Arf1 promotes porcine intestinal epithelial cell proliferation via the mTORC1 signaling pathway.

The promotion of gut health, a pervasive problem in modern animal husbandry, positively affects organismal health, productivity, and economics. Porcine intestinal epithelial cells (IPEC-J2) continuously proliferate to maintain intestinal homeostasis, including barrier, immune, and absorptive functions. Gut homeostasis is fundamental to organismal health. ADP-ribosylation factor 1 (Arf1), a small GTPase, plays a crucial role in coordinating mTORC1 in response to nutrients, especially amino acid availability in the gut. mTORC1 is the central hub of proliferation. Thus, it seems likely that Arf1 promotes IPEC-J2 cell proliferation. However, the exact role of Arf1 in the porcine gut remains unclear. Therefore, we evaluated the functional role and possible mechanisms of Arf1 in the porcine intestine through Arf1 overexpression and knockdown in IPEC-J2 cells. Arf1 overexpression and knockdown significantly enhanced and inhibited, respectively, IPEC-J2 cell viability, and PCNA expression varied with Arf1 expression. Moreover, the proportion of Ki67-positive cells was significantly greater in the Arf1-overexpressing group than in the control group. These results suggest that Arf1 improves IPEC-J2 cell proliferation. The underlying mechanism was explored by Western blotting. Arf1 overexpression and knockdown significantly enhanced and suppressed, respectively, the levels of p-S6K1 and p-RPS6, which are key downstream targets of the mTORC1 signaling pathway. Collectively, our findings reveal the role of the Arf1-mTORC1 axis in IPEC-J2 cell proliferation and its potential function in regulating intestinal homeostasis and health.

Past and present: a bibliometric study on the treatment of recurrent ovarian cancer.

Background: Ovarian cancer (OC) is a gynecological malignancy with a high mortality rate worldwide. The unfavorable prognosis of OC is mainly attributed to the recurrent propensity. Recently, mortality from OC has exhibited a downward trend. These favorable patterns are likely to be driven by advancements in novel therapeutic regimens. However, there is a lack of visualize analysis of the application of these new drugs on women with recurrent OC (ROC). Therefore, we aimed to provide a bibliometric analysis of the evolving paradigms in the ROC treatment. Methods: Documents on ROC treatment were systematically collected from the MEDLINE database and Web of Science Core Collection (WOSCC). The retrieved documents were exported in the plain text file format, and files were named and saved to the paths specified by the Java application. Microsoft Excel (version 2010), Citespace (6.2.R4) and VOSviewer (1.6.19) were used for data analysis, and included the following: 1) annual publication trend; 2) contributions of countries, institutions and authors; 3) co-citation of journals and references; and 4) co-occurrence of keywords. Results: A total of 914 documents published in the MEDLINE and 9,980 ones in WOSCC were retrieved. There has been an upward trend in the productivity of publications on ROC treatment on by years. The United States was the leading contributor in this field, and the University of Texas System stood out as the most productive institution. Giovanni Scambia and Maurie Markman were the research leaders in the field of ROC treatment. The journal Gynecologic Oncology had the highest citation frequency. The reference entitled with "Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer" got highest centrality of 0.14 in the co-citation network. Keyword analysis revealed that the focus of current ROC treatment was on platinum-based anticancer drugs, paclitaxel, angiogenesis inhibitors (AIs), immune checkpoint inhibitors (ICIs) and poly (ADP-ribose) polymerase inhibitors (PARPis). Conclusion: Scholars from a multitude of countries have been instrumental in the advancement of ROC treatment. The research hotspots and trend in the field of predominantly originated from leading international journals and specialized periodicals focused on gynecologic oncology. Maintenance therapy using AIs or (and) PARPis has emerged as a significant complement to platinum-based chemotherapy for patients with ROC.

Activity of DNA Repair Systems in the Cells of Long-Lived Rodents and Bats.

Damages of various origin accumulated in the genomic DNA can lead to the breach of genome stability, and are considered to be one of the main factors involved in cellular senescence. DNA repair systems in mammalian cells ensure effective damage removal and repair of the genome structure, therefore, activity of these systems is expected to be correlated with high maximum lifespan observed in the long-lived mammals. This review discusses current results of the studies focused on determination of the DNA repair system activity and investigation of the properties of its key regulatory proteins in the cells of long-lived rodents and bats. Based on the works discussed in the review, it could be concluded that the long-lived rodents and bats in general demonstrate high efficiency in functioning and regulation of DNA repair systems. Nevertheless, a number of questions around the study of DNA repair in the cells of long-lived rodents and bats remain poorly understood, answers to which could open up new avenues for further research.

ER and SOCE Ca2+ signals are not required for directed cell migration in human iPSC-derived microglia.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

NAD+ overconsumption by poly (ADP-ribose) polymerase (PARP) under oxidative stress induces cytoskeletal disruption in vascular endothelial cell.

