Relative Quantification Of Gene Expression Research Articles

Influence of support materials on the electroactive behavior, structure and gene expression of wild type and GSU1771-deficient mutant of Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens DL1 is a metal-reducing dissimilatory bacterium frequently used to produce electricity in bioelectrochemical systems (BES). The biofilm formed on electrodes is one of the most important factors for efficient electron transfer; this is possible due to the production of type IV pili and c-type cytochromes that allow it to carry out extracellular electron transfer (EET) to final acceptors. In this study, we analyzed the biofilm formed on different support materials (glass, hematite (Fe2O3) on glass, fluorine-doped tin oxide (FTO) semiconductor glass, Fe2O3 on FTO, graphite, and stainless steel) by G. sulfurreducens DL1 (WT) and GSU1771-deficient strain mutant (Δgsu1771). GSU1771 is a transcriptional regulator that controls the expression of several genes involved in electron transfer. Different approaches and experimental tests were carried out with the biofilms grown on the different support materials including structure analysis by confocal laser scanning microscopy (CLSM), characterization of electrochemical activity, and quantification of relative gene expression by RT-qPCR. The gene expression of selected genes involved in EET was analyzed, observing an overexpression of pgcA, omcS, omcM, and omcF from Δgsu1771 biofilms compared to those from WT, also the overexpression of the epsH gene, which is involved in exopolysaccharide synthesis. Although we observed that for the Δgsu1771 mutant strain, the associated redox processes are similar to the WT strain, and more current is produced, we think that this could be associated with a higher relative expression of certain genes involved in EET and in the production of exopolysaccharides despite the chemical environment where the biofilm develops. This study supports that G. sulfurreducens is capable of adapting to the electrochemical environment where it grows.

Differentiation-inducing effect of osteoclast microgrooves for the purpose of three-dimensional design of regenerated bone.

In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 μm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 μm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 μm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. STATEMENT OF SIGNIFICANCE: Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-μm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment.

INVESTIGATION OF THE PROTECTIVE EFFECTS OF POMEGRANATE (Punica granatum L.) PEEL EXTRACT ON LIPOPOLYSACCHARIDE-INDUCED UVEITIS IN RATS

Pomegranate peel contains bioactive ingredients such as flavonoids, ellagitannins, phenolics and proanthocyanidin compounds with high antioxidant activity. Pomegranate peel has antiapoptotic, antioxidant and anti-inflammatory effects due to its high punicalagin content. We aimed to determine the effect of pomegranate peel extract (PPE) on lipopolysaccharide (LPS)-induced uveitis. Sixty rats were seperated randomly into twelve groups (n = 5). The healthy group received intraperitoneal normal saline, the uveitis group received 200 μg/kg LPS, the dexamethasone (DEX) group received 200 μg/kg LPS plus 1 mg/kg DEX, the PPE100, PPE300 and PPE500 groups received 200 μg/kg LPS plus 100, 300 and 500 mg/kg PPE, respectively. The eye tissues were collected at 3rd and 24th hour. and investigated molecularly (Relative quantification of gene expression), biochemically (Superoxide dismutase activity, Glutathione and Malondialdehyde levels) and histopathologically (staining with Harris Hematoxylin and Eosin Y). Tumor Necrosis Factor-α, vascular endothelial growth factor, and Caspase-3 levels markedly decreased in a dose-dependent manner in the uveitic rats following PPE administration. PPE administration significantly ameliorated uveitic disorders in oxidative stress factors including Glutathione, Superoxide dismutase and Malondialdehyde, with its effects raising in a dose-dependent manner. PPE eliminated histopathological changes in eye tissues due to uveitis. PPE can be a promising agent by contributing to alternative preventive treatment methods for uveitis with its anti-inflammatory, antioxidative, antiapoptotic and antiangiogenic effects.

Expression Signature of Immune-Related MicroRNAs in Autoimmune Skin Disease: Psoriasis and Vitiligo Insights.

