Heat But Not Mechanical Hypersensitivity Depends on Voltage-Gated CaV2.2 Calcium Channel Activity in Peripheral Axon Terminals Innervating Skin.

Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor CaV2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that CaV2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral CaV2.2 channel activity, and especially a cell-type-specific CaV2.2 splice isoform. CaV2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain.SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of CaV2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral CaV2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 channels. Our findings suggest that interrupting CaV2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain.

Starch degradation in the bean fruit pericarp is characterized by an increase in maltose metabolism.

The bean fruit pericarp accumulates a significant amount of starch, which starts to be degraded 20 days after anthesis (DAA) when seed growth becomes exponential. This period is also characterized by the progressive senescence of the fruit pericarp. However, the chloroplasts maintained their integrity, indicating that starch degradation is a compartmentalized process. The process coincided with a transient increase in maltose and sucrose levels, suggesting that ß-amylase is responsible for starch degradation. Starch degradation in the bean fruit pericarp is also characterized by a large increase in starch phosphorylation, as well as in the activities of cytosolic disproportionating enzyme 2 (DPE2, EC 2.4.1.25) and glucan phosphorylase (PHO2, EC 2.4.1.1). This suggests that the rate of starch degradation in the bean fruit pericarp 20 DAA is dependent on the transformation of starch to a better substrate for ß-amylase and the increase in the rate of cytosolic metabolism of maltose.

Transcriptional Regulation of zma-MIR528a by Action of Nitrate and Auxin in Maize.

In recent years, miR528, a monocot-specific miRNA, has been assigned multifaceted roles during development and stress response in several plant species. However, the transcription regulation and the molecular mechanisms controlling MIR528 expression in maize are still poorly explored. Here we analyzed the zma-MIR528a promoter region and found conserved transcription factor binding sites related to diverse signaling pathways, including the nitrate (TGA1/4) and auxin (AuxRE) response networks. Accumulation of both pre-miR528a and mature miR528 was up-regulated by exogenous nitrate and auxin treatments during imbibition, germination, and maize seedling establishment. Functional promoter analyses demonstrated that TGA1/4 and AuxRE sites are required for transcriptional induction by both stimuli. Overall, our findings of the nitrogen- and auxin-induced zma-MIR528a expression through cis-regulatory elements in its promoter contribute to the knowledge of miR528 regulome.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Mechanisms of Neuronal Alternative Splicing and Strategies for Therapeutic Interventions.

Many cellular and physiological processes are coordinated by regulatory networks that produce a remarkable complexity of transcript isoforms. In the mammalian nervous system, alternative pre-mRNA splicing generates functionally distinct isoforms that play key roles in normal physiology, supporting development, plasticity, complex behaviors, and cognition. Neuronal splicing programs controlled by RNA-binding proteins, are influenced by chromatin modifications and can exhibit neuronal subtype specificity. As highlighted in recent publications, aberrant alternative splicing is a major contributor to disease phenotypes. Therefore, understanding the underlying mechanisms of alternative splicing regulation and identifying functional splicing isoforms with critical phenotypic roles are expected to provide a comprehensive resource for therapeutic development, as illuminated by recent successful interventions of spinal muscular atrophy. Here, we discuss the latest progress in the study of the emerging complexity of alternative splicing mechanisms in neurons, and how these findings inform new therapies to correct and control splicing defects.

The Grass was Greener: Repeated Evolution of Specialized Morphologies and Habitat Shifts in Ghost Spiders Following Grassland Expansion in South America.

While grasslands, one of Earth's major biomes, are known for their close evolutionary ties with ungulate grazers, these habitats are also paramount to the origins and diversification of other animals. Within the primarily South American spider subfamily Amaurobioidinae (Anyphaenidae), several species are found living in the continent's grasslands, with some displaying putative morphological adaptations to dwelling unnoticed in the grass blades. Herein, a dated molecular phylogeny provides the backbone for analyses revealing the ecological and morphological processes behind these spiders' grassland adaptations. The multiple switches from Patagonian forests to open habitats coincide with the expansion of South America's grasslands during the Miocene, while the specialized morphology of several grass-dwelling spiders originated at least three independent times and is best described as the result of different selective regimes operating on macroevolutionary timescales. Although grass-adapted lineages evolved towards different peaks in adaptive landscape, they all share one characteristic: an anterior narrowing of the prosoma allowing spiders to extend the first two pairs of legs, thus maintaining a slender resting posture in the grass blade. By combining phylogenetic, morphological, and biogeographic perspectives we disentangle multiple factors determining the evolution of a clade of terrestrial invertebrate predators alongside their biomes.

Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVß-dependent manner.

Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range of Ca2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCR-dependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several CaV subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of CaV channels depends on the presence of a CaVß subunit. CaVß subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.

Pulsed Field Gel Electrophoresis: Past, present, and future.

