Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Implante Inmediato en Zona Estética Anterior con Pérdida Parcial de la Cortical Vestibular: Reporte De Caso y Revisión de Literatura

El tratamiento con implantes dentales hoy en día es un procedimiento clínico de rutina que permite rehabilitar a los pacientes con prótesis fijas. En este caso presentamos un tratamiento complejo de implantación inmediata del sector anterior con pérdida parcial de la cortical vestibular en el que se realizó una regeneración ósea guiada y provisionalización en un tiempo quirúrgico en un paciente con patología renal. Complementamos el estudio con una revisión de la efectividad de las técnicas utilizadas y las posibles respuestas celular asociadas a la patología renal.

Treatment with dental implants nowadays is a routine clinical procedure that allows patient rehabilitation with fixed prostheses. In this case we present a complex treatment of immediate implantation of the anterior sector with partial loss of the vestibular cortex, in which guided bone regeneration and provisionalization was performed in surgical time in a patient with kidney pathology. The study was complemented with a review of the effectiveness of the techniques used and the possible cellular responses associated with kidney pathology.

The role of chaperone-mediated autophagy in drug resistance.

In the search for alternatives to overcome the challenge imposed by drug resistance development in cancer treatment, the modulation of autophagy has emerged as a promising alternative that has achieved good results in clinical trials. Nevertheless, most of these studies have overlooked a novel and selective type of autophagy: chaperone-mediated autophagy (CMA). Following its discovery, research into CMA's contribution to tumor progression has accelerated rapidly. Therefore, we now understand that stress conditions are the primary signal responsible for modulating CMA in cancer cells. In turn, the degradation of proteins by CMA can offer important advantages for tumorigenesis, since tumor suppressor proteins are CMA targets. Such mutual interaction between the tumor microenvironment and CMA also plays a crucial part in establishing therapy resistance, making this discussion the focus of the present review. Thus, we highlight how suppression of LAMP2A can enhance the sensitivity of cancer cells to several drugs, just as downregulation of CMA activity can lead to resistance in certain cases. Given this panorama, it is important to identify selective modulators of CMA to enhance the therapeutic response.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice.

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

Atypical teratoid/rhabdoid tumoroids reveal subgroup-specific drug vulnerabilities.

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.

Using design of experiments (DoE) to optimize performance and stability of biomimetic cell membrane-coated nanostructures for cancer therapy.

Introduction: Cell membrane-covered biomimetic nanosystems have allowed the development of homologous nanostructures to bestow nanoparticles with enhanced biointerfacing capabilities. The stability of these structures, however, still represents a challenge for the scientific community. This study is aimed at developing and optimizing cell derived membrane-coated nanostructures upon applying design of experiments (DoE) to improve the therapeutic index by homotypic targeting in cancer cells. Methods: Important physicochemical features of the extracted cell membrane from tumoral cells were assessed by mass spectrometry-based proteomics. PLGA-based nanoparticles encapsulating temozolomide (TMZ NPs) were successfully developed. The coating technology applying the isolated U251 cell membrane (MB) was optimized using a fractional two-level three-factor factorial design. All the formulation runs were systematically characterized regarding their diameter, polydispersity index (PDI), and zeta potential (ZP). Experimental conditions generated by DoE were also subjected to morphological studies using negative-staining transmission electron microscopy (TEM). Its short-time stability was also assessed. MicroRaman and Fourier-Transform Infrared (FTIR) spectroscopies and Confocal microscopy were used as characterization techniques for evaluating the NP-MB nanostructures. Internalization studies were carried out to evaluate the homotypic targeting ability. Results and Discussion: The results have shown that nearly 80% of plasma membrane proteins were retained in the cell membrane vesicles after the isolation process, including key proteins to the homotypic binding. DoE analysis considering acquired TEM images reveals that condition run five should be the best-optimized procedure to produce the biomimetic cell-derived membrane-coated nanostructure (NP-MB). Storage stability for at least two weeks of the biomimetic system is expected once the original characteristics of diameter, PDI, and ZP, were maintained. Raman, FTIR, and confocal characterization results have shown the successful encapsulation of TMZ drug and provided evidence of the effective coating applying the MB. Cell internalization studies corroborate the proteomic data indicating that the optimized NP-MB achieved specific targeting of homotypic tumor cells. The structure should retain the complex biological functions of U251 natural cell membranes while exhibiting physicochemical properties suitable for effective homotypic recognition. Conclusion: Together, these findings provide coverage and a deeper understanding regarding the dynamics around extracted cell membrane and polymeric nanostructures interactions and an in-depth insight into the cell membrane coating technology and the development of optimized biomimetic and bioinspired nanostructured systems.

