TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Investigation on the feasibility of recycled polyvinylidene difluoride polymer from used membranes for removal of methylene blue: experimental and DFT studies.

This study reports the feasibility of recycled polyvinylidene difluoride (PVDF) beads to decolourize methylene blue (MB) from aqueous streams. The beads were characterized using scanning electron microscopy (SEM), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR) for its morphological and structural analysis. The effect of various process parameters such as adsorbent dose, initial concentration, contact time, and pH was studied. The first principle density functional theory (DFT) calculations were performed to investigate the underlying mechanism behind the adsorption process. The MB dye adsorption on recycled PVDF beads followed the pseudo-second-order kinetics and Langmuir isotherm, indicating the adsorption was chemical and monolayer. The maximum adsorption capacity obtained was 27.86 mg g-1. The adsorption energy of MB-PVDF predicted from the DFT study was -64.7 kJ mol-1. The HOMO-LUMO energy gap of PVDF decreased from 9.42 eV to 0.50 eV upon interaction with MB dye due to the mixing of molecular orbitals. The DFT simulations showed that the interaction of the MB dye molecule was from the electronegative N atom of the MB dye molecule, implying that electrostatic interactions occurred between the recycled PVDF beads and the positively charged quaternary ammonium groups in MB dye. The present study demonstrates the potential of recycled PVDF beads for a low-cost dye removal technique from textile wastewater.

Cone Beam Computed Tomography Study of Root Canal Morphology of Permanent Mandibular Incisors in Indian Subpopulation.

BACKGROUND: The aim of the study was to determine the root canal morphology of permanent mandibular incisor teeth in the Indian subpopulation with the use of cone beam computed tomography (CBCT). MATERIAL/METHODS: CBCT images of 200 patients with 800 permanent mandibular incisors, fulfilling necessary inclusion criteria and aged 18 to 60 years were evaluated. The number of roots, number of root canals and canal configuration were investigated and then classified according to Vertucci's classification of root canals. The effect of gender on the incidence of root canal morphology was also investigated. RESULTS: All the permanent mandibular incisors had a single root. The majority of mandibular incisors (66.5%) had a single root with a single canal. The prevalence of second canals was as follows: right central incisor - 33.5%, left central incisor - 30%, right lateral incisors - 33.5% and left lateral incisor - 36.5%. According to gender, 15.2% of men and 20.4% of women had a second root canal. Type 1 Vertucci configuration was most prevalent, followed by type 3, type 2, type 5 and type 4 in that order. CONCLUSIONS: Type 1 Vertucci's classification (64.5%) was the most prevalent canal configuration in the mandibular anterior teeth in the Indian population. Type 5 Vertucci's classification was the most frequently observed canal configuration of the two-canalled teeth. CBCT is an excellent imaging modality for detection of different canal configurations of mandibular incisors.

Endoplasmic reticulum stress triggers gametocytogenesis in the malaria parasite.

The malaria parasite experiences a significant amount of redox stress during its growth in human erythrocytes and heavily relies on secretory functions for pathogenesis. Most certainly, the parasite is equipped with machinery to tackle perturbations in the secretory pathway, like the unfolded protein response pathway in higher eukaryotes. Our bioinformatics analysis revealed the complete absence of genes involved in the canonical unfolded protein response pathway in Plasmodium falciparum. Accordingly, the parasite was unable to up-regulate endoplasmic reticulum (ER) chaperones or ER-associated degradation in response to DTT-mediated ER stress. Global profiling of gene expression upon DTT treatment revealed a network of AP2 transcription factors and their targets being activated. The overall outcome was up-regulation of genes involved in protein export and the sexual stage of the parasite life cycle culminating in gametocytogenesis. Our results suggest that the malaria parasite uses ER stress as a cue to switch to the transmissible sexual stages.

Identification of heat shock factor binding protein in Plasmodium falciparum.

