Inkjet printable-photoactive all inorganic perovskite films with long effective photocarrier lifetimes.

Photoactive perovskite quantum dot films, deposited via an inkjet printer, have been characterized by x-ray diffraction and x-ray photoelectron spectroscopy. The crystal structure and bonding environment are consistent with CsPbBr3 perovskite quantum dots. The current-voltage (I-V) and capacitance-voltage (C-V) transport measurements indicate that the photo-carrier drift lifetime can exceed 1 ms for some printed perovskite films. This far exceeds the dark drift carrier lifetime, which is below 50 ns. The printed films show a photocarrier density 109 greater than the dark carrier density, making these printed films ideal candidates for application in photodetectors. The successful printing of photoactive-perovskite quantum dot films of CsPbBr3, indicates that the rapid prototyping of various perovskite inks and multilayers is realizable.

Low etiologic fraction for human papillomavirus in larynx squamous cell carcinoma.

BACKGROUND: Human Papillomavirus (HPV) is a cause of oropharyngeal squamous cell carcinoma (OPSCC), but its pathogenic role in larynx squamous cell carcinoma (LSCC) remains unclear. MATERIAL AND METHODS: A single-institutional, retrospective case-series was performed to estimate the etiological fraction (EF) for HPV in LSCC. Eligible cases included 436 consecutive cases of LSCC diagnosed (2005-2014) at The Ohio State University Medical Center. HPV DNA presence was detected by consensus primer PCR (Inno-LiPa) and HPV type-specific qPCR. HPV E6/E7 mRNA expression was detected by type-specific qRT-PCR. Tumor p16 expression was evaluated by immunohistochemistry (IHC). RESULTS: HPV DNA was detected by Inno-LiPa in 54 of 404 (13.4%, 95% CI 10.2-17.1) evaluable samples but was confirmed by HPV type-specific qPCR in only 14 (3.5%, 95% CI 1.9-5.7). Only 7 of 404 (1.7%, 95% CI 0.7-3.5) LSCC were positive for HPV E6/E7 mRNA expression, including HPV16 (n=4) and 1 each for 11, 26 and 33. In the HPV11-positive tumor, Sanger sequencing discovered 6 nucleotide mutations in the upstream regulation region, E6 and E7. Of 404 LSCC, 18 had strong and diffuse p16 expression. In comparison to a gold standard of HPV E6/E7 mRNA expression, p16 expression had a sensitivity of 71.4% (95% CI 29.0-96.3), specificity of 96.7% (95% CI 94.5-98.3), positive-predictive-value (PPV) of 27.8% (95% CI 9.7-53.5) and negative-predictive-value of 99.5% (95% CI 98.1-99.9). CONCLUSION: The EF for HPV in LSCC is low (1.7%) in a geographic region with high EF for OPSCC. Low-risk HPV may rarely cause LSCC. Finally, p16 expression has poor PPV for HPV in LSCC.

Australian Cattle Dogs with Neuronal Ceroid Lipofuscinosis are Homozygous for a CLN5 Nonsense Mutation Previously Identified in Border Collies.

BACKGROUND: Neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease, has been diagnosed in young adult Australian Cattle Dogs. OBJECTIVE: Characterize the Australian Cattle Dog form of NCL and determine its molecular genetic cause. ANIMALS: Tissues from 4 Australian Cattle Dogs with NCL-like signs and buccal swabs from both parents of a fifth affected breed member. Archived DNA samples from 712 individual dogs were genotyped. METHODS: Tissues were examined by fluorescence, electron, and immunohistochemical microscopy. A whole-genome sequence was generated for 1 affected dog. A TaqMan allelic discrimination assay was used for genotyping. RESULTS: The accumulation of autofluorescent cytoplasmic storage material with characteristic ultrastructure in tissues from the 4 affected dogs supported a diagnosis of NCL. The whole-genome sequence contained a homozygous nonsense mutation: CLN5:c.619C>T. All 4 DNA samples from clinically affected dogs tested homozygous for the variant allele. Both parents of the fifth affected dog were heterozygotes. Archived DNA samples from 346 Australian Cattle Dogs, 188 Border Collies, and 177 dogs of other breeds were homozygous for the reference allele. One archived Australian Cattle Dog sample was from a heterozygote. CONCLUSIONS AND CLINICAL IMPORTANCE: The homozygous CLN5 nonsense is almost certainly causal because the same mutation previously had been reported to cause a similar form of NCL in Border Collies. Identification of the molecular genetic cause of Australian Cattle Dog NCL will allow the use of DNA tests to confirm the diagnosis of NCL in this breed.

