Increased susceptibility to cortical spreading depression in the mouse model of familial hemiplegic migraine type 2

Familial hemiplegic migraine type 2 (FHM2) is an autosomal dominant form of migraine with aura that is caused by mutations of the α2-subunit of the Na,K-ATPase, an isoform almost exclusively expressed in astrocytes in the adult brain. We generated the first FHM2 knock-in mouse model carrying the human W887R mutation in the Atp1a2 orthologous gene. Homozygous Atp1a2(R887/R887) mutants died just after birth, while heterozygous Atp1a2(+/R887) mice showed no apparent clinical phenotype. The mutant α2 Na,K-ATPase protein was barely detectable in the brain of homozygous mutants and strongly reduced in the brain of heterozygous mutants, likely as a consequence of endoplasmic reticulum retention and subsequent proteasomal degradation, as we demonstrate in transfected cells. In vivo analysis of cortical spreading depression (CSD), the phenomenon underlying migraine aura, revealed a decreased induction threshold and an increased velocity of propagation in the heterozygous FHM2 mouse. Since several lines of evidence involve a specific role of the glial α2 Na,K pump in active reuptake of glutamate from the synaptic cleft, we hypothesize that CSD facilitation in the FHM2 mouse model is sustained by inefficient glutamate clearance by astrocytes and consequent increased cortical excitatory neurotransmission. The demonstration that FHM2 and FHM1 mutations share the ability to facilitate induction and propagation of CSD in mouse models further support the role of CSD as a key migraine trigger.     

Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.     

ATG16L1 polymorphisms are associated with NOD2-induced hyperinflammation

In recent years considerable advances in understanding the pathogenesis of Crohn disease have been achieved, with the identification of susceptibility variants of genes that are part of the autophagy machinery, i.e., ATG16L1 and IRGM. Subsequent functional studies have been conducted to unravel the underlying mechanism of this genetic association. For the ATG16L1 Thr300Ala polymorphism (c.898A > G, rs2241880), it was demonstrated that the risk variant is associated with a reduced capacity of innate immune cells to induce autophagy upon triggering with specific microbial structures such as peptidoglycans, that are specifically recognized by the intracellular pattern-recognition receptor nucleotide oligomerization domain-2 (NOD2). Due to the impaired autophagy activation, autophagosome formation and the subsequent antigen presentation through the major histocompatibility complex are diminished, leading to decreased immune activation. However, these findings arguing for defective host defense mechanisms in individuals bearing the ATG16L1 300Ala variant, and subsequent bacterial persistence in the gut mucosa, provide no conclusive explanation for the excessive inflammation observed in Crohn disease.     

Genome walking in eukaryotes

Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T-DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Foraging activity of bumblebees (Bombus terrestris L.) on Bt-expressing eggplants

A greenhouse experiment was setup to study foraging behavior of the bumblebee Bombus terrestris L. on Cry3Bb-expressing genetically modified (GM) eggplants and their near-isogenic control. Commonly, more bumblebees visited GM eggplants compared to near-isogenic control, but this difference was only marginally significant. The mean length of feeding bouts was similar on the two treatments. Neither the number of flowers produced nor their size could explain bumblebees?? tendency to prefer GM eggplants. Volatile compounds were extracted from five plants per genotype and separated using gas chromatography. Thirteen compounds were identified and five of them appeared significantly more abundant in GM eggplants. Six of the identified compounds [(+)-limonene, Z-jasmone, p-cymene, ??-pinene, methyl-salicilate, and (?)-limonene] were tested in electrophysiological bioassays with antennas detached from young bumblebees, and a response was recorded in all six cases. Experimental results indicate that pollination activity of bumblebees is compatible with this GM eggplant event as a food source and that chemical cues may have an important role in plant identification. The implications for environmental risk assessment of GM plants are discussed.     

Dengue virus activates polyreactive, natural IgG B cells after primary and secondary infection

Background

Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection.

Methodology/Principal Findings

We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4–7 days after fever onset was more than 50% even after primary infection.

Conclusions/Significance

Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and “innate specificities” seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.     

Energy restriction during childhood and early adulthood and ovarian cancer risk

Dietary energy restriction may protect against cancer. In parts of The Netherlands, mostly in larger cities, periods of chronically impaired nutrition and even severe famine (Hunger Winter 1944-1945) existed during the 1930s and World War II (1940-1945). We studied the association between energy restriction during childhood and early adulthood on the risk of ovarian cancer later in life. In 1986, the Netherlands Cohort Study was initiated. A self-administered questionnaire on dietary habits and other cancer risk factors was completed by 62,573 women aged 55-69 years at baseline. Follow-up for cancer was established by record linkage to the Netherlands Cancer Registry. After 16.3 years of follow-up, 364 invasive epithelial ovarian cancer cases and 2220 subcohort members (sampled from the total cohort directly after baseline) with complete information confounders were available for case-cohort analyses. In multivariable analysis, ovarian cancer risk was lower for participants with an unemployed father during the 1930s (Hazard Ratio (HR), 0.70; 95% Confidence Interval (CI), 0.47-1.06) compared to participants with an employed father as well as for participants living in a city during World War II (HR, 0.69; 95% CI, 0.54-0.90) compared to participants living in the country-side. Residence in a Western City during the famine (Hunger Winter) was not associated with a decreased risk. Our results show a relation between proxy variables for modest energy restriction over a longer period of time during childhood or early adulthood and a reduced ovarian cancer risk.     

Functional electrical stimulation of intrinsic laryngeal muscles under varying loads in exercising horses

Bilateral vocal fold paralysis (BVCP) is a life threatening condition and appears to be a good candidate for therapy using functional electrical stimulation (FES). Developing a working FES system has been technically difficult due to the inaccessible location and small size of the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle. A naturally-occurring disease in horses shares many functional and etiological features with BVCP. In this study, the feasibility of FES for equine vocal fold paralysis was explored by testing arytenoid abduction evoked by electrical stimulation of the PCA muscle. Rheobase and chronaxie were determined for innervated PCA muscle. We then tested the hypothesis that direct muscle stimulation can maintain airway patency during strenuous exercise in horses with induced transient conduction block of the laryngeal motor nerve. Six adult horses were instrumented with a single bipolar intra-muscular electrode in the left PCA muscle. Rheobase and chronaxie were within the normal range for innervated muscle at 0.55±0.38 v and 0.38±0.19 ms respectively. Intramuscular stimulation of the PCA muscle significantly improved arytenoid abduction at all levels of exercise intensity and there was no significant difference between the level of abduction achieved with stimulation and control values under moderate loads. The equine larynx may provide a useful model for the study of bilateral fold paralysis.