conserved raising a question on the functional conservation of these individual subunits. In this study, expression of Med15 of Arabidopsis (AtMed15) in gal11∆ yeast could not complement the function of ScGal11 in galactose metabolism and resistance against cycloheximide. Surprisingly, AtMed15 changed the morphology of the yeast cells. The cells adhered strongly on the surface of the agar media, and showed robust flocculation in the liquid media without affecting the growth. The AtMed15-induced adhesion and flocculation were observed in different carbon sources. Calcium-assisted cell wall-bound mannan-binding proteins were found to be involved in this flocculation, which was unaffected by wide fluctuation of pH or temperatures revealing its constitutive robust nature. Expression of few flocculation related Flo genes was up-regulated in these cells. Interestingly, there was significant increase in ethanol production by the yeast expressing AtMed15. Robust and constitutive flocculation and increased ethanol production by yeast cells harbouring AtMed15 indicate an opportunity of its important usage in biotechnology industries.

Platelet concentration in platelet concentrates and periodontal regeneration-unscrambling the ambiguity.

CONTEXT: Platelet-rich-plasma (PRP) and Platelet-rich-fibrin (PRF) are extensively used autologous platelet concentrates in periodontal regeneration, and PRF has a better efficacy as compared to PRP. The rationale for this difference has often been attributed to the difference in the structure of the fibrin matrix. However, the effect of concentration of platelets on the regenerative potential of these concentrates is obscure. AIMS: The study was conducted to evaluate and compare, clinically and radiographically, the efficacy of PRF and PRP in the treatment of periodontal endosseous defects and to assess the effect of platelet concentration on periodontal regeneration. MATERIALS AND METHODS: Twenty intrabony defects were selected and divided into two groups randomly by the coin toss method. Group I received PRP and Group II subjects were treated with PRF. The platelet counts in PRP and PRF were analyzed. Clinical and radiological parameters were assessed at baseline and 3, 6, and 9 months postoperatively. STATISTICAL ANALYSIS: Kruskal-Wallis Chi-square test, Wilcoxon signed rank test, t-test, and Spearman's rank correlation were used for statistical analysis of data. RESULTS: There was statistically significant improvement in all the parameters in the two groups except in relation to gingival recession. There was a statistically significant difference between the platelet count in Group I and Group II (P = 0.002). CONCLUSION: PRP and PRF appear to have nearly comparable effects in terms of periodontal regeneration. The concentration of platelets appears to play a paradoxical role in regeneration. The regenerative potential of platelets appears to be optimal within a limited range.

The Effect of Scaling and Root Planing on Glycaemic Control, Periodontal Status and Gingival Crevicular Fluid TNF-α Levels in an Indian Population- To Reveal the Ambivalent Link.

CONTEXT: Periodontal disease and diabetes mellitus(DM) share a two - way relationship. It can be hypothesized that successful management of periodontal infection in diabetes will lead not only to reduction of local signs and symptoms of the disease, but also to better control of glucose metabolism. AIMS: To monitor the effect of Scaling and Root planing (SRP) on glycaemic control in patients with type 2 diabetes mellitus by estimating the HbA1c and GCF TNF-α levels. SETTINGS AND DESIGN: This Interventional clinicobiochemical study was carried out over a period of 6 months from December 2010-May 2011 in Bengaluru, Karnataka, India. MATERIALS AND METHODS: Fifteen well-controlled, 15 moderately controlled and 15 poorly controlled diabetic subjects were enrolled in this study. All participants were subjected to non-surgical periodontal (SRP) therapy. GCF sampling and clinical periodontal parameters assessment were done at baseline and 3 months post-therapy. TNF-α levels in GCF were analyzed by enzyme-linked immunosorbent assay (ELISA) at baseline and 3 months post therapy. The improvement in glycaemic control was assessed using HbA1c levels at 3 months reevaluation. STATISTICAL ANALYSIS: The data obtained were statistically analysed using Kruskal-Wallis test, Mann-Whitney test and Wilcoxon Signed Rank test. RESULTS: Following periodontal treatment, all patients demon- strated a significant improvement in periodontal status. A reduction in TNF-α level and the HbA1c values were also observed. CONCLUSION: The result indicates that SRP is effective in improving metabolic control in Type 2 Diabetes Mellitus patients possibly through the reduction of TNF-α which in turn might improve the insulin resistance.

Adrenomedullin, periodontitis, diabetes-unraveling the equivocal relationship: A clinicobiochemical cross-sectional study.

CONTEXT: Antimicrobial proteins and peptides constitute a diverse class of host-defense molecules that act early to combat invasion and infection with bacteria and other microorganisms. Among the various antimicrobial peptides in the oral cavity, adrenomedullin (ADM), a cationic peptide, is found in gingival crevicular fluids (GCFs) in amounts twice as high in periodontal disease sites as healthy sites. Studies have also shown that plasma levels of ADM increased in patients with type 2 diabetes mellitus as compared with controls. AIMS: This clinico-biochemical study was undertaken to try to decipher the probable link between ADM, diabetes and periodontitis. MATERIALS AND METHODS: The study comprised of 90 patients who were divided into three groups based on community periodontal index scores and diabetes status. Probing pocket depth and clinical attachment level were measured in all subjects. GCF was collected from all the participants using micropipettes and blood samples were collected from subjects in Groups III, for analysis of glycated hemoglobin. ADM levels were measured in GCF samples by the enzyme-linked immunosorbent assay. STATISTICAL ANALYSIS USED: The data obtained were subjected to analysis of variance, Bonferroni test and Pearson's correlation. RESULTS: An increase in GCF levels of ADM from periodontal health to disease and in periodontitis patients with type 2 diabetes was noted. CONCLUSIONS: Increase in GCF levels of ADM from periodontal health to disease and in periodontitis patients with type 2 diabetes reinforces the perio-systemic interlink concept.

Structure prediction for CASP7 targets using extensive all-atom refinement with Rosetta@home.

We describe predictions made using the Rosetta structure prediction methodology for both template-based modeling and free modeling categories in the Seventh Critical Assessment of Techniques for Protein Structure Prediction. For the first time, aggressive sampling and all-atom refinement could be carried out for the majority of targets, an advance enabled by the Rosetta@home distributed computing network. Template-based modeling predictions using an iterative refinement algorithm improved over the best existing templates for the majority of proteins with less than 200 residues. Free modeling methods gave near-atomic accuracy predictions for several targets under 100 residues from all secondary structure classes. These results indicate that refinement with an all-atom energy function, although computationally expensive, is a powerful method for obtaining accurate structure predictions.