A randomized controlled trial of mindfulness-based stress reduction to prevent flare-up in patients with inactive ulcerative colitis.

BACKGROUND/AIMS: The primary therapeutic goals in ulcerative colitis (UC) are to maintain excellent quality of life (QOL) by treating flare-ups when they occur, and preventing flare-ups. Since stress can trigger UC flare-ups, we investigated the efficacy of mindfulness-based stress reduction (MBSR) to reduce flare-ups and improve QOL. METHODS: Patients with moderately severe UC, in remission, were randomized to MBSR or time/attention control. Primary outcome was disease status. Secondary outcomes were changes in markers of inflammation and disease activity, markers of stress and psychological assessments. RESULTS: 55 subjects were randomized. Absence of flares, time to flare and severity of flare over 1 year were similar between the two groups. However, post hoc analysis showed that MBSR decreased the proportion of participants with at least one flare-up among those with top tertile urinary cortisol and baseline perceived stress (30 vs. 70%; p < 0.001). MBSR patients who flared demonstrated significantly lower stress at the last visit compared to flared patients in the control group (p = 0.04). Furthermore, MBSR prevented a drop in the Inflammatory Bowel Disease Quality of Life Questionnaire during flare (p < 0.01). CONCLUSION: MBSR did not affect the rate or severity of flare-ups in UC patients in remission. However, MBSR might be effective for those with high stress reactivity (high perceived stress and urinary cortisol) during remission. MBSR appears to improve QOL in UC patients by minimizing the negative impact of flare-ups on QOL. Further studies are needed to identify a subset of patients for whom MBSR could alter disease course.

Altered gene expression in the dorsolateral prefrontal cortex of individuals with schizophrenia.

The underlying pathology of schizophrenia (SZ) is likely as heterogeneous as its symptomatology. A variety of cortical and subcortical regions, including the prefrontal cortex, have been implicated in its pathology, and a number of genes have been identified as risk factors for disease development. We used in situ hybridization (ISH) to examine the expression of 58 genes in the dorsolateral prefrontal cortex (DLPFC, comprised of Brodmann areas 9 and 46) from 19 individuals with a premorbid diagnosis of SZ and 33 control individuals. Genes were selected based on: (1) previous identification as risk factors for SZ; (2) cell type markers or (3) laminar markers. Cell density and staining intensity were compared in the DLPFC, as well as separately in Brodmann areas 9 and 46. The expression patterns of a variety of genes, many of which are associated with the GABAergic system, were altered in SZ when compared with controls. Additional genes, including C8orf79 and NR4A2, showed alterations in cell density or staining intensity between the groups, highlighting the need for additional studies. Alterations were, with only a few exceptions, limited to Brodmann area 9, suggesting regional specificity of pathology in the DLPFC. Our results agree with previous studies on the GABAergic involvement in SZ, and suggest that areas 9 and 46 may be differentially affected in the disease. This study also highlights additional genes that may be altered in SZ, and indicates that these potentially interesting genes can be identified by ISH and high-throughput image analysis techniques.

High hydrostatic pressure modification of whey protein concentrate for use in low-fat whipping cream improves foaming properties.

Whey is the inevitable by-product of cheese production. Whey can be incorporated into a variety of foods, but little has been done to investigate its suitability in whipping cream. The objective of this work was to evaluate the foaming properties of selected low-fat whipping cream formulations containing whey protein concentrate (WPC) that did or did not undergo high hydrostatic pressure (HHP) treatment. Fresh whey was concentrated by ultrafiltration, pasteurized, and standardized to 8.23% total solids and treated with HHP at 300 MPa for 15 min. Viscosity, overrun, and foam stability were determined to assess foaming properties. Sensory evaluation was conducted with 57 panelists using a duo-trio difference test. The optimal whipping time for the selected formulations was 3 min. Whipping cream containing untreated WPC and HHP-treated WPC resulted in greater overrun and foam stability than the control whipping cream without WPC. Panelists distinguished a difference between whipping cream containing untreated WPC and whipping cream containing HHP-treated WPC. High hydrostatic pressure-treated WPC can improve the foaming properties of low-fat whipping cream, which may justify expansion of the use of whey in whipping cream and application of HHP technology in the dairy industry.

Influence of salt-to-moisture ratio on starter culture and calcium lactate crystal formation.

