TaqMan Primers Research Articles

Phylogenetic and Sequence Analyses of the Variable Region in the Glycoprotein Gene of the Respiratory Syncytial Virus Isolated from Iraqi Patients.

Respiratory syncytial virus (RSV) is a common aetiological agent that causes respiratory infections, especially among infants. Identifying circulating RSV genotypes is an essential strategy for understanding the spread of the virus in a certain area. Sequencing the variable regions of the attachment glycoprotein (G) gene of RSV is a quick and direct approach for identifying the genotypes. This study was aimed to sequence the G gene region of RSV isolated from patients admitted to hospitals in Baghdad, Iraq, during the autumn of 2022 and winter of 2023. To achieve this goal, 150 patients with lower respiratory symptoms were screened for RSV infections. RSV-positive samples were detected and confirmed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approach, which involved the use of specific TaqMan primer sets targeting RSV subgroups. Then, a G gene region that included hypervariable region 2 (HVR2) was amplified and sequenced using the Sanger sequencing method. Furthermore, molecular and phylogenetic analyses were performed on the G gene region to determine the variability profile of the tested specimens. There were 41 (26.6%) RSV-positive cases. Of these, the RSV-B subgroup was the most prevalent (82.90%), while the RSV-A subgroup incidence rate was 17.07%. The phylogenetic analysis showed that the RSV-B isolates were related to the BA genotype and shared nucleotide sequence similarities with isolates from India, Australia and the UK. The RSV-A isolates belonged to the ON genotype and had some degree of similarities with isolates from Italy, Tunisia, and France. Seasonal tracking of the RSV isolates would facilitate a better understanding of virus evolution, viral pathogenesis, and genetic diversity.

Development of A Novel Multiplex TaqMan Probe qPCR Assay Distinguishing Dikaryotic ITS rDNA Types of Sclerotium rolfsii (=Agroathelia rolfsii) and S. delphinii.

Sclerotium rolfsii (=Agroathelia rolfsii) and S. delphinii are globally ubiquitous and prevalent soil-borne pathogens. These species are distinguishable by the morphology of their sclerotia formed on artificial media. However, this distinction requires prior pure isolation, complicating direct identification from diseased plants or soil samples. Additionally, each species contains two distinct ITS ribosomal DNA types in a dikaryotic cell, further complicating their identification through sequence analysis. To address this issue, we developed a novel multiplex qPCR method utilizing TaqMan probes and primers. This method specifically targets the two ITS types unique to each species, effectively distinguishing between these pathogens and differentiating them from non-target soil-borne fungal and oomycete pathogens. Its efficiency and sensitivity were confirmed through their detections in artificially inoculated plants and soil samples. Our multiplex qPCR technique offers a precise and reliable approach for the simultaneous detection and differentiation of S. rolfsii and S. delphinii, significantly improving disease surveillance and facilitating timely, effective disease management.

P-596 Impaired fatty acid desaturation in COV434 human granulosa cells leads to increased omega-6 phospholipid abundance