Vascular endothelial cytoskeletal disruption leads to increased vascular permeability and is involved in the pathogenesis and progression of various diseases. Oxidative stress can increase vascular permeability by weakening endothelial cell-to-cell junctions and decrease intracellular nicotinamide adenine dinucleotide (NAD+) levels. However, it remains unclear how intracellular NAD+ variations caused by oxidative stress alter the vascular endothelial cytoskeletal organization. In this study, we demonstrated that oxidative stress activates poly (ADP-ribose [ADPr]) polymerase (PARP), which consume large amounts of intracellular NAD+, leading to cytoskeletal disruption in vascular endothelial cells. We found that hydrogen peroxide (H2O2) could transiently disrupt the cytoskeleton and reduce intracellular total NAD levels in human umbilical vein endothelial cells (HUVECs). H2O2 stimulation led to rapid increase in ADPr protein levels in HUVECs. Pharmaceutical PARP inhibition counteracted H2O2-induced total NAD depletion and cytoskeletal disruption, suggesting that NAD+ consumption by PARP induced cytoskeletal disruption. Additionally, supplementation with nicotinamide mononucleotide (NMN), the NAD+ precursor, prevented both intracellular total NAD depletion and cytoskeletal disruption induced by H2O2 in HUVECs. Inhibition of the NAD+ salvage pathway by FK866, a nicotinamide phosphoribosyltransferase inhibitor, maintained H2O2-induced cytoskeletal disruption, suggesting that intracellular NAD+ plays a crucial role in recovery from cytoskeletal disruption. Our findings provide further insights into the potential application of PARP inhibition and NMN supplementation for the treatment and prevention of diseases involving vascular hyperpermeability.

Apurinic/apyrimidinic endonuclease 1 has major impact in prevention of suicidal covalent DNA-protein crosslink with apurinic/apyrimidinic site in cellular extracts.

DNA-protein crosslinks (DPC) are common DNA lesions induced by various external and endogenous agents. One of the sources of DPC is the apurinic/apyrimidinic site (AP site) and proteins interacting with it. Some proteins possessing AP lyase activity form covalent complexes with AP site-containing DNA without borohydride reduction (suicidal crosslinks). We have shown earlier that tyrosyl-DNA phosphodiesterase 1 (TDP1) but not AP endonuclease 1 (APE1) is able to remove intact OGG1 from protein-DNA adducts, whereas APE1 is able to prevent the formation of DPC by hydrolyzing the AP site. Here we demonstrate that TDP1 can remove intact PARP2 but not XRCC1 from covalent enzyme-DNA adducts with AP-DNA formed in the absence of APE1. We also analyzed an impact of APE1 and TDP1 on the efficiency of DPC formation in APE1-/- or TDP1-/- cell extracts. Our data revealed that APE1 depletion leads to increased levels of PARP1-DNA crosslinks, whereas TDP1 deficiency has little effect on DPC formation.

Expert consensus: Profiling and management of advanced or metastatic epithelial ovarian cancer / Consenso de expertos basado en la evidencia: perfilamiento y manejo del carcinoma epitelial de ovario de alto grado avanzado.

