Search

Abstract: Ecdysteroid hormone 20-hydroxyecdysone (20E) plays fundamental roles in insect development and reproduction, whereas the primary role of ecdysone (E) is the precursor for 20E. A cytochrome P450 monooxygenase (CYP), encoded by a Halloween gene Shade (Shd, cyp314a1), catalyzes the conversion of E into 20E in representative insect species in Diptera, Lepidoptera and Orthoptera. We describe here the cloning and characterization of LsShd in a hemipteran insect species, the small brown planthopper Laodelphax striatellus. LsSHD has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal expression pattern of LsShd was determined through the fourth-instar and the early fifth-instar stages by qPCR. LsShd showed two expression peaks in day 2 and day 5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Dietary introduction of double-stranded RNA (dsRNA) of LsShd into nymphs successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, and caused nymphal lethality and delayed development. Ingestion of 20E did not increase LsShd expression level, but almost completely rescued LsEcR mRNA level, and relieved the negative effects on the survival and development in LsShd-dsRNA-exposed nymphs. In contrast, dietary introduction of E had little rescue effects. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects, and LsSHD functions to regulate metamorphotic processes by converting E to 20E even in a hemipteran insect, L. striatellus. [Copyright &y& Elsevier]

Abstract: Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. [Copyright &y& Elsevier]

Abstract: Differential expression of ubiquitin was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding hinted at the potential involvement of ubiquitin in deltamethrin resistance, a theory that demanded further testing. Real-time PCR analyses revealed that one of the ubiquitin genes, UBL40, was overexpressed in the deltamethrin-resistant strain during the fourth instar. To investigate the functional relationship between this gene and deltamethrin resistance, RNA interference (RNAi) and cell transfection were utilised. UBL40 knockdown was observed to significantly reduce the level of resistance in RNAi-treated larvae after 48h. Conversely, overexpression of UBL40 in Drosophila Kc cells conferred a degree of protection against deltamethrin. These results represent the first evidence that UBL40 plays a role in the regulation of deltamethrin resistance in P. xylostella. [Copyright &y& Elsevier]

Abstract: Exogenous salicylic acid (SA) significantly improved abiotic tolerance in higher plants, and ascorbate (ASA) and glutathione (GSH) play important roles in abiotic tolerance. In this study, SA (0.5mM) markedly increased the contents of ASA and GSH in SA-treated plants during salt stress (250mM NaCl). The transcript levels of the genes encoding ASA and GSH cycle enzymes were measured using quantitative real-time PCR. The results indicated that, during salt stress, exogenous SA significantly enhanced the transcripts of glutathione peroxidase (GPX1), phospholipid hydroperoxide glutathione peroxidase (GPX2) and dehydroascorbate reductase (DHAR) genes at 12h, glutathione reductase (GR) at 24h, 48h and 72h, glutathione-S-transferase 1 (GST1), 2 (GST2), monodehydroascorbate reductase (MDHAR) and glutathione synthetase (GS) at the 48h and 72h after salt stress, respectively. The results implied that SA temporally regulated the transcript levels of the genes encoding ASA–GSH cycle enzymes, resulting in the increased contents of GSH and ASA and enhanced salt tolerance. [Copyright &y& Elsevier]

Abstract: We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening. [Copyright &y& Elsevier]

Abstract: Monocytes are antigen-presenting cells (APCs) in mammals. Antigen presentation by monocytes via costimulatory molecules was recently confirmed in the Japanese pufferfish Fugu rubripes (also known as Takifugu rubripes) (Sugamata et al., 2009[1]). However, no detailed investigations examining how cytokine gene expression regulates the activation/differentiation of these cells have been conducted to date. Therefore, in this study, the expression of cytokine genes in Fugu monocytes stimulated with TLR agonists (LPS, polyI:C, and IMQ) was profiled. First, the morphological changes and phagocytic activity of stimulated monocytes were examined. At 5days post-stimulation, the ratio of activated to inactivated monocytes increased (as determined by FCM analysis), and the phagocytic activity of the cells was higher (5–15%) than that of nonactivated cells. Analysis of cytokine gene expression in stimulated monocytes revealed that the genes encoding CSF-1b and IFN-γ are important cytokines in activation/differentiation of monocytes/macrophages, and that genes encoding inflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α are upregulated by stimulation. The present study revealed the types of cytokines expressed in Fugu monocytes stimulated with TLR agonists. [Copyright &y& Elsevier]

Highlights: [•] Quantitative real-time-PCR is widely used for gene expression analysis. [•] Internal reference gene selection is critical for data analysis. [•] Three reference genes are analyzed in tung tree (Vernicia fordii). [•] Rpl19b is the most stably expressed gene, followed closely by Ubl. [•] Rpl19b and Ubl are reliable reference genes for both qPCR assays. [Copyright &y& Elsevier]

