Characteristics of Vibrio vulnificus isolates from clinical and environmental sources.

Researchers have developed multiple methods to characterize clinical and environmental strains of Vibrio vulnificus. The aim of our study was to use four assays to detect virulence factors in strains from infected patients and those from surface waters/sediments/oysters of South Carolina and the Gulf of Mexico. Vibrio vulnificus strains from clinical (n = 81) and environmental (n = 171) sources were tested using three real-time PCR methods designed to detect polymorphisms in the 16S rRNA, vcg and pilF genes and a phenotypic method, the ability to ferment D-mannitol. Although none of the tests correctly categorized all isolates, the differentiation between clinical and environmental isolates was similar for the pilF, vcgC/E and 16S rRNA assays, with sensitivities of 74.1-79.2% and specificities of 77.4-82.7%. The pilF and vcgC/E assays are comparable in efficacy to the widely used 16S rRNA method, while the D-mannitol fermentation test is less discriminatory (sensitivity = 77.8%, specificity = 61.4%). Overall percent agreement for the D-mannitol fermentation method was also lower (66.7%) than overall percent agreement for the 3 molecular assays (78.0%-80.2%). This study demonstrated, using a large, diverse group of Vibrio vulnificus isolates, that three assays could be used to distinguish most clinical vs environmental isolates; however, additional assays are needed to increase accuracy.

The glucagon-receptor antagonist MK-3577 reduces glucagon-stimulated plasma glucose and insulin concentrations in metabolically healthy overweight cats.

The role of glucagon disturbances in diabetes mellitus is increasingly recognized and, hence, glucagon antagonism might aid in treatment of hyperglycemia and other metabolic disturbances. The aim of this study was to assess the pharmacokinetics of the glucagon receptor antagonist MK-3577 and its effect on plasma glucose, insulin, and glucagon concentrations in healthy cats. In a cross-over placebo-controlled study, 5 purpose-bred cats were treated with either Placebo, MK-3577 (1 mg/kg), or MK-3577 (3 mg/kg). Glucose, insulin and glucagon concentrations were measured at 0, 15, 225, 240 min post-treatment administration. Glucagon (20 mcg/kg, IM) was administered at 240 min and glucose and insulin were measured at 255, 265, 275, 285 and 300 min. Plasma MK-3577 concentrations peaked at 4.2 and 3.2 hours after 1 and 3 mg/kg dosing with a half-life of 14.8h and 15.5h respectively. Baseline glucose, insulin and glucagon concentrations did not differ significantly between treatment groups. At a dose of 3 mg/kg, MK-3577 blunted the glucagon-stimulated rise of glucose (p=0.0089) and insulin (p=0.02). Similar trends were observed with MK-3577 at the 1 mg/kg dose but the effect was smaller, and not significant. In conclusion, the GRA MK-3577 has a pharmacokinetic profile suitable for diminishing the glucagon-induced rise of glucose and insulin in healthy cats.

Measurement of the ßß decay half-life of 130Te with the NEMO-3 detector.

We report results from the NEMO-3 experiment based on an exposure of 1275 days with 661 g of (130)Te in the form of enriched and natural tellurium foils. The ßß decay rate of (130)Te is found to be greater than zero with a significance of 7.7 standard deviations and the half-life is measured to be T(½)(2ν) = [7.0 ± 0.9(stat) ± 1.1(syst)] × 10(20) yr. This represents the most precise measurement of this half-life yet published and the first real-time observation of this decay.

An influenza A (H3N2) virus outbreak in the Kingdom of Cambodia during the COVID-19 pandemic of 2020.

BACKGROUND: Global influenza virus circulation decreased during the COVID-19 pandemic, possibly due to widespread community mitigation measures. Cambodia eased some COVID-19 mitigation measures in June and July 2020. On 20 August a cluster of respiratory illnesses occurred among residents of a pagoda, including people who tested positive for influenza A but none who were positive for SARS-CoV-2. METHODS: A response team was deployed on 25 August 2020. People with influenza-like illness (ILI) were asked questions regarding demographics, illness, personal prevention measures, and residential arrangements. Respiratory swabs were tested for influenza and SARS-Cov-2 by real-time reverse transcription PCR, and viruses were sequenced. Sentinel surveillance data were analyzed to assess recent trends in influenza circulation in the community. RESULTS: Influenza A (H3N2) viruses were identified during sentinel surveillance in Cambodia in July 2020 prior to the reported pagoda outbreak. Among the 362 pagoda residents, 73 (20.2%) ILI cases were identified and 40 were tested, where 33/40 (82.5%) confirmed positive for influenza A (H3N2). All 40 were negative for SARS-CoV-2. Among the 73 residents with ILI, none were vaccinated against influenza, 47 (64%) clustered in 3/8 sleeping quarters, 20 (27%) reported often wearing a mask, 27 (36%) reported often washing hands, and 11 (15%) reported practicing social distancing. All viruses clustered within clade 3c2.A1 close to strains circulating in Australia in 2020. CONCLUSIONS: Circulation of influenza viruses began in the community following the relaxation of national COVID-19 mitigation measures, and prior to the outbreak in a pagoda with limited social distancing. Continued surveillance and influenza vaccination are required to limit the impact of influenza globally.

