R. S. Dassanayake

Isoflavonoids and a pterocarpan from Gliricidia sepium

A new isoflavan, 7,4′-dihydroxy-3′-methoxyisoflavan has been isolated from the insecticidally active hot dichloromethane extract of the heartwood of Gliricidia sepium, along with the three other isoflavonoids, isovestitol, formononetin and afrormosin, a pterocarpan, medicarpin and 4-hydroxy-3-methoxycinnamaldehyde which are new to this species.

Differential phospholipase gene expression by Candida albicans in artificial media and cultured human oral epithelium

Phospholipases B1, B2, C and D of Candida albicans play a significant role in the host invasive process. Hence we evaluated the in vitro expression of PLB1, PLB2, PLC1 and PLD1 in phospholipase‐positive (PL+) and ‐deficient (PL−) C. albicans isolates in egg yolk agar (EYA), yeast peptone dextrose broth (YPD), and in a model of oral candidiasis based on reconstituted human oral epithelium (RHOE). The growth of Candida was then determined in YPD and its cellular invasion was investigated using the RHOE model. The PL+ group demonstrated PLB1, PLB2, PLC1 and PLD1 expression in both EYA and YPD, in contrast to the PL− group, which expressed only PLB2 and PLD1. Although PL+ isolates grew profusely in the RHOE model, they expressed only PLB2, PLC1 and PLD1, and not PLB1. Gene expression investigations could not be carried out with PL− isolates due to their inability to grow in the RHOE model. Significant growth differences in YPD medium were also observed within the PL+ and PL− groups. Taken together, these findings indicate that phospholipase gene expression in C. albicans is differentially affected by their growth milieu, and this in turn may modulate the disease outcomes in vivo.

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized α/β motifs

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.

Amplification-based nucleic acid scanning techniques to assess genetic polymorphism in Candida

Opportunistic pathogen Candida causes common fungal infections that manifest both superficially and systemically, especially in compromised patients. Although C. albicans is by far the main etiological agent of candidosis, the frequency of isolation of other non-albicans species such as C. glabrata and C. krusei is increasing at an alarming rate. Therefore, the epidemiology, pathogenicity, and diagnosis of infections due to these organisms are of great importance. Of a variety of genotyping methods utilized for strain delineation of these Candida species, amplification-based techniques such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction digestion-mediated PCR (RFLP-PCR), and single-stranded conformational polymorphism (SSCP) and microsatellite PCR (interrepeat PCR, IR-PCR) are the most popular and widely used. In the last decade or so these techniques have helped unravel the clinical epidemiological features of pathogenicity, diversity, microevolution, and natural heterozygosity in Candida species. Here we review in detail the basic principles of RAPD, the nature of the primer and factors influencing its selection, and the limitations of RAPD assays as well as analysis and interpretation of banding profiles generated using the software programs. In addition, the principles of other RAPD-based amplification techniques (AFLP, RFLP-PCR, SSCP, and IR-PCR) and their application in molecular epidemiologic studies of Candida species in particular and other fungi in general are also reviewed. It is concluded that these methods have wide applicability in genotyping fungi, although they differ greatly in their resolution and have advantages and drawbacks depending on the task in question.

Molecular heterogeneity of fluconazole-resistant and -susceptible oral Candida albicans isolates within a single geographic locale.

The emergence of drug‐resistant Candida albicans in immunocompromised patients is common. A disconcerting aspect of this phenomenon is the rapid emergence of C. albicans strains that are resistant to a widely used azole drug, fluconazole (FLZ). To understand the origin of FLZ‐resistant yeast isolates, we investigated molecular profiles of 20 geographically related oral C. albicans isolates using three genotyping methods: randomly amplified polymorphic DNA‐PCR, with six different primers (OBU1, OBU2, OBU3 RSD6, RSD11 and RSD12); electrophoretic karyotyping by pulsed‐field gel electrophoresis; and HinfI restriction fragment analysis. Of the 20 isolates studied, 10 were FLZ‐ resistant and originated from patients with oral candidosis with a history of FLZ therapy, and the remainder were FLZ susceptible from individuals with oral candidosis, but without a history of FLZ therapy. A composite genotype was generated for each strain by combining molecular types derived from the three independent molecular methods. The composite profiles indicated genetic diversity amongst both the FLZ‐resistant as well as ‐sensitive isolates, and no specific features emerged distinguishing the drug‐resistant and ‐sensitive groups. These observations cast doubt on the theory of a clonal origin of FLZ‐resistant C. albicans isolates.

Characterization of the genetic diversity in superficial and systemic human isolates of Candida parapsilosis by randomly amplified polymorphic DNA (RAPD).

The application of randomly amplified polymorphic DNA (RAPD) technology for strain delineation of medically important yeasts has proved to be a valuable tool in clinico‐epidemiological studies of Candida species. Candida parapsilosis, a form species of the fungi imperfecti, is an emerging pathogen gaining recognition as an opportunistic agent, especially in the immunocompromised. Therefore, 15 clinical isolates of C. parapsilosis obtained from oral, cutaneous and systemic Candida infections were typed by RAPD analysis using four different primers. The primers RSD6 and RSD9 elicited 7 geno‐types each, whereas primers RSD7 and RSD12 revealed 6 and 10 genotypes, respectively. When the data were correlated, a higher degree of genomic heterogeneity in systemic isolates was noted compared with the oral and cutaneous isolates, which shared somewhat similar RAPD profiles. However, a single oral isolate (P5) and two systemic isolates (P13 and P15) elicited radically divergent profiles, dissimilar to their counterparts. RAPD study of the latter two isolates with three additional primers (RSD8, RSD10 and RSD11) confirmed the observed genomic disparity. These data substantiate the previous observations on the genomic heterogeneity in C. parapsilosis and point to genetic shifts which may be associated with ecodiversity, as well as the possible existence of distinct genetic groups within this form species.