Introduction and objective: The approach to patients with advanced or metastatic high-grade epithelial ovarian cancer (EOC) has evolved over time with the advent of new therapies and multimodal strategies. The objective of this consensus of experts is to generate national recommendations for the profiling and management of advanced or metastatic high-grade OEC, defined as stages III and IV of the "The International Federation of Gynecology and Obstetrics (FIGO) classification at the time of diagnosis to base on the literature review that included international evidence-based clinical practice guidelines (CPG). Material and methods: Eleven panelists (oncologists and gynecological oncologists) answered 8 questions about the profiling and management of advanced or metastatic ovarian epithelial carcinoma. The panelists were chosen for their academic profile and influence in national health institutions. Guidelines from the "ESMO Standardized Operating Procedures Consensus Conference" were used to develop the consensus. It was agreed that the level of agreement to accept a recommendation should be ≥ 80%. The document was peer reviewed. Results: Eight general recommendations are made, which are presented into five domains. Some of these recommendations are subdivided into specific recommendations. Initial treatment Recommendation 1.1 Complete primary cytoreduction (PCS) surgery is suggested as the initial therapy of choice for patients with high-grade or metastatic EOC, which should ideally be carried out in centers with experience, followed by adjuvant therapy. 1.2 Neoadjuvant chemotherapy followed by interval cytoreduction surgery (ICS) is suggested in those who are unlikely to achieve a complete cytoreduction in PCS either due to unresectable metastatic disease or who present unresectability criteria (imaging, laparoscopic and/or by laparotomy) and that have been defined by a gynecological oncologist and patients with poor functional status and comorbidities according to the criteria of the multidisciplinary team (clinical oncology, gynecological oncology, radiology, etc.). Recommendation 2. In patients with high-grade epithelial ovarian cancer (EOC), in stage III locally advanced or metastatic, who received neoadjuvant chemotherapy and achieved a complete or partial response (cytoreduction with tumor residue < 2.5 mm), the use of Hyperthermic IntraPeritoneal Chemotherapy (HIPEC) could be considered as an alternative to standard platinum-based adjuvant intravenous chemotherapy during interval cytoreductive surgery, after discussion in a multidisciplinary tumor board, at a center experienced in treating this type of patients. Use of genetic testing. Recommendation 3. It is suggested at the time of diagnosis to offer molecular genetic testing to all patients with high-grade advanced or metastatic EOC regardless of family history. Recommendation 4. It is suggested to offer genetic counseling, by qualified personnel, to all patients with high-grade advanced or metastatic EOC who are ordered genetic testing. Recommendation 5. It is suggested that all patients with advanced or metastatic high-grade EOC undergo a germ panel that includes the Breast Cancer Susceptibility Genes 1/2 genes (BRCA 1/2) and the other susceptibility genes according to with institutional protocols and the availability of genetic testing panels; If it is negative, then somatic testing should be performed that includes the homologous recombination deficiency (HRD) status, regardless of family history. Adjuvant Therapy Recommendation 6. 6.1. It is suggested that all patients with advanced stage III/IV EOC, with PSC of (0-2), got adjuvant intravenous chemotherapy as standard treatment within six weeks after Prc. It is suggested paclitaxel/carboplatin. Recommendation 6.2. It is suggested to use standard chemotherapy base on platinum plus Bevacizumab as adjuvant chemotherapy to patients with high-risk disease (EOC stage IV or stage III with suboptimal tumor cytoreduction), following by bevacizumab as maintenance. The use of bevacizumab as maintenance therapy is not recommended if bevacizumab was not included in the first line of treatment. We suggested the dose used in GOG-0218 and ICON7 trials. Recommendation 6.3 It is suggested combined intravenous/intraperitoneal chemotherapy only for selected patients, with optimal cytoreduction (residual lesions < 1 cm), especially those without residual disease (R0) and who are evaluated in a multidisciplinary meeting. It is not considered standard treatment. Recommendation 6.4. 6.4.1 It is suggested to use Poly ADP ribose polymerase (PARP) inhibitors such as olaparib or niraparib as maintenance after receiving first-line chemotherapy in patients with stage III/IV BRCA1/2 positive EOC who received platinumbased chemotherapy and obtained complete response/partial response (CR/PR), 6.4.2 It is suggested to use olaparib alone or in combination with bevacizumab or niraparib in patients with stage III/IV BRCA1/2 positive EOC who received platinum-based chemotherapy plus bevacizumab and achieved CR/PR. 6.4.3 It is suggested to use niraparibin patients with stage III/IV BRCA1/2 negative or unknown EOC who received platinum-based chemotherapy and achieved CR/PR. 6.4.4 It is suggested to use bevacizumab or olaparib plus bevacizumab in patients with EOC stage III/IV BRCA1/2 negative or unknown (HRD positive) who received platinum-based chemotherapy plus bevacizumab and obtained CR/PR. Treatment of disease relapse Recommendation 7. Secondary cytoreductive surgery followed by chemotherapy is suggested for selected patients with high-grade advanced EOC in first relapse, platinum-sensitive (platinum-free interval ≥ 6 months), positive "Arbeitsgemeinschaft Gynäkologische Onkologie ­ AGO" score or "I-model" positive (< 4.7) with a potential resection to R0 in centers with access to optimal surgical and postoperative support. Note: Platinum-free interval and AGO score have only been developed as positive predictors of complete resection and not to exclude patients from surgery. Recommendation 8. 8.1 For patients with relapse advanced high-grade EOC platinum-sensitive, the following is suggested: Platinum-based combination chemotherapy: carboplatin/liposomal doxorubicin or carboplatin/paclitaxel or carboplatin/nab-paclitaxel or carboplatin/docetaxel or carboplatin/gemcitabine) for six cycles. If combination therapy is not tolerated, give carboplatin or cisplatin alone. Combination chemotherapy (carboplatin/gemcitabine or carboplatin/paclitaxel or carboplatin/doxorubicin liposomal) plus bevacizumab followed by bevacizumab as maintenance (until progression or toxicity). Recommendation 8.2 For patients with relapsed advanced high-grade EOC platinum-resistant, it is suggested: Sequential treatment with chemotherapy, preferably with a non-platinum single agent (weekly paclitaxel or pegylated liposomal doxorubicin or docetaxel or oral etoposide or gemcitabine or trabectidine or, topotecan). Weekly paclitaxel or pegylated liposomal doxorubicin or topotecan could be administrate with or without bevacizumab. Other agents are considered potentially active (capecitabine, cyclophosphamide, ifosfamide, irinotecan, oxaliplatin, pemetrexed, vinorelbine, cyclophosphamide) could be recommended for later lines. Hormone receptor-positive patients who do not tolerate or have no response to cytotoxic regimens may receive hormone therapy with tamoxifen or other agents, including aromatase inhibitors (anastrozole and letrozole) or leuprolide acetate, or megestrol acetate. Patients with a performance score ≥ 3 should be considered only for best supportive care. Recommendation 8.3 Maintenance therapy with PARP inhibitors: It is suggested in patients with relapse advanced high-grade EOC stage III/IV BRCA1/2 (positive, negative or unknown) who have received two or more lines of platinum-based chemotherapy and have achieved CR/PR, use olaparib, niraparib or rucaparib. Niraparib could be useful in BRCA 1/2 +/-/unknown patients, as rucaparib, however, the latter does not yet have approval from the regulatory office in Colombia. Conclusions: It is expected that the recommendations issued in this consensus will contribute to improving clinical care, oncological impact, and quality of life of these women.