Abstract Study question How does fatty acid desaturation in COV434 granulosa cells influence their lipogenic pathway? Summary answer Suppressing fatty acid desaturation in human granulosa cells leads to a depletion of polyunsaturated phospholipids and an increase in omega-6 fatty acid-containing lipids. What is known already Maintaining the balance between saturated and unsaturated lipids in oocyte-surrounding granulosa cells is crucial to supporting oocyte maturation. We have shown the link between the de novo production of oleic acid through fatty acid desaturation and the main functional parameters of granulosa cells, such as cell proliferation and steroid production. A potential contributor to the observed pathophysiological outcomes lies in the dysregulation of cellular lipids as a consequence of insufficient intracellular oleic acid availability. This lipidomic study investigates the adaptive lipogenic response of granulosa cells to deficient fatty acid desaturation. Study design, size, duration We tested the influence of a chemical inhibitor against stearoyl-CoA desaturase 1 (SCD1), a major fatty acid desaturase enzyme, on cell membrane lipid packing, lipidome, and lipogenic-related genes in immortalized granulosa cells, COV434. We applied free oleic acid to conduct a rescue experiment. Three independent technical replicates were performed for each experimental condition, and mean values were compared among test conditions using one-way ANOVA analysis. Participants/materials, setting, methods The level of membrane fluidity was estimated using the membrane polarity-sensitive dye Laurdan. We used liquid chromatography–mass spectrometry to investigate the lipidome profiles. Data were obtained by calculating the peak areas of intact lipid species. We used quantitative PCR with TaqMan primers to assess the expression of mechanistic target of rapamycin (mTOR), diacylglycerol acyltransferase 1 (DGAT1), and fatty acid synthase (FASN), with 18S ribosomal RNA as the internal control. Main results and the role of chance Reduced oleic acid availability increased cholesterol content 1.2-fold (p = 0.007) and membrane packing, representing intended modifications in de novo desaturation. We used data comprising 459 lipid species. SCD1 inhibition caused a significant increase in dipalmitoyl phosphatidic acid 32:0 (7.4-fold increase vs. untreated controls, p < 0.001) and a substantial decrease in dioleoyl phosphatidic acid 36:2 (-38% reduction, p < 0.001). SCD1 inhibition also led to an elevation of omega-6 phospholipids (e.g., 38:4, 40:4), but conversely, a depletion of polyunsaturated lipids (e.g., 34:2, 36:2). In contrast, oleic acid supplementation resulted in an enrichment of oleic acid-containing lipids (e.g. 34:1, 36:1) but also caused a significant depletion of all polyunsaturated lipids, including omega-6 species (e.g., 20:3, 20:4). These findings indicate that the de novo generation of oleic acid intricately modulates polyunsaturated phospholipid metabolism, especially that of omega-6 phospholipids.Although the reductions in the expression levels of lipogenic-related genes due to SCDinhib treatment were not statistically significant, the addition of oleic acid significantly enhanced the suppression of FASN by 67% (p = 0.022). This suggests the presence of an interaction effect that potentiates the downregulation of the de novo fatty acid synthesis and diverts metabolic pathways away from omega-6 fatty acid production. Limitations, reasons for caution Examining lipidomic profile in isolated primary ovarian cells is crucial for a better understanding of their biological significance. Wider implications of the findings We identified that the role of de novo oleic acid generation in polyunsaturated phospholipid metabolism has significance for cellular physiology in oocyte-surrounding granulosa cells. Potential future applications include targeted metabolic interventions for reproductive disorders impacting oogenesis. Trial registration number not

De novo synthesis of monounsaturated fatty acids modulates exosome-mediated lipid export from human granulosa cells

BackgroundOvarian somatic cells support the maturation and fertility of oocytes. Metabolic desaturation of fatty acids in these cells has a positive paracrine impact on the maturation of oocytes. We hypothesized that the enzyme stearoyl-CoA desaturase 1 (SCD1) in granulosa cells regulates the lipid cargo of exosomes secreted from these cells by maintaining the balance between saturated and unsaturated lipids. We investigated the effect of SCD1 on exosome lipid content in a cumulus-granulosa cell model under physiologically relevant in vitro conditions. MethodsNon-luteinized human COV434 granulosa cells were subjected to treatment with an inhibitor of SCD1 (SCDinhib) alone, in combination with oleic acid, or under control conditions. Subsequently, the exosomes were isolated and characterized via nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. We used liquid chromatography mass spectrometry to investigate the lipidomic profiles. We used quantitative PCR with TaqMan primers to assess the expression of genes involved in lipogenesis and control of cell cycle progression. ResultsA trend toward exosome production was observed with a shift toward smaller exosome sizes in cells treated with SCD1inhib. This trend reached statistical significance when SCDinhib was combined with oleic acid supplementation. SCD1 inhibition led to the accumulation of saturated omega-6 lipids in exosomes. The latter effect was reversed by oleic acid supplementation, which also improved exosome production and suppressed the expression of fatty acid synthase and Cyclin D2. ConclusionThese findings underscore the critical role of de novo fatty acid desaturation in the regulation of the export of specific lipids through exosomes, with potential implications for controlling intercellular communication within the ovary.

The interplay of PTEN and AKT nexus in breast cancer: a molecular perspective.

Breast cancer is a highly prevalent and life-threatening ailment that is commonly detected among the females. The downregulation of PTEN in breast cancer is associated with a poor prognosis, aggressive tumor type, and metastasis to lymph nodes, as it activates the pro-survival pathway PI3K/AKT, which is considered the ultimate proliferative pathway. The mRNA expression of PTEN and AKT genes was investigated using RT-qPCR and TaqMan primer probe chemistry. Moreover DNA was also isolated from the same tissue samples and exonic regions of both genes were amplified for mutational analysis. The proteins expression of PTEN and AKT from seven human breast cancer cell lines was checked through western blot experiments. The study revealed a decrease in PTEN expression in 73.3% of the samples, whereas an increase in AKT expression in 40% of samples was observed when compared to the distant normal breast tissue. Conversely, the remaining 60% of samples exhibited a decrease in AKT mRNA expression. There was no observed alteration in the genetic sequence of AKT and PTEN within the targeted amplified regions ofbreast cancer samples. The high levels of PTEN protein in T-47D and MDA-MB-453 resulted in a lower p-AKT. Two cell lines ZR-75-1 and MDA-MB-468 appeared to be PTEN negative on western blot but mRNA was detected on RT-qPCR. In breast cancer the status/expression of PTEN & AKT at mRNA and protein level might be obliging in forecasting the path of disease progression, treatment and prognosis.

Applications of Environmental DNA (eDNA) in Monitoring the Endangered Status and Evaluating the Stock Enhancement Effect of Tropical Sea Cucumber Holothuria Scabra.

The tropical sea cucumber Holothuria scabra is naturally found in the Indo-West Pacific. However, due to their commercial value, natural H. scabra populations have declined significantly in recent years, resulting in its status as an endangered species. Surveys of H. scabra resource pose a challenge due to its specific characteristics, such as sand-burrowing behavior. To overcome this problem, our study established a convenient and feasible method for assessing H. scabra resources using environmental DNA (eDNA) monitoring technology. First, H. scabra-specific TaqMan primers and probe were designed based on its cox1 gene, followed by the development of an eDNA monitoring method for H. scabra in two separate sea areas (Xuwen and Daya Bay). The method was subsequently employed to investigate the distribution of H. scabra and assess the effects of aquaculture stock enhancement through juvenile releasing in the Weizhou Island sea area. The H. scabra eDNA monitoring approach was found to be more appropriate and credible than traditional methods, and a positive impact of stocking on H. scabra populations was observed. In summary, this is the first report to quantify eDNA concentration in a Holothuroidea species, and it provides a convenient and accurate method for surveying H. scabra resources. This study introduces novel concepts for eDNA-based detection of endangered marine benthic animals and monitoring their population distribution and abundance.

Expression of microRNA-590 in patients with chronic coronary heart disease, atrial fibrillation, and their combination

Background. The role of microRNAs in the processes of remodeling, proliferation, and fibrogenesis of myocardial cells is not clear enough.
 Aim. Analysis of the microRNA-590 expression level in patients with chronic coronary heart disease, atrial fibrillation, as well as their combination to assess the potential prognostic significance of the expression level.
 Material and methods. The study included 94 patients divided into three clinical groups: the first with non-valvular atrial fibrillation without coronary heart disease (39 people); the second with non-valvular atrial fibrillation and coronary heart disease (22 patients); the third with ischemic heart disease without atrial fibrillation (23 patients). The comparison group consisted of 10 people without atrial fibrillation and coronary heart disease. Venous blood was taken from all subjects, from the plasma of which microRNA was isolated. The relative level of microRNA expression was estimated based on real-time polymerase chain reaction data obtained during the reaction on a cycler using commercial TaqMan probes and primers. The statistical significance of differences between groups was determined using one-way analysis of variance, followed by post-hoc analysis using Tukey's contrasts, differences were considered statistically significant at p 0.05. The ShapiroWilk test was used to assess the normality of the distribution of residuals.
 Results. A statistically significant decrease in the expression level of microRNA-590 was registered in the third (p=0.0104) and in the second (p=0.0046) groups compared with the control group, as well as in patients with atrial fibrillation and left atrial dilatation (p=0.0313), with recurrent arrhythmias after radiofrequency ablation (p=0.0083) and with permanent atrial fibrillation (p=0.0242).
 Conclusion. Ischemic heart disease, including when combined with atrial fibrillation, and aggravating factors, such as left atrial dilatation, permanent atrial fibrillation, recurrence of arrhythmia after radiofrequency ablation, lead to a decrease in the expression level of microRNA-590.

Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections

Acute respiratory infections are widespread in vulnerable populations of all ages and are characterized by a variety of symptoms. The underlying infection can be caused by a multitude of microorganisms, including viruses and bacteria. Early detection of respiratory infections through rapid pathogen screening is vital in averting infectious respiratory disease epidemics. This study utilized a multiplex real-time PCR system to develop a three-tube reverse transcription-PCR (RT-PCR) assay, enabling simultaneously detect nine respiratory pathogens, including: influenza A and B, adenovirus, respiratory syncytial virus (RSV), Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. This technique utilizes a one-step assay, with specifically designed TaqMan primer–probe sets combined in the same tube. This assay provided rapid and simplified detection of the nine prevalent pathogens, as well as increased sensitivity and reduced cross-contamination. This assay was evaluated using 25 related viral/bacterial strains as positive references, the other 25 irrelevant strains as negative controls, and clinical specimens from 179 patients. All positive strains were detected with no amplification of the non-target microorganism mixtures and the assay’s detection limits ranged between 250–500 copies/ml (1.25–2.5 copies/reaction). A total of 167 (93.3%) samples tested positive for at least one of the pathogens identified; 109 of these samples were from patients confirmed to have RSV infections. The diagnostic accuracy of our assay was further confirmed by matching results from classical direct immunofluorescence assay and nucleotide sequencing. These data demonstrate the innovative multiplex real-time PCR assay as a promising alternative to the current approaches used for early screening of acute respiratory infections.

Regular exercise increases H2S levels in mitochondria and cardiac tissue, enhances genes expression of H2S-synthesizing enzymes and prevents mitochondrial swelling

Abstract Funding Acknowledgements Type of funding sources: None. Introduction It is known that regular exercise is important and very useful for the cardiovascular system because of decreasing the incidence of myocardial infarction and protection against chronic and acute coronary disease. It normalizes blood pressure, lowers weight, reduces vascular resistance and enhances structural adaptations through increasing number of capillaries and arteries. It was also shown that exercise has a positive effect on vasodilatation by increasing nitric oxide (NO) production via eNOS gene expression enhancing. As we know hydrogen sulfide (H2S) is also a gaseous molecule that performs many functions in the cardiovascular system. It was found that disorders of H2S synthesis are found during hypertension, atherosclerosis, diabetes, heart failure, neurodegenerative diseases and aging. Purpose The aim of this study was to investigate the effect of moderate exercise on endogenous H2S content, gene expression levels of H2S-synthesizing enzymes and opening of mitochondrial pore in adult and old rats. Methods We used adult (5-7 months) and old (22-24 months) Wistar male rats. Physical training of adult and old animals was carried out 5 days a week by forcibly swimming for six and four weeks, respectively. The first training session of adult and old rats lasted 2 minutes and the duration was increased by 4 and 2 minutes every other day respectively. Mitochondria were isolated by differential centrifugation method. H2S was determined in mitochondria and heart tissues by biochemical method using zinc acetate, N,N-DPD and FeCl3. H2S-synthesizing enzymes genes were defined by real-time PCR using TaqMan Master Mix and primers for CSE and 3-MST. Results In our experiments we showed that physical training increases the H2S level in mitochondria and heart tissue of adult and old animals. The concentration of H2S in mitochondria enhances by 2,3 times for adult (4,58±0,3 nmol/mg compare to 10,55 ±1,18 nmol/mg, Р<0,05) and by 8,8 times for old rats (2,38±0,45 nmol/mg compare to 20,91 ±3,56 nmol/mg, Р<0,05). A slight increase in these indicators was also observed in the heart tissue of both groups. It should be noted that in old animals initially H2S level was decreased twice compared to adult. At the same time the levels of H2S-synthesizing enzymes, CSE and 3-MST, was lowered by 4 and 3 times in old rat heart. Exercise increases levels of these genes by 2,6 and 3 times for adult cardiac tissue and 7,9 and 7,2 times for old heart tissue. Physical training also has positive effect on mitochondrial pore opening and reduces ROS generation in the heart of old animals. Thus, Ca2+-induced mitochondrial swelling decreased by 37% in adult and 26% in old rat cardiac tissue, and the rate of O2- generation by 85% compared to untrained rats. Conclusion Physical training increases H2S levels, genes expression of H2S-synthesizing enzymes in mitochondria and cardiac tissue and prevents mitochondrial permeability transition opening in adult and old rats.

Source Tracking based on Microbial Communities: Sample Collection and ddPCR/qPCR

Fecal contamination serves as a major public health threat, especially in drinking or recreational waters. This is particularly concerning because it promotes the exposure of communities to harmful pathogenic bacteria. Identifying the source of contamination serves as an important context when mitigating the effects of the pollution or preventing it from initially entering the water. Microbial source tracking (MST) is employed to achieve the quantification of dominant sources of fecal contamination. Specifically in this study, a library‐independent method was utilized in the sample level detection of host‐associated 16s rRNA markers. Among the most prevalent bacteria in human and animal gut microbiomes is the phylum Bacteroidetes,which prove to be extremely useful in MST assays. TaqMan primers and probes targeting host‐independent (universal) Bacteroidetes(BacUni), human‐host specific (HF183), and pig host specific (Pig2Bac) were utilized. Dye‐based SYBR assays targeting avian bacteria Helicobacter (GFD) were also investigated. Solid fecal samples were acquired from a local petting zoo and surrounding beach sites. Water samples from various lakes, streams, and wastewater treatment plants were also obtained. Samples were filtered using membrane filtration technology and DNA was isolated using DNEasy PowerSoilkit. Upon DNA isolation, the concentration of DNA was measured using NanoDrop and the samples were subjected to qPCR or ddPCR assays. Results indicate effective primer/probe selectivity. Cross‐reactivity between samples and primers demonstrated that the targets were not exclusive to the presumed host and could overlap. Future research includes testing primers on samples from a wider range of animals and water sources. Additionally, a comparison of qPCR results to those from ddPCR and quantifying any differences within or between the two.

Aberrant expression of miR-138 as a novel diagnostic biomarker in systemic sclerosis.

MicroRNAs are short nucleotide sequences that contribute to the regulation of various biological functions and therefore their roles have been investigated in many pathologic conditions such as epithelial to mesenchymal transition in cancer and fibrosis; among them, miR-138 has been mostly studied in cancer biology and is well-known for its suppressing effect on cancer progression. Being able to suppress major pathways involved in EMT, miR-138 could be a good candidate to be investigated in fibrotic responses too. Based on our previous studies, and the capability of miR-138 to target and regulate several components of the EMT pathway; we hypothesized a role for miR-138 in systemic sclerosis. Accordingly, the gene expression of miR-138 was assessed to find any alterations in the whole blood of the SSc patients. Blood was collected from 70 patients with systemic sclerosis (equally divided between 2 groups of limited and diffuse categories) and 30 healthy individuals as controls. RNA was immediately isolated from the fresh whole blood; afterward, the resulting RNA was reverse transcribed into cDNA and then the relative expression of miR-138 was compared between the patients and the controls by the means of qPCR, and specific TaqMan primer and probes. The relative expression of miR-138 was significantly lower in patients with systemic sclerosis compared to the controls. No significant difference was observed between the limited and diffuse patient groups. ROC curve analysis showed an appropriate diagnostic value of miR-138 in effectively differentiating SSc patients from the healthy controls. miR-138 is likely involved in the pathogenesis of SSc and with further evaluations may be utilized as a diagnostic biomarker in SSc. Also, targeting miR-138 in future studies could be promising for finding a novel treatment option for patients with SSc.

Molecular Detection and Quantification of Xanthomonas albilineans in Juice from Symptomless Sugarcane Stalks Using a Real-Time Quantitative PCR Assay.

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A sensitive and robust quantitative PCR (qPCR) assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis groups. The detection thresholds were 100 copies/µl of plasmid DNA, 100 fg/µl of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) of symptomless stalk samples, whereas only 28.4% (155/546) of samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi Province) and Zhanjiang (Guangdong Province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

P–208 Oleic acid rescues altered autophagy induced by palmitic acid during mouse preimplantation development

Abstract Study question The aim of the study is to identify the autophagic profile and the effects of fatty acid treatments on autophagic activity in preimplantation mouse embryos. Summary answer Autophagic activity varies significantly in early stages of mouse preimplantation development; exposure to fatty acids alters the embryonic autophagy profile. What is known already Obesity is one of the top comorbidities for infertility, and obese individuals have elevated fatty acid levels. In serum, palmitic acid (PA) and oleic acid (OA) are the most abundant saturated and unsaturated fatty acids, respectively. We recently reported that PA impairs blastocyst development, affects mitochondrial reactive oxygen species, triacylglycerol levels, and endoplasmic reticulum stress pathways during mouse preimplantation development. Interestingly, the addition of OA counteracts those effects. Autophagy plays an essential role in embryo development, as knock-out of a key autophagy protein is embryonic lethal. Little is known about the autophagic profile in fatty acid treated mouse preimplantation embryos. Study design, size, duration Pools of 20 – 25 mouse embryos were collected from gonadotrophin super-ovulated and mated CD1 female mice. Two-cell stage embryos were treated with 100 µM PA and 250 µM OA, alone and in combination, and 1.5% bovine serum albumin media (control) within KSOMaa media for 18, 24, and 48 hours in vitro. The detection of various autophagic markers were evaluated by immunofluorescence microscopy and RT-qPCR. Participants/materials, setting, methods mRNA levels of autophagic markers were measured using RT-qPCR with the Taqman primers and Universal PCR Mix. Immunofluorescence staining of LC3 puncta (marker for autophagosome formation) was performed using LC3A/B polyclonal antibody (Invitrogen PA1–16931) and DAPI (4′,6-Diamidino–2-phenylindole dihydrochloride) was used to stain for cell nuclei. Analysis of LC3 puncta was performed using ImageJ software. Images were acquired using an LSM 800 laser scanning confocal microscope. Data analysis was completed by GraphPad Prism software. Main results and the role of chance Mouse preimplantation embryos showed no change in mRNA levels of autophagic markers (Bcln1, ATG3, ATG5, and LC3) relative to the control group after 48-hours exposure of 100 µM PA and 250 µM OA treatments, alone and in combination. The number of LC3 puncta was measured and analyzed as a reflection of autophagic activity in mouse preimplantation embryos. Under the fatty acid-free condition, the average number of LC3 puncta per blastomere was significantly decreased after 18 hours of development (p < 0.005). However, the average number of LC3 puncta per blastomere at 18, 24, and 48 hours were not significantly different from each other (p = 0.2724). Following 100 µM PA and 250 µM OA treatments, alone and in combination, autophagic activity was impacted by the presence of fatty acids. Mouse preimplantation embryos exposed to control and fatty acid treatment groups demonstrated no significant differences in LC3 puncta per blastomere at 18- and 24-hours treatment time (p = 0.5381; p = 0.7829). However, embryos exposed to 48 hours of PA treatment had a significantly greater number of LC3 puncta per blastomere than embryos exposed to 48 hours of OA and PA and OA combination treatments (p < 0.05). Limitations, reasons for caution Although LC3 puncta count (autophagosome formation) is impacted by fatty acid treatment, autophagic flux must be measured to fully investigate autophagic activity during mouse preimplantation development. These processes need to be measured in human embryos cultured in vitro. Wider implications of the findings: Profiling autophagic activity in fatty acid treated mouse preimplantation embryos would guide future investigations on pharmacological modulation of autophagy as a therapeutic intervention for developmentally delayed embryos. With the information gained, we aim to develop strategies to assist overweight and obese patients with their fertility needs. Trial registration number Not applicable

Lack of Association Between ACE2 Expression and Serum Testosterone Concentrations in Peripheral Mononuclear Cells in Males

