The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.     

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.

     

Casein kinase 1 is required for efficient removal of Rec8 during meiosis I

Segregation of chromosomes during meiosis depends on separase cleavage of Rec8, the meiosis-specific alpha-kleisin subunit of cohesin. We mapped Rec8 phosphorylation sites by mass spectrometry and show that, in fission yeast, Rec8 phosphorylation is required for proper chromosome disjunction during meiosis. We further show that the fission yeast casein kinase 1 (CK1) delta/epsilon isoforms Hhp1 and Hhp2 are required for full levels of Rec8 phosphorylation and for efficient removal of Rec8 at the onset of anaphase I. Our data are consistent with the model that Hhp1/Hhp2-dependent phosphorylation of Rec8 is required for separase-mediated cleavage of Rec8 during meiosis I.Key words: meiosis, chromosome segregation, cohesin, casein kinase, fission yeast     

Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone

Ubiquitin-dependent protein degradation usually involves escort factors that target ubiquitylated substrates to the proteasome. A central element in a major escort pathway is Cdc48, a chaperone-like AAA ATPase that collects ubiquitylated substrates via alternative substrate-recruiting cofactors. Cdc48 also associates with Ufd2, an E4 multiubiquitylation enzyme that adds further ubiquitin moieties to preformed ubiquitin conjugates to promote degradation. Here, we show that E4 can be counteracted in vivo by two distinct mechanisms. First, Ufd3, a WD40 repeat protein, directly competes with Ufd2, because both factors utilize the same docking site on Cdc48. Second, Cdc48 also binds Otu1, a deubiquitylation enzyme, which disassembles multiubiquitin chains. Notably, Cdc48 can bind Otu1 and Ufd3 simultaneously, making a cooperation of both inhibitory mechanisms possible. We propose that the balance between the distinct substrate-processing cofactors may determine whether a substrate is multiubiquitylated and routed to the proteasome for degradation or deubiquitylated and/or released for other purposes.     

A knockout screen for protein kinases required for the proper meiotic segregation of chromosomes in the fission yeast Schizosaccharomyces pombe

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation after just single round of DNA replication. To identify novel proteins required for the proper segregation of chromosomes during meiosis, we analyzed the consequences of deleting Schizosaccharomyces pombe genes predicted to encode protein kinases that are not essential for cell viability. We show that Mph1, a member of the Mps1 family of spindle assembly checkpoint kinases, is required to prevent meiosis I homolog non-disjunction. We also provide evidence for a novel function of Spo4, the fission yeast ortholog of Dbf4-dependent Cdc7 kinase, in regulating the length of anaphase II spindles. In the absence of Spo4, abnormally elongated anaphase II spindles frequently overlap and thus destroy the linear order of nuclei in the ascus. Our observation that the spo4Δ mutant phenotype can be partially suppressed by inhibiting Cdc2-as suggests that dysregulation of the activity of this cyclin-dependent kinase may cause abnormal elongation of anaphase II spindles in spo4Δ mutant cells.     

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.     

Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1

The regulated degeneration of axons or dendrites (pruning) and neuronal apoptosis are widely used during development to determine the specificity of neuronal connections. Pruning and apoptosis often share similar mechanisms; for example, developmental dendrite pruning of Drosophila class IV dendritic arborization (da) neurons is induced by local caspase activation triggered by ubiquitin-mediated degradation of the caspase inhibitor DIAP1. Here, we examined the function of Valosin-containing protein (VCP), a ubiquitin-selective AAA chaperone involved in endoplasmic reticulum-associated degradation, autophagy and neurodegenerative disease, in Drosophila da neurons. Strong VCP inhibition is cell lethal, but milder inhibition interferes with dendrite pruning and developmental apoptosis. These defects are associated with impaired caspase activation and high DIAP1 levels. In cultured cells, VCP binds to DIAP1 in a ubiquitin- and BIR domain-dependent manner and facilitates its degradation. Our results establish a new link between ubiquitin, dendrite pruning and the apoptosis machinery.     

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.