Effect of aerobic exercise training and cognitive behavioural therapy on reduction of chronic fatigue in patients with facioscapulohumeral dystrophy: protocol of the FACTS-2-FSHD trial

Background

In facioscapulohumeral dystrophy (FSHD) muscle function is impaired and declines over time. Currently there is no effective treatment available to slow down this decline. We have previously reported that loss of muscle strength contributes to chronic fatigue through a decreased level of physical activity, while fatigue and physical inactivity both determine loss of societal participation. To decrease chronic fatigue, two distinctly different therapeutic approaches can be proposed: aerobic exercise training (AET) to improve physical capacity and cognitive behavioural therapy (CBT) to stimulate an active life-style yet avoiding excessive physical strain. The primary aim of the FACTS-2-FSHD (acronym for Fitness And Cognitive behavioural TherapieS/for Fatigue and ACTivitieS in FSHD) trial is to study the effect of AET and CBT on the reduction of chronic fatigue as assessed with the Checklist Individual Strength subscale fatigue (CIS-fatigue) in patients with FSHD. Additionally, possible working mechanisms and the effects on various secondary outcome measures at all levels of the International Classification of Functioning, Disability and Health (ICF) are evaluated.

Methods/Design

A multi-centre, assessor-blinded, randomized controlled trial is conducted. A sample of 75 FSHD patients with severe chronic fatigue (CIS-fatigue ≥ 35) will be recruited and randomized to one of three groups: (1) AET + usual care, (2) CBT + usual care or (3) usual care alone, which consists of no therapy at all or occasional (conventional) physical therapy. After an intervention period of 16 weeks and a follow-up of 3 months, the third (control) group will as yet be randomized to either AET or CBT (approximately 7 months after inclusion). Outcomes will be assessed at baseline, immediately post intervention and at 3 and 6 months follow up.

Discussion

The FACTS-2-FSHD study is the first theory-based randomized clinical trial which evaluates the effect and the maintenance of effects of AET and CBT on the reduction of chronic fatigue in patients with FSHD. The interventions are based on a theoretical model of chronic fatigue in patients with FSHD. The study will provide a unique set of data with which the relationships between outcome measures at all levels of the ICF could be assessed.

Trial registration

Dutch Trial Register, NTR1447.

     

Proteomic and Genetic Analyses Demonstrate that Plasmodium berghei Blood Stages Export a Large and Diverse Repertoire of Proteins

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.Malaria parasites invade and develop inside red blood cells, and extensive remodeling of the host cell is required in order for the parasite to take up nutrients and grow (1). In addition, infected red blood cells (irbcs)¹ of several Plasmodium species adhere to endothelium lining blood capillaries, and this is achieved through modification of the irbc, specifically, alteration of the irbc membrane (2, 3). This active remodeling of the erythrocyte requires the export of parasite proteins into the host cell cytoplasm and their incorporation in the irbc membrane of the host cell (1, 2). Schizont-infected red blood cells of the rodent parasite P. berghei ANKA adhere to endothelial cells of the microvasculature, leading to the sequestration of irbcs in organs such as the lungs and adipose tissue (4–6). P. berghei irbcs adhere to the class II scavenger receptor CD36 (7), which is highly conserved in mammals and is the receptor most commonly used by irbcs infected with the human parasite P. falciparum (8). These observations suggest that P. berghei may export proteins onto the surface of irbcs in a fashion analogous to the processes employed by P. falciparum that expresses PfEMP1, the protein known to be responsible for P. falciparum irbc sequestration. However, P. berghei does not contain Pfemp1 orthologs or proteins with domains with clear homology to the domains of PfEMP1 (9), and the P. berghei proteins responsible for irbc cytoadherence and proteins involved in the transport of these proteins to the irbc membrane remain largely unknown. Recently we used a proteomic analysis of P. berghei ANKA irbc membranes to identify parasite proteins associated with the erythrocyte membrane, and we have demonstrated that the deletion of a single-copy gene of P. berghei that encodes a small exported protein known as SMAC results in strongly reduced irbc sequestration (6). No evidence was found for the presence of SMAC on the irbc surface, and therefore this protein is most likely involved in the transport or anchoring of other P. berghei proteins that directly interact with host receptors on endothelial cells.For P. falciparum, a large number of exported proteins have been predicted based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) motif (10, 11). Many of these PEXEL-positive proteins belong to species-specific gene families. Comparison of PEXEL-positive proteins in different Plasmodium species suggested that P. falciparum expresses a significantly higher number of exported proteins than other Plasmodium species, which in part can be attributed to the expansion of P. falciparum–specific protein families, including those containing DnaJ or PHIST domains (12–17). One explanation for the elevated number of exported proteins in P. falciparum is that they are necessary for export of the P. falciparum–specific protein PfEMP1 to the irbc surface (10). Comparisons of different Plasmodium exportomes have mainly focused on identifying orthologs of the PEXEL-positive proteins of P. falciparum in the other species (14, 15, 18). For example, of the >500 PEXEL-positive P. falciparum proteins, only between 11 and 33 had orthologs in P. berghei (14, 15, 19). However, such an approach might underestimate the total number of exported proteins. A recent hidden Markov model (HMM) analysis of the PEXEL motif for P. berghei proteins identified at least 75 PEXEL-positive P. berghei proteins (6). Moreover, in different Plasmodium species, a number of exported proteins have been described that are PEXEL-negative, indicating that alternative export pathways might exist that are independent of the presence of a PEXEL motif (20, 21). It has been suggested that in species with a small number of PEXEL-positive proteins, PEXEL-negative exported proteins play a more prominent role in host cell remodeling (21). An example of a PEXEL-negative exported protein family is the large PIR family of proteins, which are expressed by rodent Plasmodium species (9, 22), the monkey parasite P. knowlesi (23), and the human parasite P. vivax (24, 25).To date, export to the irbc cytosol has been shown for only a few P. berghei proteins (i.e. several members of the BIR family; TIGR01590) (6), two members of the ETRAMP family (26), and two proteins encoded by a single copy gene, SMAC and IBIS1 (6, 27). In this study, comparative proteomic, genomic, and reverse-genetic approaches have been used to identify novel exported proteins of P. berghei. We report proteome analyses of samples enriched for proteins associated with membranes of irbcs from both sequestering P. berghei ANKA and non-sequestering P. berghei K173 parasites, and we also present analyses of the full genome sequence of a non-sequestering P. berghei K173 line. Fluorescent tagging of parasite proteins selected from the proteome and genome analyses identified a number of novel P. berghei ANKA proteins that are exported into the irbc cytoplasm. We report for the first time the export of members of the PEXEL-negative Pb-fam-1 gene family (pyst-a; TIGR01599) and show that two proteins are transported to the P. berghei ANKA irbc membrane. This is the first comprehensive study of exported proteins of P. berghei that has been validated via the generation of a large number of transgenic P. berghei ANKA parasites expressing tagged proteins and has shown the export of both PEXEL-positive and PEXEL-negative proteins to the irbc cytoplasm. The identification of P. berghei ANKA proteins exported to the irbc membrane and proteins involved in sequestration suggests the possibility of developing “humanized” small animal models for the in vivo analysis of the sequestration properties of P. falciparum proteins that would express (domains of) P. falciparum proteins on the surface of rodent irbcs (4, 6).     

