Relative Quantitative Real-time PCR Research Articles

Evaluation of tumor-educated platelet long non-coding RNAs (lncRNAs) as potential diagnostic biomarkers for colorectal cancer.

Cancer-derived circulating components are increasingly considered as candidate sources for non-invasive diagnostic biomarkers. This study aimed to investigate the expression of tumor-educated platelet (TEP) long non-coding RNAs (lncRNAs) in colorectal cancer (CRC) patients and determine whether it could be served as a potential tool for CRC diagnosis. Relative quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of three cancer-related platelet-derived lncRNAs CCAT1, HOTTIP, and XIST in 75 CRC patients and 42 healthy controls. Quantitative data were analyzed by SPSS (IBM Corp., Armonk, NY, USA) for comparison of cancer and non-cancer individuals. The receiver operating characteristic (ROC) curve analysis was further performed to assess the diagnostic values of lncRNAs within the CRC patients. The expression levels of lncRNAs colon cancer associated transcript 1 (CCAT1) (P = 0.006) and HOXA transcript at the distal tip (HOTTIP) (P = 0.049), but not X-inactive specific transcript (XIST) (P = 0.12), were significantly upregulated in CRC patients compared to healthy individuals. However, there were no significant correlations between platelet lncRNAs and clinicopathological characteristics, including sex, age, tumor location, differentiation, and size (all at P > 0.05). The area under the ROC curve (AUC) of the lncRNA CCAT1 was 0.61 (sensitivity, 71%; specificity, 50%). TEP lncRNA CCAT1 is detectable in the circulation of CRC patients and could be considered as a potential diagnostic biomarker.

Transcriptional alteration of NF-κB-associated long noncoding RNAs in the stomach of Helicobacter pylori-infected and non-infected patients.

Helicobacter pylori could colonize the gastric mucosa and cause gastritis, ulcers and cancer. Numerous virulence factors have been identified in this bacterium that play important roles in promoting gastric disorders. Although the interaction of long noncoding RNAs (lncRNAs) with transcription, processing, and translation of genes associated with different diseases are described, their interaction with the inflammatory genes and H. pylori infection in the gastric tissue is not well known. This study compared changes in common NF-κB-regulatory lncRNAs in the gastric tissue of H. pylori-infected and non-infected patients with gastritis. Moreover, a link between the virulence entity of the strains and the transcriptional changes was analyzed. Two groups of infected and non-infected patients with chronic gastritis were included in the study. Genotyping of the H. pylori strains was done by PCR and relative changes in the expression of NF-κB and regulatory lncRNAs, lincRNA-p21, MALAT1, NKILA, were measured by relative quantitative real time-PCR. Transcriptional levels of MALAT1, lincRNA-p21, and NKILA genes decreased in the infected patients compared with the non-infected patients, which was significantly linked with increased NF-κB gene expression. Our results showed that a hypervirulent strain of H. pylori with oipA"on"/HP-NAP+/iceA1+/iceA2+/vacA s1m1/s1m2+/cagA+ genotype can promote a higher level of NF-κB transcription in the inflamed tissue. H. pylori infection could promote down-regulation of lincRNA-p21, MALAT1 and NKILA in the infected gastric tissue in correlation with NF-κB upregulation. More detailed studies are needed to show a link between the virulence genes and their impact on the regulation of lncRNAs in the stomach.

Canine sperm motility is associated with telomere shortening and changes in expression of shelterin genes

