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Opposite temperature effect on transport activity of KCC2/KCC4 and N(K)CCs in HEK-293 cells

<p>Abstract</p> <p>Background</p> <p>Cation chloride cotransporters play essential roles in many physiological processes such as volume regulation, transepithelial salt transport and setting the intracellular chloride concentration in neurons. They consist mainly of the inward transporters NCC, NKCC1, and NKCC2, and the outward transporters KCC1 to KCC4. To gain insight into regulatory and structure-function relationships, precise determination of their activity is required. Frequently, these analyses are performed in HEK-293 cells. Recently the activity of the inward transporters NKCC1 and NCC was shown to increase with temperature in these cells. However, the temperature effect on KCCs remains largely unknown.</p> <p>Findings</p> <p>Here, we determined the temperature effect on KCC2 and KCC4 transport activity in HEK-293 cells. Both transporters demonstrated significantly higher transport activity (2.5 fold for KCC2 and 3.3 fold for KCC4) after pre-incubation at room temperature compared to 37°C.</p> <p>Conclusions</p> <p>These data identify a reciprocal temperature dependence of cation chloride inward and outward cotransporters in HEK-293 cells. Thus, lower temperature should be used for functional characterization of KCC2 and KCC4 and higher temperatures for N(K)CCs in heterologous mammalian expression systems. Furthermore, if this reciprocal effect also applies to neurons, the action of inhibitory neurotransmitters might be more affected by changes in temperature than previously thought.</p

Genome of the Asian Longhorned Beetle (\u3cem\u3eAnoplophora glabripennis\u3c/em\u3e), a Globally Significant Invasive Species, Reveals Key Functional and Evolutionary Innovations at the Beetle-Plant Interface

Background: Relatively little is known about the genomic basis and evolution of wood-feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants

Genome of the Asian longhorned beetle (Anoplophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle–plant interface

Background Relatively little is known about the genomic basis and evolution of wood-feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Famine Relief: Just a Simple Matter of Supplying Food?

In an age of unprecedented surplus, there are still over 800 million people worldwide who are subject to constant famine conditions and resultant malnutrition. The roots of famine are grounded in poverty, war and civil strife, natural disaster, drought, inequitable land distribution, and population growth rates that exceed food production. The costs of famine in terms of human suffering and loss of life are astounding. The objective of this paper is to examine the global response to famine, assess the efficacy of this response, and offer suggestions for how it might be improved. Currently, the famine relief system consists of loosely affiliated organizations without any overarching regulatory body to oversee and evaluate each organization or coordinate individual responses. Many of the criticisms of the system are age old including poor inter-agency coordination, sluggish responses to crisis situations, and technical incompetence. However, despite these shortcomings, it is certain that without an international famine relief system, the toll of famine would be greater

Famine Relief: Just a Simple Matter of Supplying Food?

In an age of unprecedented surplus, there are still over 800 million people worldwide who are subject to constant famine conditions and resultant malnutrition. The roots of famine are grounded in poverty, war and civil strife, natural disaster, drought, inequitable land distribution, and population growth rates that exceed food production. The costs of famine in terms of human suffering and loss of life are astounding. The objective of this paper is to examine the global response to famine, assess the efficacy of this response, and offer suggestions for how it might be improved. Currently, the famine relief system consists of loosely affiliated organizations without any overarching regulatory body to oversee and evaluate each organization or coordinate individual responses. Many of the criticisms of the system are age old including poor inter-agency coordination, sluggish responses to crisis situations, and technical incompetence. However, despite these shortcomings, it is certain that without an international famine relief system, the toll of famine would be greater

Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of Renibacterium salmoninarum

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a Kd of 3.0 &plusmn; 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses