DPP6 domains responsible for its localization and function.

Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K(+) channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.

Olfactory conditioning in the third instar larvae of Drosophila melanogaster using heat shock reinforcement.

Adult Drosophila melanogaster has long been a popular model for learning and memory studies. Now the larval stage of the fruit fly is also being used in an increasing number of classical conditioning studies. In this study, we employed heat shock as a novel negative reinforcement for larvae and obtained high learning scores following just one training trial. We demonstrated heat-shock conditioning in both reciprocal and non-reciprocal paradigms and observed that the time window of association for the odor and heat shock reinforcement is on the order of a few minutes. This is slightly wider than the time window for electroshock conditioning reported in previous studies, possibly due to lingering effects of the high temperature. To test the utility of this simplified assay for the identification of new mutations that disrupt learning, we examined flies carrying mutations in the dnc gene. While the sensitivity to heat shock, as tested by writhing, was similar for wild type and dnc homozygotes, dnc mutations strongly diminished learning. We confirmed that the learning defect in dnc flies was indeed due to mutation in the dnc gene using non-complementation analysis. Given that heat shock has not been employed as a reinforcement for larvae in the past, we explored learning as a function of heat shock intensity and found that optimal learning occurred around 41 °C, with higher and lower temperatures both resulting in lower learning scores. In summary, we have developed a very simple, robust paradigm of learning in fruit fly larvae using heat shock reinforcement.

Sustainability management of short-lived freshwater fish in human-altered ecosystems should focus on adult survival.

Fish populations globally are susceptible to endangerment through exploitation and habitat loss. We present theoretical simulations to explore how reduced adult survival (age truncation) might affect short-lived freshwater fish species in human-altered contemporary environments. Our simulations evaluate two hypothetical "average fish" and five example fish species of age 1 or age 2 maturity. From a population equilibrium baseline representing a natural, unaltered environment we impose systematic reductions in adult survival and quantify how age truncation affects the causes of variation in population growth rate. We estimate the relative contributions to population growth rate arising from simulated temporal variation in age-specific vital rates and population structure. At equilibrium and irrespective of example species, population structure (first adult age class) and survival probability of the first two adult age classes are the most important determinants of population growth. As adult survival decreases, the first reproductive age class becomes increasingly important to variation in population growth. All simulated examples show the same general pattern of change with age truncation as known for exploited, longer-lived fish species in marine and freshwater environments. This implies age truncation is a general potential concern for fish biodiversity across life history strategies and ecosystems. Managers of short-lived, freshwater fishes in contemporary environments often focus on supporting reproduction to ensure population persistence. However, a strong focus on water management to support reproduction may reduce adult survival. Sustainability management needs a focus on mitigating adult mortality in human-altered ecosystems. A watershed spatial extent embracing land and water uses may be necessary to identify and mitigate causes of age truncation in freshwater species. Achieving higher adult survival will require paradigm transformations in society and government about water management priorities.

Infective endocarditis caused by Klebsiella oxytoca in an intravenous drug user with cancer.

Infective endocarditis caused by Klebsiella species is rare, with most isolates being K. pneumoniae. We report the case of a 24-year-old intravenous drug user with newly diagnosed seminoma who developed K. oxytoca endocarditis. In addition to having K. oxytoca isolated from blood culture, cultures of that species were obtained from a retroperitoneal metastasis found on original presentation.

The incidence of un-indicated preoperative testing in a tertiary academic ambulatory center: a retrospective cohort study.

