Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility.

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees.

Carotid intima-media thickness and incident hypertension: the Brazilian Longitudinal Study of Adult Health.

BACKGROUND: High blood pressure (BP) increases carotid intima-media thickness (CIMT). On the other hand, it is not clear whether the vascular abnormalities reflected in high CIMT may predict incident hypertension. The present study aims to investigate the association between CIMT and incident hypertension after 4 years of follow-up in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), a multiethnic sample of middle-aged adults from six Brazilian cities. METHODS: We analyzed data from 6682 ELSA-Brasil participants (aged 35-74) without hypertension and with complete CIMT data at baseline. After 4 years of follow-up, we describe hypertension incidence, stratifying the sample according to age, sex, and race-specific CIMT quartiles. We also built crude and adjusted Poisson regression models to analyze the association between mean and maximal CIMT values and incident hypertension. RESULTS: We found incident hypertension in 987 (14.8%) participants. According to mean CIMT quartile groups, hypertension incidence varied from 10.2% (first quartile group) to 22.4% (fourth quartile group; P for trend <0.001). In fully adjusted models, 0.1 mm increments in mean CIMT values were associated with a 16% [relative risk (RR):1.16; 95% confidence interval (95% CI) 1.10-1.21; P  < 0.001] higher risk of incident hypertension, respectively. Results were similar when maximal CIMT values were considered instead of mean CIMT values. CONCLUSION: CIMT values at baseline strongly predicted incident hypertension after 4 years of follow-up in this large multiethnic cohort. This highlights the relationship between CIMT and BP and may provide important insights into the significance of this ultrasound measurement.

Pharmacokinetic assessment of ketanserin in the horse.

The purpose of this study was to determine the pharmacokinetics (PK) of the 5-HT(2A) receptor antagonist ketanserin in healthy adult horses, and to develop a computational model that could be used to optimize dosing. Plasma concentrations of ketanserin were determined using liquid chromatography with mass spectrometry after single and multiple intravenous administration in the horse. A two-compartment linear pharmacokinetic model described the plasma concentration-time profile of ketanserin after single and multiple doses in healthy horses; the terminal half-life was 11.5 h; steady-state volume of distribution was 10.5 L/kg; AUC was 115 ng · h/mL; and clearance was 0.87 L/h/kg. Model simulations followed by the examination in three healthy horses suggest 0.3 mg/kg q.8 h exhibited linear PK and produced consistent systemic blood concentrations of ketanserin above 3 ng/mL.

Evaluation of Cleaning Methods on Lithium Disilicate Glass Ceramic Surfaces After Organic Contamination.

The purposes of this study were to 1) evaluate the effectiveness of different cleaning methods from a previously etched and silanized lithium disilicate glass ceramic (EMX) surface after contact with organic fluids (saliva or human blood) and 2) assess the effect of applying a new silane layer after the cleaning methods on the microshear bond strength (mSBS) of resin cement to EMX. EMX discs were etched with 5% hydrofluoric acid (HF) and properly silanized. Three control groups were created (n=10): control (without contamination), saliva positive, and human blood positive. Later, after new contaminations, the samples were distributed into four groups according to the cleaning method (n=20): air-water spray (AWS), 35% phosphoric acid, 70% alcohol, or Ivoclean cleaning paste. After the cleaning methods, subgroups were submitted to a new silane layer application, or not (n=10). All samples received a thin layer of a bonding agent and, subsequently, three light-cured resin cement cylinders were prepared on each EMX surface for the mSBS test. This test was performed on a universal testing machine at a vertical speed of 1 mm/minute until rupture. Contaminated and cleaned silanized EMX surfaces were assessed by scanning electron microscopy (SEM) (n=1). The noncontaminated control group showed an average mSBS of 18.7 MPa, and the positive saliva and human blood control groups yielded a 34% and 42% reduction in bond strength, respectively, compared to the uncontaminated control (p<0.05). For saliva-contaminated surfaces, all cleaning methods were effective and not different from one another or the control group (p>0.05). However, for human blood contamination, only Ivoclean cleaning paste was effective in restoring µSBS to uncontaminated control group levels (p>0.05). SEM images showed a clean surface (ie, with no contaminant residues) after the cleaning methods, regardless of the organic contaminant type. All the assessed cleaning methods were effective in removing saliva from the silanized EMX surface; however, only Ivoclean was able to restore the adhesion quality when the silanized EMX surface was contaminated with human blood.

