Resistance exercise training ameliorates chronic kidney disease outcomes in a 5/6 nephrectomy model.

Chronic kidney disease (CKD) is defined by decreased glomerular filtration rate (GFR) or increased albumin excretion leading to renal injury. However, exercise training is an important non-pharmacological intervention that ameliorates and protects against Diabetes Mellitus, cardiovascular disease, and CKD. AIM: Our aim was to evaluate the capability of resistance exercise training (RET) to improve CKD outcomes and the contribution of the renal and muscular Akt/mTOR signaling pathway for RET beneficial effects on a CKD model. MAIN METHODS: Male Wistar rats were subjected to RET, followed for 10 weeks, and randomly divided into 5 groups: Sham: Sham-operated; sedentary and nephrectomy (5/6Nx) (SNS); exercising post-5/6Nx (SNE); exercising pre-5/6Nx (ENS); exercising pre- and post-5/6Nx (ENE). The systolic blood pressure (BP) was measured. Creatinine, proteinuria, and blood urea nitrogen (BUN) were evaluated. After euthanasia Renal and muscular Akt/mTOR signaling pathways were analyzed. KEY FINDING: Our study showed that the SNS presented renal injury, hypertension, weight and muscular mass loss and a higher mortality rate. SNS group also decreased renal IL-10 and increased TNF-alfa and TGF-Beta. Renal AKT, mTOR, and rpS6 pathway were increased, PTEN was decreased on SNS. And muscular Akt and mTOR were decreased on SNS. SIGNIFICANCE: The RET before and after the 5/6Nx ameliorates all these parameters mentioned above, suggesting that RET is a good non-pharmacological approach to diminish complications frequently found in CKD. We also suggest that the AKT-m-TOR pathway can play an important role in these beneficial outcomes of RET on the CKD animal model.

Link between packing morphology and the distribution of contact forces and stresses in packings of highly nonconvex particles.

An external load on a particle packing is distributed internally through a heterogeneous network of particle contacts. This contact force distribution determines the stability of the particle packing and the resulting structure. Here, we investigate the homogeneity of the contact force distribution in packings of highly nonconvex particles both in two-dimensional (2D) and three-dimensional (3D) packings. A recently developed discrete element method is used to model packings of nonconvex particles of varying sphericity. Our results establish that in 3D packings the distribution of the contact forces in the normal direction becomes increasingly heterogeneous with decreasing particle sphericity. However, in 2D packings the contact force distribution is independent of particle sphericity, indicating that results obtained in 2D packings cannot be extrapolated readily to 3D packings. Radial distribution functions show that the crystallinity in 3D packings decreases with decreasing particle sphericity. We link the decreasing homogeneity of the contact force distributions to the decreasing crystallinity of 3D packings. These findings are complementary to the previously observed link between the heterogeneity of the contact force distribution and a decreasing packing crystallinity due to an increasing polydispersity of spherical particles.

Aerobic exercise training prevents kidney lipid deposition in mice fed a cafeteria diet.

AIM: The objective of this study was to investigate the potential of aerobic exercise training (AET) to prevent kidney lipid accumulation and the contribution of renal metabolism to mediate this response. MAIN METHODS: Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 13), CHOW-TR (chow diet, trained; n = 13), CAF-SED (cafeteria diet, sedentary; n = 13) and CAF-TR (cafeteria diet, trained; n = 13). AET consisted in running sessions of 60 min at 60% of maximal speed conducted five days per week for eight weeks. KEY FINDINGS: AET prevented weight gain in both trained groups. Food intake was not different among groups, however water intake, urine output, urine potassium and osmolarity were reduced in CAF-SED and CAF-TR groups. Kidney lipid deposition increased in CAF-SED (4.12 ±â€¯0.5%/area) compared with CHOW-SED (1.7 ±â€¯0.54%/area), and the AET prevented this increase in the CAF-TR group (2.1 ±â€¯0.5%/area). The Bowman's capsule area decreased in CAF-SED and CAF-TR groups while the Bowman' space reduced in CAF-SED compared to CHOW-SED group, which was prevented by AET in the CAF-TF group. We observed a 27% increase in the p-AMPK expression in CAF-TR compared to CHOW-SED group without differences in the SIRT-1, PGC1-α, ACC and p-ACC. ß-HAD activity increased in CAF-SED (43.9 ±â€¯4.57 nmol·min-1·ug-1) and CAF-TR (44.7 ±â€¯2.6 nmol·min-1·ug-1) groups compared to CHOW-SED (35.1 ±â€¯2.9 nmol·min-1·ug-1) e CHOW-TR (36.6 ±â€¯2.7 nmol·min-1·ug-1). SIGNIFICANCE: AET prevented kidney lipid accumulation induced by cafeteria diet and this response was not associated with changes in the renal metabolic activity that favors lipid oxidation.