Incidence of Vibrio vulnificus in estuarine waters of the south Texas Coastal Bend region.

AIMS: To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. METHODS AND RESULTS: Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture-based techniques utilizing Vibrio vulnificus agar (VVA) and membrane-Enterococcus indoxyl-beta-D-glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony-blot hybridization with the species-specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9.71 degrees C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature (P = 0.002) and dissolved oxygen (P = 0.028), as well as between concentrations of V. vulnificus and enterococci (P < 0.001). CONCLUSIONS: This study demonstrated the year-round presence of V. vulnificus in coastal waters of south Texas. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen.

Progress toward sustainable influenza vaccination in the Lao Peoples' Democratic Republic, 2012-2018.

Despite global recommendations for influenza vaccination of high-risk, target populations, few low and middle-income countries have national influenza vaccination programs. Between 2012 and 2017, Lao PDR planned and conducted a series of activities to develop its national influenza vaccine program as a part of its overall national immunization program. In this paper, we review the underlying strategic planning for this process, and outline the sequence of activities, research studies, partnerships, and policy decisions that were required to build Laos' influenza vaccine program. The successful development and sustainability of the program in Laos offers lessons for other low and middle-income countries interested in initiating or expanding influenza immunization.

mir-29 regulates Mcl-1 protein expression and apoptosis.

Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies, because Mcl-1 is dysregulated in cells with the malignant phenotype. By in silico analysis, we identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated the expression of an Mcl-1 3' untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression, and thereby, apoptosis.

Cation binding to the integrin CD11b I domain and activation model assessment.

BACKGROUND: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11 b I domain and to evaluate the structural effects of divalent ion binding to this protein. RESULTS: We have determined the X-ray structure of a new crystal form of the CD11 b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straight-forward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11 a I-domain structures and a CD11 b I-domain complex with Mn2+. These structures define a majority conformation. CONCLUSIONS: Addition of the cations Mg2+, Mn2+ and Cd2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al, is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.

Patients receiving a prescription for diamorphine (heroin) in the United Kingdom.

The United Kingdom is unusual internationally in that it is one of few countries able to prescribe diamorphine for the treatment of opiate dependence. Prescribing diamorphine has been part of the UK response to drug problems since the 1920s. Despite this, little is known about who receives diamorphine and how treatment is delivered. This study aims to describe the characteristics and treatment regimes of opiate-dependent drug users receiving a prescription for diamorphine in the United Kingdom in 2000, and report on their status in 2002. A retrospective case-note review was conducted in England and Wales. Two hundred and ten (72%; 210/292) patients' sets of case-notes were reviewed at 27 of the 42 (64%) drug clinics where diamorphine was prescribed by the doctor. Patients had been receiving a prescription for diamorphine for a median length of six years. The majority were unemployed white males, with a median age of 44 years. Illicit drug use and criminal activity, while low, had not been eliminated totally. The majority were prescribed ampoules and few had significant health problems. In some cases patients had been transferred to injectable diamorphine from injectable methadone to reduce injection related problems. There were wide variations in dose. The majority of patients had no serious drug, health or social problems. Diamorphine prescribing was a long-term commitment. The experience from the United Kingdom has been one of long-term prescribing with the aim of retaining patients in treatment and reducing the harms caused by illicit drug use. Prospective studies are needed to determine the long-term consequences of receiving a diamorphine prescription.

Correction: Temporal Patterns of Influenza A and B in Tropical and Temperate Countries: What Are the Lessons for Influenza Vaccination?

[This corrects the article DOI: 10.1371/journal.pone.0152310.].

Quantitative analysis of patient-specific dosimetric IMRT verification.

Patient-specific dosimetric verification methods for IMRT treatments are variable, time-consuming and frequently qualitative, preventing evidence-based reduction in the amount of verification performed. This paper addresses some of these issues by applying a quantitative analysis parameter to the dosimetric verification procedure. Film measurements in different planes were acquired for a series of ten IMRT prostate patients, analysed using the quantitative parameter, and compared to determine the most suitable verification plane. Film and ion chamber verification results for 61 patients were analysed to determine long-term accuracy, reproducibility and stability of the planning and delivery system. The reproducibility of the measurement and analysis system was also studied. The results show that verification results are strongly dependent on the plane chosen, with the coronal plane particularly insensitive to delivery error. Unexpectedly, no correlation could be found between the levels of error in different verification planes. Longer term verification results showed consistent patterns which suggest that the amount of patient-specific verification can be safely reduced, provided proper caution is exercised: an evidence-based model for such reduction is proposed. It is concluded that dose/distance to agreement (e.g., 3%/3 mm) should be used as a criterion of acceptability. Quantitative parameters calculated for a given criterion of acceptability should be adopted in conjunction with displays that show where discrepancies occur. Planning and delivery systems which cannot meet the required standards of accuracy, reproducibility and stability to reduce verification will not be accepted by the radiotherapy community.

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.

Metabolism of an ingested serine load in psychotic and nonpsychotic subjects.