Exploring pradimicin-IRD antineoplastic mechanisms and related DNA repair pathways.

DNA-targeting agents have a significant clinical use, although toxicity remains an issue that plays against their widespread application. Understanding the mechanism of action and DNA damage response elicited by such compounds might contribute to the improvement of their use in anticancer chemotherapy. In a previous study, our research group characterized a new DNA-targeting agent - pradimicin-IRD. Since DNA-targeting agents and DNA repair are close-related subjects, the present study used in silico-modelling and a transcriptomic approach seeking to characterize the DNA repair pathways activated in HCT 116 cells following pradimicin-IRD treatment. Molecular docking analysis showed pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Furthermore, the transcriptomic study highlighted DNA repair functions related to genes modulated by pradimicin-IRD, such as nucleotide excision repair, telomeres maintenance and double-strand break repair. When validating these functions, PCNA protein levels decreased after exposure to pradimicin. Furthermore, molecular docking analysis suggested DNA-pradimicin-PCNA interaction. In addition, hTERT and POLH showed reduced mRNA levels after 6 h of treatment with pradimicin-IRD. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53-independent, unlike doxorubicin, dissimilarities between the mechanism of action and the DNA damage response of pradimicin-IRD and doxorubicin open new insights for further studies of pradimicin-IRD as a new antineoplastic compound.

DNA polymerase eta protects human cells against DNA damage induced by the tumor chemotherapeutic temozolomide.

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.

Vertical bone augmentation with guided bone regeneration: a scoping review / Aumento óseo vertical con regeneración ósea guiada: scoping review

Introduction: There are multiple techniques for vertical bone augmentation. Guided bone regeneration is one of them; however, the literature is diverse and includes different study designs, which makes it difficult to synthesize results. Objective: To analyze the general technical characteristics, clinical results, and complications of vertical bone augmentation performed with guided bone regeneration in humans. Material and Methods: This scoping review was based on the PRISMA-ScR guidelines. A search was performed in the Pubmed, Scielo, and Worldcat databases. Papers published from 1990 to April 2020 were included in the study. Research articles not conducted in humans or published in languages other than English and Spanish were excluded. Title and abstract were screened by two reviewers, then full studies were extracted, and data tabulated. Results: 89 studies were included. The highest percentage reported having obtained a vertical bone increase of less than 5 mm and having used non-resorbable membranes. The most frequent type of graft is autogenous and combinations of grafts, the most common being autogenous with xenograft. All studies that reported bone stability of implants in regenerated bone were favorable, as was implant survival, reporting values between 83.8% and 100%. Membrane exposure is the most frequently reported complication, followed by infection or abscesses, and tissue dehiscence. Conclusion: Vertical bone regeneration is a reliable technique, with high predictability and low incidence of complications compared to other vertical bone augmentation techniques.