Psoriasis and vitiligo are both chronic, skin-specific diseases classified as autoimmune diseases due to the involvement of several biochemical pathways in their pathogenesis, similar to those altered in other autoimmune diseases. The role of miRNAs in regulating skin autoimmune function has yet to be fully characterized. The aim of this study was to assess the expression profile of a panel of 11 circulating immune-related miRNAs in patients with autoimmune skin diseases, specifically psoriasis and vitiligo, and correlate their expression signature with the clinicopathological features of the diseases. Relative gene expression quantification for 11 immune-related circulating miRNAs in plasma was done for 300 subjects-100 patients with psoriasis, 100 patients with vitiligo and 100 normal healthy volunteers-followed by different modalities of bioinformatics analysis for the results. The expression levels of all the studied immune-related miRNAs were elevated in both autoimmune skin disorders, with much higher levels of expression in psoriasis than in vitiligo patients. There was a significant correlation between most of the studied miRNAs, suggesting shared target genes and/or pathways. Moreover, all the studied miRNAs showed significant results as biomarkers for autoimmune skin disease, with miRNA-145 being the best candidate. Regarding the clinicopathological data, miRNA-7, miRNA-9, miRNA-145, miRNA-148a, and miRNA-148b were positively correlated with age. All the miRNAs were inversely correlated with obesity and disease duration. This study highlights the critical role of miRNAs in skin-specific autoimmune diseases that proved to be potential biomarkers for autoimmune skin disorders, warranting their exploration as therapeutic targets.

Exercise-induced changes in gene expression do not mediate post exertional malaise in Gulf War illness

BackgroundPost-exertional malaise (PEM) is considered a characteristic feature of chronic multi-symptom illnesses (CMI) like Gulf War illness (GWI); however, its pathophysiology remains understudied. Previous investigations in other CMI populations (i.e., Myalgic Encephalomyelitis/Chronic Fatigue Syndrome) have reported associations between PEM and expression of genes coding for adrenergic, metabolic, and immune function. ObjectivesTo investigate whether PEM is meditated by gene expression in Veterans with GWI. MethodsVeterans with GWI (n = 37) and healthy control Gulf War Veterans (n = 25) provided blood samples before and after 30-min of cycling at 70% of age-predicted heart rate reserve. Relative quantification of gene expression, symptom measurements, and select cardiopulmonary parameters were compared between groups at pre-, 30 minpost-, and 24 hpost-exercise using a doubly multivariate repeated measures analysis of variance (RM-MANOVA). Mediation analyses were used to test indirect effects of changes in gene expression on symptom responses (i.e., PEM) to the standardized exercise challenge. ResultsVeterans with GWI experienced large symptom exacerbations following exercise compared to controls (Cohen's d: 1.65; p < 0.05). Expression of β-actin (ACTB), catechol-O-methyltransferase (COMT), and toll-like receptor 4 (TLR4) decreased in Veterans with GWI at 30 min (p < 0.05) and 24 h post-exercise (p < 0.05). Changes in gene expression did not mediate post-exercise symptom exacerbation in GWI (Indirect Effect Slope Coefficient: 0.06 – 0.02; 95% CI: 0.19, 0.12). ConclusionAn acute bout of moderate intensity cycling reduced the expression of select structural, adrenergic, and immune genes in Veterans with GWI, but the pathophysiological relevance to PEM is unclear.

CCR5∆32 and SDF1 3'A: Gene Variants, Expression and Influence on Biological Markers for the Clinical Progression to AIDS among HIV-1 Virus Controllers in a Mixed Population of the Amazon Region of Brazil.

CCR5Δ32 and SDF1-3'A polymorphisms were investigated in a cohort of viremia controllers, without the use of therapy, along with their influence on CD4+ T lymphocytes (TLs), CD8+ TLs, and plasma viral load (VL). The samples were analyzed from 32 HIV-1-infected individuals classified as viremia controllers 1 and 2 and viremia non-controllers, from both sexes, mostly heterosexuals, paired with 300 individuals from a control group. CCR5∆32 polymorphism was identified by PCR amplification of a fragment of 189 bp for the wild-type allele and 157 bp for the allele with the ∆32 deletion. SDF1-3'A polymorphism was identified by PCR, followed by enzymatic digestion (restriction fragment length polymorphism) with the Msp I enzyme. The relative quantification of gene expression was performed by real-time PCR. The distribution of allele and genotype frequencies did not show significant differences between the groups. The gene expression of CCR5 and SDF1 was not different between the profiles of AIDS progression. There was no significant correlation between the progression markers (CD4+ TL/CD8+ TL and VL) and the CCR5∆32 polymorphism carrier status. The 3'A allele variant was associated with a marked loss of CD4+ TLs and a higher plasma VL. Neither CCR5∆32 nor SDF1-3'A was associated with viremia control or the controlling phenotype.

