A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms.
The Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting. Of 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation. This investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.


Lead toxicosis and trace element levels in wild birds and mammals at a firearms training facility.
In May 1999, lead poisoning was diagnosed in a yellow-rumped warbler (Dendroica coronata) and a gray squirrel (Sciurus carolinensis) found at the Federal Law Enforcement Training Center (FLETC), Glynn County, GA, based on detection of 6.2 and 90.0 ppm wet weight (WW) lead in the liver of the warbler and squirrel, respectively. From October 21--26, 1999, 72 wild animals (37 mammals and 35 birds), comprised of 22 different species, were collected from a 24-ha area surrounding the FLETC outdoor firearms shooting range complex to evaluate exposure to lead and other trace elements. Ten animals were used as controls (five mammals and five birds) and were collected from areas 1.5--3 km outside the shooting range area. Kidney and liver tissues were analyzed for lead, zinc, and other trace elements. Bird gizzards and white-tailed deer abomasums were examined grossly and radiographically to detect metallic objects. Twenty-four (33.3%) animals (11 species) had kidney or liver tissue lead levels > 1.00 ppm, and 12 of these (6 species) had levels > 2.00 ppm. Carcasses of one brown-thrasher (Toxostoma rufum) and two white-tailed deer (Odocoileus virginianus) contained lead fragments. Elevated liver tissue levels of zinc (111.0 ppm) were detected in one brown thrasher that also had elevated kidney and liver tissue lead levels. In February 2000, seven yellow-rumped warblers and one solitary vireo (Vireo solitarius) found dead near the FLETC firearms shooting range also were diagnosed with lead poisoning, with liver and kidney tissue lead levels from 1.77--11.6 and 4.55--17.8 ppm WW, respectively. This frequency of elevated tissue lead levels among the animals examined, in combination with confirmed lead toxicosis in both avian and mammalian species at FLETC, indicates significant lead exposure of local wild bird and mammal communities via bullets and fragments in and on the soil surface of the four outdoor ranges. Most FLETC firearms training is being shifted to new baffled ranges (four walls with semiopen top) with bullet recovery capabilities to preclude future deposition of lead in the environment; existing outdoor ranges will be remediated to remove existing lead.


Environmental stress-dependent effects of deletions encompassing Hsp70Ba on canalization and quantitative trait asymmetry in Drosophila melanogaster.
Hsp70 genes may influence the expression of wing abnormalities in Drosophila melanogaster but their effects on variability in quantitative characters and developmental instability are unclear. In this study, we focused on one of the six Hsp70 genes, Hsp70Ba, and investigated its effects on within- and among-individual variability in orbital bristle number, sternopleural bristle number, wing size and wing shape under different environmental conditions. To do this, we studied a newly constructed deletion, Df(3R)ED5579, which encompasses Hsp70Ba and nine non-Hsp genes, in the heterozygous condition and another, Hsp70Ba(304), which deletes only Hsp70Ba, in the homozygous condition. We found no significant effect of both deletions on within-individual variation quantified by fluctuating asymmetry (FA) of morphological traits. On the other hand, the Hsp70Ba(304)/Hsp70Ba(304) genotype significantly increased among-individual variation quantified by coefficient of variation (CV) of bristle number and wing size in female, while the Df(3R)ED5579 heterozygote showed no significant effect. The expression level of Hsp70Ba in the deletion heterozygote was 6 to 20 times higher than in control homozygotes, suggesting that the overexpression of Hsp70Ba did not influence developmental stability or canalization significantly. These findings suggest that the absence of expression of Hsp70Ba increases CV of some morphological traits and that HSP70Ba may buffer against environmental perturbations on some quantitative traits.


Upregulation of GADD153 by butyrate: involvement of MAPK.
Butyrate inhibits the proliferation of cancer cells, but the early molecular events initiated by butyrate have not been fully identified. Herein, butyrate is shown to affect the growth arrest and DNA damage-inducible gene 153 (GADD153) in HCT-116 human colon adenocarcinoma cells. Despite absence of any detectable cellular DNA damage, the expression of GADD153 was upregulated before several features characteristic of apoptosis appeared. Butyrate-induced upregulation of GADD153 mRNA was attenuated by actinomycin D, but apparently not by cycloheximide. In investigating possible involvement of MAPK in mediating the effect of butyrate on GADD153 mRNA expression, the extracellular regulated kinase (ERK) inhibitor PD98059, but neither the JNK inhibitor SP600125 nor the p38 MAPK inhibitor SB203580, blunted the ability of butyrate to upregulate GADD153 mRNA expression. U0126, a selective inhibitor of upstream MEK, had a similar effect as PD98059 on butyrate-induced GADD153 mRNA upregulation. Collectively, these findings suggest that butyrate caused activation of the GADD153 gene at the level of transcription involving mainly the MEK/ERK branch of the MAPK signal transduction pathway. Moreover, these molecular events were not the result of any DNA damage and occurred before several features characteristic of apoptosis became evident.


Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-β production.
Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.


Prenatal phthalate exposure was associated with croup in Swedish infants.
This study examined whether prenatal phthalate exposure was associated with lower or upper airway inflammation in infants. From 2007 to 2010, we used liquid chromatography-tandem mass spectrometry, adjusted for creatinine, to analyse 14 phthalate metabolites and one phthalate replacement in the urine of 1062 Swedish mothers at a median of 10 weeks of pregnancy. This was used to determine any associations between prenatal phthalate exposure and croup, wheezing or otitis in their offspring until 12 months of age, using logistic regression, adjusted for potential confounders. There were significant associations between phthalate metabolites of butyl-benzyl phthalate (BBzP) and di-ethyl-hexyl phthalate (DEHP) concentrations in maternal prenatal urine and croup in 1062 infants during the first year of life, when adjusted for potential confounders. A dose-response relationship was found between prenatal phthalates exposure and maternal reported croup in the children, with a significant association in boys. There was no clear indication with regard to associations between prenatal phthalate exposure and wheezing or otitis media in the children during the first year of life. Our analysis suggests that exposure to BBzP and DEHP phthalates was associated with maternal reports of croup in infants up to 12 months of age.


Kerosene, Camphor, and Naphthalene Poisoning in Children.
Kerosene poisoning is one of the most common accidental poisoning in children in developing countries due common use of kerosene in house-hold and unsafe storage practices. Aspiration pneumonitis is the most common manifestation of kerosene ingestion due to its low viscosity, high volatility, and low surface tension. The treatment of aspiration pneumonitis due to kerosene poisoning is symptomatic including oxygen support, respiratory monitoring, and careful monitoring of fluid balance. Children with severe respiratory distress and hypoxemia unresponsive to supplemental oxygen and/or severe central nervous system involvement require early intubation and mechanical ventilation. Transfer to the pediatric intensive care unit (PICU) is required at this stage. Emesis, gastric lavage, and administration of activated charcoal are contraindicated due to risk of aspiration. There is no clear benefit of using corticosteroids or prophylactic antibiotics. Asymptomatic children should be kept under observation for atleast 6 hours after exposure. The mortality rate is low and death occurs due to pneumonitis. Camphor is used in house-hold items including vaporized or topical cold preparations, liniments, moth repellents, for performing rituals in religious ceremonies, and in antimicrobial preparations. Camphor poisoning is not very common in childhood. Even small doses of camphor can cause serious toxicity and is potentially fatal. The onset of action is very rapid (5-15 minutes). The common manifestations are confusion, restlessness, delirium, and hallucinations, muscle twitching, myoclonus, ataxia, hyperreflexia, fasciculations, and seizures. Seizures are common and serious complication in camphor toxicity. The treatment is supportive including decontamination, gastric lavage, activated charcoal, and seizure control. Naphthalene is a major constituent of mothballs which are commonly used in household to protect clothes from moths. Though the poisoning with naphthalene is uncommon in children, most of the cases with naphthalene poisoning occur in developing countries where mothballs are still commonly used. The manifestations of naphthalene toxicity are predominantly due to acute intravascular hemolysis leading to anemia, hemoglobinuria, methemoglobinemia, and acute kidney injury (AKI). The treatment of naphthalene toxicity is supportive in form of transfusion of the packed red blood cells, monitoring of fluid and electrolyte balance, administration of alkalis in presence of hemoglobinuria, and renal replacement therapy. Prevention is better than cure. The strategies should be adopted to prevent children being exposed to these toxic compounds in the house-hold. Safe storage of toxic compounds away from the reach of children, avoiding storing kerosene in cold drink and beverage bottles, community education, provision of electricity in rural areas, safe cooking practices, and parental supervision are important interventions to prevent accidental poisoning among children. Kumar S, Kavitha TK, Angurana SK. Kerosene, Camphor, and Naphthalene Poisoning in Children. Indian J Crit Care Med 2019;23(Suppl 4):S278-S281.


