Gold Standard Cholera Diagnostics Are Tarnished by Lytic Bacteriophage and Antibiotics.

A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin, detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR (qPCR) for V. cholerae (n = 78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (odds ratio [OR], 0.108; 95% confidence interval [CI], 0.002 to 0.872) and 87% (OR, 0.130; 95% CI, 0.022 to 0.649), respectively, when lytic bacteriophage were detected. The odds that an RDT or qPCR was positive was reduced by more than 99% (OR, 0.00; 95% CI, 0.00 to 0.28) and 89% (OR, 0.11; 95% CI, 0.03 to 0.44), respectively, when azithromycin was detected. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as a proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

Horizontal and vertical distribution of sea lice larvae (Lepeophtheirus salmonis) in and around salmon farms in the Bay of Fundy, Canada.

The sea louse, Lepeophtheirus salmonis, is parasitic to salmonid species in the Northern Hemisphere and has become a widespread biological and economic problem for the salmon farming industry. A better understanding is needed of their spatial distribution and early life history to disrupt the life cycle of the sea louse. In this study, sea lice larval densities within salmon farms, between salmon farms and reference sites, and at various depths were quantified using both plankton pumps and plankton nets. Farm sites exhibited significantly higher densities than reference sites; however, these densities dropped an order of magnitude at a distance of 100 m from the cages. The majority of the larvae captured in the study were nauplii (93%), and densities ranged from 0 to 10 larvae/m3 . Free-swimming sea lice larvae were found to exhibit a diel cycle where nauplii larvae were in deeper waters (10-17 m) during the day and in surface waters (1-6 m) during the night. The results of this study suggest that the early life-history stages of sea lice originate from and may remain close to active salmon farms, creating a self-sustaining population.

Strongly baryon-dominated disk galaxies at the peak of galaxy formation ten billion years ago.

In the cold dark matter cosmology, the baryonic components of galaxies-stars and gas-are thought to be mixed with and embedded in non-baryonic and non-relativistic dark matter, which dominates the total mass of the galaxy and its dark-matter halo. In the local (low-redshift) Universe, the mass of dark matter within a galactic disk increases with disk radius, becoming appreciable and then dominant in the outer, baryonic regions of the disks of star-forming galaxies. This results in rotation velocities of the visible matter within the disk that are constant or increasing with disk radius-a hallmark of the dark-matter model. Comparisons between the dynamical mass, inferred from these velocities in rotational equilibrium, and the sum of the stellar and cold-gas mass at the peak epoch of galaxy formation ten billion years ago, inferred from ancillary data, suggest high baryon fractions in the inner, star-forming regions of the disks. Although this implied baryon fraction may be larger than in the local Universe, the systematic uncertainties (owing to the chosen stellar initial-mass function and the calibration of gas masses) render such comparisons inconclusive in terms of the mass of dark matter. Here we report rotation curves (showing rotation velocity as a function of disk radius) for the outer disks of six massive star-forming galaxies, and find that the rotation velocities are not constant, but decrease with radius. We propose that this trend arises because of a combination of two main factors: first, a large fraction of the massive high-redshift galaxy population was strongly baryon-dominated, with dark matter playing a smaller part than in the local Universe; and second, the large velocity dispersion in high-redshift disks introduces a substantial pressure term that leads to a decrease in rotation velocity with increasing radius. The effect of both factors appears to increase with redshift. Qualitatively, the observations suggest that baryons in the early (high-redshift) Universe efficiently condensed at the centres of dark-matter haloes when gas fractions were high and dark matter was less concentrated.

Emerging technologies to measure neighborhood conditions in public health: implications for interventions and next steps.

Adverse neighborhood conditions play an important role beyond individual characteristics. There is increasing interest in identifying specific characteristics of the social and built environments adversely affecting health outcomes. Most research has assessed aspects of such exposures via self-reported instruments or census data. Potential threats in the local environment may be subject to short-term changes that can only be measured with more nimble technology. The advent of new technologies may offer new opportunities to obtain geospatial data about neighborhoods that may circumvent the limitations of traditional data sources. This overview describes the utility, validity and reliability of selected emerging technologies to measure neighborhood conditions for public health applications. It also describes next steps for future research and opportunities for interventions. The paper presents an overview of the literature on measurement of the built and social environment in public health (Google Street View, webcams, crowdsourcing, remote sensing, social media, unmanned aerial vehicles, and lifespace) and location-based interventions. Emerging technologies such as Google Street View, social media, drones, webcams, and crowdsourcing may serve as effective and inexpensive tools to measure the ever-changing environment. Georeferenced social media responses may help identify where to target intervention activities, but also to passively evaluate their effectiveness. Future studies should measure exposure across key time points during the life-course as part of the exposome paradigm and integrate various types of data sources to measure environmental contexts. By harnessing these technologies, public health research can not only monitor populations and the environment, but intervene using novel strategies to improve the public health.