Expression of MicroRNA-155 in Patients with Non-Hodgkin Lymphoma, Coronavirus Disease 2019, or Both: A Cross-Sectional Study.

Non-Hodgkin lymphoma (NHL) is the eleventh leading cause of cancer-related death in the world. Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL. Up to winter 2021-2022, the death toll caused by the coronavirus disease 2019 (COVID-19) has exceeded 5.6 million worldwide. Possible molecular mechanisms involved in the systemic inflammation, and cytokine storm in COVID-19 patients are still not fully understood. MicroRNA-155 (miR-155) plays a role in the post-transcriptional gene regulation of hematopoiesis, oncogenesis, and inflammation. The present study aimed to evaluate the expression of miR-155 in patients with DLBCL and/or COVID-19. A cross-sectional study was conducted from July to December 2020 in Tehran (Iran) to evaluate the expression of miR-155 in adult patients diagnosed with DLBCL and/or COVID-19. The real-time polymerase chain reaction technique was used to evaluate the expression of miR-155 in the sera of 92 adults who were either healthy or suffering from DLBCL and/or COVID-19. Relative quantification of gene expression was calculated in terms of cycle threshold (Ct) value. Data were analyzed using SPSS software, and P<0.05 was considered statistically significant. The expression of miR-155 was not associated with the sex or age of the participants. In comparison with healthy individuals (-ΔCt -1.92±0.25), the expression of miR-155 increased in patients with COVID-19 (1.95±0.14), DLBCL (2.25±0.16), or both (4.33±0.65). The expression of miR-155 increased in patients with DLBCL and/or COVID-19.

Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms.

Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determine the expression stability of commonly used RGs in mouse skeletal muscle tissue. The expression pattern of eight RGs (ACTB, GAPDH, HPRT, YWHAZ, B2M, PPIA, TUBA and 18S) were evaluated by RT-qPCR in different sample groups classified based on genetic background, muscle tissue type, and growth stage, as well as in a C2C12 myoblast cell line model. Five computational programs were included in the study (comparative ΔCq value, NormFinder, BestKeeper, geNorm, RefFinder) to evaluate the expression stability of RGs. Furthermore, the normalization effects of RGs in soleus (SOL) and gastrocnemius (GAS) muscle tissue were evaluated. Collectively, ACTB, HPRT and YWHAZ were shown to be the most stable RGs, while GADPH and 18S were the least stable. Therefore, the combined use of ACTB, HPRT and YWHAZ is recommended for the normalization of gene expression results in experiments with murine skeletal muscle. The results discussed herein provide a foundation for gene expression analysis by RT-qPCR in mammalian skeletal muscle.

