Epigenetic changes regulating the epithelial-mesenchymal transition in human trophoblast differentiation.

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial-mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome-wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

Case report: First documented case of cerebral angiostrongyliasis caused by Angiostrongylus costaricensis in a free-ranging opossum.

Angiostrongylus costaricensis is a metastrongyloid nematode that primarily infects the mesenteric arteries of wild rodents. This parasite is endemic in several regions of the American continent, and in humans, causes a disease known as abdominal angiostrongyliasis. Despite the important health implications of this nematode, there are limited studies investigating the involvement of wild animals in its life cycle. In this study, we present the clinical manifestations, pathologic findings, and molecular diagnosis, to the best of our current knowledge, of the first documented onset of cerebral angiostrongyliasis because of A. costaricensis infection in a juvenile free-ranging opossum (Didelphis marsupialis). Histopathological findings stress the presence of eosinophilic meningoencephalitis with nematodes present within the lesions, and PCR was positive for cox1 and ITS1 reactions. The obtained sequences for a 279 bp fragment of ITS1 were 100% identical to A. costaricensis from Costa Rica. This case highlights the substantial difficulties in diagnosing neuroangiostrongyliasis, yet underscores the importance of considering A. costaricensis as a potential culprit behind neurological conditions in wild marsupials. It acts as an urgent call to action to improve surveillance programs tracking infectious and parasitic diseases causing mortality in wildlife populations.

Regulation of nuclear transcription by mitochondrial RNA in endothelial cells.

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

Women in neurosurgery in Peru: current situation and representation.

The participation of women in neurosurgery in Peru began in 1974 with the first Peruvian female neurosurgeon, who was accepted into a residency program with the condition of not getting married during her training. Nowadays, the conditions are more just, and there is greater equality of opportunities between men and women, but the numbers show that the differences have not yet been fully equalized. As evidence of this, it has been observed that only 17% of the residency positions offered in 2022 were filled by women, and there has been reduced participation of female neurosurgeons in the Peruvian Society of Neurosurgery. This article discusses the importance of tracking and promoting stories of Peruvian female neurosurgeons and calls for research in the area of women in neurosurgery in latin countries, like Peru.

The humanitarian aid of neurosurgical missions in Peru: A chronicle and future perspectives.

Background: The unmet neurosurgical need has remained patent in developing countries, including Peru. However, continuous efforts to overcome the lack of affordable care have been achieved, being neurosurgical missions one of the main strategies. We chronicle the humanitarian labor of organizations from high-income countries during their visit to Peru, the contributions to local trainees' education, and the treatment of underserved patients. Furthermore, we discuss the embedded challenges from these missions and the future perspective on long-term partnerships and sustainability. Methods: This is a narrative review. We searched the literature in PubMed and Google Scholar about neurosurgical missions conducted in Peru. Results: Since 1962, twelve organizations from high-income countries have delivered humanitarian help in Peru by training local neurosurgeons, treating low-income patients, and providing surgical instrumentation. Out of the three main regions of Peru, cities on the coast and highlands have hosted most of these missions, with no reported outreach in the amazon area. About 75% of the organizations are headquartered in the United States, followed by Canada, Luxembourg, and Spain. In addition, 50% of the organizations have an active partnership. The predominant focus of these missions has been pediatrics, neuro-oncology, and spine surgery. Conclusion: Neurosurgical missions have represented a strategy to close the disparity in education and treatment in Peru. However, additional efforts must be conducted to improve long-term partnership and sustainability, such as adopting standardized indicators for progress tracking, incorporating remote technologies for continuous training and communication, and expanding partnerships in less attended areas.

Tissue-resident PSGL1loCD4+ T cells promote B cell differentiation and chronic graft-versus-host disease-associated autoimmunity.

