Reverse Transcription And Quantitative PCR Research Articles

Metabolomic analysis of the effect glutamate on fengycin-overproducing Bacillus subtilis ATCC 21332 with an enhanced fatty acid synthesis pathway

Fengycin is a of cyclic lipopeptide with antifungal, antitumor and adhesion-preventing activities, with great application potential in biological control, medicine and industry. However, the fermentation yield of fengycin is inadequate, which limits its wider application. In this study, key modules for enhanced fatty acid synthesis were cloned singly and in combination, and the suitability of these modules for B. subtilis ATCC 21332 was investigated. We constructed the engineered strain BSA034, which exhibited a 3-fold increase of fengycin titer, which reached 442.51 mg/L. Reverse transcription and quantitative PCR (RT-qPCR) revealed the effect of enhanced branched-chain fatty acid synthesis on the expression level of the fen operon in B. subtilis ATCC 21332, which significantly contributed to the increase of fengycin production. The exogenous supplementation of glutamate further increased the fengycin titer of BSA034 to 657.55 mg/L. Further comparative metabolomic analysis revealed that glutamate mainly promoted the membrane transport of BSA034, which provides a rational basis for further improving the titer of fengycin in the future.

Gene Expression in MC3T3-E1 Cells Treated with Diclofenac and Methylprednisolone.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids (GCs) are often used to treat articular-skeletal disorders. The extended use of NSAIDs and GCs have adverse effects on bone metabolism, reducing bone quality and impairing fracture healing. In the present study, we used mouse pre-osteoblast cells MC3T3-E1 to demonstrate the effects of diclofenac (DF) and methylprednisolone (MP) on cell proliferation and gene expression. Cells were incubated with three doses of DF or MP: 0.5 µM, 5 µM, and 50 µM. MP decreased cell viability even after 24 h, but DF inhibited cell viability after only seven days of treatment. The cells were lysed after one, two, three, and seven days of treatment, and gene expression was analyzed by reverse transcription and quantitative PCR (RT-qPCR) assays. DF did not significantly affect the expression of the osteogenic marker genes. MP modified the expression of Osx, Runx, and Col1a1. We concluded that MP is a more potent inhibitor of mouse pre-osteoblast differentiation and viability than is DF. Our results suggest that prolonged DF treatment could be less harmful to osteoblasts than MP treatment.

Engineering Glucose-to-Glycerol Pathway in Klebsiella pneumoniae and Boosting 3-Hydroxypropionic Acid Production Through CRISPR Interference.

The recent decline of the international biodiesel industry has led to decreased production and therefore increased the price of glycerol, which is a major by-product of biodiesel but a substrate for production of 3-hydroxypropionic acid (3-HP), that is, glycerol as a feedstock has no advantage over glucose in price. Hence, we engineered glucose to the glycerol pathway and improved 3-HP production by CRISPR interference (CRISPRi). To begin with, we cloned the genes encoding glycerol 3-phosphate dehydrogenase (gpd1) and glycerol 3-phosphatase (gpp2) from Saccharomyces cerevisiae, which jointly catalyze glucose into glycerol. The genes gpd1 and gpp2 were co-expressed in K. pneumoniae with the dCas9 gene integrated in genome, and this recombinant strain produced 2 g/L glycerol in the shake flask. To minimize the glucose consumption by competing pathways including the EMP pathway, glycerol oxidation pathway, and by-products pathways, we developed an CRISPRi system in aforementioned recombinant K. pneumoniae strain to inhibit the expression of the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) and 2,3-butanediol production gene (budA), resulting in a bi-functional strain harboring both glucose-to-glycerol pathway and CRISPRi system. Reverse transcription and quantitative PCR (RT-qPCR) results showed that this engineered CRISPRi system transcriptionally inhibited gapA and budA by 82% and 24%, respectively. In shake flask cultivation, this bi-functional strain produced 2.8 g/L glycerol using glucose as the carbon source, which was 46.6% increase compared to the strain without the engineered CRISPRi system. Moreover, this bi-functional strain produced 0.78 g/L 3-HP using glucose as the sole carbon source. In fed-batch cultivation, this bi-functional strain produced 1.77 g/L 3-HP. This study provides insights for co-utilization of distinct carbon sources.

High-quality RNA improves sensitivity of SARS-CoV-2 detection by colorimetric RT-LAMP.

The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.

