RAD51 foci as a biomarker predictive of platinum chemotherapy response in ovarian cancer

PURPOSE: To determine the ability of RAD51 foci to predict platinum chemotherapy response in high-grade serous ovarian cancer (HGSOC) patient-derived samples. EXPERIMENTAL DESIGN: RAD51 and γH2AX nuclear foci were evaluated by immunofluorescence in HGSOC patient-derived cell lines (n = 5), organoids (n = 11), and formalin-fixed, paraffin-embedded tumor samples (discovery n = 31, validation n = 148). Samples were defined as RAD51-High if \u3e10% of geminin-positive cells had ≥5 RAD51 foci. Associations between RAD51 scores, platinum chemotherapy response, and survival were evaluated. RESULTS: RAD51 scores correlated with in vitro response to platinum chemotherapy in established and primary ovarian cancer cell lines (Pearson r = 0.96, P = 0.01). Organoids from platinum-nonresponsive tumors had significantly higher RAD51 scores than those from platinum-responsive tumors (P \u3c 0.001). In a discovery cohort, RAD51-Low tumors were more likely to have a pathologic complete response (RR, 5.28; P \u3c 0.001) and to be platinum-sensitive (RR, ∞; P = 0.05). The RAD51 score was predictive of chemotherapy response score [AUC, 0.90; 95% confidence interval (CI), 0.78-1.0; P \u3c 0.001). A novel automatic quantification system accurately reflected the manual assay (92%). In a validation cohort, RAD51-Low tumors were more likely to be platinum-sensitive (RR, ∞; P \u3c 0.001) than RAD51-High tumors. Moreover, RAD51-Low status predicted platinum sensitivity with 100% positive predictive value and was associated with better progression-free (HR, 0.53; 95% CI, 0.33-0.85; P \u3c 0.001) and overall survival (HR, 0.43; 95% CI, 0.25-0.75; P = 0.003) than RAD51-High status. CONCLUSIONS: RAD51 foci are a robust marker of platinum chemotherapy response and survival in ovarian cancer. The utility of RAD51 foci as a predictive biomarker for HGSOC should be tested in clinical trials

Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works

Characterization of a Novel Interaction between Bcl-2 Members Diva and Harakiri

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding

Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first time, evidence that Puma is subject to post-translational control through phosphorylation. We show that Puma is phosphorylated at multiple sites, with the major site of phosphorylation being serine 10. Replacing serine 10 with alanine causes reduced Puma turnover and enhanced cell death. Interestingly, Puma turnover occurs through the proteasome, and substitution of serine 10 causes elevated Puma levels independently of macroautophagy, Bcl-2 family member binding, caspase activity and apoptotic death. We conclude, therefore, that phosphorylation of Puma at serine 10 promotes Puma turnover, represses Puma's cell death potential and promotes cell survival. Owing to the highly pro-apoptotic nature of Puma, these studies highlight an important additional regulatory step in the determination of cellular life or death

Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works

Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-xAK, a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-xAK may trigger apoptosis

Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production

HCV genome-wide genetic analyses in context of disease progression and hepatocellular carcinoma

<div><p>Hepatitis C virus (HCV) is a major cause of hepatitis and hepatocellular carcinoma (HCC) world-wide. Most HCV patients have relatively stable disease, but approximately 25% have progressive disease that often terminates in liver failure or HCC. HCV is highly variable genetically, with seven genotypes and multiple subtypes per genotype. This variation affects HCV’s sensitivity to antiviral therapy and has been implicated to contribute to differences in disease. We sequenced the complete viral coding capacity for 107 HCV genotype 1 isolates to determine whether genetic variation between independent HCV isolates is associated with the rate of disease progression or development of HCC. Consensus sequences were determined by sequencing RT-PCR products from serum or plasma. Positions of amino acid conservation, amino acid diversity patterns, selection pressures, and genome-wide patterns of amino acid covariance were assessed in context of the clinical phenotypes. A few positions were found where the amino acid distributions or degree of positive selection differed between in the HCC and cirrhotic sequences. All other assessments of viral genetic variation and HCC failed to yield significant associations. Sequences from patients with slow disease progression were under a greater degree of positive selection than sequences from rapid progressors, but all other analyses comparing HCV from rapid and slow disease progressors were statistically insignificant. The failure to observe distinct sequence differences associated with disease progression or HCC employing methods that previously revealed strong associations with the outcome of interferon α-based therapy implies that variable ability of HCV to modulate interferon responses is not a dominant cause for differential pathology among HCV patients. This lack of significant associations also implies that host and/or environmental factors are the major causes of differential disease presentation in HCV patients.</p></div

