A novel approach for combining the use of in vitro and in vivo data to measure and detect emerging moxidectin resistance in gastrointestinal nematodes of goats

Ivermectin and moxidectin are closely related avermectin/milbemycin anthelmintics and available data suggest that side resistance occurs with these two drugs. However, moxidectin remains effective against many species of ivermectin-resistant worms due to its higher potency. The larval development assay (LDA) is routinely used to diagnose ivermectin resistance in Haemonchus contortus but laboratory diagnosis of moxidectin resistance is hampered by the lack of any validated in vitro tests. The objective of this study was to measure the relative susceptibility/resistance of H. contortus to moxidectin on goat farms in Georgia, and to validate the DrenchRite LDA for detecting resistance to moxidectin. Fecal egg count reduction tests (FECRT) were performed at five different moxidectin dose levels and DrenchRite LDAs were performed in duplicate on nine meat goat farms in Georgia, USA. To improve our ability to make inferences on the relative levels of resistance between farms, FECRT data were first analysed using a linear mixed model, and then Tukey's sequential trend test was used to evaluate the trend in response across dose levels. LDA data were analysed using log-dose logit-response and probit models. Using these statistical results, we were able to rank the nine farms from the least to the most resistant, and to develop a set of criteria for interpreting DrenchRite LDA results so that this assay can be used to diagnose both clinically apparent moxidectin resistance, as well as sub-clinical emerging resistance. These results suggest that our novel approach for examining these types of data provides a method for obtaining an increased amount of information, thus permitting a more sensitive detection of resistance. Based on results of the LDA, moxidectin-resistant farms had resistance ratios, compared with an ivermectin-sensitive farm, ranging from 32 to 128, and had resistance ratios of 6-24 compared with an ivermectin-resistant/moxidectin naive farm. Moxidectin resistance was diagnosed both in Haemonchus and Trichostrongylus on almost half of the farms tested, despite this drug only being used on these farms for 2-3 years.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Membrane localization diversity of TPK channels and their physiological role

