Comparative-high resolution melting: a novel method of simultaneous screening for small mutations and copy number variations

Efficient and cost-effective screening for DNA sequence changes, both small mutations and copy number variations (CNVs), is a crucial aspect for routine genetic diagnostics as well as for basic research. In this study we present a development and evaluation of comparative-high resolution melting (C-HRM), a new approach for the simultaneous screening of small DNA changes and gene CNVs. In contrast to other methods, relative quantification in C-HRM is based on the results obtained during the melting process and calculations of the melting peak height ratio in the multiplex reaction. Validation of the method was conducted on DNA samples from 50 individuals from Duchenne muscular dystrophy (DMD) families, 50 probands diagnosed with familial adenomatous polyposis and a control group of 36 women and 36 men. The results of analyses conducted on fragments of the DMD and APC genes correspond completely (100 %) with the results of previous studies. C-HRM sensitivity in CNV detection was assessed through the analysis of mixed DNA samples with different proportions of a deletion carrier and wild type control. The results are presented as a linear regression with R ² of 0.9974 and imply the capability of the method to detect mosaics. C-HRM is an attractive and powerful alternative to other methods of point mutations and CNV detection with 100 % accuracy in our studied group.     

Crystal structure of a putative quorum sensing‐regulated protein (PA3611) from the Pseudomonas‐specific DUF4146 family

Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas‐specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure‐based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family. Proteins 2014; 82:1086–1092. © 2013 Wiley Periodicals, Inc.     

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.     

Relaxin Does Not Improve Angiotensin II-Induced Target-Organ Damage

Relaxin is a corpus-luteum produced protein hormone with vasodilatatory, anti-fibrotic, and angiogenic properties that are opposite to angiotensin (Ang) II. We investigated whether or not relaxin ameliorates Ang II-induced target-organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGR) that develop severe hypertension, target-organ damage, and die untreated within 7–8 weeks. Recombinant relaxin at a low (26 μg/kg/d) and a high dose (240 μg/kg/d) was given to 4 week-old dTGR and age-matched Sprague-Dawley rats (SD). Systolic blood pressure increased progressively in untreated dTGRs from 162±3 mmHg at week 5 to 225±5 mmHg at week 7. Relaxin had no effect on blood pressure whereas SD rats were normotensive (106±1 mmHg). Untreated and relaxin-treated dTGR had similarly severe cardiac hypertrophy indices. Relaxin did not ameliorate albuminuria and did not prevent matrix-protein deposition in the heart and kidney in dTGR. Finally, relaxin treatment did not reduce mortality. These data suggest that pharmacological doses of relaxin do not reverse severe effects of Ang II.     

Is the Poly (L- Lactide- Co– Caprolactone) Nanofibrous Membrane Suitable for Urinary Bladder Regeneration?

The purpose of this study was to compare: a new five-layered poly (L–lactide–co–caprolactone) (PLC) membrane and small intestinal submucosa (SIS) as a control in rat urinary bladder wall regeneration. The five-layered poly (L–lactide–co–caprolactone) membrane was prepared by an electrospinning process. Adipose tissue was harvested from five 8-week old male Wistar rats. Adipose derived stem cells (ADSCs) were seeded in a density of 3×10⁶ cells/cm² onto PLC membrane and SIS scaffolds, and cultured for 5-7 days in the stem cell culture medium. Twenty male Wistar rats were randomly divided into five equal groups. Augmentation cystoplasty was performed in a previously created dome defect. Groups: (I) PLC+ 3×10⁶ADSCs; (II) SIS+ 3×10⁶ADSCs; (III) PLC; (IV) SIS; (V) control. Cystography was performed after three months. The reconstructed urinary bladders were evaluated in H&E and Masson''s trichrome staining. Regeneration of all components of the normal urinary bladder wall was observed in bladders augmented with cell-seeded SIS matrices. The urinary bladders augmented with SIS matrices without cells showed fibrosis and graft contraction. Bladder augmentation with the PLC membrane led to numerous undesirable events including: bladder wall perforation, fistula or diverticula formation, and incorporation of the reconstructed wall into the bladder lumen. The new five-layered poly (L–lactide–co–caprolactone) membrane possesses poorer potential for regenerating the urinary bladder wall compared with SIS scaffold.     

Optimization of Plasma Sample Pretreatment for Quantitative Analysis Using iTRAQ Labeling and LC-MALDI-TOF/TOF

Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome''s complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.     

