Development of Sarcophaga dux (Diptera: Sarcophagidae) at constant temperatures and differential gene expression for age estimation of intrapuparial

Abstract BackgroundSarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly with potential forensic value in estimating minimum postmortem interval (PMImin). The basic developmental data and precise intrapuparial age estimates are significant for PMImin estimationof application entomological data in legal medicine.MethodsThe development parameters of S. dux at seven constant temperatures from 16°C to 34°C were investigated by rearing using pig lung in the artificial climate box. The appropriate reference genes and intrapuparial differentially expressed genes (DEGs) of S. dux at constant temperatures 34, 25 and 16°C were selected and analyzed using RT-qPCR for more precisely age estimations. ResultsThe developmental durations of S. dux at 16, 19, 22, 25, 28, 31 and 34°C from larviposition to adult eclosion were 1478.6±18.3, 726.1±15.8, 538.5±0.9, 394.1±9.5, 375.6±10.8, 284.1±7.3, and 252.5±6.1 h, respectively. The thermal summation constant of S. dux was 5341.71±249.29 degree hours, and the developmental threshold temperature was 12.266±0.35°C. The most reliable reference genes under intrapuparial and different temperature in our study were: GST1 and 18S rRNA for 34°C group, GST1 and RPL49 for 25 °C group, and 18S rRNA and 28S rRNA for 16 °C group，the four differential expression genes (Hsp60, A-alpha, ARP, RPL8) can be used to more precisely intrapuparial age estimation of S. dux. ConclusionsThe basic developmental data of S. dux at constant temperatures, such as body length changing of the larva period, accumulated degree hours and duration of development, to establish developmental models that can be used to estimate the PMImin. The selection and evaluation of appropriate reference genes at different experimental conditions are primary with RT-qPCR. The differentially expressed gene can contribute to more accuracy age estimations of S. dux intrapuparial. The result from this study can make contributes to the use of S.dux for estimating PMImin.

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Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety

Animals ◽

10.3390/ani11113073 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3073

Author(s):

Kelly J. Lozano-Villegas ◽

Roy Rodríguez-Hernández ◽

María P. Herrera-Sánchez ◽

Heinner F. Uribe-García ◽

Juan S. Naranjo-Gómez ◽

...

Keyword(s):

Whole Blood ◽

Reference Genes ◽

18S Rrna ◽

Relative Quantification ◽

Experimental Conditions ◽

Cattle Breeds ◽

Expression Studies ◽

Safety Alert ◽

Lower Variation ◽

The Stability

Real-time PCR is widely used to study the relative abundance of mRNA due to its specificity, sensitivity, and repeatability quantification. However, relative quantification requires a reference gene, which should be stable in its expression, showing lower variation by experimental conditions or tissues. The aim of this study was to evaluate the stability of the expression of five commonly used reference genes (actb, ywhaz, b2m, sdha, and 18s rRNA) at different physiological stages (alert and emergency) in three different cattle breeds. In this study, five genes (actb, ywhaz, b2m, sdha, and 18s rRNA) were selected as candidate reference genes for expression studies in the whole blood from three cattle breeds (Romosinuano, Gyr, and Brahman) under heat stress conditions. The transcription stability of the candidate reference genes was evaluated using geNorm and NormFinder. The results showed that actb, 18SrRNA, and b2m expression were the most stable reference genes for whole blood of Gyr and Brahman breeds under two states of livestock weather safety (alert and emergency). Meanwhile, actb, b2m, and ywhaz were the most stable reference genes for the Romosinuano breed.

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Differential Gene Expression for Age Estimation of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae) Intrapuparial

Journal of Medical Entomology ◽

10.1093/jme/tjz137 ◽

2019 ◽

Vol 57 (1) ◽

pp. 65-77 ◽

Cited By ~ 3

Author(s):

Yanjie Shang ◽

Lipin Ren ◽

Li Yang ◽

Shiwen Wang ◽

Wei Chen ◽

...