Mutational spectrum and deep intronic variants in the factor VIII gene of haemophilia A patients. Identification by next generation sequencing.

Haemophilia A (HA) is caused by a broad spectrum of different mutation types in the factor VIII gene (F8). In our patient cohort of more than 2600 HA patients as well as in other published studies, the most frequent cause are missense mutations in different F8 exons or the recurrent intron 22 inversion. Some exons and several specific nucleotide positions represent hot spots for point mutations in the examined cohort. About 4 % of cases remain without mutation after routine HA diagnostic methods including inversion PCRs, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Deep intronic mutations cannot be detected by current standard HA diagnostics but have been reported for several genetic disorders. However, next generation sequencing (NGS) of the whole genomic sequence of the F8 gene allows to identify deep intronic variants. CONCLUSION: In general, NGS provides an effective approach to screen for different HA causing mutation types in the F8 gene.

Evading Vacuum Noise: Wigner Projections or Husimi Samples?

The accuracy in determining the quantum state of a system depends on the type of measurement performed. Homodyne and heterodyne detection are the two main schemes in continuous-variable quantum information. The former leads to a direct reconstruction of the Wigner function of the state, whereas the latter samples its Husimi Q function. We experimentally demonstrate that heterodyne detection outperforms homodyne detection for almost all Gaussian states, the details of which depend on the squeezing strength and thermal noise.

Exposure to high-fat diet since post-weaning induces cardiometabolic damage in adult rats.

AIMS: This study sought to investigate the metabolic, hemodynamic and autonomic responses in adult rats exposed to high-fat diet since post-weaning. MAIN METHODS: Young male Wistar rats were assigned into groups fed with standard normal diet (3% lipids; ND, n=8) or high-fat diet (30% lipids; HD, n=8) during 8weeks. Body composition, food intake, serum triglycerides, total cholesterol, insulin, leptin and adiponectin concentrations were determined. Hemodynamic and autonomic evaluations were performed. Renin angiotensin system and nitric oxide were also studied by pharmacological blockades. KEY FINDINGS: HD group showed no difference in body weight, total cholesterol, food intake in calories and insulin concentration, but visceral fat pads weight, triglycerides and leptin were higher in HD group. Moreover, HD group decreased adiponectin level, increased 12% of mean arterial pressure (MAP) and 6% of heart rate compared with ND group. Spectral analyses showed an increase in cardiovascular sympathetic modulation in HD compared with ND group. Depressor responses after losartan were higher in HD compared with ND group: -9±0.7 vs.-3±1.6mmHg. Pressor responses after l-NAME were higher in HD compared with ND: 45±8 vs. 32±5mmHg. SIGNIFICANCE: High-fat diet consumption during early period of life can increase WAT mass and MAP. These alterations may be mediated by an augment in sympathetic activity associated with higher leptin and lower adiponectin levels. These cardiometabolic damages can lead to the development of hypertension and increase cardiovascular risk in adulthood.

A comparative reference study for the validation of HLA-matching algorithms in the search for allogeneic hematopoietic stem cell donors and cord blood units.

The accuracy of human leukocyte antigen (HLA)-matching algorithms is a prerequisite for the correct and efficient identification of optimal unrelated donors for patients requiring hematopoietic stem cell transplantation. The goal of this World Marrow Donor Association study was to validate established matching algorithms from different international donor registries by challenging them with simulated input data and subsequently comparing the output. This experiment addressed three specific aspects of HLA matching using different data sets for tasks of increasing complexity. The first two tasks targeted the traditional matching approach identifying discrepancies between patient and donor HLA genotypes by counting antigen and allele differences. Contemporary matching procedures predicting the probability for HLA identity using haplotype frequencies were addressed by the third task. In each task, the identified disparities between the results of the participating computer programs were analyzed, classified and quantified. This study led to a deep understanding of the algorithms participating and finally produced virtually identical results. The unresolved discrepancies total to less than 1%, 4% and 2% for the three tasks and are mostly because of individual decisions in the design of the programs. Based on these findings, reference results for the three input data sets were compiled that can be used to validate future matching algorithms and thus improve the quality of the global donor search process.