Introducción y objetivo: el abordaje de pacientes con cáncer epitelial de ovario (CEO) de alto grado avanzado o metastásico ha ido evolucionando a través del tiempo con el advenimiento de nuevas terapias y estrategias multimodales. El objetivo de este consenso de expertos es generar recomendaciones nacionales para el perfilamiento y manejo del CEO de alto grado avanzado o metastásico, definido como estadios III y IV de la clasificación de la Federación Internacional de Ginecología y Obstetricia (FIGO) al momento del diagnóstico, a partir de la revisión de la literatura que incluyó guías de práctica clínica (GPC) internacionales basadas en la evidencia. Materiales y métodos: once panelistas (oncólogos y ginecólogos oncólogos) respondieron ocho preguntas sobre el perfilamiento y manejo del carcinoma epitelial de ovario avanzado o metastásico. Los panelistas fueron escogidos por su perfil académico e influencia en instituciones de salud nacionales. Para el desarrollo del consenso se utilizaron los lineamientos de la "Conferencia de consenso de procedimientos operativos estandarizados de ESMO". Se definió que el nivel de acuerdo para aceptar una recomendación debía ser ≥ 80%. El documento fue revisado por pares. Resultados: Se hacen 8 recomendaciones generales, presentadas en cinco dominios; algunas de ellas se subdividen en recomendaciones específicas. Tratamiento inicial Recomendación 1 1.1. Como terapia inicial de elección para pacientes con CEO de alto grado o metastásico se sugiere la cirugía de citorreducción primaria (Cpr) completa que, idealmente, debe realizarse en centros con experiencia, seguida de terapia adyuvante. 1.2. Se sugiere quimioterapia neoadyuvante seguida de cirugía de citorreducción de intervalo (Cint) en quienes sea improbable alcanzar una citorreducción completa en la Cpr, bien sea por enfermedad metastásica no resecable o que presenten criterios de irresecabilidad (imagenológicos, laparoscópicos o por laparotomía) que hayan sido definidos por un ginecólogo oncólogo. También en pacientes con un pobre estado funcional y comorbilidades de acuerdo con el criterio del equipo multidisciplinario (oncología clínica, ginecología oncológica, radiología, etc.). Recomendación 2. En pacientes con CEO de alto grado, en estadio III localmente avanzado o metastásico, que recibieron quimioterapia neoadyuvante y alcanzaron respuesta completa o parcial (citorreducción con residuo tumoral < 2,5 mm), se podría evaluar el uso de la quimioterapia intraperitoneal hipertérmica (Hyperthermic IntraPeritoneal Chemotherapy - HIPEC) como alternativa a la quimioterapia IV adyuvante estándar basada en platinos durante la Cint, previa discusión en junta multidisciplinaria, en un centro de experiencia en este tipo de pacientes. Uso de pruebas genéticas Recomendación 3. Al momento del diagnóstico, se sugiere ofrecer testeo molecular genético a toda paciente con CEO de alto grado avanzado o metastásico, independientemente de la historia familiar. Recomendación 4. Se sugiere ofrecer asesoramiento genético, por parte de personal calificado, a toda paciente con CEO de alto grado avanzado o metastásico a quien se le ordene un testeo genético. Recomendación 5. Se sugiere que a toda paciente con CEO de alto grado avanzado o metastásico se le realice panel germinal que incluya los genes de susceptibilidad al cáncer de mama 1/2 (BRCA 1/2) y los otros genes de susceptibilidad de acuerdo con los protocolos institucionales y la disponibilidad de paneles de testeo genético; si es negativo entonces se debería realizar testeo somático que incluya el estatus de deficiencia de la recombinación homóloga (homologous recombination deficiency - HRD), independientemente de la historia familiar. Terapia adyuvante Recomendación 6 6.1. Se sugiere que a toda paciente con CEO estadios III/IV avanzado o metastásico, con estatus de desempeño (performance score care - PSC) de 0-2 se le administre como tratamiento estándar quimioterapia intravenosa (IV) adyuvante dentro de las seis semanas posteriores a la Cpr. Se sugiere administrar paclitaxel/carboplatino. 6.2. Se sugiere utilizar quimioterapia estándar basada en platino más bevacizumab como adyuvancia en pacientes con enfermedad de alto riesgo (CEO estadios IV o III con citorreducción tumoral subóptima), continuando con bevacizumab como mantenimiento. No se recomienda el uso de bevacizumab como terapia de mantenimiento si no se incluyó en la primera línea de tratamiento. Se sugiere seguir los esquemas de los estudios Gynecologic Oncology Group Study (GOG-0218) e International Collaborative Ovarian Neoplasm (ICON7). 