ΔCt Values Research Articles

Correlation Between Relative Nasopharyngeal Virus RNA Load and Lymphocyte Count Disease Severity in Patients with COVID-19.

The aim of this study was to analyze the correlation between dynamic changes in the nasopharyngeal viral load of patients infected with the new coronavirus causing pneumonia and lymphocyte count disease severity. Cases newly diagnosed with COVID-19 at the First Affiliated Hospital of Nanchang University from January 2020 to February 2020 were analyzed retrospectively. Quantitative real-time polymerase chain reaction was used to determine severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from throat swab sample ΔCT values; lymphocyte and lymphocyte subset counts, coagulation system factor levels, myocardial injury indexes, and laboratory biochemical indicators were compared between the mild group and the severe group. The correlation between the relative load of nasopharyngeal SARS-CoV-2 RNA and severe disease symptoms was analyzed. Of the 76 patients, 49 were male and 27 were female. The lymphocyte, CD4+ T lymphocyte, and CD8+ T lymphocyte counts all differed significantly between the two groups (p < 0.001), as did differences in interleukin (IL)-2R, IL-6, and IL-8 levels (p = 0.022, 0.026, and 0.012, respectively). Moreover, there were significant differences in prothrombin time, D-dimer, and fibrinogen levels between the mild group and the severe group (p = 0.029, 0.006, and <0.001, respectively), and in lactate dehydrogenase and troponin (p < 0.001 and p = 0.007, respectively). SARS-CoV-2 RNA load and lymphocyte count, CD4+ T lymphocyte count, and CD8+ T lymphocyte count were linearly negatively correlated (p < 0.001). SARS-CoV-2 RNA load was positively correlated with IL-2R, prothrombin time, lactate dehydrogenase, and hypersensitive troponin T (p = 0.002, p = 0.009, and p < 0.001, respectively). In addition, the time that it took for the nucleic acid test to turn negative was significantly shorter for patients in the mild group than for those in the severe group (Z = -6.713, p < 0.001). In conclusion, relative SARS-CoV-2 RNA load in the nasopharynx is closely related to COVID-19 severity. If the relative RNA load was higher, the lymphocyte count was lower, organ damage was greater, and the time it took for the nucleic acid test to turn negative was longer.

Effects of oral administration of 5 immunosuppressive agents on activated T-cell cytokine expression in healthy dogs.

BackgroundDogs are often adminstered >1 immunosuppressive medication when treating immune‐mediated diseases, and determining whether these different medications affect IL‐2 expression would be useful when performing pharmacodynamic monitoring during cyclosporine therapy.Hypothesis/ObjectivesTo determine the effects of 5 medications (prednisone, cyclosporine, azathioprine, mycophenolate mofetil, and leflunomide) on activated T‐cell expression of the cytokines IL‐2 and interferon‐gamma (IFN‐γ).AnimalsEight healthy dogs.MethodsRandomized, cross‐over study comparing values before and after treatment, and comparing values after treatment among drugs. Dogs were administered each drug at standard oral doses for 1 week, with a washout of at least 21 days. Activated T‐cell expression of IL‐2 and IFN‐γ mRNA was measured by quantitative reverse transcription polymerase chain reaction. Blood drug concentrations were measured for cyclosporine, mycophenolate, and leflunomide metabolites.ResultsLeast squares means (with 95% confidence interval) before treatment for IL‐2 (2.91 [2.32‐3.50] ΔCt) and IFN‐γ (2.33 [1.66‐3.00 ΔCt]) values were significantly lower (both P < .001) than values after treatment (10.75 [10.16‐11.34] and 10.79 [10.11‐11.46] ΔCt, respectively) with cyclosporine. Similarly, least squares means before treatment for IL‐2 (1.55 [1.07‐2.02] ΔCt) and IFN‐γ (2.62 [2.32‐2.92] ΔCt) values were significantly lower (both P < .001) than values after treatment (3.55 [3.06‐4.00] and 5.22 [4.92‐5.52] ΔCt, respectively) with prednisone. Comparing delta cycle threshold values after treatment among drugs, cyclosporine was significantly different than prednisone (IL‐2 and IFN‐γ both P < .001), with cyclosporine more suppressive than prednisone.Conclusions and Clinical ImportancePrednisone and cyclosporine both affected expression of IL‐2 and IFN‐γ, suggesting that both have the ability to influence results when utilizing pharmacodynamic monitoring of cyclosporine treatment.