Na(+) doping induced changes in the reduction and charge transport characteristics of Al2O3-stabilized, CuO-based materials for CO2 capture.

Chemical looping combustion (CLC) and chemical looping with oxygen uncoupling (CLOU) are emerging CO2 capture technologies that could reduce appreciably the costs associated with the capture of CO2. In CLC and CLOU, the oxygen required to combust a hydrocarbon is provided by a solid oxygen carrier. Among the transition metal oxides typically considered for CLC and CLOU, copper oxide (CuO) stands out owing to its high oxygen carrying capacity, exothermic reduction reactions and fast reduction kinetics. However, the low Tammann (sintering) temperature of CuO is a serious drawback. In this context, it has been proposed to support CuO on high Tammann temperature and low cost alumina (Al2O3), thus, reducing the morphological changes occurring over multiple CLC or CLOU redox cycles and stabilizing, in turn, the high activity of CuO. However, in CuO-Al2O3 systems, phase stabilization and avoiding the formation of the CuAl2O4 spinel is key to obtaining a material with a high redox stability and activity. Here, we report a Na(+) doping strategy to phase stabilize Al2O3-supported CuO, yielding in turn an inexpensive material with a high redox stability and CO2 capture efficiency. We also demonstrate that doping CuO-Al2O3 with Na(+) improves the oxygen uncoupling characteristics and coke resistance of the oxygen carriers. Utilizing in situ and ex situ X-ray absorption spectroscopy (XAS), the local structure of Cu and the reduction pathways of CuO were determined as a function of the Na(+) content and cycle number. Finally, using 4-point conductivity measurements, we confirm that doping of Al2O3-supported CuO with Na(+) lowers the activation energy for charge transport explaining conclusively the improved redox characteristics of the new oxygen carriers developed.

European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.

It is 30 yr since the British Journal of Anaesthesia published the first consensus protocol for the laboratory diagnosis of malignant hyperthermia susceptibility from the European Malignant Hyperthermia Group. This has subsequently been used in more than 10 000 individuals worldwide to inform use of anaesthetic drugs in these patients with increased risk of developing malignant hyperthermia during general anaesthesia, representing an early and successful example of stratified medicine. In 2001, our group also published a guideline for the use of DNA-based screening of malignant hyperthermia susceptibility. We now present an updated and complete guideline for the diagnostic pathway for patients potentially at increased risk of developing malignant hyperthermia. We introduce the new guideline with a narrative commentary that describes its development, the changes to previously published protocols and guidelines, and new sections, including recommendations for patient referral criteria and clinical interpretation of laboratory findings.

Malignant hyperthermia, a Scandinavian update.

BACKGROUND: Malignant Hyperthermia (MH) is a rare pharmacogenetic disorder, triggered by halogenated anesthetics and/or succinylcholine. In susceptible individuals, these drugs can activate an explosive life threatening clinical reaction. Leading symptoms are hypercarbia, muscle rigidity, and metabolic acidosis. MH is inherited in an autosomal-dominant manner and linked to mutations in the large ryanodine 1 gene (RYR1) gene in the majority of cases. Very few MH patients have been found to carry mutations in the CACNA1S gene. METHODS: For this review a large litterature search was carried out and the Swedish MH database consisting of 436 probands who have undergone in vitro muscle contraction test (IVCT) during 1984-2014 was analyzed. RESULTS: Twelve different MH causative mutations have been found in Swedish patients so far. These mutations lead to a disturbed calcium balance in striated muscle tissue. A muscle biopsy for the IVCT or finding of an approved causative mutation are required for the diagnosis. CONCLUSION: A Malignant Hyperthermia susceptible (MHS) patient should be anesthetized with trigger-free anesthesia. There are a few reports of MH-like reactions in patients unrelated to anesthesia. The outcome is dependent on early recognizing of the reaction and fast disconnection of the trigger agents and administration of dantrolene.

