Use of isoelectric focusing to define major histocompatibility complex class I polymorphism in goats

We have used biochemical methods to extend and improve serological class I typing using a panel of 77 Swiss goats of the Saanen breed, comprising dam-offspring combinations from six half-sib sire families and several unrelated animals. Of these animals class I molecules were precipitated from cell lysates with the mAb B1.1G6 and HC10. Immunoprecipitates were analysed by SDS-PAGE and 1D-IEF. There was a good agreement between class I serological types and IEF banding patterns. We have identified three new class I specificities and subdivided the Bel 7 specificity. IEF has enabled us to make planned immunizations to produce antisera to the new specificities. New evidence for the expression of a second class I locus product in the Be7 haplotype has been found.     

Adult spinal opioid receptor μ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long‐term changes on nociceptive processing in later life. Here, we use an established, well‐documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long‐term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ‐opioid‐receptor‐1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind‐paw from postnatal day 0–7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind‐paw incision as a model for post‐operative pain, and mechanical sensitivity was tested at multiple time‐points. Before, and 1 or 5 days post‐incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi‐quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1‐immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417–426, 2018     

Contribution of cervical cytology in the diagnostic work‐up of patients with endometrial cancer

Introduction

Abnormal cervical cytology in patients with endometrial cancer (EC) has been associated with poor outcome. The aim of this study was to evaluate whether cervical cytology could contribute to an improved preoperative identification of high‐grade EC (serous, clear cell, carcinosarcoma, high‐grade endometrioid EC) in final histology.

Methods

A retrospective cohort study was performed in five hospitals in the Netherlands. A total of 554 patients with EC that underwent primary surgical treatment between 2002 and 2010 were included. Primary outcome was defined as the contribution of abnormal cervical cytology in the preoperative identification of high‐grade EC. As secondary outcome, recurrence‐free survival (RFS) and disease‐specific survival were determined based on preoperative cervical cytology, and compared to the currently established risk factors: myometrial invasion, high‐grade and lymph vascular space invasion.

Results

Abnormal cervical cytology was present in 45.1%. For patients with preoperative inconclusive and high‐grade histology, the presence of abnormal cervical cytology contributed to an improved identification of high‐grade EC in final histology (odds ratio [OR] 6.40 [95% confidence interval {CI}: 1.92‐21.26]; OR 2.86 [95% CI: 1.14‐7.14]), respectively. Patients with abnormal cervical cytology had a significant worse 5‐year median RFS. Abnormal cervical cytology was independently related to RFS (hazard ratio 1.67 [95% CI: 1.04‐2.68]) and disease‐specific survival (hazard ratio 3.15 [95% CI: 1.74‐5.71]).

Conclusions

Abnormal cytology contributes to the preoperative identification of patients with high‐grade EC, and is associated with compromised outcome. Future studies are warranted to determine whether cervical cytology could be incorporated into preoperative prediction models for lymph node metastasis.     

Prevention of Histamine Formation in Cheese by Bacteriocin-Producing Lactic Acid Bacteria

The susceptibility of 13 amine-forming lactobacilli to several bacteriocins was investigated by an agar diffusion assay. All strains were susceptible to nisin and to five bacteriocins of enterococcal origin. Pediocin PA-1, bavaricin A, lactococcin A, and a bacteriocin from Enterococcus faecalis 1061 did not show inhibitory activity. Two bacteriocin-producing enterococci and a nisin-producing Lactococcus lactis strain were employed as starters in separate cheese-making experiments. Outgrowth of histamine producer Lactobacillus buchneri St2A, which was added to the milk at levels of up to 190 CFU/ml, was almost completely inhibited. No histamine formation was detected in the cheeses made with bacteriocin-producing starters. In the control cheese without bacteriocin, St2A reached levels of 1.1 x 10(sup8) CFU/g, and 200 mg of histamine per kg was found after 4 months of ripening. To our knowledge, this is the first report of bacteriocin-mediated inhibition of histamine formation in foods.     

Greenhouse Evaluation of Dose– and Time–Mortality Relationships of Two Nucleopolyhedroviruses for the Control of Beet Armyworm, Spodoptera exigua, on Chrysanthemum

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV) against synchronized populations of S. exigua larvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exigua target stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exigua were controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigua larvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigua larvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.     

Calibration of EMG to force for knee muscles is applicable with submaximal voluntary contractions.

PURPOSE: In this study, the influence of using submaximal isokinetic contractions about the knee compared to maximal voluntary contractions as input to obtain the calibration of an EMG-force model for knee muscles is investigated. METHODS: Isokinetic knee flexion and extension contractions were performed by healthy subjects at five different velocities and at three contraction levels (100%, 75% and 50% of MVC). Joint angle, angular velocity, joint moment and surface EMG of five knee muscles were recorded. Individual calibration values were calculated according to [C.A.M. Doorenbosch, J. Harlaar, A clinically applicable EMG-force model to quantify active stabilization of the knee after a lesion of the anterior cruciate ligament, Clinical Biomechanics 18 (2003) 142-149] for each contraction level. RESULTS: First, the output of the model, calibrated with the 100% MVC was compared to the actually exerted net knee moment at the dynamometer. Normalized root mean square errors were calculated [A.L. Hof, C.A.N. Pronk, J.A. van Best, Comparison between EMG to force processing and kinetic analysis for the calf muscle moment in walking and stepping, Journal of Biomechanics 20 (1987) 167-187] to compare the estimated moments with the actually exerted moments. Mean RMSD errors ranged from 0.06 to 0.21 for extension and from 0.12 to 0.29 for flexion at the 100% trials. Subsequently, the calibration results of the 50% and 75% MVC calibration procedures were used. A standard signal, representing a random EMG level was used as input in the EMG force model, to compare the three models. Paired samples t-tests between the 100% MVC and the 75% MVC and 50% MVC, respectively, showed no significant differences (p>0.05). CONCLUSION: The application of submaximal contractions of larger than 50% MVC is suitable to calibrate a simple EMG to force model for knee extension and flexion. This means that in clinical practice, the EMG to force model can be applied by patients who cannot exert maximal force.     

Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase

Monopolar spindle 1 (Mps1, also known as TTK) is a protein kinase crucial for ensuring that cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules. Incomplete chromosomal attachment leads to abnormal chromosome counts in the daughter cells (aneuploidy), a condition common in many solid cancers. Therefore Mps1 is an established target in cancer therapy. Mps1 kinase inhibitors include reversine (2‐(4‐morpholinoanilino)‐6‐cyclohexylaminopurine), a promiscuous compound first recognized as an inhibitor of the Aurora B mitotic kinase. Here, we present the 3.0‐Å resolution crystal structure of the Mps1 kinase domain bound to reversine. Structural comparison of reversine bound to Mps1 and Aurora B, indicates a similar binding pose for the purine moiety of reversine making three conserved hydrogen bonds to the protein main chain, explaining the observed promiscuity of this inhibitor. The cyclohexyl and morpholinoaniline moieties of reversine however, have more extensive contacts with the protein in Mps1 than in Aurora B. This is reflected both in structure‐based docking energy calculations, and in new experimental data we present here, that both confirm that the affinity of reversine towards Mps1 is about two orders of magnitude higher than towards Aurora B. Thus, our data provides detailed structural understanding of the existing literature that argues reversine inhibits Mps1 more efficiently than Aurora B based on biochemical and in‐cell assays. Proteins 2016; 84:1761–1766. © 2016 Wiley Periodicals, Inc.     

The Toll-Like Receptor 4 (TLR4) Variant rs2149356 and Risk of Gout in European and Polynesian Sample Sets

Deposition of crystallized monosodium urate (MSU) in joints as a result of hyperuricemia is a central risk factor for gout. However other factors must exist that control the progression from hyperuricaemia to gout. A previous genetic association study has implicated the toll-like receptor 4 (TLR4) which activates the NLRP3 inflammasome via the nuclear factor-κB signaling pathway upon stimulation by MSU crystals. The T-allele of single nucleotide polymorphism rs2149356 in TLR4 is a risk factor associated with gout in a Chinese study. Our aim was to replicate this observation in participants of European and New Zealand Polynesian (Māori and Pacific) ancestry. A total of 2250 clinically-ascertained prevalent gout cases and 13925 controls were used. Non-clinically-ascertained incident gout cases and controls from the Health Professional Follow-up (HPFS) and Nurses Health Studies (NHS) were also used. Genotypes were derived from genome-wide genotype data or directly obtained using Taqman. Logistic regression analysis was done including age, sex, diuretic exposure and ancestry as covariates as appropriate. The T-allele increased the risk of gout in the clinically-ascertained European samples (OR = 1.12, P = 0.012) and decreased the risk of gout in Polynesians (OR = 0.80, P = 0.011). There was no evidence for association in the HPFS or NHS sample sets. In conclusion TLR4 SNP rs2143956 associates with gout risk in prevalent clinically-ascertained gout in Europeans, in a direction consistent with previously published results in Han Chinese. However, with an opposite direction of association in Polynesians and no evidence for association in a non-clinically-ascertained incident gout cohort this variant should be analysed in other international gout genetic data sets to determine if there is genuine evidence for association.