A comparison of supination/flexion to hyperpronation in the reduction of radial head subluxations.
To compare supination at the wrist followed by flexion at the elbow (the traditional reduction technique) to hyperpronation at the wrist in the reduction of radial head subluxations (nursemaid's elbow). This prospective, randomized study involved a consecutive sampling of children younger than 6 years of age who presented to one of two urban pediatric emergency departments and two suburban pediatric ambulatory care centers with a clinical diagnosis of radial head subluxation. Patients were randomized to undergo reduction by one of the two methods and were followed every 5 minutes for return of elbow function. The initial procedure was repeated if baseline functioning did not return 15 minutes after the initial reduction attempt. Failure of that technique 30 minutes after the initial reduction attempt resulted in a cross-over to the alternate method of reduction. The alternate procedure was repeated if baseline functioning did not return 15 minutes after the alternate procedure was attempted. If the patient failed both techniques, radiography of the elbow was performed. A total of 90 patients were enrolled in the study. Five patients were removed from further analysis secondary to a final diagnosis of fracture, 84 were reduced successfully, and 1 failed both techniques. Demographic characteristics of each group were similar. Thirty-nine of 41 patients (95%) randomized to hyper-pronation were reduced successfully on the first attempt versus 34 of 44 patients (77%) randomized to supination. Two patients in the hyperpronation group required two attempts versus 10 patients in the supination group. Hyperpronation was more successful; 40 of 41 patients (97.5%) in the hyperpronation group were reduced successfully versus 38 of 44 patients (86%) in the supination group. Of the 6 patients who crossed over from supination to hyperpronation, 5 were reduced on the first attempt and 1 was reduced on the second attempt. In the reduction of radial head subluxations, the hyperpronation technique required fewer attempts at reduction compared with supination, was successful more often than supination, and was often successful when supination failed.


Different manifestations of enhanced π-acceptor ligation at every redox level of [Os(9-OP)L2]n, n = 2+, +, 0, - (9-OP- = 9-oxidophenalenone and L = bpy or pap).
The title complexes were isolated as structurally characterised compounds [OsII(9-OP)L2]ClO4, L = 2,2'-bipyridine (bpy) or 2-phenylazopyridine (pap), and were compared with ruthenium analogues. A reversible one-electron oxidation and up to three reduction processes were observed by voltammetry (CV, DPV) and spectroelectrochemistry (UV-vis-NIR, partially EPR). Supporting calculations (DFT, TD-DFT) were used to assess the oxidation state combinations of the different redox active ligands and of the metal, revealing the effects of Os versus Ru exchange and of bpy versus pap acceptor ligation. Several unexpected consequences of these variations were observed for members of the new osmium-containing redox series. Remarkably, the EPR results exhibit a clear dichotomy between the complex ion [OsIII(9-OP-)(bpy)2]2+ and the radical species [OsII(9-OP˙)(pap)2]2+, which has not been similarly observed for the analogous [RuIII(9-OP-)L2]2+ systems. This difference, unprecedented for 5dn systems, is attributed to the superior stabilisation of the OsII state by the strongly π-accepting pap ligands. The reduced forms [OsII(9-OP-)(pap˙-)(pap)] and [OsII(9-OP-)(pap˙-)2]- exhibit strong inter-ligand interactions, leading to spin isomers and electron hopping.


Artificial nutrition at the end of life: ethical issues.
Artificial nutrition is a medical treatment that first of all needs a sound scientific base before prescribing it. This base is absent for dying patients and patients in the end stage of dementia. Because feeding is a very emotional and symbolical issue, patient and family may request this treatment despite the lack of evidence. These issues should be addressed in good communication with patient and relatives. For comatose patients and patients in a persistent vegetative state artificial nutrition is a necessary support to bridge the time until either recovery is imminent or improbable. At that moment artificial nutrition no longer contributes to the life of the patient and should be ceased. Artificial nutrition has no place in patients that voluntary decide to stop eating and drinking in order to die.


Salivary cortisol mediates effects of poverty and parenting on executive functions in early childhood.
In a predominantly low-income population-based longitudinal sample of 1,292 children followed from birth, higher level of salivary cortisol assessed at ages 7, 15, and 24 months was uniquely associated with lower executive function ability and to a lesser extent IQ at age 3 years. Measures of positive and negative aspects of parenting and household risk were also uniquely related to both executive functions and IQ. The effect of positive parenting on executive functions was partially mediated through cortisol. Typical or resting level of cortisol was increased in African American relative to White participants. In combination with positive and negative parenting and household risk, cortisol mediated effects of income-to-need, maternal education, and African American ethnicity on child cognitive ability.


A reliable technique for karyotyping mouse oocytes prepared by a gradual fixation/air-drying method followed by multicolour FISH.
Chromosome segregation errors during oocyte meiosis increase with age and lead to aneuploidy; hence, the mechanism has been studied extensively. The mouse is the most widely used experimental animal for this purpose. However, the lack of a reliable and efficient technique for karyotyping mouse oocytes has limited comprehensive studies of chromosome-specific segregation errors in this animal model. Here, we developed a novel karyotyping technique for mouse oocytes by applying multicolour fluorescence in situ hybridisation (FISH) to chromosome slides prepared by a gradual fixation/air-drying method, which is best suited to avoid rupture of oocyte membrane and artificial loss of chromosomes. The success rate of karyotyping meiosis I and II oocytes was about 30%, which improved to over 90% when the oocytes were 'flattened' during fixation and the chromosome specimens were denatured at 4°C. When this technique was applied to the karyotyping of meiosis II oocytes from aged female mice and from young female mice injected with colchicine, more than 80% of the oocytes were successfully karyotyped and the number of chromosomes was identified on all aberrant chromosomes. In conclusion, our technique allows for the efficient and reliable karyotyping of mouse oocytes.


The changing face of emphysematous cholecystitis.
Emphysematous cholecystitis is a variant of acute cholecystitis characterized by the presence of gas in the gall bladder lumen, wall or pericholecystic tissues in the absence of an abnormal communication between the biliary system and the gastrointestinal tract. In the past, the diagnosis has relied on the plain abdominal radiograph (AXR), since there are no clinical features to separate this condition from simple acute cholecystitis. The apparently high mortality and morbidity associated with emphysematous cholecystitis has previously emphasized the importance of emergency cholecystectomy. We have reviewed eight cases of emphysematous cholecystitis presenting to this hospital over the last 5 years. The diagnosis was made on AXR in only one of these cases. Ultrasound (US) scans were performed in all eight cases, of which five were positive and three negative, due to non-visualization of the gall bladder. In the three negative cases, the diagnosis was made on subsequent CT scans. On initial clinical examination, only one of the eight patients appeared systemically unwell and conservative management was employed in five of the patients. The remaining three patients underwent cholecystectomy within 3-5 days because of continuing signs or symptoms. It is concluded that the AXR is relatively insensitive in the diagnosis of emphysematous cholecystitis. As a result of the regular use of US in suspected hepatobiliary disease, emphysematous cholecystitis is being diagnosed with increased frequency, uncovering a broad spectrum of disease ranging from mild to severe. Previously, failure to separate milder cases from simple acute cholecystitis may have been responsible for reports of unremitting severity and progression requiring emergency cholecystectomy. Based on clinical assessment, conservative surgical management is possible in a significant proportion of patients.


Exploring the Material-Induced Transcriptional Landscape of Osteoblasts on Bone Graft Materials.
During the past decades, there have been major advances in the field of biomaterials, thereby generating a vast variety of materials for a broad range of tissue engineering and regeneration applications. Although gene expression profiling has been used occasionally in biomaterial research, its usefulness for understanding cell-biomaterial interactions should be further explored for it to fulfill its promise as a tool to assess and improve material properties. Here, the transcriptional landscape induced by 23 materials is explored with a variety of properties within the scope of bone regeneration. An osteoblast cell line is used to identify the gene expression profiles that can be adopted in response to biophysical and chemical cues. It is shown that TGF-β and WNT signaling may be involved in the cellular response to osteoinductive materials along with differential cell adhesion kinetics via attenuated FAK signaling. The previously reported effect of calcium and phosphate on BMP2 and TGF-β signaling is confirmed and the biological effect of the addition of nanohydroxyapatite in poly (d,l-lactic acid) polymer particles is studied. Together with future applications, this approach will help researchers understand cellular responses in relation to material properties, which will promote the development of more effective biomaterials for applications in tissue regeneration.


Arsenic Uptake and Translocation in Plants.
Arsenic (As) is a highly toxic metalloid that is classified as a non-threshold class-1 carcinogen. Millions of people worldwide suffer from As toxicity due to the intake of As-contaminated drinking water and food. Reducing the As concentration in drinking water and food is thus of critical importance. Phytoremediation of soil contaminated with As and the reduction of As contamination in food depend on a detailed understanding of As uptake and transport in plants. As transporters play essential roles in As uptake, translocation and accumulation in plant cells. In this review, we summarize the current understanding of As transport in plants, with an emphasis on As uptake, mechanisms of As resistance and the long-distance translocation of As, especially the accumulation of As in grains through phloem-mediated transport.


