Feasibility of fecal microbiota transplantation via oral gavage to safely alter gut microbiome composition in marmosets

Disruption of microbial communities within human hosts has been associated with infection, obesity, cognitive decline, cancer risk and frailty, suggesting that microbiome-targeted therapies may be an option for improving healthspan and lifespan. The objectives of this study were to determine the feasibility of delivering fecal microbiota transplants (FMTs) to marmosets via oral gavage and to evaluate if alteration of the gut microbiome post-FMT could be achieved. This was a prospective study of marmosets housed at the Barshop Institute for Longevity and Aging Studies in San Antonio, Texas. Eligible animals included healthy young adult males (age 2–5 years) with no recent medication use. Stool from two donors was combined and administered in 0.5 ml doses to five young recipients once weekly for 3 weeks. Safety outcomes and alterations in the gut microbiome composition via 16S ribosomal RNA sequencing were compared at baseline and monthly up to 6 months post-FMT. Overall, significant differences in the percent relative abundance was seen in FMT recipients at the phylum and family levels from baseline to 1 month and baseline to 6 months post-FMT. In permutational multivariate analysis of variance analyses, treatment status (donor vs. recipient) (p = .056) and time course (p = .019) predicted β diversity (p = .056). The FMT recipients did not experience any negative health outcomes over the course of the treatment. FMT via oral gavage was safe to administer to young adult marmosets. The marmoset microbiome may be altered by FMT; however, progressive changes in the microbiome are strongly driven by the host and its baseline microbiome composition.     

Olfactory cell cultures on ensheathing cell monolayers

Olfactory neurons dissociated from the olfactory mucosa of 4–5-week-oldSprague - Dawley rats are plated on either monolayers of ensheathingcells or cortical astrocytes. It is found that the ensheathingcells support a slightly higher percentage of neurite-bearingolfactory neurons than the astrocytes. Scanning electron microscopyshows that some of the cytoplasmic extensions of the ensheathingcells are closely associated with the olfactory axons whileothers appear to ensheath them. Olfactory neurons grown on uncoated,poly-L-lysine or laminin-coated glass coverslips in the presenceof medium conditioned by ensheathing cells fail to grow neurites,suggesting that interaction between membrane molecules, andnot trophic factors, may be required for neurite growth. However,it is unlikely that these membrane molecules are Ll and N-cadherinbecause immunohistochemical staining shows that only a smallproportion of the cultured ensheathing cells express Ll (9%)and N-cadherin (24%).     

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla

Arginine vasopressin stimulates Na⁺-K⁺-ATPase activity located in the rat thick ascending limb of s'Henle loop. Mammalian hypothalamus appears to produce a factor capable of inhibiting Na⁺-K⁺-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na⁺-K⁺-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na⁺-K⁺-ATPase activity throughout renal segments after 10 min exposure. Na⁺-K⁺-ATPase activity stimulated by AVP (1–10 fmol l^?1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10^?7–10^?3 U ml^?1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na⁺-K⁺-ATPase activity occurred at a hypothalamic extract concentration of 10^?3 U ml^?1. Only Na⁺-K⁺-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.     

Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k_cat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

New Potential Cell Wall Glucanases of Saccharomyces cerevisiae and Their Involvement in Mating

Biotinylation of intact Saccharomyces cerevisiae cells with a nonpermeant reagent (Sulfo-NHS-LC-Biotin) allowed the identification of seven cell wall proteins that were released from intact cells by dithiothreitol (DTT). By N-terminal sequencing, three of these proteins were identified as the known proteins β-exoglucanase 1 (Exg1p), β-endoglucanase (Bgl2p), and chitinase (Cts1p). One protein was related to the PIR protein family, whereas the remaining three (Scw3p, Scw4p, and Scw10p [for soluble cell wall proteins]) were found to be related to glucanases. Single knockouts of these three potential glucanases did not result in dramatic phenotypes. The double knockout of SCW4 and the homologous gene SCW10 resulted in slower growth, significantly increased release of proteins from intact cells by DTT, and highly decreased mating efficiency when these two genes were disrupted in both mating types. The synergistic behavior of the disruption of SCW4 and SCW10 was partly antagonized by the disruption of BGL2. The data are discussed in terms of a possible counterplay of transglucosidase and glucosidase activities.     

Improved Prediction of Body Fat by Measuring Skinfold Thickness,Circumferences, and Bone Breadths

Objective: To develop improved predictive regression equations for body fat content derived from common anthropometric measurements. Research Methods and Procedures: 117 healthy German subjects, 46 men and 71 women, 26 to 67 years of age, from two different studies were assigned to a validation and a cross‐validation group. Common anthropometric measurements and body composition by DXA were obtained. Equations using anthropometric measurements predicting body fat mass (BFM) with DXA as a reference method were developed using regression models. Results: The final best predictive sex‐specific equations combining skinfold thicknesses (SF), circumferences, and bone breadth measurements were as follows: BFM_New (kg) for men = ?40.750 + [(0.397 × waist circumference) + [6.568 × (log triceps SF + log subscapular SF + log abdominal SF)]] and BFM_New (kg) for women = ?75.231 + [(0.512 × hip circumference) + [8.889 × (log chin SF + log triceps SF + log subscapular SF)] + (1.905 × knee breadth)]. The estimates of BFM from both validation and cross‐validation had an excellent correlation, showed excellent correspondence to the DXA estimates, and showed a negligible tendency to underestimate percent body fat in subjects with higher BFM compared with equations using a two‐compartment (Durnin and Womersley) or a four‐compartment (Peterson) model as the reference method. Discussion: Combining skinfold thicknesses with circumference and/or bone breadth measures provide a more precise prediction of percent body fat in comparison with established SF equations. Our equations are recommended for use in clinical or epidemiological settings in populations with similar ethnic background.     

Automated identification of RNA 3D modules with discriminative power in RNA structural alignments

Recent progress in predicting RNA structure is moving towards filling the ‘gap’ in 2D RNA structure prediction where, for example, predicted internal loops often form non-canonical base pairs. This is increasingly recognized with the steady increase of known RNA 3D modules. There is a general interest in matching structural modules known from one molecule to other molecules for which the 3D structure is not known yet. We have created a pipeline, metaRNAmodules, which completely automates extracting putative modules from the FR3D database and mapping of such modules to Rfam alignments to obtain comparative evidence. Subsequently, the modules, initially represented by a graph, are turned into models for the RMDetect program, which allows to test their discriminative power using real and randomized Rfam alignments. An initial extraction of 22 495 3D modules in all PDB files results in 977 internal loop and 17 hairpin modules with clear discriminatory power. Many of these modules describe only minor variants of each other. Indeed, mapping of the modules onto Rfam families results in 35 unique locations in 11 different families. The metaRNAmodules pipeline source for the internal loop modules is available at http://rth.dk/resources/mrm.