Autoantibodies from Patients with Scleroderma Renal Crisis Promote PAR-1 Receptor Activation and IL-6 Production in Endothelial Cells.

Background. Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc). Autoantibodies (Abs) against endothelial cell antigens have been implicated in SSc and SRC. However, their detailed roles remain poorly defined. Pro-inflammatory cytokine interleukin-6 (IL-6) has been found to be increased in SSc, but its role in SRC is unclear. Here, we aimed to determine how the autoantibodies from patients with SSc and SRC affect IL-6 secretion by micro-vascular endothelial cells (HMECs). Methods. Serum IgG fractions were isolated from either SSc patients with SRC (n = 4) or healthy individuals (n = 4) and then each experiment with HMECs was performed with SSc-IgG from a separate patient or separate healthy control. IL-6 expression and release by HMECs was assessed by quantitative reverse transcription and quantitative PCR (RT-qPCR) and immunoassays, respectively. The mechanisms underlying the production of IL-6 were analyzed by transient HMEC transfections with IL-6 promoter constructs, electrophoretic mobility shift assays, Western blots and flow cytometry. Results. Exposure of HMECs to IgG from SSc patients, but not from healthy controls, resulted in a time- and dose-dependent increase in IL-6 secretion, which was associated with increased AKT, p70S6K, and ERK1/2 signalling, as well as increased c-FOS/AP-1 transcriptional activity. All these effects could be reduced by the blockade of the endothelial PAR-1 receptor and/or c-FOS/AP-1silencing. Conclusions. Autoantibodies against PAR-1 found in patients with SSc and SRC induce IL-6 production by endothelial cells through signalling pathways controlled by the AP-1 transcription factor. These observations offer a greater understanding of adverse endothelial cell responses to autoantibodies present in patients with SRC.

Switching metabolic flux by engineering tryptophan operon-assisted CRISPR interference system in Klebsiella pneumoniae

One grand challenge for bioproduction of desired metabolites is how to coordinate cell growth and product synthesis. Here we report that a tryptophan operon-assisted CRISPR interference (CRISPRi) system can switch glycerol oxidation and reduction pathways in Klebsiella pneumoniae, whereby the oxidation pathway provides energy to sustain growth, and the reduction pathway generates 1,3-propanediol and 3-hydroxypropionic acid (3-HP), two economically important chemicals. Reverse transcription and quantitative PCR (RT-qPCR) showed that this CRISPRi-dependent switch affected the expression of glycerol metabolism-related genes and in turn improved 3-HP production. In shake-flask cultivation, the strain coexpressing dCas9-sgRNA and PuuC (an aldehyde dehydrogenase native to K. pneumoniae for 3-HP biosynthesis) produced 3.6 g/L 3-HP, which was 1.62 times that of the strain only overexpressing PuuC. In a 5 L bioreactor, this CRISPRi strain produced 58.9 g/L 3-HP. When circulation feeding was implemented to alleviate metabolic stress, biomass was substantially improved and 88.8 g/L 3-HP was produced. These results indicated that this CRISPRi-dependent switch can efficiently reconcile biomass formation and 3-HP biosynthesis. Furthermore, this is the first report of coupling CRISPRi system with trp operon, and this architecture holds huge potential in regulating gene expression and allocating metabolic flux.

N-(3',4'-dimethoxycinnamonyl) anthranilic acid alleviates severe acute pancreatitis by inhibiting intestinal barrier dysfunction and NF-κB activation.

Severe acute pancreatitis (SAP) can affect intestinal barrier with a high mortality. To date, effective therapies for SAP are still in urgently need. The purpose of this study was to investigate the role of anthranilic acid active synthetic derivative, N-(3',4'-dimethoxycinnamonyl) anthranilic acid (3,4-DAA), in intestinal barrier dysfunction of SAP. In this study, SAP mice model was induced by caerulein combined with lipopolysaccharide (LPS). SAP mice were pretreated with 3,4-DAA orally. Histological structures of pancreatic and intestinal tissues were observed via hematoxylin-eosin (H&E) staining. Pancreas myeloperoxidase (MPO), serum lipase, and amylase were detected using corresponding kits. Western blot analysis and reverse transcription and quantitative PCR (RT-qPCR) were employed to determine the levels of inflammatory factors in both pancreatic and intestinal tissues. Moreover, the levels of intestinal barrier-related proteins, NLRP3 inflammasome and NF-κB pathway were examined by western blot analysis. Result revealed that 3,4-DAA significantly attenuated pancreas and intestine damage, inhibited the release of inflammatory factors and intestinal barrier dysfunction. Moreover, the expression of NLRP3 and phospho-NF-κB p65 in pancreatic and intestinal tissues was notably suppressed by 3,4-DAA. To sum up, these results demonstrated that 3,4-DAA could ameliorate SAP, partly attributing to the inhibition of intestinal barrier dysfunction and the release of inflammatory factors. These findings may provide a new mechanism support for 3,4-DAA application in the clinical treatment of SAP.