BACKGROUND: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum. METHODS: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum. RESULTS: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite. CONCLUSIONS: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

Evaluation of apical leakage after immediate and delayed postspace preparation using different root canal sealers: An in vitro study.

BACKGROUND: Endodontically treated teeth with extensive loss of tooth structure lacks sufficient support for a permanent restoration. While restoring them with post and core it is important not to disrupt the apical seal. AIM: Evaluation of apical leakage after immediate and delayed postspace preparation using two root canal sealers. MATERIALS AND METHODS: Sixty single-rooted teeth were decoronated and roots were biomechanically prepared and obturated with gutta-percha and 2 sealers: AH Plus (Group A, n = 30) and Sure-Seal root canal sealer (Group B, n = 30). Groups A and B were subdivided into A1, A2 and B1, B2. Postspace was prepared immediately for A1 and B1. For A2 and B2 post space was prepared after storage in physiologic saline for 1 week. The samples were kept in Rhodamine B dye for 72 h and then sectioned longitudinally to observe dye penetration along the root canal wall under Stereomicroscope. The dye penetration was measured linearly and the values were subjected for statistical analysis using one-way analysis of variance and t-test. RESULTS: Statistically significant difference between Group A (1.00 mm) and B (2.71 mm) was observed (P < 0.001). However, the subgroups for immediate and delayed post space preparation did not show statistically significant difference (A1 = 0.947; A2 = 1.043; B1 = 2.306 and B2 = 3.120, P < 0.001). CONCLUSION: AH plus sealer showed lesser leakage compared to Sure-Seal Root canal sealer. The difference in leakage values was not statistically significant in delayed and immediate post space preparation groups, Time of postspace preparation has no influence on apical leakage.

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.

Aryl-alkyl-lysines: small molecular membrane-active antiplasmodial agents.

Due to emerging resistance there is a steady need for new antimalarial drugs. Here, we report a new class of water soluble, non-toxic compounds, aryl-alkyl-lysines, with promising activity against the ring stage of Plasmodium falciparum. The optimal compound perturbed the plasma membrane potential and the digestive vacuole of parasites. In the murine model of malaria (Plasmodium berghei ANKA) the compound was able to increase the survival of mice by at least 5 days by an intra-peritoneal route. Further, the compounds showed no apparent toxicity to mice at the concentration tested.

A disrupted transsulphuration pathway results in accumulation of redox metabolites and induction of gametocytogenesis in malaria.

Intra-erythrocytic growth of malaria parasite is known to induce redox stress. In addition to haem degradation which generates reactive oxygen species (ROS), the parasite is also thought to efflux redox active homocysteine. To understand the basis underlying accumulation of homocysteine, we have examined the transsulphuration (TS) pathway in the parasite, which is known to convert homocysteine to cysteine in higher eukaryotes. Our bioinformatic analysis revealed absence of key enzymes in the biosynthesis of cysteine namely cystathionine-ß-synthase and cystathionine-γ-lyase in the parasite. Using mass spectrometry, we confirmed the absence of cystathionine, which is formed by enzymatic conversion of homocysteine thereby confirming truncation of TS pathway. We also quantitated levels of glutathione and homocysteine in infected erythrocytes and its spent medium. Our results showed increase in levels of these metabolites intracellularly and in culture supernatants. Our results provide a mechanistic basis for the long-known occurrence of hyperhomocysteinemia in malaria. Most importantly we find that homocysteine induces the transcription factor implicated in gametocytogenesis namely AP2-G and consequently triggers sexual stage conversion. We confirmed this observation both in vitro using Plasmodium falciparum cultures, and in vivo in the mouse model of malaria. Our study implicates homocysteine as a potential physiological trigger of gametocytogenesis.

Identification of an exported heat shock protein 70 in Plasmodium falciparum.

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

An exported heat shock protein 40 associates with pathogenesis-related knobs in Plasmodium falciparum infected erythrocytes.

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.