Abstract: Background: Quantitative real-time PCR (qPCR) is the “gold-standard” technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. Results: We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw C q values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔC q method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized. Conclusions: We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets. [Copyright &y& Elsevier]

Highlights: [•] We reported changes in microbial community under biochar amendment in a field study. [•] Biochar soil amendment (BSA) increased bacterial but decreased fungal gene abundance. [•] Both bacterial and fungal community structure were clearly affected by BSA. [•] Changes in soil enzyme activities could partially support the above findings. [•] BSA could potentially affect the soil C and N cycling of the rice soil ecosystem. [Copyright &y& Elsevier]

Abstract: Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2−/− single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2−/− bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia. [Copyright &y& Elsevier]

Highlights: [•] TP53 mutation influences radiation responses of GBM cells in terms of transcript profiles. [•] Biological processes are differentially affected by irradiation in WT and MT TP53 GBM cell lines. [•] Irradiation of GBM cells caused down-regulation of a larger number of genes in WT TP53 than in MT TP53 cells. [ABSTRACT FROM AUTHOR]

Abstract: Studies of maternal–fetal tolerance focus on defining mechanisms for establishment of immunological privilege within the uterus during pregnancy. Fetal trophoblasts play a key role in maternal tolerance, in part through cytokines production. As a novel inhibitory cytokine, IL-35 is produced by Foxp3+ regulatory T cells (Tregs) and mediates maximal suppression of Tregs. The purpose of the study is to analyze the expression of IL-35 in first-trimester human placental trophoblasts. IL-35 expression was detected at both protein and mRNA levels by immunohistochemical staining and quantitative real-time PCR method, respectively and secretion of IL-35 was measured by ELISA assay. Our results demonstrated that human trophoblasts constitutively expressed IL-35. Ebi3 and p35 (two subunits of IL-35) mRNA was shown to be co-expressed in trophoblast cells. Moreover, large amounts of secreted IL-35 were detected in the supernatants of trophoblast cells. But we did not detect the constitutive expression of IL-35 in decidual stromal cells. Our findings confirmed for the first time that first-trimester human trophoblast cells expressed and secreted IL-35, which might contribute to their suppressive capacity to maternal immune cells. Therefore, IL-35 may be an important factor of the cytokine network regulating local immune responses during human pregnancy. [Copyright &y& Elsevier]

Purpose: We examined the role of annexins in bladder urothelium. We characterized expression and distribution in normal bladders, biopsies from patients with bladder pain syndrome, cultured human urothelium and urothelial TEU-2 cells. Materials and Methods: Annexin expression in bladder layers was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. We assessed cell survival after exposure to the pore forming bacterial toxin streptolysin O by microscopy and alamarBlue® assay. Bladder dome biopsies were obtained from 8 asymptomatic controls and 28 patients with symptoms of bladder pain syndrome. Results: Annexin A1, A2, A5 and A6 were differentially distributed in bladder layers. Annexin A6 was abundant in detrusor smooth muscle and low in urothelium, while annexin A1 was the highest in urothelium. Annexin A2 was localized to the lateral membrane of umbrella cells but excluded from tight junctions. TEU-2 cell differentiation caused up-regulation of annexin A1 and A2 and down-regulation of annexin A6 mRNA. Mature urothelium dedifferentiation during culture caused the opposite effect, decreasing annexin A1 and increasing annexin A6. Annexin A2 influenced TEU-2 cell epithelial permeability. siRNA mediated knockdown of annexin A1 in TEU-2 cells caused significantly decreased cell survival after streptolysin O exposure. Annexin A1 was significantly reduced in biopsies from patients with bladder pain syndrome. Conclusions: Several annexins are expressed in human bladder and TEU-2 cells, in which levels are regulated during urothelial differentiation. Annexin A1 down-regulation in patients with bladder pain syndrome might decrease cell survival and contribute to compromised urothelial function. [Copyright &y& Elsevier]

Quantitative real-time PCR (qPCR), as an important quantitative technique for nucleic acids, has been widely used in many fields including clinical diagnosis, molecular biology, and cancer research. However, non-specific amplification products are still a frequent problem in qPCR. In this study, we investigated the effects of QDs on real-time amplification based on either SYBR Green I or EvaGreen. It was found that QDs could raise the amplification sensitivity and thus enhance the efficiency using SYBR Green I detection system. In the case of EvaGreen detection systems, addition of QDs also led to a better correlation coefficient than without QDs. EvaGreen-based system gave sharper peaks for melting curves than SYBR Green I. The experiments indicated that the polymerase activity could be partially blocked by QDs at the pre-PCR temperatures, resulting in the improvement of PCR specificity. These results indicated that CdTe QDs could be used as a descent qPCR enhancer. Good amplification fidelity in QDs-facilitated qPCR was also a plus that has not been reported elsewhere. [ABSTRACT FROM AUTHOR]

