Free‐energy computations identify the mutations required to confer trans‐sialidase activity into Trypanosoma rangeli sialidase

Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans‐sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH‐33). They share 70% of sequence identity and their crystallographic C_α RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl‐glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans‐sialidase activity through appropriate point mutations. Recently, we calculated the free‐energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA_5mut), which has faint trans‐sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA_5mut that should create a mutant (TrSA_10mut) with high trans‐sialidase activity. Proteins 2014; 82:424–435. © 2013 Wiley Periodicals, Inc.     

Association between a Genetic Variant of Type-1 Cannabinoid Receptor and Inflammatory Neurodegeneration in Multiple Sclerosis

Genetic ablation of type-1 cannabinoid receptors (CB₁Rs) exacerbates the neurodegenerative damage of experimental autoimmune encephalomyelitis, the rodent model of multiple sclerosis (MS). To address the role on CB₁Rs in the pathophysiology of human MS, we first investigated the impact of AAT trinucleotide short tandem repeat polymorphism of CNR1 gene on CB₁R cell expression, and secondly on the inflammatory neurodegeneration process responsible for irreversible disability in MS patients. We found that MS patients with long AAT repeats within the CNR1 gene (≥12 in both alleles) had more pronounced neuronal degeneration in response to inflammatory white matter damage both in the optic nerve and in the cortex. Optical Coherence Tomography (OCT), in fact, showed more severe alterations of the retinal nerve fiber layer (RNFL) thickness and of the macular volume (MV) after an episode of optic neuritis in MS patients carrying the long AAT genotype of CNR1. MS patients with long AAT repeats also had magnetic resonance imaging (MRI) evidence of increased gray matter damage in response to inflammatory lesions of the white matter, especially in areas with a major role in cognition. In parallel, visual abilities evaluated at the low contrast acuity test, and cognitive performances were negatively influenced by the long AAT CNR1 genotype in our sample of MS patients. Our results demonstrate the biological relevance of the (AAT)_n CNR1 repeats in the inflammatory neurodegenerative damage of MS.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera,Tettigoniidae) and protein analysis of their secretions

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two‐part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the “first‐order” glands, a number of large anterior tubules, and the “second‐order” glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second‐order glands. The mesoderm‐derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron‐dense granules and globules or electron‐transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first‐order glands, and corresponds to an important protein component of the spermatophylax. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Runs of homozygosity reveal genome‐wide autozygosity in Italian sheep breeds

The availability of dense single nucleotide polymorphism (SNP) assays allows for the determination of autozygous segments based on runs of consecutive homozygous genotypes (ROH). The aim of the present study was to investigate the occurrence and distribution of ROH in 21 Italian sheep breeds using medium‐density SNP genotypes in order to characterize autozygosity and identify genomic regions that frequently appeared in ROH within individuals, namely ROH islands. After filtering, the final number of animals and SNPs retained for analyses were 502 and 46 277 respectively. A total of 12 302 ROH were identified. The mean number of ROH per breed ranged from 10.58 (Comisana) to 44.54 (Valle del Belice). The average length of ROH across breeds was 4.55 Mb and ranged from 3.85 Mb (Biellese) to 5.51 Mb (Leccese). Valle del Belice showed the highest value of inbreeding on the basis of ROH (F_ROH = 0.099), whereas Comisana showed the lowest (F_ROH = 0.016), and high standard deviation values revealed high variability in autozygosity levels within each breed. Differences also existed in the length of ROH. Analysis of the distribution of ROH according to their size showed that, for all breeds, the majority of the detected ROH were <10 Mb in length, with a few long ROH >25 Mb. The levels of ROH that we estimated here reflect the inbreeding history of the investigated sheep breeds. These results also highlight that ancient and recent inbreeding have had an impact on the genome of the Italian sheep breeds and suggest that several animals have experienced recent autozygosity events. Comisana and Bergamasca appeared as the less consanguineous breeds, whereas Barbaresca, Leccese and Valle del Belice showed ROH patterns typically produced by recent inbreeding. Moreover, within the genomic regions most commonly associated with ROH, several candidate genes were detected.     

Tolerability and efficacy of single dose albendazole,diethylcarbamazine citrate (DEC) or co-administration of albendazole with DEC in the clearance of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis>in asymptomatic microfilaraemic volunteers in Pondicherry,South India: a hospital-based study

Background  

The tolerability and efficacy of single dose albendazole (400 mg), diethylcarbamazine citrate (DEC) (6 mg/kg bodyweight) or co-administration of albendazole (400 mg) + DEC (6 mg/kg bodyweight) was studied in 54 asymptomatic Wuchereria bancrofti microfilaraemic volunteers in a double blind hospital-based clinical study.     

Stimulation of integrin-mediated cell contractility by fibronectin polymerization

Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.     

Microfluidic devices for analysis of spatial orientation behaviors in semi-restrained Caenorhabditis elegans

This article describes the fabrication and use of microfluidic devices for investigating spatial orientation behaviors in nematode worms (Caenorhabditis elegans). Until now, spatial orientation has been studied in freely moving nematodes in which the frequency and nature of encounters with the gradient are uncontrolled experimental variables. In the new devices, the nematode is held in place by a restraint that aligns the longitudinal axis of the body with the border between two laminar fluid streams, leaving the animal's head and tail free to move. The content of the fluid streams can be manipulated to deliver step gradients in space or time. We demonstrate the utility of the device by identifying previously uncharacterized aspects of the behavioral mechanisms underlying chemotaxis, osmotic avoidance, and thermotaxis in this organism. The new devices are readily adaptable to behavioral and imaging studies involving fluid borne stimuli in a wide range of sensory modalities.