scholarly journals Two microRNAs, miR-330 and miR-125b-5p, mark the juxtaglomerular cell and balance its smooth muscle phenotype

2012 ◽  
Vol 302 (1) ◽  
pp. F29-F37 ◽  
Author(s):  
Silvia Medrano ◽  
Maria C. Monteagudo ◽  
Maria Luisa S. Sequeira-Lopez ◽  
Ellen S. Pentz ◽  
R. Ariel Gomez
Keyword(s):  
Smooth Muscle ◽  
In Situ Hybridization ◽  
In Silico ◽  
Smooth Muscle Actin ◽  
In Silico Analysis ◽  
Screening Strategy ◽  
The Other ◽  
Kidney Cortex

We have shown that microRNAs (miRNAs) are necessary for renin cell specification and kidney vascular development. Here, we used a screening strategy involving microarray and in silico analyses, along with in situ hybridization and in vitro functional assays to identify miRNAs important for renin cell identity. Microarray studies using vascular smooth muscle cells (SMCs) of the renin lineage and kidney cortex under normal conditions and after reacquisition of the renin phenotype revealed that of 599 miRNAs, 192 were expressed in SMCs and 234 in kidney cortex. In silico analysis showed that the highly conserved miR-330 and miR-125b-5p have potential binding sites in smoothelin ( Smtn), calbindin 1, smooth muscle myosin heavy chain, α-smooth muscle actin, and renin genes important for the myoepithelioid phenotype of the renin cell. RT-PCR studies confirmed miR-330 and miR-125b-5p expression in kidney and SMCs. In situ hybridization revealed that under normal conditions, miR-125b-5p was expressed in arteriolar SMCs and in juxtaglomerular (JG) cells. Under conditions that induce reacquisition of the renin phenotype, miR-125b-5p was downregulated in arteriolar SMCs but remained expressed in JG cells. miR-330, normally absent, was expressed exclusively in JG cells of treated mice. In vitro functional studies showed that overexpression of miR-330 inhibited Smtn expression in SMCs. On the other hand, miR-125b-5p increased Smtn expression, whereas its inhibition reduced Smtn expression. Our results demonstrate that miR-330 and miR-125b-5p are markers of JG cells and have opposite effects on renin lineage cells: one inhibiting and the other favoring their smooth muscle phenotype.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

2020 ◽  
Vol 27 (5) ◽  
pp. 432-446
Author(s):  
Akiko Yamamoto ◽  
Ken-ichiro Matsunaga ◽  
Toyoaki Anai ◽  
Hitoshi Kawano ◽  
Toshihisa Ueda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Analysis ◽  
Protein Characterization ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Animal Cells ◽  
Dugesia Japonica ◽  
Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

In Silico Study of Potential Cross-Kingdom Plant MicroRNA Based Regulation in Chronic Myeloid Leukemia

2020 ◽  
Vol 17 (2) ◽  
pp. 125-132
Author(s):  
Marjanu Hikmah Elias ◽  
Noraziah Nordin ◽  
Nazefah Abdul Hamid
Keyword(s):  
Targeted Therapy ◽  
In Silico ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Target Prediction ◽  
In Silico Analysis ◽  
Microrna Target ◽  
Good Target

Background: Chronic Myeloid Leukaemia (CML) is associated with the BCRABL1 gene, which plays a central role in the pathogenesis of CML. Thus, it is crucial to suppress the expression of BCR-ABL1 in the treatment of CML. MicroRNA is known to be a gene expression regulator and is thus a good candidate for molecularly targeted therapy for CML. Objective: This study aims to identify the microRNAs from edible plants targeting the 3’ Untranslated Region (3’UTR) of BCR-ABL1. Methods: In this in silico analysis, the sequence of 3’UTR of BCR-ABL1 was obtained from Ensembl Genome Browser. PsRNATarget Analysis Server and MicroRNA Target Prediction (miRTar) Server were used to identify miRNAs that have binding conformity with 3’UTR of BCR-ABL1. The MiRBase database was used to validate the species of plants expressing the miRNAs. The RNAfold web server and RNA COMPOSER were used for secondary and tertiary structure prediction, respectively. Results: In silico analyses revealed that cpa-miR8154, csi-miR3952, gma-miR4414-5p, mdm-miR482c, osa-miR1858a and osa-miR1858b show binding conformity with strong molecular interaction towards 3’UTR region of BCR-ABL1. However, only cpa-miR- 8154, osa-miR-1858a and osa-miR-1858b showed good target site accessibility. Conclusion: It is predicted that these microRNAs post-transcriptionally inhibit the BCRABL1 gene and thus could be a potential molecular targeted therapy for CML. However, further studies involving in vitro, in vivo and functional analyses need to be carried out to determine the ability of these miRNAs to form the basis for targeted therapy for CML.

In-Vitro and In-Silico Characterization of Xylose Reductase from Emericella nidulans

2019 ◽  
Vol 13 (2) ◽  
pp. 159-170 ◽  
Author(s):  
Vishal Ahuja ◽  
Aashima Sharma ◽  
Ranju Kumari Rathour ◽  
Vaishali Sharma ◽  
Nidhi Rana ◽  
...  
Keyword(s):  
Production Process ◽  
In Silico ◽  
Xylose Reductase ◽  
In Silico Analysis ◽  
Emericella Nidulans ◽  
Higher Temperature ◽  
In Silico Characterization ◽  
Silico Analysis

Background: Lignocellulosic residues generated by various anthropogenic activities can be a potential raw material for many commercial products such as biofuels, organic acids and nutraceuticals including xylitol. Xylitol is a low-calorie nutritive sweetener for diabetic patients. Microbial production of xylitol can be helpful in overcoming the drawbacks of traditional chemical production process and lowring cost of production. Objective: Designing efficient production process needs the characterization of required enzyme/s. Hence current work was focused on in-vitro and in-silico characterization of xylose reductase from Emericella nidulans. Methods: Xylose reductase from one of the hyper-producer isolates, Emericella nidulans Xlt-11 was used for in-vitro characterization. For in-silico characterization, XR sequence (Accession No: Q5BGA7) was used. Results: Xylose reductase from various microorganisms has been studied but the quest for better enzymes, their stability at higher temperature and pH still continues. Xylose reductase from Emericella nidulans Xlt-11 was found NADH dependent and utilizes xylose as its sole substrate for xylitol production. In comparison to whole cells, enzyme exhibited higher enzyme activity at lower cofactor concentration and could tolerate higher substrate concentration. Thermal deactivation profile showed that whole cell catalysts were more stable than enzyme at higher temperature. In-silico analysis of XR sequence from Emericella nidulans (Accession No: Q5BGA7) suggested that the structure was dominated by random coiling. Enzyme sequences have conserved active site with net negative charge and PI value in acidic pH range. Conclusion: Current investigation supported the enzyme’s specific application i.e. bioconversion of xylose to xylitol due to its higher selectivity. In-silico analysis may provide significant structural and physiological information for modifications and improved stability.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

scholarly journals Procyanidin B2 Reduces Vascular Calcification through Inactivation of ERK1/2-RUNX2 Pathway

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 916
Author(s):  
Yingquan Liang ◽  
Guilan Chen ◽  
Feng Zhang ◽  
Xiaoxiao Yang ◽  
Yuanli Chen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Calcification ◽  
Aortic Root ◽  
Smooth Muscle Actin ◽  
Bone Morphogenetic Protein 2 ◽  
Plaque Burden ◽  
Runx2 Expression ◽  
Plaque Instability ◽  
Related Transcription Factor