Background: Male sex is a risk factor for developing severe COVID-19 illness, hospitalization, and mortality. It is possible that the male sex hormone, testosterone, contributes to the morbidity from COVID-19. SARS-CoV2 viruses use cell membrane protein Angiotensin-Converting Enzyme 2 (ACE2) receptor and undergo S protein priming by the Type II Transmembrane Serine Protease (TMPRSS2) to enter the cells. Hence, the expression level of ACE2 and TMPRSS2 may affect disease susceptibility and possible severity. TMPRSS2 is regulated by the androgen receptor. We, therefore, examined if an association exists between serum testosterone concentrations and ACE2 or TMPRSS2 expression level in men. Methods: We analyzed fasting serum samples and peripheral blood mononuclear cells (MNC) from 42 men. Total and free testosterone and estradiol were measured by liquid chromatography/equilibrium dialysis. Sex hormone binding globulin (SHBG) was measured by chemiluminescence. mRNA was prepared from MNC. Quantitative RT-PCR was conducted using commercially available, pre-designed TaqMan primers and probes targeting ACE2. The ACE2 relative level was calculated after normalization to beta Actin and GAPDH, with lowest ACE2 level being set to 1. Results: Subjects’ age ranged from 20 to 65 years. Type 2 diabetes was present in 74% of the men and the mean HbA1 was 7.2±1.6% (mean ± S.D.). Fifteen subjects had subnormal free testosterone (<50 pg/ml). Compared to the 27 subjects with normal free testosterone, they were older (49±12 vs 40±13 years, p=0.03) but had similar BMI (36±10, 35±10 kg/m2, p=0.71). As expected, they had lower total testosterone (Median [25th, 75th percentile]; 222 [171-266] vs 431 [335, 618] ng/dl, p<0.001) and free testosterone concentrations (39 [21, 44] vs 72 [59, 92], p<0.001). Total estradiol was also lower in this group (19±1 vs 29±13 pg/ml, p=0.03) but free estradiol (0.44±0.33 vs 0.56±0.34 pg/ml, p=0.44) and SHBG (27 [19, 33] vs 30 [21, 39] nmol/L, p=0.52) were similar. Quantitative PCR data showed there was a large inter-individual variation of ACE2 expression level, up to 15-fold difference. Average ACE2 level did not differ between subnormal and normal testosterone groups (3.4 [2.7, 5.0] vs 3.9 [2.5, 5.7] arbitrary units). ACE2 expression was not related to free testosterone (r=0.11), free estradiol (r=0.18) or sex hormone binding globulin (r=0.15) on linear regression analyses (p>0.30 for all). ACE2 expression was also not related to age (r= -0.15, p=0.34), BMI (r= -0.2, p=0.23) or presence of diabetes. We were not able to detect a significant expression of TMPRSS2 in MNC. Conclusion: Our results do not support a role for testosterone or estradiol as regulators of ACE2 expression in peripheral blood MNC in males.

663 Specific polycyclic aromatic hydrocarbons elicit differential expression of CYP1A1 in trophoblast cells

Prenatal exposures to polycyclic aromatic hydrocarbons (PAHs) are associated with adverse outcomes including small for gestational age infants. We have previously reported differential levels of PAHs in the placenta associated with preterm delivery. We found that levels of a specific PAH, dibenzoanthracene (DBA), was significantly higher than levels of either benzo[a]pyrene (BaP) or Benzo[b]fluoranthene (BbF). Furthermore, we reported that placental DBA levels are significantly and negatively correlated with gestational age at delivery. In this study we aimed to determine if there is a differential response in trophoblast cells with distinct PAH treatments. Immortalized choriocarcinoma cells (BeWo) were treated with 10 μM of either BaP, BbF or DBA for 24 hours. DMSO treatment served as a control. Media was collected and measured to determine levels of hCG production using an ELISA assay. RNA was extracted from cells after 24 hours of treatment, and used to make cDNA. TaqMan primers and probes were used to measure expression levels of CYP1A1, a gene involved in the processing of xenobiotic compounds, using RT-qPCR. GAPDH served as a control. While treatment with each PAH increased CYP1A1 expression compared with DMSO, DBA elicited the largest response, with a 54-fold increase in CYP1A1 expression (p<0.001). CYP1A1 expression was increased 16.5-fold with BbF treatment (p=0.001), and 7.4-fold with BaP treatment (p=0.006). Levels of hCG were not altered with any PAH treatment compared with DMSO, consistent with previous publications in trophoblast cells. In placenta tissue, levels of DBA are found at higher levels compared with BaP and BbF, and DBA levels are increased in placentae from preterm birth. In this study we find that DBA elicits a higher upregulation of CYP1A1 compared with BaP and BbF. Further experimentation is necessary using RNA-Seq analyses to help yield insight into pathways differentially responsive to DBA, yielding insight into its association with preterm delivery.