BackgroundMotion quality is a critical property for essential functions. Several endogenous and exogenous factors are involved in sperm motility. Here, we measured the relative telomere length and evaluated the gene expression of its binding-proteins, shelterin complex (TRF1, TRF2, RAP1, POT1, TIN2, and TPP1) in sperm of dogs using relative quantitative real-time PCR. We compared them between two sperm subpopulations with poor and good motion qualities (separated by swim-up method). Telomere shortening and alterations of shelterin gene expression result from ROS, genotoxic insults, and genetic predisposition.ResultsSperm kinematic parameters were measured in two subpopulations and then telomeric index of each parameter was calculated. Telomeric index for linearity, VSL, VCL, STR, BCF, and ALH were significantly higher in sperms with good motion quality than in sperms with poor quality. We demonstrated that poor motion quality is associated with shorter telomere, higher expression of TRF2, POT1, and TIN2 genes, and lower expression of the RAP1 gene in dog sperm. The levels of TRF1 and TPP1 gene expression remained consistent despite variations in sperm quality and telomere length.ConclusionData provided evidence that there are considerable changes in gene expression of many shelterin components (TRF2, TIN2, POT1and RAP1) associated with shortening telomere in the spermatozoa with poor motion quality. Possibly, the poor motion quality is the result of defects in the shelterin complex and telomere length. Our data suggests a new approach in the semen assessment and etiologic investigations of subfertility or infertility in male animals.

Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail.

Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification.

Alteration of the Inflammatory and Anti-Inflammatory Cytokine Profiles of Peripheral Blood Mononuclear Cell in Crohn's Disease Patients after Following up.

Background:Crohn’s disease (CD) has a chronic course, which its recurrence varies widely among different patients. In this study we prospectively analyzed blood samples of 19 CD patients. Alteration in transcription of inflammatory and anti-inflammatory cytokines was analyzed compared with household members after three month follow up.Methods:CD patients were diagnosed based on clinical symptoms, endoscopic and histopathologic characteristics. Nineteen CD patients and their households were evaluated from Jun 2019 to Feb 2021 at Tehran university hospitals. CD activity score, biological, clinical and demographic data of the patients were recorded at two time point intervals. Bacteriological tests were done using aerobic and anaerobic blood cultures. To investigate transcriptional alterations, peripheral blood mononuclear cells (PBMCs) were isolated using Ficol centrifugation method and relative quantitative real-time PCR was done to determine the expression level of IFN-γ, TNF-α, IL10, and FOXP3 cytokines.Results:Our results showed a correlation between fecal calprotectin level (709.8 ± 554.6), C-reactive protein concentration (18.1 ± 15.9), and erythrocyte sedimentation rate (30.4 ± 17.9) with disease activity (Flare/remission). IL10 and Foxp3 anti-inflammatory gene’s expression were significantly (P = 0.003 for IL10 and P = 0.008 Foxp3) higher during the flare and remission in patients with active disease respectively. Bacteriological examination showed infection with Streptococcus spp. and Clostridium spp. in two CD patients during flares, which was correlated with upregulation and down-regulation of IL10, TNF-α, IFN-γ and FOXP3 proteins, respectively.Conclusion:Occurrence of bacteremia, and higher amount of CAP, CRP and ESR are correlated with higher level of transcription for inflammatory cytokines, which could effectively reflect the disease activity. Raise in FoxP3 transcription proposed change in Treg sub-population in PBMC or its activity during the CD remission phase.

A single point mutation in the Plasmodium falciparum 3\u2032\u20135\u2032 exonuclease does not alter piperaquine susceptibility

BackgroundThe rise in Plasmodium falciparum resistance to dihydroartemisinin–piperaquine (DHA–PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele.MethodsTransgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility.ResultsThe transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly.ConclusionsInsertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

Evaluation of hydroalcoholic extract effects of Ferula assa-foetida on expression change of EMT and CD44-related genes in gastric cancer stem cell

