In Vitro Assay Using Proboscidea parviflora W. and Phaseolus lunatus L. Plant Extracts to Control Pythium amazonianum.

Avocado tree wilt is a disease caused by Phytophthora cinnamomi Rands. Recently, this disease has been associated to Pythium amazonianum, another causal agent. Avocado tree wilt is being currently controlled with synthetic fungicides that kill beneficial microorganisms, polluting the environment and leading to resistance problems in plant pathogens. The current research work aims to provide alternative management using extracts from Proboscidea parviflora W. and Phaseolus lunatus L. to control the development of mycelia in P. amazonianum in vitro. Raw extracts were prepared at UAAAN Toxicology Laboratory, determining the inhibition percentages, inhibition concentrations and inhibition lethal times. Several concentrations of the plant extracts were evaluated using the poisoned medium methodology, showing that both extracts control and inhibit mycelial development, in particular P. lutatus, which inhibits mycelial growth at concentrations lower than 80 mg/L, being lower than P. parviflora extracts. These extracts are promising candidates for excellent control of Pythium amazonianum.

Decidualized human decidual stromal cells inhibit chemotaxis of activated T cells: a potential mechanism of maternal-fetal immune tolerance.

Background: Numerous lines of evidence confirm that decidual stromal cells (DSCs) play a key role in maternal-fetal immune tolerance. Under the influence of progesterone and other hormones, the DSCs go through a process of differentiation (decidualization) during normal pregnancy. In mice, DSCs inhibit the expression of chemokines that attract abortigenic Th1 and Tc cells to the decidua. We have studied this phenomenon in humans. Methods: We established human DSC lines and decidualized these cells in vitro with progesterone and cAMP. We determined the expression of the chemokines CXCL9, CXCL10 and CXCL11, whose receptor CXCR3 is expressed by Th1 and Tc cells, in undifferentiated DSCs and decidualized DSCs by qRT-PCR. Activated CD3+CXCR3+ cells, including CD4+ Th1 cells and CD8+ Tc cells, were induced in vitro. The migration capacity of these activated lymphocytes was investigated in Transwell chambers with conditioned media from undifferentiated and decidualized DSCs. Results: We demonstrated that CXCL9 was not expressed by DSCs, whereas the expression of CXCL10 and CXCL11 was inhibited in decidualized cells. Conditioned media from decidualized cells significantly inhibited the migration of Th1 and Tc cells. We found that decidualized cells secrete factors of MW less than 6000-8000 Da, which actively inhibit the chemotaxis of these lymphocytes. Discussion: These results confirm in humans that decidualization of DSCs inhibits the expression by these cells of chemokines that attract Th1 and Tc cells and induces the secretion by DSCs of factors that inhibit the chemotaxis of these lymphocytes, thus preventing the arrival of abortigenic T cells in the decidua.

Symbiotic Supplementation (E. faecium and Agave Inulin) Improves Spatial Memory and Increases Plasticity in the Hippocampus of Obese Rats: A Proof-of-Concept Study.

Obesity has been linked to cognitive impairment through systemic low-grade inflammation. High fat and sugar diets (HFSDs) also induce systemic inflammation, either by induced Toll-like receptor 4 response, or by causing dysbiosis. This study aimed to evaluate the effect of symbiotics supplementation on spatial and working memory, butyrate concentration, neurogenesis, and electrophysiological recovery of HFSD-fed rats. In a first experiment, Sprague-Dawley male rats were given HFSD for 10 weeks, after which they were randomized into 2 groups (n = 10 per group): water (control), or Enterococcus faecium + inulin (symbiotic) administration, for 5 weeks. In the fifth week, spatial and working memory was analyzed through the Morris Water Maze (MWM) and Eight-Arm Radial Maze (RAM) tests, respectively, with 1 week apart between tests. At the end of the study, butyrate levels from feces and neurogenesis at hippocampus were determined. In a second experiment with similar characteristics, the hippocampus was extracted to perform electrophysiological studies. Symbiotic-supplemented rats showed a significantly better memory, butyrate concentrations, and neurogenesis. This group also presented an increased firing frequency in hippocampal neurons [and a larger N-methyl-d-aspartate (NMDA)/α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) current ratio] suggesting an increase in NMDA receptors, which in turn is associated with an enhancement in long-term potentiation and synaptic plasticity. Therefore, our results suggest that symbiotics could restore obesity-related memory impairment and promote synaptic plasticity.

