Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their ...functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
No approved targeted therapy for the treatment of patients with neuroblastoma RAS viral (v-ras) oncogene homolog (
)-mutant melanoma is currently available.
In this phase Ib escalation/expansion ...study (ClinicalTrials.gov identifier: NCT02974725), the safety, tolerability, and preliminary antitumor activity of naporafenib (LXH254), a BRAF/CRAF protein kinases inhibitor, were explored in combination with trametinib in patients with advanced/metastatic
or
-mutant non-small-cell lung cancer (escalation arm) or
-mutant melanoma (escalation and expansion arms).
Thirty-six and 30 patients were enrolled in escalation and expansion, respectively. During escalation, six patients reported grade ≥3 dose-limiting toxicities, including dermatitis acneiform (n = 2), maculopapular rash (n = 2), increased lipase (n = 1), and Stevens-Johnson syndrome (n = 1). The recommended doses for expansion were naporafenib 200 mg twice a day plus trametinib 1 mg once daily and naporafenib 400 mg twice a day plus trametinib 0.5 mg once daily. During expansion, all 30 patients experienced a treatment-related adverse event, the most common being rash (80%, n = 24), blood creatine phosphokinase increased, diarrhea, and nausea (30%, n = 9 each). In expansion, the objective response rate, median duration of response, and median progression-free survival were 46.7% (95% CI, 21.3 to 73.4; 7 of 15 patients), 3.75 (95% CI, 1.97 to not estimable NE) months, and 5.52 months, respectively, in patients treated with naporafenib 200 mg twice a day plus trametinib 1 mg once daily, and 13.3% (95% CI, 1.7 to 40.5; 2 of 15 patients), 3.75 (95% CI, 2.04 to NE) months, and 4.21 months, respectively, in patients treated with naporafenib 400 mg twice a day plus trametinib 0.5 mg once daily.
Naporafenib plus trametinib showed promising preliminary antitumor activity in patients with
-mutant melanoma. Prophylactic strategies aimed to lower the incidence of skin-related events are under investigation.
Background. Nephrotic syndrome is a condition that is clinically associated with poor outcome. In this study, we compared different techniques of urine sample preparation in order to develop a robust ...analytical protocol to define the differential urinary proteome of urinary abnormalities compared to nephrotic proteinuria. Methods. We recruited 5 normal control subjects, 16 patients with urinary abnormalities and 16 patients with nephrotic syndrome. Proteins from normal urine were processed using three different protocols acetone, ultrafiltration and trichloroacetic acid (TCA) precipitation, depletion of albumin and IgGs and then analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels and mass spectrometry. Results. Comparing the three extraction methods by visual inspection of gels after 2D gel electrophoresis, the acetone precipitation and TCA methods yielded the best quality of protein extraction, while the acetone precipitation method was the most efficient. Furthermore, we tested three commercial kits for albumin and IgG depletion. We applied the optimized acetone extraction protocol to compare urinary samples from nephrotic patients (NP) to urinary samples obtained from patients presenting with urinary abnormalities (UAP). We observed a proteolytic activity directed against albumin. This observation was more prevalent in urinary samples from NP than from UAP. Within both groups, there was some inter-individual variability in the observed proteolytic activity. An increased concentration of alpha1 antitrypsin was also observed in urine of NP. We analysed albumin fragmentation by 1D and 2D western blots in the same samples skipping the albumin and IgG depletion steps to avoid the possible confound of albumin fragment removal. The analysis confirmed a stronger proteolytic activity in the nephrotic group. Conclusions. The proteolytic activity against albumin and the anti-proteolytic activity of alpha1 antitrypsin are likely linked and could play an important role in the nephrotic process. If replicated in larger samples, this methodology may lead to a better understanding of the underlying pathophysiological process of nephrotic syndrome.
Glomerular diseases are potentially fatal, requiring aggressive interventions and close monitoring. Urine is a readily-accessible body fluid enriched in molecular signatures from the kidney and ...therefore particularly suited for routine clinical analysis as well as development of non-invasive biomarkers for glomerular diseases.
The Nephrotic Syndrome Study Network (NEPTUNE; ClinicalTrials.gov Identifier NCT01209000) is a North American multicenter collaborative consortium established to develop a translational research infrastructure for nephrotic syndrome. This includes standardized urine collections across all participating centers for the purpose of discovering non-invasive biomarkers for patients with nephrotic syndrome due to minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Here we describe the organization and methods of urine procurement and banking procedures in NEPTUNE.
