Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration - but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research

Employee Empowerment and Job Performance in the Resort Industry in Nasugbu, Batangas

Finding out what may lead to increased job performance is a central issue for the resort industry, as increased job performance will also lead to higher levels of customer satisfaction. The study aimed to determine the employee empowerment and job performance in the resort industry in Nasugbu, Batangas.  Descriptive study design with questionnaire as the main data gathering instrument was used to survey 208 rank-and-file employees of the various resorts.  The data gathered were tallied, analyzed and interpreted using frequency, percentage, mean and Pearson’s correlation coefficient.  The result of the study showed that employee empowerment practices are applied and practiced in the resort industry through participation, empowerment through involvement and empowerment through delayering.  The study also revealed that there is a significant relationship between employee empowerment practices and the job performance of the employees.  Based on the findings of the study, it is recommended that resort establishments continue practicing employee empowerment for it has a significant effect on the performance of the employees that may also enhance the organizational performance

Control of nuclear beta-dystroglycan content is crucial for the maintenance of nuclear envelope integrity and function

β-Dystroglycan (β-DG) is a plasma membrane protein that has ability to target to the nuclear envelope (NE) to maintain nuclear architecture. Nevertheless, mechanisms controlling β-DG nuclear localization and the physiological consequences of a failure of trafficking are largely unknown. We show that β-DG has a nuclear export pathway in myoblasts that depends on the recognition of a nuclear export signal located in its transmembrane domain, by CRM1. Remarkably, NES mutations forced β-DG nuclear accumulation resulting in mislocalization and decreased levels of emerin and lamin B1 and disruption of various nuclear processes in which emerin (centrosome-nucleus linkage and β-catenin transcriptional activity) and lamin B1 (cell cycle progression and nucleoli structure) are critically involved. In addition to nuclear export, the lifespan of nuclear β-DG is restricted by its nuclear proteasomal degradation. Collectively our data show that control of nuclear β-DG content by the combination of CRM1 nuclear export and nuclear proteasome pathways is physiologically relevant to preserve proper NE structure and activity

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

The study of Hutchinson–Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin‐driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1‐target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS

Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

β-Dystroglycan (β-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of β-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/β mediated nuclear import pathway has already been described for β-DG, the intracellular trafficking route by which β-DG reaches the nucleus is unknown. In this study, we demonstrated that β-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of β-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of β-DG at Tyr890 is a key stimulus for β-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that β-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE

Plasma and Urine Free Glycosaminoglycans as Monitoring Biomarkers in Nonmetastatic Renal Cell Carcinoma—A Prospective Cohort Study

Background: No liquid biomarkers are approved in renal cell carcinoma (RCC), making early detection of recurrence in surgically treated nonmetastatic (M0) patients dependent on radiological imaging. Urine- and plasma free glycosaminoglycan profiles—or free GAGomes—are promising biomarkers reflective of RCC metabolism. Objective: To explore whether free GAGomes could detect M0 RCC recurrence noninvasively. Design, setting, and participants: Between June 2016 and February 2021, we enrolled a prospective consecutive series of patients elected for (1) partial or radical nephrectomy for clinical M0 RCC (cohort 1) or (2) first-line therapy following RCC metachronous metastatic recurrence (cohort 2) at Sahlgrenska University Hospital, Gothenburg, Sweden. The study population included M0 RCC patients with recurrent disease (RD) versus no evidence of disease (NED) in at least one follow-up visit. Plasma and urine free GAGomes—consisting of 40 chondroitin sulfate (CS), heparan sulfate, and hyaluronic acid (HA) features—were measured in a blinded central laboratory preoperatively and at each postoperative follow-up visit until recurrence or end of follow-up in cohort 1, or before treatment start in cohort 2. Outcome measurements and statistical analysis: We used Bayesian logistic regression to correlate GAGome features with RD versus NED and with various histopathological variables. We developed three recurrence scores (plasma, urine, and combined) proportional to the predicted probability of RD. We internally validated the area under the curve (AUC) using bootstrap resampling. We performed a decision curve analysis to select a cutoff and report the corresponding net benefit, sensitivity, and specificity of each score. We used univariable analyses to correlate each preoperative score with recurrence-free survival (RFS). Results and limitations: Of 127 enrolled patients in total, 62 M0 RCC patients were in the study population (median age: 63 year, 35% female, and 82% clear cell). The median follow-up time was 3 months, totaling 72 postoperative visits —17 RD and 55 NED cases. RD was compatible with alterations in 14 (52%) of the detectable GAGome features, mostly free CS. Eleven (79%) of these correlated with at least one histopathological variable. We developed a plasma, a urine, and a combined free CS RCC recurrence score to diagnose RD versus NED with AUCs 0.91, 0.93, and 0.94, respectively. At a cutoff equivalent to ≥30% predicted probability of RD, the sensitivity and specificity were, respectively, 69% and 84% in plasma, 81% and 80% in urine, and 80% and 82% when combined, and the net benefit was equivalent to finding an extra ten, 13, and 12 cases of RD per hundred patients without any unnecessary imaging for plasma, urine, and combined, respectively. The combined score was prognostic of RFS in univariable analysis (hazard ratio = 1.90, p = 0.02). Limitations include a lack of external validation. Conclusions: Free CS scores detected postsurgical recurrence noninvasively in M0 RCC with substantial net benefit. External validity is required before wider clinical implementation. Patient summary: In this study, we examined a new noninvasive blood and urine test to detect whether renal cell carcinoma recurred after surgery

