Investigation of quaternary ammonium silane-coated sand filter for the removal of bacteria and viruses from drinking water.

AIMS: To develop an anti-microbial filter media using an attached quaternary ammonium compound (QAC) and evaluate its performance under conditions relevant to household drinking water treatment in developing countries. METHODS AND RESULTS: Silica sand was coated with dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride via covalent silane chemistry. Filter columns packed with coated media were challenged with micro-organisms under different water quality conditions. The anti-bacterial properties were investigated by visualizing Escherichia coli (E. coli) attachment to coated media under fluorescence microscopy combined with a live/dead stain. A 9-cm columns with a filtration velocity of 18 m h(-1) achieved log(10) removals of 1·7 for E. coli, 1·8 for MS2 coliphage, 1·9 for Poliovirus type 3 and 0·36 for Adenovirus type 2, compared to 0·1-0·3 log(10) removals of E. coli and MS2 by uncoated sand. Removal scaled linearly with column length and decreased with increasing ionic strength, flow velocity, filtration time and humic acid presence. Escherichia coli attached to QAC-coated sand were observed to be membrane-permeable, providing evidence of inactivation. CONCLUSIONS: Filtration with QAC-coated sand provided higher removal of bacteria and viruses than filtration with uncoated sand. However, major limitations included rapid fouling by micro-organisms and natural organic matter and low removal of viruses PRD1 and Adenovirus 2. SIGNIFICANCE AND IMPACT OF THE STUDY: QAC-coated media may be promising for household water treatment. However, more research is needed on long-term performance, options to reduce fouling and inactivation mechanisms.

Feather meal: a previously unrecognized route for reentry into the food supply of multiple pharmaceuticals and personal care products (PPCPs).

Antimicrobials used in poultry production have the potential to bioaccumulate in poultry feathers but available data are scarce. Following poultry slaughter, feathers are converted by rendering into feather meal and sold as fertilizer and animal feed, thereby providing a potential pathway for reentry of drugs into the human food supply. We analyzed feather meal (n = 12 samples) for 59 pharmaceuticals and personal care products (PPCPs) using EPA method 1694 employing liquid chromatography tandem mass spectrometry (LC/MS/MS). All samples tested positive and six classes of antimicrobials were detected, with a range of two to ten antimicrobials per sample. Caffeine and acetaminophen were detected in 10 of 12 samples. A number of PPCPs were determined to be heat labile during laboratory simulation of the rendering process. Growth of wild-type E. coli in MacConkey agar was inhibited by sterilized feather meal (p = 0.01) and by the antimicrobial enrofloxacin (p < 0.0001) at levels found in feather meal. Growth of a drug-resistant E. coli strain was not inhibited by sterilized feather meal or enrofloxacin. This is the first study to detect antimicrobial residues in feather meal. Initial results suggest that more studies are needed to better understand potential risks posed to consumers by drug residues in feather meal.

Arsenic species in poultry feather meal.

Organoarsenical drugs are widely used in the production of broiler chickens in the United States. Feathers from these chickens are processed into a meal product that is used as an animal feed additive and as an organic fertilizer. Research conducted to date suggests that arsenical drugs, specifically roxarsone, used in poultry production result in the accumulation of arsenic in the keratinous material of poultry feathers. The use of feather meal product in the human food system and in other settings may result in human exposures to arsenic. Consequently, the presence and nature of arsenic in twelve samples of feather meal product from six US states and China were examined. Since arsenic toxicity is highly species-dependent, speciation analysis using HPLC/ICPMS was performed to determine the biological relevance of detected arsenic. Arsenic was detected in all samples (44-4100 µg kg(-1)) and speciation analyses revealed that inorganic forms of arsenic dominated, representing 37 - 83% of total arsenic. Roxarsone was not detected in the samples (<20 µg As kg(-1)). Feather meal products represent a previously unrecognized source of arsenic in the food system, and may pose additional risks to humans as a result of its use as an organic fertilizer and when animal waste is managed.

Evaluation of RT-PCR and reverse line blot hybridization for detection and genotyping F+ RNA coliphages from estuarine waters and molluscan shellfish.

AIMS: To evaluate a PCR-based detection and typing method for faecal indicator viruses (F+ RNA coliphages) in water and shellfish, and apply the method for better understanding of the ecology and microbial source tracking potential of these viruses. METHODS AND RESULTS: Water and shellfish samples were collected over 3 years at nine estuaries in the East, West and Gulf Coasts of the USA, providing 1033 F+ RNA coliphage isolates. F+ RNA coliphage genotyping rates by reverse transcriptase-PCR-reverse line blot (RLB) hybridization ranged from 94.7% to 100% among estuaries, and were not significantly different in oysters, clams, mussels or water (P = 0.8427). Twenty samples negative by RLB were nucleotide sequenced for confirmation, and to refine RLB probes. More F+ RNA coliphages were genotyped from colder water than warmer waters, while the water salinity did not affect F+ RNA coliphage levels. CONCLUSIONS: RT-PCR-RLB was a robust method for detecting and genotyping F+ RNA coliphages from diverse coastal areas, which provided new information on the ecology of F+ RNA coliphages. SIGNIFICANCE AND IMPACT OF THE STUDY: This performance-validated F+ RNA coliphage method can be used for faecal indicator monitoring and microbial source tracking, to protect recreational bathers and shellfish consumers from exposure to pathogenic virus and their disease risks.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Calreticulin Is a receptor for nuclear export.

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739-14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes.

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.

Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export.

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

Reconstitution of HIV-1 rev nuclear export: independent requirements for nuclear import and export.