miR‐21 expression in cancer tissue has been reported to be associated with the clinical outcome and activity of gemcitabine in pancreatic cancer. However, resection is possible in only a minority of patients due to the advanced stages often present at the time of diagnosis, and safely obtaining sufficient quantities of pancreatic tumor tissue for molecular analysis is difficult at the unresectable stages. In this study, we investigated whether the serum level of miR‐21 could be used as a predictor of chemosensitivity. We tested the levels of serum miR‐21 in a cohort of 177 cases of advanced pancreatic cancer who received gemcitabine‐based palliative chemotherapy. We found that a high level of miR‐21 in the serum was significantly correlated with a shortened time‐to‐progression (TTP) and a lower overall survival (OS). The serum miR‐21 level was an independent prognostic factor for both the TTP and the OS (HR 1.920; 95% CI, 1.274–2.903, p = 0.002 for TTP and HR 1.705; 95% CI, 1.147–2.535, p = 0.008 for OS). The results from a functional study showed that gemcitabine exposure down‐regulated miR‐21 expression and up‐regulated FasL expression. The increased FasL expression following gemcitabine treatment induced cancer cell apoptosis, whereas the ectopic expression of miR‐21 partially protected the cancer cells from gemcitabine‐induced apoptosis. Additionally, we confirmed that FasL was a direct target of miR‐21. Therefore, the serum level of miR‐21 may serve as a predictor of chemosensitivity in advanced pancreatic cancer. Additionally, we identified a new mechanism of chemoresistance mediated by the effects of miR‐21 on the FasL/Fas pathway. Highlights: ► Serum miR‐21 level predicts chemosensitivity in advanced pancreatic cancer.► High level of serum miR‐21 correlates with shortened time‐to‐progression.► miR‐21 confers chemoresistance of pancreatic cancer cells to gemcitabine.► FasL is a direct target of miR‐21. [ABSTRACT FROM AUTHOR]

Abstract: The potential adverse effects of a biological control agent can be assessed from the knowledge on its environmental fate and behaviour. This study focuses on environmental fate and behaviour of Penicillium oxalicum Currie and Thom strain 212 (PO212), a promising biocontrol agent, after its application to three different soil substrates under different crop conditions in terms of the persistence, survival, vertical dispersion, and horizontal spread. Two different PO212 conidial formulations, one without any additives (F1) and one with additives (F2), were applied to the seedbeds of tomato plants seven days before their transplanting into three soil types: sterilized peat in an experimental glasshouse and the sandy loam and the loamy sand soils in two commercial orchards. The size of the Penicillium spp. population in the substrate samples was quantified using the classical microbiological method of counting of number of colony-forming units on a semi-selective culture medium. The size of PO212 biomass in the samples was quantified by a specific quantitative real-time polymerase chain reaction. We found that PO212 had a very limited vertical dispersion and horizontal spread in the three soil substrates that were tested in the study. PO212 persisted in the natural soil substrates for at least one year at very low levels (0.45–61.2ng dried PO212 biomass/g dry soil or 2.71–367.2ng fresh PO212 biomass/g dry soil). Treatment influenced the horizontal spread of the PO212 biomass in the sterilized peat and sandy loam soils. We did not find significant increases in the size of indigenous Penicillium spp. population (102–103 CFU/g dry soil) in the three soil substrates following application of the F1 or F2 conidial formulations. In the sandy loam soil of the orchard in the VO2009 field trial the Penicillium spp. population decreased at increasing depths. Although PO212 persists in the soil after its application, it does not proliferate. We conclude that PO212 is an effective and safe biocontrol agent to control soil-borne pathogens. [Copyright &y& Elsevier]

Abstract: The heart fatty acid-binding protein gene (h-fabp) plays an important role in intracellular fatty acid transport. h-fabp had been cloned and sequenced and is considered a functional candidate gene for assessing the meat quality traits in pig, chicken, and cattle. However, there have only been few studies reported on this gene in sheep. In the present study, we cloned the full-length cDNA of h-fabp in the Lanzhou fat-tailed sheep using RACE technique. Its tissue specific expression profile in the skeletal muscle, liver and 4 fat depots at seven developmental stages was determined by real-time quantitative PCR. A 425bp 5′-RACE cDNA, 231bp 3′-RACE cDNA and 177bp intermediate fragment were spliced to obtain a 748bp full-length cDNA of h-fabp (GenBank accession number JQ780322). The phylogenetic analysis revealed that the Lanzhou fat-tailed sheep is more closely related to the goat, Capra hircus. In skeletal muscle, h-fabp was expressed at a very low level in one-month-old sheep. The expression level increased in three- and five-month-old sheep, and a peak expression level was observed in seven-month-old sheep. In nine- and eleven-month-old sheep, the gene expression level was similar to the mRNA level observed in three- and five-month-old sheep. In thirteen-month-old sheep, the h-fabp mRNA level was similar to the low expression level observed in one-month-old sheep. The expression pattern observed in the skeletal muscle at different developmental stages in sheep indicates that h-fabp expression declines with age. A similar conclusion on the h-fabp expression pattern in the liver was drawn from our findings in this study. The amount of h-fabp mRNA in the subcutaneous (s.c.) and visceral (v.s.) depots at each developmental stage was very low, and there was no significant difference in the expression levels among these depots. When we added the data from the seven developmental stages together, however, the highest h-fabp expression level was observed in the s.c. fat near the wither (s.c.W), followed by the v.c. perirenal (v.c.P), v.c. omental (v.c.O), and s.c. fat near the base of tail (s.c.T). These findings provide basic data that may be useful in future studies that investigate the role h-fabp plays in fat deposition and the regulation of fatty acid metabolism in sheep. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary–adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin–proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity. [Copyright &y& Elsevier]

Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne''s disease in ruminants and may contribute to Crohn''s disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow''s milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50ml) was statistically (p <0.01) higher than the ICM (0.90–1.97 log cfu/50ml). Data suggested a direct relation (p <0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p >0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p <0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p >0.05). [Copyright &y& Elsevier]

Hantaan viruses cause two severe diseases lacking efficient treatment, yet no effective prophylactic vaccines are available. Continued exploration of alternative antiviral agents to treat hantavirus-related syndromes remains compulsory. The fluorescence-based quantitative real-time PCR (qPCR) has become the touchstone for target gene quantification. In the present study, standard curves for Hantaan virus (HTNV), mouse, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by serial 10-fold dilutions of the constructed recombinant plasmid pGEM-T/HTNV, pGEM-T/mouse-GAPDH, and pGEM-T/human-GAPDH, respectively. Comparisons between the indirect immunofluorescence assay and qPCR assay in the detection of HTNV-infected Vero E6 cells showed improved detection limit and sensitivity of latter method. To characterize the inhibitory effect of several conventional antivirals (arbidol and ribavirin) and unconventional antivirals (indomethacin and curcumin) on HTNV, the levels of viral RNAs were measured for 4 days post-treatment of HTNV-infected Vero E6 cells and 18 days post-inoculation of HTNV-infected suckling mice. Our results validated that HTNV was sensitive to ribavirin and arbidol treatment, while indomethacin and curcumin may also be therapeutically effective in treating HTNV infection. As a result, the establishment and application of qPCR may be a useful tool for the evaluation of potential antivirals for Hantaan virus infection in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Lumpy skin disease (LSD) is a severe disease of bovine characterized by nodules on the skin, mucous membranes, and profuse nasal discharge which causes severe economic losses. In October 2020, an LSD outbreak case was found in Inner Mongolia Autonomous Region, China. A total of 1,206 cattle were sold from the same imported animal quarantine field to 36 farms after the quarantine period finished, and over 30 farmers reported symptoms such as skin scabs found in newly arrived cattle shortly after that. A large-scale LSD outbreak investigation was launched after laboratory diagnosis confirmed LSD. The clinical samples of 1,206 cattle from 36 farms, including 1,206 whole blood, 1,206 oral and nose swabs, and 355 scabs, were collected for the qRT-PCR test. The result showed that 51 whole blood samples (4.23%), 580 swab samples (48.09%), and 350 skin scabs (98.59%) were lumpy skin disease virus (LSDV) positive, 33 of 36 farms were affected. This study aims to provide a basis for LSD epidemiological traceability, movement control, and measures for prevention and control., (Copyright © 2022 Li, Qi, Li, Nan, Wang, Chang, Chi, Gong, Ha, De, Ma and Wu.)

Environmental DNA (eDNA) analysis is a promising tool for the sensitive and effective monitoring of species distribution and abundance. Traditional eDNA analysis has targeted mitochondrial DNA (mtDNA) fragments due to their abundance in cells; however, the quantification may vary depending on cell type and physiology. Conversely, some recent eDNA studies have targeted multi-copy nuclear DNA (nuDNA) fragments, such as ribosomal RNA genes, in water, and reported a higher detectability and more rapid degradation than mitochondrial eDNA (mt-eDNA). These properties suggest that nuclear eDNA (nu-eDNA) may be useful for the accurate estimation of species abundance relative to mt-eDNA, but which remains unclear. In this study, we compiled previous studies and re-analyzed the relationships between mt- and nu-eDNA concentration and species abundance by comparing the R 2 values of the linear regression. We then performed an aquarium experiment using zebrafish (Danio rerio) to compare the relationships across genetic regions, including single-copy nuDNA. We found more accurate relationships between multi-copy nu-eDNA and species abundance than mt-eDNA in these datasets, although the difference was not significant upon weighted-averaging the R 2 values. Moreover, we compared the decay rate constants of zebrafish eDNA across genetic regions and found that multi-copy nu-eDNA degraded faster than mt-eDNA under pH 7, implying a quick turnover of multi-copy nu-eDNA in the field. Although further empirical studies of nu-eDNA applications are necessary to support our findings, this study provides the groundwork for improving the estimation accuracy of species abundance via eDNA analysis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Abstract: Quantitative real-time PCR (qPCR) commonly uses the fluorogenic 5′ nuclease (TaqMan) and SYBR Green I (SG) detection chemistries to enumerate biomarker genes. Dehalococcoides (Dhc) are keystone bacteria for the detoxification of chlorinated ethenes, and the Dhc 16S ribosomal RNA (rRNA) gene serves as a biomarker for monitoring reductive dechlorination in contaminated aquifers. qPCR enumeration of Dhc biomarker genes using the TaqMan or SG approach with the same primer set yielded linear calibration curves over a seven orders of magnitude range with similar amplification efficiencies. The TaqMan assay discriminates specific from nonspecific amplification observed at low template concentrations with the SG assay, and had a 10-fold lower limit of detection of ~3 copies per assay. When applied to Dhc pure cultures and Dhc-containing consortia, both detection methods enumerated Dhc biomarker genes with differences not exceeding 3-fold. Greater variability was observed with groundwater samples, and the SG chemistry produced false-positive results or yielded up to 6-fold higher biomarker gene abundances compared to the TaqMan method. In most cases, the apparent error associated with SG detection resulted from quantification of nonspecific amplification products and was more pronounced with groundwater samples that had low biomarker concentrations or contained PCR inhibitors. Correction of the apparent error using post-amplification melting curve analysis produced 2 to 21-fold lower abundance estimates; however, gel electrophoretic analysis of amplicons demonstrated that melting curve analysis was insufficient to recognize all nonspecific amplification. Upon exclusion of nonspecific amplification products identified by combined melting curve and electrophoretic amplicon analyses, the SG method produced false-negative results compared to the TaqMan method. To achieve sensitive and accurate quantification of Dhc biomarker genes in environmental samples (e.g., groundwater) and avoid erroneous conclusions, the analysis should rely on TaqMan detection chemistry, unless additional analyses validate the results obtained with the SG approach. [Copyright &y& Elsevier]

Background: High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results: We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions: Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants. [ABSTRACT FROM AUTHOR]

Abstract: There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments. [ABSTRACT FROM AUTHOR]

Abstract: Exposure of aquatic organisms to 17β-estradiol (E2) induces a variety of estrogen-responsive genes, including vitellogenin (vtg)—the precursor protein of egg yolk in oviparous animals and to date the single most used gene product in screening for estrogenic endocrine disruption. Transcription regulation of vtg by E2 is dependent on binding of the ligand (E2) to a specific nuclear receptor (estrogen receptor, ESR) which in turn binds to an estrogen responsive element (ERE) in the promoter of vtg. Since a local tilapiine, Oreochromis mossambicus (Peters), is targeted as a model for estrogenic endocrine disruption in Southern Africa, a platform of knowledge is necessary for the ontogenic and tissue specific behavior of ESR in this species before vtg levels can be interpreted in relation to such endocrine disruption. Therefore, three ESR cDNA sequences (ESR1, ESR2a and ESR2b) in O. mossambicus were isolated and QPCR protocols were developed to ascertain their quantitative transcript levels in adult brain, gonadal and hepatic tissues. ESR1 transcript levels were highest in female liver tissue compared to males and other tissues, whereas the levels for ESR2a and b were not statistically significantly different between male and female tissues. Quantitative gene levels during development demonstrated a sharp increase in ESR1 during the stage of gonad differentiation (50–60 days post-fertilization) in this species. Finally, an induction experiment in adult male liver tissue confirms the upregulation of ESR1 by E2. [Copyright &y& Elsevier]

Abstract: Gene expression studies using postmortem human brain tissue are a common tool for studying the etiology of psychiatric disorders. Quantitative real-time PCR (qPCR) is an accurate and sensitive technique used for gene expression analysis in which the expression level is quantified by normalization to one or more reference genes. Therefore, accurate data normalization is critical for validating results obtained by qPCR. This study aimed to identify genes that may serve as reference in postmortem dorsolateral–prefrontal cortices (Brodmann’s area 46) of schizophrenics, bipolar disorder (BPD) patients, and control subjects. In the exploratory stage of the analysis, samples of four BPD patients, two schizophrenics, and two controls were quantified using the TaqMan Low Density Array endogenous control panel, containing assays for 16 commonly used reference genes. In the next stage, six of these genes (TFRC, RPLP0, ACTB, POLR2a, B2M, and GAPDH) were quantified by qPCR in 12 samples of each clinical group. Expressional stability of the genes was determined by GeNorm and NormFinder. TFRC and RPLP0 were the most stably expressed genes, whereas the commonly used 18S, POLR2a, and GAPDH were the least stable. This report stresses the importance of examining expressional stability of candidate reference genes in the specific sample collection to be analyzed. [Copyright &y& Elsevier]