Atypical clinical presentations of the A3243G mutation, usually associated with MELAS.

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is a clinical syndrome associated with mitochondrial abnormalities. In approximately 80% of patients, the syndrome is associated with the A3243G mutation. However, it has been realized that the A3243G mutation is not uncommon in the general population and is found in many patients with clinical presentations other than MELAS. We present four patients who presented with rhabdomyolysis, muscle fatigue, external ophthalmoplegia and myoclonic jerks respectively. These patients were all found to have the A3243G mutation on muscle biopsy. These patients illustrate the variety of presentations associated with A3243G mutation.

Resistant Starch Production and Glucose Release from Pre-Prepared Chilled Food: The SPUD Project.

With an increasing prevalence of diabetes worldwide, effective dietary strategies for blood glucose control are crucial. As carbohydrates make up approximately 50% of the diet, it is neither practical nor advisable to avoid them altogether. Most of the carbohydrate in the diet is derived from starch, found in potatoes, pasta, rice and bread. These foods are often processed in some way before consumption, yet little is known about the effects processing, such as chilling and reheating, has on the glycaemic response, particularly when the food is consumed in the context of a mixed meal. This article introduces the SPUD project, a BBSRC DRINC-funded initiative. Taking the potato as the model carbohydrate, this project will investigate, via in vitro and in vivo studies, the effects of domestic food processing techniques on the glycaemic response. A final study, utilising intrinsically labelled potato and a dual stable isotope methodology, will model glucose flux data to determine the underlying mechanisms of action.

Human Hendra virus infection causes acute and relapsing encephalitis.

AIM: To study the pathology of two cases of human Hendra virus infection, one with no clinical encephalitis and one with relapsing encephalitis. METHODS: Autopsy tissues were investigated by light microscopy, immunohistochemistry and in situ hybridization. RESULTS: In the patient with acute pulmonary syndrome but not clinical acute encephalitis, vasculitis was found in the brain, lung, heart and kidney. Occasionally, viral antigens were demonstrated in vascular walls but multinucleated endothelial syncytia were absent. In the lung, there was severe inflammation, necrosis and viral antigens in type II pneumocytes and macrophages. The rare kidney glomerulus showed inflammation and viral antigens in capillary walls and podocytes. Discrete necrotic/vacuolar plaques in the brain parenchyma were associated with antigens and viral RNA. Brain inflammation was mild although CD68(+) microglia/macrophages were significantly increased. Cytoplasmic viral inclusions and antigens and viral RNA in neurones and ependyma suggested viral replication. In the case of relapsing encephalitis, there was severe widespread meningoencephalitis characterized by neuronal loss, macrophages and other inflammatory cells, reactive blood vessels and perivascular cuffing. Antigens and viral RNA were mainly found in neurones. Vasculitis was absent in all the tissues examined. CONCLUSIONS: The case of acute Hendra virus infection demonstrated evidence of systemic infection and acute encephalitis. The case of relapsing Hendra virus encephalitis showed no signs of extraneural infection but in the brain, extensive inflammation and infected neurones were observed. Hendra virus can cause acute and relapsing encephalitis and the findings suggest that the pathology and pathogenesis are similar to Nipah virus infection.

Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene.

AIM: To design a rapid diagnostic test to differentiate species belonging to the family Chlamydiaceae. METHODS AND RESULTS: Five oligonucleotide sets each targeting various conserved regions of the genome of six species (Chlamydia muridarum, C. suis, C. trachomatis, Chlamydophila felis, Cp. pneumoniae and Cp. psittaci) belonging to the family Chlamydiaceae were tested for their suitability for polymerase chain reaction (PCR) and high resolution melt (HRM) curve analysis to differentiate Chlamydiaceae species. Three of the oligonucleotide sets were able to detect all six reference species used in this study, but only one set (16SG) could clearly differentiate between them by HRM curve analysis. The PCR-HRM curve analysis confidence percentages correlated strongly with the nucleotide sequence identities. Clinical specimens from a number of animal species suspected of chlamydiosis were tested with the newly developed 16SG PCR-HRM curve analysis and sequenced to confirm the infecting species. It was demonstrated that PCR-HRM using the 16SG oligonucleotide set could relate the infecting Chlamydiaceae species to the most similar (based on 16S rRNA gene nucleotide sequence) reference species tested. Although Cp. pecorum was not included initially as a reference species in this assay, inclusion of a field isolate of Cp. pecorum as a reference allowed two koala specimens to be correctly identified. CONCLUSION: PCR-HRM analysis using the oligonucleotide set 16SG is a robust, simple and rapid technique for differentiation of at least the Chlamydiaceae species used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique allowed for the rapid detection and identification of the six Chlamydiaceae reference species and may be useful for identification of uncharacterized Chlamydiaceae species or for use in animal species where occurrence of the disease has not been fully investigated.

Tracheostomy protocol: experience with development and potential utility.

OBJECTIVES: To examine the feasibility and potential utility of a tracheostomy protocol based on a standardized approach to ventilator weaning. DESIGN: Prospective, observational data collection. SETTING: Academic medical center. PATIENTS: Surgical intensive care unit patients requiring mechanical ventilatory support. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Tracheostomy practice in 200 patients was analyzed in relation to spontaneous breathing trial (SBT) weaning. Decision for, and performance of, tracheostomy occurred (median [interquartile range]) 5.0 (3.75-8.0) and 7.0 (5.0-10.0) days following initiation of mechanical ventilation, respectively. Duration of mechanical ventilation was greater in tracheostomy compared with nontracheostomy patients (15.0 [11.0-19.0] vs. 6.0 [4.0-8.0], p < .001). For patients requiring ventilatory support for > or = 20 days, 100% of patients were maintained via tracheostomy. A protocol based on weaning performance, which included technical considerations, was developed. Individuals who failed preliminary weaning assessment or SBT for 3 successive days following 5 days (nonreintubated patients) or 3 days (reintubated patients) of ventilatory support met tracheostomy criteria. The protocol was implemented on a pilot basis in 125 individuals. Of the 55 (44.0%) patients undergoing tracheostomy, 25 (45.5%) did so consistent with criteria. Eighteen patients (32.7%) underwent tracheostomy before the time interval of data collection targeting weaning protocol performance, and 12 patients (21.8%) passed SBT on one or more occasions, were not extubated, and proceeded to tracheostomy. CONCLUSIONS: A standardized approach in which the decision for tracheostomy is based on objective measures of weaning performance may be a means of using this procedure more consistently and effectively.

Multicenter implementation of a consensus-developed, evidence-based, spontaneous breathing trial protocol.

OBJECTIVE: Evidence-based practice recommendations abound, but implementation is often unstructured and poorly audited. We assessed the ability of a peer network to implement an evidence-based best practice protocol and to measure patient outcomes. DESIGN: Consensus definition of spontaneous breathing trial followed by implementation in eight academic medical centers. SETTING: Six medical, two surgical, and two combined medical/surgical adult intensive care units among eight academic medical centers. STUDY POPULATION: Patients initiating mechanical ventilation through an endotracheal tube during a 12-wk interval formed the study population. INTERVENTIONS: Adoption and implementation of a common spontaneous breathing trial protocol across multiple intensive care units. MEASUREMENTS AND MAIN RESULTS: Seven hundred five patients had 3,486 safety screens for conducting a spontaneous breathing trial; 2072 (59%) patients failed the safety screen. Another 379 (11%) patients failed a 2-min tolerance screen and 1,122 (34%) patients had a full 30-120 min spontaneous breathing trial performed. Seventy percent of eligible patients were enrolled. Only 55% of passing spontaneous breathing trials resulted in liberation from mechanical ventilatory support before another spontaneous breathing trial was performed. CONCLUSIONS: Peer networks can be effective in promoting and implementing evidence-based best practices. Implementation of a best practice (spontaneous breathing trial) may be necessary for, but by itself insufficient to achieve, consistent and timely liberation from ventilator support.

Improving access to ART in low-income settings through knowledge transfer: a case study from Zimbabwe.

