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CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hoxgene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxAcluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hoxgenes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.

The interaction between organisms and their environment is central in functional morphology. Differences in habitat usage may imply divergent morphology of locomotor systems; thus, detecting which morphological traits are conservative across lineages and which ones vary under environmental pressure is important in evolutionary studies. We studied internal and external morphology in 28 species of Neotropical anurans. Our aim was to determine if internal morphology (muscle and tendons) shows lower phylogenetic signal than external morphology. In addition, we wanted to know if morphology varies in relation to the habitat use and if there are different functional groups. We found differences in the degree of phylogenetic signal on the groups of traits. Interestingly, postaxial regions of the forelimb are evolutionarily more labile than the preaxial regions. Phylomorphospace plots show that arboreal (jumpers and graspers) and swimmer frogs cluster based on length of fingers and the lack of sesamoid, also reflected by the use of habitat. These functional clusters are also related to phylogeny. Sesamoid and flexor plate dimensions together with digit tendons showed to be important to discriminate functional groups as well as use of habitat classification. Our results allow us to identify a "grasping syndrome" in the hand of these frogs, where palmar sesamoid and flexor plate are absent and a third metacarpal with a bony knob are typical. Thus, a lighter skeleton, long fingers and a prensile hand may be key for arboreality.

The motor neuron progenitor domain in the ventral spinal cord gives rise to multiple subtypes of motor neurons and glial cells. Here, we examine whether progenitors found in this domain are multipotent and which signals contribute to their cell-type-specific differentiation. Using an in vitro neural differentiation model, we demonstrate that motor neuron progenitor differentiation is iteratively controlled by Notch signaling. First, Notch controls the timing of motor neuron genesis by repressing Neurogenin 2 (Ngn2) and maintaining Olig2-positive progenitors in a proliferative state. Second, in an Ngn2-independent manner, Notch contributes to the specification of median versus hypaxial motor column identity and lateral versus medial divisional identity of limb-innervating motor neurons. Thus, motor neuron progenitors are multipotent, and their diversification is controlled by Notch signaling that iteratively increases cellular diversity arising from a single neural progenitor domain.

El TEC según la IBIA es la principal causa de convulsiones, muerte y discapacidad adquirida mundialmente. Las principales causas son las caídas en mayores de 65 años y los accidentes de tránsito en los menores. Los hematomas pos-TEC son una complicación muy frecuente, cuyas manifestaciones son variables, especialmente en los subdurales, presentando desde cefalea hasta síndromes demenciales o déficit neurológico focal, dentro de los cuales no es usual el reporte de casos con manifestaciones visuales. El presente artículo reporta un caso de discapacidad visual por déficit neurológico pos-TEC complicado con hematomas subdurales subcrónicos bilaterales parietooccipitales, y revisa la neuroanatomía funcional visual involucrada en ella.

Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate “hybrid” cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivocorrelate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering.

In this work we present experimental results that confirm the influence of an absorptive surrounding medium in the behavior of conventional SPR fiber sensors. It can be observed that when a plasmon resonance coincides with a wavelength absorbed by the medium, the depth of the dip is affected, as predicted in the simulations. The results are important from the basic point of view, since the influence of absorptive medium in plasma waves has not been sufficiently studied up to date and they can also be used as the basis for a new method of SPR-based refractometry selective to specific analytes without the need of the addition of recognizing elements to the transducer.

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.

Background: Transcription factor (TF) binding specificity is determined via a complex interplay between the transcription factor’s DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with transcription factor binding in a given cell type have been well characterized. For instance, the binding sites for a majority of transcription factors display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the transcription factor itself and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of transcription factor binding specificity, we therefore need to examine how newly activated transcription factors interact with sequence and preexisting chromatin landscapes. Results: Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of transcription factors that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced transcription factors. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some transcription factors substantially, but not others. Furthermore, by analyzing site-level predictors, we show that transcription factor binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences. Conclusions: Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.

Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vectorAphis gossypii.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novoDNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

A genetically encoded sensor of cAMP enables detection of this second messenger in live cells and tissues.

Direct cell programming via overexpression of transcription factors (TFs) aims to control cell fate with the degree of precision needed for clinical applications. However, the regulatory steps involved in successful terminal cell fate programming remain obscure. We have investigated the underlying mechanisms by looking at gene expression, chromatin states, and TF binding during the uniquely efficient Ngn2, Isl1, and Lhx3 motor neuron programming pathway. Our analysis reveals a highly dynamic process in which Ngn2 and the Isl1/Lhx3 pair initially engage distinct regulatory regions. Subsequently, Isl1/Lhx3 binding shifts from one set of targets to another, controlling regulatory region activity and gene expression as cell differentiation progresses. Binding of Isl1/Lhx3 to later motor neuron enhancers depends on the Ebf and Onecut TFs, which are induced by Ngn2 during the programming process. Thus, motor neuron programming is the product of two initially independent transcriptional modules that converge with a feedforward transcriptional logic.

A remote strain sensor with a lead fiber length of 3.3 km and an ultraweak fiber Bragg grating with reflectivity of 0.015 was demonstrated. A demodulation technique based on methods of the differential spectroscopy allows interrogating FBG with reflectance much below the Rayleigh backscattering coefficient. © 2003 Society of Photo-Optical Instrumentation Engineers.

A device and improved method based on the use of a compartmentalized Eppendorf tube that allows capture, reverse transcription and nested-PCR in a single closed-tube has been developed and patented. The main advantages of the system are the high sensitivity obtained, the simplicity, the low risk of contamination and the easy establishment of adequate conditions for nested-PCR. The method has been successfully applied to the detection and characterization of citrus tristeza closterovirus and plum pox potyvirus isolates in plant tissues and single aphids squashed on paper. This device and methodology could be easily adapted to the detection of other targets.