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Children require greater access to sound than adults as they are learning to communicate using hearing and spoken language. Yet when it comes to cochlear implant candidacy, currently approved Food and Drug Administration (FDA) criteria for adults are much less restrictive than those for children, allowing for greater levels of residual hearing and aided speech recognition in adults. Cochlear implant guidelines for children have changed very little in the 30 years since cochlear implants have been approved for pediatrics, and this lack of change has proven to be a barrier to implantation. Using evidence-based practice, centers have been providing off-label implantation for children who fall outside of current FDA criteria, including children with more residual hearing, children with single-side deafness younger than 5 years, and infants with bilateral profound loss younger than 9 months. The purpose of this article is to outline how these restrictions impede access to implants for children and describe the evidence supporting cochlear implantation in children who fall outside of current criteria.

Purpose The purpose of this study was to analyze the impact of cumulative hearing hour percentage (HHP) on pediatric cochlear implant users' speech and language development at age 3 years and to determine an evidence-based wear time recommendation that yields typical spoken language standard scores. Method A retrospective chart review of 40 pediatric cochlear implant recipients was completed. Children met the following criteria: prelingually deafened, implanted at age 2 years or younger, utilized a speech processor with datalogging capabilities, a minimum of 1 year of cochlear implant use, and language testing completed at approximately age 3 years. Exclusion criteria included significant inner ear malformation (i.e., common cavity) or developmental delay that would preclude spoken language development. Results Multiple regression analysis revealed that age and implantation and HHP were predictive of spoken language skills at age 3 years. Further analysis yielded wear time recommendations associated with age-appropriate spoken language based on the age at implantation. Conclusions When the goal is age-appropriate spoken language, wear time recommendations should reflect a child's current age, age at implantation, and the comparative daily sound access of age-matched normal-hearing peers. The HHP measurement can help provide that information. The minimum wear time recommendation should be set to 80% HHP with the ultimate goal of 100% HHP to give pediatric cochlear implant recipients enough access to sound and language to achieve their spoken language goals.

8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.

Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase–associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P

OBJECTIVE To determine factors that influence low-frequency hearing preservation following pediatric cochlear implantation and compare hearing preservation outcomes between 20 and 24 mm depth lateral wall electrodes. STUDY DESIGN Retrospective chart review. SETTING Tertiary academic referral center. PATIENTS Pediatric cochlear implant recipients (under the age of 18) who presented preoperatively with a low-frequency pure tone average (LFPTA; 125, 250 and 500 Hz) ≤ 70 dB HL. INTERVENTION Cochlear implantation MAIN OUTCOME MEASURES:: Multiple logistic regression evaluating the influence of variables on change in LFPTA including preoperative low-frequency hearing, lateral wall or perimodiolar electrode, progressive vs stable hearing, side, time from surgery, and the presence or the absence of enlarged vestibular aqueduct. A second analysis reviews the 12-month postactivation hearing preservation of a subset of subjects implanted with modern lateral wall electrodes. RESULTS A total of 105 subjects were included from the last 10 years for our multiple logistic regression analysis. This demonstrated a significant correlation of poorer preoperative low-frequency hearing with change in LFPTA. A significant negative effect of electrode type, specifically perimodiolar electrodes was also seen. Forty-five subjects from the last 3 years undergoing cochlear implantation with a lateral wall electrode demonstrated an overall 12-month preservation rate (LFPTA

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2–4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.

Sodium (Na+) electrochemical gradients established by Na+/K+ATPase activity drives the transport of ions, minerals, and sugars in both excitable and non-excitable cells. Na+-dependent transporters can move these solutes in the same direction (cotransport) or in opposite directions (exchanger) across both the apical and basolateral plasma membranes of polarized epithelia. In addition to maintaining physiological homeostasis of these solutes, increases and decreases in sodium may also initiate, directly or indirectly, signaling cascades that regulate a variety of intracellular post-translational events. In this review, we will describe how the Na+/K+ATPase maintains a Na+gradient utilized by multiple sodium-dependent transport mechanisms to regulate glucose uptake, excitatory neurotransmitters, calcium signaling, acid-base balance, salt-wasting disorders, fluid volume, and magnesium transport. We will discuss how several Na+-dependent cotransporters and Na+-dependent exchangers have significant roles in human health and disease. Finally, we will discuss how each of these Na+-dependent transport mechanisms have either been shown or have the potential to use Na+in a secondary role as a signaling molecule.