It may be assumed that infertility is not a problem in resource-poor areas where fertility rates are high. However, evidence overwhelmingly shows that childlessness is highly stigmatized in these settings and that women who are unable to bear children suffer significant social and psychological consequences. The World Health Organization has recommended that infertility be considered a global health problem and stated the need for ART to be adapted to low-resource settings. This paper describes a model for improving access to ART in low-resource settings. Experienced ART health professionals from Australia and Italy representing medical science, embryology, nursing and counselling used knowledge transfer to support a clinician, a laboratory scientist and a nurse to establish an ART service in Harare, Zimbabwe. Support and mentorship provided between October 2016 and December 2017 included: hosting the clinician and the embryologist for the new service in established ART clinics for short periods and providing them with dedicated mentorship and training during their stay; funding an experienced embryologist to travel to Zimbabwe (three times) to oversee the setting up of the lab and provide hands-on embryology training; funding a scientist and a nurse to travel to Zimbabwe to troubleshoot and establish protocols for record keeping and psychosocial care; and contributing approximately AUD $15,000 to the purchase of some equipment. By 31 March 2018, the team at IVF Zimbabwe had performed 166 ART procedures, which at time of writing had resulted in 16 births and 4 ongoing pregnancies. This case study demonstrates that with mentorship and modest financial support from ART experts from high-income settings, health professionals in low-income settings can deliver affordable ART with successful outcomes.

Pain and Complementary Therapies.

Treatment of both acute and chronic pain typically involves a combination of pharmacologic and provider-based interventions, which is effective for some patients but not for others. Use of pain medications, especially repeated and frequent usage, involves the risk of adverse reactions, overuse, and dependency. Complementary and alternative therapies (CAT) offer an alternative or adjunctive method to decrease the pain experience and enhance function and quality of life. Various evidence-based CAT methods have been proved to be effective in the management of both acute and chronic pain. Nurses are well placed to implement various CAT modalities.

Severe Hypertension and Bradycardia Secondary to Midodrine Overdose.

The objective of this case is to describe the pharmacokinetics and toxicity of midodrine in overdose. A 20 year old female ingested up to 350 mg midodrine while recovering in hospital from another overdose. She developed vomiting and severe hypertension (blood pressure [BP], 210/100 mmHg). Remarkable findings included a heart rate with a range of 43-60 beats/min, spontaneous respirations (20 breaths/min), and oxygen saturations of >95 % on FiO2 25 %, and a GS of 8. She was admitted to intensive care and had a normal non-contrast CT brain. She was treated with a glyceryl trinitrate patch (5 mg) and observed for 36 h with subsequent BP reduction to 124/81 mmHg and improved in conscious state. Midodrine and desglymidodrine concentrations were measured with liquid chromatography tandem mass spectrometry and were detected with 2-h post-ingestion at concentrations of 158.4 and 169.7 ng/mL, respectively. The parent drug concentrations rapidly decreased with an elimination of half-life of 1.6 h, and the metabolite initially increased and then decreased. The peak in blood pressure appeared to coincide with peak metabolite concentrations. Midodrine in overdose can potentially cause severe hypertension and reflex bradycardia but given its short half-life treatment with vasodilator agents and supportive care is sufficient.

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism.

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.

5-HT activates vagal afferent cell bodies in vivo: role of 5-HT2 and 5-HT3 receptors.

Occipital artery (OA) injections of 5-HT elicit pronounced reductions in heart rate and mean arterial blood pressure (MAP) in urethane-anesthetized rats by activation of vagal afferent cell bodies in the ipsilateral nodose ganglion. In contrast, internal carotid artery (ICA) and i.v. injections elicit similar cardiovascular responses by activation of peripheral vagal afferent terminals. The aim of this study was to examine the roles of 5-HT3 and 5-HT2 receptors in the 5-HT-induced activation of vagal afferent cell bodies and peripheral afferent terminals in urethane-anesthetized rats. OA, ICA and i.v. injections of 5-HT elicited dose-dependent reductions in heart rate and MAP that were virtually abolished after i.v. administration of the 5-HT3 receptor antagonists, MDL 7222 or ICS 205-930. The responses elicited by the OA injections of 5-HT were markedly diminished after i.v. injection of the 5-HT2 receptor antagonists, xylamidine or ketanserin, whereas the responses elicited by i.v. or ICA injections of 5-HT were not affected. The present findings suggest that (1) 5-HT3 and 5-HT2 receptor antagonists gain ready access to nodose ganglion cells upon i.v. administration, and (2) functional 5-HT3 and 5-HT2 receptors exist on the cell bodies of vagal afferent neurons mediating the cardiovascular responses elicited by OA injections of 5-HT. These findings also support a wealth of evidence that 5-HT3 receptors exist on the peripheral terminals of vagal afferents, and although they do not discount the possibility that 5-HT2 receptors exist on peripheral vagal afferent terminals, it appears that activation of these receptors does not have pronounced effects on 5-HT3 receptor activity on terminals that mediate the hemodynamic responses to 5-HT.