Keyword(s):

Gene Expression ◽

Differential Gene Expression ◽

Age Estimation ◽

Reference Genes ◽

Morphological Changes ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Accurate Determination ◽

28S Rrna ◽

Differential Gene

Abstract Sarcophaga peregrina is an important flesh fly species for estimating the minimum postmortem interval (PMImin) in forensic entomology. The accurate determination of the developmental age is a crucial task for using necrophagous sarcophagids to estimate PMImin. During larval development, the age determination is straight forward by the morphological changes and variation of length, weight, and width; however, the age estimation of sarcophagid intrapuparial is more difficult due to anatomical and morphological changes not being visible. The analysis of differentially expressed genes (DEGs) during sarcophagid metamorphosis is a potential method for age estimation of intrapuparial. In the present study, real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the differential gene expression level of S. peregrina intrapuparial in different constant temperatures (35°C, 25°C, and 15°C). In addition, the appropriate reference genes of S. peregrina were selected in the intrapuparial and at different temperatures to obtain reliable and valid gene expression profiles. The results indicated that two candidate genes (18S rRNA and 28S rRNA) were the most reliable reference genes, and four DEGs (Hsp90, A-alpha, AFP, AFBP) have the potential to be used to more accuracy estimate the age of S. peregrina intrapuparial.

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Development of Chrysomya rufifacies (Diptera: Calliphoridae) at Constant Temperatures Within its Colony Range in Yangtze River Delta Region of China

Journal of Medical Entomology ◽

10.1093/jme/tjz052 ◽

2019 ◽

Vol 56 (5) ◽

pp. 1215-1224 ◽

Cited By ~ 6

Author(s):

Guoliang Hu ◽

Yu Wang ◽

Yong Sun ◽

Yingna Zhang ◽

Man Wang ◽

...

Keyword(s):

Abiotic Factors ◽

Developmental Time ◽

Yangtze River Delta ◽

Postmortem Interval ◽

Threshold Temperature ◽

Insect Development ◽

Chrysomya Rufifacies ◽

Developmental Threshold ◽

Developmental Models ◽

Threshold Temperatures

Abstract The age of insects found on corpses is often used to estimate the minimum postmortem interval by forensic entomologists. Insect development is affected by biotic and abiotic factors, and temperature is the most important environmental factor that determines the length of insect development. Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) is a globally distributed fly that is commonly found on corpses, and this study investigated the development of C. rufifacies from China at various constant temperatures. At 16, 19, 22, 25, 28, 31, and 34°C, the developmental time from egg to adult was 870.17 ± 11.50, 566.20 ± 8.79, 406.38 ± 10.98, 291.14 ± 4.71, 232.59 ± 5.96, 192.47 ± 3.45, and 160.48 ± 7.15 h, respectively. We established three developmental models for C. rufifacies to estimate the age of the developing insect: the isomorphen diagram model, the isomegalen diagram model and the thermal summation model. Regression analysis was conducted to obtain the relationship between body length and development time of the larvae from hatching to wandering. The developmental threshold temperatures of the egg, 1st instar, 2nd instar, 3rd instar, and wandering larvae, and intra-puparial stages were 12.28 ± 0.30, 11.74 ± 0.95, 12.70 ± 0.55, 11.68 ± 0.96, 10.53 ± 1.53, and 12.51 ± 0.41°C, respectively. The developmental threshold temperature, and the thermal summation constant during the entire developmental period were 3759.95 ± 170.80 degree hours and 11.96 ± 0.38°C, respectively. This study provides an improved dataset to estimate the postmortem interval of corpses using C. rufifacies.

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Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua Linn

Plant Protection Science ◽

10.17221/20/2018-pps ◽

2018 ◽

Vol 55 (No. 1) ◽

pp. 61-71 ◽

Cited By ~ 4

Author(s):

Junjie Liu ◽

Peng Li ◽

Liuyang Lu ◽

Lanfen Xie ◽

Xiling Chen ◽

...

Keyword(s):

Reference Genes ◽

Developmental Stages ◽

Elongation Factor ◽

Avena Fatua ◽

28S Rrna ◽

Experimental Conditions ◽

Alpha Tubulin ◽

Herbicide Treatments ◽

Tata Box Binding Protein ◽

Suitable Reference

Eight commonly used candidate reference genes, 18S ribosomal RNA (rRNA) (18S), 28S rRNA (28S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1α), ribosomal protein L7 (RPL7), Alpha-tubulin (α-TUB), and TATA box binding protein-associated factor (TBP), were evaluated under various experimental conditions to assess their suitability in different developmental stages, tissues and herbicide treatments in Avena fatua. The results indicated the most suitable reference genes for the different experimental conditions. For developmental stages, 28S and EF1α were the optimal reference genes, both EF1α and 28S were suitable for experiments of different tissues, whereas for herbicide treatments, GAPDH and ACT were suitable for normalizations of expression data. In addition, GAPDH and EF1α were the suitable reference genes.

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Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Genes ◽

10.3390/genes11050502 ◽

2020 ◽

Vol 11 (5) ◽

pp. 502 ◽

Cited By ~ 1

Author(s):

Junming Zhao ◽

Man Zhou ◽

Yu Meng

Keyword(s):

Heavy Metal ◽

Reference Gene ◽

Reference Genes ◽

18S Rrna ◽

Target Genes ◽

Heavy Metal Contamination ◽

Internal Reference ◽

Experimental Conditions ◽

Leaves And Roots ◽

Root Tissues

Switchgrass (Panicum Virgatum L.) has been recognized as the new energy plant, which makes it ideal for the development of phytoremediation on heavy metal contamination in soils with great potential. This study aimed to screen the best internal reference genes for the real-time quantitative PCR (RT-qPCR) in leaves and roots of switchgrass for investigating its response to various heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and arsenic (As). The stability of fourteen candidate reference genes was evaluated by BestKeeper, GeNorm, NormFinder, and RefFinder software. Our results identified U2AF as the best reference gene in Cd, Hg, Cr, and As treated leaves as well as in Hg, Pb, As, and Cr stressed root tissues. In Pb treated leaf tissues, 18S rRNA was demonstrated to be the best reference gene. CYP5 was determined to be the optimal reference gene in Cd treated root tissues. The least stable reference gene was identified to be CYP2 in all tested samples except for root tissues stressed by Pb. To further validate the initial screening results, we used the different sets of combinatory internal reference genes to analyze the expression of two metal transport associated genes (PvZIP4 and PvPDB8) in young leaves and roots of switchgrass. Our results demonstrated that the relative expression of the target genes consistently changed during the treatment when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, or 18S rRNA/ACT12 were combined as the internal reference genes. However, the time-dependent change pattern of the target genes was significantly altered when CYP2 was used as the internal reference gene. Therefore, the selection of the internal reference genes appropriate for specific experimental conditions is critical to ensure the accuracy and reliability of RT-qPCR. Our findings established a solid foundation to further study the gene regulatory network of switchgrass in response to heavy metal stress.

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Evaluation of qPCR Reference Genes For Taimen (Hucho Taimen)

10.21203/rs.3.rs-646522/v1 ◽

2021 ◽

Author(s):

Xiaoxing Yang ◽

Guangxiang Tong ◽

Le Dong ◽

Guopan Tang ◽

Yongquan Zhang ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Reference Genes ◽

28S Rrna ◽

Experimental Conditions ◽

Stress Genes ◽

Qrt Pcr ◽

Processing Times ◽

Reliable Quantification ◽

Hucho Taimen

Abstract As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquacultural research. Understanding the biology of taimen (Hucho taimen) has attracted increased interest because of its ecological and economic values. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimised for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using four methods, geNorm, NormFinder, BestKeeper and RefFinder. The expression levels of the 10 genes were detected using samples from 48 experimental groups that consisted of six tissues (blood, heart, brain, gill, skin and liver) collected under five heat-stress conditions (18, 20, 22, 24 and 26°C) and four heat-stress processing times (4, 24, 48 and 96 h). RPS29 and RPL19 were the most stable genes among all the samples, whereas 28S rRNA, ARBPR and 18S rRNA were the least stable. These results were verified by an expression analysis of the heat-stress genes (hsp60 and hsp70) of taimen. In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions, and these results allow the reliable study of gene expression in taimen.

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Decoding Psoriasis: Integrated Bioinformatics Approach to Understand Hub Genes and Involved Pathways

Current Pharmaceutical Design ◽

10.2174/1381612826666200311130133 ◽

2020 ◽

Vol 26 (29) ◽

pp. 3619-3630

Author(s):

Saumya Choudhary ◽

Dibyabhaba Pradhan ◽

Noor S. Khan ◽

Harpreet Singh ◽

George Thomas ◽

...

Keyword(s):

Gene Expression ◽

Differentially Expressed Gene ◽

Therapeutic Targets ◽

Interaction Network ◽

Gene Expression Omnibus ◽

Differentially Expressed ◽

Keratinocyte Differentiation ◽

Hub Genes ◽

Lesional Skin ◽

Ncbi Gene Expression Omnibus

Background: Psoriasis is a chronic immune mediated skin disorder with global prevalence of 0.2- 11.4%. Despite rare mortality, the severity of the disease could be understood by the accompanying comorbidities, that has even led to psychological problems among several patients. The cause and the disease mechanism still remain elusive. Objective: To identify potential therapeutic targets and affecting pathways for better insight of the disease pathogenesis. Method: The gene expression profile GSE13355 and GSE14905 were retrieved from NCBI, Gene Expression Omnibus database. The GEO profiles were integrated and the DEGs of lesional and non-lesional psoriasis skin were identified using the affy package in R software. The Kyoto Encyclopaedia of Genes and Genomes pathways of the DEGs were analyzed using clusterProfiler. Cytoscape, V3.7.1 was utilized to construct protein interaction network and analyze the interactome map of candidate proteins encoded in DEGs. Functionally relevant clusters were detected through Cytohubba and MCODE. Results: A total of 1013 genes were differentially expressed in lesional skin of which 557 were upregulated and 456 were downregulated. Seven dysregulated genes were extracted in non-lesional skin. The disease gene network of these DEGs revealed 75 newly identified differentially expressed gene that might have a role in development and progression of the disease. GO analysis revealed keratinocyte differentiation and positive regulation of cytokine production to be the most enriched biological process and molecular function. Cytokines -cytokine receptor was the most enriched pathways. Among 1013 identified DEGs in lesional group, 36 DEGs were found to have altered genetic signature including IL1B and STAT3 which are also reported as hub genes. CCNB1, CCNA2, CDK1, IL1B, CXCL8, MKI 67, ESR1, UBE2C, STAT1 and STAT3 were top 10 hub gene. Conclusion: The hub genes, genomic altered DEGs and other newly identified differentially dysregulated genes would improve our understanding of psoriasis pathogenesis, moreover, the hub genes could be explored as potential therapeutic targets for psoriasis.

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Identification of the Differentially Expressed Genes Involved in the Synergistic Neurotoxicity of an HIV Protease Inhibitor and Methamphetamine

Current HIV Research ◽

10.2174/1570162x17666190924200354 ◽

2019 ◽

Vol 17 (4) ◽

pp. 290-303

Author(s):

Sangsang Li ◽

Yanfei Li ◽

Bingpeng Deng ◽

Jie Yan ◽

Yong Wang

Keyword(s):

Protease Inhibitor ◽

Growth Inhibition ◽

Quantitative Polymerase Chain Reaction ◽

Differentially Expressed Gene ◽

Antiretroviral Drugs ◽

Differentially Expressed ◽

Hiv Protease ◽

Hiv Protease Inhibitor ◽

Dopaminergic Cells ◽

Immunodeficiency Syndrome

Background: The abuse of psychostimulants such as methamphetamine (METH) is common in human immunodeficiency virus (HIV)-infected individuals. Acquired immunodeficiency syndrome (AIDS) patients taking METH and antiretroviral drugs could suffer severe neurologic damage and cognitive impairment. Objective: To reveal the underlying neuropathologic mechanisms of an HIV protease inhibitor (PI) combined with METH, growth-inhibition tests of dopaminergic cells and RNA sequencing were performed. Methods: A combination of METH and PI caused more growth inhibition of dopaminergic cells than METH alone or a PI alone. Furthermore, we identified differentially expressed gene (DEG) patterns in the METH vs. untreated cells (1161 genes), PI vs. untreated cells (16 genes), METH-PI vs. PI (3959 genes), and METH-PI vs. METH groups (14 genes). Results: The DEGs in the METH-PI co-treatment group were verified in the brains of a mouse model using quantitative polymerase chain reaction and were involved mostly in the regulatory functions of cell proliferation and inflammation. Conclusion: Such identification of key regulatory genes could facilitate the study of their neuroprotective potential in the users of METH and PIs.