Популяционный иммунитет к SARS-COV-2 населения Калиниградской области в эпидемический сезон COVID-19

Introduction. The COVID-19 pandemic was announced by WHO in February 2020. In the Kaliningrad region, the first case (imported) was registered in early March 2020, the beginning of the epidemic increase fell on the 14th week. 2020, and the peak incidence was reached in the 22nd week of the year, after which there was a steady decrease in the number of cases. The study of population immunity was carried out at the 32nd week during the period of the lowest level of intensity of the epidemic process.Purpose of the study. Assessment of the assessment of the level of population immunity to the SARS-CoV-2 virus among the population of the Kaliningrad region during the period of the epidemic incidence of the population of COVID-19.Materials and methods. The study was carried out as part of the first stage of the Rospotrebnadzor program to assess population immunity to SARS-CoV-2 among the population of the Russian Federation. The selection of volunteers for the study was carried out by a questionnaire survey and subsequent randomization. The analysis includes the results of a survey of 2675 people. The number of volunteers in age groups ranged from 314 to 493 people. The results obtained were processed by the methods of variation statistics.Results. The results obtained showed that the average seroprevalence in the population was 50.2%, while the highest seroprevalence was found in the child age group 1-17 years (66.9%) and among persons aged 18-29 (57.0). No significant gender differences were found (men – 48,3 ± 1,6%, women – 51,1 ± 1,1%). The distribution of the proportion of seropositive people in the settlements of the region varied from 33,9% to 59.6%. The largest share of seroprevalence in the representative samples was found among people engaged in art / creativity (55,3%), the smallest – among educational workers (42,0%). Among COVID-19 convalescents, the level of humoral immunity reached 94,6%. Most of the seropositive volunteers (95,2%) did not have any symptoms of COVID-19, that is, they belonged to the category of asymptomatic carriers.Output. The results of a survey of a representative cohort of volunteers in the Kaliningrad region showed that they are characterized by a high level of population immunity, which makes it possible to expect a decrease in.Введение. Пандемия COVID-19 была объявлена Всемирная организация здравоохранения в феврале 2020 г. В Калининградской области первый случай (завозной) зарегистрировали в начале марта 2020 г., начало эпидемического нарастания пришлось на 14-ю неделю 2020 г., а пик заболеваемости был достигнут на 22-й неделе года, после чего отмечалось устойчивое снижение количества заболевший. Исследование популяционного иммунитета было проведено на 32-й неделе в период самого низкого уровня напряженности эпидемического процесса.Цель. Оценка уровня популяционного иммунитета к вирусу SARS-CoV-2 среди населения Калининградской области в период эпидемической заболеваемости населения COVID-19.Материалы и методы. Исследование проведено в рамках первого этапа программы Роспотребнадзора по оценке популяционного иммунитета к SARS-CoV-2 среди населения Российской Федерации. Отбор волонтеров для исследования проводили методом анкетирования и последующей рандомизации. В анализ включены результаты обследования 2675 человек. Количество волонтеров в возрастных группах варьировало от 314 до 493 человек. Полученные результаты обрабатывали методами вариационной статистики.Результаты. Полученные результаты показали, что средняя серопревалентность по популяции составила 50,2%, при этом набольшая серопревалентность была выявлена в детской возрастной группе 1–17 лет (66,9%) и среди лиц в возрасте 18–29 лет (57,0). Достоверных половых различий не установлено (мужчины – 48,3±1,6%, женщины – 51,1±1,1%). Распределение доли серопозитивных по населенным пунктам области варьировало от 33,9% до 59,6%. Наибольшая доля серопревалентных в репрезентативных выборках выявлена среди лиц, занятых искусством/творчеством (55,3%), наименьшая – среди работников образования (42,0%). Среди реконвалесцентов COVID-19 уровень гуморального иммунитета достиг 94,6%. Большая часть серопозитивных волонтеров (95,2%) не имела каких-либо симптомов COVID-19, то есть относилась к категории бессимптомных носителей.Выводы. Результаты обследования репрезентативной когорты волонтеров Калининградской области показали, что для них характерен высокий уровень популяционного иммунитета, позволяющий ожидать снижения напряженности эпидемического процесса.