Development of species-specific molecular markers in Vanilla for seedling selection of hybrids.

Vanilla planifolia is the primary botanical source of vanilla extract used globally in various foods and beverages. V. planifolia has a global distribution based on a few foundational clones and therefore has limited genetic diversity. Many Vanilla species easily hybridize with V. planifolia and could be a source of valuable genetic traits like increased vanillin content, disease resistance, or early flowering. While breeding Vanilla hybrids may improve plant performance, basic molecular tools for this species are lacking. DNA-based molecular markers are the most efficient method to validate hybrid progeny, detect hybrids in commercial plantings, and identify unknown accessions. This study used publicly available sequence data to develop species-specific, qRT-PCR-based molecular markers for Vanilla. Over 580,000 assembled sequence fragments were filtered for species specificity and twenty-two targets were selected for qRT-PCR screening. Ten targets differentially amplified among V. planifolia, V. pompona, V. phaeantha, and V. palmarum with ΔCT values as high as 17.58 between species. The ten targets were used to validate the parentage of hybrid progeny from controlled crosses with most hybrid progeny showing amplification patterns similar to both parents. The ten targets were also used to screen sixteen Vanilla species for specificity, and supported species assignments for unknown accessions including the detection of putative hybrids. This is the first report using species-specific, qRT-PCR-based molecular markers in Vanilla. These markers are inexpensive, simple to develop, and can rapidly screen large populations. These methods will enable the further development of species-specific molecular markers when creating Vanilla interspecific hybrid populations.

Arabidopsis Seed Stored mRNAs are Degraded Constantly over Aging Time, as Revealed by New Quantification Methods.

How plant seeds age remains poorly understood and effective tools for monitoring seed aging are lacking. Dry seeds contain various stored mRNAs which are believed to be required for protein synthesis during early stages of seed germination. We reasoned that seed stored mRNAs would undergo degradation during seed aging, based on the propensity of mRNAs to degrade. We performed RT-PCR and qPCR analyses to study the changes in stored mRNA levels of Arabidopsis seeds during aging. All stored mRNAs analyzed were gradually degraded in both naturally and artificially aged seeds. The difference in Ct values between aged and control seeds (ΔCt value) was highly correlated with the mRNA fragment size and seed aging time. We derived mathematical equations for estimating the relative amount of undamaged stored mRNAs and frequency of the breakdown at one nucleotide level for individual mRNAs. Stored mRNAs were found to break down randomly. The frequency of breaks per nucleotide per day, which we named β value, remained fairly constant under the same aging conditions over aging time. This parameter should allow the effects of different conditions on the degradation of stored mRNAs to be quantitatively compared. Also, we showed that the change in stored mRNA levels could serve as a more precise biomarker for seed aging assessment than three existing methods. These methods and findings will advance the studies of stored mRNAs and seed ageing in plants, and likely slow RNA degradation in non-plant systems.

Stem-like Cells from Invasive Breast Carcinoma Cell Line MDA-MB-231 Express a Distinct Set of Eph Receptors and Ephrin Ligands.

Breast cancer cell lines consist of bulk tumor cells and a small proportion of stem-like cells. While the bulk cells are known to express a distinct combination of Eph receptors and ephrin ligands, the transcript profiles of stem-like cells in these cell lines have not been adequately characterized. The aim of this study was to determine Eph receptor/ephrin ligand profiles of cancer stem cells specific to a triple negative breast carcinoma cell line. The normal breast cell line MCF10A and the invasive breast carcinoma cell line MDA-MB-231 were used to isolate CD24+/CD24- cell populations. The profiles of Eph receptors and ephrin ligands were determined by real-time PCR and the relative abundance in bulk and stem cells were compared. Based on the mean ΔCT values, the descending order of abundance was as follows. Ephrin-A5 > EPHA2 > (EPHA8, EPHB2) > ephrin-B2 > (EPHA7, EPHB4, ephrin-A4) > ephrin-A3 > ephrin-A1 > (EPHB3, ephrin-B1) > EPHA4 > EPHA1 > EPHA10. EPHA6 and ephrin-A2 transcripts were not detectable in stem cells from either cell line. The expression of EPHA4, EPHA7, EPHA8, and ephrin-A5 in MDA-MB-231 stem cells was up-regulated by 12, 20, ~500, and 6.5-fold respectively. The up-regulation of transcripts for EPHA8 and its cognate ligand, ephrin-A5, in the stem cells isolated from MDA-MB-231, suggest their involvement in the invasiveness of this cell line. Based on literature reports, we propose the role of EPHA8 and ephrin-A5 in MDA-MB-231 stem cells via the PI3K-AKT-mTOR pathway.