Potassium (K) is one of the major nutrients that is essential for plant growth and development. The majority of cellular K⁺ resides in the vacuole and tonoplast K⁺ channels of the TPK (Two Pore K) family are main players in cellular K⁺ homeostasis. All TPK channels were previously reported to be expressed in the tonoplast of the large central lytic vacuole (LV) except for one isoform in Arabidopsis that resides in the plasma membrane. However, plant cells often contain more than one type of vacuole that coexist in the same cell. We recently showed that two TPK isoforms (OsTPKa and OsTPKb) from Oryza sativa localize to different vacuoles with OsTPKa predominantly found in the LV tonoplast and OsTPKb primarily in smaller compartments that resemble small vacuoles (SVs). Our study further revealed that it is the C-terminal domain that determines differential targeting of OsTPKa and OsTPKb. Three C-terminal amino acids were particularly relevant for targeting TPKs to their respective endomembranes. In this addendum we further evaluate how the different localization of TPKa and TPKb impact on their physiological role and how TPKs provide a potential tool to study the physiology of different types of vacuole.Key words: TPK channels, small vacuoles, vacuolar targeting, potassiumThe roles of plant vacuolar K⁺ channels are diverse and include potassium homeostasis, turgor regulation and responses to abiotic stress. Vacuolar K⁺-selective channels belong to two-pore K⁺ (TPK) channel families which have been found in genomes of many plant species such as Arabidopsis, poplar, Physcomitrella, Eucalyptus, barley, potato, rice and tobacco (Fig. 1). TPKs have structural similarity to mammalian “tandem P domain” channels with a secondary structure that contains four transmembrane domains and two pore regions (Fig. 2).^1–5 TPK channels have pore regions with a GYGD signature that endows K⁺ selectivity and a variable number of Ca²⁺ binding EF domains in the C terminus.^3–8 One of the best characterized members of the TPK family is AtTPK1 from Arabidopsis thaliana. AtTPK1 activity is voltage independent but sensitive to cytosolic Ca²⁺, cytosolic pH and N-terminal phosphorylation by 14-3-3 proteins.^5,6,8,9 In Arabidopsis, AtTPK1 expresses in the large lytic vacuole (LV) and plays roles in cellular K⁺ homeostasis, K⁺-release during stomatal closure and seed germination.^4,5 Other members of the Arabidopsis TPK family (AtTPK2, AtTPK3, AtTPK5) have been shown to localize to the LV but also showed some expression in smaller, vesicle-like, compartments.⁴ However, none of these isoforms appears to form functional channels in planta although our experiments with heterologous expression of AtTPK3 and AtTPK5 in the K⁺ uptake deficient E. coli LB2003 demonstrates complementation of bacterial growth phenotype (Isayenkov S, et al. unpublished results). Equally intriguing, is the plasma membrane localization of the Arabidopsis TPK4 isoform, in spite of its sequence being very similar to that of other TPKs.¹⁰Open in a separate windowFigure 1Phylogenetic tree of plant TPKs. The three main clusters of TPKs comprise: Cluster 1 with AtTPK1-like channels; Cluster 2 with AtTPK3/TPK5-like channels; Cluster 3 with barley HvTPKb. Bootstrap analysis was performed using ‘Molecular Evolutionary Genetics Analysis, MEGA4’ software available at www.megasoftware.net/mega4/megaOpen in a separate windowFigure 2Two-pore potassium channel secondary structure. TPK channels comprise four transmembrane domains (1–4) and two pore regions (P) per subunit. Functional channels are formed from two subunits. In most TPKs, both P regions contain a K⁺ selectivity signature, GYGD. However, the tobacco NtTPKa isoform has different motifs in the second P domain. In the N terminal region, TPKs have a 14-3-3 binding domain that impact on channel activity, with the binding of 14-3-3 protein leading to channel activation. C-termini of TPKs show a varying number of putative Ca²⁺ binding “EF hands” which may vary from zero to two.     

The postnatal development of the rat kidney,with special reference to the chemodifferentiation of the proximal tubule

Summary New nephron anlages appear in the renal cortex up to the 4th postnatal day (PD). The last anlages to be formed develop into functional nephrons by PD 10, and the cortex appears mature at PD 12 after formation of the cortex corticis. The renal medulla develops by the longitudinal growth of loops of Henle and collecting ducts. The immature medulla cannot be divided into different zones and corresponds structurally to the later inner stripe of the outer zone. The inner zone is formed by PD 8, and the outer stripe of the outer zone by PD 12. The renal medulla is mature at PD 21.From the start of its development, the renal proximal tubule consists of the pars convoluta and pars recta. In both parts the formation of the brush border is accompanied by the simultaneous appearance of brush border enzymes (alkaline phosphatase, -glutamyltranspeptidase, dipeptidylamino-peptidase IV) and lysosomal enzymes (acid phosphatase, acid -galactosidase, N-acetylglucosaminidase, dipeptidylaminopeptidase II) over the full length of the proximal tubule. During the course of proximal tubule maturation, however, the lysosomal enzyme activities decline in the pars convoluta (with constant brush border enzyme activities), while the brush border enzyme activities increase in the pars recta (with constant lysosomal enzyme activities). The two parts further differ in that they exhibit different lysosomal patterns from the outset, the pars convoluta containing numerous large, highly enzyme-active lysosomes arranged in groups, and the pars recta containing only a few very small lysosomes with low enzyme activity. Thus, even in the newborn rat, the lysosomal pattern of the pars recta already corresponds to that of the mature S₃ segment. The S₁ and S₂ segments of the pars convoluta first differentiate between PD 10 and 21, as the groups of large lysosomes are progressively broken up and the extent of the lysosomal apparatus is diminished, this proceeding in a retrograde direction from the end of the immature pars convoluta.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.