Hypoxia reduces mature hERG channels through calpain up-regulation [Research]

Human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium current (I_Kr) potassium channel, which is important for cardiac repolarization. Impairment of hERG function is the primary cause of acquired long QT syndrome, which predisposes individuals to cardiac arrhythmias and sudden death. Patients with hypoxia due to conditions such as cardiac ischemia or obstructive sleep apnea display increased incidence of cardiac arrhythmias and sudden death. We sought to understand the mechanisms that underlie hypoxia-associated cardiac arrhythmias. Using cell biology and electrophysiologic techniques, we found that hypoxic culture of hERG-expressing human embryonic kidney (HEK) cells and neonatal rat cardiomyocytes reduced hERG current/I_Kr and mature ERG channel expression with a concomitant increase in calpain expression. Calpain was actively released into the extracellular milieu and degraded cell-surface hERG. In contrast to hERG, the ether-a-go-go (EAG) channel was not reduced by hypoxic culture. By making chimeric channels between hERG and EAG, we identified that hypoxia-induced calpain degraded hERG by targeting its extracellular S5-pore linker. The scorpion toxin BeKm-1, which is known to selectively bind to the S5-pore linker of hERG, prevented hypoxia-induced hERG reduction. Our data provide novel information about hypoxia-mediated hERG dysfunction and may have biological and clinical implications in hypoxia-associated diseases.—Lamothe, S. M., Song, W., Guo, J., Li, W., Yang, T., Baranchuk, A., Graham, C. H., Zhang, S. Hypoxia reduces mature hERG channels through calpain up-regulation.

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Worcestershire hospital trust told to make urgent improvements

A hospital trust in the West Midlands has been issued with a warning notice to make urgent improvements, after inspectors found that it had become standard practice to treat emergency patients in the...

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Voice-Specialized Speech-Language Pathologist's Criteria for Discharge from Voice Therapy

No standard protocol exists to determine when a patient is ready and able to be discharged from voice therapy. The aim of the present study was to determine what factors speech-language pathologists (SLPs) deem most important when discharging a patient from voice therapy. A second aim was to determine if responses differed based on years of voice experience.

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Does Microscope Assistance in Cold Steel Tonsillectomy Reduce the Risk of Postoperative Hemorrhage? Results of a Prospective Cohort Study

Background. Posttonsillectomy hemorrhage (PTH) is the most feared complication. Dissection near the tonsillar capsule under microscopic view () could be assumed to decrease PTH compared to traditional tonsillectomy (). Methods. In this study, patients were evaluated with respect to the need for surgical control (R/N: return/no return to theater (RTT): the day of surgery or thereafter ). The findings at resection site and pain were measured. Results. 869 patients were included (183 ; 686 ). PTH requiring RTT was not seen in the group on the day of surgery (R0) while PTH requiring RTT subsequently (R1) was seen in 1.1% of the cases. In the group, hemorrhages without a need for surgical control were observed in 0.6% (N0) and 3.4% (N1), respectively. The corresponding rates for were as follows: R0, 0.3%; R1, 1.7%; N0, 0.6%; and N1, 3.6% (). Postoperative edema and local infection at resection site were proven to be predictive of PTH (). Conclusion. Microscope assistance in tonsillectomy did not statistically have an influence on the PTH even though there was a trend towards lower PTH rate in the group. Benefit for was observed in high-volume and long experienced surgeons.

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Comparison between autogenous iliac bone and freeze-dried bone allograft for repair of alveolar clefts in the presence of plasma rich in growth factors: A randomized clinical trial

This study aimed to compare the effectiveness of alveolar cleft repair using iliac bone and freeze-dried bone allograft (FDBA) in the presence of plasma rich in growth factors (PRGF).

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Cementoblastoma: an updated analysis of 258 cases reported in the literature

To investigate the recurrence rate of cementoblastomas for different variables aside from the clinical/radiologic features.

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Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle [Protein Structure and Folding]

Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi , and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation, to release of Pi . Since the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here, we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein-protein interactions during the Fe protein cycle.

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Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha). [Molecular Biophysics]

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction, with a kcat of 11 ± 0.4 s-1. We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reaction are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction so as to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme (Niks et al. (2016) J. Biol. Chem. 291, 1162- 1174), and on the basis of the present work we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalysing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.

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Site-specific inhibition of the SUMO-conjugating enzyme Ubc9 selectively impairs SUMO chain formation [Enzymology]

Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the backside of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, while mono-SUMOylation is not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.

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Quantifying enzyme activity in living cells [Enzymology]

For over a century enzymatic activity is studied in vitro, assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-β-lactamase inside living cells and compared the values to those obtained in vitro. We found the apparent in vivo catalytic efficiency, kcat/KM, to be lower than in vitro, with significant cell-to-cell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases and KM increases with increasing enzyme concentration. To rationalize these findings we measured enzyme and substrate diffusion rates in the cell and found the later to be slower than expected. Simulations showed that for attenuated diffusion the substrate flux becomes rate limiting, explaining why reaction rates in vivo can be independent on enzyme concentrations. The octanol/water partition of the substrate is 4.5, which is in the range of FDA approved drugs. This suggests substrate limited reaction rates to be common. These findings indicate that in vitro data cannot be simply extrapolated to the crowded in vivo environment.

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The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast [DNA and Chromosomes]

Cumulative evidence suggests that non-proteolytic functions of the proteasome are involved in transcriptional regulation, mRNA export and ubiquitin-dependent histone modification, and thereby modulate the intracellular levels of regulatory proteins implicated in controlling key cellular functions. To date, the non-proteolytic roles of the proteasome have been mainly investigated in euchromatin; their effects on heterochromatin are largely unknown. Here, using fission yeast as a model, we randomly mutagenized the subunits of the 19S proteasome subcomplex, and sought to uncover a direct role of the proteasome in heterochromatin regulation. We identified a non-proteolytic allele, rpt4-1. Experiments performed using rpt4-1 cells revealed that the proteasome is involved in the regulation of heterochromatin spreading to prevent its uncontrolled invasion into neighboring euchromatin regions. Intriguingly, the phenotype of the non-proteolytic rpt4-1 mutant resembled that of epe1Δ cells, which lack the Epe1 protein that counteracts heterochromatin spreading. Both mutants exhibited variegated gene-silencing phenotypes across yeast colonies, spreading of heterochromatin, bypassing of the requirement for RNAi in heterochromatin formation at the outer repeat region (otr), and upregulation of RNA polymerase II. Further analysis revealed Mst2, another factor that antagonizes heterochromatin spreading, may function redundantly with Rpt4. These observations suggest that the 19S proteasome may be involved in modulating the activities of Epe1 and Mst2. In conclusion, our findings indicate that the proteasome appears to have a heterochromatin-regulating function that is independent of its canonical function in proteolysis.

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The 3-hydroxyacyl-CoA dehydratases HACD1 and HACD2 exhibit functional redundancy and are active in a wide range of fatty acid elongation pathways [Metabolism]

Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs. However, we observed only a ~40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1 KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.

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Early hypovolemic shock and abdominal distention due to neonatal splenic rupture: urgency of diagnosis and management

Abstract

Splenic rupture in the neonatal period is a rare condition that can be complicated by hemorrhagic shock. The symptoms are not very specific, rendering the diagnosis difficult and often delayed; sometimes only discovered at autopsy. We report five cases diagnosed in the Rhône-Alpes region of France. From these observations and from a review of the literature, the circumstances of the occurrence, the clinical signs, and the therapeutic possibilities are discussed. In the presence of severe anemia with pallor and abdominal distension, particularly in the context of a difficult birth, an abdominal ultrasound must be urgently performed and surgical management promptly considered.

Conclusion: This pathology must be known to the neonatologist so that she/he can quickly evoke it, given that it can quickly become life-threatening.

What is known: • Splenic rupture in the neonatal period is a rare condition that can be complicated by hemorrhagic shock and quickly lead to the death of the newborn.

• The symptoms are not very specific, rendering the diagnosis difficult and often delayed.

What is new: • This is the first publication bringing together as many clinical cases on the subject reporting in particular very serious cases to alert the clinician on this pathology and its diagnostic urgency. • We propose a clear therapeutic behavior to help the clinician in his daily practice.

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Aberrantly methylated-differentially expressed genes and pathways in colorectal cancer

Methylation plays an important role in the etiology and pathogenesis of colorectal cancer (CRC). This study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) and pathways in CRC by ...

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Kura Oncology's (KURA) CEO Troy Wilson on Q2 2017 Results - Earnings Call Transcript - Seeking Alpha

Kura Oncology's (KURA) CEO Troy Wilson on Q2 2017 Results - Earnings Call Transcript Seeking Alpha The thyroid cohort is ongoing in the first stage. As we previously reported among the 11 evaluable patients in the first stage of the second cohort we observed two objective responses out of three patients with squamous head and neck cancer and as a ...

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Oral administration of geranylgeranylacetone to protect vestibular hair cells

Publication date: Available online 7 August 2017
Source:Auris Nasus Larynx
Author(s): Shinpei Nagato, Kazuma Sugahara, Yoshinobu Hirose, Yousuke Takemoto, Makoto Hashimoto, Hironori Fujii, Hiroshi Yamashita
ObjectiveWe recently reported that the heat shock response played a major role in the protection of hair cells against stress. Oral administration of the heat shock inducer, geranylgeranylacetone (GGA) protected hair cells against intense noise. In our present study, we investigated the effect of GGA on vestibular hair cell death induced by an aminoglycoside.MethodsWe used CBA/N mice aged 4–6 weeks. The mice were divided into two groups, GGA and control. Mice in the GGA group were fed a diet containing GGA (0.5%) for 4 weeks, and those in the control group were fed a standard diet. Immunohistochemical analyses for Hsp70 were performed in four animals. The utricles of the remaining animals were cultured in medium for 24h with neomycin to induce hair cell death. After fixation, the vestibular hair cells were immunohistochemically stained against calmodulin, and hair cell survival was evaluated.ResultsThe vestibular hair cells of mice in the GGA group expressed Hsp70. In addition, after exposure to neomycin, vestibular hair cell survival was higher in the GGA group than in the control group.ConclusionOur results demonstrated the oral administration of GGA induced the heat shock response in the vestibule and could protect sensory cells.



