On-enzyme refolding permits small RNA and tRNA surveillance by the CCA-adding enzyme.

Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menß, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates.

The structure of human argonaute-2 in complex with miR-20a.

Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported crystal structure of human Argonaute-2 (hAgo2), the "slicer" in RNA interference, in complex with a mixed population of RNAs derived from insect cells provides insight into the architecture of a eukaryotic Argonaute protein with defined biochemical and biological functions. Here, we report the structure of human Ago2 bound to a physiologically relevant microRNA, microRNA-20a, at 2.2 Å resolution. The miRNA is anchored at both ends by the Mid and PAZ domains and makes several kinks and turns along the binding groove. Interestingly, miRNA binding confers remarkable stability on hAgo2, locking this otherwise flexible enzyme into a stable conformation.

Planarians recruit piRNAs for mRNA turnover in adult stem cells.

PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Thus, PIWI proteins enable planarians to repurpose piRNAs for potentially critical roles in neoblast mRNA turnover.

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity.

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin-producing cells.

In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo-producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP-derived (REPD) cell lines. Under nonpermissive conditions, REPD cells ceased from proliferation and acquired a stem cell-like state, with strongly enhanced hypoxia-inducible factor 2 (HIF-2α), stem cell antigen 1 (SCA-1), and CD133 expression, but also enhanced alpha-smooth muscle actin (αSMA) expression, indicating myofibroblastic signaling. These cells maintained the "on-off" nature of Epo expression observed in REP cells in vivo, whereas other HIF target genes showed a more permanent regulation. Like REP cells in vivo, REPD cells cultured in vitro generated long tunneling nanotubes (TNTs) that aligned with endothelial vascular structures, were densely packed with mitochondria and became more numerous under hypoxic conditions. Although inhibition of mitochondrial oxygen consumption blunted HIF signaling, removal of the TNTs did not affect or even enhance the expression of HIF target genes. Apart from pericytes, REPD cells readily differentiated into neuroglia but not adipogenic, chondrogenic, or osteogenic lineages, consistent with a neuronal origin of at least a subpopulation of REP cells. In summary, these results suggest an unprecedented combination of differentiation features of this unique cell type.

Humidity-Driven Supercontraction and Twist in Spider Silk.

Spider silk is a protein material that exhibits extraordinary and nontrivial properties such as the ability to soften, decrease in length (i.e., supercontract), and twist upon exposure to high humidity. These behaviors stem from a unique microstructure in combination with a transition from glassy to rubbery as a result of humidity-driven diffusion of water. In this Letter we propose four length scales that govern the mechanical response of the silk during this transition. In addition, we develop a model that describes the microstructural evolution of the spider silk thread and explains the response due to the diffusion of water molecules. The merit of the model is demonstrated through an excellent agreement to experimental findings. The insights from this Letter can be used as a microstructural design guide to enable the development of new materials with unique spiderlike properties.

The association between perceived social support in adolescence and positive mental health outcomes in early adulthood: a prospective cohort study.

AIMS: The link between perceived social support and mental health has received much attention from numerous scientific fields in recent years. Most studies, however, have examined associations only over relatively short follow-up periods using global measures of perceived social support and dichotomous negative indicators of mental health. We investigated the long-term association between perceived social support from friends, parents and teachers and multiple positive hedonic and eudemonic indicators of mental health, including hope, meaningfulness and subjective well-being. METHODS: This study used questionnaire data from 2004, 2007 and 2010 from the West Jutland Birth Cohort study with linked register data. The study population consisted of 3681 adolescents born in 1989. Multiple linear and ordered logistic regression were used to analyse the association between perceived social support at age 14/15 years and mental health indicators at age 20/21 years while controlling for possible confounders, including the Big Five personality traits and baseline symptoms of depression. RESULTS: The results show that perceived social support from friends in adolescence was positively associated with all indicators of mental health in early adulthood. Furthermore, perceived paternal social support was positively associated with meaningfulness and subjective well-being, while perceived social support from teachers was positively associated with meaningfulness. CONCLUSIONS: Overall, the results from this study indicate that perceived social support in adolescence, particularly support from friends, could promote positive mental health outcomes in early adulthood.