Lentiviral vectors incorporating a human elongation factor 1alpha promoter for the treatment of canine leukocyte adhesion deficiency.

Canine leukocyte adhesion deficiency (CLAD) provides a unique large animal model for testing new therapeutic approaches for the treatment of children with leukocyte adhesion deficiency (LAD). In our CLAD model, we examined two different fragments of the human elongation factor 1alpha (EF1alpha) promoter (EF1alphaL, 1189 bp and EF1alphaS, 233 bp) driving the expression of canine CD18 in a self-inactivating (SIN) lentiviral vector. The EF1alphaS vector resulted in the highest levels of canine CD18 expression in CLAD CD34(+) cells in vitro. Subsequently, autologous CD34(+) bone marrow cells from four CLAD pups were transduced with the EF1alphaS vector and infused following a non-myeloablative dose of 200 cGy total-body irradiation. None of the CLAD pups achieved levels of circulating CD18(+) neutrophils sufficient to reverse the CLAD phenotype, and all four animals were euthanized because of infections within 9 weeks of treatment. These results indicate that the EF1alphaS promoter-driven CD18 expression in the context of a RRLSIN lentiviral vector does not lead to sufficient numbers of CD18(+) neutrophils in vivo to reverse the CLAD phenotype when used in a non-myeloablative transplant regimen in dogs.

Diversity of Glanzmann thrombasthenia in southern India: 10 novel mutations identified among 15 unrelated patients.

BACKGROUND: Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3. OBJECTIVES: To determine the molecular basis of GT in patients from southern India. PATIENTS: Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. RESULTS: Platelet surface expression of alphaIIbbeta3 was < 10%, 10%-50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no alphaIIb in 14 patients, and no beta3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface alphaIIbbeta3 expression, and normal alphaIIb, beta3 and fibrinogen levels in the lysate. Ten novel candidate disease-causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in alphaIIb, and Cys575Gly in beta3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect alphaIIbbeta3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp-16Stop and Glu715Stop in alphaIIb, and a 7-bp deletion (330-336TCCCCAG) in beta3 are predicted to result in truncated proteins. An IVS15(-1)G --> A mutation in alphaIIb induced a cryptic splice site as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Thirteen polymorphisms were also identified (five in alphaIIb and eight in beta3), among which five were novel. CONCLUSIONS: While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India.

Three novel beta-propeller mutations causing Glanzmann thrombasthenia result in production of normally stable pro-alphaIIb, but variably impaired progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa). OBJECTIVES: To determine the molecular basis of GT and characterize the mutations by in vitro expression studies. PATIENTS: We studied three unrelated patients from southern India whose diagnosis was consistent with GT. RESULTS: Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature alpha(IIb) in the G128S mutant, in contrast to 6% and 33% of the normal amount of mature alpha(IIb) in the S287L and G357S mutants, respectively. Pulse-chase analysis demonstrated pro-alpha(IIb) in the mutants comparable with the normal pro-alpha(IIb), but no conversion to mature alpha(IIb) in the G128S mutant, and only trace conversion to mature alpha(IIb) in the S287L and G357S mutants. The disappearance of pro-alpha(IIb) in the three mutants was similar to that in cells expressing normal alpha(IIb)beta3 or alpha(IIb) only. All three mutants demonstrated pro-alpha(IIb)beta3 complexes and co-localized with an ER marker by immunofluorescence. The G128S mutant showed no co-localization with a Golgi marker, and the other two mutants showed minimal and moderate co-localization with the Golgi marker. CONCLUSIONS: These three beta-propeller mutations do not affect the production of pro-alpha(IIb), its ability to complex with beta3, or its stability, but do cause variable defects in transport of pro-alpha(IIb)beta3 complexes from the endoplasmic reticulum to the Golgi.

Assessing the effectiveness of whole blood-derived platelets stored as a pool: a randomized block noninferiority trial.

BACKGROUND: Prestorage pooling of whole blood-derived platelets (PLTs) would simplify bacterial detection. This study evaluated the in vivo effect of the prestorage pooling of PLTs stored for up to 5 days, by assessing the corrected count increment (CCI) 18 to 24 hours after transfusion of the product. STUDY DESIGN AND METHODS: A randomized block noninferiority design was used. Eligible patients had chemotherapy-induced thrombocytopenia and were considered likely to need at least six PLT transfusions. For every block of two transfusion events, one consisted of PLTs stored individually and then pooled before transfusion, and the other was a product pooled before storage. The primary outcome was categorized as a successful (>4.5) or unsuccessful (

In vitro evaluation of prestorage pooled leukoreduced whole blood-derived platelets stored for up to 7 days.

BACKGROUND: Advantages to storing whole blood-derived platelets (PLTs) as a pool for 7 days would include operational efficiencies and facilitation of bacterial testing and pathogen inactivation. The in vitro quality of pre-storage pooled PLTs stored for up to 7 days was assessed. STUDY DESIGN AND METHODS: Leukoreduced PLTs were pooled before storage (5 units/pool) and stored for either 5 or 7 days. Samples were collected at the time of pooling and either on Day 5 (n=16-29) or on Day 7 (n=4-30) and tested for biochemical and activation markers and morphology and/or shape change. Control PLTs were stored individually for 5 or 7 days and then tested as indicated above. RESULTS: The mean PLT counts (x10(9)/L) were similar: control PLTs, 1344 (464 SD); and prestorage pooled PLTs, 1327 (220 SD; p=0.93). On Day 5, the pH value was significantly lower (p

Hybridization and bond-orbital components in site-specific X-ray photoelectron spectra of rutile TiO2.

We have determined the Ti and O components of the rutile TiO2 valence band using the method of site-specific x-ray photoelectron spectroscopy. Comparisons with calculations based on pseudopotentials within the local density approximation reveal the hybridization of the Ti 3d, 4s, and 4p states, and the O 2s and 2p states on each site. These chemical effects are observed due to the large differences between the angular-momentum dependent matrix elements of the photoelectron process.

Direct measurement of valence-charge asymmetry by x-ray standing waves.

By monitoring valence-photoelectron emission under condition of strong x-ray Bragg reflection, we have determined that a majority of GaAs valence charge resides on the anion sites of this heteropolar crystal, in quantitative agreement with the GaAs bond polarity as calculated from the Hartree-Fock term values. In contrast, the valence-charge distribution in Ge is found to be symmetric. In both cases, the valence emission is found to be closely coupled to the atomic cores.

A comparison of prestorage WBC-reduced whole-blood-derived platelets and bedside-filtered whole-blood-derived platelets in autologous progenitor cell transplant.

BACKGROUND: Prestorage WBC-reduced platelet concentrates (PCs) can be manufactured from platelet-rich plasma (PRP) by in-line filtration of PRP. There are few published data on the clinical use of these products, as compared to bedside-filtered pools of standard PCs (S-PCs) manufactured from PRP. STUDY DESIGN AND METHODS: A prospective, randomized trial was conducted in autologous progenitor cell transplant patients requiring platelet transfusions with each patient as his or her own control who was given a pool of 5 units of WBC-reduced PCs and a pool of 6 units of S-PCs within a 3-hour period. The pools were characterized before transfusion for platelet and WBC content, P-selectin expression, and IL-8. The patients were monitored with platelet counts and vital signs and observed for reactions. Data were analyzed using Mann-Whitney U tests. RESULTS: Thirty-three transfusions were administered to 13 patients. Median platelet content in the WBC-reduced PC pools was lower than that in the S-PC pools (3.3 vs. 4.0 x 10(11), p<0.01). Median WBC content was 4 to 5 log less in the WBC-reduced PC pools (2.5 x 10(4) vs. 4.6 x 10(8), p<0.01). Median IL-8 levels (pg/mL) were lower in the WBC-reduced PC pools (2 vs. 36, p<0.01). No differences were observed in CCI, but the median absolute increase after transfusion of the S-PC pools was higher (25 vs. 19 x 10(9)/L, p<0.01), which reflected the larger size of the S-PC pools. No overall differences in vital signs were recorded. Two reactions were observed, both in temporal association with the transfusion of pools of S-PCs. CONCLUSIONS: A pool consisting of 5 units of WBC-reduced PCs gave a median platelet increment of 19 x 10(9) per L in these thrombocytopenic patients and has a median WBC content 1 to 2 log below the accepted threshold for primary alloimmunization or CMV transmission.

