A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.
Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.


Evidence for macrophage-mediated protection against lethal Candida albicans infection.
Systemic infection of mice with a Candida albicans strain (PCA-2) incapable of yeast-mycelial conversion conferred protection against a subsequent intravenous challenge with the pathogenic strain of the parent organism, strain CA-6. Protection was nonspecific since it was also detected upon challenge of mice with Staphylococcus aureus. Moreover, the PCA-2 organisms had to be viable, their effects being most evident when they were given intravenously at a dose of 10(6) cells 7 to 14 days prior to microbial challenge. Thus, all mice pretreated with PCA-2 and challenged 14 days later with viable CA-6 cells lived through a 60-day observation period, whereas all control mice not treated with PCA-2 died within 3 days. In an attempt to correlate the immunostimulatory effects observed in vivo with possible modifications in in vitro functions, it was found that administration of PCA-2 was accompanied by an increase in the number of peripheral blood polymorphonuclear cells and by the activation in the spleen of cells with highly candidacidal activity in vitro. Moreover, the adoptive transfer of plastic-adherent cells from PCA-2-infected mice into histocompatible recipients conferred considerable protection against subsequent CA-6 challenge.


Computational Modeling of Conformer Stability in Benenodin-1, a Thermally Actuated Lasso Peptide Switch.
Benenodin-1 is a thermally actuated lasso peptide rotaxane switch with two primary translational isomers that differ in the relative position of the residue Gln15. The conversion from one conformer to the other involves substantial enthalpy-entropy compensation: one conformer is energetically favored and the other is entropically favored. Here, we take a multi-scale quantum mechanical (QM) and classical molecular dynamic (MD) approach to reveal residue-specific sources of these differences in stability. QM reveals that the two benenodin-1 conformers involve distinct hydrogen bonding networks, with the enthalpically favored conformer having more intra-peptide hydrogen bonds between the Gln15 side chain and nearby residues. The evaluation of configurational entropy over the MD-sampled geometries reveals that the entropically favored conformer has enhanced conformational flexibility. By computing the by-residue-sum entropies, we identify the role of Gln15 and neighboring Glu14 in mediating the entropic variation during the switching process. These computational insights help explain the effects of Glu14Ala and Gln15Ala mutations on the conformational population of benenodin-1 observed experimentally.


Modelling reading development through phonological decoding and self-teaching: implications for dyslexia.
The most influential theory of learning to read is based on the idea that children rely on phonological decoding skills to learn novel words. According to the self-teaching hypothesis, each successful decoding encounter with an unfamiliar word provides an opportunity to acquire word-specific orthographic information that is the foundation of skilled word recognition. Therefore, phonological decoding acts as a self-teaching mechanism or 'built-in teacher'. However, all previous connectionist models have learned the task of reading aloud through exposure to a very large corpus of spelling-sound pairs, where an 'external' teacher supplies the pronunciation of all words that should be learnt. Such a supervised training regimen is highly implausible. Here, we implement and test the developmentally plausible phonological decoding self-teaching hypothesis in the context of the connectionist dual process model. In a series of simulations, we provide a proof of concept that this mechanism works. The model was able to acquire word-specific orthographic representations for more than 25 000 words even though it started with only a small number of grapheme-phoneme correspondences. We then show how visual and phoneme deficits that are present at the outset of reading development can cause dyslexia in the course of reading development.


Recent Advances in the Biosynthesis of Polyhydroxyalkanoates from Lignocellulosic Feedstocks.
Polyhydroxyalkanoates (PHA) are biodegradable polymers that are considered able to replace synthetic plastic because their biochemical characteristics are in some cases the same as other biodegradable polymers. However, due to the disadvantages of costly and non-renewable carbon sources, the production of PHA has been lower in the industrial sector against conventional plastics. At the same time, first-generation sugar-based cultivated feedstocks as substrates for PHA production threatens food security and considerably require other resources such as land and energy. Therefore, attempts have been made in pursuit of suitable sustainable and affordable sources of carbon to reduce production costs. Thus, in this review, we highlight utilising waste lignocellulosic feedstocks (LF) as a renewable and inexpensive carbon source to produce PHA. These waste feedstocks, second-generation plant lignocellulosic biomass, such as maize stoves, dedicated energy crops, rice straws, wood chips, are commonly available renewable biomass sources with a steady supply of about 150 billion tonnes per year of global yield. The generation of PHA from lignocellulose is still in its infancy, hence more screening of lignocellulosic materials and improvements in downstream processing and substrate pre-treatment are needed in the future to further advance the biopolymer sector.


[Comparative characteristics of left ventricular remodeling in dilatation cardiomyopathy and ischemic disease].
Left ventricular remodelling plays an important pathogenetic role in progressive circulatory insufficiency both in dilated cardiomyopathy (DCMP) and in ischemic heart disease with postinfarction cardiosclerosis. In terminal cardiac failure structural changes (changed geometry of the left ventricle, in particular) become stereotypic. The dobutamin test in DCMP reveals low functional reserve of the total and segmentary contractility compared to ischemic cardiomyopathy. This may help in echocardiographic diagnostic difficulties.


Paediatric care in relation to the 2014-2015 Ebola outbreak and general reporting of deaths in Sierra Leone.
Setting: All peripheral health units countrywide in Sierra Leone and one hospital in Port Loko. Objectives: Sierra Leone was severely affected by the 2014-2015 Ebola outbreak, whose impact on paediatric care and mortality reports merits assessment. We sought to compare the periods before, during and after the Ebola outbreak, the countrywide trend in morbidities in children aged < 5 years and exit outcomes in one district hospital (Port Loko). During the Ebola outbreak period, gaps in district death reporting within the routine Health Management Information System (HMIS) were compared with the Safe and Dignified Burials (SDB) database in Port Loko. Design: This was a retrospective records analysis. Results: The average number of monthly consultations during the Ebola outbreak period declined by 27% for malaria and acute respiratory infections and 38% for watery diarrhoea, and did not recover to the pre-Ebola levels. For measles, there was an 80% increase during Ebola, which multiplied by 6.5-fold post-Ebola. The number of unfavourable hospital exit outcomes was 52/397 (13%) during Ebola, which was higher than pre-Ebola (47/496, 9%, P = 0.04). Of 6565 deaths reported in the Port Loko SDB database, only 2219 (34%) appeared in the HMIS, a reporting deficit of 66%. Conclusion: The Ebola disease outbreak was associated with reduced utilisation of health services, and appears to have triggered a measles epidemic. Almost 70% of deaths were missed by the HMIS during the Ebola outbreak period. These findings could guide health system responses in future outbreaks.


Prevalence of typical circle of Willis and the variation in the anterior communicating artery: A study of a Sri Lankan population.
To determine the extent of hypoplasia of the component vessels of the circle of Willis (CW) and the anatomical variations in the anterior communicating artery (AcomA) in the subjects who have died of causes unrelated to the brain and compare with previous autopsy studies. The external diameter of all the arteries forming the CW in 225 normal Sri Lankan adult cadaver brains was measured using a calibrated grid to determine the occurrence of "typical" CWs, where all of the component vessels had a diameter of more than 1 mm. Variations in the AcomA were classified into 12 types based on Ozaki et al., 1977. 193 (86%) showed "hypoplasia", of which 127 (56.4%) were with multiple anomalies. Posterior communicating artery (PcoA) was hypoplastic bilaterally in 93 (51%) and unilaterally in 49 (13%). Precommunicating segment of the posterior cerebral arteries (P1) was hypoplastic bilaterally in 3 (2%), unilaterally in 14 (4%), and AcomA was hypoplastic in 91 (25%). The precommunicating segment of the anterior cerebral arteries (A1) was hypoplastic unilaterally in 17 (5%). Types of variations in the AcomA were: single 145 (65%), fusion 52 (23%), double 22 (10%) [V shape, Y shape, H shape, N shape], triplication 1 (0.44%), presence of median anterior cerebral artery 5 (2%), and aneurysm 1 (0.44%). The occurrence of "typical" CW in autopsy brains was rare. Further studies would be necessary to determine if these anatomical variations could predispose to cerebral ischemia and premature stroke in the Sri Lankan population.


Reparation of Isoniazid and Rifampicin Combinatorial Therapy-Induced Hepatotoxic Effects by Bacopa monnieri.
Drug-induced liver injury is a major challenge in treating tuberculosis with isoniazid (INH) and rifampicin (RIF). This study was aimed at evaluating the protective effects of Bacopamonnieri (Brahmi) against INH and RIF-induced hepatotoxicity in a rat model and also to study the patterns of interaction between pregnane X receptor (PXR) and chosen active compounds of B. monnieri. Hepatotoxicity was induced in the experimental animals by the oral administration of INH and RIF (50 mg/kg b.w. each/day) for 28 days. The effects of co-administration of B. monnieri (500 mg/kg b.w./day) in INH- and RIF-induced rats were studied by the estimation of biochemical analyses. The standard hepatoprotective drug silymarin (25 mg/kg b.w./day) was used for the purpose of comparison. In silico docking experiments were carried out using the PatchDock server and the results were analysed on the PyMol molecular viewer. There was significant reduction in the antioxidant status of INH and RIF-induced rats. Also, there was significant elevation in the levels of serum liver function markers in the INH- and RIF-induced rats. B. monnieri was able to normalise the tested parameters. In silico studies reveal significant interaction between PXR and bacopaside I. B. monnieri exerts significant protective effects against INH and RIF-induced hepatotoxicity in rats.