The occurrence of L(+)-lactate crystals in hard cheeses continues to be an expense to the cheese industry. Salt tolerance of the starter culture and the salt-to-moisture ratio (S:M) in cheese dictate the final pH of cheese, which influences calcium lactate crystal (CLC) formation. This research investigates these interactions on the occurrence of CLC. A commercial starter was selected based on its sensitivity to salt, less than and greater than 4.0% S:M. Cheddar cheese was made by using either whole milk (3.25% protein, 3.85% fat) or whole milk supplemented with cream and ultrafiltered milk (4.50% protein, 5.30% fat). Calculated amounts of salt were added at milling (pH 5.40 +/- 0.02) to obtain cheeses with less than 3.6% and greater than 4.5% S:M. Total and soluble calcium, total lactic acid, and pH were measured and the development of CLC was monitored in cheeses. All cheeses were vacuum packaged and gas flushed with nitrogen gas and aged at 7.2 degrees C for 15 wk. Concentration of total lactic acid in high S:M cheeses ranged from 0.73 to 0.80 g/100 g of cheese, whereas that in low S:M cheeses ranged from 1.86 to 1.97 g/100 g of cheese at the end of 15 wk of aging because of the salt sensitivity of the starter culture. Concentrated milk cheeses with low and high S:M exhibited a 30 to 28% increase in total calcium (1,242 and 1,239 mg/100 g of cheese, respectively) compared with whole milk cheeses with low and high S:M (954 and 967 mg/100 g of cheese, respectively) throughout aging. Soluble calcium was 41 to 35% greater in low S:M cheeses (low-salt whole milk cheese and low-salt concentrated milk cheese; 496 and 524 mg/100 g of cheese, respectively) compared with high S:M cheeses (high-salt whole milk cheese and high-salt concentrated milk cheese; 351 and 387 mg/100 g of cheese, respectively). Because of the lower pH of the low S:M cheeses, CLC were observed in low S:M cheeses. However, the greatest intensity of CLC was observed in gas-flushed cheeses made with milk containing increased protein concentration because of the increased content of calcium available for CLC formation. These results show that the occurrence of CLC is dependent on cheese milk concentration and pH of the cheese, which can be influenced by S:M and cheese microflora.

High hydrostatic pressure modification of whey protein concentrate for improved functional properties.

Whey protein concentrate (WPC) has many applications in the food industry. Previous research demonstrated that treatment of whey proteins with high hydrostatic pressure (HHP) can enhance solubility and foaming properties of whey proteins. The objective of this study was to use HHP to improve functional properties of fresh WPC, compared with functional properties of reconstituted commercial whey protein concentrate 35 (WPC 35) powder. Fluid whey was ultrafiltered to concentrate proteins and reconstituted to equivalent total solids (8.23%) as reconstituted commercial WPC 35 powder. Solutions of WPC were treated with 300 and 400 MPa (0- and 15-min holding time) and 600 MPa (0-min holding time) pressure. After HHP, the solubility of the WPC was determined at both pH 4.6 and 7.0 using UDY and BioRad protein assay methods. Overrun and foam stability were determined after protein dispersions were whipped for 15 min. The protein solubility was greater at pH 7.0 than at pH 4.6, but there were no significant differences at different HHP treatment conditions. The maintenance of protein solubility after HHP indicates that HHP-treated WPC might be appropriate for applications to food systems. Untreated WPC exhibited the smallest overrun percentage, whereas the largest percentage for overrun and foam stability was obtained for WPC treated at 300 MPa for 15 min. Additionally, HHP-WPC treated at 300 MPa for 15 min acquired larger overrun than commercial WPC 35. The HHP treatment of 300 MPa for 0 min did not improve foam stability of WPC. However, WPC treated at 300 or 400 MPa for 15 min and 600 MPa for 0 min exhibited significantly greater foam stability than commercial WPC 35. The HHP treatment was beneficial to enhance overrun and foam stability of WPC, showing promise for ice cream and whipping cream applications.

High hydrostatic pressure modification of whey protein concentrate for improved body and texture of lowfat ice cream.

