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Germplasm banks are the most significant repository for plant genetic resources for food and agriculture (PGRFA) worldwide. Despite their strategic importance, national germplasm banks of tropical megadiverse developing countries such as Colombia have extremely limited funds. Therefore, making strategic decisions about research investment is essential. Here, we designed a data-driven approach to build an index that sorts Colombian PGRFA into three groups: high, medium, or low priority, based on four pillars of information from open-access databases and aligned with the sustainable goals of no poverty and zero hunger: Geographic origin, vulnerability status, economic benefits, and food security importance. We analyzed 345 PGRFA using the index, separating them into two groups, 275 already conserved in the Colombian germplasm bank (BGVCOL group) and 70 not currently conserved in the BGVCOL (NCB group). We used fuzzy logic to classify each PGRFA by each pillar and integrate it to obtain a priority index. Missing data for native crops were frequent in the BGVCOL group. Therefore we adopted an imputation strategy to fill the gaps and calculated the uncertainty. After applying the index, PGRFA with higher priority were 24 (8.72%) from the BGVCOL (i.e., 15 potatoes, three tomatoes, two tree tomatoes, pineapple, cocoa, papaya, and yacon) and one from NCB (i.e., coffee). We concluded that this methodology successfully prioritized PGRFA in Colombia and shows the big holes of knowledge for future research and alternatives to improve this index. The versatility of this methodology could be helpful in other genebanks with budget limitations for research investment.

During early mammalian embryonic development, trophoblast cells play an essential role in establishing cell-cell interactions at the maternal-fetal interface to ensure a successful pregnancy. In a recent study, we showed that human fibroblasts can be reprogrammed into induced trophoblast stem (iTS) cells by transcription factor-mediated nuclear reprogramming using the Yamanaka factors OCT4, KLF4, SOX2 and c-MYC (OKSM) and a selection of TS cell culture conditions. The derivation of TS cells from human blastocysts or first-trimester placenta can be limited by difficulties in obtaining adequate material as well as ethical implications. By contrast, the described approach allows the generation of iTS cells from the adult cells of individuals with diverse genetic backgrounds, which are readily accessible to many laboratories around the world. Here we describe a step-by-step protocol for the generation and establishment of human iTS cells directly from dermal fibroblasts using a non-integrative reprogramming method. The protocol consists of four main sections: (1) recovery of cryopreserved human dermal fibroblasts, (2) somatic cell reprogramming, (3) passaging of reprogramming intermediates and (4) derivation of iTS cell cultures followed by routine maintenance of iTS cells. These iTS cell lines can be established in 2-3 weeks and cultured long term over 50 passages. We also discuss several characterization methods that can be performed to validate the iTS cells derived using this approach. Our protocol allows researchers to generate patient-specific iTS cells to interrogate the trophoblast and placenta biology as well as their interactions with embryonic cells in health and diseases.

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3Δ/Δ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.

Supplementary Materials from Sequential Transcription Factor Targeting for Diffuse Large B-Cell Lymphomas

BackgroundGenome-wide association studies (GWAS) have identified > 200 loci associated with breast cancer risk. The majority of candidate causal variants are in non-coding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association, and identifying the phenotype it mediates, is a major challenge in the interpretation and translation of GWAS.ResultsHere, we show that pooled CRISPR screens are highly effective at identifying GWAS target genes and defining the cancer phenotypes they mediate. Following CRISPR mediated gene activation or suppression, we measure proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We perform 60 CRISPR screens and identify 20 genes predicted with high confidence to be GWAS targets that promote cancer by driving proliferation or modulating the DNA damage response in breast cells. We validate the regulation of a subset of these genes by breast cancer risk variants.ConclusionsWe demonstrate that phenotypic CRISPR screens can accurately pinpoint the gene target of a risk locus. In addition to defining gene targets of risk loci associated with increased breast cancer risk, we provide a platform for identifying gene targets and phenotypes mediated by risk variants.