6.3. Se sugiere la quimioterapia combinada IV/intraperitoneal (IP) solo para pacientes seleccionadas, con una citorreducción óptima (lesiones residuales < 1 cm), en especial aquellas sin enfermedad residual (R0) y que sean evaluadas en junta multidisciplinaria. La quimioterapia combinada IV/IP no se considera como tratamiento estándar. 6.4. 6.4.1. Se sugiere utilizar inhibidores de poli(ADP-ribosa) polimerasa (PARP) tales como olaparib o niraparib como mantenimiento después de recibir una primera línea de quimioterapia en pacientes con CEO estadios III/IV BRCA1/2 positivo que recibieron quimioterapia basada en platino y obtuvieron respuesta completa/respuesta parcial (RC/RP). 6.4.2. Se sugiere utilizar olaparib solo o en combinación con bevacizumab o niraparib en pacientes con CEO estadios III/IV BRCA1/2 positivo que recibieron quimioterapia basada en platino más bevacizumab y obtuvieron RC/RP. 6.4.3. Se sugiere utilizar niraparib en pacientes con CEO estadio III/IV BRCA1/2 negativo o desconocido que recibieron quimioterapia basada en platino y obtuvieron RC/RP. 6.4.4. Se sugiere utilizar bevacizumab u olaparib más bevacizumab en pacientes con CEO estadios III/IV BRCA1/2 negativo o desconocido (HRD positivo) que recibieron quimioterapia basada en platino más bevacizumab y obtuvieron RC/RP. Tratamiento de la recaída de la enfermedad Recomendación 7. Se sugiere la realización de la cirugía de citorreducción secundaria (Csec), seguida de quimioterapia, a pacientes seleccionadas con CEO de alto grado avanzado o metastásico en primera recaída, platino-sensibles (intervalo libre de platinos ≥ 6 meses), puntuación Arbeitsgemeinschaft Gynäkologische Onkologie (AGO) positiva o Integrate model (I-Model) positivo (< 4,7), y con una potencial resección a R0, en centros con acceso a soporte quirúrgico y posoperatorio óptimo. Nota: el intervalo libre de tratamiento con platinos y la puntuación AGO solo se han desarrollado como predictores positivos de resección completa y no para excluir a las pacientes de la cirugía. Recomendación 8 8.1. Para pacientes con CEO de alto grado avanzado o metastásico en recaída platino-sensibles se sugiere: Quimioterapia combinada basada en platino: carboplatino/doxorrubicina liposomal o carboplatino/paclitaxel o carboplatino/ nab-paclitaxel o carboplatino/docetaxel o carboplatino/gemcitabina, por seis ciclos. Si no se tolera la terapia combinada, dar carboplatino o cisplatino solo. Quimioterapia combinada: carboplatino/gemcitabina o carboplatino/paclitaxel o carboplatino/doxorubicina liposomal, más bevacizumab, seguida de bevacizumab como mantenimiento (hasta progresión o toxicidad). 8.2. Para pacientes con CEO de alto grado avanzado o metastásico en recaída, platino-resistentes, se sugiere: Tratamiento secuencial con quimioterapia, preferiblemente con un agente único que no sea un platino (paclitaxel semanal o doxorrubicina liposomal pegilada o docetaxel o etopósido oral o gemcitabina o trabectidina o topotecan). El paclitaxel semanal o la doxorrubicina liposomal pegilada o el topotecan pueden ser administrados con o sin bevacizumab. Existen otros agentes que se consideran potencialmente act ivos (capecitabina, ciclofosfamida, ifosfamida, irinotecán, oxaliplatino, pemetrexed, vinorelbina, ciclofosfamida), que se podrían recomendar para líneas posteriores. Las pacientes con receptores hormonales positivos que no toleran o no tienen respuesta a los regímenes citotóxicos pueden recibir terapia hormonal con tamoxifeno u otros agentes, incluidos los inhibidores de la aromatasa (anastrozol y letrozol) o acetato de leuprolide o acetato de megestrol. Pacientes con PSC ≥ 3 deberían ser consideradas solo para el mejor cuidado de soporte. 8.3. Terapia de mantenimiento con inhibidores PARP. Para pacientes con CEO de alto grado avanzado o metastásico en recaída estadios III/IV BRCA1/2 (positivo, negativo o desconocido), que hayan recibido dos o más líneas de quimioterapia basada en platino y hayan alcanzado RC/RP, se sugiere utilizar olaparib, niraparib o rucaparib. El niraparib podría ser útil en pacientes BRCA 1/2 +/-/desconocido, al igual que el rucaparib, sin embargo, este último no tiene aún aprobación del ente regulador en Colombia. Conclusiones: se espera que las recomendaciones emitidas en este consenso contribuyan a mejorar la atención clínica, el impacto oncológico y la calidad de vida de estas mujeres.

Health impact evaluation of Aspirational Districts Program in India: Evidence from National Family Health Survey.

The Aspirational District Program (ADP) is a unique initiative of Government of India launched in 2018 that aims to reduce inter-district multidimensional inequality. ADP aims to bring the least developed districts to catch up with the rest of the other districts in the country. The program is comprehensive in its scope as it targets improvement of several key development indicators spanning health and nutrition, education, agriculture and water resources, financial inclusion and skill development and basic infrastructure indicators. Aspirational districts (ADs) are eligible for enhanced funding and priority allocation of various initiatives undertaken by the central and the state governments. Our research estimates the causal impact of ADP on the targeted health and nutrition indicators using a combination of propensity score matching and difference-in-differences (PSM-DID). We use the fourth and fifth rounds of National Family Health Survey (NFHS) data collected in 2015-16 and 2019-21 respectively which serve as the pre and post-treatment data for our analysis. Moreover, we take advantage of the transparent mechanism outlined for the identification of ADs under ADP, which we use for propensity score matching for our PSM-DID. While we observe negative impact of ADP on early initiation of breastfeeding, we believe that the impact is confounded with the effects of Covid-19 since part of NFHS-5 data was collected during the pandemic. However, the negative impact of ADP on early initiation of breastfeeding disappears when we only use pre-covid data (i.e. data for districts from states surveyed before the pandemic). Additionally, using pre-covid data we find a reduction in the prevalence of underweight children younger than 5 years to an extent of 2 to 4 percentage points in ADs as an impact of ADP, which is robust across multiple specification. We do not find evidence of a positive or a negative impact of ADP on any other health and nutrition indicators. Future research efforts should be made towards impact evaluation of all the targeted indicators in order to get a comprehensive unbiased evaluation of ADP.

Acetylation, ADP-ribosylation and methylation of malate dehydrogenase.

Metabolism within an organism is regulated by various processes, including post-translational modifications (PTMs). These types of chemical modifications alter the molecular, biochemical, and cellular properties of proteins and allow the organism to respond quickly to different environments, energy states, and stresses. Malate dehydrogenase (MDH) is a metabolic enzyme that is conserved in all domains of life and is extensively modified post-translationally. Due to the central role of MDH, its modification can alter metabolic flux, including the Krebs cycle, glycolysis, and lipid and amino acid metabolism. Despite the importance of both MDH and its extensively post-translationally modified landscape, comprehensive characterization of MDH PTMs, and their effects on MDH structure, function, and metabolic flux remains underexplored. Here, we review three types of MDH PTMs - acetylation, ADP-ribosylation, and methylation - and explore what is known in the literature and how these PTMs potentially affect the 3D structure, enzymatic activity, and interactome of MDH. Finally, we briefly discuss the potential involvement of PTMs in the dynamics of metabolons that include MDH.

Structural Basis of Nucleotide Selectivity in Pyruvate Kinase.

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

An E3 ubiquitin ligase localization screen uncovers DTX2 as a novel ADP-ribosylation-dependent regulator of DNA double-strand break repair.

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Adaptive genetic mechanisms in mammalian Parp1 locus.

PARP1 is a highly conserved nuclear protein in multicellular organisms that by modulating chromatin opening facilitates gene expression during development. All reported Parp1 null knockout mice strains are viable with no developmental anomalies. It was believed that functional redundancy with other PARP family members, mainly PARP2, explain such a controversy. However, while PARP2 has similar catalytic domain to PARP1, it lacks other domains, making the absence of developmental problems in Parp1 mice knockouts unlikely. Contrary to prior assumptions, in our analysis of the best-investigated Parp1 knockout mice strain we identified persistent mRNA expression, albeit at reduced levels. Transcript analysis revealed an alternatively spliced Parp1 variant lacking exon two. Subsequent protein analysis confirmed the existence of a truncated PARP1 protein in knockout mice. The decreased level of pADPr was detected in Parp1 knockout ES cells with Western Blotting analysis, but immunofluorescence staining did not detect any difference in distribution or level of pADPr in nuclei of knockout ES cells. pADPr level in double Parp1 Parg mutant ES cells greatly exceeded its amount in normal and even in hypomorph Parg mutant ES cells, suggesting the presence of functionally active PARP1. Therefore, our findings challenge the conventional understanding of PARP1 depletion effects.