BACKGROUND: Despite existing evidence and guidelines advocating for appropriate risk stratification, ambulatory surgery in low-risk patients continues to be accompanied by a battery of routine tests prior to surgery. Using a single-center retrospective cohort study, we aimed to quantify the incidence of un-indicated preoperative testing in an academic ambulatory center by utilizing recommendations by the recently developed American Society of Anesthesiology (ASA) "Choosing Wisely" Top-5 list. METHODS: We utilized data from the EPIC medical records of 3111 patients who had ambulatory surgery at the Hospital of the University of Pennsylvania during a 6-month period. Data were abstracted from laboratory studies- complete blood count, electrolyte panel, coagulation studies, and cardiac studies-stress test, and echocardiogram obtained within 30 days prior to surgery. Preoperative tests obtained from each patient were categorized into "indicated" (ASA ≥ 3) and "un-indicated" (ASA 1 and 2) tests, and percentages were reported. RESULTS: During the study period, 52.9 % (95 % confidence interval (CI) 37.6-66.4) of all patients had at least one un-indicated laboratory test performed preoperatively. Further analysis revealed variation in the incidence of preoperative ordering between tests; 73 % of all complete blood counts (CBCs), 70 % of all metabolic panels, and 49 % of all coagulation studies were considered un-indicated by "Top-5 List" criteria. Stated differently, of the patients included in the sample, 51 % of patients received an un-indicated CBC, 41 % an un-indicated metabolic panel, and 16 % un-indicated coagulation studies. Twelve percent of "any un-indicated preoperative test" were obtained from ASA 1 healthy patients. Of the 587 patients less than 36 years old, 331 (56 %) had at least one test that was deemed un-indicated. Forty-one patients had either an echocardiogram or stress test ordered and performed within 30 days of surgery. Of these, eight (19.5 %) studies were un-indicated as determined by chart review. CONCLUSIONS: The incidence of ordering "at least one un-indicated preoperative test" in low-risk patients undergoing low-risk surgery remains high even in academic tertiary institutions. In the emerging era of optimizing patient safety and financial accountability, further studies are needed to better understand the problem of overuse while identifying modifiable attitudes and institutional influences on perioperative practices among all stakeholders involved. Such information would drive the development of feasible interventions.

Chronic symptomatic os subfibulare in children.

BACKGROUND: Some children have persistent pain and instability following inversion injuries of the ankle. Radiographs may reveal a small osseous fragment distal to the lateral malleolus, suggesting an avulsion injury of the anterior talofibular ligament and/or calcaneofibular ligament. The avulsion injury may involve an os subfibulare, causing ligamentous laxity and chronic pain resulting from nonunion. This situation must be differentiated from an asymptomatic os subfibulare, which is a normal anatomic variant in 1% of children. The purpose of this study was to evaluate the intraoperative findings and long-term outcomes of patients treated operatively for symptomatic avulsion injuries or a symptomatic os subfibulare. METHODS: Twenty-three patients presented with chronic ankle pain and instability, tenderness anterior and distal to the lateral malleolus, and imaging studies (magnetic resonance imaging and/or stress radiographs) suspicious for avulsion injury of the anterior talofibular ligament and/or calcaneofibular ligament. After unsuccessful nonoperative treatment, all patients underwent excision of the osseous fragments, anatomic reconstruction of the anterior talofibular ligament with use of drill holes through the lateral malleolus, and a modified Broström procedure. RESULTS: The mean age of the patients was 10.4 years (range, eight to thirteen years) at the time of injury and 13.6 years (range, eight to seventeen years) at the time of surgery, representing a mean delay in diagnosis and treatment of 3.2 years. At a mean follow-up of 4.5 years (range, 2.1 to 13.2 years), the mean Foot and Ankle Outcome Score was 91.4 (range, 87 to 98) out of 100, with all but one patient returning to the preinjury recreational level. Only one patient had a long-term complication. CONCLUSIONS: In children with chronic pain and instability associated with an os subfibulare, surgical excision of the os subfibulare combined with reconstruction of the anterior talofibular ligament and a modified Broström procedure was effective in restoring ankle stability, eliminating pain, and permitting return to the preinjury functional level.

A novel point mutation in the KCNJ5 gene causing primary hyperaldosteronism and early-onset autosomal dominant hypertension.