BackgroundGastric cancer (GC) with high incidence is a life-threatening malignant tumor in humans. Cancer stem cells (CSCs) due to tumor-initiating capacity, ability of invasion, metastasis, recurrence, and chemoresistance may be utilized for cancer treatment. Ferula assa-foetida as a herbal remedy in ancient Iranian medicine was shown to have various biological activities, including anti-cancer. Epithelial-mesenchymal transition (EMT) markers play an important role in migration, invasion, and metastatic dissemination of CSCs. The gastric CSCs-surface glycoprotein CD44 also responsible for high tumorigenicity, self-renewal, and metastasis through EMT. In this study, we investigated the hydroalcoholic extract effects of F. assa-foetida on the expression of EMT-related genes, anti-apoptotic factor Bcl-2 and CD44 cell-surface molecule in CSCs. MethodsCSCs were cultured in serum-free medium containing EGF and B-27 and exposed to different concentrations of F. assa-foetida hydroalcoholic extract and for different time durations. Cytotoxic effects of the extract compounds were evaluated using MTT assay. After CSCs treatment with IC50 dose of the extract, the expression level of EMT markers vimentin, Snail1, and Zeb1 and anti-apoptotic marker Bcl-2 was determined using relative quantitative real-time PCR as well as marker CD44 and CD54 were determined by flow cytometry. ResultsOur MTT assay results demonstrated that cytotoxicity effect of extract compounds was significantly dependent on time and concentration. After exposing CSCs to IC50 doses of extract in different times, mRNA expression levels of EMT markers (vimentin, Snail1, Zeb1) and anti-apoptotic marker Bcl-2, as well as protein expression of stemness marker CD44 and CD54 significantly decreased in treated CSCs compared with untreated (P < 0.05). ConclusionOur data demonstrated that extract compounds of F. assa-foetida were able to decrease EMT markers, Bcl-2 anti-apoptotic factor, surface marker of CD44 and CD54, leading to inhibition of CSCs proliferation as well as killing of them. Therefore, isolation and purification of F. assa-foetida hydroalcoholic extract could be used as a potential agent for cancer treatment.

Tumor-Educated Platelet miR-18a-3p as a Novel Liquid-Biopsy Biomarker for Early Diagnosis and Chemotherapy Efficacy Monitoring in Nasopharyngeal Carcinoma.

AimsTo evaluate the value of tumor-educated platelet (TEP) miR-18a-3p in the early diagnosis and chemotherapy efficacy monitoring of nasopharyngeal carcinoma (NPC).MethodsExpression levels of miR-18a-3p in platelets and plasma were detected by relative quantitative real-time PCR in NPC patients (n=54) and normal subjects (n=36). Diagnostic values of TEP miR-18a-3p for NPC was assessed by receiver operating characteristic (ROC) curve analysis. Follow up study was carried out to observe the dynamic changes of TEP miR-18a-3p with chemotherapy on 3 NPC patients.ResultsThe expression levels of TEP miR-18a-3p in NPC patients were significantly higher than that in healthy controls (p < 0.0001). ROC curve analysis showed that the area under the curve (AUC) value was 0.841, the sensitivity and specificity for the diagnosis of NPC were 87% and 72.7%. No correlation was found between expression levels of TEP miR-18a-3p and patients’ clinical parameters and their NPC tumor-node-metastasis (TNM) stage. The positive rate of TEP miR-18a-3p and EBV DNA for NPC diagnosis were 85.4% and 66.7%. TEP miR-18a-3p expression were down-regulated after 77.8% (7 of 9) of chemotherapy, and in 66.7% (2 of 3) patients, TEP miR-18a-3p levels decreased after 3 cycles of chemotherapy.ConclusionThe expression levels of TEP miR-18a-3p are upregulated in NPC and have a high probability to downregulated after chemotherapy, indicating a significant clinical value. TEP miR-18a-3p might serve as a novel type of liquid-biopsy biomarker for early diagnosis and chemotherapy efficacy monitoring in NPC.

Transcriptional alteration of genes linked to gastritis concerning Helicobacter pylori infection status and its virulence factors

Helicobacter pylori infection and heterogeneity in its pathogenesis could describe diversity in the expression of inflammatory genes in the gastric tissue. We aimed to investigate transcriptional alteration of genes linked to gastritis concerning the H. pylori infection status and its virulence factors. Biopsy samples of 12 infected and 12 non-infected patients with H. pylori that showed moderate chronic gastritis were selected for transcriptional analysis. Genotyping of H. pylori strains was done using PCR and relative expression of inflammatory genes was compared between the infected and non-infected patients using relative quantitative real-time PCR. Positive correlations between transcriptional changes of IL8 with TNF-α and Noxo1 in the infected and TNF-α with Noxo1, MMP7, and Atp4A in the non-infected patients were detected. Six distinct genotypes of H. pylori were detected that showed no correlation with gender, ethnicity, age, endoscopic findings, and transcriptional levels of host genes. Irrespective of the characterized genotypes, our results showed overexpression of TNF-α, MMP7, Noxo1, and ATP4A in the infected and IL-8, Noxo1, and ATP4A in the non-infected patients. A complexity in transcription of genes respective to the characterized H. pylori genotypes in the infected patients was detected in our study. The observed difference in co-regulation of genes linked to gastritis in the infected and non-infected patients proposed involvement of different regulatory pathways in the inflammation of the gastric tissue in the studied groups.

