High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.     

Chromium(III) ion and thyroxine cooperate to stabilize the transthyretin tetramer and suppress in vitro amyloid fibril formation

Transthyretin (TTR) amyloid fibril formation, which is triggered by the dissociation of tetrameric TTR, appears to be the causative factor in familial amyloidotic polyneuropathy and senile systemic amyloidosis. Binding of thyroxine (T(4)), a native ligand of TTR, stabilizes the tetramer, but the bioavailability of T(4) for TTR binding is limited due to the preferential binding of T(4) to globulin, the major T(4) carrier in plasma. Here, we show that Cr(3+) increased the T(4)-binding capacity of wild-type (WT) and amyloidogenic V30M-TTR. Moreover, we demonstrate that Cr(3+) and T(4) cooperatively suppressed in vitro fibril formation due to the stabilization of WT-TTR and V30M-TTR.     

Whole-Body Vibrations Do Not Elevate the Angiogenic Stimulus when Applied during Resistance Exercise

Knowledge about biological factors involved in exercise-induced angiogenesis is to date still scanty. The present study aimed to investigate the angiogenic stimulus of resistance exercise with and without superimposed whole-body vibrations. Responses to the exercise regimen before and after a 6-week training intervention were investigated in twenty-six healthy male subjects. Serum was collected at the initial and final exercise sessions and circulating levels of matrix metalloproteinases (MMP) -2 and -9, Vascular Endothelial Growth Factor (VEGF) and endostatin were determined via ELISA. Furthermore, we studied the proliferative effect of serum-treated human umbilical vein endothelial cells in vitro via BrdU-incorporation assay. It was found that circulating MMP-2, MMP-9, VEGF and endostatin levels were significantly elevated (P<0.001) from resting levels after both exercise interventions, with higher post-exercise VEGF concentrations in the resistance exercise (RE) group compared to the resistive vibration exercise (RVE) group. Moreover, RE provoked increased endothelial cell proliferation in vitro and higher post-exercise circulating endostatin concentrations after 6 weeks of training. These effects were elusive in the RVE group. The present findings suggest that resistance exercise leads to a transient rise in circulating angiogenic factors and superimposing vibrations to this exercise type might not further trigger a potential signaling of angiogenic stimulation in skeletal muscle.     

Inhibition of DNA Polymerases Used in Q-PCR by Structurally Different Soil-Derived Humic Substances

Real-time PCR for the quantitative assessment of microbial genes in DNA extracted from environmental samples is increasingly being used in microbial ecology studies. A significant problem with the quantitative aspect of the method is the possible inhibition of the PCR process by humic substances co-extracted with the DNA. A comparison of the inhibition exerted by five structurally different humic substances on six commercially available DNA polymerases revealed large differences in the resistance of the polymerases to inhibition. Depending on the DNA polymerases (or their formulation) the addition of Bovine Serum Albumin (BSA) to the mastermix reaction generally increased the resistance to the different humic substances and decreased differences between the polymerases. One of the tested polymerases was clearly hampered by the addition of BSA, indicating that BSA cannot be added to just any mastermix to improve enzyme performance. The structural differences in the tested humic acids suggest that the mechanism of the inhibition is not a feature of all organic structures, but mainly related to the presence of certain phenolic or quinonoid structures.     

Moderate Exercise Promotes Human RBC-NOS Activity,NO Production and Deformability through Akt Kinase Pathway

Background

Nitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs.

Methods/Findings

Fifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer¹¹⁷⁷ was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr⁴⁹⁵ were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability.

Conclusion/Significance

This human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.     

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.     

Description of Meloidoderita salina sp. n. (Nematoda,Sphaeronematidae) from a micro-tidal salt marsh at?Mont-Saint-Michel Bay in France

Meloidoderita salina sp. n. is described and illustrated from the halophytic plant Atriplex portulacoides L. (sea purslane) growing in a micro-tidal salt marsh in the Mont-Saint-Michel Bay in France. This new species is the first member of Meloidoderita Poghossian, 1966 collected from a saline environment, and is characterized by the following features: sedentary mature females having a small swollen body with a clear posterior protuberance; slightly dorsally curved stylet, 19.9 µm long, with posteriorly sloping knobs; neck region irregular in shape and twisted; well developed secretory-excretory (S–E) pore, with markedly sclerotized S-E duct running posteriorly; prominent uterus bordered by a thick hyaline wall and filled with eggs. The adult female transforms into a cystoid. Eggs are deposited in both egg-mass and cystoid. Cystoids of Meloidoderita salina sp. n. display a unique sub-cuticular hexagonal beaded pattern. Male without stylet, pharyngeal region degenerated, S-E duct prominent, deirids small, developed testis 97.5 µm long, spicules 18.4 µm long, cloacal opening ventrally protruded, small phasmids posterior to cloaca opening and situated at 5.9 (3.2–7.7) µm from tail end, and conical tail ending in a rounded terminus marked with one (rarely two) ventrally positioned mucro. Additionally, some young malesof the new species were observed enveloped in the last J2 cuticle. Second-stage juvenile body 470 µm long, with a 16.4 µm long stylet, prominent rounded knobs set off from the shaft, hemizonid anterior and adjacent to S-E pore, small deirids located just above S-E pore level, genital primordium located at 68–77% of body length, phasmids small and located at about 19 µm from tail tip, and tail 38.7 µm long, tapering to finely pointed terminus with a finger-like projection. Phylogenetic analyses based on the nearly full length small subunit ribosomal DNA sequences of Meloidoderita salina sp. n. revealed a close relationship of the new species with Sphaeronema alni Turkina & Chizhov, 1986 and placed these two species sister to the rest of Criconematina.     

Telomere Dynamics in Human Cells Reprogrammed to Pluripotency

Background

Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell “rejuvenation.”

Methodology/Principal Findings

We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres.

Conclusions/Significance

While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.     

Seed characteristics and fatty acid composition of castor (<i>Ricinus communis</i> L.) varieties in Northeast China

Oil content and fatty acid composition were investigated on 12 castor varieties and strains by using the soxhlet extraction method and capillary gas chromatography. This was made to provide a reference and theoretical basis for castorbean breeding with high oil content, determine variability of seed compounds for breeding purposes, and broaden chemical material choices. Results revealed that crude fat percentage in seeds ranged from 18.91 to 35.84% with an average of 25.91%; the absolute content of ricinoleic acid varied between 171.65 g/kg and 314.03 g/kg with an average of 222.43 g/kg, and kernel crude fat percentage was between 24.28 and 46.97% with an average of 34.30%. All these study variables were highest in the 2129 strain. The percentage of ricinoleic acid in crude fat was between 83.85 to 87.62%, and the highest value was found in the zhebi4 accession. The other fatty acids appeared in small concentrations, and showed small amplitude: 1.12 to 1.61%, 1.21 to 1.61%, 3.53 to 4.80%, 5.35 to 6.38%, 0.52 to 0.79%, 0.05 to 0.08% and 0.43 to 0.55%, for palmitic, stearic, oleic, linolic, linolenic, arachidic, and arachidonic acids, respectively. One hundred seed weight was determined for each accession. One hundred seed weight ranged from 25.7 g to 34.0 g with an average of 29.9 g. There was a significant correlation between seed weight and oil content, but the correlation value was low (r=0.51). Cluster analysis by SSPS based on the content of fatty acid composition revealed that the accessions were divided into three independent clusters. These findings will clearly provide useful information for further research in breeding and utilization of castor oil.     

Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.