Occipital artery injections of 5-HT may directly activate the cell bodies of vagal and glossopharyngeal afferent cell bodies in the rat.

The primary objective of this study was to determine whether circulating factors gain direct access to and affect the activity of vagal afferent cell bodies in the nodose ganglia and glossopharyngeal afferents cell bodies in the petrosal ganglia, of the rat. We found that the occipital and internal carotid arteries provided the sole blood supply to the nodose ganglia, and that i.v. injections of the tracer, Basic Blue 9, elicited strong cytoplasmic staining in vagal and glossopharyngeal afferent cell bodies that was prevented by prior ligation of the occipital but not the internal carotid arteries. We also found that occipital artery injections of 5-HT elicited pronounced dose-dependent reductions in heart rate and diastolic arterial blood pressure that were (1) virtually abolished after application of the local anesthetic, procaine, to the ipsilateral nodose and petrosal ganglia, (2) markedly attenuated after transection of the ipsilateral vagus between the nodose ganglion and brain and virtually abolished after subsequent transection of the ipsilateral glossopharyngeal nerve between the petrosal ganglion and the brain, (3) augmented after ipsilateral transection of the aortic depressor and carotid sinus nerves, and (4) augmented after transection of all ipsilateral glossopharyngeal and vagal afferent nerves except for vagal cardiopulmonary afferents. These findings suggest that blood-borne 5-HT in the occipital artery gains direct access to and activates the cell bodies of vagal cardiopulmonary afferents of the rat and glossopharyngeal afferents of undetermined modalities.

Utilization of fresh human tympanic membranes for structural analysis and cytokeratin immunocytochemistry implementing resin techniques.

CONCLUSIONS: The results of this study have demonstrated for the first time that tympanic membrane (TM) structure is preserved following removal of fresh, normal tissue from patients undergoing surgery. Greater clarity has been demonstrated using resin sections than in previous studies on paraffin sections. Of particular note, cytokeratin (CK) immunocytochemistry was successfully performed on resin sections, which has not been previously reported. This may have potential applications for future work involving tissues that express CKs. OBJECTIVES: To analyse the structure of normal, fresh human TM specimens after surgical removal and to evaluate their CK immunocytochemistry using resin techniques, neither of which have been demonstrated previously. MATERIAL AND METHODS: Seven TM specimens were removed during surgery and then preserved in a modified Karnovsky's fixative. Semi-thin and thin sections were examined by means of light and electron microscopy, respectively. For comparison purposes, paraffin block-embedded specimens were also sectioned. CK immunocytochemistry was performed on semi-thin sections using standard immunoperoxidase techniques, with expression being demonstrated using light microscopy. RESULTS: The three-layer architecture of the TM was preserved. The morphology of the TM was vastly superior in the semi-thin resin sections than in the thicker paraffin sections. The outer, middle and inner layers were clearly demonstrated. The integrity of the outer epithelial layer was maintained, with an outer keratinizing stratum corneum and underlying stratum granulosum, stratum spinosum and stratum basale layers resting on the basal lamina. The thin inner mucosal layer was also viable, consisting of simple squamous or cuboidal cells. Preservation of the middle lamina propria was achieved, with demonstration of the outer radial and inner circular fibres. CK immunocytochemistry utilizing resin techniques provided excellent staining of CK 7 and 8 in the inner layer, with positive staining of CK 5 and 10 in the outer layer.