Comparison of toxin-antitoxin expression among drug-susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), is a significant global public health threat. Besides extensive multidrug resistance, MTB possesses several properties for long-term viability in the host as well as stress adaptation and resistance in harsh conditions. The role of toxin-antitoxin (TA) systems in disseminating and maintaining antimicrobial resistance in bacterial populations has also been demonstrated. This study aimed to evaluate differences in expression of MazEF (a well-known TA system) related genes (mazE3, mazF3, mazE6, and mazF6) amongst drug-susceptible and resistant MTB isolates in Iran. A total of 20 confirmed clinical isolates of MTB including 10 drug-susceptible and 10 drug-resistant (nine MDR, and one XDR) species were included in this study. M. tuberculosis H37Rv was used as the standard strain. RNA extraction, cDNA synthesis, and relative quantitative real-time PCR were performed according to the standard procedures. Our analysis indicated significant enhanced expression of the mazE6 antitoxin gene in drug-susceptible isolates compared to drug-resistant isolates and the standard strain. The expression of the mazF6 toxin gene was also increased in drug-susceptible isolates compared with the standard strain. In drug-resistant isolates, the expression levels of mazF3 and mazF6 genes were significantly higher than that in the susceptible isolates and the standard strain. In this study, there was significant overexpression of mazE6 in drug-susceptible isolates. As well, mazF3 and F6 were overexpressed in drug-resistant isolates when compared with the standard strain. The changes in expression levels of MazEF6 associated genes were greater than that of MazEF3 in both groups of isolates.

Anosmic flies: what Orco silencing does to olive fruit flies

BackgroundThe olive fruit fly (Bactrocera oleae) is the most destructive pest of the olive cultivation worldwide causing significant production losses and olive fruit impoverishment, as its larvae feed exclusively on the olive fruit. Reproductive and sexual behavior, as well as host-plant recognition of the fly, are highly dependent on its chemosensory system. Therefore, exploring the role of genes that play a critical role in olfaction, could reveal potential molecular targets that determine species-specific features on chemical communication and could be used to impair sexual behavior.ResultsIn this study we identified the gene that encodes the conserved olfactory co-receptor Orco (Odorant receptor co-receptor), which interacts with all divergent insect odorant receptors, and investigated how disruption of its expression affects chemoreception. We initially searched the expression profile of Bo-Orco in both sexes during sexual maturation, as well as pre- and post-mating communication by relative quantitative real time PCR (qRT-PCR) analysis suggesting that Bo-Orco was abundantly expressed in sexually mature adults.We further investigated the functional role of Bo-Orco in mating and oviposition behavior via transient gene silencing that was performed through in vivo dsRNA hemolymph injections in sexually mature flies 7 days after eclosion. Orco-knockdown phenotypes in both sexes showed reduced copulation rates in mating competitiveness tests, possibly through impaired olfactory-mediated detection of sex pheromone. In addition, oviposition was significantly inhibited in dsRNA-Orco injected females in a post-mating behavior test.ConclusionsOur results demonstrate that Orco plays a crucial role in the reproductive behavior of the olive fruit fly, since pre- and post-mating processes were affected. This is the first report in the olive fruit fly that links the chemosensory pathway with the mating behavior and the reproductive potential at a molecular basis, rendering this gene a potential target for the improvement of the olive fruit fly population control techniques.