We discuss the rationale for urine collection and storage conditions, and demonstrate the performance of three experimental analytes (neutrophil gelatinase-associated lipocalin NGAL, retinol binding globulin, and alpha-1 microglobulin) under these conditions with and without urine preservatives (thymol, toluene, and boric acid). We also demonstrate the quality of RNA and protein collected from the urine cellular pellet and exosomes.
The urine collection protocol in NEPTUNE allows robust detection of a wide range of proteins and RNAs from urine supernatant and pellets collected longitudinally from each patient over 5 years. Combined with the detailed clinical and histopathologic data, this provides a unique resource for exploration and validation of new or accepted markers of glomerular diseases.
ClinicalTrials.gov Identifier NCT01209000.
IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the general population. There is accumulating evidence that immunosuppressive treatment is efficacious in IgAN. However, it is ...critical to define appropriate indicators for this therapy especially in the wake of potentially deleterious side effects to immunosuppressives.
This study retrospectively reviewed IgAN cases collected since 1981 to identify clinical and/or histological parameters for disease progression; 310 patients with biopsy proven IgAN, diagnosed from January 1981 to March 2004, were included.
We defined a clinical prognostic index (CPI) using multivariate analysis, which incorporated these clinical/ histological parameters. Semiquantitative scores were assigned as follows: 2 points if creatinine (Cr) was >1.4 mg/dL, 1 point if proteinuria was >1 g/24 hr, 1 point if a patient was affected by hypertension, and 1 point if a patient was older than 30 yrs. Dividing our population into two groups (scores 0-2 = low CPI group; scores 3-5 = high CPI group), we demonstrated a significantly different 10-yr renal survival rate; in the low CPI group, renal survival since time of biopsy at 10 yrs was 91.7%; in the high CPI group the renal survival at 10 yrs was 35%. We validated the CPI in an independent sample from Verona (validation group) and demonstrated similar results for the CPI.
The CPI is convenient to use for defining the risk of disease progression.
Background and purpose
Hereditary transthyretin amyloidosis (ATTRv) is a life‐threatening disease caused by mutations in the gene encoding transthyretin (TTR). The recent therapeutic advances have ...underlined the importance of easily accessible, objective biomarkers of both disease onset and progression. Preliminary evidence suggests a potential role in this respect for neurofilament light chain (NfL). In this study, the aim was to determine serum NfL (sNfL) levels in a late‐onset ATTRv population and evaluate whether it might represent a reliable biomarker of disease onset (i.e., ‘conversion’ from the asymptomatic status to symptomatic disease in TTR mutation carriers).
Methods
In all, 111 individuals harbouring a pathogenic TTR variant (61 symptomatic ATTRv patients and 50 presymptomatic carriers) were consecutively enrolled. Fifty healthy volunteers were included as the control group. Ella™ apparatus was used to assess sNfL levels.
Results
Serum NfL levels were increased in ATTRv patients compared to both presymptomatic carriers and healthy controls, whilst not differing between carriers and healthy controls. An sNfL cut‐off of 37.10 pg/mL could discriminate between asymptomatic and symptomatic individuals with high diagnostic accuracy (area under the curve 0.958; p < 0.001), sensitivity (81.4%) and specificity (100%).
Conclusions
Serum NfL seems to be a promising biomarker of peripheral nerve involvement in ATTRv amyloidosis and might become a reliable, objective measure to detect the transition from the presymptomatic stage to the onset of symptomatic disease. Further longitudinal studies are needed to confirm such a role and determine whether it could equally represent a biomarker of disease progression and response to therapy.
The ability to rapidly adapt to adverse environmental conditions represents the key of success of many pathogens and, in particular, of Mycobacterium tuberculosis. Upon exposition to heat shock, ...antibiotics or other sources of stress, appropriate responses in terms of genes transcription and proteins activity are activated leading part of a genetically identical bacterial population to express a different phenotype, namely to develop persistence. When the stress response network is mathematically described by an ordinary differential equations model, development of persistence in the bacterial population is associated with bistability of the model, since different emerging phenotypes are represented by different stable steady states. In this work, we develop a mathematical model of SigE stress response network that incorporates interactions not considered in mathematical models currently available in the literature. We provide, through involved analytical computations, accurate approximations of the system's nullclines, and exploit the obtained expressions to determine, in a reliable though computationally efficient way, the number of equilibrium points of the system. Theoretical analysis and perturbation experiments point out the crucial role played by the degradation pathway involving RseA, the anti-sigma factor of SigE, for coexistence of two stable equilibria and the emergence of bistability. Our results also indicate that a fine control on RseA concentration is a necessary requirement in order for the system to exhibit bistability.
The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal ...migration. Mutations in the
RAB7A
gene cause the autosomal dominant Charcot–Marie–Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B.
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