First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium, Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck

Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck is a common mat-forming benthic cyanobacterium from freshwater habitats. We found that two populations from southeast Queensland (Australia) produce the potent cyanotoxin cylindrospermopsin (CYN) and its analogue, deoxy-cylindrospermopsin (deoxy-CYN). The highest concentrations in environmental samples were 20 and 550 μg g⁻¹ dry weight for CYN and deoxy-CYN, respectively. A sub-sample maintained in culture for over 16 months yielded concentrations of 33 and 308 μg g⁻¹ dry weight for CYN and deoxy-CYN, respectively. The concentration of deoxy-CYN in L. wollei was 10–300 times higher than CYN, suggesting that, unlike many other CYN-producing cyanobacteria, the primary compound produced by L. wollei is deoxy-CYN. The production of CYN and deoxy-CYN by L. wollei represents a potential human health risk and an additional source of these toxins in freshwaters. This is the first report of the production of CYN and deoxy-CYN by L. wollei or any species of the Oscillatoriales.     

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(I) per CopZ and two copper(I) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(I)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(I)2CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(I)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper, from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(I) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange; a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.     

Effects of water-level fluctuation and invasive water plants on pheasant-tailed jacana (Hydrophasianus chirugus) at the Annaiwilundawa Ramsar site of northwestern Sri Lanka

The effects of alien invasive water plants (Eichhornia crassipes, Salvinia molesta, Imperata cylindrical and Mikania micrantha) on the pheasant-tailed jacana (Hydrophasianus chirugus) was studied in three reservoirs from June 2004 to May 2005 at the Annaiwilundawa Ramsar site of northwestern Sri Lanka. Jacanas were observed in significantly fewer numbers among invasive plants and open water and in significantly higher numbers among grass and floating leaved plants than predicted from the proportions of the reservoirs covered by these habitat types (Annaiwilundawa χ² = 64.35, P < 0.0001; Suruwila χ² = 45.66, P < 0.0001; Maiyawa χ² = 33.35, P < 0.0001). Feeding was the major activity of the pheasant-tailed jacana during the study period [analysis of variance (ANOVA) F = 79.58, P < 0.0001 in the morning and F = 85.53, P < 0.0001 in the evening]. Jacanas preferred to forage in grass and floating leaved vegetation compared to invasive plants and open water (ANOVA F = 168.63, P < 0.001). The jacana population was significantly positively correlated with water level in all three reservoirs. (Spearman’s rank correlation, Annaiwilundawa z = 2.035, P < 0.05; Suruwila z = 2.19, P < 0.05; Maiyawa z = 2.563, P < 0.05). The findings of this study revealed that the most critical factor for the occupation of the reservoirs by jacanas was the water level and that the spread of the invasive plants in these reservoirs were detrimental to jacanas, since these plants deprived jacanas of useful foraging habitats.     

Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes

We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.     

Histopathological diagnosis of myocarditis in a dengue outbreak in Sri Lanka, 2009

Background

In 2009, an outbreak of dengue caused high fatality in Sri Lanka. We conducted 5 autopsies of clinically suspected myocarditis cases at the General Hospital, Peradeniya to describe the histopathology of the heart and other organs.

Methods

The diagnosis of dengue was confirmed with specific IgM and IgG ELISA, HAI and RT-PCR techniques. The histology was done in tissue sections stained with hematoxylin and eosin.

Results

Of the 319 cases of dengue fever, 166(52%) had severe infection. Of them, 149 patients (90%) had secondary dengue infection and in 5 patients, DEN-1 was identified as the causative serotype. The clinical diagnosis of myocarditis was considered in 45(27%) patients. The autopsies were done in 5 patients who succumbed to shock (3 females and 2 males) aged 13- 31 years. All had pleural effusions, ascites, bleeding patches in tissue planes and histological evidence of myocarditis. The main histological findings of the heart were interstitial oedema with inflammatory cell infiltration and necrosis of myocardial fibers. One patient had pericarditis. The concurrent pulmonary abnormalities were septal congestion, pulmonary haemorrhage and diffuse alveolar damage; one case showed massive necrosis of liver.

Conclusions

The histology supports occurrence of myocarditis in dengue infection.

     

Near on-line detection of enteric bacteria using lux recombinant bacteriophage

The potential for a revolution in microbial testing can be perceived with the near on-line detection of indicator microorganisms. By definition, these are microorganisms present in significant numbers within a food which, while not pathogenic, can be related through increasing count to the increased probability of pathogen contamination. We have used recombinant lux+ bacteriophage to detect enteric indicator bacteria without recovery or enrichment in 50 min, provided that they are present at levels greater than 10(4) g-1 or cm-2. After a 4-h enrichment, samples having enteric counts of 10 g-1 or cm-2 can be distinguished from background.     

Emergence of Epidemic Dengue-1 Virus in the Southern Province of Sri Lanka

BackgroundDengue is a frequent cause of acute febrile illness with an expanding global distribution. Since the 1960s, dengue in Sri Lanka has been documented primarily along the heavily urbanized western coast with periodic shifting of serotypes. Outbreaks from 2005–2008 were attributed to a new clade of DENV-3 and more recently to a newly introduced genotype of DENV-1. In 2007, we conducted etiologic surveillance of acute febrile illness in the Southern Province and confirmed dengue in only 6.3% of febrile patients, with no cases of DENV-1 identified. To re-evaluate the importance of dengue as an etiology of acute febrile illness in this region, we renewed fever surveillance in the Southern Province to newly identify and characterize dengue.Conclusions/SignificanceWe describe the first epidemic of DENV-1 in the Southern Province of Sri Lanka in a population known to be susceptible to this serotype because of prior study. Dengue accounted for 40% of acute febrile illnesses in the current study. The emergence of DENV-1 as the foremost serotype in this densely populated but agrarian population highlights the changing epidemiology of dengue and the need for continued surveillance and prevention.     