(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.

Two genes encode the Na+ -K+ -2Cl- cotransporters, NKCC1 and NKCC2, that mediate the tightly coupled movement of 1Na+ , 1K+ , and 2Cl- across the plasma membrane of cells. Na+ -K+ -2Cl- cotransport is driven by the chemical gradient of the three ionic species across the membrane, two of them maintained by the action of the Na+ /K+ pump. In many cells, NKCC1 accumulates Cl- above its electrochemical potential equilibrium, thereby facilitating Cl- channel-mediated membrane depolarization. In smooth muscle cells, this depolarization facilitates the opening of voltage-sensitive Ca2+ channels, leading to Ca2+ influx, and cell contraction. In immature neurons, the depolarization due to a GABA-mediated Cl- conductance produces an excitatory rather than inhibitory response. In many cell types that have lost water, NKCC is activated to help the cells recover their volume. This is specially the case if the cells have also lost Cl- . In combination with the Na+ /K+ pump, the NKCC's move ions across various specialized epithelia. NKCC1 is involved in Cl- -driven fluid secretion in many exocrine glands, such as sweat, lacrimal, salivary, stomach, pancreas, and intestine. NKCC1 is also involved in K+ -driven fluid secretion in inner ear, and possibly in Na+ -driven fluid secretion in choroid plexus. In the thick ascending limb of Henle, NKCC2 activity in combination with the Na+ /K+ pump participates in reabsorbing 30% of the glomerular-filtered Na+ . Overall, many critical physiological functions are maintained by the activity of the two Na+ -K+ -2Cl- cotransporters. In this overview article, we focus on the functional roles of the cotransporters in nonpolarized cells and in epithelia. © 2018 American Physiological Society. Compr Physiol 8:871-901, 2018.

Objective To identify differences in outcomes and map characteristics in pediatric bilateral cochlear implants with modiolar conforming and lateral wall arrays in opposite ears. Study design Retrospective case series. Setting Tertiary care pediatric referral center. Patients Fourteen children who received a perimodiolar array in one ear and a slim straight array in the opposite ear in sequential surgeries. Interventions None. Main outcome measures Consonant-nucleus-consonant test (CNC) word recognition score, battery life, power levels, electrical compound action potential (ECAP) thresholds, and electrical threshold and comfort charge levels. Results Speech perception outcomes were poorer in the lateral wall ears than the perimodiolar ears, and scores in the bilateral condition were better than with the lateral wall device alone. Sequential placement was a factor with differences in preoperative candidacy time correlating with greater difference in speech perception. There was no difference in charge levels between ears, in spite of higher ECAP threshold values for the lateral wall devices. Conclusion While bilateral speech perception was good, speech perception with the lateral wall device alone was poorer. This cannot be explained solely by the device, as differences in preoperative candidacy time were a significant factor. ECAP thresholds are significantly higher for lateral wall electrodes, but that did not translate in to higher psychophysical measurements.

Purpose The aims of this study were to (a) determine a metric for describing full-time use (FTU), (b) establish whether age at FTU in children with cochlear implants (CIs) predicts language at 3 years of age better than age at surgery, and (c) describe the extent of FTU and length of time it took to establish FTU in this population. Method This retrospective analysis examined receptive and expressive language outcomes at 3 years of age for 40 children with CIs. Multiple linear regression analyses were run with age at surgery and age at FTU as predictor variables. FTU definitions included 8 hr of device use and 80% of average waking hours for a typically developing child. Descriptive statistics were used to describe the establishment and degree of FTU. Results Although 8 hr of daily wear is typically considered FTU in the literature, the 80% hearing hours percentage metric accounts for more variability in outcomes. For both receptive and expressive language, age at FTU was found to be a better predictor of outcomes than age at surgery. It took an average of 17 months for children in this cohort to establish FTU, and only 52.5% reached this milestone by the time they were 3 years old. Conclusions Children with normal hearing can access spoken language whenever they are awake, and the amount of time young children are awake increases with age. A metric that incorporates the percentage of time that children with CIs have access to sound as compared to their same-aged peers with normal hearing accounts for more variability in outcomes than using an arbitrary number of hours. Although early FTU is not possible without surgery occurring at a young age, device placement does not guarantee use and does not predict language outcomes as well as age at FTU.

