Reduction of Proteinuria through Podocyte Alkalinization

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pH_i). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pH_i.     

A new 2D-based method for myocardial velocity strain and strain rate quantification in a normal adult and paediatric population: assessment of reference values

Background

Cardiac time intervals have been described as a measure of cardiac performance, where prolongation, shortening and delay of the different time intervals have been evaluated as markers of cardiac dysfunction. A relatively recently developed method with improved ability to measure cardiac events is Tissue Doppler Imaging (TDI), allowing accurate measurement of myocardial movements.

Methods

We propose the state diagram of the heart as a new visualization tool for cardiac time intervals, presenting comparative, normalized data of systolic and diastolic performance, providing a more complete overview of cardiac function. This study aimed to test the feasibility of the state diagram method by presenting examples demonstrating its potential use in the clinical setting and by performing a clinical study, which included a comparison of the state diagram method with established echocardiography methods (E/E' ratio, LVEF and WMSI). The population in the clinical study consisted of seven patients with non ST-elevation myocardial infarction (NSTEMI) and seven control subjects, individually matched according to age and gender. The state diagram of the heart was generated from TDI curves from seven positions in the myocardium, visualizing the inter- and intraventricular function of the heart by displaying the cardiac phases.

Results

The clinical examples demonstrated that the state diagram allows for an intuitive visualization of pathological patterns as ischemia and dyssynchrony. Further, significant differences in percentage duration between the control group and the NSTEMI group were found in eight of the totally twenty phases (10 phases for each ventricle), e.g. in the transition phases (Pre-Ejection and Post-Ejection). These phases were significantly longer (> 2.18%) for the NSTEMI group than for the control group (p < 0.05). No significant differences between the groups were found for the established echocardiography methods.

Conclusion

The test results clearly indicate that the state diagram has potential to be an efficient tool for visualization of cardiac dysfunction and for detection of NSTEMI.     

Systems analysis of iron metabolism: the network of iron pools and fluxes

Background  

Every cell of the mammalian organism needs iron as trace element in numerous oxido-reductive processes as well as for transport and storage of oxygen. The very versatility of ionic iron makes it a toxic entity which can catalyze the production of radicals that damage vital membranous and macromolecular assemblies in the cell. The mammalian organism maintains therefore a complex regulatory network of iron uptake, excretion and intra-body distribution. Intracellular regulation in different cell types is intertwined with a global hormonal signalling structure. Iron deficiency as well as excess of iron are frequent and serious human disorders. They can affect every cell, but also the organism as a whole.     

Complement Receptor 3 Influences Toll-like Receptor 7/8-Dependent Inflammation: IMPLICATIONS FOR AUTOIMMUNE DISEASES CHARACTERIZED BY ANTIBODY REACTIVITY TO RIBONUCLEOPROTEINS*

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.     

Mapping quantitative trait loci (QTL) in sheep. IV. Analysis of lactation persistency and extended lactation traits in sheep

Background

In sheep dairy production, total lactation performance, and length of lactation of lactation are of economic significance. A more persistent lactation has been associated with improved udder health. An extended lactation is defined by a longer period of milkability. This study is the first investigation to examine the presence of quantitative trait loci (QTL) for extended lactation and lactation persistency in sheep.

Methods

An (Awassi × Merino) × Merino single-sire backcross family with 172 ewes was used to map QTL for lactation persistency and extended lactation traits on a framework map of 189 loci across all autosomes. The Wood model was fitted to data from multiple lactations to estimate parameters of ovine lactation curves, and these estimates were used to derive measures of lactation persistency and extended lactation traits of milk, protein, fat, lactose, useful yield, and somatic cell score. These derived traits were subjected to QTL analyses using maximum likelihood estimation and regression analysis.