Reproductive patterns of the Pacific oyster Crassostrea gigas in France

Summary

In France, national management programs focus research on understanding reproductive factors in Crassostrea gigas to confront problems of the oyster industry. However, little information has been documented in which reproductive patterns include sexual changes. The reproductive cycle of oysters at three sites of the Atlantic coast of France was examined from 1996 to 1998, and the seasonal variations in oocyte size-frequencies, and sex ratio were described. The results showed a synchronism within the population concerning reproductive behavior. Young oocytes are generated after spawning and show no apparent changes during winter. Growth of oocytes begins in spring and cells reach maturity in April-May and are ready for a single spawning season in June-July. Oocytes that were not released during spawning are reabsorbed within the gonad. The significant difference between sites is that spawning occurred 1 month later in the southern area. A modal analysis showed that oocyte populations in the sample individuals are primordially bimodal, but with polymodal occurrences in June-July, in some cases. Irregular alternative sexuality was detected at all sites, and hermaphrodites appear to be a transition phase that allows changes from male to female during early spring. Previous observations, together with the study of the development of oocyte cohorts over time, permit a hypothetical model concerning the kinetics of gametogenesis in C. gigas. The model suggests that primary oocytes are generated from energy supplied from degenerating, as well as young oocytes that do not reach the mature stage within the gonad during autumn-winter. It seems that, during vitellogenesis, there is disintegration of smaller cells coupled with transfer of energy to the larger oocytes, which continue to grow and mature.     

曲酸生产菌的复合诱变选育*

以黄曲霉(Aspergillus flavus.)为出发菌株,经3次紫外线、1次^60Co、3次亚硝基胍多重复合诱变处理,选育获得曲酸生产菌UCN7—17,配以最佳培养条件,发酵7d,曲酸产量由原来的0．926％,提高到6．3％。实验证明采用多因子复合诱变,能有效改变菌株对诱变因素敏感性,提高变异率,逐步提高突变株的产酸水平。     

Auxin perception and polar auxin transport are not always a prerequisite for differential growth