Choroid plexus hemorrhage in premature neonates: recognition by sonography.
In 34 consecutive infants admitted to the neonatal intensive care unit with birth weight of less than 1,500 g, 80 cranial real-time sonograms were obtained to determine the incidence of choroid plexus hemorrhage. Choroid plexus hemorrhage was diagnosed only in the absence of germinal matrix hemorrhage. Diagnostic criteria included choroid plexus nodularity, enlargement (greater than 12 mm in anteroposterior diameter), or asymmetry between right and left (greater than 5 mm). Ipsilateral intraventricular clots or occipital horn dilatation supported the diagnosis of choroid plexus hemorrhage in most cases. Choroid plexus hemorrhage appeared to be the sole bleeding site in 10 (59%) of the 17 patients with intracranial hemorrhage. Hemorrhage in the region of the caudate nucleus was seen in the other seven cases (41%). Ventricular dilatation and/or intraventricular hemorrhage accompanied nine (90%) of the 10 cases of choroid plexus hemorrhage. This study suggests that in very low-birth-weight premature neonates, the choroid plexus may be a more frequent site of intracranial hemorrhage than previously believed.


Westernization and non-insulin-dependent diabetes in Australian Aborigines.
When Australian Aborigines make the transition from their traditional hunter-gatherer life-style to a westernized life-style, they develop high prevalence rates of obesity (with an android pattern of fat distribution), non-insulin-dependent diabetes, impaired glucose tolerance, hypertriglyceridemia, hypertension, and hyperinsulinemia. Insulin resistance may be the common pathogenetic characteristic of this cluster of conditions associated with increased risk of cardiovascular disease. The traditional hunter-gatherer life-style, characterized by high physical activity and a diet of low energy density (low, fat, high fiber), promoted the maintenance of a very lean body weight and minimized insulin resistance. In contrast, for most Aborigines, western life-style is characterized by reduced physical activity and an energy-dense diet (high in refined carbohydrate and fat) that promotes obesity and maximizes insulin resistance. Intervention strategies aimed at prevention of insulin-resistance-related chronic disease should be directed at life-style modification. To be effective, such programs will have to be developed and controlled by Aboriginal communities.


Mechanical properties of contracted canine bronchial segments in vitro.
Airways of 2-5 mm in diameter were dissected from fresh dog lungs and mounted in a tissue bath so that they could be inflated and deflated with air. Length was held constant, and pressure and volume were monitored on an X-Y recorder. Pressure-volume curves were obtained for each airway at several difference degrees of constriction, by varying the concentration of acetylcholine (ACh) in the bath. Properties of the tissue were analyzed cy cycling airways at different speeds and by inflating them to various pressures. Circumferential length-tension curves were calculated from the inflation limbs of pressure-volume curves obtained by slow cycling (90 s for a complete cycle between -10 and 40 cmH2O). Administration of a series of increasing concentrations of ACh resulted in length-tension curves that were displaced to shorter lengths but not greatly altered in slope. These results suggest that slow inflation of the bronchus stretched only passive elements in series with a contractile element which shortened to the length determined by the concentration of ACh present.


Structure of Burkholderia cepacia UDP-glucose dehydrogenase (UGD) BceC and role of Tyr10 in final hydrolysis of UGD thioester intermediate.
Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.


Universal precautions training of preclinical students: impact on knowledge, attitudes, and compliance.
Little information exists regarding the impact of universal precautions training programs on preclinical students' knowledge, attitudes, and behavior. We developed, implemented, and assessed an educational program in universal precautions for 2nd-year medical and preclinical physician assistant students. Students (n = 170) completed pre- and post-training questionnaires to assess universal precautions knowledge and to evaluate attitudes about their perceived risk for bloodborne pathogen infection, the importance of universal precautions procedures, and their willingness to provide care for human immunodeficiency virus (HIV)-positive or acquired immune deficiency syndrome (AIDS) patients. Phlebotomy, intravenous catheter insertion, and arterial blood gas sampling techniques were demonstrated, practiced, and evaluated during practical training sessions. Outcome measures included changes in pre- and posttraining knowledge scores and attitudes, as well as observed compliance with universal precautions during practical training. Universal precautions knowledge scores increased significantly after training (P < 0.0001). Personal assessments of the risk of developing HIV due to patient care significantly decreased (P < 0.0001) and willingness to provide care for AIDS patients increased (P = 0.004) following training. Importantly, students reported that high expected rates of contact with HIV-positive and other patient groups would not significantly affect their specialty choice. Observed compliance with universal precautions procedures during practical training ranged from 95 to 99% for glove use, 76 to 77% for direct sharps disposal without needle recapping, and 56 to 78% for handwashing after glove removal during phlebotomy and intravenous catheter insertion. This program is effective in increasing students' knowledge of universal precautions. Training favorably affects students' willingness to care for HIV-positive patients and their assessed risk of developing occupational bloodborne infection.


Renal nerves and initial excretory responses to recumbency: a species comparison.
Experiments were performed in anesthetized Macaca fascicularis monkeys and mongrel dogs to determine if there are species differences concerning the involvement of the renal nerves in mediating the initial renal effects occurring with the assumption of recumbency. All animals underwent acute unilateral renal denervation with the contralateral kidney serving as the innervated control. Renal perfusion pressure was controlled throughout each experiment. Control measurements were made with all animals tilted head up 45 degrees, and recumbent measurements were made 5-15 min after the animals were lowered to 0 degree. In the monkeys, similar increases in urine flow, absolute and fractional sodium excretion, and creatinine and p-aminohippurate clearances occurred in both the innervated and denervated kidneys during recumbency. In contrast, in the dogs, renal denervation abolished these responses in that increases in these parameters occurred in the innervated kidneys only. These results demonstrate that the initial diuresis and natriuresis of recumbency are dependent on the presence of the renal nerves in the dog but not in the monkey.


Inhibition of tumor necrosis factor-alpha- and interleukin-1-induced endothelial E-selectin expression by thiol-modifying agents.
The expression of endothelial-leukocyte adhesion molecules has been postulated to be regulated by redox-sensitive events. Tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1 (IL-1)-induced E-selectin expression was analyzed after pretreating human umbilical vein endothelial cells with different thiol-modifying agents, ie, diamide, phenylarsine oxide, N-ethylmaleimide, and diethyl maleate. E-selectin protein expression was quantified by indirect immunofluorescence. All compounds suppressed the cytokine-induced E-selectin expression in a concentration-dependent manner, whereas the antioxidant N-acetylcysteine showed no effect. The inhibitory effect of diamide (100 micromol/L, 1 hour) was reversible within 6 hours when the cells were allowed to recover before application of cytokines. Reversibility was strongly delayed when cells were deprived of glutathione by buthionine sulfoximine pretreatment. Glutathione depletion alone did not influence cytokine-induced E-selectin expression. Analysis of cellular glutathione status showed a 3-fold increase in oxidized glutathione after diamide treatment. Monochlorobimane labeling also revealed a decrease in total cellular thiols. During recovery, the glutathione status was restored within 1 hour, whereas total thiol content and E-selectin expression needed at least 6 hours to return to baseline. Complete inhibition of E-selectin expression by the vicinal thiol blocker phenylarsine oxide (0.5 micromol/L) was reversed by dithiols like dithiothreitol or dimercaptopropanol, but not by the monothiol 2-mercaptoethanol. These data suggest that proteins with essential thiols, most probably vicinal thiols. are involved in the IL-1- and TNF-alpha-mediated induction of E-selectin. These thiols must be in the reduced state; oxidation or other modification thereof attenuates or abolishes the cells' response to the cytokines.


A new mutation (db3J) at the diabetes locus in strain 129/J mice. II. Studies of pancreatic alpha cell function in culture.
Monolayer cell cultures from pancreatic islets of aging 129/J strain diabetes (db3J/db3J) and lean littermate control mice were tested for differences in glucagon and insulin secretion in either serum-free Eagle's minimal essential medium (MEM) or Dulbecco's modified minimal essential medium (DMEM). There was a highly significant (p less than 0.0001) main effect of genotype and type of culture medium on glucagon secretion with time. Thus, although numbers of A-cells were not demonstrably increased in db3J/db3J cultures in DMEM, mean medium glucagon levels increased 2.7-, 18-, and 32-fold above littermate normal culture levels at days 4, 6, and 8 respectively. In MEM, the two populations could not be discriminated on the basis of glucagon secretion. By contrast, insulin secretion over culture days showed a highly significant (p less than 0.0001) dependence on genotype, but not type of medium, with the B-cell enriched db3J/db3J preparations secreting between 20 and 30 times as much insulin as controls in both medida. Analysis revealed that the heightened secretory responsiveness of mutatn A-cells in DMEM as compared to MEM was primarily elicited by the elevated DMEM amino acid concentration and specifically lysine (0.8 mmol/l in DMEM versus 0.4 mmol/l in MEM). In pulse-chase experiments using 14 day db3J/db3J cultures, incorporation of 3H-tryptophan into protein that eluted from Biogel P-10 columns in the native glucagon peak indicates that DMEM stimulated glucagon biosynthesis as well as secretion. This study reveals an augmented sensitivity of db3J/db3J A-cells to stimulation by basic amino acids in long-term culture.


