Assessment of small in-frame indels and C-terminal nonsense variants of BRCA1 using a validated functional assay.

BRCA1 (Breast Cancer 1, early onset) is linked to breast and ovarian cancer predisposition. Still, the risks conferred by a significant portion of BRCA1 variants identified in the population remains unknown. Most of these variants of uncertain significance are missense alterations. However, the functional implications of small in-frame deletions and/or insertions (indels) are also difficult to predict. Our group has previously evaluated the functional impact of 347 missense variants using an extensively validated transcriptional activity assay. Here we show a systematic assessment of 30 naturally occurring in-frame indels located at the C-terminal region of BRCA1. We identified positions sensitive and tolerant to alterations, expanding the knowledge of structural determinants of BRCA1 function. We further designed and assessed the impact of four single codon deletions in the tBRCT linker region and six nonsense variants at the C-terminus end of BRCA1. Amino acid substitutions, deletions or insertions in the disordered region do not significantly impact activity and are not likely to constitute pathogenic alleles. On the other hand, a sizeable fraction of in-frame indels at the BRCT domain significantly impact function. We then use a Bayesian integrative statistical model to derive the probability of pathogenicity for each variant. Our data highlights the importance of assessing the impact of small in-frame indels in BRCA1 to improve risk assessment and clinical decisions for carriers.

Impact of amino acid substitutions at secondary structures in the BRCT domains of the tumor suppressor BRCA1: Implications for clinical annotation.

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.

BRCA1 recruitment to damaged DNA sites is dependent on CDK9.

Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ-H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.

Characterization of LGALS3 (galectin-3) as a player in DNA damage response.

DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G 2/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3.

Biological restoration: a 4/5 crown.

Dental caries is one of the most prevalent conditions in humans; the purpose of restorative dentistry is to recreate the anatomy of the affected tooth thus the use of fragments from natural teeth as an effective restorative method. A maxillary first premolar left was prepared for an inverted 4/5 crown due to involvement of the vestibular face, after prepared received an allogeneic collage with similar color and dimension. Follow-up after 12 months indicated a stable restoration. Clinically, the site was without signs of caries, migration of the fragment or marginal infiltration. Biological restoration is a viable alternative for reestablishing function and esthetics to damaged/decayed teeth and therefore, biological restoration as an alternative to other restorative options.

Freqüência de hemolisinas anti-A e anti-B em doadores de sangue de Itapeva e Ourinhos / Anti-A and anti-B hemolysin frequencies in blood donors from Itapeva and Ourinhos

Uma transfusão de sangue com o tipo ABO incorreto pode resultar na morte dopaciente, com reação hemolítica intravascular, seguida de alterações imunológicas e bioquímicas. Considerando a pequena quantidade de estudos sobre as hemolisinas anti-A e anti-B e a importância desses anticorpos na prática transfusional, o objetivodeste trabalho foi verificar a frequência dessas hemolisinas e a associação a fatores como etnia e gênero em doadores de sangue dos bancos de sangue dos municípios de Itapeva e de Ourinhos, no interior do estado de São Paulo. Foram analisadas todas asamostras de doadores tipo O cadastradas nos bancos de sangue, com elevado nível plasmático de hemolisinas (doadores considerados perigosos). Coletaram-se os dados relativos à frequência das hemolisinas, etnia, gênero e procedência destes doadores.A frequência de doadores de sangue do grupo sanguíneo O perigosos é de 1,2%em Itapeva e de 5,3% em Ourinhos. Na cidade de Ourinhos, o risco de um doadorbranco ter hemolisina positiva é 2,16 vezes maior do que para outros doadores, e na cidade de Itapeva notou-se que o risco é menor para brancos do que para outros doadores. Em relação ao gênero, em Ourinhos o risco de um doador ter hemolisina positiva é 1,82 vezes maior para o gênero masculino, e na cidade de Itapeva, o risco foi maior para doadores do gênero feminino. A relação entre o gênero, a etnia e a frequência de hemolisinas foi diferente nas duas cidades. Mesmo assim, destaca-se a importância dos bancos de sangue estarem atentos às características de prevalência deste tipo de doador.

Blood transfusion using unmatched ABO blood types can result in patient death due to intravascular hemolytic reactions followed by immunological and biochemical changes. Considering the small number of studies on anti-A and anti-B hemolysins and the relevanceof these antibodies in the transfusional practice, this work aimed at assessing the frequency of these hemolysins and their relationship to factors such as ethnic background and gender in blood donors of blood banks located in Itapeva and Ourinhos, São Paulo State, Brazil. All samples from type O donors recorded in these two blood banks with high serum levels of hemolysins (donations considered high-risk) were analyzed. Data related to hemolysin frequencies, ethnicity, gender and origin of blood donors were collected. Thefrequency of donors belonging to the high-risk O blood group was 1.2% in Itapeva and 5.3% in Ourinhos. In Ourinhos the risk of a Caucasian donor being positive for hemolysin is 2.16 times higher than other ethnic groups and in Itapeva, it was observed that therisk is lower for Caucasians than other ethnic groups. Regarding the gender, in Ourinhos the risk of a donor being positive to hemolysin is 1.82 times greater in men and in Itapeva the risk ishigher in women. The ratios of gender and ethnic background in respect to frequency were different between the two regions. Thus it is important for blood banks to be aware of regional variations.

Antihemorrhagic, antinucleolytic and other antiophidian properties of the aqueous extract from Pentaclethra macroloba.

Several Brazilian plants have been utilized in folk medicine as active agents against various effects induced by snake venoms. The inhabitants of the Amazon region use, among others, the macerated bark of a plant popularly named "Pracaxi" (Pentaclethra macroloba Willd) to combat these effects. We report now the antihemorrhagic properties against snake venoms of the aqueous extract of Pentaclethra macroloba (EPema). EPema exhibited full inhibition of hemorrhagic and nucleolytic activities induced by several snake venoms. Additionally, partial inhibition of myotoxic, lethal, phospholipase and edema activities of snake venoms and its isolated PLA(2)s by EPema is reported. In vivo tests showed that EPema is able to totally inhibit a Bothrops jararacussu metalloprotease (BjussuMP-I) induced hemorrhage, suggesting interaction of the extract compounds with this high molecular weight protein. The extract did induce neither hemorrhage nor death in mice when administered alone by i.m. route. When administered separately by i.m. route, the extract did not induce death in mice at 12.5--300 mg/kg doses. Other assays demonstrated that EPema was unable to inhibit fibrinogenolytic and coagulant activities of Bothrops atrox venom. Although the mechanism of action of EPema is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. The search for new inhibitors of venom metalloproteases and DNAases are a relevant task. Investigation of snake venom inhibitors can provide useful tools for the elucidation of the action mechanisms of purified toxins. Furthermore, these inhibitors can be used as molecular models for development of new therapeutical agents in the treatment of ophidian accidents.