Abstract: Pathology practice is increasingly augmented with molecular tests for improved diagnostics and patient management. The polymerase chain reaction (PCR) is foremost amongst these techniques. This review explains the principles of PCR and the methodological factors that contribute to a successful assay. Key PCR technique variations, such as reverse transcription (RT)-PCR and quantitative real-time (q) PCR, are described and an overview is provided of how PCR products are analysed. The review includes examples of PCR usage in clinical practice for the detection of infectious and genetic diseases, for tumour diagnostics and in molecular forensic applications such as specimen identity confirmation. [Copyright &y& Elsevier]

Abstract: Increased levels of differentially regulated trout protein 1 (DRTP1) mRNA transcripts have been reported in fish after activation of the acute phase response. While the function of the DRTP1 protein still remains to be elucidated, this study focused on the genomic organisation of the gene, the quantification of the DRTP1 transcript in various tissues, and the isolation and analysis of the 5′ regulatory region of the DRTP1 gene in rainbow trout (Oncorhynchus mykiss). Analysis of the DRTP1 genomic and cDNA sequences showed the gene to consist of four exons separated by three introns. Tissue localisation of the DRTP1 gene was performed by Northern analysis and validated by quantitative real-time PCR (qPCR). Six tissues (liver, intestine, spleen, brain, pituitary, and hypothalamus) were analysed. The tissues with the most abundant transcripts were the liver and the pituitary, with lesser amounts detected in the intestine, hypothalamus, brain and spleen. Genome walking allowed the isolation of a 934 bp sequence of the 5′ regulatory region of the gene which was cloned, sequenced and in which potential transcription factor binding sites were identified. Promoter fragments of decreasing size were generated and transiently transfected into the human hepatoma cell line (HepG2). Inducibility of the promoter was determined by stimulation of the HepG2 cells containing the constructs with dexamethasone, polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFα). One construct, containing two potential C-EBP/β sites and two NF-κB sites, exhibited the highest promoter induction (6.34 fold ± SEM 0.5) when stimulated with human TNFα. A slightly shorter fragment containing one C-EBP/β site and one NF-κB site did not show any significant inducibility when treated with TNFα. The loss of the C-EBP/β and NF-κB in the shorter construct suggests that these sites, either individually or in combination, are critical for the induction of the DRTP1 promoter by TNFα. [Copyright &y& Elsevier]

Abstract: Longitudinal case–control studies were performed in post-weaning multisystemic wasting syndrome (PMWS) affected farms from Denmark and Spain using similar designs. Fourteen independent batches of 100–154 pigs per batch were monitored from birth to PMWS outbreak occurrence. Pigs displaying PMWS-like signs and matched healthy cohorts were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. Porcine circovirus type 2 (PCV2) quantitative PCR (qPCR) and serology techniques were applied to analyse longitudinally collected sera and/or nasal and rectal swabs. Results showed that PCV2 load increased in parallel to waning maternal antibody levels, reaching the maximum viral load concurrent with development of clinical signs. PMWS affected pigs had higher PCV2 prevalence and/or viral load than healthy pigs in all collected samples at necropsy (p <0.0001–0.05) and even in sera and nasal swabs at the sampling prior to PMWS outbreak (p <0.01–0.05). Danish farms had a higher PCV2 prevalence in young piglets as well as an earlier PMWS presentation compared to Spanish farms. PMWS diagnoses were confirmed by laboratory tests in only half of pigs clinically suspected to suffer from PMWS. Positive and significant correlations were found among PCV2 viral loads present in sera, nasal swabs, rectal swabs and lymphoid tissues (R =0.289–0.827, p <0.0001–0.01), which indicates that nasal and rectal swabs were suitable indicators of PCV2 excretion. Sensitivity and/or specificity values observed from both tests used separately or combined suggested that qPCR and/or serology tests are not apparently able to substitute histopathology plus detection of PCV2 in tissues for the individual PMWS diagnosis within PMWS affected farms. However, qPCR appears to be a potential reliable technique to diagnose PMWS on a population basis. [Copyright &y& Elsevier]

Abstract: Canine respiratory coronavirus (CRCoV) has been detected recently in dogs with canine infectious respiratory disease and is involved in the clinical disease complex. CRCoV is a group 2 coronavirus most closely related to bovine coronavirus and human coronavirus OC43. A real-time PCR assay was developed for the detection of CRCoV. The assay was validated against cell culture grown virus and shown to have a high level of sensitivity. A range of tissue samples were collected from dogs at a re-homing centre with a history of endemic respiratory disease. The samples were tested using a conventional nested PCR assay and CRCoV was quantitated by real-time PCR. CRCoV was detected most frequently in the nasal mucosa, nasal tonsil and trachea. It was also detected in the lung, and bronchial lymph node. Of the enteric tissues, only one mesenteric lymph node sample was positive. In addition two colon samples were positive for CRCoV by nested PCR only. In conclusion, CRCoV appears to infect the upper respiratory tract preferentially. The CRCoV real-time PCR assay has proved to be a highly specific and sensitive assay that can be applied for diagnostic purposes as well as to investigate further the tissue tropism of CRCoV. [Copyright &y& Elsevier]