CD4+ T cell interactions with B cells play a critical role in the pathogenesis of systemic autoimmune diseases such as systemic lupus and chronic graft-versus-host disease (cGVHD). Extrafollicular CD44hiCD62LloPSGL1loCD4+ T cells (PSGL1loCD4+ T cells) are associated with the pathogenesis of lupus and cGVHD, but their causal role has not been established. With murine and humanized MHC-/-HLA-A2+DR4+ murine models of cGVHD, we showed that murine and human PSGL1loCD4+ T cells from GVHD target tissues have features of B cell helpers with upregulated expression of programmed cell death protein 1 (PD1) and inducible T cell costimulator (ICOS) and production of IL-21. They reside in nonlymphoid tissues without circulating in the blood and have features of tissue-resident memory T cells with upregulated expression of CD69. Murine PSGL1loCD4+ T cells from GVHD target tissues augmented B cell differentiation into plasma cells and production of autoantibodies via their PD1 interaction with PD-L2 on B cells. Human PSGL1loCD4+ T cells were apposed with memory B cells in the liver tissues of humanized mice and cGVHD patients. Human PSGL1loCD4+ T cells from humanized GVHD target tissues also augmented autologous memory B cell differentiation into plasma cells and antibody production in a PD1/PD-L2-dependent manner. Further preclinical studies targeting tissue-resident T cells to treat antibody-mediated features of autoimmune diseases are warranted.

A DNA Hypomethylating Drug Alters the Tumor Microenvironment and Improves the Effectiveness of Immune Checkpoint Inhibitors in a Mouse Model of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer that has proven refractory to immunotherapy. Previously, treatment with the DNA hypomethylating drug decitabine (5-aza-dC; DAC) extended survival in the KPC-Brca1 mouse model of PDAC. Here we investigated the effects of DAC in the original KPC model and tested combination therapy with DAC followed by immune checkpoint inhibitors (ICI). Four protocols were tested: PBS vehicle, DAC, ICI (anti-PD-1 or anti-VISTA), and DAC followed by ICI. For each single-agent and combination treatment, tumor growth was measured by serial ultrasound, tumor-infiltrating lymphoid and myeloid cells were characterized, and overall survival was assessed. Single-agent DAC led to increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TIL), PD1 expression, and tumor necrosis while slowing tumor growth and modestly increasing mouse survival without systemic toxicity. RNA-sequencing of DAC-treated tumors revealed increased expression of Chi3l3 (Ym1), reflecting an increase in a subset of tumor-infiltrating M2-polarized macrophages. While ICI alone had modest effects, DAC followed by either of ICI therapies additively inhibited tumor growth and prolonged mouse survival. The best results were obtained using DAC followed by anti-PD-1, which extended mean survival from 26 to 54 days (P < 0.0001). In summary, low-dose DAC inhibits tumor growth and increases both TILs and a subset of tumor-infiltrating M2-polarized macrophages in the KPC model of PDAC, and DAC followed by anti-PD-1 substantially prolongs survival. Because M2-polarized macrophages are predicted to antagonize antitumor effects, targeting these cells may be important to enhance the efficacy of combination therapy with DAC plus ICI. SIGNIFICANCE: In a pancreatic cancer model, a DNA hypomethylating drug increases tumor-infiltrating effector T cells, increases a subset of M2 macrophages, and significantly prolongs survival in combination with immune checkpoint inhibitors.See related commentary by Nephew, p. 4610.

RADIOGRAPHIC ANATOMY OF THE FORELIMB IN THE NORTHERN TAMANDUA (TAMANDUA MEXICANA).

