The D4Z4 Macrosatellite Repeat Acts as a CTCF and A-Type Lamins-Dependent Insulator in Facio-Scapulo-Humeral Dystrophy

Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD), which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s) through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer–promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.     

Adverse effects of instrumentation in incubating Adélie penguins (Pygoscelis adeliae)

The use of data-loggers has permitted to explore the biology of free-ranging animals. However, this method has also been reported to reduce reproductive success while the reasons of this deleterious effect remain poorly documented. In this study, we aimed to identify critical periods of the breeding cycle of Adélie penguins (Pygoscelis adeliae) when the reproductive success may decrease because of instrumentation. For this purpose, we monitored 40 pairs, where one parent was instrumented before egg laying and 30 pairs without devices (controls). These pairs were followed at least during the incubation period but the majority was monitored during the entire breeding season. Reproductive success was affected in pairs where males were instrumented. This was not due to extra chick mortality during chick rearing but to a significantly lower hatching success. Moreover, the use of artificial eggs recording incubation temperatures and egg rotation indicated that in instrumented incubating males, eggs spent as much time at optimal incubation temperatures as control eggs but were rotated at a higher frequency. In Adélie penguins, males initiate incubation and it has been established that the early stage of incubation is one of the most critical periods for embryonic development. The low hatching rate observed in instrumented males was associated with a higher egg rotation rate, perhaps as a stress response to the presence of the instrument. Even though the causal effects remain unclear, instrumentation severely affected hatching success. For these reasons, we recommend equipping birds after the early incubation.     

Development of New Oomycete Taxon‐specific Mitochondrial Cytochrome b Region Primers for Use in Phylogenetic and Phylogeographic Studies

Here, we describe the development of an oomycete‐specific primer pair for amplification of the cytochrome b region in plant pathogenic species that span the order Peronosporales (Phytophthora spp., downy mildews). Because of the high number of variable sites at both inter‐ and intra‐specific levels this marker provides a powerful tool for population genetics and phylogenetic studies in this taxa. We also demonstrate its potential compared with other oomycete‐specific mitochondrial markers currently available.     

Impact of multispores in vitro subcultivation of Glomus sp. MUCL 43194 (DAOM 197198) on vegetative compatibility and genetic diversity detected by AFLP

Vegetative compatibility and amplified fragment length polymorphism (AFLP) genotyping of in vitro multispores clonal lineages, issued from the same ancestor culture of the arbuscular mycorrhizal fungal strain MUCL 43194 and subcultured several generations in different locations, was assessed. Vegetative compatibility was studied by confronting the germ tubes of two spores from the same or different clonal lineages and stained with nitrotetrazolium blue–Trypan blue and diamidinophenylindole to detect hyphal fusions and nuclei, respectively. Further AFLP analysis of single spores was performed to assess the genetic profile and Dice similarity between clonal lineages. Germ tubes of spores distant by as many as 69 generations were capable of fusing. The anastomosis frequencies averaged 69% between spores from the same clonal lineage, 57% between spores from different clonal lineages, and 0% between spores belonging to different strains. The AFLP patterns showed similarities averaging 92% within clonal lineages and 86% between clonal lineages. Each spore presented unique genotype and some of them shared more markers with spores from different lineages than within the same lineage. We showed that MUCL 43194 maintained self-recognition for long periods of subcultures in vitro and that spores involved in compatibility tests had different genotypes. Our findings suggest that MUCL 43194 maintains genetic diversity by means of anastomoses.     

Molecular characterization of β1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS)

β1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme initiating glycosaminoglycan (GAG) synthesis. Based on in vitro and ex vivo kinetics studies and structure-based modelling, we molecularly characterized β4GalT7 mutants linked to the progeroid form of Ehlers-Danlos syndrome (EDS), a severe connective tissue disorder. Our results revealed that loss of activity upon L206P substitution due to altered protein folding is the primary cause for the GAG synthesis defect in patients carrying the compound A186D and L206P mutations. We showed that R270C substitution strongly reduced β4GalT7 affinity towards xyloside acceptor, thus affecting GAG chains formation. This study establishes the molecular basis for β4GalT7 defects associated with altered GAG synthesis in EDS.     

Attenuated Bordetella pertussis Protects against Highly Pathogenic Influenza A Viruses by Dampening the Cytokine Storm

