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Background: The present research was designed to develop a nanoemulsion (NE) of triphenylphosphine-D-α-tocopheryl-polyethylene glycol succinate (TPP-TPGS1000) and paclitaxel (PTX) to effectively deliver PTX to improve breast cancer therapy. Materials & methods: A quality-by-design approach was applied for optimization and in vitro and in vivo characterization were performed. Results: The TPP-TPGS1000-PTX-NE enhanced cellular uptake, mitochondrial membrane depolarization and G2M cell cycle arrest compared with free-PTX treatment. In addition, pharmacokinetics, biodistribution and in vivo live imaging studies in tumor-bearing mice showed that TPP-TPGS1000-PTX-NE had superior performance compared with free-PTX treatment. Histological and survival investigations ascertained the nontoxicity of the nanoformulation, suggesting new opportunities and potential to treat breast cancer. Conclusion: TPP-TPGS1000-PTX-NE improved the efficacy of breast cancer treatment by enhancing its effectiveness and decreasing drug toxicity.

Aim: A novel HPLC method was developed and validated for the simultaneous estimation of paclitaxel (PTX) and baicalein (BAC). Materials & methods: The analytes were resolved in a C18 column using the aqueous solution of formic acid (0.10% v/v) and MeOH (30:70 v/v). Results: The developed method was found to be linear over the concentration ranges 0.039–10 μg/ml and 0.019–10 μg/ml for PTX and BAC, respectively. The lower limits of quantification obtained were 0.042 μg/ml and 0.361 μg/ml for PTX and BAC, respectively. Conclusion: The developed method was found to be precise and accurate as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines, for simultaneous estimation of PTX and BAC, having an application in formulation development and bioanalytical studies.

PDF file - 422KB, S1. Cell survival as measured by a clonogenic assay. S2. BRCT I domain of XRCC1. S3. Characterization of cell lines by western blotting. S4. Characterization of full-length wild-type XRCC1 and L360R mutant XRCC1 mouse proteins. S5. Inhibitory effect of olaparib (O) or veliparib (V) (10 mM) on PAR accumulation in MMS-treated (10 mM for 20 min at 4 degrees C) wild-type (red) and pol beta null cells (gray). S6. Effect of PARPi on sensitivity of wild-type (16.3, closed red symbols) and pol beta null (19.4, open black symbols) cells to MMS. S7. Sensitivity of pol beta- and XRCC1-deficient cell lines to olaparib and veliparib.

Now a Days we are designing RCC framed structure which is heart of building with consideration of factors and various and codes which is necessary and using different techniques to assess the old RCC framed Structure. In India, safety of old buildings is one of the major issues. As the strength of old hospital buildings get reduced in due course of time it creates structural defects such as unexpected over loading, material deterioration, physical damages or structural deficiency and if further utilization of such damaged structure is done, it may lead to serious damage of life and property. As Structural Audit of old building is mandatory as per municipal authorities and Government of Maharashtra has made "Structural Audit" of all old building compulsory As Prevention is better than cure, In this present dissertation we adopted Structural Auditing of Cancer hospital building which is situated at sawangi (meghe),Wardha (Maharashtra) is constructed in the year span of 1990-1992 So at the present scenario this building is 32 year Old and required to be checked in all aspect to Assure its safety. with Rebound Hammer Test, Ultrasonic Pulse Velocity Test, Half-cell test, pH and carbonation test including Visual Inspection and assessing the stability and safety of the structure to withstand for its remaining life by diagnosis and root cause of the problems by recommending strengthening and then retesting after strengthening is done to check the required strength which is expected.

In this study we had did partial replacement of fine aggregate by metal steel scrap in different percentage in concrete. In this paper, M20 and M30 grade of concrete is used and lathe metal scrap used as a fiber and added up to 30% by weight, at a gap of 10% (i.e., 0%, 10%, 20%, and 30%). In this investigation, a comparison has been made between plain cement concrete and the fiber reinforced concrete containing lathe metal scrap (metal steel scrap) in various proportions by weight. Analytical comparison is being done between the compressive strength of plain cement concrete and Lathe metal scrap reinforced concrete (LMSRC) M20 and M30. The 28 days strength of LMSRC for compressive strength is found to be increased when compared with the 28 days strength of plain cement concrete.