Comparative transcriptome analysis reveals regulatory genes involved in cold tolerance and hypoxic adaptation of high-altitude Tibetan bumblebees

China has many habitats supporting a wide diversity of bumblebees, some species of which are limited in distribution to the Tibet Plateau. The high-altitude Tibet Plateau, which has low air density as well as extremely low temperatures, strains the energy requirements for flight in most insects. However, high-altitude bumblebees have adapted to the harsh conditions of this type of habitat. Bumblebees are a particular group of insect pollinators that exclusively utilize carbohydrates from flowers for energy to sustain flight. Here, gene expression was compared between low-altitude species and Bombus longipennis, and the genes upregulated in B. longipennis were involved in aerobic metabolism, such as the oxidative phosphorylation and citric acid cycle (TCA cycle) pathways. Furthermore, a conjoint analysis of the transcriptomes of six bumblebee species from the high-altitude Tibet Plateau and two bumblebee species from the low-altitude North China Plain showed that 19 genes were commonly upregulated in high-altitude species. Among these 19 commonly upregulated genes, Pfk1 was enriched in multiple glycometabolic pathways, which are the main energy pathways in bees; this upregulation enhanced the aerobic and anaerobic glycolysis processes to produce more ATP molecules to supply energy for high-altitude bumblebee flight under severe cold conditions. In addition, glycolysis was enhanced by two other genes, Rac1 and AAC2. Relative quantitative real-time PCR was used to verify that the three genes Pfk1, Rac1, and AAC2 were upregulated in the six main bumblebee species inhabiting the Tibet Plateau.

Characterization and expression of Cm-AAT1 gene encoding alcohol acyl-transferase in melon fruit (Cucumis melo L.) ‘Hikapel’

Abstract. Wibowo WA, Fatkhurohman MI, Daryono BS. 2020. Characterization and expression of Cm-AAT1 gene encoding alcohol acyl-transferase in melon fruit (Cucumis melo L.) ‘Hikapel’. Biodiversitas 21: 3041-3046. Melon (Cucumis melo L.) is one of the horticulture commodities that have high economic value and its needs increase continuously. Many new melon cultivars have been assembled to produce a higher quality melon. Melon 'Hikapel' developed by the Laboratory of Genetics and Breeding, Faculty of Biology UGM has distinctive character in the form of a strong aroma. This aroma is a complex mixture of various kinds of volatile compound. One of the main determinant compounds is a volatile ester, synthesized by the alcohol acyl-transferase enzyme encoded by the Cm-AAT1 gene. Characterization of Cm-AAT1 began with isolation of melon rinds to get total RNAs. Synthesis cDNA was conducted with oligo-dT primer, followed by detection of Cm-AAT1 using specific primers. A specific band was sequenced to perform phylogenetic tree. Gene expression from 4 melon cultivars, ‘Hikapel’, ‘Hikadi’, ‘Sun Lady’, and ‘Luna’ analysis was performed using relative quantitative Real-Time PCR. The results of this study showed that Cm-AAT1 owned not only by aromatic cultivars ‘Hikapel’ and ‘Hikadi’, but also owned by non-aromatic cultivars ‘Sun Lady’ and ‘Luna’. Phylogenetic analysis shows a high similarity between Cm-AAT1 on 'Hikapel' and 'Hikadi'. Gene expression analysis on 'Hikapel' increases as the process of fruit ripening during the storage period and it is in contrast to 'Hikadi' at decrease when the fruit began to enter the decay process on day 7th. Expression of Cm-AAT1 on ‘Hikapel’ was higher than ‘Hikadi’ at the peak of fruit maturity.

Molecular cloning and characterization of DjRac1, a novel small G protein gene from planarian Dugesia japonica

Rac proteins are classified as a subfamily of the Rho family of small G proteins. They are important molecular switches which act as key signal transducers regulating a wide variety of processes in the cell. DjRac1, a novel Rac gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 851 bp with a putative open reading frame of 190 amino acids. It has a predicted molecular mass of 21.12 kDa and an isoelectric point of 8.42. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjRac1 from 1 to 7 days in the regenerating planarians. Results showed that the expression of DjRac1 was concentrated in the blastema and the transcription level of DjRac1 was significantly upregulated after amputation within three days, suggesting DjRac1 might participate in the process of regeneration in planarian.

Induced Thermotolerance and Expression of Three Key Hsp Genes (Hsp70, Hsp21, and sHsp21) and Their Roles in the High Temperature Tolerance of Agasicles hygrophila.

