Entamoeba invadens: Dynamics of DNA synthesis during differentiation from trophozoite to cyst

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0–8 h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8–24 h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24–72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

Biochemical composition of the marine conditioning film: implications for bacterial adhesion

The conditioning film formed on glass panels was analysed for total carbohydrates (CFCHO), total proteins (CFP) and total uronic acids (CFURA). The influence of these compounds on the adhesion of three marine bacterial cultures, Pseudomonas sp. CE-2, Pseudomonas sp. CE-10 and Bacillus sp. SS-10 was also evaluated. One-way analysis of variance suggested a significant increase in the attachment of all three cultures to conditioned glass panels. Moreover, CE-2 (r = 0.874) and CE-10 (r = 0.879) showed a significant positive correlation with CFCHO. Conversely, SS-10 (r = -0.69) showed a significant negative correlation with CFCHO. Backward multiple linear regression analysis indicated that CFCHO were the most predictive component of the conditioning film in explaining bacterial adhesion to the conditioned glass panels.     

Characterization of antibody-binding sites on proteins: development of a knowledgebase and its applications in improving epitope prediction

Immunoinformatics provides tools for reverse vaccinology and encompasses development of knowledge bases and algorithms for prediction of epitopes. AgAbDb, a database archiving molecular interactions of antigen-antibody co-crystal structures, has been developed (http://202.41.70.51:8080/agabdb2/). Analyses of antibody-binding sites on proteins helped to fine-tune the parameters for prediction of sequential and conformational B-cell epitopes.     

Species- and strain-specific probes derived from repetitive DNA for distinguishing Entamoeba histolytica and Entamoeba dispar

Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable species that are found in the human gut. Of the two, E. histolytica is considered to be pathogenic while E. dispar is nonpathogenic. To generate molecular probes to detect and distinguish between the two species, we utilized repeat sequences present in Entamoeba genome. We have developed probes and primers from rDNA episomes, and unidentified Entamoeba EST1 repeat for this purpose, and used them for dot blot hybridization and PCR amplification. To investigate the possible existence of invasive and noninvasive strains of E. histolytica, the ability to differentiate individual isolates is necessary. For this purpose, we have utilized a modification of the AFLP procedure called 'Transposon display,' which generates and displays large number of genomic bands associated with a transposon. We have used the abundant retrotransposon, EhSINE1, for this purpose,and demonstrated its potential as a marker to study strain variation in E. histolytica. This technique could suitably be employed in carrying out significant molecular epidemiological studies and large-scale typing of this parasite.     

Hyalohyphomycosis caused byPaecilomyces variotii: a case report,animal pathogenicity and ‘in vitro’ sensitivity

A case of cutaneous infection in a 25-year-old male caused byPaecilomyces variotii is described. Animal pathogenicity studies with normal and cortisone-treated mice revealed the predeliction ofP. variotii for skin and liver in both normal and cortisone-treated mice and for lungs and heart only in immunosuppressed mice. 5-fluorocytosine gave the best MIC value forP. variotii in vitro. This report documents for the first time thatP. variotii causes cutaneous infection.     

Cutaneous phaeohyphomycosis caused byPhialophora richardsiae and the effect of topical clotrimazole in its treatment

A 15 year-old girl and her 50-year-old mother from Jabalpur, India, were found to have phaeohyphomycotic skin lesions due toPhialophora richardsiae. Among the antimycotics testedin vitro, clotrimazole was found to be most active. Topical application of clotrimazole cream cured the lesions completely.     

Observations on biofilm bacteria isolated from aluminium panels immersed in estuarine waters

Microfouling biomass generally increased on aluminium panels immersed in the surface waters of a tropical estuary over a period of 30 d. A hundred bacterial colonies were randomly isolated, purified and morphologically, physiologically and biochemically characterised. There was an obvious difference in colony morphology during the period of immersion. Most of the colonies were motile, catalase and oxidase positive Gram negative rods. Pseudomonas was the most abundant initial colonizer during the first 24 h following immersion, and Flavobacterium and Alkaligenes were common throughout the immersion period. Representatives of the Enterobacteriaceae were present only during the first 7 d after immersion. Vibrio was the most predominant organism and its abundance generally increased over the period of immersion. Alkaligenes exhibited a decreasing trend over the 22 d immersion period followed by an increase in abundance at day 30 after immersion. However, no particular trend was observed for Flavobacteria.

The isolates were screened for their cell surface hydrophobicity using the BATH method. Most of the isolates were hydrophilic. However, as the period of immersion increased there was a relative increase in cell surface hydrophobicity of the isolates.     

Role of 𝛃 1-4 linked polymers in the biofilm structure of marine Pseudomonas sp. CE-2 on 304 stainless steel coupons

Pseudomonas sp CE-2 cells attach and form biofilms on 304-stainless steel (SS) coupons. A series of experiments were carried out in order to understand the role of exopolysaccharides (EPS) in the formation and maintenance of CE-2 biofilms on SS coupons. The biofilm density and EPS concentration increased over the period of incubation and the highest values for both were recorded after 72 h. Calcofluor and the lectin concanavalin A (Con A) showed a positive interaction with 72-h old biofilms, indicating the presence of β 1-4 linked polymers, and α-d-glucose and α-d-mannose in the biofilm matrix of CE-2. When the CE-2 cells were grown in the presence of calcofluor (200 μg ml^?1), biofilm formation was significantly reduced (～85%). Conversely, the lectins Con A or WGA did not influence the CE-2 biofilms on the SS coupons. Furthermore, treatment with cellulase, an enzyme specific for the degradation of β 1-4 linked polymers, removed substantial amounts of CE-2 biofilm from SS coupons. These results strongly suggest the involvement of β 1-4 linked polymers in the formation and maintenance of Pseudomonas sp. CE-2 biofilms on SS coupons.