An attempt of co-delivery of insulin and C-peptide enclosed in linseed oil globules has been made employing a protective coating of positively charged poly-L-lysine to manage diabetes-associated complications. Oral water in oil in water (w/o/w) nanoemulsion manufactured by double emulsification method showed good entrapment efficiency of 87.6 ± 7.48% for insulin and 73.4 ± 6.44% for C-peptide. The optimized uncoated nanoemulsion showed a mean globule size of 210.6 ± 9.87 nm with a good PDI of 0.145 ± 0.033 and -21.7 ± 4.5 mV ZP. The poly-L-lysine coating of the nanoemulsion resulted in the reversal of surface charge to positive i.e. 18.3 ± 2.7 mV due to the cationic nature of poly-L-lysine. In vitro drug release showed an initial burst of 15-20% release within 4 h followed by controlled release up to 24 h. The poly-L-lysine coated nanoemulsion showed an 8.28-fold higher uptake than fluorescein isothiocyanate (FITC) solution in HCT116 intestinal cell lines. In vivo studies confirmed that orally administered insulin and C-peptide bearing coated nanoemulsion has the potential to improve glycemic control confirmed by blood glucose level under 200 mg/dL for 12 h compared to that of subcutaneous administration of insulin. The formulation was found stable at 25 °C as well as 4°C for up to 3 months. These findings show a promising approach for delivering oral insulin along with C-peptide for effective glycemic control and management of complications associated with diabetes.

A Descriptive study was conducted to compare the knowledge of mothers on behavioral problems of school going children in selected urban and rural areas. The study was conducted at Solapur district among 120 mothers who were selected by using simple random sampling technique. The study result revealed that the categorization of mothers based on their knowledge regarding behavioural problems of school going children. Among the mothers, in rural area more than half of the total subjects that were 35 (58.33%) had average knowledge, 24(40%) of the mothers had low knowledge, and only 01(1.67%) of mothers had high knowledge. Whereas in urban area more than half of the total subject i.e. 39(65%) of mothers had average knowledge, 14 (23.33%) of mothers had low knowledge and 07(11.67%) of mothers had high knowledge. As per the statistical analysis the researcher concludes that urban mothers is having higher percentage of knowledge when compared to rural mothers; it may be because of education and exposure to health awareness campaigns.

Background: Congenital dyserythropoietic anemias (CDAs) are a very rare and heterogeneous group of disorders characterized by ineffective erythropoiesis. CDA II is caused by mutations in the SEC23B gene. The most common mutation reported in India is c.1385A>G, p.Y462C. There is no simple and cost-effective confirmatory diagnostic test available for CDA, and therefore, many patients remain undiagnosed. High-resolution melting (HRM) analysis is a polymerase chain reaction (PCR) based technique applied to identify genetic differences and scan nucleic acid sequences. HRM can be used to rapidly screen the common mutation causing CDA II in the Indian population. Objective: To study the use of High-Resolution Melting Curve Analysis to detect common mutation causing CDA II in the Indian population.Method:10 Indian families havingSEC23B (Y462C) mutation causing CDA II are considered for this study. HRM was used to check the presence of Y462C mutation. To verify the reliability of the HRM analysis, we compared results with the results of Sanger sequencing. This helped us to confirm the diagnosis.Results: We have described clinical, hematological, and genetic data of ten patients suffering from CDAII. According to HRM and Sanger sequencing, a homozygous SEC23B (Y462C) mutation was present in all patients, whereas a heterozygous Y462C mutation was present in their parents. Conclusion: Our data showed that High-Resolution Melting (HRM) analysis could be used to rapidly screen common SEC23B mutations that cause CDA II in the Indian population.HRM technique leads to an accurate diagnosis of CDA II patients and does not need further diagnostic workup.