Pulsed Field Gel Electrophoresis (PFGE) has been considered for many years the 'gold-standard' for characterizing many pathogenic organisms as well as for subtyping bacterial species causing infection outbreaks. This article reviews the basic principles of PFGE and it includes the main advantages and limitations of the different electrode configurations that have been used in PFGE equipment and their influence on the DNA electrophoretic separation. Remarkably, we summarize here the most relevant theoretical and practical aspects that we have learned for more than 20 years developing and using the miniaturized PFGE systems. We also discussed the theoretical aspects related to DNA migration in PFGE agarose gels. It served as the basis for simulating the DNA electrophoretic patterns in CHEF mini gels and mini-chambers during experimental design and optimization. A critical comparison between standard and miniaturized PFGE systems, as well as the enzymatic and non-enzymatic methods for intact immobilized DNA preparation, is provided throughout the review. The PFGE current applications, advantages, limitations and future challenges of the methodology are also discussed.

The influence of developmental environment on courtship song in cactophilic Drosophila.

Closely related species often differ in the signals involved in sexual communication and mate recognition. Determining the factors influencing signal quality (i.e. signal's content and conspicuousness) provides an important insight into the potential pathways by which these interspecific differences evolve. Host specificity could bias the direction of the evolution of sexual communication and the mate recognition system, favouring sensory channels that work best in the different host conditions. In this study, we focus on the cactophilic sibling species Drosophila buzzatii and D. koepferae that have diverged not only in the sensory channel used for sexual communication and mate recognition but also in the cactus species that use as primary hosts. We evaluate the role of the developmental environment in generating courtship song variation using an isofemale line design. Our results show that host environment during development induces changes in the courtship song of D. koepferae males, but not in D. buzzatii males. Moreover, we report for the first time that host rearing environment affects the conspicuousness of courtship song (i.e. song volume). Our results are mainly discussed in the context of the sensory drive hypothesis.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome.

The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process. IMPORTANCE: The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

The life and adventures of an eight-legged castaway: Colonization and diversification of Philisca ghost spiders on Robinson Crusoe Island (Araneae, Anyphaenidae).

Oceanic archipelagoes, by their young origin and isolation, provide privileged settings to study the origin and diversification of species. Here, we study the anyphaenid spider genus Philisca, endemic to the Valdivian temperate rainforest, which includes species living both on the mainland as well as on the Robison Crusoe Island in the Juan Fernández archipelago. Anyphaenids, as many spiders, are potentially good colonizers due their ability for ballooning, an airborne dispersal mediated by strands of silk that are caught in the wind. We use a molecular approach to estimate both the phylogenetic relationships and the timeframe of species diversification of Philisca, with the aim to infer its evolutionary history. We further estimate the rates of speciation on both the insular and continental Philisca species and score the microhabitat used by each species and their sizes as a proxy to evaluate ecological niche diversification within the island. Most analyses support the monophyly of Philisca, with the exclusion of Philisca tripunctata. Our results reveal colonization from a single lineage that postdated the origin of the island, followed by rapid (∼2Ma) diversification. The ancestral microhabitat was most likely leaf-dwelling but we identify two independent microhabitat shifts. Our data provides evidence that Philisca has undergone an adaptive radiation on the Robison Crusoe Island.

Colostrum proinflammatory cytokines as biomarkers of bovine immune response to bovine tuberculosis (bTB).

Bovine colostrum contains compounds, which provide passive immune protection from mother to newborn calves. Little is known about cytokine levels and their role in bovine colostrum. Moreover, the capacity of bovine colostrum cells to mount specific immune responses after natural exposure to bovine tuberculosis (bTB) antigens in dairy herds has not been studied, thus far. The purpose of this study was to identify biomarkers for bTB infection measurable in bovine colostrum. The present study reveals that isolated-immune colostrum cells can mount a specific immune response against bTB antigens, by measuring the novo IFN-γ release in cell culture. We found that IFN-γ levels in the responders (Bov+) to bTB antigen were higher than in non-responders (Bov-). On the other hand, proinflammatory cytokines contained in colostrum's whey were tested in Tuberculin Skin Test (TST) reactor (TST+) and non-reactor (TST-) animals to assess their potential role as biomarker. We observed that IFN-γ levels were lower or undetectable, as opposed to IL4 levels were measurable, the TNF-α level was higher in TST- than TST+, while IL-6 levels showed the opposite reaction and with no statistical significance. Moreover, IL-1α mRNA expression levels were higher in colostrum mononuclear cells (CMC) in Bov+ cattle. Collectively, these data suggest that the differential expression of pro and anti-inflammatory cytokines could have relevant value to diagnose bTB in cattle.

Is Ghrelin Synthesized in the Central Nervous System?

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.

Introducing Explorer of Taxon Concepts with a case study on spider measurement matrix building.

