Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LF[subscript N]) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (K[subscript d] 1 nM). The optimally oriented LF[subscript N]-PA[subscript prepore] complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (K[subscript d] 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PA[subscript prepore] into the membrane inserted pore state. This PA[subscript pore] state results in a weakened binding between the CMG2 receptor and the LF[subscript N]-PA[subscript pore] and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LF[subscript N] to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LF[subscript N]-PA[subscript prepore]-CMG2 ternary complex or the acid transitioned LF[subscript N]-PA[subscript pore] complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures

Video_2_The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms.AVI

<p>The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.</p

The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms

The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states

Video_1_The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms.AVI

<p>The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.</p

Medication-related inpatient falls: a critical review

<div><p>Abstract Falls are the second leading cause of accidental and unintentional injury deaths worldwide. Inpatient falls in hospital settings are likely to prolong the length of stay of patients in nearly 6.3 days, leading to increased hospitalization costs. The causes of fall incidents in healthcare facilities are multifactorial in nature and certain medications use could be associated with these incidents. This review seeks to critically evaluate the available literature regarding the relationship between inpatient falls and medication use. A comprehensive search was performed on MEDLINE, EMBASE and Lilacs with no time restriction. The search was filtered using English, Spanish or Portuguese languages. Our study evaluated medication use and inpatients falls that effectively happen, considering all ages and populations. An assessment of bias and quality of the studies was carried out using an adapted tool from the literature. The drugs were classified according to the Anatomic Therapeutics Chemical Code. The search strategy retrieved 563 records, among which 23 met the eligibility criteria; ninety three different pharmacological subgroups were associated with fall incidents. Our critical review suggests that the use of central nervous system drugs (including anxiolytics; hypnotics and sedatives; antipsychotics; opioids; antiepileptics and antidepressants) has a greater likelihood of causing inpatient falls. A weak relationship was found between other pharmacological subgroups, such as diuretics, cardiovascular system-related medications, and inpatient fall. Remarkably, several problems of quality were encountered with regard to the eligible studies. Among such quality problems included retrospective design, the grouping of more than one medication in the same statistical analysis, limited external validity, problems related to medication classifications and description of potential confounders.</p></div

Arsenic phytoextraction and hyperaccumulation by fern species Fitoextração e hiperacumulação de arsênio por espécies de samambaias

Arsenic (As) is an ubiquitous trace metalloid found in all environmental media. Its presence at elevated concentrations in soils derives from both anthropogenic and natural inputs. Arsenic is a toxic and carcinogenic element, which has caused severe environmental and health problem worldwide. Technologies currently available for the remediation of arsenic-contaminated sites are expensive, environmentally disruptive, and potentially hazardous to workers. Phytoextraction, a strategy of phytoremediation, uses plants to clean up contaminated soils and has been successfully applied to arsenic contaminated soils. It has the advantage of being cost-effective and environmentally friendly. A major step towards the development of phytoextraction of arsenic-impacted soils is the discovery of the arsenic hyper accumulation in ferns, first in Pteris vittata, which presented an extraordinary capacity to accumulate 2.3% arsenic in its biomass. Another fern, Pityrogramma calomelanos was found to exhibit the same hyperaccumulating characteristics. After that, screening experiments have revealed that the Pteris genus is really unique in that many species have the potential to be used in phytoextraction of arsenic. In general, these plants seem to have both constitutive and adaptive mechanisms for accumulating or tolerating high arsenic concentration. In the past few years, much work has been done to understand and improve the hyperaccumulating capability of these amazing plants. In particular, the field of molecular biology seems to hold the key for the future of the phytoremediation.<br>O arsênio e um metalóide traço encontrado basicamente em todos os ambientes. Elevadas concentrações de arsênio no solo podem acontecer naturalmente devido ao intemperismo de rochas ricas em arsênio, como também de atividades antropogênicas. O arsênio é um elemento tóxico e cancerígeno. Em muitas partes do mundo, a contaminação pelo arsênio tem causado problemas ambientais e de saude. As técnicas disponíveis para a remediação do arsênio são economicamente proibitivas, destroem a paisagem natural e ainda podem afetar a saúde de pessoas diretamente envolvidas no processo. A fitoextração, uma das estratégias da fitoremediação, utiliza plantas para descontaminar solos e tem sido aplicada com sucesso em solos contaminados com arsênio e outros elementos. Dentre muitas vantagens, essa técnica tem baixo custo quando comparada com as convencionais. Um ponto chave no desenvolvimento da fitoextração foi a constatação de que samambaias hiperacumulam arsênio. Primeiro, em Pteris vittata, que apresentou extraordinária capacidade para remover arsênio do solo, concentrando 2.3% do arsênio na biomassa. Em seguida, foi observado que a samambaia Pityrogramma calomelanos possui capacidade semelhante para acumular arsênio. Essa característica peculiar foi observada em outras samambaias do genero Pteris. Em geral, essas plantas parecem apresentar mecanismos constitutivos e adaptativos que permitem elevada absorção e sobrevivência em solos com altas concentrações de arsênio. Muitas pesquisas têm sido conduzidas no sentido de entender e aumentar a capacidade de aborção de arsênio dessas plantas. Em particular, a chave para a aplicação bem sucedida da fitoremediação parece estar na biologia molecular