Sustained release of silver from periodontal wafers for treatment of periodontitis.

Periodontal wafers intended to treat the underlying infections in patients with periodontitis have been developed. The wafers consist of poly(lactic-co-glycolic acid) as a primary bioerodible polymeric component, poly(ethylene glycol) as a plasticizer and encapsulation aid, and silver nitrate as the antimicrobial agent. The wafers are capable of sustained in vitro release of bioactive silver for at least 4 weeks. The wafers exhibit silver release that follows erosion kinetics, confirming a bulk erosion/release mechanism. In clinical evaluation, sustained release of silver at bactericidal levels for at least 21 days is observed. Staining of hard and soft tissues due to the released silver is minimal and reversible.

Role of protein kinase C in ethanol-induced activation of adenylyl cyclase.

Ethanol is known to enhance the activity of adenylyl cyclase (AC) in a number of cells and tissues. Recent work has suggested that the various isoforms of AC show differential sensitivity to ethanol, with Type VII AC being most sensitive. However, the mechanism of action of ethanol is unclear. In the present work, we investigated the effect of ethanol on AC activity in the human erythroleukemia (HEL) cell line, platelets, and AC VII-transfected HEK 293 cells. The HEL cells contain abundant amounts of mRNA for Type VII AC. We found that both ethanol and phorbol dibutyrate (PDBu) treatment enhanced agonist (prostaglandin E1; PGE1)-stimulated AC activity in HEL cells, as well as in platelets and HEK 293 cells transfected with AC VII. Inhibitors of protein kinase C (PKC) blocked the stimulatory effects of both ethanol and PDBu. However, the effects of ethanol and PDBu on AC activity were additive, suggesting that the mechanisms of action of ethanol and PDBu were not identical. Furthermore, a 30-min exposure of HEL cells to ethanol attenuated (desensitized) the ability of ethanol, but not PDBu, to enhance agonist-activated AC activity. On the other hand, a 30-min pretreatment with PDBu attenuated the AC response to the phorbol ester, but not to ethanol; but, after a 20 hr preincubation with phorbol ester, the ability of both PDBu and ethanol to enhance prostaglandin E1-stimulated AC activity was completely eliminated. Finally, pretreatment of HEL cells with pertussis toxin blocked the effect of PDBu, but not ethanol, on AC activity. The results support the involvement of phorbol ester-sensitive PKC(s) in ethanol's enhancement of agonist-activated activity of AC in HEL cells, but suggest that the mechanism of ethanol's action is different from that of PDBu. The findings with pertussis toxin suggest that PDBu activation of PKC(s) may affect AC activity through phosphorylation of a G1 protein, whereas ethanol may act by promoting phosphorylation of a different substrate (e.g., AC VII).

Fluorescence methods to assess multidrug resistance in individual cells.

PURPOSE: Microscopic methods to measure the activity of drug extrusion systems important in multidrug resistance in individual cells were developed. METHODS: Multidrug-resistant (MDR) and parental lines of hamster CHO and pituitary GH3 cells were incubated with the acetoxymethylester (AM) forms of several fluorescent calcium-sensing dyes, fura2, indo1 and fluo3. The AM forms of these compounds are hydrolyzed by intracellular esterases and then trapped in cells, and the AM forms of the dyes are excellent substrates for P-glycoprotein (Pgp). RESULTS: The fluorescent free acid forms of fura2, indol and fluo3 did not accumulate in MDR lines unless a chemosensitizer such as cyclosporin A, R(+)verapamil, quinidine, or progesterone was included during loading to prevent the cells from extruding the AM forms of the dyes before they could be hydrolyzed. Cyclosporin A increased the fluorescence due to intracellularly trapped fura2 free acid from 8- to 20-fold and was maximally effective at < 5 microM. Fluorescence microscopy was employed to measure fura2 free acid accumulation by parental and MDR cell lines using excitation at the Ca2+-insensitive wavelength. When MDR cells were incubated with rhodamine 123 and fura2/AM, no fluorescence was detectable. Cellular fluorescence was dramatically increased by inclusion of cyclosporin A, quinidine, progesterone, or R(+)verapamil. There was no measurable decline in the fura2 free acid fluorescence in 1 h while the fluorescence due to rhodamine 123 diminished rapidly in cells overexpressing Pgp. CONCLUSIONS: These fluorescence methods detect drug-extruding activity in individual cells and therefore have the potential to provide complementary information to studies quantifying protein or mRNA levels of Pgp or other efflux pumps. In addition, they provide a rapid and quantifiable method for screening multidrug resistance reversing agents.

