scholarly journals Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Dipak K. Dube ◽  
Syamalima Dube ◽  
Lynn Abbott ◽  
Ruham Alshiekh-Nasany ◽  
Charles Mitschow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Human Heart ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Adult Heart ◽  
Qrt Pcr ◽  
Long Time ◽  
Alternate Promoters ◽  
Computational Analyses

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

scholarly journals Effects of experimental periodontitis on proliferation and osteogenic differentiation of adipose-derived stem cells in rats

2019 ◽  
Author(s):  
Yan Li Huang ◽  
Le Da Cheng ◽  
Ya Jie Fan ◽  
Yi Wang ◽  
Baoyu Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Male Rats ◽  
Cell Counting ◽  
Standard Diet ◽  
Adipose Derived Stem Cells ◽  
Rt Pcr ◽  
Qrt Pcr

Abstract Background and Objective: The application of ASCs in periodontal regeneration is a good choice. Inflammatory micro-environment influenced the proliferation, mobilization, and osteogenic differentiation of ASCs in vitro.The aim of this study was to evaluate the effects of experiment periodontitis on the proliferation, wound healing and osteogenesis markers of adipose-derived stem cells (ASCs) in rats. Materials and methods: Ten male rats were divided into two groups randomly. The control (Con) group received a standard diet, and the periodontitis (Peri) group was received a standard diet with placing ligatures around the maxillary first molar. Toll like receptor 4 (TLR4), Tumor necrosis factor-α (TNF-a) and Interleukin-1β (IL-1β) were tested by immunohistochemistry (IHC) staining and quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation rate of ASCs was measured through Cell Counting Kit-8 (CCK-8) assay. The migration speed of stem cells was evaluated by using a wound healing assay. The expression of alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2) and runt related transcription factor 2 (Runx2) was evaluated by qRT-PCR analysis and western blot. Graph Pad Primer 7.0 software was used for statistical analysis.Results: After 4 weeks, periodontitis model was successfully constructed. The results of IHC and RT-PCR found that in the Peri group, the TNF-a and IL-1β levels of adipose tissues decreased compared with the Con group (P<0.05). The proliferation of Peri-ASCs significantly increased compared with Con-ASCs. Moreover, the wound healing ability of Peri-ASCs gradually increased in a time dependent manner compared with Con-ASCs. Results of RT-PCR showed that ALP and BMP2 gene levels of Peri-ASCs significantly decreased (P<0.05), while the Runx2 gene level in Peri-ASCs was increased, when compared to Con-ASCs. The ALP activity of Peri-ASCs was decreased compared to the Con-ASCs, especially the difference was significant at day 5 day (P<0.01). Western blot results showed that ALP, Runx2 and BMP2 protein levels of Peri-ASCs were significantly lower than those in Con-ASCs after osteogenic induction. Conclusion: Our study demonstrated that experiment periodontitis decreased the expression of TNF-a and IL-1β in adipose tissue in rats. Experiment periodontitis promoted the proliferation and wound-healing ability of ASCs, but obviously inhibited the osteogenic differentiation of ASCs.

scholarly journals Expression of TPM1κ, a Novel Sarcomeric Isoform of the TPM1 Gene, in Mouse Heart and Skeletal Muscle

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Syamalima Dube ◽  
Lauren Panebianco ◽  
Amr A. Matoq ◽  
Henry N. Chionuma ◽  
Christopher R. Denz ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Copy Number ◽  
Amino Acid Sequences ◽  
Expression Level ◽  
Mouse Heart ◽  
Mammalian Skeletal Muscle ◽  
Striated Muscles ◽  
Rt Pcr ◽  
Qrt Pcr

We have investigated the expression of TPM1α and TPM1κ in mouse striated muscles. TPM1α and TMP1κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1κ. However, the deduced amino acid sequences of human TPM1α and mouse TPM1α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1κ is significantly lower compared to TPM1α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1κ in mammalian skeletal muscle.

scholarly journals The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry

2012 ◽  
Vol 48 (2) ◽  
pp. 139-149 ◽  
Author(s):  
Karen Oliva ◽  
Gillian Barker ◽  
Clyde Riley ◽  
Mark J Bailey ◽  
Michael Permezel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Gel Electrophoresis ◽  
Soluble Protein ◽  
Obese Patients ◽  
Two Dimensional ◽  
2D Dige ◽  
Qrt Pcr ◽  
Difference Gel Electrophoresis ◽  
Dige Analysis

Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4–5 and 5–6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

Identification and characterization of Boone Cardiovirus, a novel virus of laboratory rats