Liver x receptor signaling pathways and atherosclerosis.
First discovered as orphan receptors, liver X receptors (LXRs) were subsequently identified as the nuclear receptor target of the cholesterol metabolites, oxysterols. There are 2 LXR receptors encoded by distinct genes: LXRalpha is most highly expressed in the liver, adipose, kidney, adrenal tissues, and macrophages and LXRbeta is ubiquitously expressed. Despite differential tissue distribution, these isoforms have 78% homology in their ligand-binding domain and appear to respond to the same endogenous ligands. Work over the past 10 years has shown that the LXR pathway regulates lipid metabolism and inflammation via both the induction and repression of target genes. Given the importance of cholesterol regulation and inflammation in the development of cardiovascular disease, it is not surprising that activation of the LXR pathway attenuates various mechanisms underlying atherosclerotic plaque development. In this brief review, we will discuss the impact of the LXR pathway on both cholesterol metabolism and atherosclerosis.


NK cell isolation from liver biopsies: phenotypic and functional analysis of low cell numbers by flow cytometry.
Natural killer (NK) cells are considered to play a critical role in liver disease. However, the available numbers of intrahepatic lymphocytes (IHL) derived from liver biopsies (LB) for ex vivo analysis of intrahepatic NK cells is very limited; and the isolation method may hamper not only yields and viability, but also phenotype and function of IHL. The aim of the present study was therefore to (1) refine and evaluate the cell yields and viability of a modified isolation protocol from standard size needle LB; and (2) to test the effects of mechanical dissociation and enzymatic tissue digestion, as well as the analysis of very low cell numbers, on the phenotype and function of intrahepatic NK cells. Peripheral blood mononuclear cells (PBMC) and IHL, freshly isolated from the peripheral blood, LB (n = 11) or partial liver resections (n = 5), were used for phenotypic analysis by flow cytometry. NK cell function, i.e., degranulation and cytokine production, was determined by staining of CD107a and intracellular IFN-γ following in vitro stimulation. The mean weight of the LB specimens was 9.1 mg, and a mean number of 7,364 IHL/mg were obtained with a viability of >90%. Exposure of IHL and PBMC to 0.5 mg/ml collagenase IV and 0.02 mg/ml DNase I for 30 min did affect neither the viability, NK cell function, nor the percentages of CD56(+), NKp46(+), and CD16(+) NK cells, whereas the level of CD56 surface expression was reduced. The phenotype of LB-derived NK cells was reliably characterized by acquiring as few as 2,500 IHL per tube for flow cytometry. The functional assay of intrahepatic NK cells was miniaturized by culturing as few as 25,000 IHL in 25 μl (10(6)/ml) using 96-well V-bottom plates with IL-2 and IL-12 overnight, followed by a 4 h stimulation with K562 cells at a NK:K562 ratio of 1:1. In summary, we report reliable phenotypic and functional analyses of small numbers of intrahepatic NK cells isolated from LB specimens providing us with a tool to better address the emerging role of human NK cell immunobiology in liver diseases.


Rhodobacter sphaeroides has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria.
The cytoplasmic pyrophosphatase from Rhodobacter sphaeroides was purified and characterized. The enzyme is a homodimer of 64 kDa. The N-terminus was sequenced and used to obtain the complete pyrophosphatase sequence from the preliminary genome sequence of Rba. sphaeroides, showing extensive sequence similarity to family II or class C pyrophosphatases. The enzyme hydrolyzes only Mg-PP(i) and Mn-PP(i) with a K(m) of 0.35 mM for both substrates. It is not activated by free Mg (2+), in contrast to the cytoplasmic pyrophosphatase from Rhodospirillum rubrum, and it is not inhibited by NaF, methylendiphosphate, or imidodiphosphate. This work shows that Rba. sphaeroides and Rhodobacter capsulatus cytoplasmic pyrophosphatases belong to family II, in contrast to Rsp. rubrum, Rhodopseudomonas palustris, Rhodopseudomonas gelatinosa, and Rhodomicrobium vannielii cytoplasmic pyrophosphatases which should be classified as members of family I. This is the first report of family II cytoplasmic pyrophosphatases in photosynthetic bacteria and in a gram-negative organism.


The mechanism of activation of plasminogen at the fibrin surface by tissue-type plasminogen activator in a plasma milieu in vitro. Role of alpha 2-antiplasmin.
The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.


Lack-of-fit testing of ion chromatographic calibration curves with inexact replicates.
Calibration studies involve the preparation and analysis of replicates for multiple concentrations of standards. Curves that are fitted through the data are evaluated for their adequacy of fit. A helpful test is a lack-of-fit procedure, which is performed easily by most statistical software. When coupled with Radj2, the procedure differentiates between data that are not linear and those that are simply noisy. The test requires data from exact replicates of the various standard levels involved. However, in ppt-level ion chromatography, the above condition may be impossible to meet. With the common anions (e.g., chloride, nitrite), the working standards must be prepared by mass and all liquids must be poured; transfer pipets contaminate at these concentrations. Since it is virtually impossible to pour out the desired mass exactly, final concentrations will vary slightly. Consequently, a different approach is needed for lack-of-fit testing. This paper discusses reasonable alternatives and applies them to actual data.


The epidemiology of ice hockey injuries.
As part of the injury profylaxes in Denmark a questionnaire investigation was undertaken in 14 randomly chosen ice hockey teams--out of 266 players, 210 answered (79%). The injury incidence per player per 1000 hours was 4.7, i.e. 1.5 in training and 38.0 in match. Half of the injuries were localised to the head (28%) and lower extremities (27%), 19% to the upper extremities and 7% to the back. Of these 48% were contusions. Knee and elbow injuries were of longest duration. The necessity for increased shock absorption in helmets and barriers as well as built-in rotational and collateral stabilizers in the existing knee protectors for injury prophylaxis is stressed.


Molecular diversity assessment of arctic and boreal Agaricus taxa.
We provide a phylogenetic diversity assessment study in genus Agaricus as part of our ongoing work to saturate ITS and LSU rDNA sequence diversity of soil-dwelling fungi in Alaska. Pairwise sequence similarity-based groupings and statistical parsimony analyses were applied to delimit operational taxonomic unit (OTU) and were compared to results of full phylogenetic analyses. Our results show that the proportion of section Arvenses taxa is particularly high in the boreal forest and hypo-arctic (low arctic) regions, whereas the genus is represented by section Agaricus in high arctic habitats. Furthermore our findings suggest that the commercially important A. bisporus occurs naturally in the boreal region of interior Alaska, substantially expanding the known northern limit of the species. Delimitations of OTU varied greatly with different methods. In general 95% similarity-based grouping proved to be the least sensitive method, often resulting in section- and subsection-level groups. The 95% connection-limit statistical parsimony separated far more groups. The 98% similarity-based groups and the 98% connection limit networks recognized respectively 11 and 13 OTU containing our specimens. The 98% connection limit statistical parsimony was the only method in which all recognized OTU consisted of members grouped by branches with significant (> .95) posterior probabilities, providing an independent support for the groups. Our results also point out that considerable additional efforts will be needed to elucidate the evolution of this diverse genus and to assess its phylogenetic diversity, given that most taxa in our analyses could not be placed convincingly within well characterized species using ITS/LSU data.


Selective induction of manganous superoxide dismutase in human monocytes.
Radioimmunoassays for both human copper-zinc and manganous superoxide dismutases (Cu-Zn SOD and Mn SOD, respectively) have been developed, validated, and utilized to measure the concentrations of these enzymes in cultured monocytes. Monocyte Mn SOD increased 4.7-fold over basal during 3 days of culture, an increase that was markedly enhanced by stimulation with bacterial lipopolysaccharide (LPS). Cu-Zn SOD showed a transient decrease over the culture period but was unaffected by LPS. Stimulation with muramyl dipeptide had minimal effect on Mn SOD and no effect on Cu-Zn SOD during culture, even at a concentration capable of activating the monocytes, as defined by zymosan-induced superoxide production.


[Fibronectin levels in the blood and cerebrospinal fluid in cerebrovascular diseases].
A total of 106 patients with different forms of cerebral circulation disorders and 28 healthy donors were investigated for the contents of fibronectin and its proteolytic degradation products in the blood and cerebrospinal fluid. In patients with cerebrovascular disorders the decrease in the blood fibronectin levels was accompanied by a decrease in its metabolite levels and increased R-protein content, irrespective of the character and degree of clinical manifestations. The fibronectin CSF levels were increased in acute stroke with unconsistent rise in R-proteins. The authors suggest that fibronectin synthesis and degradation are evidently controlled by R-proteins being the products of destruction of cellular receptors.