Identification of Ovarian Circular RNAs and Differential Expression Analysis between MeiShan and Large White Pigs

Simple SummaryChinese MeiShan pigs make up one of the oldest Chinese pig breeds. Compared with Large White pigs, MeiShans are hyper-productive and prolific, however, the expression and function of circular RNAs (circRNAs) in these two breeds of pig are not yet fully understood. We performed RNA-seq analysis of porcine ovaries from three MeiShan sows and three Large White sows, identifying 85 differentially expressed circRNAs (p < 0.05). In MeiShan pigs, 37 circRNAs were significantly up-regulated and 48 circRNAs were down-regulated. We verified that circular RNA scinderin (circSCIN) can target and sponge miR-133 and miR-148a/b, two miRNAs that affect estrogen secretion. This study highlights the circRNA profiling of ovaries and helps investigators to understand the differences in reproductive efficiency between MeiShan and Large White pigs.MeiShan and Large White pigs differ in their female fecundity. However, the mechanisms behind the gene expression and regulation that cause these differences remain unclear. In this study, we profiled circRNAs and identified 5,879 circRNAs from the ovaries of MeiShan and Large White pigs. Eighty-five circRNAs were differentially expressed between the two pig breeds. Of these, 37 were up-regulated and 48 were down-regulated in MeiShan pigs. Gene ontology enrichment analysis suggested that the differentially expressed circRNA were involved in the hormone-mediated signaling pathway. We verified that circSCIN and its parent gene, scinderin (SCIN), were differentially expressed by reverse transcription and quantitative PCR (RT-qPCR). Luciferase assays demonstrated that circSCIN can target and sponge miR-133 and miR-148a/b. The identification of differentially expressed circRNAs (DECs) and their regulatory functions increased our understanding of the differences in reproductive efficiency between MeiShan and Large White pigs.

Negative regulation of bleomycins biosynthesis by ArsR/SmtB family repressor BlmR in Streptomyces verticillus.

Bleomycin, a broad-spectrum antibiotic, has been widely used for various tumor treatments. However, its poor fermentation yield is not satisfactory for industrial production. Here, the ArsR/SmtB family regulator BlmR was characterized as a repressor of bleomycin production. As an autoregulator, BlmR was found to bind to a 12-2-12 imperfect palindrome sequence in its own promoter, and deletion of blmR led to a 34% increase of bleomycin B2 production compared with the wild-type strain. Using reverse transcription and quantitative PCR (RT-qPCR), blmT, which encoded a putative transporter, was identified as the target gene regulated by BlmR. Therefore, high-production strain was constructed by blmT overexpression in a blmR deletion strain, and the bleomycin B2 titer reached to 80 mg/L, which was 1.9-fold higher than the wild-type strain. Moreover, electrophoretic mobility shift assay (EMSA) showed neither metal-binding motifs nor redox switches in BlmR. In order to elucidate the regulatory mechanism, a model of BlmR was constructed by homology modeling and protein-protein docking. The BlmR-DNA complex was generated by protein-DNA docking with the assistance of site-directed mutagenesis and molecular dynamic (MD) simulation, which directly revealed several key amino acid residues needed for the maintenance and stabilization of the interface between BlmR and target DNA. The interface information could provide the configuration reference and seek the potential effectors that could interact with BlmR, thereby extending the regulation role of ArsR/SmtB family members on the improvement of antibiotic production.

De novo transcription of multiple Hox cluster genes takes place simultaneously in early Xenopus tropicalis embryos.

