Predictive Biophysical Neural Network Modeling of a Compendium of in vivo Transcription Factor DNA Binding Profiles for Escherichia coli.

The DNA binding of most Escherichia coli Transcription Factors (TFs) has not been comprehensively mapped, and few have models that can quantitatively predict binding affinity. We report the global mapping of in vivo DNA binding for 139 E. coli TFs using ChIP-Seq. We used these data to train BoltzNet, a novel neural network that predicts TF binding energy from DNA sequence. BoltzNet mirrors a quantitative biophysical model and provides directly interpretable predictions genome-wide at nucleotide resolution. We used BoltzNet to quantitatively design novel binding sites, which we validated with biophysical experiments on purified protein. We have generated models for 125 TFs that provide insight into global features of TF binding, including clustering of sites, the role of accessory bases, the relevance of weak sites, and the background affinity of the genome. Our paper provides new paradigms for studying TF-DNA binding and for the development of biophysically motivated neural networks.

Ectopia cordis: prenatal diagnosis, perinatal outcomes, and postnatal follow-up of an international multicenter cohort case series.

OBJECTIVE: This study aimed to analyze prenatal diagnosis, perinatal outcomes, and postnatal follow-up in fetuses with ectopia cordis (EC). METHODS: This retrospective analysis accessed 31 patients with EC who were either diagnosed or referred to a tertiary Fetal Medicine centers for EC diagnosis in Brazil, Germany, Italy, and Poland. We analyzed prenatal diagnosis, perinatal outcomes, and follow-up in these patients. RESULTS: Our study included a cohort of 31 fetuses with EC, 4 and 27 of whom had partial and complete protrusion of the heart through a ventral defect in the thoracoabdominal wall, respectively. EC was diagnosed by fetal echocardiography at a mean gestational age of 20.3 ± 8.6 weeks (range, 8-35 weeks). Of the four cases, in which the karyotype was performed, all of them had a normal result (1 - 46,XX and 3 - 46,XY). Five patients showed conotruncal abnormalities and six ventricular septal defects. Termination of pregnancy (TOP) was performed in 15 cases (48%) and seven pregnant women had spontaneous fetal demise (22.5%). Of the seven fetuses that were born alive, four of them died, and three infants underwent surgery. Among these three infants, all of them survived, one was 5 months, 13 years old and 29 years old at the time of study completion. CONCLUSIONS: Ectopia cordis is associated with high mortality rates and intracardiac/extra-cardiac defects. Ventricular septal defects and conotruncal anomalies were the more common intracardiac defects associated with EC. However, in this cohort of fetuses with EC the incidence of PC was lower than reported in the literature.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Epigenetic competition reveals density-dependent regulation and target site plasticity of phosphorothioate epigenetics in bacteria.

Phosphorothioate (PT) DNA modifications-in which a nonbonding phosphate oxygen is replaced with sulfur-represent a widespread, horizontally transferred epigenetic system in prokaryotes and have a highly unusual property of occupying only a small fraction of available consensus sequences in a genome. Using Salmonella enterica as a model, we asked a question of fundamental importance: How do the PT-modifying DndA-E proteins select their GPSAAC/GPSTTC targets? Here, we applied innovative analytical, sequencing, and computational tools to discover a novel behavior for DNA-binding proteins: The Dnd proteins are "parked" at the G6mATC Dam methyltransferase consensus sequence instead of the expected GAAC/GTTC motif, with removal of the 6mA permitting extensive PT modification of GATC sites. This shift in modification sites further revealed a surprising constancy in the density of PT modifications across the genome. Computational analysis showed that GAAC, GTTC, and GATC share common features of DNA shape, which suggests that PT epigenetics are regulated in a density-dependent manner partly by DNA shape-driven target selection in the genome.

A progesterone biosensor derived from microbial screening.

Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.

A unified resource for transcriptional regulation in Escherichia coli K-12 incorporating high-throughput-generated binding data into RegulonDB version 10.0.

BACKGROUND: Our understanding of the regulation of gene expression has benefited from the availability of high-throughput technologies that interrogate the whole genome for the binding of specific transcription factors and gene expression profiles. In the case of widely used model organisms, such as Escherichia coli K-12, the new knowledge gained from these approaches needs to be integrated with the legacy of accumulated knowledge from genetic and molecular biology experiments conducted in the pre-genomic era in order to attain the deepest level of understanding possible based on the available data. RESULTS: In this paper, we describe an expansion of RegulonDB, the database containing the rich legacy of decades of classic molecular biology experiments supporting what we know about gene regulation and operon organization in E. coli K-12, to include the genome-wide dataset collections from 32 ChIP and 19 gSELEX publications, in addition to around 60 genome-wide expression profiles relevant to the functional significance of these datasets and used in their curation. Three essential features for the integration of this information coming from different methodological approaches are: first, a controlled vocabulary within an ontology for precisely defining growth conditions; second, the criteria to separate elements with enough evidence to consider them involved in gene regulation from isolated transcription factor binding sites without such support; and third, an expanded computational model supporting this knowledge. Altogether, this constitutes the basis for adequately gathering and enabling the comparisons and integration needed to manage and access such wealth of knowledge. CONCLUSIONS: This version 10.0 of RegulonDB is a first step toward what should become the unifying access point for current and future knowledge on gene regulation in E. coli K-12. Furthermore, this model platform and associated methodologies and criteria can be emulated for gathering knowledge on other microbial organisms.