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Gene Set Correlation Analysis and Visualization Using Gene Expression Data

Current Bioinformatics ◽

10.2174/1574893615999200629124444 ◽

2020 ◽

Vol 15 ◽

Author(s):

Chen-An Tsai ◽

James J. Chen

Keyword(s):

Gene Expression ◽

Correlation Analysis ◽

Gene Expression Data ◽

Differentially Expressed Gene ◽

Differentially Expressed ◽

Superior Performance ◽

Expression Data ◽

Gene Set ◽

Gene Sets ◽

Set Correlation

Background: Gene set enrichment analyses (GSEA) provide a useful and powerful approach to identify differentially expressed gene sets with prior biological knowledge. Several GSEA algorithms have been proposed to perform enrichment analyses on groups of genes. However, many of these algorithms have focused on identification of differentially expressed gene sets in a given phenotype. Objective: In this paper, we propose a gene set analytic framework, Gene Set Correlation Analysis (GSCoA), that simultaneously measures within and between gene sets variation to identify sets of genes enriched for differential expression and highly co-related pathways. Methods: We apply co-inertia analysis to the comparisons of cross-gene sets in gene expression data to measure the costructure of expression profiles in pairs of gene sets. Co-inertia analysis (CIA) is one multivariate method to identify trends or co-relationships in multiple datasets, which contain the same samples. The objective of CIA is to seek ordinations (dimension reduction diagrams) of two gene sets such that the square covariance between the projections of the gene sets on successive axes is maximized. Simulation studies illustrate that CIA offers superior performance in identifying corelationships between gene sets in all simulation settings when compared to correlation-based gene set methods. Result and Conclusion: We also combine between-gene set CIA and GSEA to discover the relationships between gene sets significantly associated with phenotypes. In addition, we provide a graphical technique for visualizing and simultaneously exploring the associations of between and within gene sets and their interaction and network. We then demonstrate integration of within and between gene sets variation using CIA and GSEA, applied to the p53 gene expression data using the c2 curated gene sets. Ultimately, the GSCoA approach provides an attractive tool for identification and visualization of novel associations between pairs of gene sets by integrating co-relationships between gene sets into gene set analysis.

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Phylogeny of hymenolepidids (Cestoda: Cyclophyllidea) from mammals: sequences of 18S rRNA and COI genes confirm major clades revealed by the 28S rRNA analyses

Journal of Helminthology ◽

10.1017/s0022149x21000110 ◽

2021 ◽

Vol 95 ◽

Author(s):

B. Neov ◽

G.P. Vasileva ◽

G. Radoslavov ◽

P. Hristov ◽

D.T.J. Littlewood ◽

...

Keyword(s):

Ribosomal Rna ◽

18S Rrna ◽

Phylogenetic Analyses ◽

The Other ◽

Rrna Genes ◽

Host Switching ◽

28S Rrna ◽

Mitochondrial Cytochrome ◽

Rapid Radiation ◽

18S Rrna Genes

Abstract The aim of the study is to test a hypothesis for the phylogenetic relationships among mammalian hymenolepidid tapeworms, based on partial (D1–D3) nuclear 28S ribosomal RNA (rRNA) genes, by estimating new molecular phylogenies for the group based on partial mitochondrial cytochrome c oxidase I (COI) and nuclear 18S rRNA genes, as well as a combined analysis using all three genes. New sequences of COI and 18S rRNA genes were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analyses confirmed the major clades identified by Haukisalmi et al. (Zoologica Scripta 39: 631–641, 2010): Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade. While the Ditestolepis clade is associated with soricids, the structure of the other three clades suggests multiple evolutionary events of host switching between shrews and rodents. Two of the present analyses (18S rRNA and COI genes) show that the basal relationships of the four mammalian clades are branching at the same polytomy with several hymenolepidids from birds (both terrestrial and aquatic). This may indicate a rapid radiation of the group, with multiple events of colonizations of mammalian hosts by avian parasites.

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