Lymphocyte proliferation in vitro induced by hapten autologous protein conjugates. I. A study on the class of lymphocytes responding in vitro and on the nature and specificity of their receptors.
Immune lymph node cells from guinea pigs respond to soluble antigen in vitro by an increase in DNA synthesis. Optimal conditions for this proliferative response were studied in the present article. Under such conditions, immune cells showed increasing responses with increasing antigen concentration in vitro, the threshold dose of activation frequently being as low as 0.02 microg per culture. In contrast, normal lymph node cells (from FCA-stimulated animals) did only respond to antigen at very high doses (20 mg/culture), and immune cell dilution studies could be performed in normal cells without changing the kinetics of the antigen specific response of immune cells. Fractionation on anti-Ig columns indicated that purified, immune T lymphocytes were quite capable of proliferating in vitro upon antigen stimulation. However, our attempts to adsorb the proliferating cells onto chemically defined immunoadsorbants failed despite the fact that immune B cells (as measured by the rosette assay) were retained almost completely by such a procedure. Purified, immune T lymphocytes from guinea pigs immunized with different antigen concentrations in vivo and/or obtained at different times after immunization were tested for a differential sensitivity toward antigen-induced DNA synthesis in vitro. However, we were not able to demonstrate any regular increase in sensitivity to antigen in vitro, and if found, it seemed to be more dependent upon the number of antigen reactive cells in the population studied rather than upon differences in the average avidity of the receptors on the cells proliferating in vitro. The results in the present article are discussed in relation to current knowledge and hypotheses on T-lymphocyte receptors.


[Natural regeneration of young Excentrodendron hsienmu in karst mountainous region in Southwest Guangxi, China].
A field survey was conducted in the karst mountainous region in Pingguo County of Southwest Guangxi, China to explore the structural characteristics, spatial distribution pattern, and growth dynamics of young Excentrodendron hsienmu as well as the main environmental factors affecting the natural regeneration of the E. hsienmu population. In the study area, the population structure of the young E. hsienmu was stable, and exhibited a clumped spatial pattern for the seedlings and seedling sprouts. The ground diameter growth and height growth of the young E. hsienmu presented the same variation trend, i. e., the ground diameter increased with increasing height. The ground diameter growth and height growth of the E. hsienmu seedlings were limited by population density, i. e., decreased with increasing population density. The correlation analysis showed that the trees more than 2.5 m in height and the shrubs were the major stand factors affecting the natural regeneration of young E. hsienmu, while the herbs had no significant correlation with the regeneration. The percentage of covered rock also had no significant effects on the regeneration. Kruskal-Wallis ANOVA showed that there existed significant differences in the height and ground diameter of young E. hsienmu at different slope degrees and slope positions. The population density, height, and ground diameter had significant differences across slope aspects. The natural regeneration of young E. hsienmu was comprehensively affected by the species biological characteristics, intraspecific competition, interspecific competition, heterogeneous habitat, and anthropogenic disturbances.


Association of megalin genetic polymorphisms with prostate cancer risk and prognosis.
Megalin, an endocytic receptor expressed by prostate epithelial cells, can internalize biologically active androgens bound to sex hormone binding globulin. Genetic variation within megalin could potentially influence levels of steroid hormone uptake. Forty haplotype-tagging single-nucleotide polymorphisms (htSNP) were analyzed in a population-based, case-control study of 553 Caucasian men who were diagnosed with prostate cancer between the ages of 40 and 64 years from the Seattle-Puget Sound region and 534 control men. Prostate cancer risk was estimated using adjusted unconditional logistic regression for both individual SNPs and haplotypes. Risks of disease recurrence/progression and prostate-specific cancer mortality were estimated using Cox proportional hazards regression. We found no strong evidence of altered risk of developing prostate cancer for any of the htSNPs when they were assessed individually or in haplotypes. However, three htSNPs were significantly associated with both disease recurrence/progression and mortality. Risk of recurrence/progression alone was also associated with five additional htSNPs, and six other htSNPS showed evidence of modification by primary androgen deprivation therapy. Two additional htSNPs were significantly associated with altered risk of death from prostate cancer. Preliminary results suggest that common genetic variation within the megalin gene could alter both risk of recurrence/progression and prostate-specific cancer mortality. In addition, androgen deprivation therapy effectiveness may be modified by the activity of this gene. To our knowledge, this is the first study that has examined polymorphisms within the megalin gene for associations with prostate cancer risk and outcomes.


Medication reconciliation and review for older emergency patients requires improvement in Finland.
10-30% of hospital stays by older patients are drug-related. The admission phase is important for identifying drug-related problems, but taking an incorrect medication history often leads to medication errors. To enhance medication history recording and identify drug-related problems (DRPs) of older patients admitted to emergency departments (EDs). DRPs were identified by pharmacists-led medication reconciliation and review procedures in two EDs in Finland; Helsinki University Hospital (HUS), and Kuopio University Hospital (KUH). One-hundred-and-fifty patients aged ≥65-years, living at home and using ≥6 medicines were studied. 100% of patients (N = 75) in HUS and 99% in KUH (N = 75), had discrepancies in their admission-medication chart recorded by the nurse or physician. Associations between admission-diagnosis and drug-related problems were found in 12 patients (16%) in HUS and 22 patients (29%) in KUH. Of these, high-alert medications (e.g. antithrombotics, cytostatics, opioids) were linked to eight patients (11%) in HUS and six patients (8%) in KUH. Other acute DRPs were identified in 19 patients (25%) in HUS and 54 patients (72%) in KUH. Furthermore, 67 patients (89%) in HUS and all patients in KUH had non-acute DRPs. Medication reconciliation and review at admission of older ED patients requires improvement in Finland.


[Alcohol consumption patterns and associated factors among elderly Brazilians: National Health Survey (2013)].
The scope of this study was to analyze the pattern of alcohol consumption among elderly Brazilians (60 years and over) and their association with sociodemographic factors, lifestyle habits and health conditions. This is a cross-sectional study of 10,537 elderly (90.1%) participants from the National Health Survey of 2013. The consumption of alcoholic beverages was classified as non-use, mild / moderate use and risk use. The multinomial regression model was used to study the associated factors. The prevalence for mild / moderate and risk use was 9.4% (95% CI: 8.4- 10.6%) and 4.6% (95%CI: 4.0-5.3%), respectively. The two consumption patterns were inversely associated with age and more frequent among men, better schooling, smokers and physical activity practitioners. Mild / moderate consumption was less frequent among non-whites and those with a history of stroke and diabetes, whereas risk use was less frequent among the elderly diagnosed for heart disease and more frequent among those suffering from depression. This result identifies profiles of greater vulnerability, with small differences between two patterns of consumption. This information should be considered in the preparation of proposals to promote healthy habits and control of alcohol use among the elderly.


[Treatment of complications of pylorus preserving stomach resection].
Causes of unsatisfactory outcomes of pylorus preserving stomach resection are analyzed, method of prophylaxis and surgical correction is proposed. Pylorus preserving stomach resection was performed in 207 patients with chronic gastric ulcer. 2 groups of patients were compared: 166 patients who have undergone pylorus preserving stomach resection by Maky--Gorobashko (group 1); 41 patients operated according to an original method of suprapyloric stomach resection with preserving of distal Latarget nerves on serous-muscular flap formed from lesser curvature of the stomach (group 2). Of 122 examined patients of group 1 excellent and good long-term results according to Visick's criteria were achieved in 101 (82.79%), satisfactory--in 12 (9.84%), unsatisfactory--in 9 (7.37%). Bad stump evacuation due to a stable spasm of the denervated pylorus and hypokinesia of the stump were the main causes of unsatisfactory results. In group 2-39 of 41 patients were examined in long-term period. Excellent and good results were achieved in 38 patients, satisfactory--in 1. There were no unsatisfactory results.


How do we fit underground coal mining work boots?
Acceptable footwear fit, particularly width, is subjective and vaguely quantified. Proper shoe fit is important because it affects both comfort and the potential to prevent injury. Although mismatches between the feet of underground coal miners and their internal boot dimensions are known, no research has been undertaken to determine the impact of these mismatches on worker perceptions of fit, comfort and pain. This study aimed to quantitatively assess mining work boot fit relative to underground coal miners' subjectively rated work boot fit and comfort, reported foot problems, lower limb pain and lower back pain in order to develop evidence-based work boot fit recommendations. Traditional footwear fitting methods based predominantly on foot length are insufficient for underground coal mining-specific footwear. Instead, fit at the heel, instep and forefoot must be considered when fitting underground coal mining work boots, in conjunction with the traditional length measurement.Practitioner summary: Underground coal miners report their work boots fit but are uncomfortable. This study assessed actual fit relative to perceived fit, comfort, foot problems, lower limb pain and lower back pain of 197 miners. Fit at the heel, instep and forefoot must be considered when fitting mining work boots.


[Mental malaise in health professionals: an approach to its study].
A study was made of the incidence of psychiatric malaise among health workers in the health area of La Axarquía, in the province of Málaga. Goldberg's General Health Questionnaire (GHQ, 28 items) was applied to the sample population (n = 137), and the differences obtained are discussed in relation to social and job-related variables. The occurrence of psychiatric malaise among the whole sample was 29.85%, with significant differences among those above the cutoff and place of work (hospital), sex (women), tobacco habit (smokers), and pleasantness of work situation (not pleasant). There were no significant differences among those above the cutoff and profession (although higher scores were recorded among administrative personnel), nor according to contractual situation (although higher scores were recorded among contracted staff). The study concludes with a series of measures aimed at early prevention and detection of symptoms of psychiatric maladjustment within the working environment.