An attempt was made to formulate moxifloxacin loaded alginate beads incorporated into spongy wound dressing to heal chronic wounds as well as to reduce frequency of painful dressing change. Moxifloxacin loaded beads (sodium alginate:pectin, 1:1) were prepared by ionic gelation method, with entrapment efficiency 94.52%, crushing strength 25.30 N and drug release 90.52%. Beads were further incorporated into wound dressing, made of pectin and carboxymethyl tamarind seed polysaccharide (CMTSP). Spongy wound dressing was obtained by freeze drying technology, which showed good folding endurance, high wound fluid absorption and good crushing strength. Drug release was found to be 85.09%. Dressing made of CMTSP:pectin (1.5:2) showed good water vapour transmission and antibacterial activity. Porous nature of dressing absorbed exudates of wound. Excision wound model in rats revealed wound healing within 17 days: groups I (control), II (moxifloxacin beads loaded wound dressing), III (moxifloxacin beads), IV (pectin film) and V (sodium alginate film) showed 65.28, 99.09, 86.90, 66.84 and 64.30% wound closure, respectively. To conclude, moxifloxacin beads loaded spongy wound dressing has good healing and wound closing potential compared to pectin film and moxifloxacin beads. Thus, the formulation is novel for biomedical application which reduced the frequency of painful dressing change.

The NASA InSight Lander on Mars includes the Heat Flow and Physical Properties Package HPThe online version contains supplementary material available at 10.1007/s11214-022-00941-z.

Hexokinase (EC 2.7.1.1, Adenosine Tri Phosphate (ATP): D-hexose-6-phosphotransferase) is a crucial regulatory enzyme of the glycolytic pathway (Embden-Meyerhof pathway). Hexokinase deficiency is associated with chronic non-spherocytic haemolytic anaemia (HA) with some exceptional cases showing psychomotor/mental retardation and fetus death. The proband is a four-and-half-year-old female child born of a four-degree consanguineous marriage hailing from South India with autosomal recessive congenital HA associated with developmental delay. She was well till 3 months of her age post an episode of diarrhoea when she was noted to be severely anaemic and requiring regular transfusions. The common causes of HA, haemoglobinopathies, red cell membranopathies and common red cell enzymopathies (G6PD, GPI, PK and P5N) were ruled out. Targeted analysis of whole exome sequencing (WES) using an insilico gene panel for hereditary anaemia was performed to identify pathogenic variants in the patient. Next-generation sequencing revealed a novel homozygous variant in hexokinase gene c.2714CA (p. Thr905Lys) in exon-18. The pathogenic nature of the variant p. Thr905Lys in the

An attempt was made to enhance solubility of an antifungal agent lawsone, via self-microemulsifying drug delivery system (SMEDDS), formulated further into hollow pessary to treat vaginal candidiasis. Microemulsion was formed at 20% capryol 90® (oil); 80% Gelucire 44/14® (surfactant) and 20% Tween 80 (co-surfactant), which was plotted using pseudo-ternary phase diagram using aqueous titration method. Zeta potential −10.9 ± 0.54 mV and particle size 12.19 ± 1.13 nm confirmed the formation of microemulsion. Quick self-emulsification time (55 s) indicated rapid emulsification process at vaginal site. Cloud point confirmed the stability of SMEDDS (up to 60 °C). Lawsone-loaded SMEDDS exhibited improved solubility of lawsone (216 ± 2.54 mg/ml and 0.542 ± 0.04 mg/ml, respectively) and dissolution, which further helped to enhance its antifungal activity. Pessary base, consisted of Ovucire WL3460® (94, 96, 98) and beeswax (2, 4, 6), was optimized using three-level, two-factor full factorial design. Hollow pessary was further fabricated using melted base (at 60 °C), which was poured into aluminum mold fitted with center tube. This center tube was filled with lawsone-loaded SMEDDS and was solidified at 2–8 °C. Pessary was melted within 7 ± 1.1 min at 37 °C and released 96.96 ± 1.43% lawsone within 1 h at simulated vaginal fluid. Lawsone SMEDDS loaded pessary released lawsone at par with marketed clotrimazole vaginal tablet. To conclude, hollow pessary of lawsone-loaded SMEDDS can be an advanced modality to treat vulvo-vaginal candidiasis.