The thoracic limb anatomy of anteaters in the family Myrmecophagidae is specialized for accessing termite and ant nests and for defense purposes. In the case of the northern tamandua (Tamandua mexicana), the forelimbs are also adapted for arboreal and terrestrial locomotion. Unfortunately, this species faces many conservation threats, such as habitat loss and traffic accidents, and injured individuals are frequently taken to wildlife rehabilitation centers. However, lack of knowledge of the radiographic osteoanatomy of this species may prevent appropriate management of injuries and thereby reduce the chances of successful release and survival. In order to fill this knowledge gap, this article describes for the first time the radiographic anatomy of the thoracic limb of the northern tamandua using four standard views and one additional view. The additional orthogonal view helps visualize structures, such as the hamatus process and the sesamoid bone, that are otherwise difficult to visualize due to the natural forearm position of anteaters. Additionally, some fractures and physeal growth plates were identified in one juvenile individual. Further radiographic investigations should be conducted on anteaters to provide more tools for diagnosis, treatment, and rehabilitation of these animals.

Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs.

BACKGROUND: Mapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). The advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified. RESULTS: We perform whole genome methyl-seq on diverse normal cells and tissues and three cancer types. After excluding imprinting, the data pinpoint 15,112 high-confidence ASM differentially methylated regions, of which 1838 contain SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies are increased in cancers versus matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancer cells show increased allele switching at ASM loci, but disruptive SNPs in specific classes of CTCF and transcription factor binding motifs are similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations affecting these same motif classes track with de novo ASM. Allele-specific transcription factor binding from ChIP-seq is enriched among ASM loci, but most ASM differentially methylated regions lack such annotations, and some are found in otherwise uninformative "chromatin deserts." CONCLUSIONS: ASM is increased in cancers but occurs by a shared mechanism involving disruptive SNPs in CTCF and transcription factor binding sites in both normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, cardiometabolic, neuropsychiatric, and neoplastic diseases.

DNA methylation patterns in T lymphocytes are generally stable in human pregnancies but CD3 methylation is associated with perinatal psychiatric symptoms.

OBJECTIVES: To determine whether DNA methylation patterns in genes coding for selected T-lymphocyte proteins are associated with perinatal psychiatric distress or with complications of pregnancy. METHODS: T lymphocyte DNA was obtained from pregnant women across three time points in pregnancy and the postpartum period and epigenetic patterns were assessed using Illumina 450 â€‹K Methylation Beadchips. Seven selected genes critical for T cell function were analyzed for methylation changes during pregnancy and for associations of methylation patterns with psychiatric distress or with pregnancy complications, with particular attention paid to spatial aggregations of methyl groups, termed 'hotspots,' within the selected genes. RESULTS: In the candidate gene approach, DNA methylation density within a single cluster of 9 contiguous CpG loci within the CD3 gene was found to be strongly associated with anxiety and depression in mid- and late pregnancy, and weakly associated with the presence of complications of pregnancy. Average DNA methylation density across each of the seven genes examined, and assay-wide, was found to be relatively stable across pregnancy and postpartum, but methylation within the CD3 hotspot was more malleable and changes over time were coordinated across the nine cytosines in the hotspot. CD3 CpGs did not pass array-wide tests for significance, but CpG clusters in two other genes, DTNBP1 and OXSR1, showed array-wide significant associations with anxiety. CONCLUSIONS: Despite the need for tolerating the fetal hemi-allograft, overall DNA methylation patterns in T lymphocytes are generally stable over the mid to late course of human pregnancies and postpartum. However, site-specific changes in DNA methylation density in CD3 appear linked to both symptoms of depression and anxiety in pregnancy and, less strongly, to adverse pregnancy outcomes.

Correction: Genetic variants influence on the placenta regulatory landscape.

[This corrects the article DOI: 10.1371/journal.pgen.1007785.].

Genetic variants influence on the placenta regulatory landscape.