The Validation of the BCR/ABL1-like Predictor across Laboratories Shows Reproducibility of Results

The Validation of the BCR/ABL1-like Predictor across Laboratories Shows Reproducibility of Results

Quantitative Analysis of CCL5 and ep300 in Periapical Inflammatory Lesions.

In silico bioinformatical analysis suggested that the expression of two genes, CCL5 (C-C Motif Chemokine Receptor 5) and ep300 (Histone acetyltransferase p300), could be used as potential new biomarkers in differentiation between periapical granulomas and radicular cysts. Thus, we hypothesized that gene expression of CCL5 and ep300 in periapical lesions would classify the lesions as either granuloma or cyst. Patient samples (n=122) included 46 periapical granulomas, 38 radicular cysts and 38 healthy gingival samples as controls. Real-time PCR analysis of CCL5 and ep300 transcripts was compared to SDHA (Succinate dehydrogenase complex, subunit A) as the reference. Clinical parameters (e.g., intensity of inflammation and lesion size) were measured and correlated with CCL5 and ep300 expression. ROC (Receiver operating characteristic) and logistic regression analyses were used to establish the diagnostic character of ΔCt values. Granulomas and radicular cysts had significantly higher expression of CCL5 and ep300 compared to controls (P<0.05). However, no differences were observed when comparing granulomas and radicular cysts. ROC analyses showed that CCL5 and ep300 have good diagnostic accuracy, but low accuracy for distinguishing between the lesions. This study confirmed that expression of CCL5 and ep300 is relevant for the pathogenesis of periapical inflammatory lesions but cannot be used as a distinctive marker between these lesions.

Effects of pentoxifylline on whole blood IL-2 and IFN-gamma gene expression in normal dogs.

BackgroundPentoxifylline (PTX) is a methylxanthine phosphodiesterase inhibitor that is used as a hemorrheologic and anti‐inflammatory agent in veterinary and human medicine. In human studies, PTX has been shown to decrease T‐cell production of cytokines such as IL‐2 and IFN‐γ. A RT‐qPCR assay to measure activated T‐cell gene expression of IL‐2 and IFN‐γ has been validated in dogs.ObjectivesThe goal of this study was to utilize this assay to investigate the effects of PTX on in vitro cytokine gene expression in canine whole blood.MethodsWhole blood from seven healthy dogs was collected and incubated with various concentrations of PTX for 1 hr before activation. PTX concentrations spanned and exceeded blood concentrations achieved when administered at clinically relevant dosages (1, 2, 10, 50 and 200 μg/ml). Cyclosporine was used at a concentration of 500 ng/ml as a positive control. All blood samples, including untreated activated baseline samples, were then activated with phorbol myristate acetate and ionomycin for 5 hrs.ResultsAnalysis of activated whole blood by RT‐qPCR revealed that there was not a significant suppression of IL‐2 or IFN‐γ gene expression at any concentration of PTX when evaluating ΔCt values. All samples exposed to cyclosporine showed significant changes from untreated activated baseline samples, demonstrating marked suppression as the positive control. Cytokine expression, presented as a percentage of untreated activated baseline samples, was also evaluated. After exposure to the highest concentration of PTX (200 μg/ml), median percentage cytokine expression was suppressed to just below 50% of baseline values. This concentration, however, is much higher than blood concentrations reported to be achieved at standardly used pentoxifylline doses.ConclusionsPTX does not appear to significantly suppress T‐cell cytokine production in samples from most dogs at clinically relevant drug concentrations. Further testing is needed to establish the full effects of PTX on the immune system in dogs.

CT quantitative characteristics of peritumoral nodule in predicting invasiveness of thymoma

Objective: To explore the value of CT quantitative features of peritumoral nodules in predicting invasiveness of thymoma. Methods: A retrospective study of 110 patients (65 males and 45 females, with a median age of 45 years) of thymoma with complete clinical, imaging and pathological data in Peking University Third Hospital from March 2013 to October 2018. Patients were divided into high risk (23 males and 15 females, with a median age of 52 years) and low risk groups (42 males and 30 females, with a median age of 42 years) according to the 2004 WHO criteria of thymoma. The major diameter, dual phase contrast CT values, ΔCT values and distance to main tumor of peritumoral nodule were analyzed. The predictive value of CT quantitative parameters were examined with Mann-Whitney U test and ROC curves. Results: Among 110 cases of thymoma, 28 cases had peritumoral nodules.The pathological classification of thymoma were type B1 (12 cases), type B2 (7 cases), type B3 (6 cases) and type C(3 cases). No peritumoral nodules were found in type A and AB.Sixteen patients were categorized into the high risk group whereas the other 12 patients were considered low risk. For high risk group, the major diameter, dual phase contrast CT values, ΔCT values and distance to main tumor of peritumoral nodule were (9.8±3.7) mm, (48±27)HU, (51±21)HU, (17±10)HU and (6.3±6.5)mm, respectively; and for low risk group, the major diameter, dual phase contrast CT values, ΔCT values and the distance to main tumor of peritumoral nodules were (5.5±1.6)mm, (55±18)HU,(52±19)HU, (14±10)HU and (3.9±2.2)mm, respectively. Compared with the low risk group, the major diameter was significantly higher in high risk group (P<0.05). In contrast, dual phase contrast CT values, ΔCT values and the distance to main tumor only changed slightly (all P>0.05). The ROC curve analysis indicated that, the cutoff of the major diameter of peritumoral nodule predicting tumor's high risk was 6.9 mm, and the corresponding AUC, sensitivity, and specificity was 0.862, 75.0% and 75.0%, respectively. Conclusion: The major diameter of peritumoral nodule can potentially predict the invasiveness to thymoma.

Gray matter volume and microRNA levels in patients with attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder often characterized by gray matter (GM) volume reductions. MicroRNAs, which participate in regulating gene expression, potentially influence neurodevelopment. This study aimed to explore whether differential GM volume is associated with differential miRNA levels in ADHD patients. We recruited a total of 30 drug-naïve patients with ADHD (mean age 10.6years) and 25 healthy controls (mean age 10.6years) that underwent a single session of 3.0-T whole brain structural MRI scanning. RNA samples from the participants' white blood cells were collected to identify the ΔCt values of three miRNAs (miR-30e-5p, miR-126-5p, and miR-140-3p) using the real-time quantitative reverse transcription polymerase chain reaction. In comparison to the control group, ADHD patients demonstrated a significantly lower GM volume in the cingulate gyrus, left middle temporal gyrus, right middle occipital gyrus, left fusiform gyrus, and significantly higher ΔCt values of miR-30e-5p, miR-126-5p, and miR-140-3p. In the ADHD group, the GM volume of cingulate gyrus and left fusiform gyrus was negatively correlated with the ΔCt values of miR-30e-5p, miR-140-3p. The GM volume of left fusiform gyrus was negatively correlated to ADHD behavioral symptoms. Using structural equation modeling (SEM), we observed that the effect of miR-140-3p on hyperactivity/impulsivity symptoms was mediated by left fusiform gyrus. Our findings support that GM volume reduction and miRNA increases may be biomarkers for ADHD in children and adolescents. Expression levels of miRNAs may affect the development of brain structures and further participate in the pathophysiology of ADHD.

