Nerve injury affects the capillary supply in rat slow and fast muscles differently

The goal of this study was to determine the acute effects of permanent denervation on the length density of the capillary network in rat slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles and the effect of short-lasting reinnervation in slow muscle only. Denervation was performed by cutting the sciatic nerve. Both muscles were excised 2 weeks later. Reinnervation was studied 4 weeks after nerve crush in SOL muscle only. Capillaries and muscle fibres were visualised by triple immunofluorescent staining with antibodies against CD31 and laminin and with fluorescein-labelled Griffonia (Bandeira) simplicifolia lectin. A recently developed stereological approach allowing the estimation of the length of capillaries adjacent to each individual fibre (Lcap/Lfib) was employed. Three-dimensional virtual test grids were applied to stacks of optical images captured with a confocal microscope and their intersections with capillaries and muscle fibres were counted. Interrelationships among capillaries and muscle fibres were demonstrated with maximum intensity projection of the acquired stacks of optical images. The course of capillaries in EDL seemed to be parallel to the fibre axes, whereas in SOL, their preferential direction deviated from the fibre axes and formed more cross-connections among neighbouring capillaries. Lcap/Lfib was clearly reduced in denervated SOL but remained unchanged in EDL, although the muscle fibres significantly atrophied in both muscle types. When soleus muscle was reinnervated, capillary length per unit fibre length was completely restored. The physiological background for the different responses of the capillary network in slow and fast muscle is discussed. This study was supported by the Slovenian Research Agency and the Ministry of Education, Youth and Sport of the Czech Republic (KONTAKT grant no. 19/2005).     

Cathepsin D propeptide: mechanism and regulation of its interaction with the catalytic core

Propeptide blocks the active site in the inactive zymogen of cathepsin D and is cleaved off during zymogen activation. We have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin D using a kinetic assay. Our mapping of the cathepsin D propeptide indicated two domains in the propeptide involved in the inhibitory interaction with the enzyme core: the active site "anchor" domain and the N-terminus of the propeptide. The latter plays a dominant role in propeptide inhibition (nanomolar Ki), and its high-affinity binding was corroborated by fluorescence polarization measurements. In addition to the inhibitory domains of propeptide, a fragment derived from the N-terminus of mature cathepsin D displayed inhibition. This finding supports its proposed regulatory function. The interaction mechanisms of the identified inhibitory domains were characterized by determining their modes of inhibition as well as by spatial modeling of the propeptide in the zymogen molecule. The inhibitory interaction of the N-terminal propeptide domain was abolished in the presence of sulfated polysaccharides, which interact with basic propeptide residues. The inhibitory potency of the active site anchor domain was affected by the Ala38pVal substitution, a propeptide polymorphism reported to be associated with the pathology of Alzheimer's disease. We infer that propeptide is a sensitive tethered ligand that allows for complex modulation of cathepsin D zymogen activation.     

Tissue-specific effects of statins on the expression of heme oxygenase-1 in vivo

Heme oxygenase-1 (HO-1) plays a central role in antioxidant and anti-inflammatory actions, which may be mediated through its formation of biliverdin/bilirubin and carbon monoxide. HMG-CoA reductase inhibitors (statins) induce in vitro HO-1 expression and are reported to have pleiotropic benefits that reduce oxidative stress in the vasculature. We characterized the effects of statins on in vivo HO-1 expression in various extravascular tissues: liver, lung, brain, and heart. Adult mice were orally administered simvastatin, lovastatin, atorvastatin, or rosuvastatin. HO activity significantly increased in a statin- and tissue-specific manner, with all statins increasing heart and lung activity within 24 h. Significant elevations of HO-1 protein and mRNA were also observed in heart and lung after atorvastatin treatment. We conclude that in vivo HO-1 induction is statin- and tissue-specific. Through this pathway, statins may confer antioxidant and anti-inflammatory actions in the vasculature and extravascular systems.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.     

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR–Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)₃₃. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.     

Risk of infection following a visit to the emergency department: a cohort study

Background:

The risk of infection following a visit to the emergency department is unknown. We explored this risk among elderly residents of long-term care facilities.

