A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

A functional variant in NEPH3 gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population.

BACKGROUND: Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms. METHODS: We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as "Severe" or "Mild", based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population. RESULTS AND CONCLUSIONS: Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients' kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a "rare variant-strong effect" role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria.

Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae.

BACKGROUND: Transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding RNAs (lncRNAs). However, only a few lncRNAs have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. Even less lncRNAs have been annotated as essential hence implying that the majority may be functionally redundant. Therefore, the function of lncRNAs could be illuminated through systematic analysis of their synthetic genetic interactions (GIs). RESULTS: Here, we employ synthetic genetic array (SGA) in Saccharomyces cerevisiae to identify GIs between long intergenic non-coding RNAs (lincRNAs) and protein-coding genes. We first validate this approach by demonstrating that the telomerase RNA TLC1 displays a GI network that corresponds to its well-described function in telomere length maintenance. We subsequently performed SGA screens on a set of uncharacterised lincRNAs and uncover their connection to diverse cellular processes. One of these lincRNAs, SUT457, exhibits a GI profile associating it to telomere organisation and we consistently demonstrate that SUT457 is required for telomeric overhang homeostasis through an Exo1-dependent pathway. Furthermore, the GI profile of SUT457 is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. Accordingly, we show that ectopic expression of this lincRNA suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for SUT457 in telomere biology. CONCLUSIONS: Overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncRNAs in yeast and other complex organisms.

Infrared Fluorescence-Based Cancer Screening Capsule for the Small Intestine.

Infrared fluorescence endoscopy (IRFE), in conjunction with an infrared fluorescent-labelling contrast agent, is a well known technique used for efficient early-stage cancer detection. In this paper we present a cost-effective (< $500) screening capsule prototype, which is able to detect infrared (IR) fluorescence emitted by indocyanine green (ICG) fluorophore dye. Rather than image, the capsule works as a high-sensitivity fluorometer that records fluorescence levels throughout the small intestine. The presented mixed-signal system has a small size, consumes very little power (≈ 6.3 mA) and does not require an external belt and hardware for data collection. By determining fluorescence levels in the intestine, rather than collecting images, we avoid the need for labour intensive video analysis. The whole system is contained within a compact ingestible capsule, that is sized so as to come into close contact with the intestine walls during peristalsis. Ex-vivo experiments, on ICG-impregnated swine intestine, have shown that the prototype system is able to detect low concentrations of ICG in the nanomolar and micromolar region, which is required to detect early cancer in the small intestine.

Molecular and Clinical Investigation of Cystinuria in the Greek-Cypriot Population.

BACKGROUND AND AIMS: Cystinuria represents 3% of nephrolithiasis in humans. Two genes have been identified as the main genetic causes of cystinuria, SLC3A1 and SLC7A9, with an autosomal recessive mode of inheritance. In the present study, we studied for the first time, genetically and clinically, all the cystinuric families identified so far in the Greek-Cypriot population. METHODS: Discovery of mutations was performed through polymerase chain reaction (PCR)-single analysis and DNA resequencing. New families were investigated through PCR-RFLPs. Clinical data were collected through the hospital patients' records and analytical follow-up of the families. RESULTS AND DISCUSSION: We found a total of five mutations in 28 Greek-Cypriot cystinuric patients belonging in 12 families. The most frequent mutation among the 28 Greek-Cypriot patients is the SLC3A1-p.T216M, which is also the second most frequent mutation in Europe, representing a genetic founder effect. Sixteen of the 28 patients are homozygous for this mutation. Even though a consanguinity loop was obvious in only one family, other patients were from families in small villages where endogamy was practiced for many centuries. Timely clinical and genetic diagnosis, accompanied by early treatment, is significant for the good health of most of our patients. Only ∼14% of them developed chronic renal failure, and only one reached end-stage renal disease (ESRD). CONCLUSION: Five SLC3A1 and SLC7A9 mutations appear to be responsible for the genetic basis of cystinuria in the Greek-Cypriot patients; having such a limited number of causative mutations will simplify diagnostics for this population.

An ingestible, NIR-fluorometric, cancer-screening capsule.

Asymptomatic, early-stage, cancer detection is a problem in the small intestine, that is largely inaccessible. This paper presents a cost-effective screening capsule prototype, which is able to detect infrared (IR) fluorescence emitted by indocyanine green (ICG) fluorophore dye. The presented mixed-signal system has a small size, consumes little power and works as a high-sensitivity fluorometer that records fluorescence levels throughout the small intestine, rather than collecting images that need labour intensive video analysis. Ex-vivo experiments, on ICG-impregnated swine intestine, have shown that the prototype system is able to detect low concentrations of ICG in the nanomolar and micromolar region, which is required to detect early cancer in the small intestine.

Frequency of COL4A3/COL4A4 mutations amongst families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response. Focal and segmental glomerulosclerosis is a frequent development during ageing.

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.

Towards a fluoroscopic cancer screening capsule for the small intestine.