Objectives: The purpose of this study is to analyze spoken language development in the first year of cochlear implant device use for pediatric recipients under five, and to determine the impact of device wear time on receptive and expressive spoken language outcomes using objective datalogging measures.Methods: Retrospective review of 37 children utilizing Cochlear™ processors capable of datalogging, who had completed speech and language testing before implantation and one year post. Hearing Hour Percentage (HHP) was created from datalogging results and two multiple regression analysis were performed including HHP and standard language scores.Results: HHP was found to be a predictor for receptive language outcomes one year after cochlear implant use. HHP was not found to be predictive of expressive language outcomes one year after cochlear implant use.Discussion: Datalogging is a vital objective measure that yields powerful data regarding a child's device use and early receptive spoken language development.Conclusion: In the first year of cochlear implant use, datalogging using the HHP significantly predicted receptive language scores. Increased HHP yielded higher receptive language standard scores. Continued research is needed to see if HHP is predictive of expressive language outcomes after a longer period of cochlear implant use.

Na(+)/H(+) exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase–associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P

Objectives This study investigates outcomes in children fit with electric-acoustic stimulation (EAS) and addresses three main questions: (1) Are outcomes with EAS superior to outcomes with conventional electric-only stimulation in children? (2) Do children with residual hearing benefit from EAS and conventional electric-only stimulation when compared with the preoperative hearing aid (HA) condition? (3) Can children with residual hearing derive benefit from EAS after several years of listening with conventional electric-only stimulation? Design Sixteen pediatric cochlear implant (CI) recipients between 4 and 16 years of age with an unaided low-frequency pure tone average of 75 dB HL in the implanted ear were included in two study arms. Arm 1 included new recipients, and Arm 2 included children with at least 1 year of CI experience. Using a within-subject design, participants were evaluated unilaterally with the Consonant-Nucleus-Consonant (CNC) word list in quiet and the Baby Bio at a +5 dB SNR using an EAS program and a conventional full electric (FE) program. Arm 1 participants' scores were also compared with preoperative scores. Results Speech perception outcomes were statistically higher with the EAS program than the FE program. For new recipients, scores were significantly higher with EAS than preoperative HA scores for both the CNC and Baby Bio in noise; however, after 6 months of device use, results in the FE condition were not significantly better than preoperative scores. Long-term FE users benefited from EAS over their FE programs based on CNC word scores. Conclusions Whether newly implanted or long-term CI users, children with residual hearing after CI surgery can benefit from EAS. Cochlear implantation with EAS fitting is a viable option for children with HAs who have residual hearing but have insufficient access to high-frequency sounds and poor speech perception.

Two genes encode the Na

X-ray crystallography of the catalytic domain of oxidative stress response 1 (OSR1) has provided evidence for dimerization and domain swapping. However, the functional significance of dimer formation or domain swapping has yet to be addressed. In this study, we used nine glutamine residues to link the carboxyl end of one SPAK (related Ste20 kinase) monomer to the amino end of another SPAK monomer to assess the role of kinase monomers versus dimers in Na-K-2Cl cotransporter 1 (NKCC1) activation. Transport studies in Xenopus laevis oocytes show that forcing dimerization of two wild-type SPAK molecules results in cotransporter activation when calcium-binding protein 39 (Cab39) is coexpressed, indicating that the presence of Cab39 can bypass the upstream phosphorylation requirement of SPAK normally associated with kinase activation. We determined that monomers are the functional units of the kinase as concatamers consisting of an active and various inactive monomers were still functional. Furthermore, we found that two different nonfunctional SPAK mutants could be linked together in a concatamer and activated, presumably by domain swapping, indicating that dimerization and domain swapping are both important components of kinase activation. Finally, we demonstrate rescue of a nonfunctional SPAK mutant by domain swapping with wild-type OSR1, indicating that heterodimers of the two Ste20-related kinases are possible and therefore potentially relevant to the regulation of NKCC1 activity.

SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI subfamily of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant, and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration.

The conceptual breakthrough that the energy of the Na+ gradient generated by the Na+/K+ pump could be used as the driving force for another membrane transport protein has led to the functional and molecular identification of multiple secondary active transporters. We have organized this chapter to address the expression, function, regulation, and evolutionary importance of the two isoforms of the electroneutral sodium-potassium-chloride cotransporter (NKCC). The combination of basolateral expression of the sodium-potassium pump and NKCC1 in various epithelia results in salt and water secretion, whereas basolateral expression of the pump with apical expression of NKCC2 in the thick ascending limb of Henle in the kidney nephron results in salt and water reabsorption. NKCCs are regulated by serine-threonine phosphorylation of specific residues in their amino-terminal domain, and the evolutionary conservation of these cotransporters from protists to humans confirms their vital role in cellular and whole-organism physiology.

Na+–K+–2Cl− cotransport (NKCC) mediates the movement of two Cl− ions for one Na+ and one K+ ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl− uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K+ uptake with only a minimal increase in bumetanide-sensitive Cl− uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K+/K+ and Cl−/Cl− exchange. Although significant stimulation of K+/K+ exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K+/K+ exchange.

The Na-K-2Cl cotransporter (NKCC1) participates in epithelial transport and in cell volume maintenance by mediating the movement of ions and water across plasma membranes. Functional studies have previously demonstrated that NKCC1 activity is stimulated by protein phosphatase 1 (PP1) inhibitors. In this study, we utilized both in vivo (heterologous cRNA expression in Xenopus laevis oocytes) and in vitro (32P-phosphorylation assays with glutathione S-transferase fusion proteins) experiments to determine whether PP1 exerts its inhibitory effect directly on the cotransporter, or indirectly by affecting the activating kinase. We found that PP1 reduced NKCC1 activity in oocytes under both isotonic and hypertonic conditions to the same level as in water-injected controls. Interestingly, mutation of key residues in the PP1 binding motif located in the N-terminal tail of NKCC1 significantly reduced the inhibitory effect of PP1. In vitro experiments performed with recombinant PP1, SPAK (Ste20-related proline/alanine-rich kinase, which activates NKCC1), and the N terminus of NKCC1 fused to glutathione S-transferase demonstrated that PP1 dephosphorylated both the kinase and the cotransporter in a time-dependent manner. More importantly, PP1 dephosphorylation of SPAK was significantly greater when protein-protein interaction between the kinase and the N-terminal tail of NKCC1 was present in the reaction, indicating the necessity of scaffolding the phosphatase and kinase in proximity to one another. Taken together, our data are consistent with PP1 inhibiting NKCC1 activity directly by dephosphorylating the cotransporter and indirectly by dephosphorylating SPAK.

This study examines the relationship between customers' perceptions of Reliability, Customization, Overall Quality, and Customer Satisfaction in the US lodging market. The American Customer Satisfaction Index (ACSI) provides the secondary data for this research. The results provide evidence that US hotel guests are satisfied with the Overall Quality of their hotel experiences and that Overall Quality, Customization, and Reliability all have significant, positive impacts on Customer Satisfaction. This study also confirms the discriminate validity of Customization and Reliability perceptions on overall Customer Satisfaction and provides broad-based support for the contention that Customization is relatively more important than Reliability in impacting Customer Satisfaction in the US lodging market.