Vascular calcification is strongly associated with atherosclerotic plaque burden and plaque instability. The activation of extracellular signal-regulated kinase 1/2 (ERK1/2) increases runt related transcription factor 2 (RUNX2) expression to promote vascular calcification. Procyanidin B2 (PB2), a potent antioxidant, can inhibit ERK1/2 activation in human aortic smooth muscle cells (HASMCs). However, the effects and involved mechanisms of PB2 on atherosclerotic calcification remain unknown. In current study, we fed apoE-deficient (apoE−/−) mice a high-fat diet (HFD) while treating the animals with PB2 for 18 weeks. At the end of the study, we collected blood and aorta samples to determine atherosclerosis and vascular calcification. We found PB2 treatment decreased lesions in en face aorta, thoracic, and abdominal aortas by 21.4, 24.6, and 33.5%, respectively, and reduced sinus lesions in the aortic root by 17.1%. PB2 also increased α-smooth muscle actin expression and collagen content in lesion areas. In the aortic root, PB2 reduced atherosclerotic calcification areas by 75.8%. In vitro, PB2 inhibited inorganic phosphate-induced osteogenesis in HASMCs and aortic rings. Mechanistically, the expression of bone morphogenetic protein 2 and RUNX2 were markedly downregulated by PB2 treatment. Additionally, PB2 inhibited ERK1/2 phosphorylation in the aortic root plaques of apoE−/− mice and calcified HASMCs. Reciprocally, the activation of ERK1/2 phosphorylation by C2-MEK1-mut or epidermal growth factor can partially restore the PB2-inhibited RUNX2 expression or HASMC calcification. In conclusion, our study demonstrates that PB2 inhibits vascular calcification through the inactivation of the ERK1/2-RUNX2 pathway. Our study also suggests that PB2 can be a potential option for vascular calcification treatment.

scholarly journals Large intraperitoneal lipoleiomyoma in a pre-menopausal woman: a case report

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sara L. Schaefer ◽  
Amy L. Strong ◽  
Sheena Bahroloomi ◽  
Jichang Han ◽  
Michella K. Whisman ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Smooth Muscle ◽  
Case Report ◽  
Smooth Muscle Actin ◽  
Abdominal Mass ◽  
Histopathological Analysis ◽  
Menopausal Woman ◽  
En Bloc ◽  
Cross Sectional

Abstract Background Lipoleiomyoma is a rare, benign variant of the commonplace uterine leiomyoma. Unlike leiomyoma, these tumors are composed of smooth muscle cells admixed with mature adipose tissue. While rare, they are most frequently identified in the uterus, but even more infrequently have been described in extrauterine locations. Case presentation We describe a case report of a 45-year-old woman with a history of in vitro fertilization pregnancy presenting 6 years later with abdominal distention and weight loss found to have a 30-cm intra-abdominal lipoleiomyoma. While cross-sectional imaging can narrow the differential diagnosis, histopathological analysis with stains positive for smooth muscle actin, desmin, and estrogen receptor, but negative for HMB-45 confirms the diagnosis of lipoleiomyoma. The large encapsulated tumor was resected en bloc. The patients post-operative course was uneventful and her symptoms resolved. Conclusions Lipoleiomyoma should be considered on the differential diagnosis in a woman with a large intra-abdominal mass. While considered benign, resection should be considered if the mass is symptomatic, and the diagnosis is unclear or there is a concern for malignancy.

Investigation of indole functionalized pyrazoles and oxadiazoles as anti-inflammatory agents: synthesis, in-vivo, in-vitro and in-silico analysis

2021 ◽  
pp. 105068
Author(s):  
Devendra Kumar ◽  
Ravi Ranjan Kumar ◽  
Shelly Pathania ◽  
Pankaj Kumar Singh ◽  
Sourav Kalra ◽  
...  
Keyword(s):  
In Silico ◽  
In Silico Analysis ◽  
Anti Inflammatory ◽  
Silico Analysis

scholarly journals Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1502
Author(s):  
Jorge García-Hernández ◽  
Manuel Hernández ◽  
Yolanda Moreno
Keyword(s):  
In Situ Hybridization ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent In Situ Hybridization ◽  
Potential Method ◽  
Viable Count ◽  
Hybridization Technique ◽  
Fish Technique ◽  
Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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