MO060: HIF1Α and Succinate Pathway: Role in Deceased Kidney Donors Inflammation

Abstract BACKGROUND AND AIMS The type of donor has an important impact on the renal prognosis of the recipient. Thus, it has been observed that the decline of renal function is higher in deceased donor (DD) than those observed in living donors (LD). These could be related with the pre-implantation inflammation state of the donor and the development of fibrotic processes. Own data indicate that the enzymatic complex succinate dehydrogenase (SDH) has a lower gene expression in DD grafts and is associated with inflammatory markers, macrophages and purinergic receptors and a worse renal function after transplantation. The aim of this study is to determine that in renal DD donation, ischemia/hypoxia can increase renal succinate levels, inducing infiltration of inflammatory cells. METHOD Preimplantation kidneys biopsies from 47 DD and 19 LD were collected in our institution. About 159 genes from these samples were analyzed by qRT–PCR with the TaqMan gene expression assay. Relative quantification of gene expression was performed with three internal controls. Genes with high positive correlation with HIF1α (Rho &amp;gt; 0.5) have been grouped in clusters using STRING database (http://string-db.org/). Succinate levels were measured in serum from 27 renal DD and in 10 healthy volunteers with the EnzyChromTM Succinate Assay Kit. All statistical data were analyzed using GraphPad Prism 5. HIF, SDH subunits and succinate levels in serum were analyzed with a Student's T-test (P-value of 0.001). Pearson correlation Test (Rho &amp;gt;0.5) was performed to analyze interdependence of HIF1α with the rest of genes. RESULTS To determine the hypoxia status of kidney donors, transcription levels of HIF1α and HIF2α were determined from renal tissue samples. In the graphic (Figure. 1A), a significant increase in HIF1α genic expression is observed in DD compared with LD (P &amp;lt; 0.001). The next figure (Figure. 2B) shows clusters in DD that are not present in LD and have a high correlation with HIF1a (Rho &amp;gt; 0.5). Twenty-four genes showed this strong correlation and can be grouped in three different clusters: Cluster 1: Transcription factors and metalloproteinases, Cluster 2: Extracellular Matrix (ECM) proteins and integrins and finally, Cluster 3: Inflammatory and Macrophages Markers. As is shown Figure 2A, we found reduced mRNA expression of all the SDH subunits (SDHA, SDHB, SDHC and SDHD) (P &amp;lt; 0.001) in DD samples compared with LD ones. As a consequence, succinate cannot be converted to fumarate in concordance with the greater succinate levels (P = 0.002) observed in sera of DD shown in the picture (Figure. 2B). CONCLUSION These results indicate that kidneys from DD undergo a greater hypoxia compared with LD, demonstrated by the increased transcription rate of HIF1α. Hypoxia not only increases HIF1α but also may disrupt the Krebs cycle, reducing the expression of SDH subunits, increasing extracellular levels of succinate that has a role in the maintenance of HIF1α levels, which could take part in a sustained inflammatory/anti-inflammatory process in kidney from DD.

Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury).

Simple SummaryReverse transcription-quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most accessible and widely-applied method for quantifying gene transcription. Stable reference genes need to be normalized to facilitate gene expression studies and obtain more accurate qRT-PCR data. In this research, eight reference genes from different functional categories and gene families were used to assess the stability level of gene expression under different conditions in H. cunea. Finally, the expression pattern of the target gene serine proteases (HcSP1) was evaluated under different conditions to verify the precision and reliability of the results. This study provides a comprehensive list of suitable reference genes for analyses and optional reference genes for other gene function research in H. cunea.The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

Gut leakage markers in response to strenuous exercise in patients with suspected coronary artery disease

Abstract Introduction Although regular physical activity is associated with reduced risk of cardiovascular disease (CVD), acute vigorous exercise seems to transiently increase the risk of acute coronary events in patients with underlying CVD. Some studies have reported regular physical activity to associate with microbial diversity, whereas elevated levels of gut leakage markers have been shown after strenuous exercise in healthy individuals. Any predictive value of a temporary increase in gut leakage markers on the risk of coronary events in susceptible individuals is unknown. Purpose We aimed to explore gut leakage markers in response to a bout of strenuous exercise in patients with symptoms of chronic coronary syndrome (CCS). We hypothesized that gut leakage markers would increase after acute strenuous exercise, and that the increase would be higher in patients with angiographically verified CAD. Methods Patients referred to exercise stress testing or coronary angiography due to symptoms suggestive of CCS were included (n=327). A maximal exercise ECG stress test was performed using a bicycle ergometer. Venous blood samples were drawn at rest prior to the test and within 5 min after the test ended, for analysis of soluble cluster of differentiation 14 (sCD14), lipopolysaccharide-binding protein (LBP) and intestinal fatty-acid binding protein (I-FABP) by ELISAs. Quantification of lipopolysaccharide (LPS) and relative quantification of gene expression of the toll-like receptor 4 (TLR4) in circulating leukocytes was performed in a subset of patients (n=101). Patients then underwent coronary angiography, and were grouped according to the degree of CAD. Results Of the 287 patients who completed the exercise stress test and coronary angiography, 69 (24%) had no CAD, 88 (31%) had non-significant CAD and 130 (45%) had significant CAD. Mean exercise duration was 10:05±4:46 min and the duration did not differ between the groups. There were no significant differences in resting levels of gut leakage markers between the groups. In the total population, sCD14, LBP and LPS increased significantly after exercise (p&amp;lt;0.0001, all), whereas I-FABP did not. The gene expression of TLR4 decreased significantly after exercise (p&amp;lt;0.0001). There were no differences in exercise-induced changes in any of the measured markers between groups with no CAD, non-significant CAD and significant CAD. Conclusion In patients with symptoms suggestive of CCS, LPS, LBP and sCD14 increased significantly after strenuous exercise, suggesting that even short bouts of vigorous exercise are associated with gut leakage. The decrease in gene expression of TLR4 may be discussed to be compensatory to the increase in LPS or possibly reflecting an increase in TLR4 translation in response to LPS. The presence of CAD or not did not seem to impact exercise-induced increase in gut leakage markers. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Stein Erik Hagen Foundation for Clinical Heart Research, Olso, Norway