The CASE (Council for the Advancement and Support of Education) Professor of the Year program recognizes outstanding professors for their dedication to teaching, commitment to students, and innovative instructional methods. It is the only national program to recognize college and university professors for their teaching skills. Three marketing professors were chosen for this award in 2005 and now share their teaching philosophies. Their philosophies focus on innovation, experimentation, creating a culture of learning, the first day of class, and maintaining high academic standards.

Faced with increasing needs and limitations on resources for water-quality monitoring, emerging tools complement the traditional procedure based on sampling and laboratory analysis. Apart from their simple (on-site) use and affordability, they can be considered as sustainable development tools. We propose sustainability criteria for water-quality monitoring and assess some emerging tools for this purpose.

Based on the alignment of 12 sequences of protein motifs that interact with the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress response 1), we performed genome-wide searches of the sequence [S/G/V]RF x[V/I]xx[V/I/T/S]xx, where x represents any amino acid. The “Mus musculus” search resulted in the identification of 131 mouse proteins containing 137 SPAK/OSR1 putative binding motifs. Similar numbers were found for human, zebrafish, fruit fly, and worm. A little more than half of the mouse proteins containing SPAK/OSR1 binding domains (53%) were also identified in the human search, whereas ∼17–18% of these common hits were identified in the zebrafish search. The mouse proteins could be divided into two broad categories: 2/3 had an identified function, whereas 1/3 were either predicted or of unknown function. The known proteins were grouped as transport proteins, other membrane proteins, kinases, phosphatases, cytoskeletal, ribosomal, nuclear, enzymes, and others. Analysis of the location of the SPAK/OSR1 binding motif within the protein sequence revealed distribution throughout the entire length, but with preference to the extreme amino- or carboxyl termini for a large number of proteins. Analysis of the amino acid composition of the motifs revealed a preponderance of serine residues at positions 5, 6, 7, and 8. In summary, our new search found and thus confirms the 12 proteins previously shown to interact with the kinases and identifies 119 potential new targets for SPAK and OSR1 in the mouse proteome.

High-resolution computed tomography (CT) imaging of a live animal within a lead-lined synchrotron light hutch presents several unique challenges. In order to confirm that the animal is under a stable plane of anaesthesia, several physiological parameters (e.g.heart rate, arterial oxygen saturation, core body temperature and respiratory rate) must be remotely monitored from outside the imaging hutch. In addition, to properly scan the thoracic region using CT, the animal needs to be held in a vertical position perpendicular to the fixed angle of the X-ray beam and free to rotate 180°–360°. A new X-ray transparent mouse restraint designed and fabricated using computer-aided design software and three-dimensional rapid prototype printing has been successfully tested at the Biomedical Imaging and Therapy bending-magnet (BMIT-BM) beamline at the Canadian Light Source.

An increasing number of hotel organizations endeavor to achieve long-term competitive advantages in the marketplace through the implementation of strategic performance measurement systems. These systems are designed to integrate and monitor strategic objectives throughout the business processes. This paper investigates the causal relationships between the strategic performance measures of hotel organizations, using The Balanced Scorecard (BSC) framework. This comparative study illustrates how hotel organizations plan, improve and monitor their strategic performance targets through the use of comprehensive performance measurement systems. The paper also recommends specific approaches for the implementation of the BSC for hotel organizations.

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn2+ rather than Mg2+. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl−cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl−cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.

Succession of soil microbial communities proceeds interdependently with crop development under the control of edaphic, agricultural, and climatic conditions. Soil microbial community composition and activity were characterized in two managed cropping systems (continuous corn or mixed forage) receiving liquid hog manure (LHM) or inorganic fertilization (IF) since 1989. Microbial community composition was determined from patterns of phospholipid fatty acids (PLFA). Soil microbial biomass C, enzymatic activity, and total phospholipids were greater under forage than under continuous corn. Principal component analysis (PCA) revealed a high degree of variation in PLFA composition and microbial community structure due to treatments and time of sampling. The first two PCA axes explained 80 to 90% of the variability on all sampling dates. Two main groups were created along the first component, the first comprising all forage treatments and a second with LHM-treated continuous corn. The IF and control-treated continuous corn were differentiated from all others on the second component. All groups were characterized by specific microbial communities. The most significant shift in specific groups of fatty acids was caused by LHM addition to continuous corn, especially due to the relative abundance of the fungal fatty acid 18:29,12. Our findings indicated that the addition of organic materials, rich in available C and N, induced development of diverse microbial populations, which can be successfully monitored by phospholipid fatty acid prof iles. Key words: Microbial community, phospholipid fatty acid profiles, hog manure, corn, forage

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide.

