A program of moderate physical training for Wistar rats based on maximal oxygen consumption

Moderate physical training is often associated with improved cardiorespiratory fitness in athletes and the general population. In animals, studies are designed to investigate basic physiology that could be invasive and uncomfortable for humans. The standardization of an exercise training protocol for rats based on maximal consumption of oxygen (VO(2)max) is needed. This study validated a program of moderate physical training for Wistar rats based on VO(2)max determined once a week. A 10-stage treadmill running test was developed to measure VO(2)max through an indirect, open circuit calorimeter. Thirty male Wistar rats (210-226 g) were randomly assigned to either a nontrained group or a trained group. The animals were evaluated weekly to follow their VO(2)max during 8 weeks of moderate training and to adjust the intensity of the protocol of training. The soleus muscle was removed for determination of citrate synthase activity. Trained animals maintained their values of VO(2)max during a moderate running training and showed a significant less body weight gain. An increase of 42% in citrate synthase activity of the soleus muscle from trained rats was found after the training program. Our study presents a protocol of moderate physical training for Wistar rats based on VO(2)max. Peripheral adaptations such as the values of citrate synthase activity also responded to the moderate training program imposed as observed for VO(2)max. Other studies can use our protocol of moderate training to study the physiologic adaptations underlying this specific intensity of training. It will provide support for study with humans.     

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L–AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.     

Antimycotic Activity Potentiation of Allium sativum Extract and Silver Nanoparticles against Trichophyton rubrum

A natural and biocompatible extract of garlic as a support, decorated with silver nanoparticles, is a proposal to generate an effective antifungal agent against dermatophytes at low concentrations. Silver nanoparticles (AgNPs) with a diameter of 26±7 nm were synthesized and their antimycotic activity was examined against Trichophyton rubrum (T. rubrum), inhibiting 94 % of growth at a concentration of 0.08 mg ml^?1. Allium sativum (garlic) extract was also obtained (AsExt), and its MIC was 0.04 mg ml^?1. To increase the antifungal capacity of those systems, AsExt was decorated with AgNPs, obtaining AsExt‐AgNPs. Using an AsExt concentration of 0.04 mg ml^?1 in independent experiments with concentrations from 0.01 to 0.08 mg ml^?1 of AgNPs, it was possible to inhibit T. rubrum at all AgNPs concentrations; it proves a synergistic effect between AgNPs and AsExt. Even if 1 % of the minimum inhibitory concentration of AsExt (0.0004 mg ml^?1) is used, it was possible to inhibit T. rubrum at all concentrations of AgNPs, demonstrating the successful antimycotic activity potentiation when combining AsExt and AgNPs.     

Deep soil inventories reveal that impacts of cover crops and compost on soil carbon sequestration differ in surface and subsurface soils

Increasing soil organic carbon (SOC) via organic inputs is a key strategy for increasing long‐term soil C storage and improving the climate change mitigation and adaptation potential of agricultural systems. A long‐term trial in California's Mediterranean climate revealed impacts of management on SOC in maize‐tomato and wheat–fallow cropping systems. SOC was measured at the initiation of the experiment and at year 19, at five depth increments down to 2 m, taking into account changes in bulk density. Across the entire 2 m profile, SOC in the wheat–fallow systems did not change with the addition of N fertilizer, winter cover crops (WCC), or irrigation alone and decreased by 5.6% with no inputs. There was some evidence of soil C gains at depth with both N fertilizer and irrigation, though high variation precluded detection of significant changes. In maize?tomato rotations, SOC increased by 12.6% (21.8 Mg C/ha) with both WCC and composted poultry manure inputs, across the 2 m profile. The addition of WCC to a conventionally managed system increased SOC stocks by 3.5% (1.44 Mg C/ha) in the 0–30 cm layer, but decreased by 10.8% (14.86 Mg C/ha) in the 30–200 cm layer, resulting in overall losses of 13.4 Mg C/ha. If we only measured soil C in the top 30 cm, we would have assumed an increase in total soil C increased with WCC alone, whereas in reality significant losses in SOC occurred when considering the 2 m soil profile. Ignoring the subsoil carbon dynamics in deeper layers of soil fails to recognize potential opportunities for soil C sequestration, and may lead to false conclusions about the impact of management practices on C sequestration.     

Use of biotechnological approaches to add value to rice hulls

One of the most common agricultural wastes generated in rice producing countries, rice hull (RH) is considered an environmental problem due to increased rice production and RH accumulation, especially because natural degradation in the environment is very difficult and time-consuming. Currently, RH is mostly used as bed for broiler chickens or burned for energy generation, two processes that prevent environmental accumulation in a sustainable way, without adding value to the RH. To diversificate its use and effectively add some value to the RH, a pretreatment is frequently needed, allowing the application of several biotechnological approaches. In this review, we gather information about biotechnological uses of crude and processed RH, including their use as fertilizers, filler material in natural rubber and incorporation in cement for civil construction purposes, along with their use in processes as silica extraction and adsorption/removal of environmental contaminants as heavy metals and dyes. Finally, we critically evaluate the data published in the literature, and based on our own findings, we point future directions related to RH biodegradation and further methane production.     

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca²⁺ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca²⁺ release response in HEK293 cells and bound the RyR-specific ligand [³H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K⁺ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca²⁺ release in HEK293 cells, low [³H]ryanodine binding levels, and channels that were not regulated by Ca²⁺ and did not conduct Ca²⁺ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.     

Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

The cardiac Ca²⁺ release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca²⁺ sparks showed that Ca²⁺ sparks originated exclusively from RyR2 clusters. Ca²⁺ sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca²⁺ level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.     

The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion

SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet β- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP⁺: glucagon⁺ cells from KO mice, revealed recombination in ∼30% of α-cells, of which ∼50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn²⁺ levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca²⁺ increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca²⁺-independent mechanisms.     

Fungi colonizing hair-baits from three coastal beaches of Mexico

The abundance of hair-bait colonizing fungi was investigated on the beaches of Caracoles, Mocambo, and lcacos, located on the coasts of the Caribbean Sea, Gulf of Mexico and Pacific Ocean, respectively. On each beach a sample of sandy soil was collected. The samples were analyzed by two selective isolation methods for keratinophilic fungi resulting in a total of 544 occurrences. A total of 17 species was found, of which 4 were ascomycetes and 13 hyphomycetes.Gymnascella dankaliensis andAspergillus terreus were the most abundant.Arthroderma curreyi andChrysosporium tropicum were found in low percentages in this survey. From the three beaches sampled, Icacos beach, on the Pacific Ocean coast had the highest number of isolated species.