Our previous studies have shown that in psychotics, the plasma serine level is abnormally high and that plasma serine hydroxymethyltransferase (which cleaves serine to glycine) activity is abnormally low as compared with that in nonpsychotic subjects. In this study, psychotic and nonpsychotic subjects ingested a large bolus of L-serine (4 mM/kg) at breakfast and blood was drawn before breakfast, 2 hr, 4 hr, and 6 hr after serine ingestion. Baseline serine and SHMT activity differentiated between psychotics and nonpsychotics with high degrees of significance (p less than 0.0001) and p less than 0.01, respectively). Plasma serine levels 2 hr after serine ingestion were significantly higher (p less than 0.01) in nonpsychotics as compared with psychotics. Elimination of serine in psychotics was bimodal and was significantly different from that of nonpsychotics (p less than 0.0079, Moses test). These findings provide additional evidence for abnormal serine metabolism in psychotic patients.

Biochemical diversity in a phage display library of random decapeptides.

Detailed knowledge and understanding of the structure and function of biologically important macromolecules is frequently insufficient to permit rational, de novo design of recognition molecules and therapeutics. Traditional drug discovery has, thus, focused on screening and identifying native molecules as drugs or as templates for genetic engineering or organic synthesis. The number and novelty of lead compounds for drug discovery might be expanded significantly, however, by the ability to express and screen large libraries of peptide structures with phage display technologies [Scott and Smith, Science 249 (1990) 386-390]. The significance of such libraries as sources of novel biological ligands will depend in part on the depth and degree of biochemical diversity they comprise. We have prepared a phage display library of greater than 2 x 10(6) individual decapeptides produced as N-terminal fusions to the pIII surface protein of fd filamentous phage. The decapeptides were expressed from a degenerate DNA insert sequence that was chemically synthesized with an equal mixture of all four nucleotide bases at the three positions in each of the ten codons. Fifty-two clones were picked randomly and without prior selection from the population and the sequences of their peptide inserts were determined. Our results confirm and document the broad representation at the primary amino acid sequence level that is expected in a library expressed from random DNA inserts. More significantly, biochemical characterization shows these insert sequences correspond to structures comprising a wide range and combination of isoelectric, hydropathic, and biochemical properties necessary in drug discovery to access a significant percentage of the repertoire of possible peptide structures by affinity or activity screening.

Primary structures of two homologous subunits of PA28, a gamma-interferon-inducible protein activator of the 20S proteasome.

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28 alpha and PA28 beta, are about 50% identical to one another and are highly conserved between rat and human. PA28 alpha and PA28 beta are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28 alpha, but neither PA28 beta nor Ki antigen, contains a 'KEKE motif', which has been postulated to promote the binding of proteins having this structural feature. PA28 alpha and PA28 beta were coordinately regulated by gamma-interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to gamma-interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

Relation between body fat and age in 4 ethnic groups.

BACKGROUND: Previous studies of the relation between age and body fat reached differing conclusions concerning the question of whether body fat is lower in the elderly than in middle-aged persons. OBJECTIVE: The objectives of this study were to characterize the relation between age and body fat in 4 ethnic groups and test the hypothesis that body fat is lower in the elderly than in middle-aged persons. DESIGN: Body fat was measured in a sample of 1324 volunteers aged 20-94 y by using a 4-component model of body composition. Four ethnic groups were studied: Asians, blacks, Puerto Ricans, and whites. Regression models were developed for fat mass and fat percentage as functions of age. RESULTS: In all but one of the groups, a highly significant curvilinear relation between age and body fat was found, indicating a peak amount of body fat in late middle age and lower amounts of body fat at younger and older ages (P < 0.001). The age at which maximum body fat was predicted in the various groups ranged from 53 to 61 y for fat mass and from 55 to 71 y for fat percentage. In Puerto Rican men there was no significant relation between age and fat mass, and the relation between age and fat percentage was linear and positively correlated. CONCLUSIONS: This study provided data on the relation between age and body fat in 4 ethnic groups and supported the hypothesis that body fat is lower in the elderly than in middle-aged persons.

Effects of glutaminase inhibition on release of endogenous glutamic acid.

The effects of four inhibitors of glutamine hydrolysis on synaptosomes derived from several regions of the brain were studied. The calcium-specific release of endogenous glutamic acid was determined in the presence of varying concentrations of 6-diazo-5-oxo-norleucine (DON), N-ethyl-maleimide (NEM), 2-chloroadenosine (2-CA) or haloperidol. Both DON and NEM reduced the calcium-specific release in a concentration-dependent manner, equally in all regions tested. 2-Chloroadenosine also decreased release and the effect was most evident in the amygdala. As reported earlier, haloperidol blocked release of glutamic acid only in the amygdala. In synaptosomes from the amygdala, both DON and NEM failed to affect the calcium-specific release of aminobutyric acid (GABA), glycine or serotonin at concentrations which reduced release of glutamate by 50%; NEM, but not DON, elevated the release of dopamine. Dopamine itself affected neither the release of glutamate nor its blockade by haloperidol even in extremely large concentrations.