Mental Health Status and Its Associated Factors Related to Pulmonary Tuberculosis Patients in Primary Health Care Centre in Surabaya, Indonesia.
Mental disorders in TB patients are due to long-term treatment, drug side effects, and relapse. This study aimed to analyse the mental health status among TB patients and its associated factors. The study was carried out on 107 Pulmonary TB patients from 5 Primary Healthcare centres in Surabaya, Indonesia. Furthermore, Mental Health Inventory (MHI-18) was used to measure the mental health status. The MHI-18 has four subscales including, anxiety, depression, behaviour control, and positive affection. In addition, the score range of MHI and its subscales is 0-100, where the higher score showed a better mental health status. The results showed no difference in the score of mental health status, anxiety, depression, and positive affect in all factors. However, behaviour control depicted a significant difference between sex and marital status. In conclusion, mental health problems can occur in all TB patients irrespective of their characteristics. Screening is required for the prevention of severe disease in the early treatment phase and various factors related to mental health should be considered during the implementation of TB management to optimize treatment outcomes.


Comparison of mortality risk for dialysis patients and cadaveric first renal transplant recipients in Ontario, Canada.
In population-based studies, renal transplantation has been shown to improve survival compared to dialysis patients awaiting transplantation in the United States. However, dialysis mortality in the United States is higher than in Canada. Whether transplantation offers a survival advantage in regions where dialysis survival is superior to that in the United States is uncertain. This study examines a cohort of 1156 patients who started end-stage renal disease (ESRD) therapy and were wait-listed for cadaveric renal transplantation in the province of Ontario, Canada between January 1, 1990 and December 31, 1994. Patients were followed from wait-listing for renal transplant (n = 1156), to cadaveric first renal transplant (n = 722), to death, or to study end (December 31, 1995). The annual crude mortality rates for wait-listed dialysis patients and transplanted patients were 5.0 and 3.4%, respectively. In Cox proportional hazards models, mortality in wait-listed patients was associated with increased age and diabetes, but not time from onset of ESRD to wait-listing. Factors associated with death following transplantation include older age, diabetes, and longer time spent on the waiting list before transplantation. In a time-dependent Cox regression model, the relative risk of death after transplantation compared to dialysis varied in a time-dependent manner. Covariates associated with increased risk included older age, diabetes, and time from onset of ESRD to wait-listing. The average relative risk (RR) of dying was 2.91 (95% confidence interval [CI], 1.34 to 6.32) in the first 30 d after transplantation, but was significantly lower 1 yr after transplantation (RR 0.25; 95% CI, 0.14 to 0.42), indicating a beneficial long-term effect when compared to wait-listed dialysis patients. This long-term benefit was most evident in subgroups of patients with diabetes (RR 0.38; 95% CI, 0.17 to 0.87) and glomerulonephritis (RR 0.13; 95% CI, 0.04 to 0.39) as the cause of ESRD. The survival advantage associated with renal transplantation is evident in this cohort of patients with a lower wait-listed dialysis mortality than that reported previously in the United States. The magnitude of the treatment effect is consistent across studies.


A reanalysis of IgM Western blot criteria for the diagnosis of early Lyme disease.
A two-step approach for diagnosis of Lyme disease, consisting of an initial EIA followed by a confirmatory Western immunoblot, has been advised by the Centers for Disease Control and Prevention (CDC). However, these criteria do not examine the influence of the prior probability of Lyme disease in a given patient on the predictive value of the tests. By using Bayesian analysis, a mathematical algorithm is proposed that computes the probability that a given patient's Western blot result represents Lyme disease. Assuming prior probabilities of early Lyme disease of 1%-10%, the current CDC minimum criteria for IgM immunoblot interpretation yield posttest probabilities of 4%-32%. The value of the two-step approach for diagnosis of early Lyme disease may be limited in populations at lower risk of disease or when patients present with atypical signs and symptoms.


Treatment of acrodermatitis continua of Hallopeau with TNF-blocking agents: case report and review.
Acrodermatitis continua of Hallopeau (ACH) is a rare acropustular eruption, characterized by sterile pustules, paronychia and atrophic skin changes, onychodystrophy and osteolysis of the distal phalanges of the fingers and toes. It is considered to be a variant of pustular psoriasis with a chronic relapsing course and frequent refractoriness to many therapeutic modalities, which can be amenable to successful treatment by tumor necrosis factor alpha antagonists. We report 1 patient with pustular psoriasis and ACH whom we have treated successfully with etanercept (for 30 months) and then adalimumab (for 13 months and ongoing). Blanching was initially achieved with etanercept 50 mg twice a week, but suppression of periungual inflammation then required combination therapy with etanercept 50 mg twice a week and methotrexate 10 mg weekly; lower doses of both drugs did not allow complete control of the disease. Eventually, adalimumab 40 mg every 2 weeks has provided the most cost-effective response in this patient, allowing maintenance of response with partial nail regrowth under monotherapy.


On the Use of Phylogeographic Inference to Infer the Dispersal History of Rabies Virus: A Review Study.
Rabies is a neglected zoonotic disease which is caused by negative strand RNA-viruses belonging to the genus Lyssavirus. Within this genus, rabies viruses circulate in a diverse set of mammalian reservoir hosts, is present worldwide, and is almost always fatal in non-vaccinated humans. Approximately 59,000 people are still estimated to die from rabies each year, leading to a global initiative to work towards the goal of zero human deaths from dog-mediated rabies by 2030, requiring scientific efforts from different research fields. The past decade has seen a much increased use of phylogeographic and phylodynamic analyses to study the evolution and spread of rabies virus. We here review published studies in these research areas, making a distinction between the geographic resolution associated with the available sequence data. We pay special attention to environmental factors that these studies found to be relevant to the spread of rabies virus. Importantly, we highlight a knowledge gap in terms of applying these methods when all required data were available but not fully exploited. We conclude with an overview of recent methodological developments that have yet to be applied in phylogeographic and phylodynamic analyses of rabies virus.


Clinically actionable genotypes among 10,000 patients with preemptive pharmacogenomic testing.
Since September 2010, more than 10,000 patients have undergone preemptive, panel-based pharmacogenomic testing through the Vanderbilt Pharmacogenomic Resource for Enhanced Decisions in Care and Treatment program. Analysis of the genetic data from the first 9,589 individuals reveals that the frequency of genetic variants is concordant with published allele frequencies. Based on five currently implemented drug-gene interactions, the multiplexed test identified one or more actionable variants in 91% of the genotyped patients and in 96% of African American patients. Using medication exposure data from electronic medical records, we compared a theoretical "reactive," prescription-triggered, serial single-gene testing strategy with our preemptive, multiplexed genotyping approach. Reactive genotyping would have generated 14,656 genetic tests. These data highlight three advantages of preemptive genotyping: (i) the vast majority of patients carry at least one pharmacogenetic variant; (ii) data are available at the point of care; and (iii) there is a substantial reduction in testing burden compared with a reactive strategy.


Construction and expression of a single chain antibody mimicing human ovarian cancer antigen CA125.
One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody WJ02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02 could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.


Transcranial sonography findings in a large family with homozygous and heterozygous PINK1 mutations.
To investigate substantia nigra (SN) echogenicity in members of a family with homozygous and heterozygous PTEN induced kinase (PINK1) mutations with or without signs of Parkinson's disease (PD). Transcranial sonography (TCS) was used to investigate 20 members of a family with PINK1 mutations, including four homozygous and 11 heterozygous mutation carriers and five individuals with no mutation. For comparison, a healthy control group of 18 subjects without a positive family history of PD (control group) and a healthy control group of 15 subjects with a positive family history of sporadic PD (relative group) were investigated. For statistical analysis, the larger area of the two SNs echogenicity (aSNmax) of each individual was selected. A significantly increased aSNmax was found for all subgroups compared with the control group. The group of homozygous carriers of a PINK1 mutation had a significantly increased aSNmax compared with all of the other subgroups, except the group of heterozygous mutation carriers. These findings in carriers of a PINK1 mutation are comparable with those in carriers of Parkin mutations and non-genetic PD. The increased aSNmax in family members without a mutation suggests an additional contributing factor independent of the PINK1 mutation that may also play a role in relatives of patients with sporadic PD.


Food allergen detection by mass spectrometry: From common to novel protein ingredients.
Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.


The interplay between homeostatic synaptic plasticity and functional dendritic compartments.
Homeostatic synaptic plasticity (HSP) is an important mechanism attributed with the slow regulation of the neuron's activity. Whenever activity is chronically enhanced, HSP weakens the weights of the synapses in the dendrites and vice versa. Because dendritic morphology and its electrical properties partition the dendritic tree into functional compartments, we set out to explore the interplay between HSP and dendritic compartmentalization. For this purpose, we used a detailed model of a CA1 pyramidal neuron receiving a large number of activity-dependent plastic synapses and developed a novel approach for specifying functional dendritic subunits. We found that the degree of dendritic compartmentalization and the location-specificity of HSP are strongly tied. A local HSP mechanism, operating at the level of the individual synapse, will regard the neuron as a multiunit distributed system, each unit consisting of many synapses, and will thus support dendritic compartmentalization, whereas a global HSP mechanism, modifying all synapses in unison, will treat the neuron as a single centralized unit. Both local and global HSP can successfully counterbalance persistent, cell-wide perturbations of dendritic activity. The spatial distribution of synaptic weights throughout the dendrites will markedly differ under the local versus global HSP mechanisms. We suggest an experimental paradigm to unravel which type of HSP mechanism operates in the dendritic tree. The answer to this question will have important implications to our understanding of the functional organization of the neuron.


