scholarly journals An Integrated Centrifugal Degassed PDMS-Based Microfluidic Device for Serial Dilution

Micromachines ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 482
Author(s):  
Anyang Wang ◽  
Samaneh Moghadasi Boroujeni ◽  
Philip J. Schneider ◽  
Liam B. Christie ◽  
Kyle A. Mancuso ◽  
...  
Keyword(s):  
Centrifugal Force ◽  
Microfluidic Device ◽  
Quantitative Polymerase Chain Reaction ◽  
Surface Condition ◽  
Hybrid Approach ◽  
Serial Dilution ◽  
Chamber Volume ◽  
Polymerase Chain ◽  
Good Agreement ◽  
Serial Dilutions

We propose an integrated serial dilution generator utilizing centrifugal force with a degassed polydimethylsiloxane (PDMS) microfluidic device. Using gas-soluble PDMS as a centrifugal microfluidic device material, the sample can be dragged in any arbitrary direction using vacuum-driven force, as opposed to in a single direction, without adding further actuation components. The vacuum-driven force allows the device to avoid the formation of air bubbles and exhibit high tolerance in the surface condition. The device was then used for sample metering and sample transferring. In addition, centrifugal force was used for sample loading and sample mixing. In this study, a series of ten-fold serial dilutions ranging from 100 to 10−4 with about 8 μL in each chamber was achieved, while the serial dilution ratio and chamber volume could easily be altered by changing the geometrical designs of the device. As a proof of concept of our hybrid approach with the centrifugal and vacuum-driven forces, ten-fold serial dilutions of a cDNA (complementary DNA) sample were prepared using the device. Then, the diluted samples were collected by fine needles and subject to a quantitative polymerase chain reaction (qPCR), and the results were found to be in good agreement with those for samples prepared by manual pipetting.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1759-1767 ◽  
Author(s):  
Kyu-Tae Kim ◽  
Kristin Baird ◽  
Joon-Young Ahn ◽  
Paul Meltzer ◽  
Michael Lilly ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Quantitative Polymerase Chain Reaction ◽  
Colony Growth ◽  
Dominant Negative ◽  
Internal Tandem Duplication ◽  
Downstream Target ◽  
Before And After ◽  
Flt3 Expression ◽  
Polymerase Chain ◽  
Expression Microarrays

AbstractConstitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling. (Blood. 2005;105: 1759-1767)

scholarly journals Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 352
Author(s):  
Wei Wei ◽  
Valeria Trivellone ◽  
Christopher H. Dietrich ◽  
Yan Zhao ◽  
Kristi D. Bottner-Parker ◽  
...  
Keyword(s):  
Host Range ◽  
Quantitative Polymerase Chain Reaction ◽  
Rflp Analysis ◽  
Natural Habitats ◽  
Genetic Lineages ◽  
Genetic Subgroup ◽  
Polymerase Chain ◽  
Phloem Feeding ◽  
Fresh Insight

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

scholarly journals Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

2021 ◽  
Author(s):  
Sara Keränen ◽  
Santeri Suutarinen ◽  
Rahul Mallick ◽  
Johanna P. Laakkonen ◽  
Diana Guo ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Rank Correlation ◽  
Inflammatory Cells ◽  
Vessel Remodeling ◽  
Brain Avm ◽  
Inflammatory Drugs ◽  
Polymerase Chain ◽  
The Brain ◽  
Cox2 Expression

Abstract Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

Gonadotrophins modulate cell death-related genes expression in human endometrium

2020 ◽  
Vol 41 (2) ◽  
Author(s):  
Sandro Sacchi ◽  
Paola Sena ◽  
Chiara Addabbo ◽  
Erika Cuttone ◽  
Antonio La Marca
Keyword(s):  
Cell Death ◽  
Quantitative Polymerase Chain Reaction ◽  
Hypoxia Inducible Factor ◽  
Endometrial Cells ◽  
Microtubule Associated Proteins ◽  
Genes Expression ◽  
Associated Proteins ◽  
Polymerase Chain ◽  
Cytochrome P450 Family ◽  
Apoptosis Marker

AbstractBackgroundGonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival.Materials and methodsCollected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed.ResultsGonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced.ConclusionsDifferent gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.

scholarly journals Estimating epidemiologic dynamics from cross-sectional viral load distributions

Science ◽  
2021 ◽  
pp. eabh0635
Author(s):  
James A. Hay ◽  
Lee Kennedy-Shaffer ◽  
Sanjat Kanjilal ◽  
Niall J. Lennon ◽  
Stacey B. Gabriel ◽  
...  
Keyword(s):  
Population Distribution ◽  
Quantitative Polymerase Chain Reaction ◽  
Cross Sectional ◽  
Outbreak Management ◽  
Viral Loads ◽  
Time Estimates ◽  
Load Distributions ◽  
Random Samples ◽  
Combining Data ◽  
Polymerase Chain

Estimating an epidemic’s trajectory is crucial for developing public health responses to infectious diseases, but case data used for such estimation are confounded by variable testing practices. We show that the population distribution of viral loads observed under random or symptom-based surveillance, in the form of cycle threshold (Ct) values obtained from reverse-transcription quantitative polymerase chain reaction testing, changes during an epidemic. Thus, Ct values from even limited numbers of random samples can provide improved estimates of an epidemic’s trajectory. Combining data from multiple such samples improves the precision and robustness of such estimation. We apply our methods to Ct values from surveillance conducted during the SARS-CoV-2 pandemic in a variety of settings and offer alternative approaches for real-time estimates of epidemic trajectories for outbreak management and response.

scholarly journals Type III Collagen is Required for Adipogenesis and Actin Stress Fibre Formation in 3T3-L1 Preadipocytes

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 156
Author(s):  
Mohammad Al Hasan ◽  
Patricia E. Martin ◽  
Xinhua Shu ◽  
Steven Patterson ◽  
Chris Bartholomew
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Type Iii Collagen ◽  
Type Iii ◽  
Actin Stress Fibre ◽  
Matrix Gene ◽  
Gene Transcripts ◽  
Polymerase Chain ◽  
Oil Red O Staining ◽  
Oil Red O

GPR56 is required for the adipogenesis of preadipocytes, and the role of one of its ligands, type III collagen (ColIII), was investigated here. ColIII expression was examined by reverse transcription quantitative polymerase chain reaction, immunoblotting and immunostaining, and its function investigated by knockdown and genome editing in 3T3-L1 cells. Adipogenesis was assessed by oil red O staining of neutral cell lipids and production of established marker and regulator proteins. siRNA-mediated knockdown significantly reduced Col3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. Col3a1−/− 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ2 (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). Col3a1−/− 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin (Fn) and collagen (Col4a1, Col6a1) extracellular matrix gene transcripts. Col3a1−/− 3T3-L1 cells also had dramatically reduced actin stress fibres. We conclude that ColIII is required for 3T3-L1 preadipocyte adipogenesis as well as the formation of actin stress fibres. The phenotype of Col3a1−/− 3T3-L1 cells is very similar to that of Gpr56−/− 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.

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