Association of perceived work pace and physical work demands with occupational accidents: a cross-sectional study of ageing male construction workers in Denmark.

BACKGROUND: Occupational accidents continue to be a significant public health challenge worldwide. Construction workers in particular are at high risk of occupational accidents, and thus it is of major importance to identify possible predictors of occupational accidents among construction workers. We aimed to investigate the association between self-reported work pace and physical work demands and occupational accidents among ageing male construction workers in Denmark. METHODS: Data on perceived work pace, physical work demands, and occupational accidents was acquired from questionnaires sent to ageing construction workers in Denmark in 2016 as part of the ALFA project (ALdring og Fysisk Arbejde; Ageing and Physical Work). A sample of 1270 Danish male construction workers above 50 years of age was included in the present study. Multiple logistic regression models were applied, with adjustments for age, smoking, body mass index, musculoskeletal disorders, occupation, work experience, and support at work. RESULTS: Of 1270 construction workers, 166 (13.1%) reported an occupational accident within the last 12 months. There was no significant association between perceived work pace and occupational accidents, but physical work demands were associated with higher odds for occupational accidents, with an odds ratio of 2.27 (95% confidence interval 1.26-4.10) for medium physical work demands and 2.62 (95% confidence interval 1.50-4.57) for high physical work demands. CONCLUSIONS: Ageing male construction workers with high physical work demands had statistically significant higher odds of having an occupational accident. By contrast, perceived work pace was not associated with occupational accidents in this large cross-sectional study.

On the Origin of Supercontraction in Spider Silk.

Spider silk is a protein material that exhibits extraordinary and nontrivial properties such as the ability to soften and decrease its length by up to ∼60% upon exposure to high humidity. This process is commonly called supercontraction and is the result of a transition from a highly oriented glassy phase to a disoriented rubbery phase. In this work, we derive a microscopically motivated and energy-based model that captures the underlying mechanisms that give rise to supercontraction. We propose that the increase in relative humidity and the consequent wetting of a spider silk have two main consequences: (1) the dissociation of hydrogen bonds and (2) the swelling of the fiber. From a mechanical viewpoint, the first consequence leads to the formation of rubbery domains. This process is associated with an entropic gain and a loss of orientation of chains in the silk network, which motivates the contraction of the spider silk. The swelling of the fiber is accompanied by the extension of chains in order to accommodate the influx of water molecules. Supercontraction occurs when the first consequence is more dominant than the second. The model presented in this work allows us to qualitatively track the transition of the chains from glassy to rubbery states and determine the increase in entropy, the loss of orientation, and the swelling as the relative humidity increases. We also derive explicit expressions for the stiffness and the mechanical response of a spider silk under given relative humidity conditions. To illustrate the merit of this model, we show that the model is capable of capturing several experimental findings. The insights from this work can be used as a microstructural design guide to enable the development of new materials with unique spider-like properties.

The importance of adverse childhood experiences for labour market trajectories over the life course: a longitudinal study.

BACKGROUND: Transitioning from school to work is important in influencing people's trajectories throughout their life course. This study investigated the extent to which adverse childhood experiences (ACEs) were associated with differences in labour market trajectories for young adults in the context of a Nordic child care regime with low levels of child poverty. METHODS: Information on labour market participation, educational events, and public transfer records was recoded into seven state spaces for each month between ages 16 and 32 for a cohort of Danish adolescents born in a rural county in 1983 (N = 3373). Cluster analysis of the sequences using the optimal matching algorithm was used to identify groups with similar trajectories. Multinomial regression was used to assess the association between self-reported ACEs and cluster membership, taking gender and family of origin into account. RESULTS: 'In employment' was the state space in which the young adults spent the most time over their early life courses (mean: 85 out of 204 months; 42%). Cluster analysis identified three clusters. Cluster 3 was most distinct, where the mean time 'outside the labour market' was 149 months (73%), and only 17 months (8%) were spent 'in employment'. Cumulative ACEs increased the probability of being included in Cluster 3 (OR: 1.51). Experiencing parental divorce (OR: 3.05), witnessing a violent event (OR: 3.70), and being abused (OR: 5.64) were most strongly associated with Cluster 3 membership. CONCLUSIONS: Labour market trajectories among adolescents with a higher number of ACEs consisted of more time outside the labour market, compared to adolescents who had experienced fewer adversities. The lasting consequences of childhood adversity should be taken more into account in welfare policies, even in countries such as Denmark, with high social security levels and high-quality universal childcare.