Habitat loss and fragmentation are main causes for Asian elephant population declines. We mapped wildlands - large, unfragmented and undeveloped areas - asking: (1) Where are the largest wildlands that constitute elephant habitats? (2) What proportion of these wildlands is protected? (3) What is their potential for elephant conservation? Our study demonstrates that wildlands constitute only 51% of the Asian elephant range. Myanmar has the largest wildland (∼170,000 km2), followed by Thailand and India. In Principal Components Analysis (PCA), the first two components explained 73% of the variation in fragmentation among ranges. We identified three fragmentation clusters from the PCA. Cluster A contains large ranges with unfragmented wildlands; cluster B includes ranges with well-developed transportation networks and large human populations; and cluster C contains ranges with severely fragmented wildlands. In cluster A, we identified four ranges with elephant populations >1000 animals: ARYO, MYUC, BNMH and BITE. Together with ranges that support >1000 elephants in cluster B, these A ranges have great potential for long-term elephant conservation. We propose that fragmentation clusters and population size can be used to identify different elephant monitoring and management zones.

Information on the different forms and availability of P following compost addition to soil may help to better manage manure in respect to plant growth and the environment. An experiment was conducted to investigate through a sequential extraction procedure the availability of P of fresh dairy manure and several on-farm compost-soil mixtures after a 13 wk incubation in glass jars at 35°C. Materials were mixed at a rate of 200 mg N kg-1 with an Arago sandy loam (Humo-Ferric Podzol), supplying from 64 to 301 mg P kg-1. Fresh dairy manure gave the highest net increase of resin-P and labile P fractions in terms of percentage of total P added, whereas poultry litter compost was the most efficient in increasing NaHCO3-inorganic P (-Pi). Among compost materials, poultry litter, vegetable residue and sheep manure increased labile P fraction the most. The contribution of the young dairy manure compost to this fraction was largely negative, and lower than those of fresh manure or partially and well-decomposed manure composts. A large part of added P was found in the moderately labile P fraction. The organic P (Po) fractions in the soil were less affected by manure or compost addition. This study indicated that the material P availability was reduced by composting, and was more affected by the origin of residue than by manure management. Key words: Composting, farm manure, soil P fractionation

Among the over 300 members of the solute carrier (SLC) group of integral plasma membrane transport proteins are the nine electroneutral cation-chloride cotransporters belonging to the SLC12 gene family. Seven of these transporters have been functionally described as coupling the electrically silent movement of chloride with sodium and/or potassium. Although in silico analysis has identified two additional SLC12 family members, no physiological role has been ascribed to the proteins encoded by either the SLC12A8 or the SLC12A9 genes. Evolutionary conservation of this gene family from protists to humans confirms their importance. A wealth of physiological, immunohistochemical, and biochemical studies have revealed a great deal of information regarding the importance of this gene family to human health and disease. The sequencing of the human genome has provided investigators with the capability to link several human diseases with mutations in the genes encoding these plasma membrane proteins. The availability of bacterial artificial chromosomes, recombination engineering techniques, and the mouse genome sequence has simplified the creation of targeting constructs to manipulate the expression/function of these cation-chloride cotransporters in the mouse in an attempt to recapitulate some of these human pathologies. This review will summarize the three human disorders that have been linked to the mutation/dysfunction of the Na-Cl, Na-K-2Cl, and K-Cl cotransporters (i.e., Bartter's, Gitleman's, and Andermann's syndromes), examine some additional pathologies arising from genetically modified mouse models of these cotransporters including deafness, blood pressure, hyperexcitability, and epithelial transport deficit phenotypes.