Coordinated regulation of acid resistance in Escherichia coli.

BACKGROUND: Enteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored. RESULTS: We utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally. CONCLUSIONS: Our data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.

Clinical and Electrodiagnostic Findings in Patients with Peripheral Neuropathy and Inflammatory Bowel Disease.

BACKGROUND: Several neurological diseases, especially different types of peripheral neuropathy (PN) are common in inflammatory bowel disease (IBD). METHODS: We prospectively evaluated the presence of PN in 121 patients with IBD (51 with Crohn's disease [CD] and 70 with ulcerative colitis [UC]) and 50 controls (gastritis and dyspepsia) over 3.5 years. RESULTS: A total of 15 patients (12.4%) with small-fiber neuropathy and IBD (7 CD and 8 UC) and 24 patients (19.8%) with large-fiber PN (12 CD and 12 UC) were diagnosed. Small-fiber neuropathy affected 6% and large-fiber PN affected 4% of the control patients. Patients with CD with PN were older, had more metabolic complications and more severe motor involvement than patients with UC with PN. Carpal tunnel syndrome was more common in patients with UC. Sural and median sensory nerves were the most commonly and severely affected sensory responses. Tibial, peroneal, median, and ulnar compound muscle action potential amplitudes were also significantly decreased in patients with CD and UC. In general, sensory and motor amplitudes were a more sensitive marker for PN in patients with IBD than conduction velocities. CONCLUSIONS: In summary, PN is common in patients with IBD. It may be primarily related to IBD, phenotypically modified by metabolic complications. Its phenotype is diverse (most commonly small to predominantly axonal sensory large-fiber), but usually more severe in CD. It also includes ataxic and demyelinating forms. Results from our 10-year follow-up will elucidate the PN clinical course and the real impact of the comorbidities and new therapies.

Pioderma gangrenoso / Pyoderma gangrenosum

É descrito caso de paciente de sete meses de idade com Pioderma gangrenoso em coxa direita. Não há associação com doença sistêmica. O tratamento foi feito com corticóide oral e sulfadiazina de prata. Obteve-se remissão da lesão.

The case is described of a 7 months old patient that presented Pyoderma gangrenosum in the right thigh. There isn't association with systemic desease. It was treated with oral corticoid and silver sulfadiazine.

Tinea capitis em João Pessoa: visão socioeconômica / Tinea capitis in João Pessoa: a social and economic view

FUNDAMENTOS: A distribuição das espécies de dermatófitos varia ao longo do tempo e de acordo com a região, refletindo as condições socioeconômicas da populaçãoOBJETIVOS: Estudar a Tinea Capitis quanto ao agente etiológico, ao sexo, à idade e ao grupo étnicodos doentes em João Pessoa, PB, Brasil e salientar a importância socioeconômica desses dados, com-parando-os aos de regiões mais ricas do país.MÉTODOS: Foram avaliados o perfil e os exames micológicos direto e cultura de 82 pacientes com suspeita clínica de T. capitis em João Pessoa.RESULTADOS: A freqüência de T. capitis incluiu 64,6 por cento das suspeitas clínicas. O dermatófito isolado com maior freqüência foi o T. rubrum (37,7 por cento), seguido por T. tonsurans (28,3 por cento), M. canis (24,5 por cento), T. verru c o s u m (7,5 por cento) e T. mentagrophytes (1,9 por cento). Não houve predileção quanto ao sexo. A faixa etária mais acometida foi a de 0 a 10 anos, e 71,7 por cento dos doentes são caucasóides.CONCLUSÃO: Comparando os resultados obtidos com publicações anteriores da Região Sudeste, os autores salientam as diferenças das variantes socioeconômicas na epidemiologia da doença.

Estudo retrospectivo de 145 casos de Tinea capitis na população de João Pessoa - Paraíba / Retrospective study in 145 cases by Tinea capitis from the population in João Pessoa - Paraíba

Foram analisados 145 casos de pacientes atendidos no Hospital Universitário Lauro Wanderley, da Universidade Federal da Paraíba, em João Pessoa - PB, Brasil, com diagnóstico de Tinea capitis conforme idade, sexo, número de casos/ano e doenças associadas. Houve maior incidência no sexo feminino e na faixa escolar. A média de casos/ano foi de 14,5. Verificou-se associação de Tinea capitis com dermatite seborréica, Tinea corporis e pediculose.

We studied 145 patients with Tinea capitis from Lauro Wanderley's University Hospital of Paraíba Federal University, João Pessoa-PB, Brasil, considering age, sex, number of cases/year and associated diseases...