Previous research demonstrated that application of high hydrostatic pressure (HHP), particularly at 300 MPa for 15 min, can enhance foaming properties of whey protein concentrate (WPC). The purpose of this research was to determine the practical impact of HHP-treated WPC on the body and texture of lowfat ice cream. Washington State University (WSU)-WPC was produced by ultrafiltration of fresh separated whey received from the WSU creamery. Commercial whey protein concentrate 35 (WPC 35) powder was reconstituted to equivalent total solids as WSU-WPC (8.23%). Three batches of lowfat ice cream mix were produced to contain WSU-WPC without HHP, WSU-WPC with HHP (300 MPa for 15 min), and WPC 35 without HHP. All lowfat ice cream mixes contained 10% WSU-WPC or WPC 35. Overrun and foam stability of ice cream mixes were determined after whipping for 15 min. Ice creams were produced using standard ice cream ingredients and processing. The hardness of ice creams was determined with a TA-XT2 texture analyzer. Sensory evaluation by balanced reference duo-trio test was carried out using 52 volunteers. The ice cream mix containing HHP-treated WSU-WPC exhibited the greatest overrun and foam stability, confirming the effect of HHP on foaming properties of whey proteins in a complex system. Ice cream containing HHP-treated WSU-WPC exhibited significantly greater hardness than ice cream produced with untreated WSU-WPC or WPC 35. Panelists were able to distinguish between ice cream containing HHP-treated WSU-WPC and ice cream containing untreated WPC 35. Improvements of overrun and foam stability were observed when HHP-treated whey protein was used at a concentration as low as 10% (wt/wt) in ice cream mix. The impact of HHP on the functional properties of whey proteins was more pronounced than the impact on sensory properties.

A comparison between improvers and non-improvers among children with anemia enrolled in the WIC program.

OBJECTIVES: To investigate differences between children ages one to five enrolled in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) in the state of Texas whose anemia status improved and those whose anemia status did not improve. METHODS: The study involved administration of a survey to a randomly selected stratified sample of parents and guardians of WIC children residing in the eleven public health regions in Texas. Subjects volunteered to complete the survey during a scheduled WIC appointment. A total of 459 usable surveys were completed. Fifty-one percent of the surveys were completed by parents/guardians of children classified as anemia improvers and 49% were completed by parents/guardians of children classified as anemia non-improvers. RESULTS AND CONCLUSIONS: A majority of the respondents were the child's parent (97%). Most of the children were Hispanic/Latino (77%). More non-improvers than improvers were of young age (p < 0.01), had a parent/guardian who was 18-30 years of age (p < 0.01), lived in a household with four or more other children (p = 0.05) and were from households with a very low income (p = 0.03). Compared to improvers, non-improvers ate fewer snacks (p = 0.01) and a greater percentage never consumed dried fruits (p < 0.01). Knowledge level of the parents/guardians was similar for the two groups. A high percentage of parents/guardians of non-improvers incorrectly believed that their child's condition had improved. WIC staff and other public health educators can utilize these findings to identify those children with anemia who are at higher risk for non-improvement.

Cheese pH, protein concentration, and formation of calcium lactate crystals.

The occurrence of calcium lactate crystals (CLC) in hard cheeses is a continual expense to the cheese industry, as consumers fail to purchase cheeses with this quality defect. This research investigates the effects of the protein concentration of cheese milk and the pH of cheese on the occurrence of CLC. Atomic absorption spectroscopy was used to determine total and soluble calcium concentrations in skim milk (SM1, 8.7% total solids), and skim milk supplemented with nonfat dry milk (CSM1, 13.5% total solids). Calcium, phosphorus, lactic acid, and citrate were determined in cheeses made with skim milk (SM2, 3.14% protein), skim milk supplemented with ultrafiltered milk (CSM2, 6.80% protein), and nonfat dry milk (CSM3, 6.80% protein). Supplementation with nonfat dry milk increased the initial total calcium in CSM1 (210 mg/100 g of milk) by 52% compared with the total calcium in SM1 (138 mg/100 g of milk). At pH 5.4, soluble calcium concentrations in CSM1 were 68% greater than soluble calcium in SM1. In cheeses made from CSM2 and CSM3, total calcium was 26% greater than in cheeses made from SM2. As the pH of cheeses made from SM2 decreased from 5.4 to 5.1, the concentration of soluble calcium increased by 61.6%. In cheeses made from CSM2 and CSM3, the concentrations of soluble calcium increased by 41.4 and 45.5%, respectively. Calcium lactate crystals were observed in cheeses made from SM2 at and below pH 5.1, whereas CLC were observed in cheeses from CSM2 and CSM3 at and below pH 5.3. The increased presence of soluble calcium can potentially cause CLC to occur in cheese manufactured with increased concentrations of milk solids, particularly at and below pH 5.1.

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese.

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10 degrees C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm2. The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10 degrees C compared with that stored at 7.2 degrees C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10 degrees C compared with 7.2 degrees C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of D(-)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.

Detecting avian malaria: an improved polymerase chain reaction diagnostic.