PARP-1 is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activated synthesizing PAR polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase β (pol β) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol β−/− and Xrcc1−/− cells resulted in hypersensitivity to the PARP inhibitor 4-AN and reexpression of pol β or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and veliparib also exhibited hypersensitivity in both pol β−/− and Xrcc1−/− BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency. Implications: BER deficiency represents a new therapeutic opportunity to enhance PARPi efficacy. Visual Overview: http://mcr.aacrjournals.org/content/12/8/1128/F1.large.jpg. Mol Cancer Res; 12(8); 1128–39. ©2014 AACR.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Human computer communication has growing demand recent days. The new generation of autonomous technology aspires to give computer interfaces emotional states that relate and consider user as well as system environment considerations. In the existing computational model is based an artificial intelligent and externally by multi-modal expression augmented with semi human characteristics. But the main problem with is multi-model expression is that the hardware control given to the Artificial Intelligence (AI) is very limited. So, in our project we are trying to give the Artificial Intelligence (AI) more control on the hardware. There are two main parts such as Speech to Text (STT) and Text to Speech (TTS) engines are used accomplish the requirement. In this work, we are using a raspberry pi 3, a speaker and a mic as hardware and for the programing part, we are using python scripting.

The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase beta (Pol beta). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE.Pol beta.DNA that formed specifically at BER intermediates containing a 5'-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol beta gap-filling and much stronger 5'-deoxyribose phosphate lyase activities than was observed with the Pol beta.DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol beta, although coordination also may be facilitated by APE.Pol beta.DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair.

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.

Background: Antibiotics are important in the management and prophylaxis of infections in patients at a risk of experiencing microbial disease. Uses of systemic antibiotics in dentistry are limited since management of acute dental conditions is primarily based upon extraction of teeth or extirpation of the pulp. However, the literature provides evidence of inappropriate prescribing practices by practitioners, due to a number of factors from inadequate knowledge to social factors. Aim: The aim was to assess the therapeutic prescription of antibiotics in the dental office. Materials and Methods: In the current study, 42 faculty members of two dental colleges in the same vicinity were included. A questionnaire was drafted and sent to the dentists to collect data pertaining to the conditions in which antibiotics were prescribed and most commonly prescribed antibiotic. Results: During the study period, 42 faculty members from various departments in the institutes were surveyed, of which 41 questionnaires were completely filled. Amoxicillin was the most commonly prescribed antibiotic followed by other amoxicillin combinations; Metronidazole was most widely prescribed antibiotic for anaerobic infections. Conclusion: We have entered an era where cures may be few due to increasing microbial resistance. The biggest force for change will be if all practicing dentists looked at their prescribing and made it more rational.

Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.

All of the synthesized thiazolidinedione (IX) and (XI) show promising antibacterial, antitubercular and antidiabetic activities in comparison to the standard drugs norfloxacin, streptomycin and glibenclamide, respectively.

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.

Treatment of base excision repair–proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G2-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti–PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor. Mol Cancer Res; 10(3); 360–8. ©2012 AACR.

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.

We have analyzed the long-period (∼1000 s) part of the Earth's free oscillation spectrum of the 1989 Macquarie Ridge earthquake. This event stands out as the largest in the last decade and as the first great earthquake to have been recorded digitally on the new generation of very broadband seismic sensors around the globe. Its spectrum was modeled for five spectral peaks, 0S4, 0S6, 0S9, 1S8, and 5S3, taking into account the effects of lateral heterogeneity, rotation and ellipticity which cause coupling within the spectral peaks (self coupling) and between different multiplets (multiplet coupling). The effects of self coupling were calculated following the formulation of Woodhouse and Girnius (1982). Multiplet coupling only weakly affected the spectral peaks that were used. A large discrepancy in phase and amplitude between the calculated spectrum and the data was observed, which suggests that the seismic moment was ∼50% larger than that inferred from surface wave analysis (Satake and Kanamori, 1990). An error analysis was performed in order to detect inaccuracies that might have resulted in this discrepancy; centroid time, source finiteness correction, and directivity effects are likely to cause a significant bias both in the moment and phase determination. We saw no direct evidence that a precursory event could have been responsible for the additional energy release, as was suggested by Ihmle et al. (1993).

Benzimidazole is a bicyclic heterocycle system consisting of two nitrogen atoms and fused phenyl ring shows wide range of biological activities. Benzimidazole can be synthesized using ophenylenediamine and carboxylic acid. Benzimidazole posses wide spectrum of biological activities like including antibacterial, antifungal, antiviral, anti-inflammatory, anticonvulsant, antidepressant, antihypertensive, analgesic, and hypoglycemic properties. The present reviews attempted to gather the various developments in synthesis and biological activities of Benzimidazole derivatives.

The hypothesis was tested that cancer, by virtue of it being a thrombotic diathesis, may enhance ischemic heart disease. Because cancer therapy may precipitate thromboembolism, the authors focused on patients with occult neoplasia before therapy. Occult cancer was defined as a time period of 2 years before a cancer diagnosis was established. Data obtained from the files of patients with the diagnosis of malignant tumors and admitted to a general hospital during a 3-year period were reviewed for coronary risk factors, coronary events, and characteristics of cancer. Indices of coronary instability were studied: the incidence of first coronary events, the incidence of all coronary events, and the coronary events burden. These indices were calculated for 366 patients with cancer (from the Department of Medicine files of 166 consecutive patients with cancer of several primary sites and from the surgical ward files of 100 consecutive patients with colorectal cancer and 100 consecutive patients with cancer of the prostate or bladder) and for 100 patients with benign prostatic hypertrophy. The patients with benign prostatic hypertrophy served as controls. A steep and statistically significant increase in coronary instability indices was observed in all groups of patients with cancer in the 2-year period before cancer diagnosis in comparison with the coronary instability indices of control patients (P less than or equal to 0.05 to 0.0001). Patients with colorectal cancer presented the highest indices in the 2-year period before cancer diagnosis, with unstable ischemic heart disease being reported in 18% and first coronary events in 10%. The coronary events burden was 0.92. The lowest indices among patients with cancer were recorded in those with prostatic and bladder cancer. Unstable ischemic heart disease occurred in 6% and first coronary events in 4%. The coronary events burden was 0.36. The indices were several times lower for control patients than for patients with cancer. Unstable ischemic heart disease occurred in 3% of control patients, and first coronary events in 2%. The coronary events burden was 0.15. Other possible etiologic factors, particularly the known coronary risk factors and anemia, were not statistically related with an increased risk of coronary events in the 2-year period before cancer diagnosis. Based on these epidemiologic data, it appears that there may be an association between occult cancer and coronary events.

DNA polymerase beta (pol beta) is a constitutively expressed DNA repair enzyme in vertebrate cells. Yet, it had been shown previously that the pol beta mRNA level increases in Chinese hamster ovary (CHO) cells within 4 h after treatment with several monofunctional DNA damaging agents, notably, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Herein we report that a transfected pol beta promoter fusion gene is activated by MNNG treatment of CHO cells; mRNA from the transfected gene is approximately 10-fold higher in treated cells than in untreated cells 16 h after treatment. This activation is mediated through the decanucleotide palindromic element GTGACGTCAC at positions -49 to -40 in the "TATA-less" core promoter. This element, which is similar to the ATF/CREB transcription factor-binding site in a number of mammalian genes, forms the center of a strong protein-binding site for CHO cell nuclear extract proteins. Mutated pol beta promoter fusion genes lacking the element fail to bind protein at this site and fail to respond to MNNG treatment of cells.

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.

