In Vitro Antifungal Susceptibility of <Emphasis Type="Italic">Neoscytalidium dimidiatum</Emphasis> Clinical Isolates from Malaysia

Neoscytalidium dimidiatum is an opportunistic fungus causing cutaneous infections mostly, which are difficult to treat due to antifungal resistance. In Malaysia, N. dimidiatum is associated with skin and nail infections, especially in the elderly. These infections may be mistaken for dermatophyte infections due to similar clinical appearance. In this study, Neoscytalidium isolates from cutaneous specimens, identified using morphological and molecular methods (28 Neoscytalidium dimidiatum and 1 Neoscytalidium sp.), were evaluated for susceptibility towards antifungal agents using the CLSI broth microdilution (M38-A2) and Etest methods. Amphotericin B, voriconazole, miconazole and clotrimazole showed high in vitro activity against all isolates with MIC ranging from 0.0313 to 1 µg/mL. Susceptibility towards fluconazole and itraconazole was noted in up to 10% of isolates, while ketoconazole was inactive against all isolates. Clinical breakpoints for antifungal drugs are not yet available for most filamentous fungi, including Neoscytalidium species. However, the results indicate that clinical isolates of N. dimidiatum in Malaysia were sensitive towards miconazole, clotrimazole, voriconazole and amphotericin B, in vitro.     

Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so‐called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide‐rhamnose synthase/epimerase‐reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy‐thymidine diphosphate (dTDP)‐rhamnose, a precursor of L‐rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal–host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)‐rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.     

Influence of UV-B Supplemental Radiation on Growth and Pigment Content in Suaeda Maritima L.

In a field experiment with a mangrove species Suaeda maritima L. grown under ambient and supplementary UV-B radiation corresponding to 20 % ozone depletion, changes in growth and contents of photosynthetic and UV-absorbing pigments were determined. Supplemental UV-B irradiation for 9 d significantly reduced the growth and concentration of photosynthetic pigments. However, anthocyanin and flavonoid contents were significantly increased in UV-treated plants and which could be reduce the UV-B penetration and damage to the underlying tissues.     

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.     

Protein evolution: The deciphering of latent facets — correlation of synthesis profiles of ribosomally directed proteins and enzyme directed peptides

Key facets pertaining to the evolution of proteins have been probed, using as springboard, the relevant data bases constructed from (i) 60 ribosomally directed proteins, whose 3D structures are known and having 10,000 residues and (ii) from 73 enzyme directed peptides, comprising of 524 residues. The preference profiles, both in terms of the choice of neighbours and the placement of the peptide bonds, have been delineated with respect to each of the 20 coded amino acids. By and large, the preference profile from both the sets are similar, thus giving importance to the nature of the side chains of the coded amino acids. The predictive power of the preference profile has been tested with good results, thus demonstrating the evidence of common preference pathways for peptide formation during evolution. The ribosomally directed protein synthesis, controlled by the genome, proceeds by the addition of single residues at a time. On the other hand, the enzyme directed peptide synthesis largely operates in a more energy conscious block mode, where each constituent of a large enzyme ensemble is engaged in precisely assembling the modules and transfering them to the adjacent one, thus realizing a sequence specific peptide synthesis. Of significance is the fact that, in spite of such divergence in assembly, predictions for neighbour preferences in ribosomally directed protein synthesis work well when applied to enzyme directed peptide synthesis. The findings here are significant since they provide (i) a clear picture of directed peptide synthesis in the absence of direct genomic control, (ii) evidence for the preferred formation of peptide bonds using protein templates, (iii) they also provide evidence for the presence of protein like structures, with catalytic activity, prior to the freezing of the genetic code arising from dominance of the information system and (iv) a logical approach to the evolution of a hierarchical pattern. Presented at the National Symposium on Evolution of Lite.     

Effect of individual ingredients of fortified skim milk as suspending media on survival of freeze-dried cells of Streptococcus lactis

The viability of freeze-dried cells of Streptococcus lactis C-2 was studied by suspending washed cells in reconstituted skim milk and in solutions of ascorbic acid, thiourea and ammonium chloride used alone or in various combinations. Low cell survival was observed with the use of reconstituted skim milk. Fortification of this menstruum with ascorbic acid and thiourea gave maximum survival of freeze-dried cells.