Next-generation DNA sequencing of a Swedish malignant hyperthermia cohort.

Malignant hyperthermia (MH)-related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next-generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype-like MH, the pre-selection of genes poses problems in variant interpretation.

Expression studies of mutant factor VIII alleles with premature termination codons with regard to inhibitor formation.

About 10% of mutations in haemophilia A cases generate a premature termination codon in the factor VIII gene (F8). Upon therapeutic FVIII substitution, it was noted that the risk of developing inhibitors is higher when the nonsense mutation is located in the light chain (LC) of the factor VIII (FVIII) protein than in the heavy chain (HC). We analysed the impact of six different nonsense mutations distributed over the six FVIII domains on recombinant FVIII expression to elucidate the process of inhibitor formation in haemophilic patients. Full-length F8 mRNA was transcribed from all constructs despite the presence of nonsense mutations. Polyclonal antigen assays revealed high antigen levels in transfection experiments with constructs truncated in LC whereas low antigen was detected from constructs truncated in HC. Those results were supported by FVIII localization experiments. These findings suggest that F8 transcription occurs in a usual way despite nonsense mutations, whereas translation appears to be interrupted by the premature stop codon. We hypothesize that the inclusion of the B domain enables proteins truncated in LC to accumulate in the ER. Proteins truncated in HC are mainly degraded or may pass through the ER and be secreted into the blood circulation, thus presumably preventing inhibitor formation after therapeutic FVIII substitution. The LC is known to have higher immunogenicity than the HC. Moreover, translation of the F8B gene comprising F8 exons 23-26 may be dependent on the position of the premature stop codon and thus contributes to the immune response of truncated FVIII proteins.

Comparative genetics of warfarin resistance.

Warfarin and other 4-hydroxycoumarin-based oral anticoagulants targeting vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) are administered to humans, mice and rats with different purposes in mind - to act as pesticides in high-dosage baits for killing rodents, but also to save lives when administered in low dosages as antithrombotic drugs in humans. However, high-dosage warfarin used to control rodent populations has resulted in numerous mutations causing warfarin resistance. Currently, six single missense mutations in mice, 12 distinct missense mutations in rats, as well as compound heterozygous or homozygous mutations with up to six distinct missense mutations per Vkorc1 allele have been described. Warfarin resistance missense mutations for human VKORC1 have also been found world-wide, but differ characteristically from those in rodents. In humans, 26 distinct mutations have been characterized, but occur only rarely either in heterozygous or, even rarer, in homozygous form. In this review, we summarize the known VKORC1 missense mutations causing warfarin and other 4-hydroxycoumarin drug resistance, identify genomics databases as new sources of data, explore possible underlying genetic mechanisms, and summarize similarities and differences between warfarin resistant VKORC1 variants in humans and rodents.

Methylation analysis of the promoter region and intron 1 of the factor VIII gene in haemophilia A patients.

Methylation, CpG island, promoter, intron 1 Haemophilia A is the most common X-linked inherited coagulation disorder caused by a deficiency of the factor VIII protein (FVIII). A plethora of different mutations in the factor VIII gene (F8) have been identified as causative for this bleeding disease including a few promoter mutations. However, in approximately 2-5% of all haemophilic patients, the causal mutation still remains unknown. To our knowledge, epigenetic abnormalities in regulatory regions of the F8 gene have not yet been implicated in the disease pathogenesis. We therefore developed bisulfite pyrosequencing assays to screen patients with unknown mutation status for their methylation patterns in presumed regulative regions of the F8 gene (5'UTR and intron 1). The methylation patterns of haemophilia A patients did not differ from that of controls. In three patients, chromosomal aberrations were identified which could be associated with a defective FVIII synthesis.

An update to the HLA Nomenclature Guidelines of the World Marrow Donor Association, 2012.