Acidosis metabólica neonatal por síndrome de enterocolitis inducido por proteína de alimentos: reporte de caso / Neonatal metabolic acidosis caused by food proteininduced enterocolitis syndrome: a case report

El síndrome de enterocolitis inducido por proteínas de los alimentos (FPIES, por su sigla en inglés) es una reacción alérgica no mediada por inmunoglobulina E (IgE) con síntomas gastrointestinales, como vómitos y diarrea. El diagnóstico se basa en criterios clínicos y en una prueba de provocación para confirmarlo. Es una enfermedad desconocida en las unidades neonatales, debido a la inespecificidad de los síntomas en los recién nacidos. La cifra de metahemoglobina elevada es una opción sencilla de aproximación diagnóstica. Se describe el caso clínico de un recién nacido que ingresa al servicio de urgencias por deshidratación, letargia, vómitos, diarrea y acidosis metabólica grave con elevación de metahemoglobina, con mejora clínica y recuperación total tras el inicio del aporte de fórmula elemental. La sospecha diagnóstica se confirmó tras la prueba de provocación positiva.

Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE mediated allergic reaction with gastrointestinal symptoms, such as vomiting and diarrhea. FPIES diagnosis is based on clinical criteria and on a food challenge test. It is an unknown disease in neonatal units due to its nonspecific symptoms in newborn infants. An elevated methemoglobin level is a simple way to approach diagnosis. Here we describe a clinical case of a newborn admitted to the emergency department because of dehydration, lethargy, vomiting, diarrhea, severe metabolic acidosis, and a high methemoglobin level. Clinical improvement and complete recovery was achieved after initiation of elemental formula. The diagnostic suspicion was confirmed after a positive challenge test.

Influence of the ectopic location on the antigen expression and functional characteristics of endometrioma stromal cells.

RESEARCH QUESTION: Are the alterations observed in the endometriotic cells, such as progesterone resistance, already present in the eutopic endometrium or acquired in the ectopic location? DESIGN: The response to decidualization with progesterone and cyclic AMP for up to 28 days was compared in different endometrial stromal cell (EnSC) lines established from samples of endometriomas (eEnSC), eutopic endometrium from women with endometriosis (eBEnSC), endometrial tissue from healthy women (BEnSC) and menstrual blood from healthy donors (mEnSC). RESULTS: Usual features of decidualized cells, such as changes in cell morphology and expression of prolactin, were similarly observed in the three types of eutopic EnSC studied, but not in the ectopic cells upon decidualization. Among the phenotypic markers analysed, CD105 was down-regulated under decidualization in all cell types (mEnSC, P = 0.005; BEnSC, P = 0.029; eBEnSC, P = 0.022) except eEnSC. mEnSC and BEnSC underwent apoptosis during decidualization, whereas eBEnSC and eEnSC were resistant to the induction of cell death. Lastly, migration studies revealed that mEnSC secreted undetermined factors during decidualization that inhibited cell motility, whereas eEnSC showed a significantly lower ability to produce those migration-regulating factors (P < 0.0001, P  < 0.001 and P = 0.0013 for the migration of mEnSC at 24, 48 and 72 h, respectively; P  < 0.0001 for the migration of eEnSC at all times studied). CONCLUSIONS: This study provides novel insights into the differences between endometriotic and eutopic endometrial cells and reinforces the idea that the microenvironment in the ectopic location plays additional roles in the acquisition of the alterations that characterize the cells of the endometriotic foci.

Neonatal metabolic acidosis caused by food proteininduced enterocolitis syndrome: a case report. / Acidosis metabólica neonatal por síndrome de enterocolitis inducido por proteína de alimentos: reporte de caso.

Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE mediated allergic reaction with gastrointestinal symptoms, such as vomiting and diarrhea. FPIES diagnosis is based on clinical criteria and on a food challenge test. It is an unknown disease in neonatal units due to its nonspecific symptoms in newborn infants. An elevated methemoglobin level is a simple way to approach diagnosis. Here we describe a clinical case of a newborn admitted to the emergency department because of dehydration, lethargy, vomiting, diarrhea, severe metabolic acidosis, and a high methemoglobin level. Clinical improvement and complete recovery was achieved after initiation of elemental formula. The diagnostic suspicion was confirmed after a positive challenge test.

El síndrome de enterocolitis inducido por proteínas de los alimentos (FPIES, por su sigla en inglés) es una reacción alérgica no mediada por inmunoglobulina E (IgE) con síntomas gastrointestinales, como vómitos y diarrea. El diagnóstico se basa en criterios clínicos y en una prueba de provocación para confirmarlo. Es una enfermedad desconocida en las unidades neonatales, debido a la inespecificidad de los síntomas en los recién nacidos. La cifra de metahemoglobina elevada es una opción sencilla de aproximación diagnóstica. Se describe el caso clínico de un recién nacido que ingresa al servicio de urgencias por deshidratación, letargia, vómitos, diarrea y acidosis metabólica grave con elevación de metahemoglobina, con mejora clínica y recuperación total tras el inicio del aporte de fórmula elemental. La sospecha diagnóstica se confirmó tras la prueba de provocación positiva.

Pre-clinical models to study abnormal uterine bleeding (AUB).