PS1452 GLI‐1, EFFECTOR OF CANONICAL HEDGEHOG SIGNALING PATHWAY, DOES NOT SEEM TO BE DYSREGULATED IN PATIENTS WITH MYELOFIBROSIS

Background:Although activation of Hedgehog signaling pathway was reported to be present in murine bone marrow transplant model of primary myelofibrosis (PMF) and increased expression of Hedgehog target genes was reported in granulocytes isolated from unselected patients with myeloproliferative neoplasms, therapeutic Hedgehog inhibition showed only limited results in myelofibrosis so far. Also, inconsistent effects of such therapy on Gli1 (effector of hedgehog signaling) were observed.Aims:We aimed to investigate Gli‐1 expression in bone‐marrow tissues of patients with primary and secondary myelofibrosis (SMF) and to assess its clinical correlations.Methods:We retrospectively investigated Gli‐1 mRNA expression in 32 diseased (28 PMF, 4 SMF) and 16 healthy (limited stage agressive Non‐Hodgkin lymphoma) bone marrow samples using real‐time‐polymerase‐chain‐reaction (GLI1 Hs01110766_m1 Thermo Fischer Scientific TaqMan assay). Expression was normalized to Abl and expressed as a ΔCT value. Correlations with clinical parameters were made. Study was approved by the institutional review board. All procedures were in accordance with the Helsinki Declaration. All patients provided written informed consent.Results:Median age of our patients was 70 years, there were 21 (66%) males and 11 (34%) females. Gli‐1 expression did not significantly differ between PMF, SMF and controls (median ΔCT 7.2, 7.3 and 6.9 respectively; P = 0.864).Higher Gli‐1 bone marrow expression was not significantly associated with neither age, gender, JAK2, CALR, MPL mutational status nor degree of bone marrow fibrosis (P&gt;0.05 for all analyses). Only statistically significant associations were present between higher Gli‐1 expression and higher transferrin saturation (P = 0.039; Rho = 0.4) and higher absolute lymphocyte count (P = 0.032; Rho = 0.4).Gli‐1 expression separated at median (ΔCT 7.25) had neutral effect on overall survival in our cohort of patients (P = 0.651) as shown in a Figure.Summary/Conclusion:Our findings indicate Gli‐1 mRNA expression might not be dysregulated in patients with myelofibrosis and has neutral impact on prognosis of these patients. Although limited by small number of patients, retrospective study design and single center experience, our results are in line with previous observations of modest clinical effects of Hedgehog inhibition in patients with myelofibrosis.image

Expression of osa-miR7695 against the blast fungus Magnaporthe oryzae in Vietnamese rice cultivars

Magnaporthe oryzae causes rice blast disease, which leads to devastating yield losses in Vietnam and other rice-growing countries. Recent studies indicated that 30% of rice- growing areas are affected by rice blast disease. MicroRNAs (miRNAs) are short non-coding RNAs that are related to plant development and biotic stress tolerance. Osa-miR7695 as one of the common miRNAs, and its target the OsNramp6 gene were evaluated as factors that contribute to the pathogenic activity of different rice cultivars. The expression profile of microRNA osa-miR7695 was evaluated to identify blast resistance or susceptibility in rice cultivars. Results of RT-PCR and real-time qPCR indicated the presence of osa-miR7695 in most Oryzae sativa Indica group cultivars in Vietnam. Upregulation of osa-miR7695 observed in blast resistant rice cultivars at different infection time intervals was higher than in blast non-resistant rice cultivars. Expression of the OsNramp6 gene increased in blast non-resistant cultivars, particularly at 72 h post inoculation. Average ΔCt values of osa-miR7695 in blast resistant rice cultivars were higher by approximately 4-fold than in blast non-resistant rice cultivars. Results suggested osa-miR7695 as a potential biomarker for microRNA based detection of blast resistance and non-resistance in Vietnamese rice cultivars to improve understanding of the biological role of microRNA in rice immunity.

Analysis of microRNA expression in brush cytology specimens improves the diagnosis of pancreatobiliary cancer

Background/ObjectivesMalignant pancreatobiliary strictures are in many cases clinically indistinguishable and present a major problem to endoscopy specialists. Intraductal sampling procedures such as brush cytology are commonly used for diagnosis with a sensitivity that is low for a diagnostic test used in daily clinical practice. MicroRNA (miR) alterations detected in many cancers are disease-specific, which can be utilized in clinical applications. The aim of the present study was to analyze whether determination of miR expression levels in intraductal brush cytology specimens is a feasible approach to improve the diagnosis of pancreatobiliary cancer. MethodsBrush cytology specimens have been collected during endoscopic retrograde cholangio-pancreatography (ERCP) and analyzed by routine cytology and ancillary miR assays. Total RNA was extracted using the miRNeasy Mini Kit and the expression of miRs frequently dysregulated in pancreatobiliary cancer (miR-16, miR-21, miR-196a, miR-221) were analyzed by quantitative real-time PCR using RNU6B as internal control. ResultsRoutine cytology resulted in no false positive diagnoses, however, the combined sensitivity remained at 53.8%. Expression (ΔCt values) of miR-16 (p = 0.0039), miR-196a (p = 0.0003) and miR-221 (p = 0.0049) showed a clear statistical significance between malignant and benign pancreatobiliary specimens (n = 35). Malignancy could be detected combining routine cytology and the miR-196a single marker expression levels with a sensitivity of 84.6% (92.9% in biliary strictures) with no false positives. ConclusionsThe results offer the first direct demonstration that microRNAs are readily detectable in brush cytology specimens obtained during ERCP, and have the potential to help the cytological diagnosis of pancreatobiliary malignancy.

Detection of circulating microRNAs with Ago2 complexes to monitor the tumor dynamics of colorectal cancer patients during chemotherapy.

Because of the different forms of circulating miRNAs in plasma, Argonaute2 (Ago2)‐miRNAs and extracellular vesicles (EV‐miRNAs), we examined the two forms of extracellular miRNAs in vitro and developed a unique methodology to detect circulating Ago2‐miRNAs in small volumes of plasma. We demonstrated that Ago2‐miR‐21 could be released into the extracellular fluid by active export from viable cancer cells and cytolysis in vitro. As miR‐21 and miR‐200c were abundantly expressed in both metastatic liver sites and primary lesions, we evaluated Ago2‐miR‐21 as a candidate biomarker of both active export and cytolysis while Ago2‐miR‐200c as a biomarker of cytolysis in plasma obtained from colorectal cancer (CRC) patients before treatment and in a series of plasma obtained from CRC patients with liver metastasis who received systemic chemotherapy. The measurement of Ago2‐miR‐21 allowed us to distinguish CRC patients from subjects without CRC. The trend in ΔCt values for Ago2‐miR‐21 and ‐200c during chemotherapy could predict tumor response to ongoing treatment. Thus, capturing circulating Ago2‐miRNAs from active export can screen patients with tumor burdens, while capturing them from passive release by cytolysis can monitor tumor dynamics during chemotherapy treatment.

Antitumoral potential of microvesicles extracted from human adipose-derived mesenchymal stem cells on human breast cancer cells.

One of the new methods that have promising results is the use of cell-derived microvesicles (MVs) to kill tumor cells. Given that MVs contain apoptotic materials, genes, and proteins, they can interfere with the fate of adjacent cells. In the present study, after adipose tissue-derived mesenchymal stem cells (AT-MSCs) isolation and characterization, MVs were derived from AT-MSCs and then characterized morphologically by standard error of the mean and size determination by DLS, and after that, the influence of MVs on human breast cancer cells (MCF-7) was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and apoptosis-related gene expression. The raw data were analyzed in SPSS.17 software. The results indicated that MVs have a size range of 500-1500 nm, and the viability of MCF-7 was significantly decreased when treated by different concentrations of MVs and it was confirmed when apoptosis-related genes' expression level was measured by real-time reverse transcription polymerase chain reaction whereas demonstrated that apoptosis genes including Bax, P53, P21, and EP300 (2- ΔΔ CT) and ΔCT values were expressed significantly in MCF-7 treated by MVs higher than those nontreated, and decrease of Bcl-2 expression level in MVs-treated MCF-7 was also significant as an antiapoptosis-related gene. Taking together, AT-MSC-derived MVs demonstrated anticancer or antitumoral properties on MCF-7 cells, and it could also be effective for other types of cancer cells.

The expression level of hsa-miR-146a-5p in plasma-derived exosomes of patients with diffuse large B-cell lymphoma.

Background:The standard treatment for patients with diffuse large B-cell lymphoma (DLBCL) had been rituximab-based immunochemotherapy. However, the biological and clinical heterogeneity within DLBCL seems to affect treatment outcome. Therefore, the evaluation of miRNA levels might be useful in predicting treatment response and relapse risk. miR-146a is a modulator of innate and acquired immunity and may play an important role in predicting treatment response. The aim of the present study was to compare the expression level of miR-146a in plasma-derived exosomes of responsive DLBCL patients (response to R-CHOP (Rituximab, and Cyclophosphamide, Hydroxydaunorubicin, Oncovine and Prednisone)), refractory DLBCL patients (resistant to R-CHOP), patients receiving R-CHOP, and healthy donors.Materials and Methods:After the preparation of plasma and isolation of exosomes, the presence of plasma-derived exosome was confirmed by Zetaseizer, electron microscope, and Western blot. The patients’ medical records were collected and analyzed. The expression level of exosomal miR-146a was evaluated in DLBCL patients and healthy donors using real-time polymerase chain reaction (PCR). The −ΔCt values of miR-146a were compared among responsive patients (n = 17), refractory patients (n = 16), patients receiving R-CHOP therapy (n = 15), and healthy donors (n = 6).Results:The presence and size of plasma-derived exosomes were confirmed. Our findings did not show any significant difference in the expression level of exosomal miR-146a between DLBCL patients and healthy donors (P = 0.48). As well, the clinical and histopathological parameters were not correlated with the expression level of exosomal miR-146a or plasma miR-146a. The expression level of plasma miR-146 was lower than the expression level of exosomal miR-146 (P = 0.01).Conclusion:Exosomal miR-146a might be useful as a promising “liquid biopsy” biomarker in predicting treatment response and relapse risk; however, we could not find significant differences due to small sample size.

Evaluation of Estrogen Receptor α and ß Genes Expression in Normal and Neoplastic Mammary Gland in Dogs by Real-time PCR

Background: Estrogen and progesterone play a key role in mammary tumorigenesis of female dogs. Malignant tumors are considered hormone-dependent neoplasms, which may reappear after surgical removal or might spread to other organs such as liver, lymph nodes, and lungs. Objective: The purpose of this study was to evaluate the expression of estrogen receptor α and ß genes in normal and neoplastic mammary glands of female dogs. Methods: Ten neoplastic tumors (benign and malignant) and 10 normal samples were selected. After RNA extraction and cDNA synthesis, ERα and ERβ genes were amplified by Real-time PCR based on SYBR green related to β-actin reference gene. Results: The obtained Ct and ΔCt values were analyzed by GLM procedure of SAS software v9.2. The results showed that the expression of ERα and ERβ genes in neoplastic cases was over- and down-expressed respectively as compared with normal samples (p < 0.01). Conclusion: The findings indicated that these genes could be used as reference genes for screening and also for early diagnosis of patients, and this method could be helpful for patient selection and monitoring of the anti-estrogen therapy.

Detection and Monitoring of Insecticide Resistance Mutations in Anopheles gambiae: Individual vs Pooled Specimens.

Bioassays and molecular diagnostics are routinely used for the monitoring of malaria vector populations to support insecticide resistance management (IRM), guiding operational decisions on which insecticides ought to be used for effective vector control. Previously developed TaqMan assays were optimised to distinguish the wild-type L1014 from the knockdown resistance (kdr) point mutations 1014F and 1014S (triplex reaction), and the N1575 wild-type from the point mutation 1575Y (duplex reaction). Subsequently, artificial pools of Anopheles gambiae (An. gambiae) specimens with known genotypes of L1014F, L1014S, and N1575Y were created, nucleic acids were extracted, and kdr mutations were detected. These data were then used to define a linear regression model that predicts the allelic frequency within a pool of mosquitoes as a function of the measured ΔCt values (Ct mutant − Ct wild type probe). Polynomial regression models showed r2 values of >0.99 (p < 0.05). The method was validated with populations of variable allelic frequencies, and found to be precise (1.66–2.99%), accurate (3.3–5.9%), and able to detect a single heterozygous mosquito mixed with 9 wild type individuals in a pool of 10. Its pilot application in field-caught samples showed minimal differences from individual genotyping (0.36–4.0%). It allowed the first detection of the super-kdr mutation N1575Y in An. gambiae from Mali. Using pools instead of individuals allows for more efficient resistance allele screening, facilitating IRM.

Multislice computed tomography performance in differential diagnosis of high-density thymic cyst and thymoma in lesions less than 3 cm.

BackgroundThis study was conducted to evaluate the performance of multislice computed tomography (MSCT) to diagnose high‐density thymic cysts and thymomas in lesions measuring < 3 cm.MethodsThe records of 42 patients admitted to Ningbo No. 2 Hospital with an anterior mediastinal mass (diameter < 3 cm) suspected of carcinoma originating from the thymus were retrospectively analyzed. All patients underwent surgery and pathological examination. Twenty‐eight were diagnosed with thymic cysts and 14 with thymoma. The features of thymic cysts and thymomas revealed on MSCT were compared.ResultsMediastinal masses with a triangular or teardrop shape and a straight mediastina–lung border were more likely to be diagnosed as thymic cysts (P < 0.05), while those with a bulging nodule–lung border were more likely thymomas (P < 0.05). Using the CT value as a reference for differential diagnosis, the diagnostic sensitivity, specificity, and area under the receiver operating characteristic curve for CT values of non‐contrast CT images, enhanced CT values, and ΔCT values were 0.65 (0.47–0.84), 0.95(0.86–1.00), and 0.96 (0.89–1.00), respectively.ConclusionMSCT of high‐density thymus gland nodules (maximum diameter ≤ 3 cm) can reveal specific features that could be helpful for differential diagnosis.

Quantitative study on grading activity of Crohn disease with CT enterography

Objective To explore the feasibility and the value of CT enterography (CTE) in the quantitative evaluation of Crohn's disease (CD). Methods Retrospectively analyzed 49 patients diagnosed as CD by clinical, enteroscopy, pathology, and imaging from April 2016 to June 2017 in Zhongnan Hospital of Wuhan University. All patients underwent routine enteroscopy and standardized CTE. The interval between the two examinations was less than 2 weeks. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were measured at intervals. The ESR ranged from 2.0 to 97.0 mm/1 h, with an average of (30.6±26.5) mm/1 h, CRP from 0.3 to 143.3 mg/L, and a median of 27.7 mg/L. CD patients were classified into inactive, mild and moderate-severe according to Crohn's disease simplified endoscopic activity score(SES-CD) after enteroscopy. CTE evaluated the site of lesion, the thickness and enhanced patterns of the most severe lesions of the bowel wall, mural enhanced CT value in the portal vein phase, ΔCT value, stenosis, perienteric inflammation, mesenteric hypervascularity (comb sign), enlarged lymph nodes, abdominal abscesses, fistulas, etc. The Chi-square test was used to compare the differences of enhanced patterns among different groups. ANOVA (Analysis of Variance) was used to compare the differences in bowel wall thickness, plain CT values, mural enhanced CT value in the portal vein phase and ΔCT values among different groups. The correlation between CTE parameters and SES-CD or laboratory data was analyzed by Pearson (normal distribution data) or Spearman (skewed distribution data). Results SES-CD ranged from 1 to 15, with an average of 5.1±2.6. Among 49 cases, 13 were inactive, 19 were mild, and 17 were moderate-severe. There were 25 cases of intestinal obstruction or stenosis, including 24 cases in the active group and 1 case in the non-active group, and the difference was statistically significant (χ2=13.3, P 0.05). Bowel wall thickness, mural enhanced CT value in the portal vein phase and ΔCT values were all moderately correlated with SES-CD (r were 0.564, 0.585, 0.533, respectively; P all 0.05). Conclusion The bowel wall thickness, mural enhanced CT value in the portal vein phase and the ΔCT value can be used to accurately assess the activity of CD lesions. Key words: Crohn disease; Tomography,X-ray computed; Simplified endoscopic activity score for Crohn disease