We describe a polymerase chain reaction (PCR) assay that detects avian malarial infection across divergent host species and parasite lineages representing both Plasmodium spp. and Haemoproteus spp. The assay is based on nucleotide primers designed to amplify a 286-bp fragment of ribosomal RNA (rRNA) coding sequence within the 6-kb mitochondrial DNA malaria genome. The rRNA malarial assay outperformed other published PCR diagnostic methods for detecting avian infections. Our data demonstrate that the assay is sensitive to as few as 10(-5) infected erythrocytes in peripheral blood. Results of avian population surveys conducted with the rRNA assay suggest that prevalences of malarial infection are higher than previously documented, and that studies based on microscopic examination of blood smears may substantially underestimate the extent of parasitism by these apicomplexans. Nonetheless, because these and other published primers miss small numbers of infections detected by other methods, including inspection of smears, no assay now available for avian malaria is universally reliable.

Low-fat set yogurt made from milk subjected to combinations of high hydrostatic pressure and thermal processing.

The combined use of high hydrostatic pressure (300 to 676 MPa, 5 min) and thermal treatment (85 degrees C, 30 min) in milk for the manufacture of low-fat yogurt was studied. The objective was to reduce syneresis and improve the rheological properties of yogurt, reducing the need for thickeners and stabilizers. The use of high hydrostatic pressure alone, or after thermal treatment, reduced the lightness and increased the viscosity of skim milk. However, milk recovered its initial lightness and viscosity when thermal treatment was applied after high hydrostatic pressure. The MALDI-TOF spectra of skim milk presented monomers of whey proteins after a treatment of 676 MPa for 5 min. Yogurts made from skim milk subjected to 400 to 500 MPa and thermal treatment showed increased yield stress, resistance to normal penetration, and elastic modulus, while having reduced syneresis when compared to yogurts from thermally treated or raw milks. The combined use of thermal treatment and high hydrostatic pressure assures extensive whey protein denaturation and casein micelle disruption, respectively. Although reaggregation of casein submicelles occurs during fermentation, the net effect of the combined HHP and thermal treatment is the improvement of yogurt yield stress and reduction of syneresis.

Food processing by high hydrostatic pressure.

The use of high hydrostatic pressures (HHP) for food processing is finding increased application within the food industry. One of the advantages of this technology is that because it does not use heat, sensory, and nutritional attributes of the product remain virtually unaffected, thus yielding products with better quality than those processed traditional methods. HHP have the ability to inactivate microorganisms as well as enzymes responsible for shortening the life of a product. In addition to lengthening the shelf-life of food products, HHP can modify functional properties of components such as proteins, which in turn can lead to the development of new products. Equipment for large-scale production of HHP processed products are commercially available nowadays. Guacamole, sliced ham, oysters, and fruit juices are some of the products currently available on the market. HHP technology is one of the most promising nonthermal processes.

Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa.

The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.

Beta-lactoglobulin molten globule induced by high pressure.

Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.

Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections.

The ability of Pseudomonas aeruginosa to secrete specific toxins using the type III-mediated pathway has been reported. To determine the association of this phenotype with human illness, immunoblot analysis was used to detect expression of type III secretory proteins in P. aeruginosa isolates from respiratory tract or blood cultures of 108 consecutive patients. Relative risk of mortality was 6-fold greater with expression of the type III secretory proteins ExoS, ExoT, ExoU, or PcrV. Phenotype was independently correlated with toxicity in cellular and murine models. Prevalence of this phenotype was significantly higher in acutely infected patients than in chronically infected patients with cystic fibrosis. These results suggest that the type III protein secretion system is integral to increased P. aeruginosa virulence. A positive phenotype is a predictor of poor clinical outcome. In the future, such analyses may help distinguish potentially lethal infection from colonization and help determine appropriate therapy for critically ill patients.

Detection of multivalent interactions through two-tiered energy transfer.

A method based on two-tiered fluorescence resonant energy transfer (FRET) has been developed for selective and sensitive detection of species involved in a multivalent interaction. Pentavalent binding between cholera toxin and ganglioside GM1 is used as a model system to demonstrate the advantage of the two-tiered FRET over one-stage FRET in both conventional fluorimeter and flow cytometer. In the system, three fluorescent probes (namely, fluorescence donor, acceptor, and intermediate) are covalently tagged to receptors, and the intermediate is used to bridge the energy transfer between the donor and acceptor even though the donor's fluorescence spectrum does not overlap with absorption spectrum of the acceptor. One of the most significant improvements of the scheme over one-stage FRET is a dramatic decrease in the background fluorescence of the acceptor fluorescence, which, theoretically and practically, increases the detection sensitivity.