The piRNA pathway in planarian flatworms: new model, new insights.

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that associate with members of the PIWI clade of the Argonaute superfamily of proteins. piRNAs are predominantly found in animal gonads. There they silence transposable elements (TEs), regulate gene expression and participate in DNA methylation, thus orchestrating proper germline development. Furthermore, PIWI proteins are also indispensable for the maintenance and differentiation capabilities of pluripotent stem cells in free-living invertebrate species with regenerative potential. Thus, PIWI proteins and piRNAs seem to constitute an essential molecular feature of somatic pluripotent stem cells and the germline. In keeping with this hypothesis, both PIWI proteins and piRNAs are enriched in neoblasts, the adult stem cells of planarian flatworms, and their presence is a prerequisite for the proper regeneration and perpetual tissue homeostasis of these animals. The piRNA pathway is required to maintain the unique biology of planarians because, in analogy to the animal germline, planarian piRNAs silence TEs and ensure stable genome inheritance. Moreover, planarian piRNAs also contribute to the degradation of numerous protein-coding transcripts, a function that may be critical for neoblast differentiation. This review gives an overview of the planarian piRNA pathway and of its crucial function in neoblast biology.

N-Metallocenoylsphingosines as targeted ceramidase inhibitors: Syntheses and antitumoral effects.

Three N-metallocenoylsphingosines with variance in the central metal (Fe, Co, Ru), the charge (neutral or cationic), and the arene ligands (Cp2, Cp*Ph) were synthesized from serine and metallocene carboxylic acids as substrate-analogous inhibitors of human acid ceramidase (AC). Their inhibitory potential was examined using the recombinant full length ASAH1 enzyme, expressed and secreted from High Five insect cells, and the fluorescent substrate Rbm14-12. All complexes inhibited AC, most strongly so ruthenium(II) complex 13a. Some antitumoral effects of the complexes, such as the interference with the microtubular and F-actin cytoskeleton of cancer cells, were correlated to their AC-inhibition, whereas others, e.g. their cytotoxicity and their induction of caspase-3/-7 activity in cancer cells, were not. All complexes accumulated preferentially in the lysosomes of cancer cells like their target AC, arrested the cells in G1 phase of the cell cycle, and displayed cytotoxicity with mostly single-digit micromolar IC50 values while inducing cancer cell apoptosis.

Dimensionality of the system usability scale among professionals using internet-based interventions for depression: a confirmatory factor analysis.

BACKGROUND: The System Usability Scale (SUS) is used to measure usability of internet-based Cognitive Behavioural Therapy (iCBT). However, whether the SUS is a valid instrument to measure usability in this context is unclear. The aim of this study is to assess the factor structure of the SUS, measuring usability of iCBT for depression in a sample of professionals. In addition, the psychometric properties (reliability, convergent validity) of the SUS were tested. METHODS: A sample of 242 professionals using iCBT for depression from 6 European countries completed the SUS. Confirmatory Factor Analysis (CFA) was conducted to test whether a one-factor, two-factor, tone-model or bi-direct model would fit the data best. Reliability was assessed using complementary statistical indices (e.g. omega). To assess convergent validity, the SUS total score was correlated with an adapted Client Satisfaction Questionnaire (CSQ-3). RESULTS: CFA supported the one-factor, two-factor and tone-model, but the bi-factor model fitted the data best (Comparative Fit Index = 0.992, Tucker Lewis Index = 0.985, Root Mean Square Error of Approximation = 0.055, Standardized Root Mean Square Residual = 0.042 (respectively χ2diff (9) = 69.82, p < 0.001; χ2diff (8) = 33.04, p < 0.001). Reliability of the SUS was good (ω = 0.91). The total SUS score correlated moderately with the CSQ-3 (CSQ1 rs = .49, p < 0.001; CSQ2 rs = .46, p < 0.001; CSQ3 rs = .38, p < 0.001), indicating convergent validity. CONCLUSIONS: Although the SUS seems to have a multidimensional structure, the best model showed that the total sumscore of the SUS appears to be a valid and interpretable measure to assess the usability of internet-based interventions when used by professionals in mental healthcare.