Plasticity in the contribution of T cell receptor variable region residues to binding of peptide-HLA-A2 complexes.
One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this "germline codon" hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs.


Alcohol marketing and youth alcohol consumption: a systematic review of longitudinal studies published since 2008.
Youth alcohol consumption is a major global public health concern. Previous reviews have concluded that exposure to alcohol marketing was associated with earlier drinking initiation and higher alcohol consumption among youth. This review examined longitudinal studies published since those earlier reviews. Peer-reviewed papers were identified in medical, scientific and social science databases, supplemented by examination of reference lists. Non-peer-reviewed papers were included if they were published by organizations deemed to be authoritative, were fully referenced and contained primary data not available elsewhere. Papers were restricted to those that included measures of marketing exposure and alcohol consumption for at least 500 underage people. Multiple authors reviewed studies for inclusion and assessed their quality using the National Heart, Lung and Blood Institute's Quality Assessment Tool for Observation Cohort and Cross-Sectional Studies. Twelve studies (ranging in duration from 9 months to 8 years), following nine unique cohorts not reported on previously involving 35 219 participants from Europe, Asia and North America, met inclusion criteria. All 12 found evidence of a positive association between level of marketing exposure and level of youth alcohol consumption. Some found significant associations between youth exposure to alcohol marketing and initiation of alcohol use (odds ratios ranging from 1.00 to 1.69), and there were clear associations between exposure and subsequent binge or hazardous drinking (odds ratios ranging from 1.38 to 2.15). Mediators included marketing receptivity, brand recognition and alcohol expectancies. Levels of marketing exposure among younger adolescents were similar to those found among older adolescents and young adults. Young people who have greater exposure to alcohol marketing appear to be more likely subsequently to initiate alcohol use and engage in binge and hazardous drinking.


Variations in anxiety and depression as a function of ADHD subtypes defined by DSM-IV: do subtype differences exist or not?
Concerns have been raised about the ability of diagnostic criteria for attention-deficit/hyperactivity disorder (ADHD) to distinguish subtypes that are clearly distinct from each other with regard to clinical correlates. One area of concern is that research regarding differences in anxiety and depression as a function of ADHD subtype has produced discrepant findings. This study was designed to systematically evaluate whether the ADHD subtypes differ with regard to level of internalizing symptoms. From a large pool of children referred to an ADHD center based in a pediatric hospital, children were differentiated into three groups: ADHD, Combined Type (ADHD/COM); ADHD, Inattentive Type (ADHD/I); and a non-ADHD, comparison group (COMP). Parent- and child-report measures using both dimensional and categorical methods were used to assess internalizing symptoms. The results indicated that children with ADHD/COM and ADHD/I had similar levels of anxiety and depression. Subtype differences related to parent-reported depression were accounted for by group differences in level of externalizing problems. The results were discussed with regard to their implications for refining the criteria used to differentiate children with ADHD into subtypes.


Engineering terpenoid production through transient expression in Nicotiana benthamiana.
Terpenoids are the most structurally diverse class of plant natural products with a huge range of commercial and medical applications. Exploiting this enormous potential has historically been hindered due to low levels of these compounds in their natural sources, making isolation difficult, while their structural complexity frequently makes synthetic chemistry approaches uneconomical. Engineering terpenoid biosynthesis in heterologous host production platforms provides a means to overcome these obstacles. In particular, plant-based production systems are attractive as they provide the compartmentalisation and cofactors necessary for the transfer of functional pathways from other plants. Nicotiana benthamiana, a wild relative of tobacco, has become increasingly popular as a heterologous expression platform for reconstituting plant natural product pathways, because it is amenable to Agrobacterium-mediated transient expression, a scalable and highly flexible process that enables rapid expression of genes and enzymes from other plant species. Here, we review recent work describing terpene production in N. benthamiana. We examine various strategies taken to engineer this host for increased production of the target metabolite. We also look at how transient expression can be utilised for rapid generation of molecular diversity, including new-to-nature products. Finally, we highlight current issues surrounding this expression platform and discuss the future directions and developments which will be needed to fully realise the potential of this system.


A randomized study of the prevention of sudden death in patients with coronary artery disease. Multicenter Unsustained Tachycardia Trial Investigators.
Empirical antiarrhythmic therapy has not reduced mortality among patients with coronary artery disease and asymptomatic ventricular arrhythmias. Previous studies have suggested that antiarrhythmic therapy guided by electrophysiologic testing might reduce the risk of sudden death. We conducted a randomized, controlled trial to test the hypothesis that electrophysiologically guided antiarrhythmic therapy would reduce the risk of sudden death among patients with coronary artery disease, a left ventricular ejection fraction of 40 percent or less, and asymptomatic, unsustained ventricular tachycardia. Patients in whom sustained ventricular tachyarrhythmias were induced by programmed stimulation were randomly assigned to receive either antiarrhythmic therapy, including drugs and implantable defibrillators, as indicated by the results of electrophysiologic testing, or no antiarrhythmic therapy. Angiotensin-converting-enzyme inhibitors and beta-adrenergic-blocking agents were administered if the patients could tolerate them. A total of 704 patients with inducible, sustained ventricular tachyarrhythmias were randomly assigned to treatment groups. Five-year Kaplan-Meier estimates of the incidence of the primary end point of cardiac arrest or death from arrhythmia were 25 percent among those receiving electrophysiologically guided therapy and 32 percent among the patients assigned to no antiarrhythmic therapy (relative risk, 0.73; 95 percent confidence interval, 0.53 to 0.99), representing a reduction in risk of 27 percent). The five-year estimates of overall mortality were 42 percent and 48 percent, respectively (relative risk, 0.80; 95 percent confidence interval, 0.64 to 1.01). The risk of cardiac arrest or death from arrhythmia among the patients who received treatment with defibrillators was significantly lower than that among the patients discharged without receiving defibrillator treatment (relative risk, 0.24; 95 percent confidence interval, 0.13 to 0.45; P<0.001). Neither the rate of cardiac arrest or death from arrhythmia nor the overall mortality rate was lower among the patients assigned to electrophysiologically guided therapy and treated with antiarrhythmic drugs than among the patients assigned to no antiarrhythmic therapy. Electrophysiologically guided antiarrhythmic therapy with implantable defibrillators, but not with antiarrhythmic drugs, reduces the risk of sudden death in high-risk patients with coronary disease.


Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities.
Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.


In-vitro propagation and antimycotic potential of extracts and essential oil of roots of Aristolochia bracteolata Linn. (Aristolochiaceae).
In spite of the therapeutic importance of Aristolochia bracteolata Linn. in Nigerian ethnomedicine, it is largely collected from the wild. Owing to the acclaimed potency of the plant and the difficulty in treating candidiasis, the anticandidal activity and in vitro propagation of the plant were investigated. Phytochemical screening and preparation of extracts of the roots were done using standard procedures. Clinical isolates of Candida albicans were screened against extracts and essential oil of Aristolochia bracteolata root using agar-well diffusion method. Minimum Inhibitory Concentration (MIC) of the ethanol extract was determined using broth dilution method. The nodal cuttings of A. bracteolata were cultured on Murashige and Skoog (MS) basal media. A. bracteolata contained alkaloids, saponins and cardenolides. The water extract was inactive on all isolates. The ethanol extract (500 mg/ml) and essential oil (undiluted) exhibited anticandidal activity on 9 out of 10 isolates at 10(1) - 10(6) cfu/ml inoculums concentration. Green growth and callus formation were observed in explants cultured on MS basal media after 30 days. A. bracteolata could be a source of anticandidal phytomedicine and the in vitro propagation confirmed its sustainability as anticandidal agent.


Bioactive biomaterials.
The most important advances in the field of biomaterials over the past few years have been in bioactive biomaterials. Materials have been developed to incorporate bioactivity through biological recognition, including incorporation of adhesion factors, polyanionic sites that mimic the electrostatics of biological regulatory polysaccharides, and cleavage sites for enzymes involved in cell migration. Materials have also been developed to be active in biological environments by undergoing phase changes in situ, including transformations from liquid precursors to solids and from soluble materials to materials that are immobilised on tissue surfaces.


