scholarly journals Variability of human fasted venous plasma metabolomic profiles with tourniquet induced hemostasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sarita Devi ◽  
Roshni M. Pasanna ◽  
Nikhil Nadiger ◽  
Santu Ghosh ◽  
Anura V. Kurpad ◽  
...  
Keyword(s):  
Venous Blood ◽  
Interindividual Variation ◽  
Intra Class Correlation ◽  
Highly Sensitive ◽  
Healthy Female ◽  
Health And Disease ◽  
Plasma Metabolite ◽  
Extreme Care ◽  
Venous Plasma

AbstractVenous plasma metabolomics is a potent and highly sensitive tool for identifying and measuring metabolites of interest in human health and disease. Accurate and reproducible insights from such metabolomic studies require extreme care in removing preanalytical confounders; one of these is the duration of tourniquet application when drawing the venous blood sample. Using an untargeted plasma metabolomics approach, we evaluated the effect of varying durations of tourniquet application on the variability in plasma metabolite concentrations in five healthy female subjects. Tourniquet application introduced appreciable variation in the metabolite abundances: 73% of the identified metabolites had higher temporal variation compared to interindividual variation [Intra-Class Correlation (ICC) > 0.50]. As such, we recommend tourniquet application for minimal duration and to wait for 5 min with the needle in situ after removing the tourniquet, to reduce hemostasis-induced variability and false flags in interpretation.

scholarly journals Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

2019 ◽  
Author(s):  
J.C.W. Hildyard ◽  
F. Rawson ◽  
D.J. Wells ◽  
R.J. Piercy
Keyword(s):  
Transcript Level ◽  
Vital Role ◽  
Dystrophic Muscle ◽  
Transcriptional Dynamics ◽  
Novel Approach ◽  
Highly Sensitive ◽  
Health And Disease ◽  
Single Transcript ◽  
Long Genes

AbstractDystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcripts at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We present a novel approach, instead using RNAscope probes targeted to 5’ and 3’ regions of the same transcript: labelling muscle dystrophin mRNA in this manner allows transcriptional dynamics to be deciphered in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs (the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggests this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

Ultrastructure of rat alveolar macrophages activated in situ by poly:IC

1987 ◽  
Vol 45 ◽  
pp. 768-769
Author(s):  
A. M. Klinkner ◽  
R. A. Weiss ◽  
A. Kelley ◽  
P. J. Bugelski
Keyword(s):  
Venous Blood ◽  
Intratracheal Instillation ◽  
Buffy Coat ◽  
Fischer 344 Rats ◽  
Blood Monocytes ◽  
Fischer 344 ◽  
Polyinosinic:Polycytidylic Acid ◽  
Macrophage Activator ◽  
Endogenous Peroxidase

Polyinosinic:polycytidylic acid is an inducer of interferon and a macrophage activator. We have found that intratracheal instillation of polyI:C (IT-pI:C) activates rat bronchoalveolar lavage cells (BAL) for a variety of functions. Examination of Giemsa stained, cytocentrifuge preparations showed that IT-pI:C induced a population of BAL not seen in resident BAL. The morphology of these cells suggested that they might be derived from blood monocytes. To test this hypothesis we have examined several populations of macrophages that had been stained for endogenous peroxidase activity as a marker of cells derived from the monocyte-macrophage lineage.Macrophages were obtained from Fischer 344 rats. Peritoneal exudate cells (PEC) were collected by lavage 4 days after i.p. injection of 20 ml 3% thioglycolate. Buffy coat monocytes were separated from venous blood from naive rats.

scholarly journals Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lenka Ulrychová ◽  
Pavel Ostašov ◽  
Marta Chanová ◽  
Michael Mareš ◽  
Martin Horn ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Rna Sequencing ◽  
Serine Proteases ◽  
Expression Patterns ◽  
High Sensitivity ◽  
Host Parasite Interactions ◽  
Highly Sensitive ◽  
Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

scholarly journals Distribution of Potato spindle tuber viroid in Reproductive Organs of Petunia During Its Developmental Stages

2014 ◽  
Vol 104 (9) ◽  
pp. 964-969 ◽  
Author(s):  
Yosuke Matsushita ◽  
Shinya Tsuda
Keyword(s):  
Developmental Stages ◽  
Cell Movement ◽  
Seed Transmission ◽  
Potato Spindle Tuber Viroid ◽  
Reproductive Organs ◽  
Infected Male ◽  
Infected Female ◽  
Reproductive Tissues ◽  
Healthy Female

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

A highly sensitive chlorine gas sensor and enhanced thermal DC electrical conductivity from polypyrrole/silicon carbide nanocomposites

RSC Advances ◽  
2016 ◽  
Vol 6 (87) ◽  
pp. 84200-84208 ◽  
Author(s):  
Adil Sultan ◽  
Sharique Ahmad ◽  
Faiz Mohammad
Keyword(s):  
Electrical Conductivity ◽  
Silicon Carbide ◽  
Gas Sensor ◽  
Dodecylbenzenesulfonic Acid ◽  
Chemical Polymerization ◽  
Dc Electrical Conductivity ◽  
Highly Sensitive ◽  
Chlorine Gas

We report the synthesis of polypyrrole (PPy) and polypyrrole/silicon carbide nanocomposites (PPy/SiC) and PPy/SiC/dodecylbenzenesulfonic acid (DBSA) by in situ chemical polymerization and their application as sensors for the detection of highly toxic chlorine gas.

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