Using time-lapse photography, we studied the response kinetics of low light intensity-induced upward leaf-movement, called hyponastic growth, in Arabidopsis thaliana. This response is one of the traits of shade avoidance and directs plant organs to more favorable light conditions. Based on mutant- and pharmacological data we demonstrated that among other factors, functional auxin perception and polar auxin transport (PAT) are required for the amplitude of hyponastic growth and for maintenance of the high leaf angle, upon low light treatment. Here, we present additional data suggesting that auxin and PAT antagonize the hyponastic growth response induced by ethylene treatment. We conclude that ethylene- and low light-induced hyponastic growth occurs at least partly via separate signaling routes, despite their strong similarities in response kinetics.Key words: hyponastic growth, petiole, Arabidopsis, ethylene, low light, auxin, polar auxin transport, differential growthUpward leaf movement (hyponastic growth) is a trait of several plant species to escape from growth-limiting conditions.^1,2 Interestingly, Arabidopsis thaliana induces a marked hyponastic growth response triggered by various environmental stimuli, including complete submergence, high temperature, canopy shade and spectral neutral low light intensities (Fig. 1).^3–6 The paper of Millenaar et al. in the New Phytologist 2009,⁷ provides a detailed analysis of low light intensity-induced hyponastic growth and components of the signal transduction are characterized using time-lapse photography. Low light intensity-induced hyponastic growth is a component of the so-called shade avoidance syndrome. Light-spectrum manipulations and mutant analyses indicated that predominantly the blue light wavelength region affects petiole movement and fast induction of hyponastic growth to low light conditions involves the photoreceptor proteins Cryptochrome 1 (Cry1), Cry2, Phytochrome-A (PhyA) and PhyB. Moreover, we show that also photosynthesis-derived signals can induce differential growth.Open in a separate windowFigure 1Typical hyponastic growth phenotype of Arabidopsis thaliana. Side view of Columbia-0 plants treated 10 h with ethylene (5 µl l⁻¹) or low light (20 µmol m⁻² s⁻¹). Plants in control light conditions were in 200 µmol m⁻² s⁻¹. Both stimuli induce a clear leaf inclination (hyponasty) relative to the horizontal by differential growth of the petioles. Plants kept in control conditions only show modest diurnal leaf movement and leaf angles gradually decline over time due to maturation of the leaves. Note that the paint droplets were applied to facilitate quantitative measurement of leaf angle kinetics in a time-lapse camera setup.⁷The hyponastic growth response to low light intensity was not affected in several ethylene-insensitive mutant lines. Moreover, low light did not affect expression of ethylene inducible marker genes nor differences in ethylene release were noted. Therefore, we concluded that low light-induced hyponastic growth is independent of ethylene signaling. This is perhaps surprising, because ethylene is the main trigger of hyponastic growth induced by complete submergence in several species. Interestingly, both ethylene and low light can induce hyponastic growth in Arabidopsis with similar kinetics.³We showed that plants mutant in auxin perception components (transport inhibitor response1 (tir1) and tir1 afb1 afb2 afb3 quadruple, containing additional mutant alleles of TIR1 homologous F-box proteins) and plants mutant in (polar) auxin transport (tir3-1, pin-formed3 (pin3) and pin7) components had a lower hyponastic growth amplitude in low light conditions.⁷ Moreover, these mutants were less able to maintain the high leaf angles after the response maximum. Both characteristics were also noted in plants pre-treated with the polar auxin transport (PAT) inhibitor 2,3,5-triiodobenzoic acid (TIBA). We therefore concluded that auxin perception and PAT are involved in the regulation of low light-induced hyponastic growth.⁷ Interestingly, we observed that TIBA pretreatment did not inhibit ethylene-induced hyponastic growth. In fact, the response upon ethylene treatment was even modestly enhanced. In agreement with this observation, we show here that the above mentioned auxin perception and PAT mutants also showed a slightly enhanced hyponastic growth response upon ethylene treatment (Fig. 2).Open in a separate windowFigure 2Auxin involvement in ethylene induced hyponasty. Effect of exposure to ethylene (5 µl l⁻¹) on the kinetics of hyponastic petiole growth (A) in Arabidopsis thaliana Columbia-0 plants treated with 50 µm TIBa (open circles) or a mock treatment (line) adapted from Supporting Information Figure S3 of Millenaar et al. (2009)⁷ and (B–F) in Arabidopsis auxin signaling and polar auxin transport mutants (closed circles), compared to the wild type response to low light (lines). Petiole angles are pair wise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions, treatments, data acquirement and analysis see.^7,13 Error bars represent standard errors; n ≥ 12. mutants were obtained from the Nottingham Arabidopsis Stock Center (accession numbers are shown between brackets) or from the authors describing the lines. tir1-1 (n3798,¹⁴), tir1-1 afb1-1 afb2-1 (in a mixed Columbia/Wassilewskija background),¹⁵ tir3-1,¹⁴ pin3-4 (n9363,¹⁶) and pin7-1 (n9365,¹⁰).Despite that auxin and PAT are required for many differential growth responses such as phototropism and gravitropism,^8,11 these data indicate that auxin perception and PAT are not obligatory for ethylene-induced hyponasty in Arabidopsis per se. In fact, one might even conclude that auxin and PAT antagonizes ethylene-induced hyponasty. These results are partly in agreement with observations on the wetland species Rumex palustris, were pretreatment with the auxin-efflux carrier 1-naphthylphthalamic acid (NPA) resulted in doubling of the lag-phase for hyponastic growth under water, but hardly affected the amplitude of the response.¹²Together, this indicates that auxin is not always a prerequisite for differential growth responses. Based on the apparent contrasting effects of auxin perception and PAT in low light- and ethylene-induced hyponastic growth, we conclude that ethylene and low light induce hyponastic growth, at least partly, via separate signaling routes.     