Thermal adaptation plays a fundamental role in the expansion and distribution of insects, and heat shock proteins (Hsps) play important roles in the temperature adaptation of various organisms. To determine the roles of Hsp genes (Hsp70, Hsp21, and sHsp21) on the high temperature tolerance of Agasicles hygrophila, we obtained complete cDNA (complementary DNA) sequences for Hsp70, Hsp21, and sHsp21 by rapid amplification of cDNA ends (RACE), analyzed their expression profiles under different high temperature treatments by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and performed functional verification by RNA interference (RNAi). The open reading frames of Hsp70, Hsp21, and sHsp21 were 1940, 543, and 567 bp, encoding 650, 180, and 188 amino acids, respectively. Their molecular weights (MWs) were 71.757, 20.879, and 21.510 kDa, and the isoelectric points were 5.63, 6.45, and 6.24, respectively. Phylogenetic tree analysis showed that the Hsp70, Hsp21, and sHsp21 genes of A. hygrophila were relatively conserved in evolution. The Hsp70 and Hsp21 genes in A. hygrophila were homologous to those in Leptinotarsa decemlineata (87 and 79% similarity, respectively), and the sHsp21 gene in A. hygrophila was homologous to that in Lissorhoptrus oryzophilus (74% similarity). The amino acid polypeptide chain had highly conserved sequences of DLGGGTFD, VLVGGSTR, and GPTIEEVD. The sequence of EEVD was the characteristic motif of cytoplasmic Hsp70, and the highly conserved sequences of MALFR and MSLLP were characteristic sequences of Hsp2 and sHsp21, respectively. Relative quantitative real time PCR showed that the three Hsps could be induced by 4-h treatment at high temperatures. Significant upregulation of these Hsps was observed when the temperature was further increased. The RNAi results showed that the injection of the three Hsps’ dsRNA could suppress the expression at the gene level significantly. Compared with the control group, high temperature heat shock reduced the fecundity of A. hygrophila significantly, and the fecundity decreased with the increase in temperature. Our results suggest that Hsp70, Hsp21, and sHsp21 might play key roles in high temperature adaptation of A. hygrophila and help improve our understanding of their mechanism of thermotolerance.

Specific role of the tight junction proteins occludin and claudin-5 on the blood–brain barrier during Listeria monocytogenes infection

To investigate the blood-brain barrier (BBB) permeability of mice after Listeria monocytogenes infection for further study on the mechanism of L. monocytogenes crossing the BBB, a mouse model was established and Evans blue assay was performed to assess the BBB disruption. Using relative quantitative real-time PCR, the RNA expression of Zonula occludens-1 (ZO-1), occludin and claudin-5 were detected. In addition, the protein expression level of ZO-1, occludin and claudin-5 were detected by immunohistochemistry and western blot. The extravasation of Evans blue dye was significantly different between 24 h and 96 h (P &lt; 0.05). The mRNA expression of occludin and claudin-5 were down-regulated than that of the control group at each sampling point (P &lt; 0.05) and ZO-1 showed a significant change at 96 h (P &lt; 0.05). In addition, the protein expression level of occludin and claudin-5 decreased significantly at 48 h and 96 h (P &lt; 0.05) by immunohistochemistry and western blot, compared with the control, while ZO-1 was almost unchanged (P &gt; 0.05). All results indicating that the tight junction integrity of endothelial cells was destroyed and BBB permeability was enhanced in the process of L. monocytogenes infection, and this change was related to the decrease of the expression occludin and claudin-5.

Moderate hyperthermic heating encountered during thermal ablation increases tumor cell activity

