Role of Alpha-actinin-3 in Contractile Properties of Human Single Muscle Fibers: A Case Series Study in Paraplegics

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3^−/− (KO) mouse suggest a shift towards ‘slow’ metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an ‘equal environment condition’ what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier’s biopsy was the only one that presented type I fibers with a complete lack of type II_x fibers. Properties of hybrid type II_a/II_x fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II_a/II_x fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm²) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type II_x fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity.     

Fc Gamma Receptor IIIB (FcγRIIIB) Polymorphisms Are Associated with Clinical Malaria in Ghanaian Children

Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p = 0.0061; recessive: p = 0.097; dominant: p = 0.0076) of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Oral Antibiotic Treatment of Mice Exacerbates the Disease Severity of Multiple Flavivirus Infections

1. Download high-res image (163KB)
2. Download full-size image

     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.      

In situ deprotection: a method for covalent immobilization of peptides with well-defined orientation for use in solid phase immunoassays such as enzyme-linked immunosorbent assay.

The orientation of an immobilized antigen is important for recognition by, e.g., an antibody. When noncovalent passive adsorption is used for immobilization, the number of ways that the antigen can attach to the surface is numerous and control of how the antigen orientates on the surface is limited. Covalent immobilization restricts the number of the ways that the antigen can be immobilized to the number of reactive groups on the antigen and, hence, the orientation of the immobilized antigen is more predictable. Peptide antigens were synthesized and purified with protection groups on the lysine and cysteine side chains. These peptides, which have only one good nucleophilic group (the N-terminal alpha-amino group), were immobilized covalently in microtiter plates supplied with tresyl groups on the surface and the protection groups were cleaved off in situ after immobilization. The controlled orientation of these peptides resulted in enhanced recognition by antibodies in general. An enzyme-linked immunosorbent assay for detection of antibodies against a peptide derived from outer surface protein C from Borrelia burgdorferi, found in Lyme borreliosis patients, was established using this strategy. Lyme borreliosis suspect patient sera showed up to a 10-fold increase in the signal when the orientation of the peptide antigen was controlled by the in situ deprotection strategy.     

Response of plant species richness and primary productivity in shrublands along a north–south gradient in Europe to seven years of experimental warming and drought: reductions in primary productivity in the heat and drought year of 2003

We used a nonintrusive field experiment carried out at six sites – Wales (UK), Denmark (DK), the Netherlands (NL), Hungary (HU), Sardinia (Italy – IT), and Catalonia (Spain – SP) – along a climatic and latitudinal gradient to examine the response of plant species richness and primary productivity to warming and drought in shrubland ecosystems. The warming treatment raised the plot daily temperature by ca. 1 °C, while the drought treatment led to a reduction in soil moisture at the peak of the growing season that ranged from 26% at the SP site to 82% in the NL site. During the 7 years the experiment lasted (1999–2005), we used the pin‐point method to measure the species composition of plant communities and plant biomass, litterfall, and shoot growth of the dominant plant species at each site. A significantly lower increase in the number of species pin‐pointed per transect was found in the drought plots at the SP site, where the plant community was still in a process of recovering from a forest fire in 1994. No changes in species richness were found at the other sites, which were at a more mature and stable state of succession and, thus less liable to recruitment of new species. The relationship between annual biomass accumulation and temperature of the growing season was positive at the coldest site and negative at the warmest site. The warming treatment tended to increase the aboveground net primary productivity (ANPP) at the northern sites. The relationship between annual biomass accumulation and soil moisture during the growing season was not significant at the wettest sites, but was positive at the driest sites. The drought treatment tended to reduce the ANPP in the NL, HU, IT, and SP sites. The responses to warming were very strongly related to the Gaussen aridity index (stronger responses the lower the aridity), whereas the responses to drought were not. Changes in the annual aboveground biomass accumulation, litterfall, and, thus, the ANPP, mirrored the interannual variation in climate conditions: the most outstanding change was a decrease in biomass accumulation and an increase in litterfall at most sites during the abnormally hot year of 2003. Species richness also tended to decrease in 2003 at all sites except the cold and wet UK site. Species‐specific responses to warming were found in shoot growth: at the SP site, Globularia alypum was not affected, while the other dominant species, Erica multiflora, grew 30% more; at the UK site, Calluna vulgaris tended to grow more in the warming plots, while Empetrum nigrum tended to grow less. Drought treatment decreased plant growth in several studied species, although there were some species such as Pinus halepensis at the SP site or C. vulgaris at the UK site that were not affected. The magnitude of responses to warming and drought thus depended greatly on the differences between sites, years, and species and these multiple plant responses may be expected to have consequences at ecosystem and community level. Decreases in biodiversity and the increase in E. multiflora growth at the SP site as a response to warming challenge the assumption that sensitivity to warming may be less well developed at more southerly latitudes; likewise, the fact that one of the studied shrublands presented negative ANPP as a response to the 2003 heat wave also challenges the hypothesis that future climate warming will lead to an enhancement of plant growth and carbon sequestration in temperate ecosystems. Extreme events may thus change the general trend of increased productivity in response to warming in the colder sites.     