CONTEXT: Aldosterone production in the adrenal zona glomerulosa is mainly regulated by angiotensin II, [K(+)], and ACTH. Genetic deletion of subunits of K(+)-selective leak (KCNK) channels TWIK-related acid sensitive K(+)-1 and/or TWIK-related acid sensitive K(+)-3 in mice results in primary hyperaldosteronism, whereas mutations in the KCNJ5 (potassium inwardly rectifying channel, subfamily J, member 5) gene are implicated in primary hyperaldosteronism and, in certain cases, in autonomous glomerulosa cell proliferation in humans. OBJECTIVE: The objective of the study was to investigate the role of KCNK3, KCNK5, KCNK9, and KCNJ5 genes in a family with primary hyperaldosteronism and early-onset hypertension. PATIENTS AND METHODS: Two patients, a mother and a daughter, presented with severe primary hyperaldosteronism, bilateral massive adrenal hyperplasia, and early-onset hypertension refractory to medical treatment. Genomic DNA was isolated and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. RESULTS: Sequencing of the KCNJ5 gene revealed a single, heterozygous guanine to thymine (G → T) substitution at nucleotide position 470 (n.G470T), resulting in isoleucine (I) to serine (S) substitution at amino acid 157 (p.I157S). This mutation results in loss of ion selectivity, cell membrane depolarization, increased Ca(2+) entry in adrenal glomerulosa cells, and increased aldosterone synthesis. Sequencing of the KCNK3, KCNK5, and KCNK9 genes revealed no mutations in our patients. CONCLUSIONS: These findings explain the pathogenesis in a subset of patients with severe hypertension and implicate loss of K(+) channel selectivity in constitutive aldosterone production.

KCNJ5 mutations in the National Institutes of Health cohort of patients with primary hyperaldosteronism: an infrequent genetic cause of Conn's syndrome.

KCNJ5 mutations were recently described in primary hyperaldosteronism (PH or Conn's syndrome). The frequency of these mutations in PH and the way KCNJ5 defects cause disease remain unknown. A total of 53 patients with PH have been seen at the National Institutes of Health over the last 12 years. Their peripheral and tumor DNAs (the latter from 16 that were operated) were screened for KCNJ5 mutations; functional studies on the identified defects were performed after transient transfection. Only two mutations were identified, and both in the tumor DNA only. There were no germline sequencing defects in any of the patients except for known synonymous variants of the KCNJ5 gene. One mutation was the previously described c.G451C alteration; the other was a novel one in the same codon: c.G451A; both lead to the same amino acid substitution (G151R) in the KCNJ5 protein. Functional studies confirmed previous findings that both mutations caused loss of channel selectivity and a positive shift in the reversal potential. In conclusion, the KCNJ5 protein was strongly expressed in the zona glomerulosa of normal adrenal glands but showed variable expression in the aldosterone-producing adenomas with and without mutation. The rate of KCNJ5 mutations among patients with PH and/or their tumors is substantially lower than what was previously reported. The G151R amino acid substitution appears to be the most frequent one so far detected in PH, despite additional nucleotide changes. The mutation causes loss of this potassium channel's selectivity and may assist in the design of new therapies for PH.

A low concentration of ethanol impairs learning but not motor and sensory behavior in Drosophila larvae.

Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (∼7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects.

Primary musculoskeletal sarcoidosis.

Sarcoidosis is a well-described clinical entity with several types of presentation among different organ systems. However, primary musculoskeletal manifestation is a rare and seldom-described manifestation. A retrospective review of patients seen at our institution from 1999 to 2009 was performed to identify patients with such presentation. Eleven patients with musculoskeletal sarcoidosis were identified. Seven patients presented with solitary lesions, while 4 presented with multiple foci of disease. Histologically, all lesions revealed granulomatous inflammation consistent with sarcoidosis. Cultures and gram stain were negative in all patients. One patient was found to have evidence of pulmonary sarcoidosis after diagnosis of musculoskeletal sarcoidosis. Eight patients had primarily soft tissue involvement with visible masses, while 3 patients had primarily osseous involvement with no mass. Pain was reported by 9 of 11 patients. On magnetic resonance imaging, all lesions were dark on T1, bright on T2, and poorly marginated. Magnetic resonance imaging was noted to be more accurate in revealing the pathology than radiographic imaging. Four patients underwent complete excision and 1 received a steroid injection. The remaining patients required no further treatment after diagnosis. Musculoskeletal sarcoidosis is rare and can present as single or multiple lesions. Definitive diagnosis is made by biopsy. The decision to operate should be based on symptoms, issues with cosmesis, impending fractures, growth, and/or involvement of neuro-vascular structures. Patients should be evaluated for pulmonary involvement.