Characterization of a short isoform of the kidney protein podocin in human kidney

BACKGROUND: Steroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking. METHODS: We used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin. RESULTS: Mass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform. CONCLUSIONS: A second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum

MTOR regulates endocytosis and nutrient transport in proximal tubular cells

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR–dependent regulatory network for nutrient transport in renal proximal tubular cells

Uncertainty in Calibration, Detection and Estimation of Metal Concentrations in Engine Plumes Using OPAD

Improvements in uncertainties in the values of radiant intensity (I) can be accomplished mainly by improvements in the calibration process and in minimizing the difference between the background and engine plume radiance. For engine tests in which the plume is extremely bright, the difference in luminance between the calibration lamp and the engine plume radiance can be so large as to cause relatively large uncertainties in the values of R. This is due to the small aperture necessary on the receiving optics to avoid saturating the instrument. However, this is not a problem with the SSME engine since the liquid oxygen/hydrogen combustion is not as bright as some other fuels. Applying the instrumentation to other type engine tests may require a much brighter calibration lamp

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination

Acute physical-activity related increases in interoceptive ability are not enhanced with simultaneous interoceptive attention.

Interoception, the sense of the internal body, is proposed to support self-regulation, and consequently influence mental health. Researchers have therefore shown interest in improving the ability to accurately monitor internal signals (i.e., interoceptive accuracy, IAcc). Research suggests that cardiac IAcc is modifiable by both manipulations of interoceptive attention (guided attention towards the internal body), and interoceptive exposure (strategically inducing somatic signals e.g., via physical activity). Whilst successful in isolation, it is unclear whether a combined approach (i.e., directing attention towards the internal body when signals are more salient) could elicit greater benefits. In a 2 × 2 within-subject design, 48 healthy adults (Mage = 25.98 ± 4.73 years, 50% female) completed four 20-min conditions varying in both attentional focus (interoceptive vs exteroceptive) and physical activity (active vs rest), with cardiac IAcc measured immediately after. Results revealed a main effect for physical activity (p < 0.001), however, there was no effect for attentional focus (p = 0.397), and no interaction effect (p = 0.797). Differential analyses showed that a higher sporting background increased sensitivity to physical activity-related increases in cardiac IAcc (p = 0.031). Findings indicate that (irrespective of attentional focus) moderate-vigorous physical activity-based interventions have the potential to increase cardiac IAcc, with certain individuals potentially benefiting more

Leave the Screen: The Influence of Everyday Behaviors on Self-reported Interoception.

The influence of physical activity on interoception is apparent, however little is known about within-person variability following physical activity and sedentary behavior in daily life. To test this, 70 healthy adults (Mage 21.67±2.50) wore thigh-mounted accelerometers for 7-days, with self-reported interoception recorded on movement-triggered smartphones. Participants additionally reported the predominant activity type performed across the last 15minutes. Investigating this timeframe, multi-level analyses revealed that each one-unit increase in physical activity was associated with an increase in self-reported interoception (B = 0.0025, p =.013), whereas contrastingly, each one-minute increase in sedentary behavior was associated with a decrease (B = -0.06. p =.009). Investigating the influence of different activity types in comparison to screen time behavior, both partaking in exercise (B = 4.48, p <.001) and daily-life physical activity (B = 1.21, p <.001) were associated with an increase in self-reported interoception. Regarding other behavior categories, non-screen time behavior both with (B = 1.13, p <.001) and without (B = 0.67, p =.004) social interaction were also associated with an increase in self-reported interoception compared to screen-time behavior. Extending from previous laboratory-based studies, these findings indicate that physical activity influences interoceptive processes in real-life, further supplemented by the novel and contrasting findings regarding sedentary behavior. Furthermore, associations with activity type reveal important mechanistic information, highlighting the importance of reducing screen-time behavior to preserve and support interoceptive perceptions. Findings can be used to inform health recommendations for reducing screen-time behavior and guiding evidence-based physical activity interventions to promote interoceptive processes

Environmental determinants of macroinvertebrate diversity in small water bodies: insights from tank-bromeliads

The interlocking leaves of tank-forming bromeliads (Bromeliaceae) collect rainwater and detritus, thus creating a freshwater habitat for specialized organisms. Their abundance and the possibility of quantifying communities with accuracy give us unparalleled insight into how changes in local to regional environments influence community diversity in small water bodies. We sampled 365 bromeliads (365 invertebrate communities) along a southeastern to northwestern range in French Guiana. Geographic locality determined the species pool for bromeliad invertebrates, and local environments determined the abundance patterns through the selection of traits that are best adapted to the bromeliad habitats. Patterns in community structure mostly emerged from patterns of predator species occurrence and abundance across local-regional environments, while the set of detritivores remained constant. Water volume had a strong positive correlation with invertebrate diversity, making it a biologically relevant measure of the pools' carrying capacity. The significant effects of incoming detritus and incident light show that changes in local environments (e.g., the conversion of forest to cropping systems) strongly influence freshwater communities. Because changes in local environments do not affect detritivores and predators equally, one may expect functional shifts as sets of invertebrates with particular traits are replaced or complemented by other sets with different traits

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

Exchange Rates, Energy Prices, Unemployment, Money, and Inflation: A Further Test

Energy; Exchange Rates; Inflation; Money; Prices; Unemployment