Decreased miRNA-320e correlates with allergy in children with otitis media with effusion

ObjectiveOtitis media with effusion (OME) is a common childhood disease and the main cause of conductive hearing loss in this age group. Many factors predispose to OME but allergy is still widely disputed. The answer may lay in the molecular mechanisms of ear exudate formation and the recent studies showed miRNAs might take part in it. MiRNAs are also potent regulators of allergic response. As miRNAs are present in the middle ear, we hypothesized their expression differs between allergic and non-allergic patients and reflects the difference in pathomechanism of effusion formation between these two groups. Materials and methodsThis study aimed to establish the expression of 5 different miRNAs (miR-223-3p, miR-451a, miR-16-5p, miR-320e, miR-25-3p) in ear exudates in children diagnosed with OME. The allergy group consisted of 18 patients whereas the non-allergic group had 36 patients. MicroRNA was isolated from the middle ear fluid collected during myringotomy and transcribed into cDNA. MiRNA expression was measured with TaqMan™ MicroRNA Assays and analyzed with DataAssist software. The comparative CT method was used for calculating the relative quantification of gene expression based on the endogenous control gene expression (U6 snRNA-001973). ResultsMiR-320e expression was significantly decreased in allergic children with OME. Other studied miRNAs also showed reduced expression in allergic children, but the decrease was not significant. ConclusionsMiRNA expression differs between children with and without allergy in the course of OME, but further studies are needed to explain the exact role of miR-320e and its target genes in OME pathology in allergic patients.

RETRACTED: Healing potential of Spirulina platensis for skin wounds by modulating bFGF, VEGF, TGF-ß1 and α-SMA genes expression targeting angiogenesis and scar tissue formation in the rat model

Blue-green microalga Spirulina platensis (SP) gained more attention for its antioxidant and/or anti-inflammatory properties magnifying its beneficial effects as a feed additive and for cosmetic and biomedical applications. This study was performed to examine the impact of SP on the cutaneous wound and burn healing and to develop an understanding of the correlation between the sequelae of wound healing and the molecular expression patterns of wound healing-related genes as angiogenic basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and fibrosis-related genes as transforming growth factor-β (TGF-β) and α-smooth muscle actin (α-SMA) in rat wound models. To achieve these goals, two experiments were performed on 32 Wister male rats that were divided into 4 groups of 8 rats each. Each experiment was represented by 2 groups; the control group (CG) and the Spirulina group (SG). A full-thickness wound (1.5×1.5cm) and burn wound (2×2cm) were made on the back of each generally anaesthetized rat and the areas of wound and burn were measured on days of 0, 3, 6, 9, 12, and 15 and 0, 3, 6, 9, 12, 15, 18, and 21 post-wound and post-burn respectively. In both experiments, SP was topically applied on the backs of wounded and burned rats in Spirulina treated groups. The phases of wound granulation tissues were detected histopathologically. Immunohistochemistry was used to determine the expressions of (TGF-B1) and (VEGF). Furthermore, the relative quantification of gene expression was implemented using the (bFGF), (VEGF), (TGF-Ɓ1), and (α-SMA) as target genes. Histopathological examination revealed inflammatory cell infiltration, angiogenesis, epithelialization, and extracellular matrix deposition and wound contraction in SG as compared to CG in both experiments. Immunohistochemistry results showed a significant improvement in the VEGF and TGF-β1 expression levels of SG in both experiments. Interestingly, SG in both experiments revealed upregulation of angiogenic genes (bFGF and VEGF) and downregulation of fibrotic genes (TGF-β1 and α-SMA). In conclusion, our findings suggest that the topically applied Spirulina promoted wound healing. Thus, SP can be used as a biomedical application to treat various skin wounds and may reveal a potential molecular basis for future promising antifibrotic agents against scar formation.

Notch expression profile and satellite cell stimulation in masseter muscle before and after orthognathic surgery

The aim of this prospective study was to compare the expression of the Notch receptor family with the biomarker for stimulation of satellite cells (SC), which are responsible for functional adaptation.Tissue samples from the masseter muscle were taken presurgically and 7 months later. Samples from controls came from the extraction of third molars. The expression of Notch 1 to 4 and the satellite cell markers CD34, Pax7, and MyoD1 were investigated. PCR was used for relative quantification of gene expression, which was calculated with the ΔΔCT method.The study involved 38 white patients — 10 prognathic, 18 retrognathic, and 10 orthognathic controls. The median value for Notch 1 was significantly reduced presurgically for prognathic (0.46, SD 0.45) and retrognathic (0.57, SD 0.35) patients compared with the controls. Postsurgically, Notch 2 was significantly upregulated in the prognathic group (0.55, SD 0.28/1.37, SD 0.85). Similarly, there was upregulation of Notch 3 in the prognathic group (0.33, SD 0.42/0.59, SD 1.37) and downregulation in retrognathic patients (0.59, SD 0.79/0.52, SD 0.97). Upregulations for the satellite cell markers CD34 and Pax7 were also found in prognathic patients.The significant upregulation of Notch 1–3 and CD34 in prognathics, but unchanged MyoD expression, signals high stimulation for SC and maintenance of the regeneration cell pool. A lower expression of Notch and SC in retrognathic patients could be responsible for weak functional adaptation.

Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae).

The relative quantification of gene expression is mainly realized through reverse transcription-quantitative PCR (RT-qPCR). However, the accuracy of this technique is deeply influenced by the expression stability of the reference genes used for data normalization. Therefore, the selection of suitable reference genes for a given experimental condition is a prerequisite in gene expression studies. Dichelops melacanthus (Hemiptera: Pentatomidae) is an important phloem sap-sucking insect pest of soybean, wheat, and maize in Brazil. Most of the genetic and molecular biology studies require gene expression analysis. Nevertheless, there are no reports about reference genes for RT-qPCR data normalization in D. melacanthus. In this study, we evaluated the expression stability of nine candidate reference genes (nadh, sdhb, gapdh, fau, ef1a, rpl9, ube4a, gus and rps23) in different developmental stages, body parts, sex, starvation-induced stress and dsRNA exposure by RefFinder software that integrates the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt method. Our results showed that ef1a and nadh are the most stable reference genes for developmental stages, fau and rps23 for sex, ube4a and rps23 for body parts, rpl9 and fau for starvation stress, and nadh and sdhb for dsRNA exposure treatment. The reference genes selected in this work will be useful for further RT-qPCR analyses on D. melacanthus, facilitating future gene expression studies that can provide a better understanding of the developmental, physiological, and molecular processes of this important insect pest. Moreover, the knowledge gained from these studies can be helpful to design effective and sustainable pest management strategies.

Satellite cell capacity for functional adaptation of masseter muscle in Class II and Class III patients after orthognathic surgery-a pilot study.