Introducción: Existen múltiples técnicas para el aumento óseo vertical siendo una opción la regeneración ósea guiada, sin embargo, la literatura es diversa y con distintos diseños que dificultan la síntesis de resultados. Objetivo: Analizar las características generales técnicas, resultados clínicos y complicaciones del aumento óseo vertical realizado con regeneración ósea guiada en humanos. Material y Métodos: Esta revisión de alcance se basó en la guía PRISMA-ScR. Se realizó una búsqueda en las bases de datos Pubmed, Scielo y Worldcat. Fueron incluidos aquellos publicados desde el año 1990 hasta abril de 2020. Se excluyeron los estudios no realizados en humanos o publicados en idiomas distintos al inglés y español. Dos revisores examinaron título y resumen, luego los estudios completos se extrajeron y se ordenaron los datos en tablas. Resultados: 89 estudios fueron incluidos. El mayor porcentaje reportó haber obtenido un aumento óseo vertical menor a 5 mm y haber utilizado membranas no reabsorbibles. El tipo de injerto que más frecuente es el autógeno y las combinaciones de injertos, siendo el más común autógeno con xenoinjerto. Todos los estudios que reportaron estabilidad ósea de implantes en hueso regenerado fueron favorables, al igual que la supervivencia de implantes, reportando valores entre 83,8% y 100%. La exposición de membrana es la complicación que más se repite en los estudios, seguido por infección o abscesos y dehiscencia de tejidos. Conclusión: La regeneración ósea vertical es una técnica confiable, con alta predictibilidad y baja incidencia de complicaciones en comparación a otras técnicas de aumento óseo vertical.

Smarcb1 Loss Results in a Deregulation of esBAF Binding and Impacts the Expression of Neurodevelopmental Genes.

The murine esBAF complex plays a major role in the regulation of gene expression during stem cell development and differentiation. As one of its core subunits, Smarcb1 is indispensable for its function and its loss is connected to neurodevelopmental disorders and participates in the carcinogenesis of entities such as rhabdoid tumours. We explored how Smarcb1 regulates gene programs in murine embryonic stem cells (mESC) and in this way orchestrates differentiation. Our data underline the importance of Smarcb1 expression and function for the development of the nervous system along with basic cellular functions, such as cell adhesion and cell organisation. Using ChIP-seq, we were able to portray the consequences of Smarcb1 knockdown (kd) for the binding of esBAF and PRC2 as well as its influence on histone marks H3K27me3, H3K4me3 and H3K27ac. Their signals are changed in gene and enhancer regions of genes connected to nervous system development and offers a plausible explanation for changes in gene expression. Further, we describe a group of genes that are, despite increased BAF binding, suppressed after Smarcb1 kd by mechanisms independent of PRC2 function.

Single-cell transcriptomics identifies potential cells of origin of MYC rhabdoid tumors.

Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we establish and characterize different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets. From all models only Sox2-positive progenitor cells give rise to murine RT. Using single-cell analyses, we identify distinct cells of origin for the SHH and MYC subgroups of RT, rooting in early stages of embryogenesis. Intra- and extracranial MYC tumors harbor common genetic programs and potentially originate from fetal primordial germ cells (PGCs). Using PGC specific Smarcb1 knockout mouse models we validate that MYC RT originate from these progenitor cells. We uncover an epigenetic imbalance in MYC tumors compared to PGCs being sustained by epigenetically-driven subpopulations. Importantly, treatments with the DNA demethylating agent decitabine successfully impair tumor growth in vitro and in vivo. In summary, our work sheds light on the origin of RT and supports the clinical relevance of DNA methyltransferase inhibitors against this disease.

Mutagenicity Profile Induced by UVB Light in Human Xeroderma Pigmentosum Group C Cells.

Nucleotide excision repair (NER) is one of the main pathways for genome protection against structural DNA damage caused by sunlight, which in turn is extensively related to skin cancer development. The mutation spectra induced by UVB were investigated by whole-exome sequencing of randomly selected clones of NER-proficient and XP-C-deficient human skin fibroblasts. As a model, a cell line unable to recognize and remove lesions (XP-C) was used and compared to the complemented isogenic control (COMP). As expected, a significant increase of mutagenesis was observed in irradiated XP-C cells, mainly C>T transitions, but also CC>TT and C>A base substitutions. Remarkably, the C>T mutations occur mainly at the second base of dipyrimidine sites in pyrimidine-rich sequence contexts, with 5'TC sequence the most mutated. Although T>N mutations were also significantly increased, they were not directly related to pyrimidine dimers. Moreover, the large-scale study of a single UVB irradiation on XP-C cells allowed recovering the typical mutation spectrum found in human skin cancer tumors. Eventually, the data may be used for comparison with the mutational profiles of skin tumors obtained from XP-C patients and may help to understand the mutational process in nonaffected individuals.

Aproximações entre a Ginástica para Todos e conceitosda Gerontologia / Approximations between Gymnastics for All and concepts of Gerontology / Aproximaciones entre Gimnasia para Todos y conceptos de Gerontología

Objetivo:Analisar e refletir sobre as possíveis aproximações entre os fundamentos da Ginástica para Todos (GPT) e alguns termos próprios da área da Gerontologia, dentre os quais destacamos: life span, autonomia, geratividade e criatividade. Método:Trata-se de um trabalho analítico, de cunho bibliográfico e qualitativo. Resultados e discussão:O termo life span refere-se a uma forma de observar o desenvolvimento humano, no seu trajeto do nascimento à morte, considerando-o como um processo multidimensional e multidirecional, que envolve ganhos e perdas e é influenciado por variáveis biológicas, sociais e psicológicas. A autonomiapodeser definida como uma combinação da capacidade funcional com o senso individual de independência, com o senso de autodeterminação e domínio, e com a identidade. A geratividade diz respeito à motivação e ao envolvimento com a continuidade e com o bem-estar individual e do grupo social. A criatividade pode ser definida como a capacidade que nos permite organizar conhecimentos de formas novas e diferentes daquelas usuais. Considerações finais: Os conceitos destacados neste trabalho se relacionam com os fundamentos da GPT de forma bastante estreita, mostrando-se esta uma prática com muito potencial para promover o desenvolvimento da pessoa idosa.É importante destacar que o fortalecimento das relações aqui propostas é condicionado às abordagens e processos pedagógicos utilizados 1Universidade de São Paulo,Escola de Artes, Ciências e Humanidades,São Paulo-SP, Brasil.2Universidade de São Paulo,Faculdade de Filosofia, Letras e Ciências Humanas, Departamento de Letras Orientais,São Paulo-SP, Brasil.Correspondência: Mariana Harumi Cruz Tsukamoto. EACH-USP, Rua Arlindo Béttio, 1000, Vila Guaraciaba, São Paulo -SP, CEP 03828-000. Email: maharumi@usp.br 2Conexões, Campinas: SP, v. 20, e022035, 2022. ISSN: 1983-9030durante as práticas, considerando que algumas podem oferecer mais força e amplitude e outras podem limitar o desenvolvimento dos pontos levantados.

Objective:To analyze and reflect on possible approximations between the fundamentals of Gymnastics for All (GPT) and some specific terms in the field of Gerontology, among which we highlight: life span, autonomy, generativity and creativity. Method:This is an analytical, bibliographic and qualitative work. Results and discussion: The term life span refers to a way of observing human development, in its path from birth to death, considering it as a multidimensional and multidirectional process, which involves gains and losses and is influenced by biological variables, social and psychological. Autonomy can be defined as a combination of functional capacity with an individual's sense of independence, with a sense of self-determination and mastery, and with identity. Generativity concerns motivation and involvement with continuity and with individual and social group well-being. Creativity can be defined as the ability that allows us to organize knowledge in new and different ways from the usual ones. Final considerations:The concepts highlighted in this work relate to the fundamentals of GPT in a very narrow way, showing that this practice has a lot of potential to promote the development of the elderly. It is important to highlight that the strengthening of the relationships proposed here is conditioned to the pedagogical approaches and processes used during the practices, considering that some canoffer more strength and amplitude and others can limit the development of the points raised.

