The R345W mutation in EFEMP1 is pathogenic and causes AMD-like deposits in mice

Age-related macular degeneration (AMD) is the most common cause of vision loss in developed countries. A defining characteristic of this disorder is the accumulation of material between Bruch's membrane and the retinal pigment epithelium (RPE), first as microscopic basal deposits and later as clinically evident drusen. The pathogenesis of these deposits remains to be defined. Biochemical and genetic studies have suggested that inflammation and complement activation may play roles in AMD. Several lines of evidence also suggest that alterations to the extracellular matrix (ECM) of the RPE and choroid contribute to the development of AMD. The inherited macular degeneration Doyne honeycomb retinal dystrophy/Malattia Leventinese is thought to be caused by an R345W mutation in the EFEMP1 gene (also called fibulin-3). The pathogenicity of this mutation has been questioned because all individuals identified to date with the R345W mutation have shared a common haplotype. We investigated the pathogenicity of this mutation in families with early-onset macular degeneration and by generating Efemp1-R345W knockin mice. Genetic studies show that one of the identified families with the R345W mutation has a novel haplotype. The mutant Efemp1-R345W mice develop deposits of material between Bruch's membrane and the RPE, which resemble basal deposits in patients with AMD. These basal deposits contain Efemp1 and Timp3, an Efemp1 interacting protein. Evidence of complement activation was detected in the RPE and Bruch's membrane of the mutant mice. These results confirm that the R345W mutation in EFEMP1 is pathogenic. Further, they suggest that alterations in the ECM may stimulate complement activation, demonstrating a potential connection between these two etiologic factors in macular degeneration.     

Monoclonal antibodies against recombinant GAPDH of Edwardsiella tarda reveal the conserved nature of the protein

The outer membrane protein, encoded by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, of Edwardsiella tarda is a highly conserved immunogenic protein. The GAPDH was cloned and expressed in Escherichia coli. The purified protein was used to produce mouse monoclonal antibodies (MAbs). Four stable hybridomas producing MAbs (3G12, 4E9, 5A11 and 9G1) against rGAPDH were obtained. The heavy chains of antibodies produced by the hybridomas were of the isotypes IgG1 and IgM. Cross reactivity of MAbs (3G12 and 9G1) was observed with GAPDH of Aeromonas hydrophila and Micrococcus luteus. MAbs 3G12 and 4E9 reacted with Vibrio cholerae, Salmonella enterica and Penaeus monodon tissues but not with vertebrate GAPDH. None of the MAbs reacted with Staphylococcus aureus. The results indicate that the level of conservation of GAPDH is high among evolutionarily close species. The MAbs developed will be a useful tool to study the evolutionarily conserved and functionally diverse GAPDH.     

Important Contribution of the Novel Locus comEB to Extracellular DNA-Dependent Staphylococcus lugdunensis Biofilm Formation

The coagulase-negative species Staphylococcus lugdunensis is an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability of S. lugdunensis to form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation in S. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917 insertion within the competence gene comEB. Site-directed mutagenesis and subsequent complementation with a functional copy of comEB verified the importance of comEB in biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in the comEB mutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of the comEB mutation on biofilm integrity. The comEB mutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in the comEB mutant biofilms did not result from elevated levels or activity of the S. lugdunensis thermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence gene comEB in staphylococcal biofilm formation. Our findings indicate that comEB stimulates biofilm formation via a lysis-independent mechanism of DNA release.     

Development of radiopaque,biocompatible, antimicrobial,micro-particle fillers for micro-CT imaging of simulated periodontal pockets

Objectives

Approximately 10⁹ bacteria can be harbored within periodontal pockets (PP) along with inflammatory byproducts implicated in the pathophysiology of systemic diseases linked to periodontitis (PD). Calculation of this inflammatory burden has involved estimation of total pocket surface area using analog data from conventional periodontal probing which is unable to determine the three-dimensional (3-D) nature of PP. The goals of this study are to determine the radiopacity, biocompatibility, and antimicrobial activity of transient micro-particle fillers in vitro and demonstrate their capability for 3-D imaging of artificial PP (U.S. Patent publication number: 9814791 B2).