The vertebrate main-body axis is laid down during embryonic stages in an anterior-to-posterior (head-to-tail) direction, driven and supplied by posteriorly located progenitors. Whilst posterior expansion and segmentation appears broadly uniform along the axis, there is developmental and evolutionary support for at least two discrete modules controlling processes within different axial regions: a trunk and a tail module. Here, we identify Nuclear receptor subfamily 6 group A member 1 (Nr6a1) as a master regulator of trunk development in the mouse. Specifically, Nr6a1 was found to control vertebral number and segmentation of the trunk region, autonomously from other axial regions. Moreover, Nr6a1 was essential for the timely progression of Hox signatures, and neural versus mesodermal cell fate choice, within axial progenitors. Collectively, Nr6a1 has an axially-restricted role in all major cellular and tissue-level events required for vertebral column formation, supporting the view that changes in Nr6a1 levels may underlie evolutionary changes in axial formulae.

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.

Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology.

La pandemia de la COVID-19 determinó la suspensión de clases y el cierre de los locales de las universidades para evitar el contagio y muerte de docentes y estudiantes, y obligó a los gobiernos de los países a implementar medidas rápidas que permitieran la adaptación de los procesos de enseñanza-aprendizaje a la educación remota. Finalizado el 2020, se considera necesario cumplir con el objetivo de analizar las estrategias que se implementaron, sus resultados y determinar así los retos futuros que deberán enfrentar los gobiernos de cara a la realidad. Para ello, se consideró aplicar una metodología de investigación cualitativa, de análisis de contenido documental en una muestra de 28 textos que tratan sobre el tema; algunos de ellos fueron de investigación y otros, documentos de naturaleza legal de tres países seleccionados: Perú, Colombia y Chile. Se establecieron cinco categorías de análisis para ordenar los resultados, los cuales evidenciaron que los tres países pusieron en práctica estrategias educativas similares, como son: adoptar la educación a distancia virtual utilizando plataformas virtuales como entornos de aprendizaje, capacitación de docentes y estudiantes en competencias digitales y distribución de tablets y celulares con servicio de Internet a los estudiantes, entre otras.

[ES] Introducción: El objetivo es evaluar la eficacia y seguridad de los neuroestimuladores periféricos del ganglio esfenopalatino (GEP) para el tratamiento de la cefalea en racimos crónica refractaria al tratamiento. Desarrollo: Revisión sistemática de la literatura científica. Se identificaron estudios mediante una búsqueda en diferentes bases de datos. Las estrategias de búsqueda se realizaron hasta el 31 de octubre de 2016, incluyendo ensayos clínicos, revisiones sistemáticas o metaanálisis, informes de evaluación de tecnologías sanitarias y guías de práctica clínica que recogieran medidas de eficacia/efectividad o efectos adversos asociados al tratamiento. Se excluyeron estudios de cohortes, casos y controles, series de casos, revisiones narrativas, cartas al director, artículos de opinión, editoriales y estudios duplicados o desfasados por estudios posteriores de la misma institución. Respecto a la eficacia, los resultados son positivos tras la estimulación del GEP en relación con el alivio de dolor, el número de episodios, el uso de la medicación o la calidad de vida del paciente. En relación con la seguridad, hay un número importante de efectos adversos en los primeros 30 días de la intervención y en algunos pacientes fue necesaria la retirada del dispositivo. Los datos de seguimiento son a corto plazo y escasos. Conclusiones: Los resultados resultan prometedores a pesar de que la evidencia disponible es limitada. Consideramos fundamental continuar con la investigación sobre la seguridad y eficacia de los neuroestimuladores del GEP en la cefalea en racimos crónica. En aquellos casos en que pueda estar indicada la intervención, el tratamiento debería realizarse supervisado en un estudio de monitorización. [EN] Introduction: This study aimed to assess the safety and effectiveness of peripheral neurostimulation of the sphenopalatine ganglion (SPG) in the treatment of refractory chronic cluster headache. Development: Various medical databases were used to perform a systematic review of the scientific literature. The search for articles continued until 31 October 2016, and included clinical trials, systematic reviews and/or meta-analyses, health technology assessment reports, and clinical practice guidelines that included measurements of efficiency/effectiveness or adverse effects associated with the treatment. The review excluded cohort studies, case–control studies, case series, literature reviews, letters to the editor, opinion pieces, editorials, and studies that had been duplicated or outdated by later publications from the same institution. Regarding effectiveness, we found that SPG stimulation had positive results for pain relief, attack frequency, medication use, and patients’ quality of life. In the results regarding safety, we found a significant number of adverse events in the first 30 days following the intervention. Removal of the device was necessary in some patients. Little follow-up data, and no long-term data, is available. Conclusions: These results are promising, despite the limited evidence available. We consider it essential for research to continue into the safety and efficacy of SPG stimulation for patients with refractory chronic cluster headache. In cases where this intervention may be indicated, treatment should be closely monitored. Sí