Abstract: In this study, we explored whether SYBR Green-based quantitative real-time PCR (qPCR) could be used to determine the copy number of a plasmid and whether the method was broadly applicable to chromosomally encoded genetic elements often occurring in multiple copies, such as rRNA genes and insertion sequences (IS). Three different template sources (whole cells, total DNA, and restriction-enzyme digested total DNA) derived from the bacterium Comamonas sp. strain JS46 were analyzed by qPCR using primer-pairs targeting plasmid pJS46 and three chromosomally encoded sequences (16S rDNA, ISCsp1, and IS1071). The difference between threshold cycle values, C T, of amplicons targeting these elements and of an amplicon targeting the single-copy reference element mnbA (chromosomally encoded) was used to establish ΔC T. ΔC T values were then used to derive copy number. For pJS46, qPCR analyses of whole cells and total DNA underestimated the copy number of pJS46 ∼7-fold and ∼2.5-fold, respectively, whereas copy number values derived from qPCR analyses of digested total DNA were comparable to those derived from Southern blot (SB) analyses. In contrast, for the chromosomally encoded elements, qPCR analyses of all three template sources gave copy number values that were virtually identical to or differed by ∼2 from copy number values derived by SB analysis. These data indicate that qPCR can be used to estimate the copy number of various genetic elements but that the accuracy of qPCR-derived values is affected by the template source. [Copyright &y& Elsevier]

Scrub typhus is a potentially severe rickettsiosis, caused by Orientia tsutsugamushi in the Asia-Pacific region. Recently, however, two distinct pathogens, " Candidatus Orientia chuto" and " Candidatus Orientia chiloensis", have been discovered in the Middle East and South America, respectively. Since the novel pathogens differ significantly from O. tsutsugamushi , many established diagnostic methods are unreliable. This work describes the development and validation of a new quantitative real-time PCR (qPCR) assay (Orien16S) for the detection of all known Orientia species. Based on a 94 bp sequence of the 16S rRNA gene ( rrs ), Orien16S recognized DNA samples from O. tsutsugamushi ( n = 41), Ca. O. chiloensis ( n = 5), and Ca. O. chuto ( n = 1), but was negative for DNA preparations from closely related rickettsiae and other members of the order Rickettsiales ( n = 22) as well as unrelated bacterial species ( n = 11). After its implementation in Chile, the assay was verified, correctly identifying all tested eschar and buffy coat samples ( n = 28) of clinical suspected cases. Furthermore, Orien16S detected Orientia DNA in trombiculid mites collected in endemic regions in southern Chile. The presented novel qPCR assay provides a useful tool for detecting Orientia and diagnosing scrub typhus from all geographical regions., Competing Interests: JJ and AR are employed by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jiang, Martínez-Valdebenito, Weitzel, Farris, Acosta-Jamett, Abarca and Richards.)

Enniatins (ENs) are depsipeptide mycotoxins produced by Fusarium fungi. They are known for their capacity to modulate cell membrane permeability and disruption of ionic gradients, affecting cell homeostasis and initiating oxidative stress mechanisms. The effect of the acute toxicity of ENs A, A1, B and B1 at two different concentrations after 8 h of exposure was analysed in Wistar rats by a transcriptional approach. The following key mitochondrial and nuclear codified genes related to the electron transport chain were considered for gene expression analysis in stomach, liver, kidney and lower intestine by quantitative Real-Time PCR: mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded cytochrome c oxidase 1 (MT-COX1), succinate dehydrogenase flavoprotein subunit A and ATP synthase F1 subunit alpha, respectively. Moreover, the expression of markers involved in oxidative stresssuperoxide dismutase 1 (SOD1), glutathione peroxidase 1 (Gpx1), heme oxygenase 1, apoptosis B-cell lymphoma 2, Bcl2 Associated protein X (Bax), tumor suppressor protein (p53), inhibition of apoptosis nuclear factor kappa of activated B cells, immune system interleukin 1β and intestinal tight junction Occludin merely in lower intestine tissues have been investigated. For mitochondrial genes, the main differences were observed for MT-ND1 and MT-COX1, showing its deficiency in all selected organs. With regard to the intestinal barrier's cellular response to oxidative stress, the activity of the antioxidant gene SOD1 was decreased in a dose-dependent manner. Similarly, the catalytic enzyme GPx1 was also downregulated though merely at medium dose employed. On the contrary, the pro-apoptotic Bax and p53 regulators were activated after ENs exposure, reporting a significant increase in their expression. Furthermore, the alteration of intestinal permeability was assessed by the abnormal activity of the tight junction protein occludin. In summary, ENs may generate mitochondrial disorders and induce oxidative stress in intestinal barrier function. [ABSTRACT FROM AUTHOR]