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.

Genomic diversity of oral Candida krusei isolates as revealed by DNA fingerprinting and electrophoretic karyotyping

Candida krusei is receiving increasing attention as an important human pathogen, especially in compromised patients, who frequently manifest with multiepisodes of candidosis. As there is scant information on the genetic diversity of this pathogen the present study was undertaken to establish its genetic profiles using three different typing methods: PFGE (pulsed‐field gel electrophoresis), RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA). When 11 oral isolates of C. krusei were molecular typed by PFGE, 3 to 5 chromosomes with sizes ranging from 1000 kb to 3000 kb per isolate were revealed. All isolates produced a single bright band at approximately 1,100 kb and two to three bands between 2,500 kb and 3,000 kb, demonstrating 5 different karyotypes. RFLP with Hinfl yielded 9 different genotypes, while DNA fingerprinting by RAPD with 3 primers (RSD6 (5′GCGATCCCCA3′), RSD7 (5′AGTGAATTCG CGGTGA‐GATGCC3′) and RSD12 (5′GCATATCAATAAGC GCAGGAAAAG 3′)), resulted in 8, 3 and 11 different genotypes, respectively. This study provides evidence hitherto unavailable on the genetic polymorphism of C krusei isolates colonizing the oral niche under different clinical conditions. Such genotypic polymorphism should help strain delineation in epidemiologic surveillance of either nosocomial or community outbreaks of C. krusei infections.

Phenotypic diversity of oral C. albicans isolated on single and sequential visits in an HIV-infected Chinese cohort.

HIV‐infected individuals maintain multiple oral C. albicans strains over time that are thought to undergo microevolution in terms of both phenotypic and genotypic features. To study this phenomenon, a 12‐month prospective study was conducted in a cohort of 16 HIV‐infected ethnic Chinese individuals with (A) and without (B) symptoms of oropharyngeal candidiasis to evaluate the phenotype distribution among oral C. albicans isolates during disease progression. Oral rinse samples were obtained and up to five C. albicans colony‐forming units were selected per each visit, during the one year period of multiple visits. The isolates were phenotyped using two commercially available biotyping kits, the API 20C system, API ZYM system, and a plate test for resistance to boric acid. A total of 261 C. albicans strains in group A were differentiated into 67 biotypes, while 42 biotypes were seen amongst the 182 isolates from group B. The major biotypes in the two groups were similar and were in decreasing order of prevalence J1R, J1S, J6S, J6R, J2S, K1S, J10R, K1R, and K6R; 48 different biotypes were seen in group A and 24 in group B, with some uniquely represented in each group, leading to a significant association between the prevalence of the biotypes J1S and J2S and symptomatic candidiasis (p<0.05). Taken together this study illustrates the wide phenotypic spectrum of oral C. albicans associated with HIV‐infection.

A putative nuclear growth factor-like globular nematode specific protein.

Expressed sequence tags (ESTs) are an effective approach for discovery of novel genes. In the current study, approximately 250 ESTs of the cattle parasitic nematode Setaria digitata were examined and a cDNA clone identified whose coding sequence could not be functionally annotated by searching over publicly available genome, protein, EST and STS databases. Here, we report the extensive characterization of this ORF (UP) and its homologues using a bioinformatic approach. Uncharacterized protein (SDUP) of S. digitata consists of 204 amino acids with a predicted molecular weight and isoelectric point of 22.8KDa and 9.94, respectively. A search carried out using SDUP over nucleotide, EST and protein databases at NCBI, NEMBASE3 and Parasite Genome Database (PGD) identified homologous counterparts from the human parasitic nematodes Wuchereria bancrofti (WB), Brugia malayi (BM), Onchocerca volvulus (OV), the mouse filarial worm Litomosoides sigmodontis (LS), swine parasitic nematodes Ascaris suum (AS) and diverged counterparts from the plant parasitic nematode Meloidogyne hapla (MH) and free living nematodes Caenorhabditis elegans (CE) and Caenorhabditis briggsae (CB). Phylogenetic analyses revealed the UPs to be undergoing divergent evolution. A search of the ESTs at PGD showed that UP is expressed in all the stages of BM. Secondary structure analyses of multiply-aligned sequences of homologues using Jpred server indicated UPs to be rich in beta-pleated structures. TMMHH server and beta barrel finder programme indicated, UPs to be neither transmembrane or beta barrels proteins but are likely to be globular proteins. Further, the Motif discovery tool of MEME identified three novel potential motifs for UPS, of which only two are present in CE, CB & MH. Analyses of UPs using Signal IP, TargetP, Psort servers predicted this group of proteins to be devoid of signal peptide cleavage sites, are not mitochondrial targeting peptides but appear to be localized to the nucleus, respectively. Further analyses of the UPs using ScanProsite server for phosphorylation revealed potential sites for cAMP and cGMPdependent protein kinase, Protein kinase C and Casein kinase II. Putative functional analysis using ProtFun 2.1 Server indicated UPs to be nonenzymatic, growth factor like protein. Finally, collating all the information derived from bioinformatic analyses, we conclude that the UPs of nematodes are most likely to be expressed at all stages in the life cycle, localized to the nucleus, regulated by phosphorylation, rich in betapleated strands and are growth factor like nematode specific proteins.