Repercussions of the COVID-19 pandemic on athletes: a cross-sectional study

The COVID-19 pandemic has presented significant challenges and implications for the sports community. Thus, this study aimed to describe the prevalence of COVID-19 in Brazilian athletes and identify the epidemiological, clinical, athletic, life and health factors associated with the disease in these individuals. A cross-sectional study was performed involving 414 athletes from 22 different sports using an online questionnaire from August to November 2020. The association between the athletes’ characteristics and COVID-19 was evaluated using a logistic regression model. The prevalence of COVID-19 was 8.5%, although only 40% of athletes reported having been tested. Being under 27 years of age (3-fold), having children (~5-fold), having a teammate test positive for COVID-19 (2.5-fold), and smoking (14-fold) were associated with a possible higher risk of disease. Almost 20% of athletes self-reported musculoskeletal injuries during the period of the pandemic that was studied. Athletes with a university education (P = 0.02), a profession other than sports (P < 0.001), those from a low-income family (P = 0.01), and public health system users (P = 0.04) were significantly less frequently tested for COVID-19, whereas international competitors, athletes who received a wage, and athletes who had a teammate who tested positive for COVID-19 were 2-, 3-, and 15-fold more likely to be tested for COVID-19, respectively. Approximately 26% of the athletes who tested negative or were untested reported more than three characteristic COVID-19 symptoms, and 11% of athletes who tested positive for COVID-19 were asymptomatic. The identification of modifiable (have children, smoking, and teammates positively tested) and non-modifiable (age under 27 years) factors related to COVID-19 in athletes can contribute to implementing surveillance programmes to decrease the incidence of COVID-19 in athletes and its negative impacts in sports.     

Screening of epoxide hydrolase producing microorganisms for biotransformation of deoxynivalenol

Deoxynivalenol (DON) transformation products from selected time course experiments were analyzed by thin-layer chromatography. With the strainAlternaria alternata f. sp.lycopersici AS27-3, one major metabolite of DON in ethyl acetate was observed. This unidentified metabolite was more polar than DON and has a R_f value of 0.71. Derivatization indicated that this metabolite was probably an unidentified trichothecene. Screening of 29 other microbial isolates (bacteria, yeast, filamentous fungi) for DON transformation did not result in any active organism. Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May 17–19, 2004     

A Genotype-First Approach for the Molecular and Clinical Characterization of Uncommon De Novo Microdeletion of 20q13.33

Background

Subtelomeric deletions of the long arm of chromosome 20 are rare, with only 11 described in the literature. Clinical features of individuals with these microdeletions include severe limb malformations, skeletal abnormalities, growth retardation, developmental and speech delay, mental retardation, seizures and mild, non-specific dysmorphic features.

Methodology/Principal Findings

We characterized microdeletions at 20q13.33 in six individuals referred for genetic evaluation of developmental delay, mental retardation, and/or congenital anomalies. A comparison to previously reported cases of 20q13.33 microdeletion shows phenotypic overlap, with clinical features that include mental retardation, developmental delay, speech and language deficits, seizures, and behavior problems such as autistic spectrum disorder. There does not appear to be a clinically recognizable constellation of dysmorphic features among individuals with subtelomeric 20q microdeletions.

Conclusions/Significance

Based on genotype-phenotype correlation among individuals in this and previous studies, we discuss several possible candidate genes for specific clinical features, including ARFGAP1, CHRNA4 and KCNQ2 and neurodevelopmental deficits. Deletion of this region may play an important role in cognitive development.