Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of Dasypyrum villosum L. (H. villosa) by a Single DNA Probe

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.     

Point-of-care assays for tuberculosis: Role of nanotechnology/microfluidics

Tuberculosis (TB) remains one of the most devastating infectious diseases and its eradication is still unattainable given the limitations of current technologies for diagnosis, treatment and prevention. The World Health Organization's goal to eliminate TB globally by 2050 remains an ongoing challenge as delayed diagnosis and misdiagnosis of TB continue to fuel the worldwide epidemic. Despite considerable improvements in diagnostics for the last few decades, a simple and effective point-of-care TB diagnostic test is yet not available. Here, we review the current assays used for TB diagnosis, and highlight the recent advances in nanotechnology and microfluidics that potentially enable new approaches for TB diagnosis in resource-constrained settings.     

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Secretion of alpha-amylase from Pseudoalteromonas haloplanktis TAB23: two different pathways in different hosts

Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for alpha-amylase secretion in Escherichia coli. The occurrence of two different secretion pathways in different hosts is proposed.     

Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules

Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.     

Beta-endorphin-like and alpha-MSH-like immunoreactivities in human milk

We measured with radioimmunoassay the beta-endorphin-like and alpha-MSH-like immunoreactivities in milk and plasma of 8 lactating women. Mean beta-endorphin concentrations ( +/- SD) were 16.6 +/- 6.7 fmol/ml in milk and 9.9 +/- 4.1 fmol/ml in plasma. alpha-MSH concentrations (mean +/- SD) were 39.4 +/- 15.5 pg/ml in milk and 18.2 +/- 8.4 pg/ml in plasma. The concentrations of both peptides in milk were significantly (p less than 0.05) higher than in plasma. No significant correlation between milk and plasma concentrations of these peptides was found.     

Three Sesquiterpenoid Dimers from Chloranthus japonicus: Absolute Configuration of Chlorahololide A and Related Compounds

A novel sesquiterpenoid dimer, named multistalide C ( 1 ), together with two known congeners, shizukaols C ( 2 ) and D ( 3 ), was isolated from the whole plant of Chloranthus japonicus Sieb. The structures of compounds 1 , 2 , 3 were elucidated by extensive HR‐ESI‐MS, 1D, and 2D NMR spectroscopic analysis. Compounds 1 , 2 , 3 exhibited significant toxic effects on brine shrimp larvae (Artemia salina). The absolute configuration of 1 was established by CD/TDDFT calculations. The related compound chlorahololide A was also reinvestigated. The previous assignment of the absolute configuration of chlorahololide A and several related sesquiterpenoid dimers, based on an incorrect application of the exciton chirality method, is criticized. Chirality 28:158–163, 2015. © 2015 Wiley Periodicals, Inc.     

Good Computational Practice in the Assignment of Absolute Configurations by TDDFT Calculations of ECD Spectra

Quantum‐mechanical calculations of chiroptical properties have rapidly become the most popular method for assigning absolute configurations (AC) of organic compounds, including natural products. Black‐box time‐dependent Density Functional Theory (TDDFT) calculations of electronic circular dichroism (ECD) spectra are nowadays readily accessible to nonexperts. However, an uncritical attitude may easily deliver a wrong answer. We present to the Chirality Forum a discussion on what can be called good computational practice in running TDDFT ECD calculations, highlighting the most crucial points with several examples from the recent literature. Chirality 28:466–474, 2016. © 2016 Wiley Periodicals, Inc.