Niraparib in Japanese patients with platinum-sensitive recurrent ovarian cancer: final results of a multicenter phase 2 study.

OBJECTIVE: This study evaluated the long-term safety and efficacy of niraparib in Japanese patients with platinum-sensitive recurrent ovarian cancer. METHODS: This was a follow-up analysis of a phase 2, multicenter, open-label, single-arm study in Japanese women with platinum-sensitive, relapsed ovarian cancer. Participants received niraparib (starting dose 300 mg) once daily in continuous 28-day cycles. The primary endpoint was the incidence of Grade 3 or 4 thrombocytopenia-related events (defined as the overall incidence of the MedDRA Preferred Terms "thrombocytopenia" and "platelet count decreased") occurring in the 30 days after initial administration of niraparib, and secondary endpoints included evaluation of treatment-emergent adverse events and progression-free survival. RESULTS: Nineteen patients (median age, 62 years; median body weight, 53.9 kg) were enrolled. As previously reported, the incidence of Grade 3 or 4 thrombocytopenia-related events during the first 30 days of treatment was 31.6%. At data cutoff, median (range) treatment exposure was 504.0 (56-1,054) days and mean ± standard deviation dose intensity was 154.4±77.5 mg/day. The most common treatment-emergent adverse events were nausea (n=14, 73.7%), decreased platelet count (n=12, 63.2%), decreased neutrophil count (n=11, 57.9%), anemia, vomiting, and decreased appetite (all n=9, 47.4%). One patient was diagnosed with treatment-related leukemia, which resulted in death. Median (95% confidence interval) progression-free survival was 18.0 (5.6-26.7) months. CONCLUSION: Overall, the safety profile of niraparib was considered manageable in this study population of Japanese patients with platinum-sensitive, relapsed ovarian cancer and was consistent with that observed in studies of non-Japanese patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03759587.

Intraocular kinetics of pathological ATP after photoreceptor damage in rhegmatogenous retinal detachment.

PURPOSE: Extracellular Adenosine triphosphate (ATP) released by dying cells may cause a secondary cell death in neighboring cells in retinal degeneration. We investigated intraocular ATP kinetics to gain mechanical insights into the pathology in rhegmatogenous retinal detachment (RRD). STUDY DESIGN: Retrospective clinical study. METHODS: Vitreous or subretinal fluids (SRF) were obtained from patients with RRD (n=75), macular hole (MH; n=20), and epiretinal membrane (ERM; n=35) during vitrectomy. ATP levels in those samples were measured by luciferase assay. RESULTS: Mean ATP levels in the vitreous from RRD patients were significantly higher compared to those from MH and ERM patients (2.3 and 0.3 nM, respectively. P<0.01). Mean ATP levels in the SRF from RRD (11.7 nM) were higher than those in the vitreous from RRD (P<0.01). Mean ATP levels in the vitreous with short durations (1-8 days) of RRD were higher compared to those with long durations (>8 days) (3.2 and 1.4 nM, respectively. P<0.05). Similarly, ATP in SRF with short durations were higher than those with long durations (23.8 and 3.6 nM, respectively. P<0.05). Furthermore, the concentrations of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), a major ATP degradative enzyme, in the vitreous from RRD were higher than those from MH/ERM (1.2 and 0.2 ng/ml, respectively. P<0.01). ENTPD1 expression was localized in the cytoplasm of CD11b-positive infiltrating cells in the vitreous and retinal cells. CONCLUSION: ATP increased in the vitreous and SRF in RRD and decreased over time with an upregulation of ENTPD1. The kinetics indicate the pathological mechanism of the excessive extracellular ATP after RRD.

General ADP-Ribosylation Mechanism Based on the Structure of ADP-Ribosyltransferase-Substrate Complexes.

ADP-ribosylation is a ubiquitous modification of proteins and other targets, such as nucleic acids, that regulates various cellular functions in all kingdoms of life. Furthermore, these ADP-ribosyltransferases (ARTs) modify a variety of substrates and atoms. It has been almost 60 years since ADP-ribosylation was discovered. Various ART structures have been revealed with cofactors (NAD+ or NAD+ analog). However, we still do not know the molecular mechanisms of ART. It needs to be better understood how ART specifies the target amino acids or bases. For this purpose, more information is needed about the tripartite complex structures of ART, the cofactors, and the substrates. The tripartite complex is essential to understand the mechanism of ADP-ribosyltransferase. This review updates the general ADP-ribosylation mechanism based on ART tripartite complex structures.