Neuroinflammation has a major role in several brain disorders including Alzheimer’s disease (AD), yet at present there are no effective anti-neuroinflammatory therapeutics available. Copper(II) complexes of bis(thiosemicarbazones) (CuII(gtsm) and CuII(atsm)) have broad therapeutic actions in preclinical models of neurodegeneration, with CuII(atsm) demonstrating beneficial outcomes on neuroinflammatory markers in vitro and in vivo. These findings suggest that copper(II) complexes could be harnessed as a new approach to modulate immune function in neurodegenerative diseases. In this study, we examined the anti-neuroinflammatory action of several low-molecular-weight, charge-neutral and lipophilic copper(II) complexes. Our analysis revealed that one compound, a thiosemicarbazone–pyridylhydrazone copper(II) complex (CuL5), delivered copper into cells in vitro and increased the concentration of copper in the brain in vivo. In a primary murine microglia culture, CuL5 was shown to decrease secretion of pro-inflammatory cytokine macrophage chemoattractant protein 1 (MCP-1) and expression of tumor necrosis factor alpha (Tnf), increase expression of metallothionein (Mt1), and modulate expression of Alzheimer’s disease-associated risk genes, Trem2 and Cd33. CuL5 also improved the phagocytic function of microglia in vitro. In 5xFAD model AD mice, treatment with CuL5 led to an improved performance in a spatial working memory test, while, interestingly, increased accumulation of amyloid plaques in treated mice. These findings demonstrate that CuL5 can induce anti-neuroinflammatory effects in vitro and provide selective benefit in vivo. The outcomes provide further support for the development of copper-based compounds to modulate neuroinflammation in brain diseases.

Purpose: The aim of this study was to analyze the performance of multivariate machine learning (ML) models applied to a speech-in-noise hearing screening test and investigate the contribution of the measured features toward hearing loss detection using explainability techniques. Method: Seven different ML techniques, including transparent (i.e., decision tree and logistic regression) and opaque (e.g., random forest) models, were trained and evaluated on a data set including 215 tested ears (99 with hearing loss of mild degree or higher and 116 with no hearing loss). Post hoc explainability techniques were applied to highlight the role of each feature in predicting hearing loss. Results: Random forest (accuracy = .85, sensitivity = .86, specificity = .85, precision = .84) performed, on average, better than decision tree (accuracy = .82, sensitivity = .84, specificity = .80, precision = .79). Support vector machine, logistic regression, and gradient boosting had similar performance as random forest. According to post hoc explainability analysis on models generated using random forest, the features with the highest relevance in predicting hearing loss were age, number and percentage of correct responses, and average reaction time, whereas the total test time had the lowest relevance. Conclusions: This study demonstrates that a multivariate approach can help detect hearing loss with satisfactory performance. Further research on a bigger sample and using more complex ML algorithms and explainability techniques is needed to fully investigate the role of input features (including additional features such as risk factors and individual responses to low-/high-frequency stimuli) in predicting hearing loss.

Emotions processing is a complex mechanism that involves different physiological systems. In particular, the Central Nervous System (CNS) is considered to play a key role in this mechanism and one of the main modalities to study the CNS activity is the Electroencephalographic signal (EEG). To elicit emotions, different kinds of stimuli can be used e.g.: audio, visual or a combination of the two. Literature studies focus more on the correct classification of the different types of emotions or which kind of stimulation gives the best performance in terms of classification accuracy. However, it is still unclear how the different stimuli elicit the emotions and which are the results in terms of brain activity. In this paper, we analysed and compared EEG signals given by eliciting emotions using audio and visual stimuli or a combination of the latter two. Data were collected during experiments conducted in our laboratories using IAPS and IADS dataset. Our study confirmed literature physiological studies about emotions highlighting higher brain activity in the frontal and central regions and in the δ and θ bands for each kind of stimulus. However, audio stimulation was found to have higher responses when compared to the other two modalities of stimulation in almost all the comparisons performed. Higher values of the δ/β ratios, an index related to negative emotions, have been achieved when using only sounds as stimuli. Moreover, the same type of stimuli, resulted in higher δ-β coupling, suggesting a better attention control. We concluded that stimulating subjects without letting them know (seeing) what is actually happening may give a higher perception of emotions, even if this mechanism remains highly subjective. Clinical Relevance- This paper suggests that audio stimuli may give higher perception of the elicited emotion resulting in higher brain activity in the physiological areas and more focused subjects. Thus using only audio in emotion related studies may give more reliable and consistent results.