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES + S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).     

Expansion of the Protein Repertoire in Newly Explored Environments: Human Gut Microbiome Specific Protein Families

The microbes that inhabit particular environments must be able to perform molecular functions that provide them with a competitive advantage to thrive in those environments. As most molecular functions are performed by proteins and are conserved between related proteins, we can expect that organisms successful in a given environmental niche would contain protein families that are specific for functions that are important in that environment. For instance, the human gut is rich in polysaccharides from the diet or secreted by the host, and is dominated by Bacteroides, whose genomes contain highly expanded repertoire of protein families involved in carbohydrate metabolism. To identify other protein families that are specific to this environment, we investigated the distribution of protein families in the currently available human gut genomic and metagenomic data. Using an automated procedure, we identified a group of protein families strongly overrepresented in the human gut. These not only include many families described previously but also, interestingly, a large group of previously unrecognized protein families, which suggests that we still have much to discover about this environment. The identification and analysis of these families could provide us with new information about an environment critical to our health and well being.     

Fluoride inhibition of enolase: crystal structure and thermodynamics

Enolase is a dimeric metal-activated metalloenzyme which uses two magnesium ions per subunit: the strongly bound conformational ion and the catalytic ion that binds to the enzyme-substrate complex inducing catalysis. The crystal structure of the human neuronal enolase-Mg2F2P(i) complex (enolase fluoride/phosphate inhibitory complex, EFPIC) determined at 1.36 A resolution shows that the combination of anions effectively mimics an intermediate state in catalysis. The phosphate ion binds in the same site as the phosphate group of the substrate/product, 2-phospho-D-glycerate/phosphoenolpyruvate, and induces binding of the catalytic Mg2+ ion. One fluoride ion bridges the structural and catalytic magnesium ions while the other interacts with the structural magnesium ion and the ammonio groups of Lys 342 and Lys 393. These fluoride ion positions correspond closely to the positions of the oxygen atoms of the substrate's carboxylate moiety. To relate structural changes resulting from fluoride, phosphate, and magnesium ions binding to those that are induced by phosphate and magnesium ions alone, we also determined the structure of the human neuronal enolase-Mg2P(i) complex (enolase phosphate inhibitory complex, EPIC) at 1.92 A resolution. It shows the closed conformation in one subunit and a mixture of open and semiclosed conformations in the other. The EPFIC dimer is essentially symmetric while the EPIC dimer is asymmetric. Isothermal titration calorimetry data confirmed binding of four fluoride ions per dimer and yielded Kb values of 7.5 x 10(5) +/- 1.3 x 10(5), 1.2 x 10(5) +/- 0.2 x 10(5), 8.6 x 10(4) +/- 1.6 x 10(4), and 1.6 x 10(4) +/- 0.7 x 10(4) M(-1). The different binding constants indicate negative cooperativity between the subunits; the asymmetry of EPIC supports such an interpretation.     

Biocidal effect of (<Emphasis Type="Italic">E</Emphasis>)-anethole on the cyanobacterium <Emphasis Type="Italic">Aphanizomenon gracile</Emphasis> Lemmermann

Biocidal natural substances of botanical origin offer a promising ecofriendly option for controlling toxic cyanobacteria. Herein, we study 11 essential oils and some of their major components for their activity on Aphanizomenon gracile. On the basis of our results we support that Origanum vulgare and O. dictamnus, Ocimum basilicum, Eucalyptus meliodora, Melissa officinalis, and Pimpinella anisum exhibited the strongest activities, and the IC_50/1d values of the extracts were calculated to be between 168.43 and 241.97 μg mL^?1. When the major components of the biocidal essential oils were tested individually, (E)-anethole was found active, exhibiting an IC_50/1d value of 71.35 μg mL^?1. On the other hand, the half-life (t _1/2 ) of (E)-anethole was calculated at 1 h. A preliminary attempt of (E)-anethole microencapsulation was conducted, in order to slowly release this biocidal agent, increasing the residual life under open air conditions and thus the biological activity. Results were promising since the microencapsulated product exhibited better activity than did the non-formulated (E)-anethole. This is a first report on the biocidal activity of EOs and (E)-anethole on A. gracile and a preliminary indication of the microencapsulated (E)-anethole potential use as a natural biocidal in fresh waters.

Open image in new window

Graphical abstract Filaments of Aphanizomenon gracile capable of bloom forming (strain SAG 31.79, in a batch culture) and (E)-anethole