From genomic association studies, quantitative trait loci analysis, and epigenomic mapping, it is evident that significant efforts are necessary to define genetic-epigenetic interactions and understand their role in disease susceptibility and progression. For this reason, an analysis of the effects of genetic variation on gene expression and DNA methylation in human placentas at high resolution and whole-genome coverage will have multiple mechanistic and practical implications. By producing and analyzing DNA sequence variation (n = 303), DNA methylation (n = 303) and mRNA expression data (n = 80) from placentas from healthy women, we investigate the regulatory landscape of the human placenta and offer analytical approaches to integrate different types of genomic data and address some potential limitations of current platforms. We distinguish two profiles of interaction between expression and DNA methylation, revealing linear or bimodal effects, reflecting differences in genomic context, transcription factor recruitment, and possibly cell subpopulations. These findings help to clarify the interactions of genetic, epigenetic, and transcriptional regulatory mechanisms in normal human placentas. They also provide strong evidence for genotype-driven modifications of transcription and DNA methylation in normal placentas. In addition to these mechanistic implications, the data and analytical methods presented here will improve the interpretability of genome-wide and epigenome-wide association studies for human traits and diseases that involve placental functions.

Diagnóstico morfológico y molecular de Cyclocoelum mutabile (Trematoda: Cyclocoelidae) en el Perú

Cyclocoelum mutabile , un digeneo de la familia Cyclocoelidae, fue hallado parasitando los sacos aéreos de una polla de agua común (Gallinula chloropus), proveniente de alrededores del Refugio de Vida Silvestre Pantanos de villa, localizada en el distrito de Chorrillos en Lima, Perú. Un total de 7 parásitos fueron colectados e identificados por métodos morfológicos como C. mutabile. El diagnóstico fue confirmado por análisis molecular, amplificando los genes mitocondriales citocromo c oxidasa subunidad 1 (cox1) y deshidrogenasa NADH subunidad 1 (nad1). Las secuencias de nucleótidos de los aislados se compararon con secuencias previas de GenBank, y mostraron una similitud entre ellas (> 96%). Este hallazgo constituye el primer registro de C. mutabile para el Perú. Además, el trabajo realiza una breve descripción del parásito, así como la discusión de sus hospederos y distribución geográfica en Sudamérica.

Cyclocoelum mutabile , a digenea of the family Cyclocoelidae, was found parasitizing the air sacs of a common moorhen (Gallinula chloropus), from the surroundings of the Pantanos de Villa Wildlife Refuge, located in Chorrillos, a district of Lima, Peru. A total of 7 parasites were collected and identified as C. mutabile by morphological methods. Diagnosis was confirmed by molecular analysis and partially amplified mitochondrial genes cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1). Nucleotide sequences from the isolates were compared with previous sequences from GenBank, and they showed a high similarity between them (> 96%). This finding constitutes the first record of C. mutabile in Peru. Also, this work makes a brief description of the parasite, as well as the discussion of its hosts and geographic distribution in South America.

Trans effects of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models.

BACKGROUND: Trisomy 21 causes Down syndrome (DS), but the mechanisms by which the extra chromosome leads to deficient intellectual and immune function are not well understood. RESULTS: Here, we profile CpG methylation in DS and control cerebral and cerebellar cortex of adults and cerebrum of fetuses. We purify neuronal and non-neuronal nuclei and T lymphocytes and find biologically relevant genes with DS-specific methylation (DS-DM) in each of these cell types. Some genes show brain-specific DS-DM, while others show stronger DS-DM in T cells. Both 5-methyl-cytosine and 5-hydroxy-methyl-cytosine contribute to the DS-DM. Thirty percent of genes with DS-DM in adult brain cells also show DS-DM in fetal brains, indicating early onset of these epigenetic changes, and we find early maturation of methylation patterns in DS brain and lymphocytes. Some, but not all, of the DS-DM genes show differential expression. DS-DM preferentially affected CpGs in or near specific transcription factor binding sites (TFBSs), implicating a mechanism involving altered TFBS occupancy. Methyl-seq of brain DNA from mouse models with sub-chromosomal duplications mimicking DS reveals partial but significant overlaps with human DS-DM and shows that multiple chromosome 21 genes contribute to the downstream epigenetic effects. CONCLUSIONS: These data point to novel biological mechanisms in DS and have general implications for trans effects of chromosomal duplications and aneuploidies on epigenetic patterning.

