Analysis of localized cAMP perturbations within a tissue reveal the effects of a local, dynamic gap junction state on ERK signaling.

Beyond natural stimuli such as growth factors and stresses, the ability to experimentally modulate at will the levels or activity of specific intracellular signaling molecule(s) in specified cells within a tissue can be a powerful tool for uncovering new regulation and tissue behaviors. Here we perturb the levels of cAMP within specific cells of an epithelial monolayer to probe the time-dynamic behavior of cell-cell communication protocols implemented by the cAMP/PKA pathway and its coupling to the ERK pathway. The time-dependent ERK responses we observe in the perturbed cells for spatially uniform cAMP perturbations (all cells) can be very different from those due to spatially localized perturbations (a few cells). Through a combination of pharmacological and genetic perturbations, signal analysis, and computational modeling, we infer how intracellular regulation and regulated cell-cell coupling each impact the intracellular ERK response in single cells. Our approach reveals how a dynamic gap junction state helps sculpt the intracellular ERK response over time in locally perturbed cells.

The Maize MID-COMPLEMENTING ACTIVITY Homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF Coordinates Organ Growth and Tissue Patterning.

Organogenesis occurs through cell division, expansion, and differentiation. How these cellular processes are coordinated remains elusive. The maize (Zea mays) leaf provides a robust system to study cellular differentiation due to its distinct tissues and cell types. The narrow odd dwarf (nod) mutant displays defects at both the cellular and tissue level that increase in severity throughout growth. nod mutant leaves have reduced size due to fewer and smaller cells compared with the wild type. The juvenile-to-adult transition is delayed, and proximal distal-patterning is abnormal in this mutant. Differentiation of specialized cells such as those forming stomata and trichomes is incomplete. Analysis of nod-1 sectors suggests that NOD plays a cell-autonomous function in the leaf. We cloned nod positionally and found that it encodes CELL NUMBER REGULATOR13 (CNR13), the maize MID-COMPLEMENTING ACTIVITY homolog. CNR13/NOD is localized to the membrane and is enriched in dividing tissues. Transcriptome analysis of nod mutants revealed overrepresentation of cell wall, hormone metabolism, and defense gene categories. We propose that NOD coordinates cell activity in response to intrinsic and extrinsic cues.

In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells.

Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. Here we quantify dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. We identify phosphorylation of histone H3 at Ser 28 as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. We propose that regulation of the kinetic properties of PcG-chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities.

Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin.

The Polycomb (PcG) and Trithorax (TrxG) group proteins work antagonistically on several hundred developmentally important target genes, giving stable mitotic memory, but also allowing flexibility of gene expression states. How this is achieved in quantitative terms is poorly understood. Here, we present a quantitative kinetic analysis in living Drosophila of the PcG proteins Enhancer of Zeste, (E(Z)), Pleiohomeotic (PHO) and Polycomb (PC) and the TrxG protein absent, small or homeotic discs 1 (ASH1). Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy reveal highly dynamic chromatin binding behaviour for all proteins, with exchange occurring within seconds. We show that although the PcG proteins substantially dissociate from mitotic chromatin, ASH1 remains robustly associated with chromatin throughout mitosis. Finally, we show that chromatin binding by ASH1 and PC switches from an antagonistic relationship in interphase, to a cooperative one during mitosis. These results provide quantitative insights into PcG and TrxG chromatin-binding dynamics and have implications for our understanding of the molecular nature of epigenetic memory.

Postoperative pain: What can we do?

Chronic postoperative pain (CPOP) is a potentially devastating consequence of a surgical procedure. It leads to increased medical costs, painful, and stress experience to the patients. After a surgical decompression performed in a patient with a non-traumatic compartment syndrome, a muscle biopsy confirmed McArdle disease, and after surgery, severe pain of neuropathic characteristics developed in the arm decompressed. Advanced techniques up to neuromedullary stimulation failed to improve the clinical status, after which repeated treatment with capsaicin patch ameliorated the patient's condition. This case report illustrates the need for a high index of suspicion for metabolic diseases in patients who present compartment syndrome without prior history of trauma and also the challenges in treating neuropathic pain after surgery.

Orthogonal control of mean and variability of endogenous genes in a human cell line.

Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells.

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods for the assembly of mammalian DNA circuits are laborious, slow, and expensive. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible, and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. We showcase the capabilities of the MTK by using it to generate single-integration landing pads, create and deliver libraries of protein variants and sgRNAs, and iterate through dCas9-based prototype circuits. As a biological proof of concept, we demonstrate how the MTK can speed the generation of noninfectious viral circuits to enable rapid testing of pharmacological inhibitors of emerging viruses that pose a major threat to human health.

[Cultural adaptation and validation of the "Fibromyalgia Impact Questionnaire"--Portuguese version]. / Adaptação cultural e validação do "Fibromyalgia Impact Questionnaire" versão Portuguesa.

The aim of this study was to translate the Fibromyalgia Impact Questionnaire (FIQ) into Portuguese (Portugal) and to evaluate its reliability and validity by use with Portuguese--speaking patients with Fibromyalgia. After translating the FIQ into Portuguese we administered it to 68 patients with Fibromyalgia together with an informed consent, a Portuguese version of the Health Assessment Questionnaire (HAQ) and a formulary with the socio-demographic characteristics and duration of the complaints. The content validity was assessed with a panel of experts, with high consensus. In the concurrent validity, we obtained significant correlations between the FIQ first item and the HAQ [r = 0,531 (p = 0,001)]. Cronbach's alpha was 0,814, indicating an acceptable level of internal consistency. In conclusion, the Portuguese version of the FIQ is a reliable and valid instrument for measuring health status and physical functioning in Portuguese patients with Fibromyalgia. This instrument is available for use in the clinical practice.

Avaliaçäo da eficácia da associaçäo gentamicina e cloranfenicol no tratamento de endometrites puerperais / Evaluation of the efficacy of the combination of gentamicin plus cloramphenicol in the treatment of puerperal endometritis

Os autores avaliam um esquema antibiótico no tratamento das endometrites puerperais, visando eficácia terapêutica e diminuiçäo dos custos. Baseado na gênese bifásica do abcesso, vem sendo utilizado nos EUA o esquema de um aminoglicosídeo, geralmente a gentamicina, e um anaerobicida, a clindamicina, na endometrite pós-cesárea, com pleno sucesso. Os autores avaliaram os resultados da substituiçäo da clindamicina por outro antibiótico eficaz contra anaeróbios e de custo muito menor, o cloranfenicol. Foi realizado um inquérito exaustivo com a populaçäo que teve parto na Maternidade Odete Valadares-BH e desenvolveu a infecçäo no período de janeiro de 1990 a março de 1991, totalizando 116 casos. Foram consideradas as seguintes variáveis: tipo de parto, resposta ao tratamento, tipo de alta, duraçäo do tratamento, remissäo da febre e custos dos medicamentos. Os resultados foram comparados com aqueles da literatura com gentamicina/clindamicina e gentamicina/penicilina. O esquema gentamicina/cloranfenicol teve eficácia semelhante ao primeiro, com custos inferiores; foi mais eficaz e de custos inferiores ao segundo. Os autores concluem que este esquema apresenta alta eficácia, tem baixo custo e sugerem sua escolha como primeira linha nas endometrites puerperais.