The threat of a pandemic spread of highly virulent influenza A viruses currently represents a top global public health problem. Mass vaccination remains the most effective way to combat influenza virus. However, current vaccination strategies face the challenge to meet the demands in a pandemic situation. In a mouse model of severe influenza virus-induced pneumonitis, we observed that prior nasal administration of an attenuated strain of Bordetella pertussis (BPZE1) provided effective and sustained protection against lethal challenge with two different influenza A virus subtypes. In contrast to most cross-protective effects reported so far, the protective window offered upon nasal treatment with BPZE1 lasted up to at least 12 weeks, suggesting a unique mechanism(s) involved in the protection. No significant differences in viral loads were observed between BPZE1-treated and control mice, indicating that the cross-protective mechanism(s) does not directly target the viral particles and/or infected cells. This was further confirmed by the absence of cross-reactive antibodies and T cells in serum transfer and in vitro restimulation experiments, respectively. Instead, compared to infected control mice, BPZE1-treated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration, and strong suppression of the production of major proinflammatory mediators in their bronchoalveolar fluids (BALFs). Our findings thus indicate that protection against influenza virus-induced severe pneumonitis can be achieved through attenuation of exaggerated cytokine-mediated inflammation. Furthermore, nasal treatment with live attenuated B. pertussis offers a potential alternative to conventional approaches in the fight against one of the most frightening current global public health threats.Influenza virus pandemics are unpredictable but recurring events that can have severe consequences on societies worldwide. In the 20th century, three novel influenza virus strains emerged, causing the 1918, 1957, and 1968 pandemics, the most devastating being the 1918 Spanish flu that led to an estimated 50 million deaths (47). The recent spread of highly pathogenic avian influenza (HPAI) H5N1 virus across parts of Asia, Europe, and the Middle East, with an overall fatality rate of over 60% for humans, as well as the rapid pandemic spread of a novel influenza A virus of the H1N1 subtype, has caused worldwide concern about a potential remake of the 1918 disaster (8).Severe complications arising from pandemic influenza or HPAI H5N1 viruses are associated with rapid, massive inflammatory cell infiltration, resulting in acute respiratory distress, and reactive hemophagocytosis with multiple organ involvement. Both the 1918 Spanish influenza virus and HPAI H5N1 induce a cytokine storm characterized by an exaggerated production of inflammatory cytokines and chemokines in the serum and lungs caused by uncontrolled activation of the host''s innate immune system. This triggers massive pulmonary edema, primary and/or secondary pneumonia, and alveolar hemorrhage with acute bronchopneumonia (4, 12, 24, 27, 37, 40, 43, 44).The relationship between mortality, viral load, and immunopathology during influenza virus infection remains elusive and somewhat controversial. Some studies suggest that severe lung immunopathology is a direct consequence of a high viral load that the host is unable to resolve (12, 13), whereas others have reported that influenza virus-induced mortality is not a direct function of viral burden but a consequence of immune-mediated pathology (9, 11). Moreover, the picture is further complicated by the fact that different highly virulent influenza A viruses may induce distinct pathological signatures and lead to different courses of acute respiratory distress syndrome, refuting the hypothesis of a single, universal cytokine storm underlying all fatal influenza virus diseases (16).Currently, vaccination remains the cornerstone of influenza virus prevention. However, due to constant antigenic drift and shift of the two major viral surface proteins hemagglutinin (HA) and neuraminidase (NA) (7), influenza virus vaccines must be reformulated each year in order to match the circulating subtypes (41). The potential emergence of an influenza virus pandemic at any time, combined with limited vaccine supplies, has rendered global vaccination strategies difficult. Therefore, a universal influenza virus vaccine that can provide protection against different variants or strains and thus not require frequent updates is highly desirable.Here, we report that nasal administration of a recently developed live attenuated Bordetella pertussis vaccine strain, named BPZE1 (35), provides effective and sustained protection against lethal challenge with mouse-adapted H3N2 or H1N1 (A/PR/8/34) influenza A viruses. We demonstrate that the protective mechanism(s) does not target the viral particles or the infected host cells but controls the influenza virus-mediated inflammation by dampening the cytokine storm. As BPZE1 has recently entered phase I safety trials with humans (http://www.child-innovac.org), our observations support the potential application of this vaccine strain as a universal prophylactic treatment against highly pathogenic influenza A viruses.     

Development of antibodies against secoisolariciresinol--application to the immunolocalization of lignans in Linum usitatissimum seeds

Lignans are widely distributed plant metabolites associated with a large range of biological activities. In order to gain insight into their biosynthesis and their spatio-temporal accumulation an immunological probe was developed. Secondary metabolites generally have too small molecular weight to be antigenic and have to be associated with a carrier protein. Secoisolariciresinol was chosen as the hapten and was linked to bovine serum albumin via a spacer arm, the p-aminohippuric acid. The artificial antigen was injected to New Zealand rabbits. The successful production of polyclonal antibodies against secoisolariciresinol was assessed with indirect enzyme immunosorbent assay (ELISA) by comparison with pre-immune serum and by competitive assays using dilutions of secoisolariciresinol standards. The antibodies had an IC(50) value of 94 μg/ml and showed moderate cross-reactivities with structurally related compounds. They were thus used to immunolocalize lignans in flaxseed (Linum usitatissimum), one of the richest sources of lignans. The immunohistochemical labeling allowed us to localize for the first time lignans in planta. They are mainly localized in the secondary wall of the sclerite cells of the outer integument of the seed. A very light labeling is also observed in cytoplasmic inclusions of the endosperm. The results were correlated with HPLC analytical results which enabled to evaluate the relative lignan quantities: in flaxseed about 90% of the metabolites are localized in the outer integument.