Toward an Alignment-Free Method for Feature Extraction and Accurate Classification of Viral Sequences.
The classification of pathogens in emerging and re-emerging viruses represents major interests in taxonomic studies, functional genomics, host-pathogen interplay, prevention, and disease treatments. It consists of assigning a given sequence to its related group of known sequences sharing similar characteristics and traits. The challenges to such classification could be associated with several virus properties including recombination, mutation rate, multiplicity of motifs, and diversity. In domains such as pathogen monitoring and surveillance, it is important to detect and quantify known and novel taxa without exploiting the full and accurate alignments or virus family profiles. In this study, we propose an alignment-free method, CASTOR-KRFE, to detect discriminating subsequences within known pathogen sequences to classify accurately unknown pathogen sequences. This method includes three major steps: (1) vectorization of known viral genomic sequences based on k-mers to constitute the potential features, (2) efficient way of pattern extraction and evaluation maximizing classification performance, and (3) prediction of the minimal set of features fitting a given criterion (threshold of performance metric and maximum number of features). We assessed this method through a jackknife data partitioning on a dozen of various virus data sets, covering the seven major virus groups and including influenza virus, Ebola virus, human immunodeficiency virus 1, hepatitis C virus, hepatitis B virus, and human papillomavirus. CASTOR-KRFE provides a weighted average F-measure >0.96 over a wide range of viruses. Our method also shows better performance on complex virus data sets than multiple subsequences extractor for classification (MISSEL), a subsequence extraction method, and the Discriminative mode of MEME patterns extraction tool.


Aspergillus collected in specific indoor settings: their molecular identification and susceptibility pattern.
Exposure to Aspergillus conidia is an increased risk factor for the development of respiratory symptoms. The emergence of azole resistance in Aspergillus fumigatus is a major concern for the scientific community. The aim of this study was to perform the molecular identification of Aspergillus species collected from different occupational and non-occupational indoor settings and to study the azole susceptibility profile of the collected Fumigati isolates. The selected Aspergillus isolates were identified as belonging to the sections Fumigati, Nigri Versicolores, Terrei, Clavati and Nidulantes. All the Aspergillus fumigatus were screened for azole resistance using an agar media supplemented with itraconazole, voriconazole and posaconazole. None of the tested isolates showed resistance to those azoles. Knowledge of Aspergillus epidemiology in specific indoor environments allows a better risk characterization regarding Aspergillus burden. This study allowed the analysis of the molecular epidemiology and the determination of the susceptibility pattern of Aspergillus section Fumigati found in the studied indoor settings.


Cancer patient navigator tasks across the cancer care continuum.
Cancer patient navigation (PN) programs have been shown to increase access to and utilization of cancer care for poor and underserved individuals. Despite mounting evidence of its value, cancer patient navigation is not universally understood or provided. We describe five PN programs and the range of tasks their navigators provide across the cancer care continuum (education and outreach, screening, diagnosis and staging, treatment, survivorship, and end-of-life). Tasks are organized by their potential to make cancer services understandable, available, accessible, affordable, appropriate, and accountable. Although navigators perform similar tasks across the five programs, their specific approaches reflect differences in community culture, context, program setting, and funding. Task lists can inform the development of programs, job descriptions, training, and evaluation. They also may be useful in the move to certify navigators and establish mechanisms for reimbursement for navigation services.


Diabetic patients with essential hypertension treated with amlodipine: blood pressure and arterial stiffness effects of canagliflozin or perindopril.
Hypertension control reduces cardiovascular and renal risks in type 2 diabetes. Sodium-glucose cotransporter-2 inhibitors prevent renal glucose reabsorption and decrease glucose plasma levels, blood pressure (BP) and weight reduction. Treatment of hypertension and sodium-glucose cotransporter-2 are able to improve arterial stiffness. To evaluate, in patients with type 2 diabetes and hypertension, the effects of 6 months treatment with canagliflozin, or perindopril, an angiotensin converting enzyme inhibitor, on central BP and carotid-femoral pulse wave velocity (cfPWV). Thirty type 2 diabetic patients with hypertension taking amlodipine, 10 mg daily, and metformin, 750-2000 mg daily, were randomized and a third medication was added: canagliflozin, 300 mg daily (n = 15, nine women, mean age: 63 ± 8 years), or perindopril, 10 mg daily (n = 15, five women, mean age 59 ± 4 years), for 6 months. Ambulatory BP monitoring was assessed at baseline and after 3 and 6 months of treatment, whereas cfPWV was measured before and after 6 months of treatment. Plasma fasting glucose, glycated hemoglobin, creatinine, plasma and urinary sodium and potassium were also measured. Both treatments significantly reduced BP and cfPWV. Only canagliflozin maintained the PWV action after adjusting for BP values and reduced glycemia, glycated hemoglobin and 24 h urinary sodium. Other security laboratory parameters, including gluthamic oxaloacetic transaminase, gluthamic piruvic transaminase; and bilirubin failed to show any change. Canagliflozin reduced BP and improve arterial stiffness, independently of the BP effect. These two conditions could explain the cardiovascular protection observed with canagliflozin compared with perindopril.


Extraction of the essential oil from endemic Origanum bilgeri P.H.Davis with two different methods: comparison of the oil composition and antibacterial activity.
The antibacterial activity and chemical composition of the essential oils (EOs) isolated from Origanum bilgeri P.H.Davis by two different extraction methods, i.e., hydrodistillation (HD) and solvent-free microwave extraction (SFME), were examined. This endemic Origanum species had shown very good antibacterial activity. The composition of the O. bilgeri EOs obtained by SFME and HD was investigated by GC/MS analysis. The main components of the oils obtained by both methods were carvacrol (90.20-84.30%), p-cymene (3.40-5.85%), γ-terpinene (0.47-1.20%), and thymol (0.69-1.08%). The EO isolation by SFME offered many important advantages, including a higher extraction yield, a shorter extraction time, and a higher content of the active component carvacrol. The carvacrol-rich oils obtained by both HD and SFME showed a good antibacterial activity. The largest inhibition zones were observed for the O. bilgeri EO obtained by SFME. Our study suggests that O. bilgeri EO has the potential to be used as preventative against bacterial contamination in many foods, instead of the common synthetic antimicrobial products.


[Role of macromycetes as accumulators of 137Cs in forest ecosystems].
One of the problems of forest radioecology concerning the role of macromycetes in accumulation of caesium radionuclides in ground ecosystems is discussed in the paper. The main positions of the researchers working in this area are stated, and their arguments relating to the given problem are compared. The findings of the analysis of the literary data on a 137Cs content in fruit bodies and mycelium of fungi are represented. Similar findings on the behaviour of its chemical analog--potassium, studies of fungi physiology and effects of physicochemical processes in soils on the behaviour of this radionuclide are also used when examining the behaviour of 137Cs in fungi.


Effect of heparin on vascular smooth muscle cells. I. Cell metabolism.
Previous work from our laboratory has shown that heparin inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. The mechanism of action of this glycosaminoglycan is unknown. In this communication, we have examined the antiproliferative effect of heparin on smooth muscle and other cell types, and have investigated several aspects of heparin on smooth muscle cell metabolism. Smooth muscle and closely related cell types from several species, including human, were much more sensitive to heparin than any other cell type tested, including primary and established cell lines, normal and transformed cell pairs, fibroblasts, epithelial, and endothelial cells. Flow microfluorimetric analysis of cell cycle distribution indicated that heparin blocked either the G0----S transition or a very early S-phase event in smooth muscle cells. Heparin rapidly inhibited DNA and RNA synthesis, but did not affect the rate of protein synthesis. The decrease in nucleic acid synthesis could be accounted for by an inhibition of thymidine and uridine uptake. Interestingly, heparin did not block amino acid or glucose transport. Although no change in the overall rate of protein synthesis was observed in the presence of heparin, we noted at least two changes in the synthesis of specific proteins by smooth muscle cells: two 35,000-dalton proteins which appeared in the culture medium of heparin-treated cells, and the transient disappearance of a 48,000-dalton protein in the substrate attached material of smooth muscle cells exposed to heparin. The role of the observed changes in smooth muscle cell metabolism is yet to be determined, but they may provide valuable clues to the molecular mechanisms controlling the antiproliferative activity of heparin.


Crystal structure and spectroscopic studies of a stable mixed-valent state of the hemerythrin-like domain of a bacterial chemotaxis protein.
The bacterial chemotaxis protein of Desulfovibrio vulgaris DcrH (DcrH-Hr) functions as an O(2)-sensing protein. This protein has a hemerythrin-like domain that includes a nonheme diiron center analogous to the diiron center of the hemerythrin (Hr) family. Interestingly, the O(2) affinity of DcrH-Hr is 3.3 × 10(6) M(-1), a value 25-fold higher than that of the Pectinaria gouldii Hr. This high affinity arises from the fast association of the O(2) ligand with DcrH-Hr (k(on) = 5.3 × 10(8) M(-1) s(-1)), which is made possible by a hydrophobic tunnel that accelerates the passage of the O(2) ligand to the diiron site. Furthermore, the autoxidation kinetics indicate that the rate of autoxidation of DcrH-Hr is 54-fold higher than that of P. gouldii Hr, indicating that the oxy form of DcrH-Hr is not stable toward autoxidation. More importantly, a mixed-valent state, semimet(R), which was spectroscopically observed in previous Hr studies, was found to be stable for over 1 week and isolable in the case of DcrH-Hr. The high-resolution crystal structures of the semimet(R)- (1.8 Å) and met-DcrH-Hr (1.4 Å) indicate that the semimet(R)- and met-DcrH-Hr species have very similar coordination geometry at the diiron site.