Chronic unpredictable stress regulates visceral adipocyte-mediated glucose metabolism and inflammatory circuits in male rats.

Chronic psychological stress is a prominent risk factor involved in the pathogenesis of many complex diseases, including major depression, obesity, and type II diabetes. Visceral adipose tissue is a key endocrine organ involved in the regulation of insulin action and an important component in the development of insulin resistance. Here, we examined for the first time the changes on visceral adipose tissue physiology and on adipocyte-associated insulin sensitivity and function after chronic unpredictable stress in rats. Male rats were subjected to chronic unpredictable stress for 35 days. Total body and visceral fat was measured. Cytokines and activated intracellular kinase levels were determined using high-throughput multiplex assays. Adipocyte function was assessed via tritiated glucose uptake assay. Stressed rats showed no weight gain, and their fat/lean mass ratio increased dramatically compared to control animals. Stressed rats had significantly higher mesenteric fat content and epididymal fat pad weight and demonstrated reduced serum glucose clearing capacity following glucose challenge. Alterations in fat depot size were mainly due to changes in adipocyte numbers and not size. High-throughput molecular screening in adipocytes isolated from stressed rats revealed activation of intracellular inflammatory, glucose metabolism, and MAPK networks compared to controls, as well as significantly reduced glucose uptake capacity in response to insulin stimulation. Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress-related disease states.

Creation and characterization of BAC-transgenic mice with physiological overexpression of epitope-tagged RCAN1 (DSCR1).

The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). To develop a physiologically relevant model for studying the biochemistry of RCAN1 and its contribution to DS, we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope tag incorporated by recombineering. The BAC-Tg was expressed at levels only moderately higher than the native Rcan1 gene: approximately 1.5-fold in RCAN1 (BAC-Tg1) and twofold in RCAN1 (BAC-Tg2). Affinity purification of the RCAN1 protein complex from brains of these mice revealed a core complex of RCAN1 with CaN, glycogen synthase kinase 3-beta (Gsk3b), and calmodulin, with substoichiometric components, including LOC73419. The BAC-Tg mice are fully viable, but long-term synaptic potentiation is impaired in proportion to BAC-Tg dosage in hippocampal brain slices from these mice. RCAN1 can act as a tumor suppressor in some systems, but we found that the RCAN1 BAC-Tg did not reduce mammary cancer growth when present at a low copy number in Tp53;WAP-Cre mice. This work establishes a useful mouse model for investigating the biochemistry and dose-dependent functions of the RCAN1 protein in vivo.

Hypomethylating therapy in an aggressive stroma-rich model of pancreatic carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that resists current treatments. To test epigenetic therapy against this cancer, we used the DNA demethylating drug 5-aza-2'-deoxycytidine (DAC) in an aggressive mouse model of stromal rich PDAC (KPC-Brca1 mice). In untreated tumors, we found globally decreased 5-methyl-cytosine (5-mC) in malignant epithelial cells and in cancer-associated myofibroblasts (CAF), along with increased amounts of 5-hydroxymethyl-cytosine (5-HmC) in CAFs, in progression from pancreatic intraepithelial neoplasia to PDAC. DAC further reduced DNA methylation and slowed PDAC progression, markedly extending survival in an early-treatment protocol and significantly though transiently inhibiting tumor growth when initiated later, without adverse side effects. Escaping tumors contained areas of sarcomatoid transformation with disappearance of CAFs. Mixing-allografting experiments and proliferation indices showed that DAC efficacy was due to inhibition of both the malignant epithelial cells and the CAFs. Expression profiling and immunohistochemistry highlighted DAC induction of STAT1 in the tumors, and DAC plus IFN-γ produced an additive antiproliferative effect on PDAC cells. DAC induced strong expression of the testis antigen deleted in azoospermia-like (DAZL) in CAFs. These data show that DAC is effective against PDAC in vivo and provide a rationale for future studies combining hypomethylating agents with cytokines and immunotherapy.