Objective:To analyze and reflect on possible approximations between the fundamentals of Gymnastics for All (GPT) and some specific terms in the field of Gerontology, among which we highlight: life span, autonomy, generativity and creativity. Method:This is an analytical, bibliographic and qualitative work. Results and discussion: The term life span refers to a way of observing human development, in its path from birth to death, considering it as a multidimensional and multidirectional process, which involves gains and losses and is influenced by biological variables, social and psychological. Autonomy can be defined as a combination of functional capacity with an individual's sense of independence, with a sense of self-determination and mastery, and with identity. Generativity concerns motivation and involvement with continuity and with individual and social group well-being. Creativity can be defined as the ability that allows us to organize knowledge in new and different ways from the usual ones. Final considerations:The concepts highlighted in this work relate to the fundamentals of GPT in a very narrow way, showing that this practice has a lot of potential to promote the development of the elderly. It is important to highlight that the strengthening of the relationships proposed here is conditioned to the pedagogical approaches and processes used during the practices, considering that some canoffer more strength and amplitude and others can limit the development of the points raised.

A therapeutic DNA vaccine and gemcitabine act synergistically to eradicate HPV-associated tumors in a preclinical model.

Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation.

Resección intestinal por endometriosis severa, diagnóstico y tratamiento luego de la introducción de la Unidad de Endometriosis: revisión de 18 años en el Hospital Clínico de la Universidad de Chile / Bowel resection for severe endometriosis, diagnosis and treatment after the introduction of Endometriosis Unit: 18 years review, Clinical Hospital of the University of Chile

INTRODUCCIÓN: La endometriosis intestinal afecta en gran medida la calidad de vida de una mujer joven y habitualmente requiere un tratamiento quirúrgico con resección intestinal. Esta cirugía es técnicamente compleja por las adherencias firmes del intestino a la vagina, el útero y los ovarios. OBJETIVO: Describir y analizar los resultados quirúrgicos e histopatológicos de las resecciones intestinales por endometriosis grave durante los últimos 18 años en el Hospital Clínico de la Universidad de Chile, en relación con la introducción de la unidad multidisciplinaria de endometriosis, a partir del año 2011, y las experiencias publicadas en la literatura chilena y extranjera. MÉTODO: Trabajo retrospectivo realizado en un hospital terciario desde el año 2001 hasta el año 2019. Las pacientes se asignaron a dos grupos según el período de cirugía: grupo 2001-2010 y grupo 2011-2019, luego de la introducción de la unidad de endometriosis. Se recopilaron todas las pacientes a las que se realizó una resección intestinal (discoidal o segmentaria) por endometriosis, por laparotomía o laparoscopía. Los datos distribuidos normalmente se presentan como promedio ± DE y los datos no paramétricos como mediana (rango). Las comparaciones demográficas de variables continuas se hicieron con la prueba t de Student y las de las variables categóricas con las pruebas de ji al cuadrado o de Fisher. La significación estadística se estableció en p < 0,05. RESULTADOS: Se recopilaron 52 casos. El 94,2% de las cirugías fueron electivas. El 5,8% fueron de urgencia por obstrucción intestinal (todas entre 2001 y 2010). Un 75% de las cirugías fueron laparoscópicas. Se realizó resección segmentaria en el 67,3%, resección discoidal simple en el 28,8%, resección discoidal doble en el 1,9% y resección segmentaria y una discoidal en el 1,9%. La histopatología demostró compromiso de la lesión hasta la mucosa intestinal en un 7,7%. Hubo franca disminución del dolor en el seguimiento de las pacientes. El 24% de las pacientes con deseo de embarazo y endometriosis intestinal lograron un parto de término mediante fecundación in vitro o espontáneamente. Hubo cuatro complicaciones posoperatorias, tres de ellas de categoría II según la clasificación de Clavien-Dindo y una de categoría IV A con reintervención a las 72 horas. Al comparar ambos periodos, en 2001-2010 los exámenes diagnósticos utilizados fueron ecografía transvaginal (0%), enema baritado (60%), tomografía computarizada de abdomen y pelvis (45%) y resonancia magnética pelviana (20%), mientras que en 2011-2019 fueron ecografía transvaginal (100%), enema baritado (3%), tomografía computarizada (3%) y resonancia magnética pelviana (66%). En 2001-2010, las lesiones fueron más más infiltrativas (mayor compromiso mucoso y submucoso) (75 vs. 16% de las resecciones intestinales; p < 0,05), estenóticas (cirugías de urgencia por obstrucción), con mayor porcentaje de resecciones segmentarias (100 vs. 46,9%; p < 0,05) y más días de hospitalización (5,8 ± 2,3 vs. 4,1 ± 0,9 días) que en 2011-2019. CONCLUSIONES: A nuestro entender, esta es la serie más grande publicada en Chile de resecciones intestinales por endometriosis. Estos hallazgos demuestran cómo la introducción de la unidad multidisciplinaria de endometriosis permite un diagnóstico precoz y un tratamiento quirúrgico eficaz y oportuno, tal como se decribe en la literatura.