Closing the loop: Novel quantitative fMRI approach for manipulation of the sensorimotor loop in tremor.
Tremor is thought to be an effect of oscillatory activity within the sensorimotor network. To date, the underlying pathological brain networks are not fully understood. Disentangling tremor activity from voluntary motor output and sensorimotor feedback systems is challenging. To better understand the intrinsic sensorimotor fingerprint underlying tremor, we aimed to disentangle the sensorimotor system into driving (motor) and feedback/compensatory (sensory) neuronal involvement, and aimed to pinpoint tremor activity in essential tremor (ET) and tremor-dominant Parkinson's disease (PD) with a novel closed-loop approach. Eighteen ET patients, 14 tremor-dominant PD patients, and 18 healthy controls were included. An MR-compatible wrist manipulator was employed during functional MRI (fMRI) while muscle activity during (in)voluntary movements was concurrently recorded using electromyography (EMG). Tremor was quantified based on EMG and correlated to brain activity. Participants performed three tasks: an active wrist motor task, a passive wrist movement task, and rest (no wrist movement). The results in healthy controls proved that our experimental paradigm activated the expected motor and sensory networks separately using the active (motor) and passive (sensory) task. ET patients showed similar patterns of activation within the motor and sensory networks. PD patients had less activity during the active motor task in the cerebellum and basal ganglia compared to ET and healthy controls. EMG showed that in ET, tremor fluctuations correlated positively with activity in the inferior olive region, and that in PD tremor fluctuations correlated positively with cerebellar activity. Our novel approach with an MR-compatible wrist manipulator, allowed to investigate the involvement of the motor and sensory networks separately, and as such to better understand tremor pathophysiology. In ET sensorimotor network function did not differ from healthy controls. PD showed less motor-related activity. Focusing on tremor, our results indicate involvement of the inferior olive in ET tremor modulation, and cerebellar involvement in PD tremor modulation.


Synovial facet joint cysts after lumbar posterior decompression surgery.
Numerous studies have reported on synovial facet joint cysts of the spine as a primary lesion. The exact pathogenesis of those cysts is still controversial, but degeneration and destabilization seem to be underlying mechanisms. However, only a few reports have thus far investigated synovial cysts of the spine as postoperative complications of decompression surgery. In this retrospective clinical study, the authors focused on synovial cysts of the lumbar facet joints as complications after lumbar decompression surgery, with the aim of elucidating their pathophysiology. A total of 326 patients with 384 segments treated with posterior decompression surgery without fusion were included in the study. Of these segments, 107 were surgically decompressed unilaterally and 277 were decompressed bilaterally. After surgery, 18 of the 384 segments developed a complication of symptomatic facet synovial cyst. The anatomical and morphological evaluations of these segments were performed using functional plain radiographs, CT scans, and MR images. All of the 18 segments with postoperative lumbar facet synovial cyst were treated with bilateral lumbar posterior decompression. There was no significant radiological lumbar spinal instability in any segments, although 17 of 18 segments demonstrated facet articular spondylotic changes. Moreover, 12 of 18 patients demonstrated lumbar retrolisthesis in the neutral position. Based on the authors' results, they propose that patients with lumbar degenerative disease who have a potential biomechanical lumbar instability such as disruption of the facet articular surface and hydrarthrosis or lumbar facet tropism might have a high risk for formation of lumbar facet synovial cyst after bilateral posterior decompression surgery.


Cumulative pregnancy rates of two strategies: Day 3 fresh embryo transfer followed by Day 3 or Day 5/6 vitrification and embryo transfer: a randomized controlled trial.
Are cumulative pregnancy rates better if supernumerary embryos are vitrified on Day 5/6 instead of Day 3? The results do not show a significant difference in cumulative pregnancy rates between the Day 3 and Day 5/6 vitrification groups. Pregnancy and live birth rates following IVF or ICSI treatment are higher after extended embryo culture and blastocyst transfer (Day 5/6) compared to cleavage-stage (Day 3) transfer. Cumulative pregnancy rates from one oocyte retrieval (OR) cycle show no significant difference after fresh and frozen embryo transfers, but only one study has used vitrification for the cryopreservation of supernumerary embryos while four studies have used a slow freezing protocol. Our prospective randomized controlled trial was performed in an academic centre between January 2018 and August 2020. Patients were randomized into vitrification Day 3 (n = 80) or Day 5/6 (n = 81) groups. The primary outcome was the cumulative ongoing pregnancy rate (cOPR), considering only the first pregnancy for each couple. The power calculation revealed that 75 patients were required in each group, when assuming a 50% cOPR with four embryo transfers in the vitrification Day 3 group vs two transfers in the vitrification Day 5/6 group. Patients <38 years undergoing their first or second OR cycles were randomized at the start of the first cycle. Up to two cycles were included in the analysis. A fresh embryo transfer was performed on Day 3. Supernumerary embryos (with ≥6 cells, <25% fragmentation, and equal blastomeres) or blastocysts (with expansion grade ≥2 with inner cell mass and trophectoderm score A/B) were vitrified on Day 3 or Day 5/6, respectively, and then transferred at a later date. A time-to-event analysis was performed with the patient's first ongoing pregnancy as the event of interest and the number of embryo transfers as the time component. The statistical comparison was performed by a Cox proportional hazards model. Cumulative costs of vitrification on Day 3 vs Day 5/6 were explored and compared using Mann-Whitney U tests. By December 2021, 233 transfers (96 fresh and 137 frozen) in 77 patients were performed in the vitrification Day 3 group and 201 transfers (88 fresh and 113 frozen) in 77 patients were performed in the vitrification Day 5/6 group. The time-to-event analysis did not show a difference between the two arms with regard to the patient's first ongoing pregnancy as the primary study outcome (hazard ratio [HR] 1.25, 95% CI 0.82; 1.92, P = 0.30). The cumulative ongoing pregnancy rate after eight transfers (from one or two ORs) was 57% in the vitrification Day 3 group vs 58% in the vitrification Day 5/6 group. The median number of embryo transfers until a pregnancy was achieved was five vs four, respectively, in the vitrification Day 3 group vs the Day 5/6 group. Similar results were found for the secondary study outcome, i.e. clinical pregnancy with foetal heart rate (HR 1.19, 95% CI 0.78; 1.80, P = 0.41). The cumulative clinical pregnancy rate (cCPR) after eight embryo transfers was 62% in the vitrification Day 3 group vs 59% in the vitrification Day 5/6 group. The median number of transfers until a pregnancy was achieved was four in both groups. The healthcare consumption pattern differed between the two groups and we observed higher costs for the vitrification Day 3 group compared to the vitrification Day 5/6 group, although these differences were not statistically significant. Although our power calculation revealed that only 75 patients were needed in each study group (β = 0.87, α < 0.05), the numbers were low. Also, different numbers of single and double embryo transfers were performed between the two groups, which may have affected the results. The cost analysis was performed on a subset of the patients and is therefore exploratory. Our study shows no difference in the cumulative pregnancy rate nor costs after fresh and frozen embryo transfers of at most two sequential OR cycles between the Day 3 and Day 5/6 vitrification groups; however, obstetric and perinatal outcomes should be taken into account to determine the best strategy. This study was funded as an investigator-sponsored study of S.D. by Merck nv/sa Belgium, an affiliate of Merck KGaA, Darmstadt, Germany, and by Gedeon Richter Benelux (PA18-0162). The authors declare no conflict of interest related to this study. NCT04196036. 15 January 2018. 15 January 2018.


Addressing the elephant in the room: a possible new way to increase patient adherence to medical advice.
Lack of patient adherence to medical advice (PAMA) is recognized as an area of interest. None of the previous initiatives to improve PAMA, such as patient centered care and shared decision making, have proved to be successful in terms of improving patient adherence. The aim of the present study is to assess beliefs about priorities in public health care, and adherence to medical advice, to establish a novel approach to increase PAMA. The present study is based on responses to two questions in an experimental survey from the Norwegian Citizen Panel, addressing people's attitudes to priorities in public health care and adherence to medical advice. The questions on priorities in the health care sector are organized into six groups. The questions on adherence are organized into three groups. All questions are answered on a 7-point Likert scale. This study is the first to use experimental surveys to assess PAMA. The results indicate that if health care providers refer to national expertise and patient organizations' recommendations on a given treatment, PAMA could improve. Although technical and methodological interventions in health care have, to some extent, improved PAMA, medical adherence is still low. In the present study, it is shown that integrating either national expertise or collaborated messages with other health professions and patient organizations' recommendations in everyday care may help improve patients adherence to medical advice. A minor change in how treatment suggestions are presented could improve PAMA.


[Electron-microscopic study of neuroendocrine tumors of the lung].
An electron-microscopic study was made of carcinoids, malignant carcinoids and small-cell lung cancer. These neoplasms are shown to represent one histogenetic group--neuroendocrine tumours (apudomas) of the lung. One can distinguish 3 main structural forms among them, i.e. epithelioid, sarcoma-like and symplastic. According to the ultrastructural criteria the neoplasms mentioned can be classified by their differentiation degree, as well, intermediately and poorly differentiated variants. This classification correlates well enough with the catamnesis of patients and may serve for prognosis of the disease.