Purpose The aim of this study was to determine whether moderate hyperthermic doses, routinely encountered in the periablational zone during thermal ablation, activate tumor cells sufficiently to secrete pro-tumorigenic factors that can induce increased proliferation. Material and methods R3230 rat mammary tumor cells and human cancer cell lines, MCF7 breast adenocarcinoma, HepG2 and Huh7 HCC, and HT-29 and SW480 colon adenocarcinoma, were heated in to 45 ± 1 °C or 43 ± 1 °C in vitro for 5-10 min and incubated thereafter at 37 °C for 1.5, 3 or 8 hr (n = 3 trials each; total N = 135). mRNA expression profiles of cytokines implicated in RF-induced tumorigenesis including IL-6, TNFα, STAT3, HGF, and VEGF, were evaluated by relative quantitative real-time PCR. HSP70 was used as control. c-Met and STAT3 levels were assessed by Western blot. Finally, naïve cancer cells were incubated with medium from R3230 and human cancer cells that were subjected to 43–45 °C for 5 or 10 min and incubated for 3 or 8 h at 37 °C in an xCELLigence or incuCyte detection system. Results Cell-line-specific dose and time-dependent elevations of at least a doubling in HSP70, IL-6, TNFα, STAT3, and HGF gene expression were observed in R3230 and human cancer cells subjected to moderate hyperthermia. R3230 and several human cell lines showed increased phosphorylation of STAT3 3 h post-heating and increased c-Met following heating. Medium of cancer cells subject to moderate hyperthermia induced statistically significant accelerated cell growth of all cell lines compared to non-heated media (p < 0.01, all comparisons). Conclusion Heat-damaged human tumor cells by themselves can induce proliferation of tumor by releasing pro-tumorigenic factors.

Comparison of αLEPRA and αLELY Co-Inheritance with a SPTA1 Whole Gene Deletion, a Family Study

Background: Genetic variants resulting in quantitative defects in the production of α-spectrin are associated with autosomal recessive hereditary spherocytosis (HS). Because erythroid cells produce α-spectrin in a 3-4:1 ratio in relation to β-spectrin, one defective allele is typically well-tolerated and a single null allele combined with a second partially defective allele may display a sub-clinical phenotype. Protein expression can be reduced by nonsense, frameshift, missense or splicing variants but reported large deletion events are extraordinarily rare (n = 1). Splicing variants include the low expression alleles αLELY - Low Expression LYon (SPTA1:c.6531-12C&gt;T, p.?) and αLEPRA-Low Expression PRAgue (SPTA1:c.4339-99C&gt;T, p.?) which reduce protein expression by 50% and 16%, respectively. Here, we present a family study with a whole gene SPTA1 deletion that characterizes the patient phenotype when a full gene mutation is co-inherited with the αLEPRA and αLELY low expression alleles. Methods: The 6 month-old male proband had a history of congenital Coombs negative hemolytic anemia requiring transfusion from 3 months of age and in whom a red cell membrane disorder was clinically suspected. Pretransfusion CBC and peripheral blood smear results were unknown. Transfused CBC indices were as follows: RBC 4 x 1012/L, HGB 11 g/dL, MCV 76 fL, MCH 27 pg, MCHC 36 g/dL and a reticulocyte count of 1.8%. The proband's mother, of Cuban descent, and father, of European descent, had no history of anemia or HS. Targeted high throughput sequencing of genes related to hereditary hemolytic anemia was performed for the detection of single nucleotide variants (SNVs) and small insertion/deletion variants (indels). Relative quantitative real time PCR (RT-PCR) was utilized to confirm large structural variants. Results: Sequencing identified a large heterozygous deletion that spanned the entire SPTA1 gene (NC_000001.11:g.(?_158656337)_(158581024_?) in the proband and his mother. In addition, the proband had inherited hemizygous αLEPRA from his father. The mother had hemizgous αLELY. The father showed heterozygous αLEPRA and no other SPTA1 variants (Table 1). Conclusion: To our knowledge, only Iolascon et al in 2011 has reported a SPTA1 gene large deletion (SpectrinExeter, exon 11-exon 12 deletion, loss of 83 amino acids) which resulted in a structurally abnormal protein and ellipto-poikilocytosis phenotype when in trans to αLELY. Our current case is the first reported SPTA1 full gene deletion and our family study allows comparison of the effects of coinheritance of the low expression alleles αLEPRA and αLELY. When in combination with complete absence of the SPTA1 gene in trans configuration, αLELY manifests in subclinical effects and αLEPRA causes a recessive HS phenotype, which resulted in severe anemia. This contrast further demonstrates that, unlike αLEPRA, αLELY does not decrease the α-spectrin concentration sufficiently to contribute to the baseline phenotype regardless of the severity of the second allele mutation for quantitative disorders such as HS. Disclosures No relevant conflicts of interest to declare.