The innate immune receptor MDA5 limits rotavirus infection but promotes cell death and pancreatic inflammation

Melanoma differentiation‐associated protein 5 (MDA5) mediates the innate immune response to viral infection. Polymorphisms in IFIH1, the gene coding for MDA5, correlate with the risk of developing type 1 diabetes (T1D). Here, we demonstrate that MDA5 is crucial for the immune response to enteric rotavirus infection, a proposed etiological agent for T1D. MDA5 variants encoded by minor IFIH1 alleles associated with lower T1D risk exhibit reduced activity against rotavirus infection. We find that MDA5 activity limits rotavirus infection not only through the induction of antiviral interferons and pro‐inflammatory cytokines, but also by promoting cell death. Importantly, this MDA5‐dependent antiviral response is specific to the pancreas of rotavirus‐infected mice, similar to the autoimmunity associated with T1D. These findings imply that MDA5‐induced cell death and inflammation in the pancreas facilitate progression to autoimmune destruction of pancreatic β‐cells.     

Taphonomy and compositional fidelity of Quaternary fossil assemblages of terrestrial gastropods from carbonate-rich environments of the Canary Islands

Quaternary aeolian deposits of the Canary Islands contain well‐preserved terrestrial gastropods, providing a suitable setting for assessing the taphonomy and compositional fidelity of their fossil record over ~13 kyr. Nine beds (12, 513 shells) have been analysed in terms of multivariate taphonomic and palaeoecological variables, taxonomic composition, and the stratigraphic and palaeontological context. Shells are affected by carbonate coatings, colour loss and fragmentation. Shell preservation is size‐specific: juveniles are less fragmented and show colour preservation more commonly than adults. In palaeosols, the adult shell density correlates negatively with the proportion of fragmented adults, negatively with the proportion of juveniles, and positively with the proportion of adults with coatings. High bioturbation intensity in palaeosols is associated with low shell fragmentation and high proportion of shells with coatings. These relationships imply that high adult density in palaeosols was driven by an increase in shell production rate (related to a decrease in predation rates on adults and a decrease in juvenile mortality) and a decrease in shell destruction rate (related to an increase in durability enhanced by carbonate precipitation). In dunes, the relationships between taphonomic alteration, shell density and bioturbation are insignificant. However, dune assemblages are characterized by a lower frequency of shells with coatings and higher rates of colour loss, indicating lower shell durability in dunes than in palaeosols. Additionally, non‐random differences in the coating proportion among palaeosols imply substantial temporal variation in the rate of carbonate crust formation, reflecting long‐term changes in bioturbation intensity that covaries positively with shell preservation. Dunes and palaeosols do not differ in species abundances despite differences in the degree of shell alteration, suggesting that both weakly and strongly altered assemblages offer data with a high compositional fidelity. Carbonate‐rich terrestrial deposits originating in arid conditions can enhance the preservation of gastropods and result in fossil assemblages that are suitable for palaeoecological and palaeoenvironmental studies of terrestrial ecosystems.     

Efficacy of neem and diatomaceous earth against cowpea aphids and their deleterious effect on predating Coccinelidae

Philippine vegetable farmers commonly use synthetic insecticides to control insect pests on yardlong beans ( Vigna unguiculata ssp. sesquipedalis ). An important pest on yardlong beans is the cowpea aphid, Aphis craccivora Koch. Overuse of chemical insecticides and the adverse consequences for farmer health and for the environment have been reported. The natural enemies of A. craccivora , such as the coccinelidae beetle, Menochilus sexmaculatus (Fabr.), do not provide economic control on their own. In the present study the efficacy of the biological insecticide neem (both commercial and homemade) alone, and in combination with diatomaceous earth against A. craccivora was evaluated. The same insecticides were also examined to investigate their deleterious effect on M. sexmaculatus . The efficacies of different treatments with biological insecticides were compared with the use of the synthetic insecticide Hostathion (triazophos). Experiments were conducted under Philippine lowland conditions during the dry season when the occurrence of pest problems in yardlong beans is very great. Commercial neem, NeemAzal-T/S (Trifolio-M GmbH, Lahnau, Germany), significantly reduced the number of A. craccivora . NeemAzal-T/S and diatomaceous earth in combination produced the best control of A. craccivora and were less toxic to M. sexmaculatus than treatment with Hostathion (triazophos). Aqueous homemade neem solution did not control the A. craccivora populations.