BACKGROUND: Taxonomic descriptions are traditionally composed in natural language and published in a format that cannot be directly used by computers. The Exploring Taxon Concepts (ETC) project has been developing a set of web-based software tools that convert morphological descriptions published in telegraphic style to character data that can be reused and repurposed. This paper introduces the first semi-automated pipeline, to our knowledge, that converts morphological descriptions into taxon-character matrices to support systematics and evolutionary biology research. We then demonstrate and evaluate the use of the ETC Input Creation - Text Capture - Matrix Generation pipeline to generate body part measurement matrices from a set of 188 spider morphological descriptions and report the findings. RESULTS: From the given set of spider taxonomic publications, two versions of input (original and normalized) were generated and used by the ETC Text Capture and ETC Matrix Generation tools. The tools produced two corresponding spider body part measurement matrices, and the matrix from the normalized input was found to be much more similar to a gold standard matrix hand-curated by the scientist co-authors. Special conventions utilized in the original descriptions (e.g., the omission of measurement units) were attributed to the lower performance of using the original input. The results show that simple normalization of the description text greatly increased the quality of the machine-generated matrix and reduced edit effort. The machine-generated matrix also helped identify issues in the gold standard matrix. CONCLUSIONS: ETC Text Capture and ETC Matrix Generation are low-barrier and effective tools for extracting measurement values from spider taxonomic descriptions and are more effective when the descriptions are self-contained. Special conventions that make the description text less self-contained challenge automated extraction of data from biodiversity descriptions and hinder the automated reuse of the published knowledge. The tools will be updated to support new requirements revealed in this case study.

Human population genetic structure detected by pain-related mu opioid receptor gene polymorphisms.

Several single nucleotide polymorphisms (SNPs) in the Mu Opioid Receptor gene (OPRM1) have been identified and associated with a wide variety of clinical phenotypes related both to pain sensitivity and analgesic requirements. The A118G and other potentially functional OPRM1 SNPs show significant differences in their allele distributions among populations. However, they have not been properly addressed in a population genetic analysis. Population stratification could lead to erroneous conclusions when they are not taken into account in association studies. The aim of our study was to analyze OPRM1 SNP variability by comparing population samples of the International Hap Map database and to analyze a new population sample from the city of Corrientes, Argentina. The results confirm that OPRM1 SNP variability differs among human populations and displays a clear ancestry genetic structure, with three population clusters: Africa, Asia, and Europe-America.

EscO, a functional and structural analog of the flagellar FliJ protein, is a positive regulator of EscN ATPase activity of the enteropathogenic Escherichia coli injectisome.

Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein.

Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

Parallel artery and vein: sign of benign nature of breast masses.

OBJECTIVE: The purpose of this study was to assess the parallel artery and vein sign at color Doppler breast ultrasound as a predictor of the benign nature of breast masses. SUBJECTS AND METHODS: A prospective study was performed to identify evidence of a parallel artery and vein in breast lesions consecutively biopsied with a 14-gauge needle under ultrasound guidance. Sensitivity, specificity, positive and negative predictive values, likelihood ratios, and 95% CIs for the parallel artery and vein sign were calculated. RESULTS: The parallel artery and vein sign was identified in 142 of the 1074 masses (13.2%). The specificity for benignity was 99.3% (CI 95%, 98.3-100.0%); sensitivity, 17.6% (CI 95%, 15.0-20.3%); positive predictive value, 99.0% (CI 95%, 96.7-100); negative predictive value, 30.0% (CI 95%, 27.0-32.9); positive likelihood ratio, 24.7 (CI 95%, 21.2-28.7); and negative likelihood ratio, 0.83 (CI 95%, 0.80-0.86). Among masses found to have the parallel artery and vein sign, all BI-RADS ultrasound category 3 and 95.1% of BI-RADS 4 lesions were determined to be benign. CONCLUSION: Although the parallel artery and vein sign is an uncommon finding, it has a significant association with benign pathologic results (96.5%) with a positive likelihood ratio of 24.7. The presence of this color Doppler ultrasound finding in breast masses in BI-RADS ultrasound categories 3 and 4 reinforces the benign nature and may allow follow-up rather than biopsy in the care of some patients.

Tricarbonyl(η5-cyclopentadienyl)(trifluorohydroxyborato)tungsten.

The title compound, [W(C(5)H(5))(HOBF(3))(CO)(3)], has a four-legged piano-stool geometry which is typically found for C(5)H(5)W(CO)(3)X complexes. The HOBF(3)(-) anion is the hydrolysis product of BF(4)(-) and is coordinated via oxygen.

Time to Wake Up: Epigenetic and Small-RNA-Mediated Regulation during Seed Germination.

Plants make decisions throughout their lifetime based on complex networks. Phase transitions during seed growth are not an exception. From embryo development through seedling growth, several molecular pathways control genome stability, environmental signal transduction and the transcriptional landscape. Particularly, epigenetic modifications and small non-coding RNAs (sRNAs) have been extensively studied as significant handlers of these processes in plants. Here, we review key epigenetic (histone modifications and methylation patterns) and sRNA-mediated regulatory networks involved in the progression from seed maturation to germination, their relationship with seed traits and crosstalk with environmental inputs.