Methods:

We compared the rates of new respiratory and gastrointestinal infections among elderly residents aged 65 years and older of 22 long-term care facilities. We used standardized surveillance definitions. For each resident who visited the emergency department during the study period, we randomly selected two residents who did not visit the emergency department and matched them by facility unit, age and sex. We calculated the rates and proportions of new infections, and we used conditional logistic regression to adjust for potential confounding variables.

Results:

In total, we included 1269 residents of long-term care facilities, including 424 who visited the emergency department during the study. The baseline characteristics of residents who did or did not visit the emergency department were similar, except for underlying health status (visited the emergency department: mean Charlson Comorbidity Index 6.1, standard deviation [SD] 2.5; did not visit the emergency department: mean Charlson Comorbidity index 5.5, SD 2.7; p < 0.001) and the proportion who had visitors (visited the emergency department: 46.9%; did not visit the emergency department: 39.2%; p = 0.01). Overall, 21 (5.0%) residents who visited the emergency department and 17 (2.0%) who did not visit the emergency department acquired new infections. The incidence of new infections was 8.3/1000 patient-days among those who visited the emergency department and 3.4/1000 patient-days among those who did not visit the emergency department. The adjusted odds ratio for the risk of infection following a visit to the emergency department was 3.9 (95% confidence interval 1.4–10.8).

Interpretation:

A visit to the emergency department was associated with more than a threefold increased risk of acute infection among elderly people. Additional precautions should be considered for residents following a visit to the emergency department.Infections associated with health care are an important health risk. A recent survey by the World Health Organization reported that 8.7% of patients in hospital developed such infections.^1,2 The third leading cause of death in the United States is health care–associated deaths, with over 100 000 people dying from infections associated with health care each year.³ In Canada, a point-prevalence survey found that 11.6% of adults in hospital experience a health care–associated infection.⁴Little attention has been paid to infections acquired in other health care settings. Visiting an emergency department has been identified as a risk for disease during outbreaks of measles^5,6 and SARS,^7,8 but little is known about the potential risk of endemic infection from exposure in this setting. A visit to the emergency department differs from a stay in hospital: exposure and duration of contact with other patients is shorter, but the number and density of patients with acute illness with whom there could be contact is higher.Elderly residents of long-term care facilities are likely to be at the greatest risk of morbidity and mortality from communicable diseases acquired in the emergency department. When residents are transferred to the emergency department for assessment, they are likely to have longer stays and to be cared for in multibed observation areas and corridors.⁹ If they acquire an infection while in the emergency department, these residents may be the source of an outbreak upon return to their facility; this can lead to increases in workload and costs. A Canadian study estimated the cost of an influenza outbreak to be over $6000 per 30-day period, with an estimated incidence of death of 0.75/100 residents during the same period.¹⁰ In this study, we explored the risk of acute respiratory and gastrointestinal infection associated with a visit to the emergency department among elderly residents of long-term care facilities.     

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1^G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.     

Architecture of the trypanosome RNA editing accessory complex, MRB1

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.     

Effect of nitrogen,phosphorus and potassium availability on emergence,nodulation and growth of acidicole Trifolium arvense L. in alkaline soil

The acidicole behavior of many species is given by their inability to acquire enough P in alkaline soil, but reasons for acidicole (syn. calcifuge) behavior of Trifolium arvense have never been studied experimentally. We asked how emergence of seedlings, the survival of plants, the growth of aboveground organs, the nodulation and the growth of roots of this species is affected by different N, P and K supply in alkaline soil. In the years 2010 and 2011, we performed a pot experiment (ten N, P and K fertilizer treatments) with seeding of T. arvense into alkaline soil. The acidicole behavior of T. arvense proved to be connected with the inability of seedlings to acquire enough P during their emergence. In all treatments, T. arvense was not able to flower in the seeding year and in the second year it flowered only in P treatments. This indicates the biennial character of the species in the case of late seeding and points to a strong P limitation of flowering and seeds production in plants grown on alkaline soil. Without P addition, T. arvense was not able to develop beyond the seedling stage. Nodulation was positively affected by P application and negatively by N application. Although T. arvense is a typical species for P poor soils, it does not suffer from P toxicity under high P supply. We concluded that the acidicole behavior of T. arvense is based on its P limitation when growing on alkaline soil.