Abnormal Uterine Bleeding (AUB) is a common debilitating condition that significantly reduces quality of life of women across the reproductive age span. AUB creates significant morbidity, medical, social, and economic problems for women, their families, workplace, and health services. Despite the profoundly negative effects of AUB on public health, advancement in understanding the pathophysiology of AUB and the discovery of novel effective therapies is slow due to lack of reliable pre-clinical models. This review discusses currently available laboratory-based pre-clinical scientific models and how they are used to study AUB. Human and animal in vitro, ex vivo, and in vivo models will be described along with advantages and limitations of each method.

Stromal cells of the endometrium and decidua: in search of a name and an identity†.

Human endometrial and decidual stromal cells are the same cells in different environments (nonpregnancy and pregnancy, respectively). Although some authors consider decidual stromal cells to arise solely from the differentiation of endometrial stromal cells, this is a debatable issue given that decidualization processes do not end with the formation of the decidua, as shown by the presence of stromal cells from both the endometrium and decidua in both undifferentiated (nondecidualized) and decidualized states. Furthermore, recent functional and transcriptomic results have shown that there are differences in the decidualization process of endometrial and decidual stromal cells, with the latter having a greater decidualization capacity than the former. These differences suggest that in the terminology and study of their characteristics, endometrial and decidual stromal cells should be clearly distinguished, as should their undifferentiated or decidualized status. There is, however, considerable confusion in the designation and identification of uterine stromal cells. This confusion may impede a judicious understanding of the functional processes in normal and pathological situations. In this article, we analyze the different terms used in the literature for different types of uterine stromal cells, and propose that a combination of differentiation status (undifferentiated, decidualized) and localization (endometrium, decidua) criteria should be used to arrive at a set of accurate, unambiguous terms. The cell identity of uterine stromal cells is also a debatable issue: phenotypic, functional, and transcriptomic studies in recent decades have related these cells to different established cells. We discuss the relevance of these associations in normal and pathological situations.

Endometrial macrophages in health and disease.

Macrophages are present in the endometrium throughout the menstrual cycle and are most abundant during menstruation. Endometrial macrophages contribute to tissue remodeling during establishment of pregnancy and are thought to play key roles in mediating tissue breakdown and repair during menstruation. Despite these important roles, the phenotype and function of endometrial macrophages remains poorly understood. In this review, we summarize approaches used to characterize endometrial macrophage phenotype, current understanding of the functional role of macrophages in normal endometrial physiology as well as the putative contribution of macrophage dysfunction to women's reproductive health disorders.

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization.

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

Metallothionein1A Regulates Rhizobial Infection and Nodulation in Phaseolus vulgaris.

Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.

Decidualization modulates the mesenchymal stromal/stem cell and pericyte characteristics of human decidual stromal cells. Effects on antigen expression, chemotactic activity on monocytes and antitumoral activity.

Decidual stromal cells (DSCs) are the most abundant cellular component of human decidua and play a central role in maternal-fetal immune tolerance. Antigen phenotyping and functional studies recently confirmed the relationship of DSCs with mesenchymal stem/stromal cells (MSCs) and pericytes, the latter two cell types being closely related or identical. The present study investigated the effect of decidualization, a process of cell differentiation driven by progesterone (P4) and other pregnancy hormones, on the MSC/pericyte characteristics of DSCs. To this end we isolated undifferentiated DSC (preDSC) lines that were decidualized in vitro (dDSC) by the effect of P4 and cAMP. Using flow cytometry, we found significant downmodulation of the expression of the MSC/pericyte markers α-smooth muscle actin, nestin, CD140b, CD146 and SUSD2 in dDSCs. The dDSCs did not differ, compared to preDSCs, in the expression of angiogenic factors (characteristic of pericytes) HGF, FGF2, ANGPT1 or VEGF according to RT-PCR results, but had significantly increased PGF expression. In migration assays, preDSC-conditioned media had a chemotactic effect on the THP-1 monocytic line (characteristic of pericytes), and this effect was significantly greater in dDSC-conditioned media. Media conditioned with dDSC, but not with preDSC, induced apoptosis in 4 out of 6 different tumor cell lines (characteristic of MSCs) according to propidium iodide staining and flow cytometry results. Our findings show that decidualization induces phenotypic and functional changes in the MSC/pericyte properties of DSCs that may have a role in the normal development of pregnancy.

The Menstrual Endometrium: From Physiology to Future Treatments.

