Cloning and characterisation of a putative ST2L homologue from Atlantic salmon (Salmo salar)

The ST2L receptor is a member of the interleukin-1 (IL-1) receptor family and has previously been cloned from human, mouse, rat and chicken. This orphan receptor has no known physiological role but has been implicated in T helper cell type 2 effector function. We describe in this report the cloning and characterisation of a cDNA encoding a homologue of ST2L in Atlantic salmon (Salmo salar). The salmon ST2L cDNA is 2364bp in length and has an open reading frame encoding a polypeptide of 582 amino acids. Similar to other members of the IL-1 receptor (IL-1R) family, the predicted protein has a potential signal peptide, extracellular immunoglobulin-like domains, a short transmembrane region and a characteristic cytoplasmic Toll-IL-1R domain. The predicted protein shows 33% identity and 44% similarity to the chicken ST2L homologue. Phylogenetic analyses cluster the putative salmon ST2L with the chicken and the mammalian ST2L homologues, away from the other members of the IL-1R family. Salmon ST2L is constitutively expressed in brain, white and red blood cells, head kidney, liver, gills and muscle, with highest level of expression in spleen. In vivo stimulation of salmon with lipopolysaccaride does not appear to have a significant effect on expression of the ST2L homologue.     

Exposure of Candida to p-anisaldehyde inhibits its growth and ergosterol biosynthesis

p-Anisaldehyde (4-methoxybenzaldehyde), an extract from Pimpinella anisum seeds, is a very common digestive herb of north India. Antifungal activity of p-anisaldehyde was investigated on 10 fluconazole-resistant and 5 fluconazole-sensitive Candida strains. Minimum inhibitory concentrations (MIC(90)) ranged from 250 μg/ml to 600 μg/ml for both sensitive and resistant strains. Ergosterol content was drastically reduced by p-anisaldehyde-62% in sensitive and 66% in resistant strains-but did not corelate well with MIC(90) values. It appears that p-anisaldehyde exerts its antifungal effect by decreasing NADPH routed through up-regulation of putative aryl-alcohol dehydrogenases. Cellular toxicity of p-anisaldehyde against H9c2 rat cardiac myoblasts was less than 20% at the highest MIC value. These findings encourage further development of p-anisaldehyde.     

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion)

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non- Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site- 2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba- like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.      

Transport genes and chemotaxis in Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is a Gram-negative, sea gull-shaped rod associated with community-acquired gastroenteritis. The bacterium has been found in diverse freshwater environments including fish, frogs and drinking water reservoirs. Using the complete genome sequence data of L. hongkongensis, we performed a comprehensive analysis of putative transport-related genes and genes related to chemotaxis, motility and quorum sensing, which may help the bacterium adapt to the changing environments and combat harmful substances.

Results

A genome-wide analysis using Transport Classification Database TCDB, similarity and keyword searches revealed the presence of a large diversity of transporters (n = 457) and genes related to chemotaxis (n = 52) and flagellar biosynthesis (n = 40) in the L. hongkongensis genome. The transporters included those from all seven major transporter categories, which may allow the uptake of essential nutrients or ions, and extrusion of metabolic end products and hazardous substances. L. hongkongensis is unique among closely related members of Neisseriaceae family in possessing higher number of proteins related to transport of ammonium, urea and dicarboxylate, which may reflect the importance of nitrogen and dicarboxylate metabolism in this assacharolytic bacterium. Structural modeling of two C⁴-dicarboxylate transporters showed that they possessed similar structures to the determined structures of other DctP-TRAP transporters, with one having an unusual disulfide bond. Diverse mechanisms for iron transport, including hemin transporters for iron acquisition from host proteins, were also identified. In addition to the chemotaxis and flagella-related genes, the L. hongkongensis genome also contained two copies of qseB/qseC homologues of the AI-3 quorum sensing system.

Conclusions

The large number of diverse transporters and genes involved in chemotaxis, motility and quorum sensing suggested that the bacterium may utilize a complex system to adapt to different environments. Structural modeling will provide useful insights on the transporters in L. hongkongensis.     

Cataract Surgery Visual Outcomes and Associated Risk Factors in Secondary Level Eye Care Centers of L V Prasad Eye Institute,India

Purpose

To evaluate cataract surgery visual outcomes and associated risk factors in rural secondary level eye care centers of L V Prasad Eye Institute (LVPEI), India.

Methods

The Eye Health pyramid of LVPEI has a network of rural secondary care centres (SCs) and attached vision centres (VCs) that provide high quality comprehensive eye care with permanent infrastructure to the most disadvantaged sections of society. The most common procedure performed at SCs is cataract surgery. We audited the outcome of a random sample of 2,049 cataract surgeries done from October 2009-March 2010 at eight rural SCs. All patients received a comprehensive ophthalmic examination, both before and after surgery. The World Health Organization recommended cataract surgical record was used for data entry. Visual outcomes were measured at discharge, 1–3 weeks and 4–11 weeks follow up visits. Poor outcome was defined as best corrected visual acuity <6/18.