For more than two decades, international cooperation and information technology have been playing key roles in the identification of suitable unrelated donors and cord blood units for hematopoietic SCT. To ensure consistent coding and interpretation of HLA data among the linked computer systems, the World Marrow Donor Association has standardized the extensions of the World Health Organization (WHO) Nomenclature for factors of the HLA system applied in practice. The first version of this report published in 2007 has become the reference for the technical validation of HLA information on donors and patients in the context of search and matching and is used by registries of volunteer unrelated hematopoietic stem cell donors and umbilical cord blood banks throughout the world. The present update became necessary after the major revision of the WHO HLA nomenclature in April 2010. It now covers issues arising when alleles are withdrawn or renamed because of the continuous updating of the WHO HLA nomenclature. In addition, formal validation and interpretation rules for the so-called 'multiple allele codes' have been added.

Comparative validation of computer programs for haplotype frequency estimation from donor registry data.

Estimation of human leukocyte antigen (HLA) haplotype frequencies from unrelated stem cell donor registries presents a challenge because of large sample sizes and heterogeneity of HLA typing data. For the 14th International HLA and Immunogenetics Workshop, five bioinformatics groups initiated the 'Registry Diversity Component' aiming to cross-validate and improve current haplotype estimation tools. Five datasets were derived from different donor registries and then used as input for five different computer programs for haplotype frequency estimation. Because of issues related to heterogeneity and complexity of HLA typing data identified in the initial phase, the same five implementations, and two new ones, were used on simulated datasets in a controlled experiment where the correct results were known a priori. These datasets contained various fractions of missing HLA-DR modeled after European haplotype frequencies. We measured the contribution of sampling fluctuation and estimation error to the deviation of the frequencies from their true values, finding equivalent contributions of each for the chosen samples. Because of patient-directed activities, selective prospective typing strategies and the variety and evolution of typing technology, some donors have more complete and better HLA data. In this setting, we show that restricting estimation to fully typed individuals introduces biases that could be overcome by including all donors in frequency estimation. Our study underlines the importance of critical review and validation of tools in registry-related activity and provides a sustainable framework for validating the computational tools used. Accurate frequencies are essential for match prediction to improve registry operations and to help more patients identify suitably matched donors.

A new cell culture-based assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay.

BACKGROUND: Warfarin directly inhibits the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) enzyme to effect anticoagulation. VKORC1 function has historically been assessed in vitro using a dithiothreitol (DTT)-driven vitamin K 2,3-epoxide reductase (VKOR) assay. Warfarin inhibits wild-type VKORC1 function by the DTT-VKOR assay. However, VKORC1 variants with warfarin resistance-associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance. OBJECTIVES: A cell culture-based, indirect VKOR assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant VKORC1 proteins. METHODS: Human coagulation factor (F)IX and VKORC1 variants were coexpressed in HEK 293T cells under standardized conditions at various warfarin concentrations. Secreted FIX activity served as surrogate marker to report wild-type and variant VKORC1 inhibition by warfarin. RESULTS AND CONCLUSIONS: Warfarin dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild-type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC50 values were 24.7, 136.4, 152.0 and 1226.4 nm, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild-type VKORC1. Ranked IC50 values from the cell culture-based assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation-associated warfarin resistance.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations.

Mutation screenings in haemophilia A (HA) patients identified a great variety of mutations in the factor VIII gene (F8): intron 22 or intron 1 inversions, missense mutations, nonsense mutations, small or large deletions, insertions, duplications and splice site mutations. Mutations which do not result in amino acid substitutions (silent mutations) and intronic variants located outside the splice site consensus sequences cannot be easily classified as causative for HA. In these cases, special prediction software algorithms are applied to estimate their impact on splicing. Here, we present mRNA analysis of novel F8 mutations with possible impact on splicing in four HA patients with silent mutations and seven patients with intronic variants close to or within splice site consensus sequences. Seven of eleven mutations examined in vitro could be shown to have an effect on F8 mRNA splicing and the results were compared to in silico predictions. In addition, to validate the splice site prediction software Alamut v2.0 (Interactive Biosoftware), we compared published F8 mRNA analyses with the results of the in silico prediction. In general, the results of the splice site prediction tools of Alamut were in good accordance with the experimental F8 mRNA analyses, but a fundamental discrepancy between in silico and in vitro analyses was obtained in some cases. In conclusion, this study shows that the functional classification of potential splicing mutations should not only rely on prediction software, but be rather based on mRNA analysis experiments.