Abnormal uterine bleeding (AUB) is experienced by up to a third of women of reproductive age. It can cause anaemia and often results in decreased quality of life. A range of medical and surgical treatments are available but are associated with side effects and variable effectiveness. To improve the lives of those suffering from menstrual disorders, delineation of endometrial physiology is required. This allows an increased understanding of how this physiology may be disturbed, leading to uterine pathologies. In this way, more specific preventative and therapeutic strategies may be developed to personalise management of this common symptom. In this review, the impact of AUB globally is outlined, alongside the urgent clinical need for improved medical treatments. Current knowledge of endometrial physiology at menstruation is discussed, focusing on endocrine regulation of menstruation and local endometrial inflammation, tissue breakdown, hypoxia and endometrial repair. The contribution of the specialised endometrial vasculature and coagulation system during menstruation is highlighted. What is known regarding aberrations in endometrial physiology that result in AUB is discussed, with a focus on endometrial disorders (AUB-E) and adenomyosis (AUB-A). Gaps in existing knowledge and areas for future research are signposted throughout, with a focus on potential translational benefits for those experiencing abnormal uterine bleeding. Personalisation of treatment strategies for menstrual disorders is then examined, considering genetic, environmental and demographic characteristics of individuals to optimise their clinical management. Finally, an ideal model of future management of AUB is proposed. This would involve targeted diagnosis of specific endometrial aberrations in individuals, in the context of holistic medicine and with due consideration of personal circumstances and preferences.

HYPOXIA AND REPRODUCTIVE HEALTH: The presence and role of hypoxia in the endometrium.

The endometrium is a multicellular tissue that is exquisitely responsive to the ovarian hormones. The local mechanisms of endometrial regulation to ensure optimal function are less well characterised. Transient physiological hypoxia has been proposed as a critical regulator of endometrial function. Herein, we review the literature on hypoxia in the non-pregnant endometrium. We discuss the pros and cons of animal models, human laboratory studies and novel in vivo imaging for the study of endometrial hypoxia. These research tools provide mounting evidence of a transient hypoxic episode in the menstrual endometrium and suggest that endometrial hypoxia may be present at the time of implantation. This local hypoxia may modify the inflammatory environment, influence vascular remodelling and modulate endometrial proliferation to optimise endometrial function. Finally, we review current knowledge of the impact of this hypoxia on endometrial pathologies, with a focus on abnormal uterine bleeding. Throughout the manuscript areas for future research are highlighted with the aim of concentrating research efforts to maximise future benefits for women and society.

Menstrual blood-derived stromal cells modulate functional properties of mouse and human macrophages.

Menstrual blood-derived stromal cells (MenSCs) are emerging as a strong candidate for cell-based therapies due to their immunomodulatory properties. However, their direct impact on innate immune populations remains elusive. Since macrophages play a key role in the onset and development of inflammation, understanding MenSCs implication in the functional properties of these cells is required to refine their clinical effects during the treatment of inflammatory disorders. In this study, we assessed the effects that MenSCs had on the recruitment of macrophages and other innate immune cells in two mouse models of acute inflammation, a thioglycollate (TGC)-elicited peritonitis model and a monobacterial sepsis model. We found that, in the TGC model, MenSCs injection reduced the percentage of macrophages recruited to the peritoneum and promoted the generation of peritoneal immune cell aggregates. In the sepsis model, MenSCs exacerbated infection by diminishing the recruitment of macrophages and neutrophils to the site of infection and inducing defective bacterial clearance. Additional in vitro studies confirmed that co-culture with MenSCs impaired macrophage bactericidal properties, affecting bacterial killing and the production of reactive oxygen intermediates. Our findings suggest that MenSCs modulate the macrophage population and that this modulation must be taken into consideration when it comes to future clinical applications.

Author Correction: High-throughput microCT scanning of small specimens: preparation, packing, parameters and post-processing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-throughput microCT scanning of small specimens: preparation, packing, parameters and post-processing.

High-resolution X-ray microcomputed tomography, or microCT (µCT), enables the digital imaging of whole objects in three dimensions. The power of µCT to visualize internal features without disarticulation makes it particularly valuable for the study of museum collections, which house millions of physical specimens documenting the spatio-temporal patterns of life. Despite the potential for comparative analyses, most µCT studies include limited numbers of museum specimens, due to the challenges of digitizing numerous individuals within a project scope. Here we describe a method for high-throughput µCT scanning of hundreds of small (< 2 cm) specimens in a single container, followed by individual labelling and archival storage. We also explore the effects of various packing materials and multiple specimens per capsule to minimize sample movement that can degrade image quality, and hence µCT investment. We demonstrate this protocol on vertebrate fossils from Queensland Museum, Australia, as part of an effort to track community responses to climate change over evolutionary time. This system can be easily modified for other types of wet and dry material amenable to X-ray attenuation, including geological, botanical and zoological samples, providing greater access to large-scale phenotypic data and adding value to global collections.

COVID 19: Síndrome febril y clínica neurológica en neonato / Covid 19: fever syndrome and neurological symptoms in a neonate

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Endometrial and decidual stromal precursors show a different decidualization capacity.

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context - that is, non-pregnancy vs pregnancy - of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.