Quick and reliable method to analyze meiotic segregation patterns in Coprinus cinereus using the polymerase chain reaction

It is well known that multiple auxotrophic markers impede fruiting in Coprinus cinereus. Restriction fragment length polymorphisms have been used to advantage (Cassidy et al. 1984 Curr. Genet. 8:607-613; Zolan et al. 1992 Nucl. Acids Res. 20:3993-3999) but the required Southern analysis is both time consuming and laborious

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species

Stimulation of Host Immune Defenses by a Small Molecule Protects C. elegans from Bacterial Infection

The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans–based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens

Effect of salt reduction on consumer acceptance and sensory quality of food

Reducing salt (NaCl) intake is an important public health target. The food industry and catering services are searching for means to reduce the salt content in their products. This review focuses on options for salt reduction in foods and the sensory evaluation of salt-reduced foods. Simple salt reduction, mineral salts and flavor enhancers/modifiers (e.g., umami compounds) are common options for salt reduction. In addition, the modification of food texture and odor-taste interactions may contribute to enhanced salty taste perception. Maintaining consumer acceptance of the products is a challenge, and recent examples of the consumer perception of salt-reduced foods are presented.</p

Novel Proteins Required for Meiotic Silencing by Unpaired DNA and siRNA Generation in Neurospora crassa

During meiosis in the filamentous fungus Neurospora crassa, unpaired genes are identified and silenced by a process known as meiotic silencing by unpaired DNA (MSUD). Previous work has uncovered six proteins required for MSUD, all of which are also essential for meiotic progression. Additionally, they all localize in the perinuclear region, suggesting that it is a center of MSUD activity. Nevertheless, at least a subset of MSUD proteins must be present inside the nucleus, as unpaired DNA recognition undoubtedly takes place there. In this study, we identified and characterized two new proteins required for MSUD, namely SAD-4 and SAD-5. Both are previously uncharacterized proteins specific to Ascomycetes, with SAD-4 having a range that spans several fungal classes and SAD-5 seemingly restricted to a single order. Both genes appear to be predominantly expressed in the sexual phase, as molecular study combined with analysis of publicly available mRNA-seq datasets failed to detect significant expression of them in the vegetative tissue. SAD-4, like all known MSUD proteins, localizes in the perinuclear region of the meiotic cell. SAD-5, on the other hand, is found in the nucleus (as the first of its kind). Both proteins are unique compared to previously identified MSUD proteins in that neither is required for sexual sporulation. This homozygous-fertile phenotype uncouples MSUD from sexual development and allows us to demonstrate that both SAD-4 and SAD-5 are important for the production of masiRNAs, which are the small RNA molecules associated with meiotic silencing

Twist and snai1 expression in pharyngeal squamous cell carcinoma stroma is related to cancer progression

<p>Abstract</p> <p>Background</p> <p>Epithelial-mesenchymal transition (EMT) is a crucial process in tumorigenesis since tumor cells attain fibroblast-like features enabling them to invade to surrounding tissue. Two transcription factors, <it>TWIST </it>and <it>SNAI1</it>, are fundamental in regulating EMT.</p> <p>Methods</p> <p>Immunohistochemistry was used to study the expression of TWIST and SNAI1 in 109 pharyngeal squamous cell carcinomas.</p> <p>Results</p> <p>Tumors with intense stromal staining of TWIST relapsed more frequently (p = 0.04). Tumors with both positive TWIST and SNAI1 immunoreactivity in the stroma were at least Stage II (p = 0.05) and located more often in hypopharynx (p = 0.035). Tumors with negative immunostaining of TWIST and SNAI1 in the stromal compartment were smaller (T1-2) (p = 0.008), less advanced (SI-II) (p = 0.031) and located more often in the oropharynx (p = 0.007). Patients with negative SNAI1 and TWIST immunostaining in tumor stroma had a better 5-year disease-specific and overall survival (p = 0.037 and p = 0.014 respectively).</p> <p>Conclusion</p> <p>TWIST and SNAI1 expression in stromal cells is associated with clinical and histopathological characteristics that indicate progressive disease. Negative expression of these EMT-promoting transcription factors predicts a better outcome.</p

Candida albicans Infection of Caenorhabditis elegans Induces Antifungal Immune Defenses

Candida albicans yeast cells are found in the intestine of most humans, yet this opportunist can invade host tissues and cause life-threatening infections in susceptible individuals. To better understand the host factors that underlie susceptibility to candidiasis, we developed a new model to study antifungal innate immunity. We demonstrate that the yeast form of C. albicans establishes an intestinal infection in Caenorhabditis elegans, whereas heat-killed yeast are avirulent. Genome-wide, transcription-profiling analysis of C. elegans infected with C. albicans yeast showed that exposure to C. albicans stimulated a rapid host response involving 313 genes (124 upregulated and 189 downregulated, ∼1.6% of the genome) many of which encode antimicrobial, secreted or detoxification proteins. Interestingly, the host genes affected by C. albicans exposure overlapped only to a small extent with the distinct transcriptional responses to the pathogenic bacteria Pseudomonas aeruginosa or Staphylococcus aureus, indicating that there is a high degree of immune specificity toward different bacterial species and C. albicans. Furthermore, genes induced by P. aeruginosa and S. aureus were strongly over-represented among the genes downregulated during C. albicans infection, suggesting that in response to fungal pathogens, nematodes selectively repress the transcription of antibacterial immune effectors. A similar phenomenon is well known in the plant immune response, but has not been described previously in metazoans. Finally, 56% of the genes induced by live C. albicans were also upregulated by heat-killed yeast. These data suggest that a large part of the transcriptional response to C. albicans is mediated through “pattern recognition,” an ancient immune surveillance mechanism able to detect conserved microbial molecules (so-called pathogen-associated molecular patterns or PAMPs). This study provides new information on the evolution and regulation of the innate immune response to divergent pathogens and demonstrates that nematodes selectively mount specific antifungal defenses at the expense of antibacterial responses

The invertebrate lysozyme effector ILYS-3 is systemically activated in response to danger signals and confers antimicrobial protection in C. elegans

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material

Gymnemic acids inhibit hyphal growth and virulence in Candida albicans

Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine