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31.
Mathis Hjort Hjelms? Lars Hestbjerg Hansen Jacob B?lum Louise Feld William E. Holben Carsten Suhr Jacobsen 《Applied and environmental microbiology》2014,80(12):3568-3575
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. 相似文献
32.
Frank Suhr Julian Brenig Rebecca Müller Hilke Behrens Wilhelm Bloch Marijke Grau 《PloS one》2012,7(9)
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. 相似文献33.
René Günther Martin Suhr Jan C. Koch Mathias B?hr Paul Lingor Lars T?nges 《Journal of visualized experiments : JoVE》2012,(61)
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. 相似文献
34.
Steven T. Suhr Eun Ah Chang Ramon M. Rodriguez Kai Wang Pablo J. Ross Zeki Beyhan Shashanka Murthy Jose B. Cibelli 《PloS one》2009,4(12)
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. 相似文献35.
Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples 下载免费PDF全文
Line Fredslund Flemming Ekelund Carsten Suhr Jacobsen Kaare Johnsen 《Applied microbiology》2001,67(4):1613-1618
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. 相似文献
36.
Purpose
Transthyretin V30M (ATTR V30M) amyloidosis is a phenotypically diverse disease with symptoms ranging from predominant neuropathy to exclusive cardiac manifestations. The aims of this study were to determine the dispersion of the two types of fibrils found in Swedish ATTR V30M patients -Type A consisting of a mixture of truncated and full length ATTR fibrils and type B fibrils consisting of full length fibrils, and to estimate the severity of cardiac dysfunction in relation to fibril composition and sex.Material and Methods
Echocardiographic data were analysed in 107 Swedish ATTR V30M patients with their fibril composition determined as either type A or type B. Measurements of left ventricular (LV) dimensions and evaluation of systolic and diastolic function including speckle tracking derived strain were performed. Patients were grouped according to fibril type and sex. Multivariate linear regression was utilised to determine factors of significant impact on LV thickness.Results
There was no significant difference in proportions of the two types of fibrils between men and women. In patients with type A fibrils, women had significantly lower median septal (p = 0.007) and posterior wall thicknesses (p = 0.010), lower median LV mass indexed to height (p = 0.008), and higher septal strain (p = 0.037), as compared to males. These differences were not apparent in patients with type B fibrils. Multiple linear regression analysis revealed that fibril type, sex and age all had significant impact on LV septal thickness.Conclusion
This study demonstrates a clear difference between sexes in the severity of amyloid heart disease in ATTR V30M amyloidosis patients. Even though type A fibrils were associated with more advanced amyloid heart disease compared to type B, women with type A fibrils generally developed less cardiac infiltration than men. The differences may explain the better outcome for liver transplanted late-onset female patients compared to males. 相似文献37.
Parkin accumulation in aggresomes due to proteasome impairment 总被引:16,自引:0,他引:16
Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and by the presence of ubiquitinated cytoplasmic inclusions known as Lewy bodies. Alpha-synuclein and Parkin are two of the proteins associated with inherited forms of PD and are found in Lewy bodies. Whereas numerous reports indicate the tendency of alpha-synuclein to aggregate both in vitro and in vivo, no information is available about similar physical properties for Parkin. Here we show that overexpression of Parkin in the presence of proteasome inhibitors leads to the formation of aggresome-like perinuclear inclusions. These eosinophilic inclusions share many characteristics with Lewy bodies, including a core and halo organization, immunoreactivity to ubiquitin, alpha-synuclein, synphilin-1, Parkin, molecular chaperones, and proteasome subunit as well as staining of some with thioflavin S. We propose that the process of Lewy body formation may be akin to that of aggresome-like structures. The tendency of wild-type Parkin to aggregate and form inclusions may have implications for the pathogenesis of sporadic PD. 相似文献
38.
Sato T Ando Y Susuki S Mikami F Ikemizu S Nakamura M Suhr O Anraku M Kai T Suico MA Shuto T Mizuguchi M Yamagata Y Kai H 《FEBS letters》2006,580(2):491-496
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. 相似文献
39.
Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. 总被引:30,自引:43,他引:30 下载免费PDF全文
A D Miller J V Garcia N von Suhr C M Lynch C Wilson M V Eiden 《Journal of virology》1991,65(5):2220-2224
We have constructed hybrid retrovirus packaging cell lines that express the gibbon ape leukemia virus env and the Moloney murine leukemia virus gag-pol proteins. These cells were used to produce a retrovirus vector at over 10(6) CFU/ml, with a host range that included rat, hamster, bovine, cat, dog, monkey, and human cells. The gag-pol and env expression plasmids were separately transfected to reduce the potential for helper virus production, which was not observed. The NIH 3T3 mouse cells from which the packaging lines were made are not infectable by gibbon ape leukemia virus; thus, the generation and spread of possible recombinant viruses in the packaging cells is greatly reduced. These simian virus-based packaging cells extend the host range of currently available murine and avian packaging cells and should be useful for efficient gene transfer into higher mammals. 相似文献
40.