Germplasm banks are the most significant repository for plant genetic resources for food and agriculture (PGRFA) worldwide. Despite their strategic importance, national germplasm banks of tropical megadiverse developing countries such as Colombia have extremely limited funds. Therefore, making strategic decisions about research investment is essential. Here, we designed a data-driven approach to build an index that sorts Colombian PGRFA into three groups: high, medium, or low priority, based on four pillars of information from open-access databases and aligned with the sustainable goals of no poverty and zero hunger: Geographic origin, vulnerability status, economic benefits, and food security importance. We analyzed 345 PGRFA using the index, separating them into two groups, 275 already conserved in the Colombian germplasm bank (BGVCOL group) and 70 not currently conserved in the BGVCOL (NCB group). We used fuzzy logic to classify each PGRFA by each pillar and integrate it to obtain a priority index. Missing data for native crops were frequent in the BGVCOL group. Therefore we adopted an imputation strategy to fill the gaps and calculated the uncertainty. After applying the index, PGRFA with higher priority were 24 (8.72%) from the BGVCOL (i.e., 15 potatoes, three tomatoes, two tree tomatoes, pineapple, cocoa, papaya, and yacon) and one from NCB (i.e., coffee). We concluded that this methodology successfully prioritized PGRFA in Colombia and shows the big holes of knowledge for future research and alternatives to improve this index. The versatility of this methodology could be helpful in other genebanks with budget limitations for research investment.

According to Denis Noble, one of the greatest illusions of the Modern Synthesis is embodied in the Central Dogma, a principle first formulated by Francis Crick in 1958. The principle (roughly) holds that DNA makes protein, not the other way around. For Noble, the Dogma has contributed to the illusion that genes alone are responsible for the development and evolution of an organism’s phenotype. Though I am largely sympathetic to Noble’s critique, I argue that there may be alternative grounds for accepting the plausibility of the Central Dogma and also the illusion it perpetuates.

There is no denying the Central Dogma's impact on the biological sciences. Since the Dogma's formulation by Francis Crick in 1958, however, many have debated the Dogma's empirical adequacy. My aim is to move beyond these discussions, and instead consider the Central Dogma's significance to contemporary biological practice. To do this, I consider four distinct approaches for determining the non-descriptive methodological significance of a scientific principle. I argue that these approaches fail to vindicate the Central Dogma, and that, under many of these approaches, the Dogma amounts to a triviality.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1–4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization. Human fibroblasts are reprogrammed to generate blastocyst-like structures called iBlastoids, which recapitulate aspects of embryo implantation.

Nuclear transporter Importin (Imp, Ipo) 13 is known to transport various mammalian cargoes into/out of the nucleus, but its role in directing cell-fate is unclear. Here we examine the role of Imp13 in the maintenance of pluripotency and differentiation of embryonic stem cells (ESCs) for the first time, using an embryonic body (EB)-based model. When induced to differentiate, Ipo13-/- ESCs displayed slow proliferation, reduced EB size, and lower expression of the proliferation marker KI67, concomitant with an increase in the number of TUNEL+ nuclei compared to wildtype ESCs. At days 5 and 10 of differentiation, Ipo13-/- EBs also showed enhanced loss of the pluripotency transcript OCT3/4, and barely detectable clusters of OCT3/4 positive cells. Day 5 Ipo13-/- EBs further exhibited reduced levels of the mesodermal markers Brachyury and Mixl1, correlating with reduced numbers of haemoglobinised cells generated. Our findings suggest that Imp13 is critical to ESC survival as well as early post-gastrulation differentiation.

