Messenger RNA (mRNA)-based age determination using skin-specific markers of saliva epithelial cells

Abstract Background Age determination is a vital factor in biological identification in forensics. This study was carried out to determine the expression levels of three target genes (Keratin 9 (KRT9), Loricrin (LOR) and Corneodesmosin (CDSN)) in salivary epithelial cells and how they can be used in age determination using reference gene, β-actin. Thirty young adults participated in the study and were divided into three groups according to their ages (16–20, 21–25, and 26–30). Ribonucleic acid (RNA) extraction, complementary deoxyribonucleic acid (cDNA) synthesis and quantitative polymerase chain reaction (qPCR) were performed. Data analysis was done using IBM SPSS Version 26 and the comparative Ct method (2−∆∆Ct method). Results CDSN was detected in all the sampled age groups. Though the age group 16–20 had the highest (0.4237) expression of CDSN among the three age groups, there was no significant difference (p > 0.05) in the expression of the gene among the three age groups. The LOR gene was lowly expressed across all age groups used in the study. The expression of the gene did not significantly differ (p > 0.05) between the control and 26–30 years age group, but they were however significantly higher (F = 36.47, p ≤ 0.05) than the expression of the gene in both 16–20 and 21–25 years age groups. The KRT9 gene was expressed only in age groups 16–20 and 26–30 and the expression of the gene did not significantly (p > 0.05) differ between these age groups. Though the expression of all the target genes was low, it was observed that the LOR gene expression varied among 21–25 and 26–30 age groups; therefore, more data and further analyses are still required since this experimental approach for age determination using gene expression is still at an emerging stage. Conclusion Although RNA concentration was low and the expression values of the genes were low and could not be used in comparing the expression levels among the three age groups, it can be concluded that the three messenger ribonucleic acid (mRNA) markers CDSN, LOR and KRT9, as well as the ACTB reference mRNA marker analysed via the described qPCR assays, are suitable for identifying epithelial cells in saliva.

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Identification of reference genes for gene expression studies during seed germination and seedling establishment in Ricinus communis L.

Seed Science Research ◽

10.1017/s0960258514000294 ◽

2014 ◽

Vol 24 (4) ◽

pp. 341-352 ◽

Cited By ~ 15

Author(s):

Paulo R. Ribeiro ◽

Bas J. W. Dekkers ◽

Luzimar G. Fernandez ◽

Renato D. de Castro ◽

Wilco Ligterink ◽

...

Keyword(s):

Gene Expression ◽

Seed Germination ◽

Reference Genes ◽

Target Genes ◽

Ricinus Communis ◽

Quantitative Polymerase Chain Reaction ◽

Optimal Combination ◽

Expression Levels ◽

Gene Expression Studies ◽

Gene Expression Levels

AbstractReverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

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Association of Mandible Anatomy with Age, Gender, and Dental Status: A Radiographic Study

ISRN Radiology ◽

10.5402/2013/453763 ◽

2013 ◽

Vol 2013 ◽

pp. 1-4 ◽

Cited By ~ 11

Author(s):

Revant H. Chole ◽

Ranjitkumar N. Patil ◽

Swati Balsaraf Chole ◽

Shailesh Gondivkar ◽

Amol R. Gadbail ◽

...

Keyword(s):

Age Groups ◽

Age Determination ◽

Dental Status ◽

Age Group ◽

Radiographic Study ◽

Mandibular Angle ◽

Panoramic Radiographs ◽

Gonial Angle ◽

Significant Difference ◽

Males And Females

Introduction. Gonial angle and antegonial region are important landmarks in mandible which is influenced by gender, age, and dental status. The objective of this study was to evaluate the gonial angle, antegonial angle, and antegonial depth and to investigate their relationship to gender, age group, and dental status. Materials and Methods. A total of 1060 panoramic radiographs were evaluated: the dentulous group, 854 subjects and the edentulous group, 206 subjects. The patients were grouped into six age groups of 10-years each. Gonial angle, antegonial angle, and antegonial depth were measured from panoramic radiographs. Results and Discussion. Corelation of age with gonial angle, antegonial angle and antegonial depth was not significant. Significant difference in mandibular angle was found between males and females. Males had significantly smaller antegonial angle and greater antegonial depth than females. Significant difference was found for gonial angle, antegonial angle, and antegonial depth between right and left sides of mandible. Conclusion. Gonial angle, antegonial angle, and antegonial depth can be implicated as a forensic tool for gender determination but not suitable for age determination.

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There is no magic bullet: the importance of testing reference gene stability in RT-qPCR experiments across multiple closely related species

PeerJ ◽

10.7717/peerj.9618 ◽

2020 ◽

Vol 8 ◽

pp. e9618

Author(s):

Bert Foquet ◽

Hojun Song

Keyword(s):

Gene Expression ◽

Related Species ◽

Reference Genes ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

Magic Bullet ◽

Experimental Conditions ◽

Closely Related Species ◽

Expression Levels ◽

Gene Stability

Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) is the current gold standard tool for the study of gene expression. This technique is highly dependent on the validation of reference genes, which exhibit stable expression levels among experimental conditions. Often, reference genes are assumed to be stable a priori without a rigorous test of gene stability. However, such an oversight can easily lead to misinterpreting expression levels of target genes if the references genes are in fact not stable across experimental conditions. Even though most gene expression studies focus on just one species, comparative studies of gene expression among closely related species can be very informative from an evolutionary perspective. In our study, we have attempted to find stable reference genes for four closely related species of grasshoppers (Orthoptera: Acrididae) that together exhibit a spectrum of density-dependent phenotypic plasticity. Gene stability was assessed for eight reference genes in two tissues, two experimental conditions and all four species. We observed clear differences in the stability ranking of these reference genes, both between tissues and between species. Additionally, the choice of reference genes clearly influenced the results of a gene expression experiment. We offer suggestions for the use of reference genes in further studies using these four species, which should be taken as a cautionary tale for future studies involving RT-qPCR in a comparative framework.

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ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200721101904 ◽

2020 ◽

Vol 20 (18) ◽

pp. 2274-2284

Author(s):

Faroogh Marofi ◽

Jalal Choupani ◽

Saeed Solali ◽

Ghasem Vahedi ◽

Ali Hassanzadeh ◽

...

Keyword(s):

Gene Expression ◽

Zoledronic Acid ◽

Mrna Expression ◽

Promoter Methylation ◽

Methylation Level ◽

Target Genes ◽

Osteoblastic Differentiation ◽

Therapeutic Effects ◽

Significant Difference ◽

Scheduled Time

Objective: Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation (OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood. It seems that the methylation of the promoter region of key genes might be an important factor involved in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment with ZA. Materials and Methods: MSCs were induced to be differentiated into the osteoblastic cell lineage using routine protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and real.time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the related markers to this stage. Results: Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes, while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result for FRA1 was not significant in both groups at the same scheduled time (days 0-21). Conclusion: The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1 may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined that promoter-methylation is an important process in the regulation of OSD. However, yet there was no significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation- independent pathway might be involved in the regulation of target genes during OSD of MSCs.

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Different macrophages equally induce EMT in endometria of adenomyosis and normal

Reproduction ◽

10.1530/rep-17-0174 ◽

2017 ◽

Vol 154 (1) ◽

pp. 79-92 ◽

Cited By ~ 7

Author(s):

Min An ◽

Dong Li ◽

Ming Yuan ◽

Qiuju Li ◽

Lu Zhang ◽

...

Keyword(s):

Epithelial Cells ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Quantitative Polymerase Chain Reaction ◽