In response to NASA's announced requirement for Earth hazard monitoring sensor-web technology, a multidisciplinary team involving sensor-network experts (Washington State University), space scientists (JPL), and earth scientists (USGS cascade volcano observatory (CVO)), is developing a prototype dynamic and scaleable hazard monitoring sensor-web and applying it to volcano monitoring. The combined optimized autonomous space - in-situ sensor-web (OASIS) will have two-way communication capability between ground and space assets, use both space and ground data for optimal allocation of limited power and bandwidth resources on the ground, and use smart management of competing demands for limited space assets. It will also enable scalability and seamless infusion of future space and in-situ assets into the sensor-web. The prototype will be focused on volcano hazard monitoring at Mount St. Helens, which has been active since October 2004. The system is designed to be flexible and easily configurable for many other applications as well. The primary goals of the project are: 1) integrating complementary space (i.e., Earth observing one (EO-1) satellite) and in-situ (ground-based) elements into an interactive, autonomous sensor-web; 2) advancing sensor-web power and communication resource management technology; and 3) enabling scalability for seamless infusion of future space and in-situ assets into the sensor-web. To meet these goals, we are developing: 1) a test-bed in-situ array with smart sensor nodes capable of making autonomous data acquisition decisions; 2) efficient self-organization algorithm of sensor-web topology to support efficient data communication and command control; 3) smart bandwidth allocation algorithms in which sensor nodes autonomously determine packet priorities based on mission needs and local bandwidth information in real-time; and 4) remote network management and reprogramming tools. The space and in-situ control components of the system will be integrated such that each element is capable of autonomously tasking the other. Sensor-Web data acquisition and dissemination will be accomplished through the use of the open geospatial consortium sensor-web enablement protocols. The three-year project will demonstrate end-to-end system performance with the in-situ test-bed at Mount St. Helens and NASA's EO-1 platform.

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.

Primer and dNTP recognition by purified HIV reverse transcriptase have been investigated. Earlier kinetic studies suggested that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence [Majumdar et al. (1988) J. Biol. Chem. 263, 15657-15665], and through use of a particularly high affinity template-primer analogue [r(I)n.Sd(C)28], rate values for formation of the first complex were calculated [Majumdar et al. (1989) Biochemistry 28, 1340-1346]. We now report rate values for first complex formation in the usual model replication system with poly[r(A)].oligo [d(T)] as template-primer. We find that 3'-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis against the substrate dNTP (dTTP) in the poly[r(A)].oligo[d(T)] replication system. This suggests that 3'-azido-dTTP and dTTP combine with the same form of the enzyme in the reaction scheme, i.e., the enzyme-primer complex. This is not trivial, since a second analogue, 3'-amino-dTTP, also is an inhibitor against dTTP, but the mechanism in this case is linear noncompetitive. Because the inhibition by 3'-azido-dTTP is linear competitive, the KD for physical binding to the enzyme is assumed to be the same as the Ki for inhibition (20 nM). Substrate kinetic studies of DNA synthesis using 3'-azido-dTTP as substrate revealed that the Michaelis constant is 3 microM. Therefore, the Km for this substrate analogue is 100-fold higher than the KD for binding of the analogue to the enzyme-primer complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.

Summary Deoxyribose phosphate (dRP) removal by DNA polymerase β (Pol β) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol β null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1 −/− mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.

Human fibroblasts, capable of expressing a kinase-dead form of ATR (ATRkd), can be sensitized to the cytotoxic effects of methyl methanesulfonate (MMS) by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN). The combination of MMS + 4-AN results in accumulation of cells in S-phase of the cell cycle and activation of Chk1. Inhibition of ATR activity by expression of ATRkd suppresses the S-phase accumulation and partially reverses the Chk1 phosphorylation. The results confirm involvement of an ATR-mediated damage response pathway in the MMS + 4-AN-induced S-phase cell cycle checkpoint in human fibroblasts. Consistent with this hypothesis, the inhibitors caffeine and UCN-01 also abrogate the ATR- and Chk1-mediated delay in progression through S-phase. In the absence of ATR-mediated signaling, MMS + 4-AN exposure results in a G2/M arrest, rather than an S-phase checkpoint. Thus, whereas ATR mediates the S-phase response, it is not critical for arrest of cells in G2/M.

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.