Predictive modelling of fatigue failure in concentrated lubricated contacts.
Reducing frictional losses in response to the energy agenda will require use of less viscous lubricants causing hydrodynamically-lubricated bearings to operate with thinner films leading to "mixed lubrication" conditions in which a degree of direct interaction occurs between surfaces protected only by boundary tribofilms. The paper considers the consequences of thinner films and mixed lubrication for concentrated contacts such as those occurring between the teeth of power transmission gears and in rolling element bearings. Surface fatigue in gears remains a serious problem in demanding applications, and its solution will become more pressing with the tendency towards thinner oils. The particular form of failure examined here is micropitting, which is identified as a fatigue phenomenon occurring at the scale of the surface roughness asperities. It has emerged recently as a systemic difficulty in the operation of large scale wind turbines where it occurs in both power transmission gears and their support bearings. Predictive physical modelling of these contacts requires a transient mixed lubrication analysis for conditions in which the predicted lubricant film thickness is of the same order or significantly less than the height of surface roughness features. Numerical solvers have therefore been developed which are able to deal with situations in which transient solid contacts occur between surface asperity features under realistic engineering conditions. Results of the analysis, which reveal the detailed time-varying behaviour of pressure and film clearance, have been used to predict fatigue and damage accumulation at the scale of surface asperity features with the aim of improving understanding of the micropitting phenomenon. The possible consequences on fatigue of residual stress fields resulting from plastic deformation of surface asperities is also considered.


Process versus outcome in hypertension: a positive result.
We studied the association between the outcome of antihypertensive care and three items of that care among 230 hypertensive steelworkers who were referred to 83 physicians. The first item was the decision to treat some patients but not others: 63% of the patients were prescribed antihypertensive drugs and the mean decrease in their diastolic blood pressure (DBP) was greater than that among untreated patients (12.2 +/- 0.84 vs 7.8 +/- 0.83 mm Hg [+/- SEM], p less than 0.001). The second item was the vigor of prescribed medication: Patients prescribed more vigorous treatments had lower DBP (p less than 0.005). Third, patient compliance was related to achieving a goal DBP of less than 90 mm Hg (p less than 0.05) and the product of prescribed vigor and compliance was highly associated with DBP response (p less than 0.0001). These results stand in contrast to those of previous studies that failed to detect associations between various other items of the care process and the outcome of antihypertensive care.


[Relationships between Medicine and Spirituality].
This review aims at analysing the links between medicine and spirituality, two seemingly distant concepts. Medicine at its beginnings was imbued with rituals that invoked the intervention of supernatural powers, as man were unable to treat diseases and struggled to bear the suffering caused by them and the fragility of their own bodies. Today, in the post-genomic era, medicine has gained great benefits from new and extraordinary scientific and technological achievements, permitting sophisticated therapeutic and diagnostic approaches, which assure cures not previously possible. Even considering these great accomplishments in medicine and technology, it should be borne in mind that diseases not only induce bodily changes in sufferers, but also affect their emotional state and social interactions. Illness, especially when serious and in presence of a poor prognosis, raises profound questions around the meaning of life, affections, suffering and death. In the last few decades scientists, doctors, theologians, psychologists and others, in considering these questions, have emphasized the importance of spirituality as a relevant factor in the care of the sick and their illnesses. Drawing from some thoughts expressed in the book, "When the Breath Becomes Air," authored by the physician Paul Kalanithi, we claim that spirituality should be perceived as an important contributing factor in the therapeutic path. Our aim is to deepen the meaning of spirituality, differentiating it from religion, faith and mysticism, and to understand how it should be integrated with post-genomic medicine to enhance its positive aspects and effects.


Sympathetic activity in patients with panic disorder at rest, under laboratory mental stress, and during panic attacks.
The sympathetic nervous system has long been believed to be involved in the pathogenesis of panic disorder, but studies to date, most using peripheral venous catecholamine measurements, have yielded conflicting and equivocal results. We tested sympathetic nervous function in patients with panic disorder by using more sensitive methods. Sympathetic nervous and adrenal medullary function was measured by using direct nerve recording (clinical microneurography) and whole-body and cardiac catecholamine kinetics in 13 patients with panic disorder as defined by the DSM-IV, and 14 healthy control subjects. Measurements were made at rest, during laboratory stress (forced mental arithmetic), and, for 4 patients, during panic attacks occurring spontaneously in the laboratory setting. Muscle sympathetic activity, arterial plasma concentration of norepinephrine, and the total and cardiac norepinephrine spillover rates to plasma were similar in patients and control subjects at rest, as was whole-body epinephrine secretion. Epinephrine spillover from the heart was elevated in patients with panic disorder (P=.01). Responses to laboratory mental stress were almost identical in patient and control groups. During panic attacks, there were marked increases in epinephrine secretion and large increases in the sympathetic activity in muscle in 2 patients but smaller changes in the total norepinephrine spillover to plasma. Whole-body and regional sympathetic nervous activity are not elevated at rest in patients with panic disorder. Epinephrine is released from the heart at rest in patients with panic disorder, possibly due to loading of cardiac neuronal stores by uptake from plasma during surges of epinephrine secretion in panic attacks. Contrary to popular belief, the sympathetic nervous system is not globally activated during panic attacks.


Association between distress and displacement settings: a cross-sectional survey among displaced Yazidis in northern Iraq.
Globally 70.8 million people have been forcibly displaced from their homes and are at disproportionally high risk for trauma. At the time of this study, there was an estimated 1.6 million internally displaced persons (IDP) in Iraq, more than two-thirds of whom reside in private, urban settings. This study aims to understand the impact of post-displacement accommodation on mental well-being of the Yazidi minority group displaced in Iraq. Multi-stage stratified sampling was used to randomly select IDPs in camp and out of camp settlements in northern Iraq. Standardized questionnaires evaluated factors including exposure to violence and self-reported distress symptoms (measured by Impact of Event Scale-Revised). A multi-variate linear model assessed the relationship between settlement setting and distress symptoms. One thousand two hundred fifty-six displaced Yazidi participants were included in the study: 63% in camps and 37% out of camps. After controlling for exposure to violence, social cohesion, unemployment, and access to basic services, IDPs in camps were predicted to have a 19% higher mean distress symptom score compared to those out of camps. This study provides a framework to investigate post-displacement accommodation as a potential intervention to improve well-being for displaced populations. With a shift towards new models of emergency and long-term housing, it is important to understand the potential and limitations of more decentralized models, and identify effective methods to maintain access to basic services while improving living conditions for both displaced populations and their host communities.


IGF-1R expression is associated with HPV-negative status and adverse survival in head and neck squamous cell cancer.
Head and neck squamous cell carcinomas (HNSCC) are treated with surgery, radiotherapy and cisplatin-based chemotherapy, but survival from locally-advanced disease remains poor, particularly in patients whose tumors are negative for Human papillomavirus (HPV). Type 1 IGF receptor (IGF-1R) is known to promote tumorigenesis and resistance to cancer therapeutics. Here, we assessed IGF-1R immunohistochemistry on tissue microarrays containing 852 cores from 346 HNSCC patients with primary tumors in the oropharynx (n = 231), larynx (85), hypopharynx (28), oral cavity (2). Of these, 236 (68%) were HPV-negative, 110 (32%) positive. IGF-1R was detected in the cell membrane of 36% and cytoplasm of 92% of HNSCCs; in 64 cases with matched normal tonsillar epithelium, IGF-1R was overexpressed in the HNSCCs (P < 0.001). Overall survival (OS) and disease-specific survival (DSS) were reduced in patients whose tumors contained high membrane IGF-1R [OS: hazard ratio (HR) = 1.63, P = 0.006; DSS: HR = 1.63, P = 0.016], cytoplasmic IGF-1R (OS: HR = 1.58, P = 0.009; DSS: HR = 1.58, P = 0.024) and total IGF-1R (OS: HR = 2.02, P < 0.001; DSS: HR = 2.2, P < 0.001). High tumor IGF-1R showed significant association with high-tumor T-stage (P < 0.001) and HPV-negativity (P < 0.001), and was associated with shorter OS when considering patients with HPV-positive (P = 0.01) and negative (P = 0.006) tumors separately. IGF-1R was independently associated with survival in multivariate analysis including HPV, but not when lymphovascular invasion, perineural spread and T-stage were included. Of these factors, only IGF-1R can be manipulated; the association of IGF-1R with aggressive disease supports experimental incorporation of anti-IGF-1R agents into multimodality treatment programs for HPV-negative and high IGF-1R HPV-positive HNSCC.


New approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccines.
The preparation of inactivated polio and rabies vaccine in tissue culture has been developed into a "Unit Process" at our laboratory during recent years. The process comprises trypsinization of animal kidney cells by the perfusion method, cell/virus cultivation in microcarrier culture and concentration and purification of the virus suspension followed by inactivation. Also, the control tests on the virus suspensions and vaccines are performed, to the greatest possible extent, according to the same procedures. In the meantime, both vaccines thus prepared have been tested in man. High antibody titres have been obtained and no adverse reactions have been observed. The results of a field trial with inactivated poliovaccine, administered at different concentrations, will be presented by Dr Salk. Preliminary concepts for further development of the production and control of these inactivated virus vaccines are discussed.


Rapid densitometric determination of cell migration and cell adhesion in a microchemotaxis chamber.
A new rapid staining and measuring method has been developed for the quantification of migrated cells in a microchemotaxis chamber. The migrated cells were, after staining, evaluated by a transmission densitometer. The method introduced here is more accurate and faster than those described previously. In addition the technique can be used to determine the adherent capacity of cells.