Immunohistochemical Analysis ◽

Secretory Phase ◽

Normal Endometrium ◽

Mesenchymal Transition ◽

Expression Levels ◽

Endometrial Cells

Endometrial cells and microenvironment are two important factors in the pathogenesis of adenomyosis. Our previous study demonstrated that macrophages can induce eutopic epithelial cells of adenomyosis to suffer from epithelial–mesenchymal transition (EMT). The aim of this study is to detect whether macrophages interacting with epithelial cells equally induce the EMT process in normal and eutopic endometria of healthy and adenomyotic patients; and whether macrophages parallelly polarize to M2. We investigated the expression levels of epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), cytokeratin7 (CK7), vimentin, transforming growth factor-β1 (TGFB1), SMAD3 and pSMAD3 using immunohistochemistry and western blot, and then estimated the genetic levels of CD163, IL10 and MMP12 using real-time quantitative polymerase chain reaction (RT-PCR) in macrophages. Eutopic and normal endometrial tissues were obtained from 20 patients with adenomyosis and 11 control patients without adenomyosis, respectively. The immunohistochemical analysis shows distinct EMT in eutopic endometria in secretory phase; the expression levels of TGFB1, SMAD3 and pSMAD3 that indicate signal pathway of EMT were also higher in secretory phase. Macrophages can induce EMT process in primary endometrial epithelial cells derived from normal and eutopic endometria. After co-culturing, THP-1-derived macrophages polarized to M2. Compared with the eutopic endometrium group, further polarization to M2 was observed in the normal endometrium group. These results indicate that adenomyosis may be promoted by the pathologic EMT of epithelial cells, which is induced by macrophages that incapably polarize to M2.

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Constructing the Logical Regression Model to Predict the Target of Jianpi Jiedu Decoction in the Treatment of Hepatocellular Carcinoma

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/8859558 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Rongjie Zhang ◽

Yan Chen ◽

Ge Zhou ◽

Baoguo Sun ◽

Yue Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Survival Analysis ◽

Regression Model ◽

Target Genes ◽

Cox Regression ◽

Risk Group ◽

Cox Regression Analysis ◽

Expression Levels ◽

Prognostic Analysis ◽

Significant Difference

Objectives. The purpose of this study was to identify the molecular mechanism and prognosis-related genes of Jianpi Jiedu decoction in the treatment of hepatocellular carcinoma. Methods. The gene expression data of hepatocellular carcinoma samples and normal tissue samples were downloaded from TCGA database, and the potential targets of drug composition of Jianpi Jiedu decoction were obtained from TCMSP database. The genes were screened out in order to obtain the expression of these target genes in patients with hepatocellular carcinoma. The differential expression of target genes was analyzed by R software, and the genes related to prognosis were screened by univariate Cox regression analysis. Then, the LASSO model was constructed for risk assessment and survival analysis between different risk groups. At the same time, independent prognostic analysis, GSEA analysis, and prognostic analysis of single gene in patients with hepatocellular carcinoma were performed. Results. 174 compounds of traditional Chinese medicine were screened by TCMSP database, corresponding to 122 potential targets. 39 upregulated genes and 9 downregulated genes were screened out. A total of 20 candidate prognostic related genes were screened out by univariate Cox analysis, of which 12 prognostic genes were involved in the construction of the LASSO regression model. There was a significant difference in survival time between the high-risk group and low-risk group ( p < 0.05 ). Among the genes related to prognosis, the expression levels of CCNB1, NQO1, NUF2, and CHEK1 were high in tumor tissues ( p < 0.05 ). Survival analysis showed that the high expression levels of these four genes were significantly correlated with poor prognosis of HCC ( p < 0.05 ). GSEA analysis showed that the main KEGG enrichment pathways were lysine degradation, folate carbon pool, citrate cycle, and transcription factors. Conclusions. In the study, we found that therapy target genes of Jianpi Jiedu decoction were mainly involved in metabolism and apoptosis in hepatocellular carcinoma, and there was a close relationship between the prognosis of hepatocellular carcinoma and the genes of CCNB1, NQO1, NUF2, and CHEK1.

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Characteristics of Helicobacter pylori infection in a National Referral Hospital in Bhutan

Bhutan Health Journal ◽

10.47811/bhj.93 ◽

2020 ◽

Vol 6 (1) ◽

pp. 6-10

Author(s):

Chhabi Lal Adhikari ◽

Guru Prasad Dhakal ◽

Nongluck Suwisith ◽

Sonam Dargay ◽

Krishna P Sharma

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Age Groups ◽

Referral Hospital ◽

Upper Gastrointestinal Endoscopy ◽

Age Group ◽

Exact Test ◽

Significant Difference ◽

H Pylori ◽

National Referral Hospital

Introduction: Helicobacter pylori (H. pylori) is a bacterium causing chronic gastric infection and may cause gastric cancer. It was necessary to see the trend of infection, especially in symptomatic patients. This retrospective descriptive study was aimed to describe the characteristics of H. pylori infection in Bhutanese patients referred for an endoscopy to the National Referral Hospital, Thimphu. Methods: The sample of the study was randomized 380 medical records of the patients who underwent upper gastrointestinal endoscopy and Rapid Urea Test for symptomatic dyspepsia and peptic ulcer. Data was collected using a survey form designed by the researchers. Data analysis was done using descriptive statistics and either Chi-square or Fisher’s exact test. Results: The prevalence of H. pylori infection was very high (76.6%). The mean age of the infection was 42 with a range from 15 to 84 years. The highest prevalence of infection was observed in the age group 20-29 years (82.7%) and lowest in the oldest age group 70-84 years (66.7%). The analysis showed no significant difference in infection amongst age groups, gender, and endoscopic findings to the positive results at 5% significant level except for monthly prevalence (p<0.001). Gastritis was the commonest endoscopy finding (153/380) and gastro-duodenitis had the highest positivity rate (88.9%). Conclusion: The prevalence of infection was relatively high compared with previous studies. Young and middle-aged adults had a high prevalence and this group needs to be given priority for screening and eradication treatment considering limited resources to prevent associated gastric cancer in Bhutan.

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Activation of the JAK1 / STAT1 Signaling Pathway is Associated with Prdx6 Expression Levels in Human Epididymis Epithelial Cells

10.21203/rs.3.rs-824964/v1 ◽

2021 ◽

Author(s):

Jianyuan Li ◽

Hui Shi ◽

Xiaoyu Liu ◽

Yanwei Wang ◽

Haiyan Wang ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Signaling Pathway ◽

Expression Patterns ◽

Digital Gene Expression ◽

Protective Role ◽

Expression Levels ◽

Human Epididymis ◽

Total Antioxidant ◽

Stat1 Signaling

Abstract I. Background: Peroxiredoxin 6 (Prdx6) is widely expressed in mammalian tissues. Our previous study demonstrated that Prdx6 was expressed in human epididymis and spermatozoa, and the protective role of Prdx6 in human spermatozoa was also reported. In this study, we demonstrate the potential role and mechanism of Prdx6 in human epididymis epithelial cells (HEECs).II. Methods and Results: Western blotting was used to measure expression levels of key proteins in the JAK / STAT signaling pathway. Digital gene expression analysis (DGE) was used to identify gene expression patterns in control HECs and in HECs after Prdx6-RNA interference (P6-RNAi). The DGE analysis identified 589 up-regulated and 314 down-regulated genes (including Prdx6) in Prdx6-RNAi (P6-RNAi) HEECs. Thirteen significantly different pathways were identified between the two groups, with the majority different expressed genes belonging to the CCL, CXCL, IL, and IFIT families. In particular, the expression levels of IL6, IL6ST, and eighteen IFN related genes were significantly increased in the condition of the down-regulated expression of Prdx6. Compared to control HEECs, the expression levels of JAK1, STAT1, phosphorylated JAK1 and STAT1 were significantly increased, while the expression levels of SOCS3 was significantly decreased in P6-RNAi HEECs. The Malondialdehyde (MDA) level and total antioxidant capacity in P6-RNAi HEECs were significantly increased and decreased compared to that of control, respectively. III. Conclusions: We speculated that knockdown of Prdx6 resulted in higher levels of ROS in HEECs, which in turn, activated the JAK1 / STAT1 signaling pathway induced by IL-6 receptor and IFN.

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The Dynamics of Cullin-1 Expression in Preeclamptic Placenta and its Association with Pregnancy Termination Time

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2020.3665 ◽

2020 ◽

Vol 8 (B) ◽

pp. 210-215

Author(s):

Makbruri Makbruri ◽

Isabella Kurnia Liem ◽

Ahmad Aulia Jusuf ◽

Tantri Hellyanti

Keyword(s):

Gestational Age ◽

Pregnancy Termination ◽

Age Groups ◽

Trophoblast Invasion ◽

Age Group ◽

Placental Development ◽

Very Preterm ◽

Significant Difference ◽

Cellular Factors ◽

Termination Time

BACKGROUND: Preeclampsia is a systemic syndrome occurring in 3–5% of pregnancies, caused by disorders of cellular factors resulting in the disruption of trophoblast differentiation and invasion which is important for the placental development and maintaining pregnancy. Cullin-1 is a protein that plays a role in the process of maintaining pregnancy, development, and trophoblast invasion in the placenta. Until now, there have been no studies linking the expression of cullin-1 in preeclamptic patients with the timing of pregnancy termination. AIM: This study analyzed cullin-1 expression in preeclamptic patients and their relationship to the timing of pregnancy termination was carried out. METHODS: Placental samples were taken from preeclampsia patients consisting of three gestational age groups, then immunohistochemical staining was performed to see the dynamics of expression and distribution in each age group of pregnancy and to find out their relationship with the timing of pregnancy termination. RESULTS: Cullin-1 was expressed in syncytiotrophoblasts and cytotrophoblasts. The lowest cullin-1 level was obtained in the very preterm age group, and the highest was found in the moderate preterm gestational age group. There was a significant difference between cullin-1 optical density (OD) expression and termination time of pregnancy, and there was a significant difference (OD) in cullin-1 preeclamptic patients with very preterm gestational age with moderate preterm gestational age. CONCLUSION: Cullin-1 was expressed both in syncytiotrophoblasts and cytotrophoblasts and was associated with the timing of pregnancy termination.

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Spatial regulation of gene expression in nonsyndromic sagittal craniosynostosis

Journal of Neurosurgery Pediatrics ◽

10.3171/2018.6.peds18229 ◽

2018 ◽

Vol 22 (6) ◽

pp. 620-626 ◽

Cited By ~ 2

Author(s):

Garrett N. Cyprus ◽

Jefferson W. Overlin ◽

Rafael A. Vega ◽

Ann M. Ritter ◽

René Olivares-Navarrete

Keyword(s):

Gene Expression ◽

Wnt Signaling ◽

Transforming Growth Factor ◽

Quantitative Polymerase Chain Reaction ◽

Regulation Of Gene Expression ◽

Micro Ct ◽

Sagittal Suture ◽

Sagittal Craniosynostosis ◽

Significant Difference ◽

Suture Fusion

OBJECTIVECranial suture patterning and development are highly regulated processes that are not entirely understood. While studies have investigated the differential gene expression for different sutures, little is known about gene expression changes during suture fusion. The aim of this study was to examine gene expression in patent, fusing, and fused regions along sagittal suture specimens in nonsyndromic craniosynostosis patients.METHODSSagittal sutures were collected from 7 patients (average age 4.5 months) who underwent minimally invasive craniotomies at the Children’s Hospital of Richmond at VCU under IRB approval. The sutures were analyzed using micro-CT to evaluate patency. The areas were classified as open, fusing, or fused and were harvested, and mRNA was isolated. Gene expression for bone-related proteins, osteogenic and angiogenic factors, transforming growth factor–β (TGF-β) superfamily, and Wnt signaling was analyzed using quantitative polymerase chain reaction and compared with normal sutures collected from fetal demise tissue (control).RESULTSMicro-CT demonstrated that there are variable areas of closure along the length of the sagittal suture. When comparing control samples to surgical samples, there was a significant difference in genes for Wnt signaling, TGF-β, angiogenic and osteogenic factors, bone remodeling, and nuclear rigidity in mRNA isolated from the fusing and fused areas of the sagittal suture compared with patent areas (p < 0.05).CONCLUSIONSIn nonsyndromic sagittal craniosynostosis, the affected suture has variable areas of being open, fusing, and fused. These specific areas have different mRNA expression. The results suggest that BMP-2, FGFR3, and several other signaling pathways play a significant role in the regulation of suture fusion as well as in the maintenance of patency in the normal suture.

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