Can We Reproduce the Latch-State in Vitro at the Molecular Level?

Human adipose tissue-derived stem cells (hADSCs) are highly suitable for regeneration therapies being easily collected and propagated in vitro. The effects of different external factors and culturing conditions are able to affect hADSC proliferation, senescence, differentiation, and migration, even at the molecular level. In the present paper, we exposed hADSCs to an exhausted medium from the breast cancer cell line (MCF-7) to evaluate whether the soluble factors released by these cells may be able to induce changes in stem cell behavior. In particular, we investigated the expression of stemness-related genes (OCT4; Sox 2; Nanog), the cell-cycle regulators p21 (WAF1/CIP1) p53, epigenetic markers (DNMT1 and Sirt1), and autophagy-related proteins. From our results, we can infer that the exhausted medium from MCF-7 is able to influence the hADSCs behavior increasing the expression of stemness-related genes, cell proliferation, and autophagy. Polyamines detectable in MCF-7 exhausted medium could be related to the higher proliferation capability observed in hADSCs, suggesting direct crosstalk between these molecules and the observed changes in stem cell potency.

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Desethylamiodarone antagonizes the effect of thyroid hormone at the molecular level

Acta Endocrinologica ◽

10.1530/eje.0.1450059 ◽

2001 ◽

pp. 59-64 ◽

Cited By ~ 18

Author(s):

F Bogazzi ◽

L Bartalena ◽

S Brogioni ◽

A Burelli ◽

F Raggi ◽

...

Keyword(s):

Thyroid Hormone ◽

Molecular Mechanisms ◽

Thyroid Hormone Receptor ◽

Molecular Level ◽

Inhibitory Effect ◽

Mobility Shift ◽

Down Regulation ◽

Nih3t3 Cells ◽

Peripheral Tissues

OBJECTIVE: To evaluate the molecular mechanisms of the inhibitory effects of amiodarone and its active metabolite, desethylamiodarone (DEA) on thyroid hormone action. MATERIALS AND METHODS: The reporter construct ME-TRE-TK-CAT or TSHbeta-TRE-TK-CAT, containing the nucleotide sequence of the thyroid hormone response element (TRE) of either malic enzyme (ME) or TSHbeta genes, thymidine kinase (TK) and chloramphenicol acetyltransferase (CAT) was transiently transfected with RSV-TRbeta into NIH3T3 cells. Gel mobility shift assay (EMSA) was performed using labelled synthetic oligonucleotides containing the ME-TRE and in vitro translated thyroid hormone receptor (TR)beta. RESULTS: Addition of 1 micromol/l T4 or T3 to the culture medium increased the basal level of ME-TRE-TK-CAT by 4.5- and 12.5-fold respectively. Amiodarone or DEA (1 micromol/l) increased CAT activity by 1.4- and 3.4-fold respectively. Combination of DEA with T4 or T3 increased CAT activity by 9.4- and 18.9-fold respectively. These data suggested that DEA, but not amiodarone, had a synergistic effect with thyroid hormone on ME-TRE, rather than the postulated inhibitory action; we supposed that this was due to overexpression of the transfected TR into the cells. When the amount of RSV-TRbeta was reduced until it was present in a limited amount, allowing competition between thyroid hormone and the drug, addition of 1 micromol/l DEA decreased the T3-dependent expression of the reporter gene by 50%. The inhibitory effect of DEA was partially due to a reduced binding of TR to ME-TRE, as assessed by EMSA. DEA activated the TR-dependent down-regulation by the negative TSH-TRE, although at low level (35% of the down-regulation produced by T3), whereas amiodarone was ineffective. Addition of 1 micromol/l DEA to T3-containing medium reduced the T3-TR-mediated down-regulation of TSH-TRE to 55%. CONCLUSIONS: Our results demonstrate that DEA, but not amiodarone, exerts a direct, although weak, effect on genes that are regulated by thyroid hormone. High concentrations of DEA antagonize the action of T3 at the molecular level, interacting with TR and reducing its binding to TREs. This effect may contribute to the hypothyroid-like effect observed in peripheral tissues of patients receiving amiodarone treatment.

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Establishment of Highly Efficient Agrobacterium Rhizogenes-mediated Transformation for Stevia Rebaudiana Bertoni Explants

Acta Biologica Cracoviensia s Botanica ◽

10.1515/abcsb-2016-0003 ◽

2016 ◽

Vol 58 (1) ◽

pp. 113-118 ◽

Cited By ~ 5

Author(s):

Żaneta Michalec-Warzecha ◽

Laura Pistelli ◽

Francesca D’Angiolillo ◽

Marta Libik-Konieczny

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Roots ◽

Molecular Level ◽

Stevia Rebaudiana ◽

Culture Conditions ◽

Light Conditions ◽

Inoculum Concentration ◽

Stevia Rebaudiana Bertoni ◽

Pcr Method

Abstract Leaves and internodes from Stevia rebaudiana Bertoni plants growing in different conditions were used for transformation with two strains of Agrobacterium rhizogenes: ATCC 15384 and LBA 9402. Hairy roots formation was observed and the percentage of the transformed explants depended on the type of explant, time of inoculation and inoculum concentration. Inoculation of explants from ex vitro and in vitro plants with LBA 9402 strain led to higher efficiency of transformation than inoculation with ATCC 15384 strain. Growth rate of hairy roots in liquid culture was assessed under light and dark conditions. It was found that the growth of hairy roots decreased significantly under light conditions. Transformation of hairy roots growing in different culture conditions was confirmed at the molecular level using PCR method with primers constructed against rolB and rolC genes from A. rhizogenes.

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Subinhibitory Antimicrobial Concentrations: A Review of In Vitro and In Vivo Data

Canadian Journal of Infectious Diseases ◽

10.1155/1992/793607 ◽

1992 ◽

Vol 3 (4) ◽

pp. 193-201 ◽

Cited By ~ 14

Author(s):

George G Zhanel ◽

Daryl J Hoban ◽

Godfrey KM Harding

Keyword(s):

Antimicrobial Activity ◽

Animal Studies ◽

Molecular Level ◽

Bacterial Virulence ◽

Antibacterial Effects ◽

Wide Range ◽

Immune Defences

Antimicrobial activity is not an ‘all or none’ effect. An increase in the rate and extent of antimicrobial action is usually observed over a wide range of antimicrobial concentrations. Subinhibitory antimicrobial concentrations are well known to produce significant antibacterial effects, and various antimicrobials at subinhibitory concentrations have been reported to inhibit the rate of bacterial growth. Bacterial virulence may be increased or decreased by subinhibitory antimicrobial concentrations by changes in the ability of bacteria to adhere to epithelial cells or by alterations in bacterial susceptibility to host immune defences. Animal studies performed in rats, hamsters and rabbits demonstrate decreased bacterial adherence, reduced infectivity and increased survival of animals treated with subinhibitory antimicrobial concentrations compared to untreated controls. The major future role of investigation of subinhibitory antimicrobial concentrations will be to define more fully, at a molecular level, how antimicrobials exert their antibacterial effects.

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Age-associated features of the expression level of apoptosis markers in cardiomyocytes of patients with dilated cardiomyopathy

Успехи геронтологии ◽

10.34922/ae.2021.34.6.010 ◽

2022 ◽

pp. 885-890

Author(s):

К.П. Кравченко ◽

К. Л. Козлов ◽

А.О. Дробинцева ◽

Д.С. Медведев ◽

В.О. Полякова

Keyword(s):

Cell Death ◽

Dilated Cardiomyopathy ◽

Molecular Mechanisms ◽

Molecular Level ◽

Confocal Laser ◽

Control Group ◽

Cellular Mechanisms ◽

Apoptosis Pathway ◽

Bax Protein

Для понимания патогенеза дилатационной кардиомиопатии (ДКМП) необходимо установить молекулярно-клеточные механизмы старения миокарда, в том числе связанные с программируемой клеточной гибелью, молекулярные механизмы которого практически не изучены. Цель работы - изучение маркеров апоптоза в кардиомиоцитах у пациентов с ДКМП in vitro. В работе использовали метод первичных диссоциированных клеточных культур и метод иммунофлюоресцентной конфокальной лазерной микроскопии. Для моделирования клеточного старения использовали клетки 3-го и 14-го пассажей, соответствующие «молодым» и «старым» культурам. На молекулярном уровне старение клеток кардиомиоцитов сопровождалось повышением экспрессии р16 в 2 раза по сравнению с «молодыми культурами» как в контрольной, так и в группе с ДКМП. Также установлено, что экспрессия р16 в культурах, взятых от пациентов с патологией, была в 2 раза выше, чем в аналогичных культурах от здоровых пациентов. Экспрессия р21 была повышена в группе с ДКМП по сравнению с контрольной группой, однако при старении культуры экспрессия p21 не изменялась, оставаясь на высоком уровне. Наиболее значимые различия были получены при сравнении экспрессии Bax в культуре клеток кардиомиоцитов из группы с ДКМП в «молодой» культуре с нормой - в 3,2 раза. Старение клеток миокарда на молекулярном уровне проявлялось в повышении экспрессии белка Baх, именно он является запускающим механизмом митохондриального пути апоптоза. Возможно, этот путь клеточной гибели является превалирующем при ДКМП. To understand the pathogenesis of dilated cardiomyopathy (DCMP), it is necessary to establish the molecular-cellular mechanisms of myocardial aging, including those associated with programmed cell death, the molecular mechanisms of which have not been practically studied. The aim of this work is to study markers of apoptosis in cardiomyocytes of patients with DCMP in vitro. We used the method of primary dissociated cell cultures and the method of immunofluorescence confocal laser microscopy. Cells of the 3 and 14 passages, corresponding to «young» and «old» cultures, were used to simulate cellular senescence. Results. At the molecular level, aging of cardiomyocyte cells was accompanied by a twofold increase in the expression of p16 compared to «young cultures» both in the control group and in the group with DCMP. It was also found that the expression of p16 in cultures taken from patients with pathology was 2 times higher than in similar cultures from healthy patients. The expression of p21 was increased in the group with DCMP compared to the control; however, with aging of the culture, the expression of p21 did not change, remaining at a significant level. The most significant differences were obtained when comparing the expression of Bax in the cell culture of cardiomyocytes from the group with DCMP in a «young» culture compared with the norm, 3,2 times. Aging of myocardial cells at the molecular level was manifested in an increase in the expression of the Bax protein, which is the triggering mechanism of the mitochondrial apoptosis pathway. It is possible that this pathway of cell death is prevalent in DCMP.

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Abstract 211: Myocardial Hypertrophy and Circulating RNAs

Circulation Research ◽

10.1161/res.121.suppl_1.211 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Ravi V Shah ◽

Olivia Ziegler ◽

Kahraman Tanriverdi ◽

Jian Rong ◽

Martin Larson ◽

...

Keyword(s):

Biological Process ◽

Molecular Level ◽

Myocardial Hypertrophy ◽

Clinical Phenotype ◽

Left Ventricular ◽

Loss Of Function ◽

Heart Study ◽

Non Coding Rnas

While increased left ventricular mass (LVM) is strongly associated with incident heart failure (HF), events during transition from increased LVM to HF remain unclear. Extracellular non-coding RNAs (ex-RNAs) have been implicated in cardiac hypertrophy, though whether these ex-RNAs reflect important pathways in HF in humans is underexplored. In >2,000 individuals with concomitant M-mode echocardiography and ex-RNA measurements in the Framingham Heart Study, we found that lower circulating concentrations of three ex-RNAs—miR-20a-5p, miR-106b-5p, miR-17-5p—were associated with (1) greater LVM (+ one other pre-clinical phenotype, e.g., left atrial dimension or LVEDV) and (2) greater incident HF risk over a median follow-up 7.7 years ( Fig. A ). These 3 miRNAs were members of a tight cluster, regulating 883 mRNAs in common, associated with “hypertension” (OMIM) and biological process relevant to HF, including TGF-β signaling. We observed an increase in myocardial expression of these miRNAs during different phases of hypertrophy/HF development ( Fig. C, D ). Using gain and loss of function in vitro , our preliminary results suggest up-regulation of cardiomyocyte miR-106b expression abrogates expression of pathologic hypertrophy markers (ANP and BNP) during phenylephrine treatment, consistent with in silico results suggesting broad connections between miR-106b targets and natriuretic peptide signaling ( Fig. B, E-F ). These results provide translational evidence that circulating miRNAs associated with hypertrophy in patients may be protective in the transition from hypertrophy to HF at the molecular level.

