Upper Limb Evaluation and One-Year Follow Up of Non-Ambulant Patients with Spinal Muscular Atrophy: An Observational Multicenter Trial

Assessment of the upper limb strength in non-ambulant neuromuscular patients remains challenging. Although potential outcome measures have been reported, longitudinal data demonstrating sensitivity to clinical evolution in spinal muscular atrophy patients are critically lacking. Our study recruited 23 non-ambulant patients, 16 patients (males/females = 6/10; median age 15.4 years with a range from 10.7 to 31.1 years) with spinal muscular atrophy type II and 7 patients (males/females = 2/5; median age 19.9 years with a range from 8.3 to 29.9 years) with type III. The Brooke functional score was on median 3 with a range from 2 to 6. The average total vital capacity was 46%, and seven patients required non-invasive ventilation at night. Patients were assessed at baseline, 6 months, and 1 year using the Motor Function Measure and innovative devices MyoGrip, MyoPinch, and MoviPlate, which assess handgrip strength, key pinch strength, and hand/finger extension-flexion function, respectively. The study demonstrated the feasibility and reliability of these measures for all patients, and sensitivity to negative changes after the age of 14 years. The younger patients showed an increase of the distal force in the follow-up period. The distal force measurements and function were correlated to different functional scales. These data represent an important step in the process of validating these devices as potential outcome measures for future clinical trials.

Trial Registration

ClinicalTrials.gov NCT00993161     

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.     

Clustering of monosialyl-Gb5 initiates downstream signalling events leading to invasion of MCF-7 breast cancer cells

Invasion is a complex process controlled by secretion and activation of proteases, alteration of integrin levels and GSL (glycosphingolipid) patterns. Differential organization of GSLs with specific membrane proteins and signal transducers in GEMs (GSL-enriched microdomains), initiates signalling events to modify cellular phenotype. Although the GSL monosialyl-Gb5 has been linked with invasion, its functional role in invasion is poorly described and understood. To investigate this problem, we induced the invasion of human breast cancer cells and subsequently explored the underlying mechanism. In the present study, the invasion of human MCF-7 breast cancer cells is highly dependent on clustering of monosialyl-Gb5, and the subsequent activation of monosialyl-Gb5-associated focal adhesion kinase and cSrc in GEM leading to the downstream activation of extracellular-signal-regulated kinase (ERK). As a result, we observed increased expression levels and activity of matrix metalloproteinases-2 and -9, which correlated with decreased expression of integrins alpha1 and beta1. Together these results suggest that the organization of crucial molecules in GEMs of MCF-7 cells is critical for their invasive properties.     

Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca²⁺dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.     

Burkholderia cepacia Genomovar III Is a Common Plant-Associated Bacterium

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with “cepacia syndrome” in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.     

Isolation and characterization of 18 microsatellite loci in the Egyptian vulture (Neophron percnopterus)

We developed 18 new microsatellite loci for the endangered Egyptian vulture (Neophron percnopterus). Microsatellite loci were screened for variation in two different populations belonging to separate subspecies: the nominal N. p. percnopterus and the Canarian N. p. majorensis. Mean expected heterosygosities were respectively 0.51 and 0.46, while the mean number of alleles per locus was 4.7 and 3.9. These new markers allow further genetic studies for the endangered Canarian Egyptian Vulture.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Human susceptibility and resistance to Norwalk virus infection

Infectious diseases have influenced population genetics and the evolution of the structure of the human genome in part by selecting for host susceptibility alleles that modify pathogenesis. Norovirus infection is associated with approximately 90% of epidemic non-bacterial acute gastroenteritis worldwide. Here, we show that resistance to Norwalk virus infection is multifactorial. Using a human challenge model, we showed that 29% of our study population was homozygous recessive for the alpha(1,2)fucosyltransferase gene (FUT2) in the ABH histo-blood group family and did not express the H type-1 oligosaccharide ligand required for Norwalk virus binding. The FUT2 susceptibility allele was fully penetrant against Norwalk virus infection as none of these individuals developed an infection after challenge, regardless of dose. Of the susceptible population that encoded a functional FUT2 gene, a portion was resistant to infection, suggesting that a memory immune response or some other unidentified factor also affords protection from Norwalk virus infection.