INTRODUCTION: Bowel endometriosis severely affects a young woman's quality of life and often requires surgical treatment with bowel resection. This surgery is technically complex due to the tight adhesions of the intestine to the vagina, uterus, and ovaries. The objective of this work is to describe and analyze the surgical and histopathological results of intestinal resections for severe endometriosis during the last 18 years at the Clinical Hospital University of Chile, in relation to the implementation of the multidisciplinary endometriosis unit, based on the year 2011 and the experiences published in Chilean and foreign literature. METHOD: Retrospective work carried out in a tertiary hospital from 2001 to 2019. The patients were assigned to two groups according to the surgery period: group 2001-2010 and group 2011-2019, after endometriosis unit formation. All patients who underwent bowel resection (discoidal or segmental) for endometriosis by laparotomy or laparoscopy were collected. Normally distributed data are presented as mean ± SD and nonparametric data as median (range). Demographic comparisons of continuous variables are compared using Student's t test and categorical variables using chi squared or Fisher's test. Statistical significance was established at p < 0.05. RESULTS: 52 cases were collected. 94.2% of the surgeries were elective. 5.8% were urgent due to intestinal obstruction (all between 2001 and 2010). 75% of the surgeries were laparoscopic. Segmental resection 67.3%, simple discoidal resection 28.8%, double discoidal resection 1.9% and segmental resection and a discoidal resection 1.9%. Histopathology showed involvement of the lesion up to the intestinal mucosa in 7.7%. A marked decrease in pain in the follow-up of the patients. 24% of the patients with a desire for pregnancy and intestinal endometriosis achieved a full-term delivery by IVF or spontaneously. There were four postoperative complications, three of them category II according to the Clavien-Dindo classification, and one category IV A complication with reoperation at 72 h. When comparing both periods, between 2001-2010 the diagnostic tests used were: transvaginal ultrasound (ECO TV) (0%), barium enema (BE) (60%), abdomen pelvis CT (45%) and pelvic resonance (MRI) (20%). Between 2011 and 2019 ECO TV (100%), EB (3%), TAC (3%) RM (66%). In the period 2001 to 2010, the lesions were more infiltrative (greater mucosal and submucosal involvement) (75% vs 16% of intestinal resections (P <0.05)), stenotic (urgent surgery for obstruction), with a higher percentage of resections segmental (100% vs 46.9% (P <0.05) and more days of hospitalization (5.8 ± 2.3 SD vs 4.1 ± 0.9 SD) than in the period from 2011 to 2019. CONCLUSIONS: To our knowledge, this is the largest series published in Chile of intestinal resections for endometriosis. These findings demonstrate how the introduction of the multidisciplinary endometriosis unit allows early diagnosis and effective and timely surgical treatment as described in the literature.

Postoperative neurosensory impairment perception using ultrasonic BoneScalpel and conventional rotary instruments after bilateral split sagittal osteotomy.