Summary Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general., Graphical Abstract, Highlights • TINC allows the isolation of a specific locus for molecular analyses • TINC identified hundreds of proteins at the Nanog promoter • RCOR2 is a component of the pluripotency network in embryonic stem cells • RCOR2 is required for efficient differentiation, Here, Knaupp and colleagues describe TINC, an epigenetic method that allows interrogation of mammalian regulatory complexes at a single-locus resolution. TINC was applied to dissect the transcriptional complex at the Nanog promoter in embryonic stem cells, revealing hundreds of interactors, including RCOR2, hence redefining how this gene is regulated in pluripotency.

The capability to generate induced pluripotent cell (iPSC) lines, in combination with the CRISPR-Cas9 DNA editing technology, offers great promise to understand the underlying genetic mechanisms of human disease. However, technical impediments including, but not limited to, low transfection efficiency, single-cell survival, and high clonal heterogeneity, limit the potential of these techniques. Here we provide an efficient methodology addressing these challenges, resulting in high transfection efficiency exceeding 97% with an increased single cell clone survival rate (up to 70%). These enhancements were accompanied by a high editing efficiency in the range of 48.6 to 57.5%, comparable to existing methods.

Postnatal maturation of the immune system is poorly understood, as is its impact on illnesses afflicting term or preterm infants, such as bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension. These are both cardiopulmonary inflammatory diseases that cause substantial mortality and morbidity with high treatment costs. Here, we characterized blood samples collected from 51 preterm infants longitudinally at five time points, 20 healthy term infants at birth and age 3 to 16 weeks, and 5 healthy adults. We observed strong associations between type 2 immune polarization in circulating CD3 + CD4 + T cells and cardiopulmonary illness, with odds ratios up to 24. Maternal magnesium sulfate therapy, delayed hepatitis B vaccination, and increasing fetal, but not maternal, chorioamnionitis severity were associated with attenuated type 2 polarization. Blocking type 2 mediators such as interleukin-4 (IL-4), IL-5, IL-13, or signal transducer and activator of transcription 6 (STAT6) in murine neonatal cardiopulmonary disease in vivo prevented changes in cell type composition, increases in IL-1β and IL-13, and losses of pulmonary capillaries, but not gains in larger vessels. Thereby, type 2 blockade ameliorated lung inflammation, protected alveolar and vascular integrity, and confirmed the pathological impact of type 2 cytokines and STAT6. In-depth flow cytometry and single-cell transcriptomics of mouse lungs further revealed complex associations between immune polarization and cardiopulmonary disease. Thus, this work advances knowledge on developmental immunology and its impact on early life disease and identifies multiple therapeutic approaches that may relieve inflammation-driven suffering in the youngest patients.

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.

The vertebrate main-body axis is laid down during embryonic stages in an anterior-to-posterior (head-to-tail) direction, driven and supplied by posteriorly located progenitors. For the vertebral column, the process of axial progenitor cell expansion that drives elongation, and the process of segmentation which allocates progenitor-descendants into repeating pre-vertebral units, occurs seemingly uninterrupted from the first to the last vertebra. Nonetheless, there is clear developmental and evolutionary support for two discrete modules controlling processes within different axial regions: a trunk and a tail module. Here, we identify Nuclear receptor subfamily 6 group A member 1 (Nr6a1) as a master regulator of elongation, segmentation, patterning and lineage allocation specifically within the trunk region of the mouse. Both gain- and loss-of-function in vivo analyses revealed that the precise level of Nr6a1 acts as a rheostat, expanding or contracting vertebral number of the trunk region autonomously from other axial regions. Moreover, Nr6a1 was found to be required for segmentation, but only for trunk-forming somites, with the timely clearance of Nr6a1 critical in supporting tail formation. In parallel with these morphological outcomes, we reveal Nr6a1 as a novel regulator of global Hox signatures within axial progenitors, preventing the precocious expression of multiple posterior Hox genes as the trunk is being laid down and thus reinforcing that patterning and elongation are coordinated. Finally, our data supports a crucial role for Nr6a1 in regulating gene regulatory networks that guide cell lineage choice of axial progenitors between neural and mesodermal fate. Collectively, these data reveal an axially-restricted role for Nr6a1 in all major cellular and tissue-level events required for vertebral column formation, supporting the view that modulation of Nr6a1 expression level or function is likely to underpin evolutionary changes in axial formulae that exclusively alter the trunk region.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development 1–6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas 7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

Summary Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies., Highlights • Integrated molecular characterization reveals differences in four iMGL protocols • A dual tdTomato and nanoluciferase reporter tool was generated to track iMGLs via CX3CR1 • Kinetics of surface marker expression during iMGL differentiation • Co-culture with glia/neurons restores ex vivo microglial transcription factors, In this article, Jose Polo and colleagues molecularly compare existing microglia differentiation methods from stem cells, generate a new dual fluorescent and enzymatic reporter tool to study microglia, and show that human glia/neuron co-culture pushes the regulatory landscape of in vitro microglia-like cells to more closely resemble bona fide microglia.