The effect of the 2-week wait referral system on the detection of and mortality from colorectal cancer: protocol of a systematic review and meta-analysis.
Colorectal cancer represents the fourth most common cancer in England and Wales; survival is high for early stage disease but declines sharply with advanced stage. UK figures suggest that cancer survival rates are lower than those of other Western European countries. Current 5-year survival is around 50 %. A rapid access strategy was introduced through the Department of Health in 2000. This 2-week wait (TWW) referral pathway was devised to streamline referral for suspected cancer, allow diagnosis at an earlier stage, reduce cancer survival inequality and reduce cancer-related mortality. However, only around half of patients with colorectal cancer have symptoms that fit the TWW criteria plus there is a fourfold difference in referral rates across England and Wales. High-quality evidence of TWW outcome measures for colorectal cancer is lacking. This systematic review will collate and evaluate the latest evidence on colorectal cancer detection rate, stage at diagnosis and mortality. English-language publications from 2000 reporting outcomes on the TWW referral system for suspected colorectal cancer will be eligible for inclusion. Cochrane, EMBASE, MEDLINE via PubMed, NHS Evidence, Trip and the British Library Catalogue databases will be searched. Two paired reviewers will independently screen all titles/abstracts and full text for eligibility, then extract data and assess for bias using standardised formats. They will hand review reference lists of eligible articles. Disagreement will be resolved via third party adjudication. Summary effect measures for post-referral diagnosis and mortality rates will be calculated and expressed as relative risk, hazard rate ratio or risk difference with corresponding 95 % confidence intervals. Where possible summary effect measures will be pooled, heterogeneity and its extent for pooled estimates will be assessed via visual inspection of forest plots and explored via sub-group analysis. In this systematic review, we aim to summarise the relevant evidence on cancer detection rate, cancer stage at diagnosis and disease-related mortality rates for patients with suspected colorectal cancer investigated through the TWW referral system in England and Wales. We will highlight gaps in the evidence and provide a better understanding of whether it is meeting its desired effect. PROSPERO CRD42016037368.


Ultrafast fluorescence dynamics of DNA-based silver clusters.
Atomic-level understanding of the nature of the electronically excited states in ligand-stabilized metal nanoclusters (NCs) is a prerequisite for the design of new NCs with desired properties. In this study, we investigate the emission dynamics of a Ag-DNA complex using the fluorescence up-conversion technique. We show that most of the relaxation from the Franck-Condon state to the emissive state takes place in less than 100 fs, in spite of a relatively large Stokes shift of 4500 cm-1. This relaxation is much faster than typical solvent/DNA relaxation rates. A further small relaxation occurs with time constants ranging from a few to hundreds of picoseconds. We also calculate the Stokes shift for model complexes of a small three-atom Ag3+ cluster with cytosine and guanine. The results of our calculations show that a substantial geometry change of the Ag3+ cluster is observed in the S1 state of both complexes, which results in Stokes shifts comparable with the experimental value. We conclude that the Stokes shift in the Ag-DNA complex arises mostly due to the change in the geometry of the Ag cluster in the excited state rather than to the solvent/DNA reorganization. Also, a different structure of the Ag-DNA complex ("dark cluster"), the excited state of which decays in 200 fs, is observed. The nature of this ultrafast deactivation is unclear, which requires further investigations.


Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry.
We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1(-/-) skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K(1/2)) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K(1/2) for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.


Shake-It-Up: health promotion and capacity building for people with spinal cord injuries and related neurological disabilities.
This is a description of a model demonstration project called Project Shake-It-Up. This project promotes the health and empowerment of people with spinal cord injuries and related neurological disabilities. It also builds the capacity of community organizations that provide services to this population. Participants are involved in interactive seminars concerning health and advocacy on topics like nutrition and being assertive with healthcare providers. They take part in physical and recreational activities such as seat kayaking, kite-flying and hand cycling. Case coordinators support their project participation and personal goal attainment. Community organizations and university researchers developed a successful partnership. Individual members of the first cohort supported one another and benefited physically and psychologically. Project-Shake-It-Up provides positive initial signs of the value of combining the resources of universities and community agencies. Working together, these organizations can develop distinctive, multi-faceted programmes to support the health and empowerment of people with spinal cord injuries and other related neurological disabilities.


[Postpartum and early postoperative anemia after gynecological surgery: treatment with intravenous iron].
To assess the efficacy and safety of treatment with intravenous iron for postpartum anemia or postoperative anemia after gynecological surgery. A prospective study enrolling patients entering our recovery care unit from June through December 2004 with hemoglobin levels less than 10 g x dL(-1) after vaginal childbirth or cesarean section or after another form of gynecological surgery. Three 200 mg doses of intravenous iron sucrose (Venofer) were administered on consecutive days. Fifteen days after the last dose, the patient came for follow-up tests and was asked about side effects. The results were analyzed with a Student t test for matched samples. A total of 250 obstetric and 52 gynecological surgery patients were enrolled; 156 and 33 completed the study in each group, respectively. Hemoglobin increased after treatment by 3.2 g x dL(-1) in the obstetric patients and by 2.7 g x dL(-1) in patients who underwent gynecological surgery. The increase was significant in both groups (P<0.001); the 95% confidence interval was 2.918-3.519 for the obstetric patients and 2.220-3.071 for the gynecological surgery patients. The incidence of side effects was low (13 obstetric patients and 1 gynecological surgery patient). Most side effects were related to pain at the injection site (in 12 of the 14 women). Intravenous iron sucrose is safe and effective for treating puerperal anemia and following gynecological surgery. The low incidence of serious side effects and the rapid recovery of hemoglobin levels make this a safe, effective drug for treating anemia.


Effects of a frontal lesion on the cortical potentials associated with voluntary movement in the awake monkey.
An accidental, punctate lesion to a medial portion of the frontal lobe, including supplementary motor cortex and anterior commissure, produced a change in the cortical potentials preceding voluntary movement in a cynomolgus monkey. At the time of the lesion, the animal was highly trained in a repetitive voluntary movement task, and baseline mapping of specific movement-related potentials (MRPs) was complete. Prelesion MRPs were characterized by a well-defined N2 response, which began approximately 100 msec before the onset of movement. This response was centered over the contralateral precentral gyrus and believed to reflect the phasic portion of the corticospinal discharge. After the lesion, the animal fully recovered the ability to perform the movement task. However, the amplitude of N2 on the side ipsilateral to the lesion was significantly reduced. This decrease was not associated with any deficit in movement execution. EMG measurements did not exhibit any diminished or abnormal patterns of activity. These data suggest that portions of the frontal lobe not directly involved in the genesis of MRPs may still provide a supportive role in organizing the sequence and pattern of neuroelectric activity associated with movement. Furthermore, the findings document the sensitivity of MRPs for detecting subtle changes in the pattern of efferent electrical activity associated with a lesion within the brain, even in the absence of overt movement disorders.


Fornix transection impairs visuospatial memory acquisition more than retrieval.
It has been hypothesized that some fornical fibres may instantiate a neuromodulatory reinforcement signal supporting memory acquisition in medial temporal cortical regions. This suggests that fornix transection should impair postoperative new learning more severely than the recall of preoperatively acquired information. Here, postoperative recall of 288 concurrent visuo-spatial discrimination problems acquired preoperatively was unaffected after fornix transection in the macaque, whereas new postoperative learning of 72 problems was impaired. This and other recent evidence supports the idea that the main function of the fornix in macaque monkeys is to support new learning about spatio-temporal context.


Pancreatitis during intravenous pentamidine therapy in an AIDS patient with prior exposure to didanosine.
To report a case of an HIV-positive man who received sequential didanosine and pentamidine treatment and subsequently developed acute clinical pancreatitis. In June 1992 didanosine 200 mg po bid was initiated in a 30-year-old man with AIDS. After a 22-week course of didanosine, the patient was hospitalized and didanosine was discontinued on day 4. The patient then received 8 days of treatment for a presumed Pneumocystis carinii pneumonia (PCP) with pentamidine 4 mg/kg/d iv. As the patient responded clinically to therapy, he was discharged home to complete a 21-day course of pentamidine. On day 14 of therapy, the patient experienced nausea, vomiting, diarrhea, fatigue, and was hypotensive. The dosage of pentamidine was reduced by 50 percent. After receiving 18 doses of pentamidine, treatment was discontinued, as symptoms had worsened and serum amylase and lipase concentrations were elevated. The patient was hospitalized and the diagnosis of acute clinical pancreatitis was made. After a 21-day hospitalization, the patient was discharged home in fair condition on hyperalimentation. Potential causes of pancreatitis, including opportunistic infections, neoplasms, and drugs, are discussed. The most probable factors associated with pancreatitis in our patient are didanosine and pentamidine therapy. As our patient developed pancreatitis following sequential administration of didanosine and pentamidine, it would be prudent to monitor for signs and symptoms of pancreatitis in similar cases. In addition, didanosine should be discontinued during and for one week following treatment of PCP when pentamidine is used.