Tumor-educated platelet miR-34c-3p and miR-18a-5p as potential liquid biopsy biomarkers for nasopharyngeal carcinoma diagnosis.

Background: Nasopharyngeal carcinoma (NPC) is the common malignant tumor of nasopharynx in southern China and other southeastern Asian countries. MicroRNAs (miRNAs) have been shown to play important roles in carcinogenesis. Recently, miR-34c-3p and miR-18a-5p have been found to be involved in carcinogenesis of NPC. Furthermore, platelets in NPC patients may acquire RNAs from NPC cells and turn into “tumor-educated platelet (TEP)”, which may serve as potential biomarkers for a diagnosis of NPC. However, the expression profiles of TEP miR-34c-3p and miR-18a-5p in NPC patients and their diagnostic values are yet to be determined.Aims: To investigate expression levels of TEP miR-34c-3p and miR-18a-5p and determine their diagnostic values for NPC.Materials and methods: Relative quantitative real-time PCR was used to determine the expression levels of TEP miR-34c-3p and miR-18a-5p in NPC patients (n=54) as compared to normal subjects (n=36). The receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic values of TEP miR-34c-3p and miR-18a-5p for NPC.Results: The expression levels of TEP miR-34c-3p and miR-18a-5p were significantly higher in NPC patients as compared to healthy subjects. The ROC analysis showed that the area under the ROC curve (AUC), sensitivity, specificity and accuracy for TEP miR-34c-3p, miR-18a-5p, or a combination of both miRNAs for NPC diagnostic tests were 0.952, 94.44%, 86.11%, 91.11%, or 0.884, 85.19%, 86.11%, 85.55%, or 0.954, 92.59%, 86.11%, 90.00%, respectively. No correlation was found between expression levels of TEP miR-34c-3p or miR-18a-5p and patients’ demographic variables and their NPC tumor/node/metastasis stages. The positive rates of TEP miR-34c-3p and miR-18a-5p for NPC diagnosis were 93.8% and 87.5%, respectively, which were significantly higher than Epstein-Barr virus DNA with a positive rate of 66.7%.Conclusion: The expression levels of TEP miR-34c-3p and miR-18a-5p are upregulated in NPC, rendering a significant clinical value for NPC diagnosis. The TEP miRNAs might serve as a novel type of liquid biopsies for NPC diagnosis.

Yulangsan polysaccharide inhibits 4T1 breast cancer cell proliferation and induces apoptosis in vitro and in vivo

Yulangsan polysaccharide (YLSPS) is derived from the root of Millettia pulchra (Benth.) Kurz var. Recent studies have postulated YLSPS as a regimen for cancer treatment. However, the underlying mechanism anti-breast cancer is still poorly unknown. The aim of this study was to examine the suppressive and apoptosis effect of YLSPS on the growth of breast cancer cell 4T1 and its possible underlying mechanism. In this study, breast cancer cell 4T1 viability and apoptosis were assessed by CCK-8 and flow cytometry, relative quantitative real-time PCR and western blot after treated with drug-serum of YLSPS. Furthermore, therapy experiments were conducted using a Balb/c mouse transplanted tumor model of breast cancer. The number of apoptotic cells and microvascular density (MVD) in the tumor tissues were assessed by TUNEL and CD34 immunostaining. Immunohistochemical assays and ELISA were used to detect the expression of VEGF, Bcl-2, Bax and Caspase-3 in the tissues. The in vitro studies showed that the drug-serum of YLSPS significantly inhibition of proliferation and effectively induced apoptosis of 4T1 cells. Oral administration of YLSPS in the breast cancer models significantly reduced the tumor volume and weight. The enhanced antitumor efficacy was associated with decreased angiogenesis, an enhanced antioxidant capacity, an increased induction of apoptosis and an inhibition of lung metastasis. These findings indicate that YLSPS significantly inhibited mouse breast cancer growth in vitro and in vivo. These data suggest that YLSPS may serve as a potential therapeutic agent for breast cancer.