Results

Overall, one highly significant (LOD > 3.0), four significant (2.0 < LOD < 3.0) and five suggestive (1.7 < LOD < 2.0) QTL were detected across all traits in common by both mapping methods. One additional suggestive QTL was identified using maximum likelihood estimation, and four suggestive (0.01 < P < 0.05) and two significant (P < 0.01) QTL using the regression approach only. All detected QTL had effect sizes in the range of 0.48 to 0.64 SD, corresponding to QTL heritabilities of 3.1 to 8.9%. The comparison of the detected QTL with results in cattle showed conserved linkage regions. Most of the QTL identified for lactation persistency and extended lactation did not coincide. This suggests that persistency and extended lactation for the same as well as different milk yield and component traits are not controlled by the same genes.

Conclusion

This study identified ten novel QTL for lactation persistency and extended lactation in sheep, but results suggest that lactation persistency and extended lactation do not have a major gene in common. These results provide a basis for further validation in extended families and other breeds as well as targeting regions for genome-wide association mapping using high-density SNP arrays.     

Synthesis of a Nano-Silver Metal Ink for Use in Thick Conductive Film Fabrication Applied on a Semiconductor Package

The success of printing technology in the electronics industry primarily depends on the availability of metal printing ink. Various types of commercially available metal ink are widely used in different industries such as the solar cell, radio frequency identification (RFID) and light emitting diode (LED) industries, with limited usage in semiconductor packaging. The use of printed ink in semiconductor IC packaging is limited by several factors such as poor electrical performance and mechanical strength. Poor adhesion of the printed metal track to the epoxy molding compound is another critical factor that has caused a decline in interest in the application of printing technology to the semiconductor industry. In this study, two different groups of adhesion promoters, based on metal and polymer groups, were used to promote adhesion between the printed ink and the epoxy molding substrate. The experimental data show that silver ink with a metal oxide adhesion promoter adheres better than silver ink with a polymer adhesion promoter. This result can be explained by the hydroxyl bonding between the metal oxide promoter and the silane grouping agent on the epoxy substrate, which contributes a greater adhesion strength compared to the polymer adhesion promoter. Hypotheses of the physical and chemical functions of both adhesion promoters are described in detail.     

Supervised Lowess normalization of comparative genome hybridization data – application to lactococcal strain comparisons

Background

Visualising the evolutionary history of a set of sequences is a challenge for molecular phylogenetics. One approach is to use undirected graphs, such as median networks, to visualise phylogenies where reticulate relationships such as recombination or homoplasy are displayed as cycles. Median networks contain binary representations of sequences as nodes, with edges connecting those sequences differing at one character; hypothetical ancestral nodes are invoked to generate a connected network which contains all most parsimonious trees. Quasi-median networks are a generalisation of median networks which are not restricted to binary data, although phylogenetic information contained within the multistate positions can be lost during the preprocessing of data. Where the history of a set of samples contain frequent homoplasies or recombination events quasi-median networks will have a complex topology. Graph reduction or pruning methods have been used to reduce network complexity but some of these methods are inapplicable to datasets in which recombination has occurred and others are procedurally complex and/or result in disconnected networks.

Results

We address the problems inherent in construction and reduction of quasi-median networks. We describe a novel method of generating quasi-median networks that uses all characters, both binary and multistate, without imposing an arbitrary ordering of the multistate partitions. We also describe a pruning mechanism which maintains at least one shortest path between observed sequences, displaying the underlying relations between all pairs of sequences while maintaining a connected graph.

Conclusion

Application of this approach to 5S rDNA sequence data from sea beet produced a pruned network within which genetic isolation between populations by distance was evident, demonstrating the value of this approach for exploration of evolutionary relationships.     

Cell-free DNA ultra-low-pass whole genome sequencing to distinguish malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion: A cross-sectional study

BackgroundThe leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors.Methods and findingsThis multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort.ConclusionsTumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.

Jeffrey J. Szymanski and colleagues investigate the use of cell-free DNA ultra-low-pass whole genome sequencing to distinguish the malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion in patients with Neurofibromatosis type 1 in United States.