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Garidisan: Improving the Quality of Ulcer Healing in Rats with Ulcerative Colitis

Garidisan, commonly used in Mongolia to treat ulcerative colitis (UC), contains wild poppy and Artemisia frigida Willd. Clinical evidence shows that Garidisan can effectively treat UC and that recurrence is low. Thus, we evaluated the effects of Garidisan on ulcer healing quality and the regulation of immune balance in rats with experimental UC. UC was induced by immunization with TNBS and Garidisan significantly reduced DAI, CMDI, and HS. H&E staining, SEM, and VG staining showed that Garidisan repaired damaged intestinal mucosa and significantly reduced expression of ICAM-1 and CD105 in regenerated tissues of UC rats. Collagen fibers were significantly fewer as well after treatment. Garidisan elevated EGF, VEGF, bFGF, VEGFR2, and FGFR1 of UC rats, reduced CD3+CD4+/CD3+CD8+ T cell ratios, and increased CD4+Th1/CD4+Th2 cell ratios and IFN-r/IL-4 ratios in peripheral blood of UC rats. In conclusion, Garidisan promoted tissue maturation of regenerated tissues by regulating the immune balance and improved functional maturity of regenerated tissues by reducing collagen formation, promoting maturation of new blood vessels, and increasing expression of growth factors and their receptors.

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Silica Based Superhydrophobic Nanocoatings for Natural Rubber Surfaces

Silica based nonfluorinated superhydrophobic coatings for natural rubber surfaces have been developed. The coating was synthesized using nanosilica dispersion and a polychloroprene type binder as a compatibilizer. This nanocoating of silica was applied on to the surface of finished natural rubber gloves, by spray coating or dipped coating methods. The nanocoating demonstrates a water contact angle of more than 150° and sliding angle of 7°. The morphological features of the coating have been studied using scanning electron microscopy and atomic force microscopy while Fourier transform infrared spectroscopy was used to understand the nature of surface functional groups. Both imaging techniques provided evidence for the presence of nanosized particles in the coating. Coated gloves demonstrated comparable mechanical properties and significantly better alcohol resistivity when compared to those of the uncoated gloves.

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A Novel Electric Bicycle Battery Monitoring System Based on Android Client

The battery monitoring system (BMS) plays a crucial role in maintaining the safe operation of the lithium battery electric bicycle and prolonging the life of the battery pack. This paper designed a set of new battery monitoring systems based on the Android system and ARM single-chip microcomputer to enable direct management of the lithium battery pack and convenient monitoring of the state of the battery pack. The BMS realizes the goal of monitoring the voltage, current, and ambient temperature of lithium batteries, estimating the state of charge (SOC) and state of health (SOH), protecting the battery from abuse during charging or discharging, and ensuring the consistency of the batteries by integrating the passive equalization circuit. The BMS was proven effective and feasible through several tests, including charging/discharging, estimation accuracy, and communication tests. The results indicated that the BMS could be used in the design and application of the electric bicycle.

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Does Microscope Assistance in Cold Steel Tonsillectomy Reduce the Risk of Postoperative Hemorrhage? Results of a Prospective Cohort Study

Background. Posttonsillectomy hemorrhage (PTH) is the most feared complication. Dissection near the tonsillar capsule under microscopic view () could be assumed to decrease PTH compared to traditional tonsillectomy (). Methods. In this study, patients were evaluated with respect to the need for surgical control (R/N: return/no return to theater (RTT): the day of surgery or thereafter ). The findings at resection site and pain were measured. Results. 869 patients were included (183 ; 686 ). PTH requiring RTT was not seen in the group on the day of surgery (R0) while PTH requiring RTT subsequently (R1) was seen in 1.1% of the cases. In the group, hemorrhages without a need for surgical control were observed in 0.6% (N0) and 3.4% (N1), respectively. The corresponding rates for were as follows: R0, 0.3%; R1, 1.7%; N0, 0.6%; and N1, 3.6% (). Postoperative edema and local infection at resection site were proven to be predictive of PTH (). Conclusion. Microscope assistance in tonsillectomy did not statistically have an influence on the PTH even though there was a trend towards lower PTH rate in the group. Benefit for was observed in high-volume and long experienced surgeons.

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