Prediction-Based Energy Saving Mechanism in 3GPP NB-IoT Networks.
The current expansion of the Internet of things (IoT) demands improved communication platforms that support a wide area with low energy consumption. The 3rd Generation Partnership Project introduced narrowband IoT (NB-IoT) as IoT communication solutions. NB-IoT devices should be available for over 10 years without requiring a battery replacement. Thus, a low energy consumption is essential for the successful deployment of this technology. Given that a high amount of energy is consumed for radio transmission by the power amplifier, reducing the uplink transmission time is key to ensure a long lifespan of an IoT device. In this paper, we propose a prediction-based energy saving mechanism (PBESM) that is focused on enhanced uplink transmission. The mechanism consists of two parts: first, the network architecture that predicts the uplink packet occurrence through a deep packet inspection; second, an algorithm that predicts the processing delay and pre-assigns radio resources to enhance the scheduling request procedure. In this way, our mechanism reduces the number of random accesses and the energy consumed by radio transmission. Simulation results showed that the energy consumption using the proposed PBESM is reduced by up to 34% in comparison with that in the conventional NB-IoT method.


Regulation of epidermal-growth-factor-receptor signal transduction by cis-unsaturated fatty acids. Evidence for a protein kinase C-independent mechanism.
The effect of acute treatment with non-esterified fatty acids (NEFA) on transmembrane signalling has been investigated in three different cell lines. In EGFR T17 cells, pretreatment with cis-unsaturated (oleic and palmitoleic acids) NEFA, but not with saturated or trans-unsaturated NEFA, inhibited the epidermal-growth-factor (EGF)-induced increases in cytosolic [Ca2+], membrane potential and Ins(1,4,5)P3 generation. The blocking effect was found to be time- and dose-dependent and rapidly reversible after washout. However, oleic acid treatment did not block either binding of 125I-EGF to its receptor or EGF-induced autophosphorylation of the EGF receptor. The mechanism of action of NEFA could not be attributed to protein kinase C activation, since (i) down-regulation of the enzyme by long-term treatment with phorbol esters did not prevent blockade by oleic acid, and (ii) the effects of acutely administered phorbol ester and oleic acid were additive. In this cell line, signalling at bradykinin and bombesin receptors was also impaired by oleic acid. In A431 cells, oleic acid also blocked signal transduction at the EGF and B2 bradykinin receptors. Finally, in PC12 cells, oleic acid blocked the Ca2+ influx mediated by the activation of B2 bradykinin receptors. (1) NEFA block signal transduction by interfering with receptor-phospholipase C or phospholipase C-substrate interaction without preventing ligand binding; (2) NEFA do not act by a protein kinase C-mediated mechanism; (3) the effect of NEFA is dependent on their configuration rather than hydrophobicity or chain length; (4) this effect is evident in several different cell lines and receptor systems.


The ankyrin repeats and DHHC S-acyl transferase domain of AKR1 act independently to regulate switching from vegetative to mating states in yeast.
Signal transduction from G-protein coupled receptors to MAPK cascades through heterotrimeric G-proteins has been described for many eukaryotic systems. One of the best-characterised examples is the yeast pheromone response pathway, which is negatively regulated by AKR1. AKR1-like proteins are present in all eukaryotes and contain a DHHC domain and six ankyrin repeats. Whilst the DHHC domain dependant S-acyl transferase (palmitoyl transferase) function of AKR1 is well documented it is not known whether the ankyrin repeats are also required for this activity. Here we show that the ankyrin repeats of AKR1 are required for full suppression of the yeast pheromone response pathway, by sequestration of the Gβγ dimer, and act independently of AKR1 S-acylation function. Importantly, the functions provided by the AKR1 ankyrin repeats and DHHC domain are not required on the same molecule to fully restore WT phenotypes and function. We also show that AKR1 molecules are S-acylated at locations other than the DHHC cysteine, increasing the abundance of AKR1 in the cell. Our results have important consequences for studies of AKR1 function, including recent attempts to characterise S-acylation enzymology and kinetics. Proteins similar to AKR1 are found in all eukaryotes and our results have broad implications for future work on these proteins and the control of switching between Gβγ regulated pathways.


Asbestos exposure and psychic injury--a review of 48 claims.
Asbestos exposure has been a common occupational risk resulting in much litigation. Where pulmonary dysfunction has been minimal or even absent, psychic injury has been made an element in claimed damages. Analysis of psychiatric and psychologic claims in 48 cases reveals that diagnoses often do not conform to professional standards, are based on insufficient data sampling, lack adequate overall history as well as medical history, and do not comport with the standard of probability usually required for litigation. The group studied was elderly (mean age--62.6), mostly retired (71%), with some significant medical illnesses (18% on medical retirement). None were retired for pulmonary reasons. As expected, conflict in opinion between the opposing professional medical participants was frequent. Commonly the patients did not substantiate the complaints reported in the medico-legal reports; some ridiculed statements made on their behalf. Many psychological reports reflected simplistic or erroneous concepts of medicine or ignored relevant medical data. This study indicates that in this group claims of psychic injury due to asbestos exposure have little justification and supports the view that the current system of utilization of expert opinions is not reliable or in conformity with reasonable professional standards. Correspondingly, these claims did not result in augmented awards.


Male partner selectivity, romantic confidence, and media depictions of partner scarcity.
An experiment was conducted to explore the effects of exposure to partner scarcity or abundance messages on men's partner selectivity, romantic confidence, and self-assessed attractiveness. Undergraduate male participants watched a soap opera narrative featuring either two men competing over one potential female partner (partner scarcity) or two women competing over one potential male partner (partner abundance). Relative to control subjects, watching either narrative reduced romantic confidence. Experimental condition also affected partner selectivity and self-assessed attractiveness, though both effects were moderated by endorsement of traditional masculine ideology. Viewing the abundance narrative resulted in greater selectivity and self-assessed attractiveness for men high in endorsement of traditional masculinity but diminished selectivity and self-assessed attractiveness for men low in endorsement of traditional masculine identity.


Attrition of older adults in longitudinal surveys: detection and correction of sample selection bias using multigenerational data.
The purpose of this study was (a) to investigate whether attrition due to death and nonresponse leads to bias in estimated growth-decline trajectories when only complete data are used in longitudinal research, and (b) to examine the extent of the bias and possible solutions. The study sample was a subset of the Longitudinal Study of Generations and included data from 208 G1-G2 parent-child dyads and 538 G2-G3 dyads over 30 years. We used a latent growth-decline curve model based on full information maximum likelihood estimation in order to compare parents' and adult children's reports on older respondents' health and intergenerational solidarity by parents' attrition status. Results indicated that attrition due to mortality biased estimates of respondents' assessments of their functional health status over time, and parents' perceptions of the quality of the parent-child relationship deteriorated more rapidly among those who died by Time 7, but nonresponse did not seriously bias estimates of these measures. Using proxies, we found that functional impairment increased more rapidly when children reported about parents, especially in advanced old age. These results support the use of full information in estimating growth curves where mortality is present but raise concerns when using child proxies to evaluate parental health or the quality of intergenerational relationships.


Hemilaminectomy and Bilateral Decompression for Thoracic Spinal Stenosis.
To assess the effectiveness and reliability of hemilaminectomy and bilateral decompression (HLBD) for the treatment of thoracic spinal stenosis (TSS) in selected patients. Clinical data of 21 consecutive adult patients who underwent HLBD were investigated. Patients diagnosed with TSS by computed tomography or magnetic resonance imaging, with stenosis secondary to posterior element hypertrophy, and who underwent HLBD for the treatment of narrow spinal canal were included in this study. Patients were evaluated based on their pre- and postoperative modified Japanese Orthopedic Association (JOA) scores using their medical records. Recovery rates were evaluated using the Hirabayashi?s method. The mean follow-up period, age, and preoperative JOA score were 37.6 months, 61.6 years, and 5.0 (range: 1?10), respectively. The mean JOA score improved postoperatively, i.e., 10.0 (range: 7?11), during follow-up. The recovery rate was 83.3%. Operation-related transfusion, neurological deterioration, and postoperative instability did not occur during the follow-up examination. HLBD is a suitable surgical technique for patients with stenosis primarily caused by posterior element hypertrophy, such as ligamentum flavum (LF) and facet joints because it has a relatively less complication ratio, protects the posterior anatomical structures, prevents post-laminectomy kyphosis and postoperative instability, and promotes successful clinical improvement.


Performance of recidivism risk assessment instruments in U.S. correctional settings.
With the population of adults under correctional supervision in the United States at an all-time high, psychologists and other professionals working in U.S. correctional agencies face mounting pressures to identify offenders at greater risk of recidivism and to guide treatment and supervision recommendations. Risk assessment instruments are increasingly being used to assist with these tasks; however, relatively little is known regarding the performance of these tools in U.S. correctional settings. In this review, we synthesize the findings of studies examining the predictive validity of assessments completed using instruments designed to predict general recidivism risk, including committing a new crime and violating conditions of probation or parole, among adult offenders in the United States. We searched for studies conducted in the United States and published between January 1970 and December 2012 in peer-reviewed journals, government reports, master's theses, and doctoral dissertations using PsycINFO, the U.S. National Criminal Justice Reference Service Abstracts, and Google. We identified 53 studies (72 samples) conducted in U.S. correctional settings examining the predictive validity of 19 risk assessment instruments. The instruments varied widely in the number, type, and content of their items. For most instruments, predictive validity had been examined in 1 or 2 studies conducted in the United States that were published during the reference period. Only 2 studies reported on interrater reliability. No instrument emerged as producing the "most" reliable and valid risk assessments. Findings suggest the need for continued evaluation of the performance of instruments used to predict recidivism risk in U.S. correctional agencies. (PsycINFO Database Record


Sperm allocation in relation to female size in a semelparous salmonid.
To maximize reproductive success, males have to adaptively tailor their sperm expenditure in relation to the quality of potential mates because they require time to replenish their sperm supply for subsequent mating opportunities. Therefore, in mating contexts where males must choose among females in a short period of time, as is the case with semelparous species (which die after one intensely competitive short duration breeding season), selection on sperm allocation can be expected to be a powerful selective agent that shapes the male reproductive success. We quantitatively investigated sperm allocation patterns in chum salmon in relation to perceived female quality by developing a novel method for determining the amount of sperm allocated per ejaculate during spawning bouts. We examined the relationship between sperm expenditure and the body size of paired females (a proxy of egg number and egg quality) in the absence of male-male competition in an experimental channel. The estimated amount of sperm released per spawning event was positively correlated with the size of paired females. However, the number of spawning events a female participated in, which reduces the number of eggs she spawns in each subsequent bout, did not affect this relationship. These results provide support for predictions arising from the sperm allocation hypothesis, male salmon do economize their sperm expenditure in accordance with paired female body size as predicted for their first spawning event, but males overestimate or are unable to assess the quality of females beyond size and provide more sperm than they should in theory when paired with a female that spawned previously. Overall, the observed sperm allocation pattern in chum salmon appears to be adapted to maximize reproductive success assuming female size is an honest indicator of quality, although temporal changes in a female's quality during a reproductive season should be considered when examining sperm allocation strategies.


Defective B cell tolerance in adult (NZB X MZW)F1 mice.
Hapten-specific tolerance was induced in vitro by trinitrophenyl-human gamma globulin (TNP32HGG) to a comparable degree in B cells from adult autoimmune (NZB X NZW)F1 (B/W) mice and normal BDF1, CBA/J, and DBA/1J mice. When a lower epitope density tolerogen (TNP7HGG) was used, B/W mice were significantly less sensitive than normal mice to the induction of B cell tolerance. This finding of defective B cell tolerance in adult B/W mice is consistent with previous reports that document other B cell abnormalities that may relate to the expression of autoimmune disease.


A method to estimate the contribution of regional genetic associations to complex traits from summary association statistics.
Despite considerable efforts, known genetic associations only explain a small fraction of predicted heritability. Regional associations combine information from multiple contiguous genetic variants and can improve variance explained at established association loci. However, regional associations are not easily amenable to estimation using summary association statistics because of sensitivity to linkage disequilibrium (LD). We now propose a novel method, LD Adjusted Regional Genetic Variance (LARGV), to estimate phenotypic variance explained by regional associations using summary statistics while accounting for LD. Our method is asymptotically equivalent to a multiple linear regression model when no interaction or haplotype effects are present. It has several applications, such as ranking of genetic regions according to variance explained or comparison of variance explained by two or more regions. Using height and BMI data from the Health Retirement Study (N = 7,776), we show that most genetic variance lies in a small proportion of the genome and that previously identified linkage peaks have higher than expected regional variance.


Serological precursors of cancer: serum hormones and risk of subsequent prostate cancer.
A population-based nested case-control study was conducted to determine the relation of prediagnostic serum levels of testosterone, dihydrotestosterone, prolactin, follicle-stimulating hormone, luteinizing hormone, estrone, and estradiol to the risk of subsequent prostate cancer. Serum specimens of study subjects were available from a blood collection campaign in Washington County, Maryland, in 1974. Serum hormone levels of 98 histologically confirmed prostate cancer cases diagnosed in the subsequent 13 years were compared to those of 98 controls who were individually matched to cases on the basis of age (within weeks), sex, and race. There were no significant differences in levels of these hormones between cases and controls, although elevated levels of luteinizing hormone and of testosterone:dihydrotestosterone ratios were associated with mild increased risks of prostate cancer.


Report of the 4th European Zebrafish Principal Investigator Meeting.
The European Zebrafish Principal Investigator Meeting (EZPM) is an ideal forum for group leaders using this fantastic animal model not only to discuss science but also to strengthen their interactions, to push forward technological advances, and to define guidelines for the use of this fish in research. The city of Lisbon (Portugal) was voted by the European group leaders to be the setting for the 4th EZPM, and the organizing committee, composed by Leonor Saúde (iMM Lisboa, PT), Susana Lopes (CEDOC, PT), Michael Orger (Champalimaud Foundation, PT), Rui Oliveira (ISPA, PT), and António Jacinto (CEDOC, PT), was very enthusiastic to organize a productive event. The 4th EZPM took place from March 15 to 19 at Pavilhão do Conhecimento, a Science Museum and Educational Center winner of The Great Prize FAD of Arquitecture 1999 and The Society for Environmental Graphic Design Award 2011. Over 5 days, 135 group leaders (89 men and 46 women) coming from 19 different European countries and also from the United States, Turkey, Israel, Chile, and Singapore presented and discussed their recent research achievements. In addition to the scientific oral and poster presentations, the group leaders gathered in very lively community sessions on morphants versus mutants (chaired by Didier Stainier, Max Planck Institute for Heart and Lung Research, DE), funding issues (chaired by Uwe Strahle, KIT-ITG, DE), and gender equality (chaired by Corinne Houart, KCL, United Kingdom). One of the highlights of the 4th EZPM was the guided visit to Oceanário de Lisboa, an international award-winning place that celebrates life with a stunning display of living aquatic creatures.


Properties of the linear canonical integral transformation.
We provide a general expression and different classification schemes for the general two-dimensional canonical integral transformations that describe the propagation of coherent light through lossless first-order optical systems. Main theorems for these transformations, such as shift, scaling, derivation, etc., together with the canonical integral transforms of selected functions, are derived.


Clinical outcomes in adult patients with aplastic anemia: A single institution experience.
Newer treatment modalities are being investigated to improve upon historical outcomes with standard immunosuppressive therapy (IST) in aplastic anemia (AA). We analyzed outcomes of adult patients with AA treated with various combinatorial anti-thymoglobulin-based IST regimens in frontline and relapsed/refractory (R/R) settings. Pretreatment and on-treatment clinical characteristics were analyzed for relationships to response and outcome. Among 126 patients reviewed, 95 were treatment-naïve (TN) and 63, R/R (including 32 from the TN cohort); median ages were 49 and 50 years, respectively. Overall survival (OS) was superior in IST responders (P < .001). Partial response to IST was associated with shorter relapse-free survival (RFS), as compared with complete response (P = .03). By multivariate analysis, baseline platelet and lymphocyte count predicted for IST response at 3 and 6 months, respectively. While additional growth factor interventions led to faster count recovery, there were no statistically significant differences in RFS or OS across the various frontline IST regimens (i.e., with/without G-CSF or eltrombopag). While marrow cellularity did not correlate with peripheral-blood counts at 3 months, cytomorphological assessment revealed dyspoietic changes in all nonresponders with hypercellular-marrow indices. Covert dysplasia, identified through early bone marrow assessment, has implications on future therapy choices after IST failure. Salvage IST response depended upon prior response to ATG: prior responders (46%) vs. primary refractory (0%) (P < .01). In the R/R setting, there was no survival difference between IST and allogeneic stem cell transplant groups, with a trend toward superior OS in the former. Transplant benefits in the R/R setting may be underrealized due to transplant-related mortality.


Evaluating and Validating GBI Mania and Depression Short Forms for Self-Report of Mood Symptoms.
Objective: To evaluate short forms of free self-report mania and depression scales, evaluating their reliability, content coverage, criterion validity, and diagnostic accuracy.Method: Youths age 11 to 18 years seeking outpatient mental health services at either an Academic medical clinic (N = 427) or urban Community mental health center (N = 313), completed the General Behavior Inventory (GBI) and other rating scales. Youths and caregivers completed semi-structured interviews to establish diagnoses and mood symptom severity, with GBI scores masked during diagnosis. Ten- and seven-item short forms, psychometric projections, and observed performance were tested first in the Academic sample and then externally cross-validated in the Community sample.Results: All short forms maintained high reliability (all alphas >.80 across both samples), high correlations with the full-length scales (r.85 to.96), excellent convergent and discriminant validity with mood, behavior, and demographic criteria, and diagnostic accuracy undiminished compared to using the full-length scales. Ten-item scales showed advantages in terms of coverage; the 7 Up showed slightly weaker performance.Conclusions: Present analyses evaluated and externally cross-validated short forms that maintain high reliability and content coverage, and show strong criterion validity and diagnostic accuracy - even when used in an independent sample with very different demographics and referral patterns. The short forms appear useful in clinical applications including initial evaluation, as well as in research settings where they offer an inexpensive quantitative score. Short forms are available in more than two dozen languages. Future work should further evaluate sensitivity to treatment effects and cultural invariance.