Background The adverse effects of air pollutants including particulate matter (PM) on the central nervous system is increasingly reported by epidemiological, animal and post-mortem studies in the last decade. Oxidative stress and inflammation are key consequences of exposure to PM although little is known of the exact mechanism. The association of PM exposure with deteriorating brain health is speculated to be driven by PM entry via the olfactory system. How air pollutants affect this key entry site remains elusive. In this study, we investigated effects of urban size-segregated PM on a novel cellular model: primary human olfactory mucosal (hOM) cells. Results Metabolic activity was reduced following 24-h exposure to PM without evident signs of toxicity. Results from cytometric bead array suggested a mild inflammatory response to PM exposure. We observed increased oxidative stress and caspase-3/7 activity as well as perturbed mitochondrial membrane potential in PM-exposed cells. Mitochondrial dysfunction was further verified by a decrease in mitochondria-dependent respiration. Transient suppression of the mitochondria-targeted gene, neuronal pentraxin 1 (NPTX1), was carried out, after being identified to be up-regulated in PM2.5–1 treated cells via RNA sequencing. Suppression of NPTX1 in cells exposed to PM did not restore mitochondrial defects resulting from PM exposure. In contrast, PM-induced adverse effects were magnified in the absence of NPTX1, indicating a critical role of this protein in protection against PM effects in hOM cells. Conclusion Key mitochondrial functions were perturbed by urban PM exposure in a physiologically relevant cellular model via a mechanism involving NPTX1. In addition, inflammatory response and early signs of apoptosis accompanied mitochondrial dysfunction during exposure to PM. Findings from this study contribute to increased understanding of harmful PM effects on human health and may provide information to support mitigation strategies targeted at air pollution.

Background: This case study aimed to identify and describe meaningful physical occupations used by a cancer survivor to increase or maintain levels of participation during active chemotherapy and subsequent cancer related fatigue. Method: A case study approach was used to develop an in-depth description and analysis based on one participant’s experience with breast cancer and associated treatments. A semi-structured interview was conducted. The data were analyzed through description of the case, categories, and themes. It also included categorical aggregation in efforts to seek a collection of instances from the data to explore any issue-relevant meanings. Results: Following data analysis, one overarching theme, return to normalcy, was identified with three subthemes: (a) prioritization of meaningful activities, (b) modifications to activities or routines, (c) lack of referral for immediate needs. Conclusion: Personal knowledge of occupational therapy practice provided support for enhancing engagement in daily meaningful occupations. Lessons learned from this experience could be applied to clients experiencing cancer related side effects to improve engagement in daily occupations.

A conceptual biodiesel production homogeneous process was developed, considering the typical reaction conditions, appropriate thermodynamic models as well as the kinetics of a representative reaction for vegetable oils, under assumption to be mass-produced and provide a continuous way the raw material for the production of the biofuel. Reasonable assumptions and the use of a rigorous simulator of processes were employed; the process was simulated for the treatment of 1000 mol/h of oil operating continuously, at 45 and 55 °C, 1.0 wt% catalyst and 6 mol methanol by mol of oil. The processes of catalyst preparation, raw material pumping, heating, isothermal reaction, separation of byproducts by density and distillation were considered, and also auxiliary services of heating and cooling during the process. The convergence of each device was achieved by properly choosing the specifications of the unit operation models. Material and energy balances were obtained at each point in the process and in a global manner. For space-time of 30 min in single and series reactors global efficiencies from ~ 80 to 85% biodiesel was calculated for all explored alternatives. The ratio of energy production of biodiesel to the energy consumed by the process, within the battery limits used in this study, was found to be more than 6 times, thus ensuring the sustainability of the process from energetic point of view. It was found that, for the highest purity and better ratio of energy produced/consumed, the global efficiency process was the lowest.