Age-related studies of SIg, Leu-4 and concanavalin A receptor densities and capping in human lymphocytes.
We compared the cell surface antigen density and capping of three antigens in lymphocytes obtained from healthy, young (mean age 27 years) and elderly (mean age 76), population. There were no differences in the expression of surface immunoglobulin (SIg), concanavalin A (con A) receptors and Leu-4 antigen between the two groups. Kinetic analysis of these molecules revealed a slight decrease in capping in the older population, but the differences were not statistically significant. In order to test the possibility that subjecting the cells to metabolic stress might bring out the differences, we performed a kinetic analysis of SIg and con A capping in the presence of various concentrations of the metabolic inhibitor sodium azide. Although the capping in cells from elderly subjects was slightly more sensitive to azide, no statistical difference was found. Analysis of con A capping by a flow cytometric method yielded similar results, confirming the data obtained by visual capping experiments. We conclude that although a trend toward decreased capping was observed, there is little alteration in the surface molecule capping phenomenon in the age-groups studied.


Highly selective agonists for substance P receptor subtypes.
The existence of a third tachykinin receptor (SP-N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N-methylation of individual peptide bonds in the C-terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl-[Asp6,Me-Phe8]SP6-11, elicits half-maximal contraction of the guinea pig ileum through the neuronal SP-N receptor at a concentration of 0.5 nM. At least 60,000-fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP-P and SP-E). The action of selective SP-N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP-N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.


Chromotropic acid-functionalized polyurethane foam: A new sorbent for on-line preconcentration and determination of cobalt and nickel in lettuce samples.
A chromotropic acid-functionalized polyurethane foam has been developed for use in an on-line preconcentration system for cobalt and nickel determination. The packing material was prepared by covalent coupling of chromotropic acid with the polyurethane foam through an azo group. Co and Ni ions were sorbed in the mini-column, from which they could be eluted directly to the nebulizer-burner system of a flame atomic absorption spectrometer. Elution of cobalt and nickel from the mini-column can be accomplished with 0.50 and 0.75 M HCl solutions, respectively. The enrichment factors obtained were 22 (Co) and 27 (Ni), for 60 s preconcentration time, and 57 (Co) and 59 (Ni), if a preconcentration time of 180 s was used. Under the optimum conditions, the proposed procedure allowed the determination of metals with detection limits of 0.43 (cobalt) and 0.52 microg/L (nickel), respectively, on using preconcentration periods of 180 s. The accuracy of the developed procedure was evaluated by analysis of the certified reference materials NIST 1515 Apple Leaves and NIST 1570a Spinach Leaves. The method was applied to the analysis of lettuce samples. The contents of cobalt in the samples analyzed varied from 0.75 to 0.98 microg/g. Nickel was not detected in the lettuce samples.


Longitudinal Outcomes of an Institutionally Developed Nurse Residency Program.
Nurse residency programs are widely implemented to enhance integration of new graduate nurses entering the workforce. This article presents a retrospective analysis of 10 years of residency data from an internally developed residency program that used the Casey-Fink Graduate Nurse Experience Survey. Outcomes of this program were similar to those from studies using commercially available products, suggesting that an internally developed residency curricula may be equally beneficial to the development of new graduate nurses.


Restricted fluid bolus versus current practice in children with septic shock: the FiSh feasibility study and pilot RCT.
There has been no randomised controlled trial (RCT) of fluid bolus therapy in paediatric sepsis in the developed world despite evidence that excess fluid may be associated with harm. To determine the feasibility of the Fluids in Shock (FiSh) trial - a RCT comparing restricted fluid bolus (10 ml/kg) with current practice (20 ml/kg) in children with septic shock in the UK. (1) Qualitative feasibility study exploring parents' views about the pilot RCT. (2) Pilot RCT over a 9-month period, including integrated parental and staff perspectives study. (1) Recruitment took place across four NHS hospitals in England and on social media. (2) Recruitment took place across 13 NHS hospitals in England. (1) Parents of children admitted to a UK hospital with presumed septic shock in the previous 3 years. (2) Children presenting to an emergency department with clinical suspicion of infection and shock after 20 ml/kg of fluid. Exclusion criteria were receipt of > 20 ml/kg of fluid, conditions requiring fluid restriction and the patient not for full active treatment (i.e. palliative care plan in place). Site staff and parents of children in the pilot were recruited to the perspectives study. (1) None. (2) Children were randomly allocated (1 : 1) to 10- or 20-ml/kg fluid boluses every 15 minutes for 4 hours if in shock. (1) Acceptability of FiSh trial, proposed consent model and potential outcome measures. (2) Outcomes were based on progression criteria, including recruitment and retention rates, protocol adherence and separation between the groups, and collection and distribution of potential outcome measures. (1) Twenty-one parents were interviewed. All would have consented for the pilot study. (2) Seventy-five children were randomised, 40 to the 10-ml/kg fluid bolus group and 35 to the 20-ml/kg fluid bolus group. Two children were withdrawn. Although the anticipated recruitment rate was achieved, there was variability across the sites. Fifty-nine per cent of children in the 10-ml/kg fluid bolus group and 74% in the 20-ml/kg fluid bolus group required only a single trial bolus before shock resolved. The volume of fluid (in ml/kg) was 35% lower in the first hour and 44% lower over the 4-hour period in the 10-ml/kg fluid bolus group. Fluid boluses were delivered per protocol (volume and timing) for 79% of participants in the 10-ml/kg fluid bolus group and for 55% in the 20-ml/kg fluid bolus group, mainly as a result of delivery not being completed within 15 minutes. There were no deaths. Length of hospital stay, paediatric intensive care unit (PICU) transfers, and days alive and PICU free did not differ significantly between the groups. Two adverse events were reported in each group. A questionnaire was completed by 45 parents, 20 families and seven staff were interviewed and 20 staff participated in focus groups. Although a minority of site staff lacked equipoise in favour of more restricted boluses, all supported the trial. Even though a successful feasibility and pilot RCT were conducted, participants were not as unwell as expected. A larger trial is not feasible in its current design in the UK. Further observational work is required to determine the epidemiology of severe childhood infection in the UK in the postvaccine era. Current Controlled Trials ISRCTN15244462. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 51. See the NIHR Journals Library website for further project information.


Contact activation of human plasma prorenin in vitro.
Acid activation of plasma prorenin occurs during dialysis to pH 3.3. and also during subsequent dialysis to pH 7.4. The latter, alkaline phase, involves Hageman factor-dependent formation of kallikrein, which in turn activates prorenin. The present study evaluates whether prorenin activation always occurs whenever kallikrein is activated in plasma. TAME esterase activity was used as a measure of plasma kallikrein activity an increase was observed during the alkaline phase of acid activation of prorenin. TAME esterase activity was absent when Hageman factor- or prekallikrein-deficient plasmas were similarly assayed and prorenin was not activated. Kaolin treatment of normal plasma rapidly increased TAME esterase activity at both 25 degrees and -4 degrees C, but no prorenin activation occurred. Similar changes in TAME esterase activity were observed in acid-treated plasma, in which setting prorenin was activated. No change in TAME esterase or renin activity occurred after addition of kaolin to acid-treated plasma deficient in Hageman factor; however, both enzymatic activities increased slightly in acidified prekallikrein-deficient plasma. Mixtures of these deficient plasmas exhibited normal kaolin activation of both TAME esterase and prorenin after acidification. Thus both Hageman factor and prekallikrein are needed for optimal contact activation of prorenin. These results demonstrate that prorenin activation does not always occur when active kallikrein is present in plasma. Prior acidification appears to be a prerequisite. Acidified prorenin may be more susceptible to cleavage; alternatively, competing substrates and/or inhibitors of kallikrein may be destroyed at acid pH, thereby permitting kallikrein to activate prorenin. Under normal conditions, activation of the plasma kallikrein-kinin system appears unlikely to result in activation of prorenin in vivo.


Out-of-office blood pressure monitoring in chronic kidney disease.
Blood pressure (BP) control is vital to the management of patients with chronic kidney disease (CKD) yet most treatment decisions use BPs obtained in the clinic. The purpose of this report is to review the importance of self-measured and automatic ambulatory BPs in the management of patients with CKD. Compared with clinic-obtained BPs, self-measured BP more accurately defines hypertension in CKD. Masked hypertension seems to be associated with higher risk of end-stage renal disease in CKD patients. Conversely, white-coat hypertension seems to be associated with better renal outcomes than those who have persistent hypertension. Ambulatory BP monitoring is the only tool to monitor BP during sleep, diagnose nondipping, and, as self-measured BPs, have greater prognostic power in CKD compared with clinic BP. In hemodialysis patients, self-measured BP, but not pre/post-dialysis BP, shares the combination of high sensitivity and high specificity of greater than 80% to make a diagnosis of hypertension with the reference standard of ambulatory BP monitoring. In addition, self-measured and ambulatory BPs seem to be better correlates of left-ventricular hypertrophy and mortality in hemodialysis patients compared with pre/post-dialysis BP. Emerging data suggest that out-of-office BP monitoring is superior to BP obtained in the clinic when predicting target-organ damage and prognosis. Out-of-office BP monitoring is recommended for the management of hypertension in all stages of CKD.


Structure of the HIV-1 full-length capsid protein in a conformationally trapped unassembled state induced by small-molecule binding.
The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site ("H site"), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by "conformationally trapping" CA in assembly-incompetent conformational states induced by H-site binding.