Characterization of the calcium response to thyrotropin-releasing hormone in lactotrophs and GH cells.

Thyrotropin-releasing hormone (TRH) acts via a G-protein-coupled receptor on lactotrophs to increase the intracellular free calcium ion concentration, [Ca(2+)](i). The [Ca(2+)](i) response depends on both TRH concentration and the duration of TRH exposure. An initial, short-lived [Ca(2+)](i) spike results from release of Ca(2+) from intracellular stores, whereas a later sustained [Ca(2+)](i) increase, often characterized by [Ca(2+)](i) oscillations, results from an influx of extracellular Ca(2+) through both voltage-gated and non-voltage-gated, store-operated Ca(2+) channels. The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca(2+)](i) response to TRH are discussed.

Continuous gastric suctioning decreases measured esophageal temperature during general anesthesia.

OBJECTIVE: This study sought to determine whether continuous gastric suctioning influences esophageal temperature measurements. METHODS: This study evaluated 21 patients scheduled for extremity or lower abdominal surgery. After induction of general endotracheal anesthesia, an orogastric tube, and esophageal and nasopharyngeal temperature probes were placed in functional positions. Baseline esophageal (Tes) and nasopharyngeal (Tnas) temperatures were recorded and the orogastric tube was placed on continuous suction. After the first 11 patients (Group I) were studied, 10 additional patients (Group II) were studied with more frequent data collection to improve the time resolution of temperature changes. Temperatures were recorded for patients in Group I at 2 and 10 min with suctioning and 10 min after cessation of suctioning. In Group II, temperatures were recorded at 1, 2, 5 and 10 min with suctioning and 10 min after cessation of suctioning. Analysis of data was performed using repeated measures analysis of variance and paired t-tests with the Bonferroni correction. RESULTS: In Group I, Tes decreased significantly from 35.9 +/- 0.2 degrees C (mean +/- SE) to 35.1 +/- 0.4 degrees C at 2 min and 34.8 +/- 0.3 degrees C at 10 min of suctioning (p < 0.01). Ten minutes after cessation of suctioning, Tes was not significantly different from the baseline measurement. Tnas did not change significantly over the 20 min observation period. In Group II, Tes continually decreased from 36.2 +/- 0.1 degrees C to 34.8 +/- 0.3 degrees C after 10 min of suctioning (p < 0.006) and returned to near baseline 10 min after cessation of suctioning. There was no significant change in Tnas over the 20 min observation period. CONCLUSION: We conclude that continuous gastric suctioning decreases esophageal temperature measurements. This phenomenon should be recognized as an artifactual change in esophageal temperature and not a reflection of core temperature.

Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling.

Endocytosis and recycling of both thyrotropin-releasing hormone (TRH) and its G-protein-coupled receptor were visualized by conventional and confocal fluorescence microscopy in pituitary cells using a rhodamine-labeled TRH analog (Rhod-TRH) and indirect immunofluorescent staining of cells stably transfected with an epitope-tagged TRH receptor (TRHR). The epitope-tagged TRHR was confined to the cell surface prior to agonist treatment. Both Rhod-TRH and TRHR were also localized on the plasma membrane after agonist binding at 0 degrees C. Ligand binding at 37 degrees C resulted in rapid endocytosis, and both Rhod-TRH and the epitope-tagged TRHR appeared in cytoplasmic vesicles within 5 min. Fluorescently labeled TRH and transferrin colocalized in the same endocytotic vesicles, and internalization of Rhod-TRH and TRHR was inhibited by hypertonic medium, suggesting that endocytosis occurred by a clathrin-dependent mechanism. Internalized TRHRs returned to the membrane within 20 min after removal of TRH, and cycloheximide did not block receptor recycling. A mutant TRHR truncated at Cys335 signaled but did not internalize Rhod-TRH, confirming the importance of the carboxyl terminus of the TRHR in receptor-mediated endocytosis. Thus, the TRH-TRHR complex is endocytosed via clathrin-coated vesicles and the receptor is recycled to the plasma membrane.