The contribution of overweight, obesity, and lack of physical activity to exit from paid employment: a meta-analysis.
The objective of this review was to analyze systematically the association between overweight, obesity, and lack of physical activity (PA) and exit from paid employment through disability pension, unemployment, and early retirement. We also aimed to identify the influence of study population and study design on the magnitude of this association. We searched PubMed and Embase for English language, longitudinal, quantitative studies that described the relationship between overweight, obesity, or lack of PA and exit from work. A short checklist was used to assess the internal and external validity of the studies. We first estimated the pooled effects using a random effects model and then analyzed the influence of study and population characteristics on associations by stratified meta-analyses. In total, 28 out of 1097 publications met the inclusion criteria. Obese [relative risk (RR)=1.53) and, to a lesser extent, overweight (RR=1.16) individuals had an increased likelihood of exit from paid employment through disability pension, but were not at statistically significant increased risk for unemployment or early retirement. Of 17 associations between a lack of PA and disability pension, 8 were statistically significant; this was also the case for 2 of 3 for unemployment. No associations were statistically significant for early retirement. Obesity is a risk factor for exit from paid employment through disability pension. There are also indications that a lack of PA is related to an increased risk of disability pension and unemployment. To protect workers against premature exit from paid employment, long-term interventions to prevent overweight and obesity and promote PA in the working population should be considered for implementation.


Cigarette smoking and steroid hormones in women.
Epidemiological evidence has suggested that cigarette smoking has an anti-oestrogenic effect in women, but the effects of smoking on steroid hormone metabolism are not fully understood. We compared serum concentrations of oestradiol, progesterone (luteal phase) and dehydroepiandrosterone sulphate (DHEA-S), and urinary excretion rates of six steroids of predominantly adrenal origin, in healthy premenopausal and postmenopausal female smokers and non-smokers. Serum concentrations of oestradiol, progesterone and DHEA-S did not differ between smokers and non-smokers by greater than 5%, and none of these differences was statistically significant. Mean urinary excretion rates of androsterone, aetiocholanolone, DHEA, 11-keto-aetiocholanolone, 11-hydroxyandrosterone and 11-hydroxyaetiocholanolone were very similar in smokers and non-smokers in premenopausal women, but were from 2-44% higher in smokers than non-smokers in postmenopausal women. The difference was statistically significant only for 11-hydroxyandrosterone. These results confirm previous reports that cigarette smoking does not affect serum oestradiol in premenopausal or postmenopausal women, but provide only weak evidence to support previous findings of increased levels of some adrenal steroids in postmenopausal women smokers. The mechanism for the apparent anti-oestrogenic effect of cigarette smoking remains unclear.


InternationaL cross-sectIonAl and longItudinal assessment on aSthma cONtrol in European adult patients--the LIAISON study protocol.
According to international guidelines, the goal of asthma management is to achieve and maintain control of the disease, which can be assessed using composite measures. Prospective studies are required to determine how these measures are associated with asthma outcomes and/or future risk. The 'InternationaL cross-sectIonAl and longItudinal assessment on aSthma cONtrol (LIAISON)' observational study has been designed to evaluate asthma control and its determinants, including components of asthma management. The LIAISON study will be conducted in 12 European countries and comprises a cross-sectional phase and a 12-month prospective phase. Both phases will aim at assessing asthma control (six-item Asthma Control Questionnaire, ACQ), asthma-related quality of life (Mini Asthma Quality of Life Questionnaire, Mini-AQLQ), risk of non-adherence to treatment (four-item Morisky Medication Adherence Scale, MMAS-4), potential reasons for poor control, treatment strategies and associated healthcare costs.The cross-sectional phase will recruit > 8,000 adult patients diagnosed with asthma for at least 6 months and receiving the same asthma treatment in the 4 weeks before enrolment.The prospective phase will include all patients with uncontrolled/poorly controlled asthma at the initial visit to assess the proportion reaching control during follow-up and to examine predictors of future risk. Visits will take place after 3, 6 and 12 months. The LIAISON study will provide important information on the prevalence of asthma control and on the quality of life in a broad spectrum of real-life patient populations from different European countries and will also contribute to evaluate differences in management strategies and their impact on healthcare costs over 12 months of observation. ClinicalTrials.gov identifier, NCT01567280.


Evaluation of a guinea pig model to assess interference in the immunogenicity of different components of a combination vaccine comprising diphtheria, tetanus and acellular pertussis (DTaP) vaccine and haemophilus influenzae type b capsular polysaccharide conjugate vaccine.
A guinea pig model to assess the immunogenicity of a combination vaccine containing diphtheria, tetanus and acellular pertussis (DTaP) vaccine and Haemophilus influenzae type b (Hib) capsular polysaccharide conjugated to tetanus toxoid (HibT) was evaluated comparatively with the mouse immunogenicity test to study the effect of combining these antigens on the immunogenicity of various components. The immunogenicity test in mice was performed by subcutaneous injection of groups of 10 animals twice at an interval of four weeks with 1/10 of a single human dose of various formulations of combination vaccines, DTaP or HibT vaccine. The animals were bled at 4 and 6 weeks and IgG or total antibodies to various components were determined by ELISA or RIA. The guinea pig immunogenicity model included groups of animals injected subcutaneously twice at an interval of six weeks with 1.5 times the single human dose of various formulations. The animals were bled at 4, 6 and 8 weeks and serum samples were tested for antibodies to various components by ELISA, RIA and/or neutralization tests. Additionally, potency of tetanus and diphtheria components was assessed as per the US Food and Drug Administration's regulations. Aluminium phosphate (AIPO(4)) adsorbed HibT vaccine or HibT as a combination with AIPO(4)adsorbed DTaP vaccine showed significant increases in IgG antibodies to tetanus toxin in mice as well increased tetanus antitoxin levels in guinea pigs as compared to soluble HibT vaccine. In general, combining DTaP and HibT vaccines did not affect the antibody levels to tetanus and diphtheria toxoids whereas DTaP-HibT combination vaccine elicited significantly lower IgG antibodies to pertussis toxin and filamentous haemagglutinin than DTaP vaccine alone, particularly after first injection. Mice showed similar Hib antibody responses for the combination and HibT alone whereas guinea pigs consistently showed lower anamnestic responses to Hib for combination formulations than for HibT alone. Reducing the amount of HibT and/or tetanus toxoid in the combination formulations reduced this suppression of Hib antibody response in guinea pigs. Suppression of Hib antibody response in combination vaccines has also been reported from recent clinical trials. Based on the results from this study, it appears that the guinea pig model may be able to predict the human response to various components of combination vaccines.


Activation of the Two-Component System LisRK Promotes Cell Adhesion and High Ampicillin Tolerance in Listeria monocytogenes.
Listeria monocytogenes is a foodborne pathogen which can survive in harsh environmental conditions. It responds to external stimuli through an array of two-component systems (TCS) that sense external cues. Several TCS, including LisRK, have been linked to Listeria's ability to grow at slightly elevated antibiotic levels. The aim of this study was to determine if the TCS LisRK is also involved in acquiring the high antibiotic tolerance that is characteristic of persister cells. LisRK activates a response that leads to remodeling of the cell envelope, and we therefore hypothesized that activation of LisRK could also increase in the cells' adhesiveness and initiate the first step in biofilm formation. We used a ΔlisR mutant to study antibiotic tolerance in the presence and absence of LisRK, and a GFP reporter strain to visualize the activation of LisRK in L. monocytogenes LO28 at a single-cell level. LisRK was activated in most cells in stationary phase cultures. Antimicrobial susceptibility tests showed that LisRK was required for the generation of ampicillin tolerance under these conditions. The wildtype strain tolerated exposure to ampicillin at 1,000 × inhibitory levels for 24 h, and the fraction of surviving cells was 20,000-fold higher in the wildtype strain compared to the ΔlisR mutant. The same protection was not offered to other antibiotics (vancomycin, gentamicin, tetracycline), and the mechanism for antibiotic tolerance is thus highly specific. Furthermore, quantification of bacterial attachment rates and attachment force also revealed that the absence of a functional LisRK rendered the cells less adhesive. Hence, LisRK TCS promotes multiple protective mechanisms simultaneously.


Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68.
All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.