The aim of the prospective pilot study was to analyze the biomarkers CD34, Pax7, Myf5, and MyoD for stimulation of satellite cells (SCs), which are responsible for functional adaptation. Forty-five Caucasian patients were consecutively recruited from the Maxillo-Facial-Surgery at TU Dresden. Eleven orthognathic Class III patients, 24 Class II patients, and 10 controls with Class I were involved in the study. Tissue samples from masseter muscle were taken from the patients pre-surgically (T1) and 7 months later (T2). Samples from controls were taken during the extraction of third molars in the mandible. Polymerase chain reaction (PCR) for relative quantification of gene expression was calculated with the delta delta cycle threshold (ΔΔCT) method. The results show significant differences for the marker of SC stimulation between the controls, the patient groups, males, and females. The gene expression of CD34 was post-surgically upregulated for Class III (0.35-0.77, standard deviation [SD] = 0.39, P < 0.05) in comparison with controls. For Pax7, there was a significant difference shown between the retrognathic and the prognathic group because of downregulation in Class II patients (1.64-0.76, SD = 0.55, P < 0.05). In Class III patients, there was a significant upregulation for Myf5 (0.56-1.05, SD = 0.52, P < 0.05) after surgery too. The significant decline of Pax7 in Class II patients indicates a deficiency of stimulated SC post-surgically. The expression of CD34 and Myf5 in Class II stayed unchanged. In contrast, there was an upregulation for all Class III patients, mainly in females, shown post-surgically. This may be one reason for weak functional adaptation and relapse in Class II patients.

From structure to function for the characterization of ERAP1 active site in Behçet syndrome. A novel polymorphism associated with known gene variations

IntroductionERAP1 has been recently proposed as risk marker of Behçet syndrome (BS). Gene single nucleotide polymorphisms (SNPs) could affect the enzymatic activity and the conserved active site is pivotal for the aminopeptidase function. This study aims to characterize the ERAP1 active site in a cohort of BS patients vs healthy controls (HC) integrating genomics, transcriptomics and bioinformatics approach. Materials and methodsWe recruited 109 consecutive Italian BS patients (63M:46 F; mean age: 45.07 ± 12.28 years) and 106 matched HC (55M:51 F; mean age: 42.57 ± 12.29 years). DNA was isolated and amplified using PCR with home made-primer pairs. PCR products were directly sequenced and computational analyses were performed to search active site SNPs (NCBI-BlastN tool), to predict SNPs functional effect (PolyPhen-2 software) and to obtain protein 3D modelling (Protean3D software). In a second phase of analysis, RNA was extracted and reverse transcribed. Quantitative Real-Time PCR (qPCR) was performed to assess ERAP1 mRNA level in presence (target) and in absence (control) of gene polymorphisms. The Fold change was calculated for the relative quantification of gene expression. ResultsA novel coding variation (NG_027839.1:g.25637 T > G; NP_057526.3:p.Phe360Cys, HGSV nomenclature) was found in heterozygosity state in 5/109 BS patients (4.59 % of cases) and none of HC. It was recognized in association with rs2287987, rs30187, rs17482078, and rs27044 BS-related polymorphisms for 4 out of 5 patients. All patients carrying the novel SNP were HLA-B*51-positive. The novel SNP was released in GenBank database with MK140632.1 ID. The SNP was predicted to be damaging and resides within the Zn-binding HEXXH(X)18E region of the active site, changing the structurally conserved region for the amminopeptidase function. In fact, the change in energy (ΔE) score between wild-type and SNP-containing protein showed a less stable protein in presence of p.Cys360 (ΔE:3.584) (Protean3D prediction). Preliminary qPCR results underlined a significant difference in fold change value when target and control values were compared (p < 0.05), suggesting a reduced expression of ERAP1 mRNA in presence of the novel SNP. ConclusionsOur study strengthens the association between ERAP1 and BS. The most significant point was the localization of the novel p.Phe360Cys SNP within the Zn-binding region of protein active site that was predicted to affect its function, causing protein destabilization. Our findings need to be tested in larger genetic studies.

Suitable reference genes determination for real-time PCR using induced sputum samples.