Efficient microcancer detection in the small intestine can be realised by infrared fluorescence endoscopy (IRFE). The affected areas can be visualised through that technique in conjunction with an infrared fluorescent-labeling contrast agent, which is selectively uptaken by cancerous cells. In this paper we present a screening capsule prototype that is able to measure IR fluorescence levels emitted by fluorophore indocyanine green (ICG) of different concentrations. The mixed-signal system presented has small area footprint, and very little power requirements. In-vitro experiments have shown that the system is able to detect and discriminate low concentrations of ICG in the micromolar region, which is required to detect early cancer in the small intestine.

Epistatic role of the MYH9/APOL1 region on familial hematuria genes.

Familial hematuria (FH) is explained by at least four different genes (see below). About 50% of patients develop late proteinuria and chronic kidney disease (CKD). We hypothesized that MYH9/APOL1, two closely linked genes associated with CKD, may be associated with adverse progression in FH. Our study included 102 thin basement membrane nephropathy (TBMN) patients with three known COL4A3/COL4A4 mutations (cohort A), 83 CFHR5/C3 glomerulopathy patients (cohort B) with a single CFHR5 mutation and 15 Alport syndrome patients (cohort C) with two known COL4A5 mild mutations, who were categorized as "Mild" (controls) or "Severe" (cases), based on renal manifestations. E1 and S1 MYH9 haplotypes and variant rs11089788 were analyzed for association with disease phenotype. Evidence for association with "Severe" progression in CFHR5 nephropathy was found with MYH9 variant rs11089788 and was confirmed in an independent FH cohort, D (cumulative p value = 0.001, odds ratio = 3.06, recessive model). No association was found with APOL1 gene. Quantitative Real time PCR did not reveal any functional significance for the rs11089788 risk allele. Our results derive additional evidence supporting previous reports according to which MYH9 is an important gene per se, predisposing to CKD, suggesting its usefulness as a prognostic marker for young hematuric patients.

Familial C3 glomerulopathy associated with CFHR5 mutations: clinical characteristics of 91 patients in 16 pedigrees.

BACKGROUND AND OBJECTIVES: Complement factor H and related proteins (CFHR) are key regulators of the alternative complement pathway, where loss of function mutations lead to a glomerulopathy with isolated mesangial C3 deposits without immunoglobulins. Gale et al. (12) reported on 26 patients with the first familial, hematuric glomerulopathy caused by a founder mutation in the CFHR5 gene in patients of Cypriot descent living in the United Kingdom. CFHR5 nephropathy is clinically characterized by continuous microscopic hematuria whereas some patients present with additional episodes of synpharyngitic macrohematuria, associated with infection and pyrexia. A subgroup of patients, particularly men, develop additional proteinuria, hypertension, and chronic renal disease or ESRD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We herewith expand significantly on the study by Gale et al., reporting on histologic, molecular, and clinical findings in 91 patients from 16 families with the same founder mutation. RESULTS: Eighty-two patients (90%) exhibited microscopic hematuria; 51 (62%), exhibited only microscopic hematuria, whereas the remaining 31 additionally had proteinuria (38%); 28 proteinuric patients developed chronic renal failure (CRF). Among carriers of CFHR5 mutation aged >50 years, 80% of the men and 21% of the women developed CRF; 18 developed ESRD (14 men [78%], 4 women [22%]). CONCLUSIONS: The diagnosis of CFHR5-related, isolated C3 glomerulopathy was established in 2009 using newly described mutation analysis after decades of follow-up with unclear diagnoses, occasionally confused with IgA nephropathy. This larger patient cohort establishes the clinical course, significant variable expressivity, and marked gender difference regarding the development of CRF and ESRD.

Founder mutations in the ATP6V1B1 gene explain most Cypriot cases of distal renal tubular acidosis: first prenatal diagnosis.

AIMS: To investigate clinically and genetically all the distal renal tubular acidosis (dRTA) cases in Cyprus, to study one more family from Greece and to perform the first dRTA prenatal diagnosis. We also tried to find any association with sensorineural hearing loss (SNHL) onset and particular mutations. METHODS: Nine dRTA families from Cyprus and one from Greece were analyzed for mutations in ATP6V1B1 gene by DNA resequencing and PCR-RFLPs. Clinical diagnosis was performed by standard criteria. Prenatal diagnosis was performed for one Cypriot family. RESULTS: Results show that 7/9 dRTA cases in Cyprus are caused by 229+1G>T and R157C founder mutations in ATP6V1B1 gene. 229+1G>T mutation was estimated to be older than 400 years. No genotype- phenotype correlation was found with SNHL. A known (L81P) and a novel mutation (912delT) were found in the Greek family. Prenatal diagnosis was performed for one Cypriot family, after parents' demand, showing that the embryo was a heterozygous carrier. CONCLUSION: Existence of only two ATP6V1B1 mutations in the Cypriot population is a diagnostic advantage. The age of onset of SNHL varies in our patients and probably is not related to ATP6V1B1 genotypes. Effective therapy for most of the syndrome symptoms is not satisfactory for some parents who choose prenatal diagnosis to ensure their child's health.