Development and Characterization of Recombinant ADP-Ribose Binding Reagents that Allow Simultaneous Detection of Mono and Poly ADP-Ribose.

ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADP-ribosylation. Recently we developed a set of recombinant antibody-like ADP-ribose binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally-occuring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunogical tools, recognize specific ADP-ribose moieties, and can be used for simultaneous detection of MAR and PAR at single cell resolution in various antibody-based assays . The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.

Pathological and physiological roles of ADP-ribosylation: established functions and new insights.

The posttranslational modification of proteins with poly(ADP-ribose) was discovered in the sixties. Since then, we have learned that the enzymes involved, the so-called poly(ADP-ribosyl)polymerases (PARPs), are transferases which use cofactor NAD+ to transfer ADP-ribose to their targets. Few PARPs are able to create poly(ADP-ribose), whereas the majority transfers a single ADP-ribose. In the last decade, hydrolases were discovered which reverse mono(ADP-ribosyl)ation, detection methods were developed and new substrates were defined, including nucleic acids. Despite the continued effort, relatively little is still known about the biological function of most PARPs. In this review, we summarise key functions of ADP-ribosylation and introduce emerging insights.

Assessment of ATP metabolism to adenosine by ecto-nucleotidases carried by tumor-derived small extracellular vesicles.

Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression.Here, the ATP pathway of ADO production (ATP →  ADP →  AMP →  ADO) by ecto-nucleotidases carried on the sEV surface was evaluated by a method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates for enzymatic activity. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL).Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP. MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation by both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by ecto-nucleotidases located on the sEV surface.The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of ecto-nucleotidase activity primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.

ADP-ribosylome analysis reveals homogeneous DNA-damage-induced serine ADP-ribosylation across wild-type and BRCA-mutant breast cancer cell lines.

ADP-ribosylation (ADPr) signaling plays a crucial role in DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage, poly(ADP-ribose) polymerase 1 (PARP1), are used to treat patients with breast cancer harboring BRCA1/2 mutations. However, resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To understand the role of ADPr in PARPi sensitivity, we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze ADPr in six breast cancer cell lines exhibiting different PARPi sensitivities. We identify 1,632 sites on 777 proteins across all cell lines, primarily on serine residues, with site-specific overlap of targeted residues across DNA-damage-related proteins across all cell lines, demonstrating high conservation of serine ADPr-signaling networks upon DNA damage. Furthermore, we observe site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants and unique ADPr sites in PARPi-resistant BRCA-mutant HCC1937 cells, which have low poly(ADP-ribose) glycohydrolase (PARG) levels and longer ADPr chains on PARP1.

Tankyrase1/2 inhibitor XAV-939 reverts EMT and suggests that PARylation partially regulates aerobic activities in human hepatocytes and HepG2 cells.

The maintenance of a highly functional metabolic epithelium in vitro is challenging. Metabolic impairments in primary human hepatocytes (PHHs) over time is primarily due to epithelial-to-mesenchymal transitioning (EMT). The immature hepatoma cell line HepG2 was used as an in vitro model to explore strategies for enhancing the hepatic phenotype. The phenotypic characterization includes measuring the urea cycle, lipid storage, tricarboxylic acid-related metabolites, reactive oxygen species, endoplasmic reticulum calcium efflux, mitochondrial membrane potentials, oxygen consumptions rate, and CYP450 biotransformation capacity. Expression studies were performed with transcriptomics, co-immunoprecipitation and proteomics. CRISPR/Cas9 was also employed to genetically engineer HepG2 cells. After confirming that PHHs develop an EMT phenotype, expression of tankyrase1/2 was found to increase over time. EMT was reverted when blocking tankyrases1/2-dependent poly-ADP-ribosylation (PARylation) activity, by biochemical and genetic perturbation. Wnt/ß-catenin inhibitor XAV-939 blocks tankyrase1/2 and treatment elevated several oxygen-consuming reactions (electron-transport chain, OXHPOS, CYP450 mono-oxidase activity, phase I/II xenobiotic biotransformation, and prandial turnover), suggesting that cell metabolism was enhanced. Glutathione-dependent redox homeostasis was also significantly improved in the XAV-939 condition. Oxygen consumption rate and proteomics experiments in tankyrase1/2 double knockout HepG2 cells then uncovered PARylation as master regulator of aerobic-dependent cell respiration. Furthermore, novel tankyrase1/2-dependent PARylation targets, including mitochondrial DLST, and OGDH, were revealed. This work exposed a new mechanistic framework by linking PARylation to respiration and metabolism, thereby broadening the current understanding that underlies these vital processes. XAV-939 poses an immediate and straightforward strategy to improve aerobic activities, and metabolism, in (immature) cell cultures.