Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle

Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective  The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods  In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results  Follistatin treatment resulted in smaller muscles after 3 months denervation ( p  = 0.019) and reduced force after 3 months sham denervation ( p  < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p  = 0.066). Follistatin increased satellite cell counts after denervation ( p  < 0.001) but reduced satellite cell counts after sham denervation ( p  = 0.037). Conclusion  Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.     

Responses of two semiarid conifer tree species to reduced precipitation and warming reveal new perspectives for stomatal regulation

Relatively anisohydric species are predicted to be more predisposed to hydraulic failure than relatively isohydric species, as they operate with narrower hydraulic safety margins. We subjected co‐occurring anisohydric Juniperus monosperma and isohydric Pinus edulis trees to warming, reduced precipitation, or both, and measured their gas exchange and hydraulic responses. We found that reductions in stomatal conductance and assimilation by heat and drought were more frequent during relatively moist periods, but these effects were not exacerbated in the combined heat and drought treatment. Counter to expectations, both species exhibited similar g_s temporal dynamics in response to drought. Further, whereas P. edulis exhibited chronic embolism, J. monosperma showed very little embolism due to its conservative stomatal regulation and maintenance of xylem water potential above the embolism entry point. This tight stomatal control and low levels of embolism experienced by juniper refuted the notion that very low water potentials during drought are associated with loose stomatal control and with the hypothesis that anisohydric species are more prone to hydraulic failure than isohydric species. Because direct association of stomatal behaviour with embolism resistance can be misleading, we advocate consideration of stomatal behaviour relative to embolism resistance for classifying species drought response strategies.     

Processing and characterization of porous structures from chitosan and starch for tissue engineering scaffolds

Natural biodegradable polymers were processed by different techniques for the production of porous structures for tissue engineering scaffolds. Potato, corn, and sweet potato starches and chitosan, as well as blends of these, were characterized and used in the experiments. The techniques used to produce the porous structures included a novel solvent-exchange phase separation technique and the well-established thermally induced phase separation method. Characterization of the open pore structures was performed by measuring pore size distribution, density, and porosity of the samples. A wide range of pore structures ranging from 1 to 400 microm were obtained. The mechanisms of pore formation are discussed for starch and chitosan scaffolds. Pore morphology in starch scaffolds seemed to be determined by the initial freezing temperature/freezing rate, whereas in chitosan scaffolds the shape and size of pores may have been determined by the processing route used. The mechanical properties of the scaffolds were assessed by indentation tests, showing that the indentation collapse strength depends on the pore geometry and the material type. Bioactivity and degradation of the potential scaffolds were assessed by immersion in simulated body fluid.     

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.     

Atorvastatin modifies the protein profile of circulating human monocytes after an acute coronary syndrome

Aggressive treatment with high‐dose atorvastatin reduces more effectively the incidence of cardiovascular events than moderate statin therapy. The mechanism of this benefit has not been fully elucidated. In order to know the potential effects of statin treatment on the protein expression of circulating monocytes in acute coronary syndrome (ACS) patients, a proteomic analysis of these cells was carried out by 2‐DE and MS. Twenty‐five patients with non‐ST‐elevation acute coronary syndrome (NSTEACS) were randomized, the fourth day after admission, to receive ATV 80 mg/dL (n = 14) or conventional treatment (CT) (n = 11), for two months. Blood was withdrawn at the end of the treatment, and monocytes were extracted for proteomic analysis and their protein expression patterns determined. Age, sex, total cholesterol, LDL, HDL, triglycerides, body mass index, presence of hypertension, diabetes, and smoking status were not significantly different between the two groups of patients. The expression of 20 proteins was modified by intensive ATV. Among the most relevant results stand out the normalization by intensive ATV treatment of the expression of proteins that modulate inflammation and thrombosis such as protein disulfide isomerase ER60 (PDI), Annexin I, and prohibitin, or that have other protective effects as HSP‐70. Thus, this approach shed light at the molecular level of the beneficial mechanisms of anti‐atherothrombotic drugs.     

Invasion biology of Australian ectomycorrhizal fungi introduced with eucalypt plantations into the Iberian Peninsula

In the last two centuries, several species of Australian eucalypts (e.g. Eucalyptus camaldulensis and E.␣globulus) were introduced into the Iberian Peninsula for the production of paper pulp. The effects of the introduction of exotic root-symbitotic fungi together with the eucalypts have received little attention. During the past years, we have investigated the biology of ectomycorrhizal fungi in eucalypt plantations in the Iberian Peninsula. In the plantations studied, we found fruit bodies of several Australian ectomycorrhizal fungi and identified their ectomycorrhizas with DNA molecular markers. The most frequent species were Hydnangium carneum, Hymenogaster albus, Hysterangium inflatum, Labyrinthomyces donkii, Laccaria fraterna, Pisolithus albus, P. microcarpus, Rhulandiella berolinensis, Setchelliogaster rheophyllus, and Tricholoma eucalypticum. These fungi were likely brought from Australia together with the eucalypts, and they seem to have facilitated the establishment of eucalypt plantations and their naturalization. The dispersion of Australian fungal propagules may be facilitating the spread of eucalypts along watercourses in semiarid regions increasing the water lost. Because ectomycorrhizal fungi are obligate symbionts, their capacity to persist after eradication of eucalypt stands, and/or to extend beyond forest plantations, would rely on the possibility to find compatible native host trees, and to outcompete the native ectomycorrhizal fungi. Here we illustrate the case of the Australasian species Laccaria fraterna, which fruits in Mediterranean shrublands of ectomycorrhizal species of Cistus (rockroses). We need to know which other Australasian fungi extend to the native ecosystems, if we are to predict environmental␣risks associated with the introduction of Australasian ectomycorrhizal fungi into the Iberian Peninsula. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthetic antimicrobial β‐peptide in dual‐treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans

Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β‐peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β‐peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole‐resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA‐based method and the mass‐action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole‐resistant biofilms and fluconazole resistant C. albicans strain was obtained. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily

Cyclic N⁶-threonylcarbamoyladenosine (‘cyclic t⁶A’, ct⁶A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct⁶A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct⁶A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t⁶A to form ct⁶A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K⁺ and Na⁺ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t⁶A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct⁶A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.     

Challenges and opportunities of the bio-pesticides production by solid-state fermentation: filamentous fungi as a model

In recent years, production and use of bio-pesticides have increasing and replacing some synthetic chemical pesticides applied to food commodities. In this review, biological control is focused as an alternative, to some synthetic chemical treatments that cause environmental, human health, and food quality risks. In addition, several phytopathogenic microorganisms have developed resistance to some of these synthetic chemicals and become more difficult to control. Worldwide, the bio-pesticides market is growing annually at a rate of 44% in North America, 20% in Europe and Oceania, 10% in Latin and South American countries and 6% in Asia. Use of agro-industrial wastes and solid-state fermentation (SSF) technology offers an alternative to bio-pesticide production with advantages versus conventional submerged fermentations, as reduced cost and energy consumption, low production of residual water and high stability products. In this review, recent data about state of art regarding bio-pesticides production under SSF on agroindustrial wastes will be discussed. SSF can be defined as a microbial process that generally occurs on solid material in the absence of free water. This material has the ability to absorb water with or without soluble nutrients, since the substrate must have water to support the microorganism’s growth and metabolism. Changes in water content are analyzed in order to select the conditions for a future process, where water stress can be combined with the best spore production conditions, obtaining in this way an inexpensive biotechnological option for modern agriculture in developing countries.