Folate deficiency promotes differentiation of vascular smooth muscle cells without affecting the methylation status of regulated genes.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

Coping strategies in adolescence and labour-market participation in young adulthood: A prospective birth cohort study.

Aims: The aim of this study was to investigate whether coping strategies in adolescence (14-15 years of age) were associated with labour-market participation (LMP) in young adulthood (25-26 years of age) and whether the association differed by sex. Methods: A birth cohort from the former county of Ringkjoebing, Denmark, consisting of 2826 individuals, comprised the study population. In 2004, the study population completed a questionnaire from which information about coping and covariates were gathered. Coping strategies were measured using five sub-scales of the Brief COPE Scale, which were combined into two overall coping strategies: active coping and avoidant coping. Ten years later, the participants were followed for a 52-week period in a register on social benefits. Logistic regression was applied to data, with adjustment for covariates: sex, parents' socio-economic status (education and income) and self-rated health. Results: A total of 2203 (78%) participants were categorised as high LMP at follow-up. No significant associations were found between active coping in adolescence and LMP in 2014/2015. For avoidant coping, in the fully adjusted model, medium-level avoidant coping was associated with higher odds (odds ratio (OR)=1.02 (95% confidence interval (CI) 0.83-1.25) of high LMP. For low avoidant coping, the OR was 1.37 (95% 1.07-1.75). For both coping strategies, sex did not modify the association. Conclusions: Findings showed that avoidant coping was significantly associated with high LMP. Further research is needed to investigate coping in relation to specific problem areas.

A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN ß long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.

Efficient depletion of ribosomal RNA for RNA sequencing in planarians.

BACKGROUND: The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed. RESULTS: In this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium. CONCLUSIONS: The ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.

The effect of early-life and adult socioeconomic position on development of lifestyle-related diseases.

BACKGROUND: Early-life socioeconomic position (SEP) is associated with lifestyle-related diseases in adulthood. However, evidence is lacking on the extent to which adult SEP mediates this association. METHODS: Time to either chronic obstructive pulmonary disease (COPD), cardiovascular disease or diabetes were assessed in the Danish population born between 1961 and 1971 (n = 793 674) from age 30 until 2015. Early-life position was assessed in 1981 (by parental) and again at age 30 (own) by four markers; income, occupation, education-divided into high, middle, low-and a combined score for all markers. Using a counterfactual approach, we estimated the total effect of early-life position on disease onset and the degree to which adult position mediated this effect. RESULTS: Results of the time-to-event analysis showed a gradient of all early-life markers on the risk of developing all lifestyle-related diseases. Notably, comparing those in the lowest to the highest educational position, the hazard of COPD was 130% higher for women [hazard ratio = 2.30(95% confidence interval = 2.20-2.41)] and 114% higher for men [2.14 (2.05-2.25)]. About 67%(63-70%) of the effect of educational position was mediated through adult position for COPD, 55% for cardiovascular disease and 50% for diabetes. For the combined score 44, 29 and 33%, respectively, was mediated. CONCLUSION: About one-tenth to two-thirds of the effect of early-life position is mediated by the position attained in adulthood. The degree mediated depend on the outcome investigated, gender and the social position marker used indicating that alternative pathways may play a key role in developing effective policies targeting early-life behaviours.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

BACKGROUND: The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. METHODS: To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. RESULTS: Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. CONCLUSIONS: Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Accuracy enhanced and synthetic wavelength adjustable optical metrology via spectrally resolved digital holography.

This paper demonstrates the usefulness of spectrally resolved digital holography for dual-wavelength optical metrology. Based on the large degree of phase information available, multiple de-correlated dual-wavelength phase maps can be generated, which, when averaged, result in a signal-to-noise-ratio improvement. Compared with single-wavelength averaging, no further post-processing of the reconstructed dual-wavelength phase map is required. Moreover, the constraint imposed on the wavelength stability, as experienced in the conventional dual-wavelength method, can be relaxed, and the corresponding synthetic wavelength is adapted to the object under investigation. In addition, the possibility of optical sectioning based on the narrow-width coherence envelope is also demonstrated in transmission mode.