Longitudinal studies in South Indian villages on Japanese encephalitis virus infection in mosquitoes and seroconversion in goats

Japanese encephalitis (JE) is endemic in Cuddalore district, Tamil Nadu, where Culex tritaeniorhynchus Giles was the major vector. We screened 45 100 adult female Cx. tritaeniorhynchus (902 pools) by enzyme-linked immunosorbent assay and isolated and confirmed JE virus (JEV) by using an insect bioassay system. We had 69 isolates of which 62 (90%) were identified as JEV. The average vector abundance per man hour for Cx. tritaeniorhynchus was 324.5 per month for the period June 1998-May 2000. The average minimum infection rate (MIR) per month in Cx. tritaeniorhynchus was 1.4 (range 0.0-5.6). Every year, a new batch of goats, 20 in the first year and 31 in the second year, born during the non-JE transmission period (January-June), aged <6 months and negative for haemagglutination inhibition (HI) antibodies were procured and placed in the villages as sentinels. Fortnightly, blood specimens were collected from these goats and tested for JE antibodies by HI test. Seroconversions (SCs) were recorded in 14 goats (70%) in the first year and 23 goats (74%) in the second year. JE HI antibody titres in goats were low (1:10-1:80) and these levels declined to undetectable levels in about 4 weeks following SCs. The time sequence of events indicated that four of five peaks of MIR in mosquitoes were followed 1-3 months later by peaks in the proportion of seroconverted goats. We suggest the screening of goats and cattle as a more feasible tool to stratify areas according to JE infection risk to the human population through the regular health system rather than screening mosquitoes using monoclonal antibodies, which is possible only in specialized laboratories.     

Na(+)-dependent fluid absorption in intact perfused rat colonic crypts

BACKGROUND & AIMS: The traditional paradigm of fluid movement in the mammalian colon is that fluid absorption and secretion are present in surface and crypt cells, respectively. We have recently demonstrated Na(+)-dependent fluid absorption in isolated crypts that are devoid of neurohumoral stimulation. We now explore the mechanism of Na(+)-dependent fluid absorption in isolated rat colonic crypts. METHODS: Net fluid absorption was determined using microperfusion techniques and methoxy[(3)H]inulin with ion substitutions and transport inhibitors. RESULTS: Net fluid absorption was reduced but not abolished by substitution of either N-methyl-D-glucamine- Cl(-) or tetramethylammonium for Na(+) and by lumen addition of 5-ethylisopropyl amiloride, an amiloride analogue that selectively inhibits Na(+)-H(+) exchange. Net fluid absorption was also dependent on lumen Cl(-) because removal of lumen Cl(-) significantly (P < 0.001) reduced net fluid absorption. DIDS at 100 micromol/L, a concentration at which DIDS is an anion exchange inhibitor, minimally reduced net fluid absorption (P < 0.05). In contrast, either 500 micromol/L DIDS, a concentration at which DIDS is known to act as a Cl(-) channel blocker, or 10 micromol/L NPPB, a Cl(-) channel blocker, both substantially inhibited net fluid absorption (P < 0.001). Finally, both the removal of bath Cl(-) and addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl(-) secretion, resulted in a significant increase in net fluid absorption. CONCLUSIONS: (1) Net Na(+)-dependent net fluid absorption in the isolated colonic crypt represents both a larger Na(+)-dependent absorptive process and a smaller secretory process; and (2) the absorptive process consists of a Na(+)-dependent, HCO(3)(-)-independent process and a Na(+)-independent, Cl(-)-dependent, HCO(3)(-)-dependent process. Fluid movement in situ represents these transport processes plus fluid secretion induced by neurohumoral stimulation.     

HCO(3)(-) secretion in the rat colonic crypt is closely linked to Cl(-) secretion

BACKGROUND & AIMS: The mechanism of colonic HCO(3)(-) secretion has not been established largely because of a lack of experimental methods for its detailed study. The present studies were designed to establish whether the isolated, perfused crypt of the rat distal colon is an excellent model to study HCO(3)(-) movement and the mechanism of colonic HCO(3)(-) secretion. METHODS: HCO(3)(-) secretion was determined in isolated, microperfused crypts by measuring [HCO(3)(-)] by microcalorimetry on nanoliter samples. RESULTS: Net HCO(3)(-) absorption was observed during lumen and bath perfusion with an HCO(3)(-)-Ringer solution. Vasoactive intestinal polypeptide (60 nmol/L), acetylcholine (100 nmol/L), or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mmol/L) induced active HCO(3)(-) secretion that required bath but not lumen HCO(3)(-)/CO(2). DBcAMP-stimulated HCO(3)(-) secretion was not affected by acetazolamide, an inhibitor of carbonic anhydrase. Removal of lumen Cl(-) did not alter DBcAMP-stimulated HCO(3)(-) secretion but reduced fluid secretion. DBcAMP-stimulated HCO(3)(-) secretion was closely linked to active Cl(-) secretion because HCO(3)(-) secretion was substantially reduced by removal of bath Cl(-), by addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl(-) secretion, and by addition of lumen NPPB, a Cl(-) channel inhibitor. CONCLUSIONS: These studies establish that colonic crypt HCO(3)(-) secretion (1) is not a result of an apical membrane Cl(-)-HCO(3)(-) exchange, (2) is tightly associated with Cl(-) secretion, and (3) primarily occurs via an apical membrane Cl(-) channel.