Abstract Background High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

The fungus Magnaporthiopsis maydis is a soil-borne, seed-borne vascular wilt pathogen that causes severe damage to sensitive Zea mays L. (maize) hybrids throughout Egypt, Israel, India, Spain, and other countries. It can undergo virulence variations and survive as spores, sclerotia, or mycelia on plant residues. Maize, Lupinus termis L. (lupine) and Gossypium hirsutum L. (cotton) are the only known hosts of M. maydis. Identification of new plant hosts that can assist in the survival of the pathogen is an essential step in restricting disease outbreak and spread. Here, by field survey and growth chamber pathogenicity test, accompanied by real-time PCR analysis, the presence of the fungal DNA inside the roots of cotton (Pima cv.) plants was confirmed in infested soil. Moreover, we identified M. maydis in Setaria viridis (green foxtail) and Citrullus lanatus (watermelon, Malali cv.). Infected watermelon sprouts had delayed emergence and development, were shorter, and had reduced root and shoot biomass. M. maydis infection also affected root biomass and phenological development of cotton plants but caused only mild symptoms in green foxtail. No M. maydis DNA was detected in Hordeum vulgare (barley, Noga cv.) and the plants showed no disease symptoms except for reduced shoot weight. These findings are an important step towards uncovering the host range and endophytic behavior of M. maydis, encouraging expanding this evaluation to other plant species. [ABSTRACT FROM AUTHOR]

The complexity of natural soils presents a challenge to the systematic identification and disentanglement of governing processes that shape natural bacterial communities. Studies have highlighted the critical role of the soil aqueous phase in shaping interactions among soil bacterial communities. To quantify and improve the attributability of soil aqueous-phase effects, we introduced a synthetic and traceable bacterial community to simple porous microcosms and subjected the community to constant or dynamic hydration conditions. The results were expressed in terms of absolute abundance and show species-specific responses to hydration and nutrient conditions. Hydration dynamics exerted a significant influence on the fraction of less-abundant species, especially after extended incubation periods. Phylogenetic relationships did not explain the group of most abundant species. The ability to quantify species-level dynamics in a bacterial community offers an important step toward deciphering the links between community composition and functions in dynamic terrestrial environments. IMPORTANCE The composition and activity of soil bacteria are central to various ecosystem services and soil biogeochemical cycles. A key factor for soil bacterial activity is soil hydration, which is in a constant state of change due to rainfall, drainage, plant water uptake, and evaporation. These dynamic changes in soil hydration state affect the structure and function of soil bacterial communities in complex ways often unobservable in natural soil. We designed an experimental system that retains the salient features of hydrated soil yet enables systematic evaluation of changes in a representative bacterial community in response to cycles of wetting and drying. The study shows that hydration cycles affect community abundance, yet most changes in composition occur with the less-abundant species (while the successful ones remain dominant). This research offers a new path for an improved understanding of bacterial community assembly in natural environments, including bacterial growth, maintenance, and death, with a special focus on the role of hydrological factors., (Copyright © 2019 Kleyer et al.)

Colorectal cancer (CRC) is one of the most common malignancies, giving rise to serious financial burden globally. This study was designed to explore the potential mechanisms implicated with CRC and identify some key biomarkers. CRC-associated gene expression dataset (GSE32323) was downloaded from GEO database. The differentially expressed genes (DEGs) were selected out based on the GEO2R tool. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to search the enriched pathways of these DEGs. Additionally, a protein-protein interaction (PPI) network was also constructed to visualize interactions between these DEGs. Quantitative Real-time PCR (qPCR) was further performed to valid the top5 up-regulated and top5 down-regulated genes in patients with CRC. Finally, the survival analysis of the top5 up-regulated and top5 down-regulated genes was conducted using GEPIA, aiming to clarify their potential effects on CRC. In this study, a total of 451 DEGs were captured (306 down-regulated genes and 145 up-regulated genes). Among these DEGs, the top5 up-regulated genes were DPEP1, KRT23, CLDN1, LGR5 and FOXQ1 while the top5 down-regulated genes were CLCA4, ZG16, SLC4A4, ADH1B and GCG. GO analysis revealed that these DEGs were mainly enriched in cell adhesion, cell proliferation, RNA polymerase II promoter and chemokine activity. KEGG analysis disclosed that the enriched pathway included mineral absorption, chemokine signaling pathway, transcriptional misregulation in cancer, pathways in cancer and PPAR signaling pathway. Survival analysis showed that the expression level of ZG16 may correlate with the prognosis of CRC patients. Furthermore, according to the connectivity degree of these DEGs, we selected out the top15 hub genes, namely MYC, CXCR1, TOP2A, CXCL12, SST, TIMP1, SPP1, PPBP, CDK1, THBS1, CXCL1, PYY, LPAR1, BMP2 and MMP3, which were expected to be promising therapeutic target in CRC. Collectively, our analysis unveiled potential biomarkers and candidate targets in CRC, which could be helpful to the diagnosis and treatment of CRC.