Have mangrove restoration projects worked? An in‐depth study in Sri Lanka

This study investigated the effectiveness of mangrove planting initiatives in Sri Lanka. All the lagoons and estuaries in Sri Lanka were included in the study. We documented all agencies and locations, involved in mangrove planting efforts, along with the major drivers of these planting initiatives, their extents, and the possible causes of the success or failure of planting. An adapted three‐step framework and a field survey consisting of vegetation and soil surveys and questionnaires were used to evaluate the objectives. We found that about 1,000–1,200 ha of mangroves, representing 23 project sites with 67 planting efforts, have been under restoration with the participation of several governmental and nongovernmental organizations. However, about 200–220 ha showed successful mangrove restoration. Nine out of 23 project sites (i.e. 36/67 planting efforts) showed no surviving plants. The level of survival of the restoration project sites ranged from 0 to 78% and only three sites, that is, Kalpitiya, Pambala, and Negombo, showed a level of survival higher than 50%. Survival rates were significantly correlated with post‐care. Planting mangrove seedlings at the incorrect topography often entails inappropriate soil conditions for mangroves. Survival rates showed significant correlations with a range of soil parameters except soil pH. Disturbance and stress caused by cattle trampling, browsing, algal accumulation, and insect attacks, factors that may themselves relate to choosing sites with inappropriate topography and hydrology, were common to most sites. The findings are a stark illustration of the frequent mismatch between the purported aims of restoration initiatives and the realities on the ground.     

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl⁻-HCO₃⁻ exchange and Cl⁻ currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl⁻ currents with only a modest affect on nCl⁻-HCO₃⁻ exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.Slc26 genes and proteins have attracted the attention of physiologists and geneticists. Why? Slc26a1 (Sat-1) was characterized as a Na⁺-independent SO₄²⁻ transporter (1). Given the transport characteristics of the founding member of the gene family, Slc26 proteins were assumed to be sulfate transporters. Disease phenotypes, clone characterization, and family additions demonstrate that the Slc26 proteins are anion transporters or channels (2–4). These proteins have varied tissue expression patterns. At one extreme, Slc26a5 in mammals is found in the hair cells of the inner ear (5), whereas Slc26a2 (DTDST) is virtually ubiquitous in epithelial tissues (2).Several Slc26 proteins are found in the epithelia of the lung, intestine, stomach, pancreas, and kidney, usually in apical membranes. Interestingly these are also tissues and membranes in which the cystic fibrosis transmembrane conductance regulator (CFTR)⁵ has been found functionally or by immunohistochemistry. Ko and co-workers (6–8) examined the distribution of Slc26a3 and Slc26a6 in HCO₃⁻ secretory epithelia, and asked if an interaction might occur between these Slc26 proteins and CFTR. In particular, these studies indicate that in expression systems, there is a reciprocal-stimulatory interaction of the STAS (sulfate transporter anti-sigma) domains of Slc26a3 and Slc26a6 with the regulatory region (R-region) of CFTR. These investigators hypothesized that this stimulatory interaction could account for the differences in pancreatic insufficiency and sufficiency observed in cystic fibrosis patients. Nevertheless, knock-out Slc26a6 mouse studies reveal more complicated cell and tissue physiology (see “Discussion”).Slc26a9 has been reported to be a Cl⁻-HCO₃⁻ exchanger (9, 10) or a large Cl⁻ conductance (3, 11, 12). Loriol and co-workers (12) indicated that SLC26A9 has a Cl⁻ conductance that may be stimulated by HCO₃⁻. Two other groups have indicated that the Cl⁻ conductance is not affected by the presence of HCO₃⁻ (10, 11). We have recently demonstrated that Slc26a9 functions as both an electrogenic nCl⁻-HCO₃⁻ exchanger and a Cl⁻ channel (10). Dorwart and colleagues (11) found that WNK kinases inhibited the SLC26A9 Cl⁻ conductance but that this effect was independent of kinase activity. One group has a preliminary report indicating that WNK3 decreased Cl⁻ uptake, whereas WNK4 increased Cl⁻ uptake via Slc26a9 expressed in Xenopus oocytes (13).Slc26a9 and CFTR are also co-expressed in several tissues. Slc26a9 protein has been localized to epithelia of the stomach and lung (9, 10, 14), although mRNA is also detectable in brain, heart, kidney, small intestine, thymus, and ovary (10). The R-region of CFTR was previously shown to increase the activity of Slc26a3 and Slc26a6 by interaction with STAS domains (6, 15, 16). Because Slc26a9 displays several different modes of ion transport, we asked if the R-region of CFTR would also increase the activity of Slc26a9. Our results indicate that the R-region of CFTR does interact with the STAS domain of Slc26a9. However, in the case of Slc26a9 this apparently similar interaction results in inhibition of Slc26a9 ion transport.