Results

Mean age was 61.8 years (SD: 8.9 years) and 1,133 (55.3%) surgeries were performed on female patients. Pre-existing ocular co-morbidity was present in 165 patients (8.1%). The most common procedure was small incision cataract surgery (SICS) with intraocular lens (IOL) implantation (91.8%). Intraoperative complications were seen in 29 eyes (1.4%). At the 4–11 weeks follow-up visit, based on presenting visual acuity (PVA), 61.8% had a good outcome and based on best-corrected visual acuity (BCVA), 91.7% had a good outcome. Based on PVA and BCVA, those with less than 6/60 were only 2.9% and 1.6% respectively. Using multivariable analysis, poor visual outcomes were significantly higher in patients aged ≥70 (OR 4.63; 95% CI 1.61, 13.30), in females (OR 1.58; 95% CI 1.04, 2.41), those with preoperative comorbidities (odds ratio 4.68; 95% CI 2.90, 7.57), with intraoperative complications (OR 8.01; 95% CI 2.91, 22.04), eyes that underwent no IOL or anterior chamber-IOL (OR 12.63; 95% CI 2.65, 60.25) and those undergoing extracapsular cataract extraction (OR 9.39; 95% CI 1.18, 74.78).

Conclusions

This study demonstrates that quality cataract surgeries can be achieved at rural SCs. The concept of the LVPEI SCs can be applied to other developing countries, allowing rural patients to attain better vision through cataract surgery. Despite improvements in quality of cataract surgery, gender discrimination in terms of outcome continues to be an issue and needs further investigation.     

Medaka fish exhibits longevity gender gap,a natural drop in estrogen and telomere shortening during aging: a unique model for studying sex-dependent longevity

Introduction

Females having a longer telomere and lifespan than males have been documented in many animals. Such linkage however has never been reported in fish. Progressive shortening of telomere length is an important aging mechanism. Mounting in vitro evidence has shown that telomere shortening beyond a critical length triggered replicative senescence or cell death. Estrogen has been postulated as a key factor contributing to maintenance of telomere and sex-dependent longevity in animals. This postulation remains unproven due to the lack of a suitable animal system for testing. Here, we introduce a teleost model, the Japanese medaka Oryzias latipes, which shows promise for research into the molecular mechanism(s) controlling sex difference in aging.

Results

Using the medaka, we demonstrate for the first time in teleost that (i) sex differences (female?>?male) in telomere length and longevity also exist in fish, and (ii) a natural, ‘menopause’-like decline of plasma estrogen was evident in females during aging. Estrogen levels significantly correlated with telomerase activity as well as telomere length in female organs (not in males), suggesting estrogen could modulate telomere length via telomerase activation in a sex -specific manner. A hypothetical in vivo ‘critical’ terminal restriction fragment (TRF, representing telomere) length of approximately 4 kb was deduced in medaka liver for prediction of organismal mortality, which is highly comparable with that for human cells. An age conversion model was also established to enable age translation between medaka (in months) and human (in years). These novel tools are useful for future research on comparative biology of aging using medaka.

Conclusion

The striking similarity in estrogen profile between aging female O. latipes and women enables studying the influence of “postmenopausal” decline of estrogen on telomere and longevity without the need of invasive ovariectomy. Medaka fish is advantageous for studying the direct effect of increased estrogen on telomere length and longevity without the breast cancer complications reported in rodents. The findings strongly support the notion that O. latipes is a unique non-mammalian model for validation of estrogenic influence on telomere and longevity in vertebrates. This laboratory model fish is of potential significance for deciphering the ostensibly conserved mechanism(s) of sex-associated longevity in vertebrates.

     

Eucalyptus microsatellites mined in silico: survey and evaluation

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.     

Development of SCAR marker in Casuarina equisetifolia for species authentication

Casuarina equisetifolia is one of the most extensively introduced tree species outside its natural range and is a true multipurpose species, providing a range of services and products for industrial and local end users. Natural hybrids of C. equisetifolia have been reported and a putative hybrid of C. junghuhniana and C. equisetifolia is commercially cultivated in Thailand and India. In Taiwan, studies have also revealed that most plants of Casuarina are the result of introgressive hybridization involving C. equisetifolia, C. glauca, and C. cunninghamiana causing difficulty in species identification. The present study was aimed at developing DNA markers for the identification of C. equisetifolia. Seven ISSR primers were amplified in 120 individuals belonging to three species of Casuarina (C. equisetifolia, C. glauca and C. junghuhniana) and two Allocasuarina species (A. littoralis and A. huegeliana). One species-specific amplicon at 650 bp amplified in all individuals of C. equisetifolia was cloned, sequenced and primer pairs were developed and designated as IFGTBCE01. The sequence characterized amplified region (SCAR) marker was multiplexed with internal SSR primer pair (positive control) and amplified in ten randomly selected individuals of C. equisetifolia, C. junghuhniana, C. glauca, A. littoralis and A. huegeliana. The species-specific band amplified only in C. equisetifolia at 500 bp. Subsequently, the SCAR marker was validated in 30 individuals collected from 10 provenances belonging to 9 countries and in 10 locally selected clones. The marker was validated in the natural male hybrid of C. equisetifolia × C. junghuhniana which is widely planted in India for fast growth, wide adaptability and good form. Hence, this marker can be used for accurate and rapid identification of the species during certification and determination of putative hybrids in breeding programs.     

Transgenic Tobacco and Apple Plants Expressing Biotin-binding Proteins are Resistant to two Cosmopolitan Insect Pests,Potato Tuber Moth and Lightbrown Apple Moth,Respectively

Tobacco (Nicotiana tabacum cv. Samsun) and apple (Malus x domestica cv. Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation. ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple. Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam. Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam. Tortricidae). More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation. Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days. Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.