Zebrafish Model as a Screen to Prevent Cyst Inflation in Autosomal Dominant Polycystic Kidney Disease.

In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer's vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knockdown by showing that it causes a 73% reduction in the number of KV cilia expressing PC2.

CiliarMove: new software for evaluating ciliary beat frequency helps find novel mutations by a Portuguese multidisciplinary team on primary ciliary dyskinesia.

Evaluation of ciliary beat frequency (CBF) performed by high-speed videomicroscopy analysis (HVMA) is one of the techniques required for the correct diagnosis of primary ciliary dyskinesia (PCD). Currently, due to lack of open-source software, this technique is widely performed by visually counting the ciliary beatings per a given time-window. Our aim was to generate open-source, fast and intuitive software for evaluating CBF, validated in Portuguese PCD patients and healthy volunteers.

Pkd2 Affects Cilia Length and Impacts LR Flow Dynamics and .

The left-right (LR) field recognizes the importance of the mechanism involving the calcium permeable channel Polycystin-2. However, whether the early LR symmetry breaking mechanism is exclusively Polycystin-2 has not been tested. For that purpose, we need to be able to isolate the effects of decreasing the levels of Pkd2 protein from any eventual effects on flow dynamics.

Notch/Her12 signalling modulates, motile/immotile cilia ratio downstream of in zebrafish left-right organizer.

Foxj1a is necessary and sufficient to specify motile cilia. Using transcriptional studies and slow-scan two-photon live imaging capable of identifying the number of motile and immotile cilia, we now established that the final number of motile cilia depends on Notch signalling (NS). We found that despite all left-right organizer (LRO) cells express and the ciliary axonemes of these cells have dynein arms, some cilia remain immotile.

New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression.

Aim: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection.

Methods: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene.

Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.

Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA.

Singlet oxygen in antimicrobial photodynamic therapy: photosensitizer-dependent production and decay in E. coli.

Several families of photosensitizers are currently being scrutinized for antimicrobial photodynamic therapy applications. Differences in physical and photochemical properties can lead to different localization patterns as well as differences in singlet oxygen production and decay when the photosensitizers are taken up by bacterial cells. We have examined the production and fate of singlet oxygen in Escherichia coli upon photosensitization with three structurally-different cationic photosensitizers, namely New Methylene Blue N (NMB), a member of the phenothiazine family, ACS268, a hydrophobic porphyrin with a single cationic alkyl chain, and zinc(II)-tetramethyltetrapyridinoporphyrazinium salt, a phthalocyanine-like photosensitizer with four positive charges on the macrocycle core.

Ulcerogenic Helicobacter pylori strains isolated from children: a contribution to get insight into the virulence of the bacteria.

Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence.

A nonionic porphyrin as a noninterfering DNA antibacterial agent.

The increasing interest in clinical bacterial photodynamic inactivation has led to the search for photosensitizers with higher bactericidal efficiency and less side effects on the surrounding tissues. We present a novel nonionic porphyrin, the 5,10,15-tris(2,6-dichlorophenyl)-20-[4-N-(6-amino-hexyl)sulfonamido)phenyl]-porphyrin (ACS769F4) with substantial improvements in the efficiency of nonionic sensitizers. This porphyrin causes eradication of both Escherichia coli and Staphylococcus aureus by the photodynamic effect but in higher concentrations compared with 5,10,15,20-tetrakis (4-N,N,N-trimethylammoniumphenyl)-porphyrin p-tosylate (TTAP(4+)), a known bactericidal tetracationic porphyrin.

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori.

Background:  The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H.

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms.

The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking.

Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease.

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins.

Proteomics techniques for cystic fibrosis research.

Numerous factors, other than mutations in the CFTR gene, affect the phenotypic variability of cystic fibrosis (CF). With a two-dimensional electrophoresis (2-DE) analysis of total protein expression profiles (proteomics) of CF versus non-CF cells it is possible to obtain an integrative picture of CF cellular alterations. Through this approach, proteins that interact differently with wild type- and mutant-CFTR can also be identified (interactomics).

Biochemical methods to assess CFTR expression and membrane localization.

Detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein is usually a difficult task to accomplish due to the low levels of expression and high turnover that this membrane protein is submitted to in the cell. Common biochemical methods can be used for the detection of CFTR but several critical points must be taken into account. The scope of this article is to outline biochemical methods commonly used to assess CFTR expression, processing and membrane localization.

Antibodies for CFTR studies.

For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells/tissues. Here, we review the applicability of most Abs currently in use in CF research for the biochemical and/or immunocytochemical detection of CFTR.

A comparison of 14 antibodies for the biochemical detection of the cystic fibrosis transmembrane conductance regulator protein.

Interest in the biochemical detection of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein followed soon after cloning of the gene and prediction of the protein structure. Ever since, antibodies (Abs) have been produced and used to detect CFTR in both heterologously and endogenously expressing cells and tissues. Although designed to be sensitive and specific, these Abs produce, in most cases, unsatisfactory results when used for the biochemical detection of CFTR either by Western blot or by immunoprecipitation.

Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein.

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism.