Assessing potentiation of the (α4)3(β2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity “canonical” acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity “noncanonical” ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod–Wyman–Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.     

CpG oligonucleotides can prophylactically immunize against Th2-mediated schistosome egg-induced pathology by an IL-12-independent mechanism

Using a Schistosoma mansoni egg-induced granuloma model, we examined the ability of CpG oligodeoxynucleotides (ODN) to suppress Th2-type cytokine expression and to prophylactically immunize against Th2-dependent pulmonary pathology. The mechanism was examined by studying Th2 response regulation in cytokine-deficient mice. Surprisingly, our findings revealed several functions of CpG DNA that were completely IL-12 independent. Most striking was the marked suppression in Th2 cytokine expression and granulomatous inflammation observed in egg/CpG-sensitized IL-12-deficient mice. Immune deviation was not dependent on NK or B cells. However, a role for IL-10, B7.1, and CD40 expression in Th2 response inhibition was suggested. Indeed, CpG ODN up-regulated all three elements in both wild-type and IL-12-deficient mice. The role of IL-10 was demonstrated in mice exhibiting combined deficiencies in IL-12 and IL-10. Here, a marked increase in egg-specific IL-4/IL-5-producing cells confirmed a role for both cytokines in Th2 response inhibition. Nevertheless, the frequency of Th2-producing cells was again reduced by CpG ODN. However, in marked contrast to IL-12-deficient animals, a significant increase in IFN-gamma-producing cells likely explains the reduced Th2 response in IL-10/IL-12-deficient mice. Thus, a novel IL-12-independent type 1-inducing pathway was revealed in the combined absence of IL-12 and IL-10. Together, these data demonstrate 1) that the Th1-promoting activity of CpG DNA is controlled by IL-12 and IL-10, and 2) that Th2 response inhibition by CpG ODN involves IL-12-independent changes in IL-10 and costimulatory molecule expression. These findings illustrate the utility of CpG DNA as adjuvants for vaccines designed to prevent Th2-dependent immunopathology.     

Production of autoantibodies associated with polyclonal activation in Yersinia enterocolitica O: 8-infected mice

Polyclonal lymphocyte stimulation is one of the immunomodulatory mechanisms induced by arthritogenic pathogens. In this study we examined the polyclonal activation potential of a virulent strain of Y. enterocolitica serotype O: 8 (WA 2707(+)) and its plasmidless isogenic pair (WA 2707(-)). SPF Swiss mice were infected intragastrically and spleen cells were obtained on days 7, 14, 21, 28, 35 and 42 after infection. The number of cells secreting nonspecific immunoglobulins of IgG, IgM and IgA isotypes was determined by the ELISPOT technique. The presence of serum-specific antibodies was investigated by ELISA and the presence of autoantibodies by dot-blot assay. Although the patterns of infection of the two bacterial strains were almost the same, only the animals infected with the virulent strain presented clinical anomalies. Neither arthritic nor inflammatory signs were observed in the joints of the infected animals. The greatest activation observed was that of the nonspecific IgM-secreting cells, and their peak of secretion occurred between the 28th and the 42nd day after infection, for both strains of Y. enterocolitica O: 8. Only the animals infected with the virulent strain (WA 2707(+)) produced IgG-specific antibodies in the serum, from the 28th day after infection. The serum of animals infected with either strain showed reactivity to all the autologous constituents tested, mainly on the 28th and 42nd day after infection. It was concluded that infection of mice with either the virulent strain of Y. enterocolitica O: 8 or with its plasmidless isogenic pair resulted in the polyclonal activation of the splenic B lymphocytes including some autoreactive clones.     

Telethonin protein expression in neuromuscular disorders

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G).We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression.     

Face Orientation and Motion Differently Affect the Deployment of Visual Attention in Newborns and 4-Month-Old Infants

Orienting visual attention allows us to properly select relevant visual information from a noisy environment. Despite extensive investigation of the orienting of visual attention in infancy, it is unknown whether and how stimulus characteristics modulate the deployment of attention from birth to 4 months of age, a period in which the efficiency in orienting of attention improves dramatically. The aim of the present study was to compare 4-month-old infants’ and newborns’ ability to orient attention from central to peripheral stimuli that have the same or different attributes. In Experiment 1, all the stimuli were dynamic and the only attribute of the central and peripheral stimuli to be manipulated was face orientation. In Experiment 2, both face orientation and motion of the central and peripheral stimuli were contrasted. The number of valid trials and saccadic latency were measured at both ages. Our results demonstrated that the deployment of attention is mainly influenced by motion at birth, while it is also influenced by face orientation at 4-month of age. These findings provide insight into the development of the orienting visual attention in the first few months of life and suggest that maturation may be not the only factor that determines the developmental change in orienting visual attention from birth to 4 months.     

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F₂ screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies.     

Exploratory screening for Fabry’s disease in young adults with cerebrovascular disorders in northern Sardinia

Background

The etiologic determinants of stroke in young adults remain a diagnostic challenge in up to one-fourth of cases. Increasing evidences led to consider Fabry’s disease (FD) as a possible cause to check up. We aimed at evaluating the prevalence of unrecognized FD in a cohort of patients with juvenile stroke in northern Sardinia.

Methods

For this study, we enrolled 178 patients consecutively admitted to our Neurological Ward for ischemic stroke, transient ischemic attack, intracerebral haemorrhage, neuroradiological evidence of silent infarcts, or white matter lesions possibly related to cerebral vasculopathy at brain MRI, and cerebral venous thrombosis. The qualifying events have to occur between 18 and 55 years of age.

Results

We identified two patients with an α-galactosidase A gene variant, with a prevalence of 0.9 %. According to recent diagnostic criteria, one patient, included for the occurrence of multiple white matter lesions at brain MRI, had a diagnosis of definite FD, the other, included for ischemic stroke, had a diagnosis of uncertain FD.

Conclusions

Our study places in a middle position among studies that found a prevalence of FD up to 4 % and others that did not find any FD patients. Our findings confirm that FD should be considered in the differential diagnosis of patients with juvenile stroke, particularly those with a personal or familial history positive for cerebrovascular events, or evidence of combined cardiologic and/or renal impairment. All types of cerebrovascular disorders should be screened for FD, including patients with white matter lesions possibly related to cerebral vasculopathy at brain MRI.

     

High connectivity and migration potentiate the invasion of Limnoperna fortunei (Mollusca: Mytilidae) in South America

Even after almost 30 years of Limnoperna fortunei introduction into South America, it is still unclear how the source and propagules are connected. Here, we present genetic evidence of population connectivity and gene flow of L. fortunei propagules from Asia into South America, proposing the main invasion routes into South America. To achieve that we expanded the sampling effort to cover all occurrence points of L. fortunei in South America. We sequenced the mtDNA COI gene and genotyped eight microsatellite loci (ML), and we evaluated the genetic source of the recently introduced population in Sobradinho hydroelectric power plant reservoir in Northeast Brazil. Our results revealed that China is the main genetic source of propagules for the Sobradinho population. We also found COI haplotypes and ML genotypes unique to South American populations, demonstrating a bridgehead effect likely caused by local mutation, adaptation, and admixture patterns that are maintained by high levels of gene flow among them. However, two genetic barriers were also detected. We concluded that L. fortunei is a well-established invader and is still rapidly expanding in Brazil, and the Amazon hydrographic basin is under an alarming threat of invasion.

     

Time course of stretching-induced changes in mechanomyogram and force characteristics

To evaluate the time-course of stretching-induced changes in mechanical properties of the muscle-tendon unit (MTU), 11 participants (age 22 ± 1 yr; body mass 77 ± 5 kg; stature 1.78 ± 0.05 m; mean ± SD) underwent tetanic electrical stimulations of the medial gastrocnemius muscle before and after (up to 2 h) stretching administration. During contractions, surface electromyogram (EMG), mechanomyogram (MMG) and force were recorded simultaneously. From MMG, peak-to-peak (p–p) and root mean square (RMS) were calculated during the on-phase and plateau phase of tetanic contraction, respectively. After stretching: (i) no differences were found in EMG parameters; (ii) MMG p–p and slope decreased (−16% and −10%, respectively; P < 0.05) and remained depressed for the entire recovery period; (iii) MMG RMS increased (+20%; P < 0.05), returning to pre-stretching values within 15 min; and (iv) peak force (pF), with its first (dF/dt) and second (d²F/dt²) derivative, decreased significantly by 32%, 35% and 54%, respectively, and remained depressed for the entire recovery period. The lack of MMG p–p and pF recovery could be ascribable to a reduced muscle force generating capacity due to persisting changes in viscoelastic characteristics of series elastic components. The early return of MMG RMS to pre-stretching values suggests that changes in viscoelastic parallel components recovered after few minutes.