PURPOSE: Although bilateral sagittal split osteotomy (BSSO) is the most widely used surgical technique for the correction of mandibular dentofacial anomalies, it is associated with lesion of inferior alveolar nerve (IAN) and unwanted neurosensory disorders. The aim of this study was to document the perception of changes in sensitivity and mean recovery time after BSSO, using an ultrasonic BoneScalpel versus the conventional rotary instruments. PATIENTS AND METHODS: This retrospective observational study included all patients with diagnosis of skeletal anomaly who underwent advancement or setback BSSO of less than 10 mL, using the ultrasonic osteotome or conventional rotary instruments. The patients were operated on at the Hospital Universitario Clínica San Rafael, Bogotá Colombia, between 2017 and 2018. The primary predictor variable was the osteotomy technique. The primary outcome was the presence or absence of postoperative sensory alteration, whereas secondary outcomes were time of appearance and recovery, affected anatomical region, laterality, and disturbance in daily activities. Data were analyzed using Chi-square, Mann-Whitney U, and Fisher's exact test. RESULTS: Data of 38 patients were retrieved, of which 23 were operated with BoneScalpel and 13 with the conventional technique. Twenty patients were women and 18 were men. All patients reported experiencing at least one type of sensory disturbance immediately after the surgical procedure. There was a significant difference (p = 0.0001) in the time that the alteration was present between the two groups, in favor of the BoneScalpel group. The chin and the lower lip were the anatomical regions with the greatest alteration in sensitivity and persistence of it. CONCLUSIONS: The results of this study indicate that BoneScalpel is effective in performing BSSO. They also suggest that it may reduce the occurrence of nerve damage during BSSO, although more research on this topic is required.

A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons.

Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

An extracellular vesicle-related gene expression signature identifies high-risk patients in medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts. METHODS: We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche. RESULTS: Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes. CONCLUSIONS: With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.

Inflammation response, oxidative stress and DNA damage caused by urban air pollution exposure increase in the lack of DNA repair XPC protein.

Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1ß, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM2.5.

Reversine exerts cytotoxic effects through multiple cell death mechanisms in acute lymphoblastic leukemia.

PURPOSE: Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited therapeutic options for adult patients. Aurora kinases have drawn attention as potential targets in hematological neoplasms due to their high expression and biological functions. Aurora kinase A (AURKA) and AURKB are essential for a successful mitosis, acting in spindle mitotic organization and cytokinesis. Reversine is a synthetic purine analog that acts as a multi-kinase inhibitor with anti-neoplastic activity by targeting AURKA and AURKB. METHODS: ALL patient gene expression data were retrieved from the Amazonia! DATABASE: For functional assays, Jurkat (T-ALL) and Namalwa (B-ALL) cells were exposed to increasing concentrations of reversine and submitted to various cellular and molecular assays. RESULTS: We found that AURKB expression was higher in ALL patient samples compared to normal lymphocytes (p < 0.0001). The ALL cell lines tested displayed aberrant AURKA and AURKB expression. In Jurkat and Namalwa cells, reversine reduced cell viability in a dose- and time-dependent manner (p < 0.05). Reversine also significantly reduced the viability of primary ALL cells. Reversine induced apoptosis and autophagy, and reduced cell proliferation in both cell lines (p < 0.05). Mitotic catastrophe markers, including cell cycle arrest at G2/M, increased cell size and DNA damage, were observed upon reversine exposure. Short- and long-term treatment with reversine inhibited autonomous clonogenicity (p < 0.05). At the molecular level, reversine reduced AURKB activity, induced SQSTM1/p62 consumption, and increased LC3BII and γ-H2AX levels. In Namalwa cells, reversine modulated 25 out of 84 autophagy-related genes, including BCL2, BAD, ULK1, ATG10, IRGM and MAP1LC3B, which indicates that reversine acts by initiating and sustaining autophagy signals in ALL cells. CONCLUSIONS: From our data we conclude that reversine reduces the viability of ALL cells by triggering multiple cell death mechanisms, including apoptosis, mitotic catastrophe, and autophagy. Our findings highlight reversine as a potential anticancer agent for ALL.