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Human Na+-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.5.c1215 ◽

1998 ◽

Vol 274 (5) ◽

pp. C1215-C1225 ◽

Cited By ~ 12

Author(s):

Francesca Porcellati ◽

Tommy Hlaing ◽

Masaki Togawa ◽

Martin J. Stevens ◽

Dennis D. Larkin ◽

...

Keyword(s):

Molecular Level ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Transcriptional Unit ◽

Cdna Sequencing ◽

Alternate Splicing ◽

Reading Frame ◽

Hypertonic Stress ◽

Exon Splicing

Na+- myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na+- myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na+-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and ∼1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.

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Decellularized pericardium tissues at increasing glucose, galactose and ribose concentrations and at different time points studied using scanning X-ray microscopy

IUCrJ ◽

10.1107/s2052252521005054 ◽

2021 ◽

Vol 8 (4) ◽

Author(s):

Cinzia Giannini ◽

Liberato De Caro ◽

Alberta Terzi ◽

Luca Fusaro ◽

Davide Altamura ◽

...

Keyword(s):

Molecular Level ◽

Wide Angle ◽

Collagen Structure ◽

Tissue Samples ◽

X Ray ◽

X Ray Scattering ◽

Aging Populations ◽

Secondary Diseases

Diseases like widespread diabetes or rare galactosemia may lead to high sugar concentrations in the human body, thereby promoting the formation of glycoconjugates. Glycation of collagen, i.e. the formation of glucose bridges, is nonenzymatic and therefore cannot be prevented in any other way than keeping the sugar level low. It relates to secondary diseases, abundantly occurring in aging populations and diabetics. However, little is known about the effects of glycation of collagen on the molecular level. We studied in vitro the effect of glycation, with D-glucose and D-galactose as well as D-ribose, on the structure of type 1 collagen by preparing decellularized matrices of bovine pericardia soaked in different sugar solutions, at increasing concentrations (0, 2.5, 5, 10, 20 and 40 mg ml−1), and incubated at 37°C for 3, 14, 30 and 90 days. The tissue samples were analyzed with small- and wide-angle X-ray scattering in scanning mode. We found that glucose and galactose produce similar changes in collagen, i.e. they mainly affect the lateral packing between macromolecules. However, ribose is much faster in glycation, provoking a larger effect on the lateral packing, but also seems to cause qualitatively different effects on the collagen structure.

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MARCKS affects cell motility and response to BTK inhibitors in CLL

Blood ◽

10.1182/blood.2020009165 ◽

2021 ◽

Author(s):

Laura Beckmann ◽

Valeska Berg ◽

Clarissa Dickhut ◽

Clare Sun ◽

Olaf Merkel ◽

...

Keyword(s):

Molecular Level ◽

Cell Receptor ◽

Akt Signaling ◽

B Cell Receptor ◽

Nodal Disease ◽

Highly Active ◽

Migratory Capacity ◽

Phosphorylation Level ◽

Btk Inhibitors

BTK inhibitors are highly active drugs for the treatment of CLL. To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines, threonines and tyrosines (pS:pT:pY). Expression of 83 proteins differed between unmutated IGHV (UM)-CLL and mutated IGHV (M)-CLL. Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition and survival. One protein, MARCKS, showed striking differences in expression and phosphorylation level in UM-CLL versus M-CLL. MARCKS sequesters PIP2, thereby affecting central signaling pathways and clustering of the B cell receptor. Genetically induced loss of MARCKSignificantly increased AKT signaling and the migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitiors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in CLL patients treated with acalabrutinib.

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Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

Protocols used in Molecular Biology ◽

10.2174/9789811439315120010006 ◽

2020 ◽

pp. 15-34

Keyword(s):

Gene Expression ◽

Real Time ◽

Tissue Regeneration ◽

Real Time Pcr ◽

Molecular Level ◽

Northern Blotting ◽

Tissue Constructs ◽

Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

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