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay

H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was recently discovered and proposed to cause runting-stunting syndrome and urate deposition in geese. At present, however, there is a lack of epidemiological information pertaining to the dynamics and distribution of H146-like GCV. The development of novel molecular diagnostic approaches capable of rapidly and accurately detecting this virus would support the strengthening, the prevention, and control of H146-like GCV infection. In the present study, we therefore used a TaqMan probe and primers specific for the viral nonstructural (NS) gene to develop a highly sensitive and specific PCR assay capable of detecting this H146-like GCV. The assay reproducibly detected 5.07 × 102 copies of a recombinant DNA plasmid containing the NS gene, with a dynamic range of 8 orders of magnitude (102–109 copies). Importantly, no cross-reactivity was observed with common viruses that affected waterfowl, and when we used this assay to evaluate clinical samples, we found it to be more sensitive and faster than traditional PCR. In summary, herein, we developed a novel TaqMan-based real-time PCR approach that could reliably detect and diagnose H146-like GCV. This tool will allow for the real-time diagnosis of H146-like GCV infections, enabling researchers to better understand the epidemiology and clinical presentation of this disease.

Copy number variation of IL17RA gene and its association with the ankylosing spondylitis risk in Iranian patients: a case-control study

BackgroundAnkylosing spondylitis (AS) is considered as a subtype of spondyloarthritis (SpA) that mainly leads to fatigue, stiffness, spinal ankylosis, and impaired physical functions with reduced quality of life. Interleukin (IL)-17A provokes additional inflammatory mediators and recruits immune cells to the inflamed site. IL17 expression increased in various inflammatory disorders including psoriasis, rheumatoid arthritis, multiple sclerosis, crohn’s disease, and ankylosing spondylitis. The current study aimed to evaluate the association of IL17RA copy number changes with the susceptibility to AS and their correlation to IL17RA expression in Iranian population.MethodsIL17RA copy number genotyping assessments were carried out in 455 AS patients and 450 healthy controls, using custom TaqMan CNV assays. TaqMan primers and probe were located in Chr.22:17109553 based on pre-designed IL17RA Copy Number Assay ID, Hs02339506_cn. mRNA expression of IL17RA was also measured by SYBR Green real-time polymerase chain reaction (PCR).ResultsA IL17RA copy number loss (< 2) was associated with AS compared to 2 copies as reference (OR:2.18, 95% CI: (1.38–3.44), P-value < 0.001) and increased the risk of AS. IL17RA mRNA expression showed a significant increase in peripheral blood mononuclear cells (PBMCs) of all AS individuals than controls. The mRNA expression level of 2 copies was significantly higher in AS patients.ConclusionsOur findings revealed that a low copy number of IL17RA might confer a susceptibility risk to AS. However, it is probably not directly involved in the regulation of IL17RA mRNA expression. Epigenetic mechanisms like DNA methylation, post-transcriptional, and -translational modifications that regulate the expression of the genes may contribute in upregulation of IL17RA mRNA expression in the loss of gene copy number condition.

Quantitative PCR assay for the simultaneous identification and enumeration of multiple Karenia species.

Quantitative PCR (qPCR) is the method of choice for specific detection and quantification of harmful algal bloom (HAB) species. Development of qPCR assay for simultaneous enumeration of species that frequently co-exist in HABs is required. A high sensitivity TaqMan qPCR assay, using probe and primers, located at ITS1-5.8S-ITS2 rDNA region, detecting, specifically, Karenia selliformis, K. bidigitata, and K. mikimotoi, was designed. ITS1-5.8S-ITS2 rDNA region copy numbers per Karenia cell genome were estimated to 217.697 ± 67.904, allowing cell quantification. An application of the designed methodology in field samples has been conducted, and it showed high sensitivity (detection of around 10-1 cell/100 mg of bivalve mollusk tissue, equivalent to about 20 copies of the target sequence). We suggest that the optimized method could contribute to early detection of three closely related Karenia species in seafood cultivating areas to promote control quality, guarantee a fast and effective intervention, and improve public health prevention.

Intravenous Injection of Muse Cells as a Potential Therapeutic Approach for Epidermolysis Bullosa

Intravenous Injection of Muse Cells as a Potential Therapeutic Approach for Epidermolysis Bullosa