The Rev protein of HIV-1 actively shuttles between nucleus and cytoplasm and mediates the export of unspliced retroviral RNAs. The localization of shuttling proteins such as Rev is controlled by the relative rates of nuclear import and export. To study nuclear export in isolation, we generated cell lines expressing a green fluorescent protein-labeled chimeric protein consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence. Steroid removal switches off import thus allowing direct visualization of the Rev export pathway in living cells. After digitonin permeabilization of these cells, we found that a functional nuclear export sequence (NES), ATP, and fractionated cytosol were sufficient for nuclear export in vitro. Nuclear pore-specific lectins and leptomycin B were potent export inhibitors. Nuclear export was not inhibited by antagonists of calcium metabolism that block nuclear import. These data further suggest that nuclear pores do not functionally close when luminal calcium stores are depleted. The distinct requirements for nuclear import and export argue that these competing processes may be regulated independently. This system should have wide applicability for the analysis of nuclear import and export.

Leishmania amazonensis: the phagocytosis of amastigotes by macrophages.

In the present study, we examine the cell biology of Leishmania amastigote uptake by mammalian cells and compare this process to the phagocytosis of IgG-opsonized erythrocytes. We report that many aspects of amastigote uptake into macrophages resemble classical receptor-mediated phagocytosis. Parasite uptake requires energy expenditure by macrophages but not by parasites. Treating macrophages to prevent either energy metabolism or actin polymerization prevents amastigote uptake. The uptake of amastigotes by macrophages involves the colocalization of f-actin, paxillin, and talin to phagocytic cups that are formed around amastigotes during internalization. Treatment of macrophages with genestein, to inhibit protein phosphorylation, prevents amastigote uptake, indicating that this process, like receptor-mediated phagocytosis, depends on protein tyrosine phosphorylation. However, the amount and the pattern of protein tyrosine phosphorylation observed during amastigote uptake by macrophages is reduced relative to that observed during IgG-erythrocyte phagocytosis. The uptake of viable, but not heat-killed amastigotes, is associated with a decrease in the intensity of several specific macrophage proteins that are phosphorylated on tyrosine residues. In summary, although many features of amastigote uptake by macrophages resemble classical receptor-mediated phagocytosis, differences in macrophage protein phosphorylation during amastigote phagocytosis may contribute to the unique aspects of amastigote uptake and intracellular survival in macrophages.

High-molecular-weight proteins of nontypeable Haemophilus influenzae mediate bacterial adhesion to cellular proteoglycans.

A family of high-molecular-weight (HMW) surface-exposed proteins of nontypeable Haemophilus influenzae (NT H. influenzae) mediated adherence of these organisms to human epithelium. To better understand the molecular basis for this adherence, the role of glycosaminoglycans (GAGs), substances commonly expressed on cell surfaces, was examined. Bacterial adherence to cells with specific deficiencies in GAG biosynthesis was measured. HMW protein-dependent bacterial adherence to normal cells was significantly greater than adherence to cells deficient in sulfated GAGs or to cells deficient in heparan sulfate but overexpressing chondroitin sulfate. Cells expressing undersulfated heparan sulfate exhibited intermediate levels of bacterial adherence. The addition of exogenous dextran sulfate or heparin inhibited over 70% of the adherence of NT H. influenzae to normal cells, whereas hyaluronic acid and chondroitin sulfate tested at the same concentration (100 micrograms/ml) inhibited bacterial adherence by less than 11%. Treatment of cells with heparinase significantly reduced bacterial adherence. Following electrophoretic separation, HMW proteins were shown to bind directly to radiolabeled heparin. These results indicate that HMW protein-dependent adherence of NT H. influenzae is mediated by cellular sulfated GAGs and that heparan sulfate may be the predominant GAG involved in this process. However, the decreased adherence of bacteria to cells expressing undersulfated heparan sulfate and the inhibition of bacterial adherence by the addition of exogenous dextran sulfate suggest that bacterial adhesion to mammalian cells is likely to be influenced by a variety of factors, including the degree of sulfation and the specificity of the carbohydrate moieties contained in the cellular proteoglycans.

A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans.

The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes.

Preliminary results on the seasonality and life cycle of the parasitic dinoflagellate causing bitter crab disease in Alaskan tanner crabs (Chionoecetes bairdi).

Tanner crabs (Chionoecetes bairdi) from the Sullivan Island area of southeast Alaska were sampled for 1 year to determine the prevalence and intensity of the parasitic dinoflagellate which causes bitter crab disease (BCD). The prevalence and intensity of infection were the greatest in the summer, declined in the fall and winter, and increased again in the spring. A possible relationship between softer, newer shells and higher levels of parasitism was also observed. In vivo transmission studies in the laboratory suggested there are several morphologically different forms of the vegetative cell of the BCD dinoflagellate which occur prior to sporulation of the parasite. In addition, it appears that both the two spore types produced by the parasite are infectious by injection and that there is no ploidy difference between the two spore types and the vegetative cell, suggesting that the two spore types may not represent separate sexes.

Long-term follow-up of argon laser trabeculoplasty for uncontrolled open-angle glaucoma.

Between May 1978 and October 1981, 82 phakic eyes in 72 patients with uncontrolled open-angle glaucoma underwent 360 degrees argon laser trabeculoplasty. Continued long-term follow-up has shown a decreasing pressure-lowering effect. The peak pressure lowering was 9.7 mm Hg at two months, 7.3 mm Hg at two years, 6.8 mm Hg at four years, and 4.9 mm Hg at five years. In 1982, we reported a 77% success rate, but, after five years of observation, the success rate is 46%. However, our clinical population is unique in that the majority of our patients (57%) are black. The most important factor in the long-term success rate appears to be race. Only 32% of cases involving black patients were successful, while 65% of cases involving white patients were successful. A Kaplan-Meier survival curve analysis revealed that the median time to an intraocular pressure greater than 21 mm Hg was 12 months for black patients and 60 months for white patients.