2012 ◽  
Author(s):  
◽  
Judith Diane Gohndrone
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Western Blot ◽  
Amino Acid Identity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Acid Identity ◽  
Laboratory Rats ◽  
Persistent Infections ◽  
Qrt Pcr

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cardioviruses are members of the Picornaviridae family capable of causing several forms of disease including myocarditis, encephalitis, diabetes, fetal death, and neuron degeneration. A wide range of hosts are susceptible to cardioviruses with rodents frequently being suspected as the viral reservoir following outbreaks of infection. In this study we discovered a novel Cardiovirus, provisionally designated Boone Cardiovirus (BCV). BCV was identified in the feces of laboratory rats using a pan picornavirus-PCR assay. Phylogenetic analysis revealed that BCV is a new species within the Cardiovirus genus distinct from both Encephalomyelitis Virus (EMCV) and Theilovirus. With these two species, BCV shares [less-than]45% amino acid identity in the polyprotein region and [less-than]50% amino acid identity in the capsid proteins. To assess the prevalence of BCV in laboratory rodents a sensitive hemi-nested RT-PCR assay was developed to screen fecal samples. Screening revealed that 20% of rat samples and 30% of research institutions tested positive for BCV. During the screening process a second isolate of BCV was also identified. Phylogenetic analysis of the second isolate determined it shared 91% amino acid identity with the original strain in the polyprotein region. Identification of additional BCV strains aids in the understanding of BCV variability and provides additional information for the development of comprehensive PCR and serologic screening assays. As no definitive clinical disease has been observed with BCV, a quantitative RT-PCR (qRT-PCR) assay was developed to screen rat tissues for sites of replication. BCV was found predominately localized to the gastrointestinal tract with the highest titers in the duodenum. Screening animals of various ages also revealed that BCV causes persistent infections in laboratory rats. In addition, to the RT-PCR and qRT-PCR assays the VP2 protein was expressed and purified for use in Western blot analysis of rat serum for preliminary serologic data on BCV. Collectively, the RT-PCR and Western blot assays provide a foundation for BCV detection and will enable researchers to screen animals prior to experiments. These assays will also make it possible to establish BCV-free rat colonies as virally infected research animals can confound and invalidate research findings. Preliminary data from infections of nude rats suggest that BCV is capable of replication and the immune system of immunocompetent animals plays a role in modulating infections as once T-cell are eliminated viral titers are approximately 4 logs higher. Furthermore, the discovery of BCV may lead to the establishment of research models that can provide valuable information including host-viral interactions during persistent infections.

Resolution of a protein sequence ambiguity by X-ray crystallographic and mass spectrometric methods

1992 ◽  
Vol 25 (2) ◽  
pp. 205-210 ◽  
Author(s):  
L. J. Keefe ◽  
E. E. Lattman ◽  
C. Wolkow ◽  
A. Woods ◽  
M. Chevrier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Electron Density ◽  
Mass Spectrometric ◽  
Amino Acid Sequences ◽  
Data Matrix ◽  
Protein Crystal ◽  
Insertion Mutant ◽  
Laser Desorption Mass Spectrometry ◽  
X Ray

Ambiguities in amino acid sequences are a potential problem in X-ray crystallographic studies of proteins. Amino acid side chains often cannot be reliably identified from the electron density. Many protein crystal structures that are now being solved are simple variants of a known wild-type structure. Thus, cloning artifacts or other untoward events can readily lead to cases in which the proposed sequence is not correct. An example is presented showing that mass spectrometry provides an excellent tool for analyzing suspected errors. The X-ray crystal structure of an insertion mutant of Staphylococcal nuclease has been solved to 1.67 Å resolution and refined to a crystallographic R value of 0.170 [Keefe & Lattman (1992). In preparation]. A single residue has been inserted in the C-terminal α helix. The inserted amino acid was believed to be an alanine residue, but the final electron density maps strongly indicated that a glycine had been inserted instead. To confirm the observations from the X-ray data, matrix-assisted laser desorption mass spectrometry was employed to verify the glycine insertion. This mass spectrometric technique has sufficient mass accuracy to detect the methyl group that distinguishes glycine from alanine and can be extended to the more common situation in which crystallographic measurements suggest a problem with the sequence, but cannot pinpoint its location or nature.

scholarly journals The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics

2021 ◽  
pp. 1-8
Author(s):  
Tiange Wu ◽  
Xiaoning Wang ◽  
Kai Ren ◽  
Xiaochen Huang ◽  
Jiankai Liu
Keyword(s):  
Mass Spectrometry ◽  
Methylene Blue ◽  
Western Blot ◽  
Blue Light ◽  
Differentially Expressed ◽  
Enzyme Linked Immunosorbent Assay ◽  
Differentially Expressed Protein ◽  
Significant Difference ◽  
Modified Proteins ◽  
Frozen Plasma

Introduction: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. Methods: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). Results: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). Conclusion: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

scholarly journals Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 730
Author(s):  
Magda Rybicka ◽  
Ewa Miłosz ◽  
Krzysztof Piotr Bielawski
Keyword(s):  
Mass Spectrometry ◽  
Gold Standard ◽  
Viral Genome ◽  
False Negative ◽  
Rt Pcr ◽  
Rna Detection ◽  
Maldi Tof ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Test

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.

scholarly journals Knockdown of lncRNA X inactive specific transcript (XIST) radiosensitizes non-small cell lung cancer (NSCLC) cells through regulation of miR-16-5p/WEE1 G2 checkpoint kinase (WEE1) axis

2021 ◽  
Vol 35 ◽  
pp. 205873842096608
Author(s):  
Ran Du ◽  
Feng Jiang ◽  
Yanhua Yin ◽  
Jinfen Xu ◽  
Xia Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Western Blot ◽  
Small Cell ◽  
Luciferase Reporter ◽  
Small Cell Lung ◽  
Luciferase Reporter Gene ◽  
G2 Checkpoint ◽  
Qrt Pcr ◽  
Specific Transcript

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) is reported to play an oncogenic role in non-small cell lung cancer (NSCLC). However, the role of XIST in regulating the radiosensitivity of NSCLC cells remains unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expressions of XIST and miR-16-5p in NSCLC in tissues and cells, and Western blot was used to assess the expression of WEE1 G2 checkpoint kinase (WEE1). Cell counting kit-8 (CCK-8), colony formation and flow cytometry assays were used to determine cell viability and apoptosis after NSCLC cells were exposed to different doses of X-rays. The interaction between XIST and miR-16-5p was confirmed by StarBase database, qRT-PCR and dual-luciferase reporter gene assays. TargetScan database was used to predict WEE1 as a target of miR-16-5p, and their targeting relationship was further validated by Western blot, qRT-PCR and dual-luciferase reporter gene assays. XIST was highly expressed in both NSCLC tissue and cell lines, and knockdown of XIST repressed NSCLC cell viability and cell survival, and facilitated apoptosis under the irradiation. MiR-16-5p was a target of XIST, and rescue experiments demonstrated that miR-16-5p inhibitors could reverse the role of XIST knockdown on radiosensitivity in NSCLC cells. WEE1 was validated as a target gene of miR-16-5p, and WEE1 could be negatively regulated by XIST. XIST promotes the radioresistance of NSCLC cells by regulating the expressions of miR-16-5p and WEE1, which can be a novel target for NSCLC therapy.

scholarly journals Overview of Safety Measures at Selected Airports during the COVID-19 Pandemic

2021 ◽  
Vol 13 (15) ◽  
pp. 8499
Author(s):  
Monika Blišťanová ◽  
Michaela Tirpáková ◽  
Ľubomíra Brůnová
Keyword(s):  
Time Frame ◽  
Organizational Changes ◽  
Rt Pcr ◽  
Final Phase ◽  
Chain Reaction ◽  
Scoring Method ◽  
Safety Measures ◽  
Long Time ◽  
Polymerase Chain ◽  
Cleaning And Disinfection

The year 2020 was very challenging for the whole world, given the outbreak of the ongoing coronavirus-related pandemic, and was marked in particular by overcoming new hitherto unknown obstacles. For air transport, in particular, airlines stopped flying altogether and were forced to ground hundreds of planes worldwide involuntarily. Airports had to close their terminals for a long time, wholly suspend operations, and its resumption required significant organizational changes. This article summarizes the measures related to the COVID-19 pandemic adopted by airports to minimize the risk of spreading the disease. The article focuses on countermeasures and their implementation at selected airports in a specific time frame and airports’ behavior during a pandemic which varies depending on country and time of the year. The results demonstrated that steps being taken at airports include the use of face coverings or masks, social distance, enhanced cleaning and disinfection, or temperature checks and/or symptoms (fever, loss of smell, chills, cough, shortness of breath), RT-PCR (reverse transcription-polymerase chain reaction) screening and data collection with health declaration. These measures have now become an essential standard for the operation of airports and can, therefore, be used to assess the level of airport safety achieved. In the final phase, the article evaluates the level of achieved airport safety based on the proposed scoring method.

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