Subcellular distribution of cell surface-biotinylated β-dystroglycan in C2C12 myoblasts overexpressing active ezrin.

<p><b>A.</b> Cells were transfected to transitory express either ET567D-GFP, Ez-T567A-GFP or GFP alone and incubated with biotin 24 h post-transfection to label cell surface proteins, as described in Material and Methods. Cytosolic and nuclear fractions isolated from biotinylated cells were pulled-down using streptavidin-agarose beads and precipitated proteins were subjected to SDS-PAGE/Western analysis using an anti-β-DG antibody (7D11). Input, biotinylated cytosolic and nuclear extracts subjected to SDS-PAGE/Western analysis without previous streptavidin-mediated precipitation. Membranes were stripped and reprobed for calnexin (Clnx) and Sp3, loading controls for cytoplasmic and nuclear lysates respectively. <b>B.</b> Nuclear to cytoplasmic levels (n/c) of β-DG were quantified as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a> and results plotted represent the mean +/- SD from a series of three separate experiments, with significant differences determined by Student t-test.</p

β-DG interacts with ezrin in C2C12 myoblasts.

<p><b>A.</b> Schematic of the domain structure of β-DG. EC, extracellular domain; TM, transmembrane domain; NLS, nuclear localization signal; CYT, cytoplasmic tail. The NLS of β-DG serves as binding site for both importin α2/β1 and ezrin. <b>B.</b> C2C12 cells grown on glass coverslips were fixed and double stained with anti-ezrin (green) and anti- β-DG antibodies prior to be analyzed by confocal laser scanning microscopy (CLSM). Merged images show colocalization between ezrin and β-DG (yellow), specifically at plasma membrane projections (Inset); scale bar is 10 µm. <b>C.</b> C2C12 total extracts were immunoprecipitated (IP) using an anti-β-DG antibody or control antibody (IgG0), and precipitated proteins subjected to SDS-PAGE/Western analysis employing anti-β-DG or anti-ezrin antibodies. <b>D.</b> The IMPα2/β1 heterodimer interacts with the NLS of β-DG with higher affinity than ezrin. Increasing concentrations (0-60 nm) of GST-tagged ezrin, GST-tagged predimerized IMPα2/β1 or GST alone as a control were incubated with a GFP-β-DGNLS fusion protein and an ALPHA-Screen assay performed as described in Materials and Methods. Sigmoidal curves were fitted using the SigmaPlot software to determine the apparent dissociation constants (Kd) as indicated. Each data point represents the average of three measurements from a single typical experiment from a series of three separate experiments.</p

Nuclear translocation of endogenous β-DG induced by active ezrin is dependent on IMPβ1.

<p>C2C12 myoblasts stably transfected with vector expressing either the control or importin β1 (IMPβ1) RNAi were cultured on glass coverslips, fixed and immunostained for IMPβ1 <b>A</b> or β-DG <b>B</b>, using FITC-conjugated secondary antibody (green), with nuclei stained using DAPI (blue). Cells were imaged by CLSM, with typical single Z-sections shown (scale bar is 10 µm). <b>B.</b> Quantitative analysis to determine the nuclear to cytoplasmic ratio (Fn/c) of β-DG was performed in control- and RNAi IMPβ1-transfected cells (bottom panel), as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD (n &gt; 50 cells) from a series of three separate experiments, with significant differences between cells expressing the control or IMPβ1 RNAi determined by Student t-test. <b>C.</b> Cytoplasmic and nuclear fractions obtained from cells stably expressing either the control or IMPβ1 RNAi and transiently expressing GFP or Ez-T567D-GFP fusion proteins were analyzed by SDS-PAGE/Western using an anti-β-DG antibody (upper panels). Membranes were stripped and reprobed with antibodies against calnexin (Clnx) and Sp3, loading controls for cytoplasmic and nuclear lysates respectively. Nuclear to cytoplasmic ratio (n/c) of β-DG levels were quantified and plotted (bottom panel), as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD from a series of three separate experiments, with significant differences between cells expressing the control or IMPβ1 RNAi determined by Student t-test.</p