Induced sputum is a non-invasive method of collecting cells from airways. Gene expression analysis from sputum cells has been used to understand the underlying mechanisms of airway diseases such as asthma or chronic obstructive pulmonary disease (COPD). Suitable reference genes for normalisation of target mRNA levels between sputum samples have not been defined so far.The current study assessed the expression stability of nine common reference genes in sputum samples from 14 healthy volunteers, 12 asthmatics and 12 COPD patients.Using three different algorithms (geNorm, NormFinder and BestKeeper), we identified HPRT1 and GNB2L1 as the most optimal reference genes to use for normalisation of quantitative reverse transcriptase (RT) PCR data from sputum cells. The higher expression stability of HPRT1 and GNB2L1 were confirmed in a validation set of patients including nine healthy controls, five COPD patients and five asthmatic patients. In this group, the RNA extraction and RT-PCR methods differed, which attested that these genes remained the most reliable whatever the method used to extract the RNA, generate complementary DNA or amplify it.Finally, an example of relative quantification of gene expression linked to eosinophils or neutrophils provided more accurate results after normalisation with the reference genes identified as the most stable compared to the least stable and confirmed our findings.

Identification of robust reference genes for studies of gene expression in FFPE melanoma samples and melanoma cell lines.

There is an urgent need for novel diagnostic melanoma biomarkers that can predict increased risk of metastasis at an early stage. Relative quantification of gene expression is the preferred method for quantitative validation of potential biomarkers. However, this approach relies on robust tissue-specific reference genes. In the melanoma field, this has been an obstacle due to lack of validated reference genes. Accordingly, we aimed to identify robust reference genes for normalization of gene expression in melanoma. The robustness of 24 candidate reference genes was evaluated across 80 formalin-fixed paraffin-embedded melanomas of different thickness, -/+ ulceration, -/+ reported cases of metastases and of different BRAF mutation status using quantitative real-time PCR. The expression of the same genes and their robustness as normalizers was furthermore evaluated across a number of melanoma cell lines. We show that housekeeping genes like GAPDH do not qualify as stand-alone normalizers of genes expression in melanoma. Instead, we have as the first identified a panel of robust reference genes for normalization of gene expression in melanoma tumors and cultured melanoma cells. We recommend using a geometric mean of the expression of CLTA, MRPL19 and ACTB for normalization of gene expression in melanomas and a geometric mean of the expression of CASC3 and RPS2 for normalization of gene expression in melanoma cell lines. Normalization, according to our recommendation will allow for quantitative validation of potential novel melanoma biomarkers by quantitative real-time PCR.

Excreted and secreted products (72/60 kDa) from Haemonchus placei larvae induce in vitro peripheral blood mononuclear cell proliferation and activate the expression of cytokines and FCεR1A receptor

The aim of the present study was to assess the expression of cytokines and FCεR1A receptor stimulated by Haemonchus placei larval excretory and secretory (ES) products associated with the pathogenesis in calves. Bovine peripheral blood mononuclear cells (PBMC) were stimulated in in vitro assays with H. placei L4 ES product at 8, 12, 16 and 24 h. ES products were collected in in vitro assays at 48 h with molecular weight of 72/60 kDa and isoelectric point of 7.2 pI. Specific IgG for infected and control calves, positive and negative, were employed to recognise H. placei larval ES products by indirect ELISA, showing a mean of 1.8, 0.83 and 0.28 OD, respectively, (p ≤ 0.001). The quantification of relative gene expression was performed using a set of cytokines (IL-2, IFNγ, TGFβ, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13), FCεR1A receptor and housekeeping (GAPDH, β-actin and β-2-microglobulin) by RT-qPCR. An early increased expression, 2.2- to 3.4-fold change, of IL-2 (p ≤ 0.001), IL-5 and TGFβ (p ≥ 0.05) was determined, followed by TGFβ (30.7 and 14.14), IL-8 (102.8 and 1504.4) and IL-10 (60.4 and 1.7) (p ≤ 0.05) after 12 and 16 h, respectively, and reducing the expression level at 24 h. In addition, IL-6, IL-13 and FCεR1A receptor also displayed mild expression level, 2.1 – to 7.60-fold change, at 24 h (p ≥ 0.05). We conclude that ES products of 72/60 kDa collected in vitro from H. placei larvae are recognised by infected hosts and have the ability to induce diverse immune factors to modulate the nematode damage.