ABSTRACThox genes are found as clusters in the genome in most bilaterians. The order of genes in the cluster is supposed to be correlated with the site of expression along the anterior-posterior body axis and the timing of expression during development, and these correlations are called spatial and temporal collinearity, respectively. Here we studied the expression dynamics of all hox genes of the diploid species Xenopus tropicalis in four Hox clusters (A–D) by analyzing high-temporal-resolution RNA-seq databases and the results showed that temporal collinearity is not supported, which is consistent with our previous data from allotetraploid Xenopus laevis. Because the temporal collinearity hypothesis implicitly assumes the collinear order of gene activation, not mRNA accumulation, we determined for the first time the timing of when new transcripts of hox genes are produced, by detecting pre-spliced RNA in whole embryos with reverse transcription and quantitative PCR (RT-qPCR) for all hoxa genes as well as several selected hoxb, hoxc and hoxd genes. Our analyses showed that, coinciding with the RNA-seq results, hoxa genes started to be transcribed in a non-sequential order, and found that multiple genes start expression almost simultaneously or more posterior genes could be expressed earlier than anterior ones. This tendency was also found in hoxb and hoxc genes. These results suggest that temporal collinearity of hox genes is not held during early development of Xenopus.

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

Cell-Specific RNA Quantification in Human SN DA Neurons from Heterogeneous Post-mortem Midbrain Samples by UV-Laser Microdissection and RT-qPCR.

Cell specificity of gene expression analysis is from particular relevance when the abundance of target cells is not homogeneous in the compared tissue samples, like it is the case, e.g., when comparing brain tissues from controls and in neurodegenerative disease states. While single-cell gene expression profiling is already a methodological challenge per se, it becomes even more prone to artifacts when analyzing individual cells from human post-mortem samples. Not only because human samples can never be matched as precisely as those from animal models, but also, because the RNA-quality that can be obtained from human samples usually displays a high range of variability. Here, we detail our most actual method for combining contact-free UV-laser microdissection (UV-LMD) with reverse transcription and quantitative PCR (RT-qPCR) that addresses all these issues. We specifically optimized our protocols to quantify and compare mRNA as well as miRNA levels in human neurons from post-mortem brain tissue. As human post-mortem tissue samples are never perfectly matched (e.g., in respect to distinct donor ages and RNA integrity numbers RIN), we refined data analysis by applying a linear mixed effects model to RT-qPCR data, which allows dissecting and subtracting linear contributions of distinct confounders on detected gene expression levels (i.e., RIN, age). All these issues were considered for comparative gene expression analysis in dopamine (DA) midbrain neurons of the Substantia nigra (SN) from controls and Parkinson's disease (PD) specimens, as the preferential degeneration of SN DA neurons in the pathological hallmark of PD. By utilizing the here-described protocol we identified that a variety of genes-encoding for ion channels, dopamine metabolism proteins, and PARK gene products-display a transcriptional dysregulation in remaining human SN DA neurons from PD brains compared to those of controls. We show that the linear mixed effects model allows further stratification of RT-qPCR data, as it indicated that differential gene expression of some genes was rather correlated with different ages of the analyzed human brain samples than with the disease state.

Odk-1201, One-Step RT-qPCR Major BCR-ABL/ABL mRNA Kit for the International Scale, with High Sensitivity to Detect Deeper MR

Abstract Background: Molecular monitoring by the measurement of major BCR-ABL mRNA levels is important for the evaluation of the therapeutic efficacy of TKI in CML patients. However, there is no commercial kit easily available in each laboratory or hospital, which can estimate deeper responses below MR4.5that is 4.5-log reduction by the international scale (IS) 0.0032%. Methods: An ODK-1201 kit (Otsuka Pharmaceutical Co. Japan) is based on a HawkZ05 Fast One-Step RT-PCR Kit (Roche, Indianapolis, IN, USA). In this kit, reverse transcription and quantitative PCR (RT-qPCR) are performed in one tube, of which products are measured by ABI 7500 fast Dx (Life Technologies Japan, Japan). To isolate enough amount of RNA for the evaluation of molecular responses below MR4.5in CML patients, 7 ml of peripheral blood is required for this kit. To get the conversion factor (CF) for the IS, the panel of WHO International Standard was obtained from NIBSC, which comprises four ampoules each containing freeze-dried BCR-ABL-positive and -negative cells at various ratios from 0.01% to 10%. RNAs were isolated from each ampoule with a QIAamp RNA Blood Mini Kit (QIAGEN) and were subjected to the measurement of major BCR-ABL and ABL mRNAs with an ODK-1201 kit. Since %BCR-ABL/ABL in the WHO standard panel was already defined, CF was calculated by comparing the values of %BCR-ABL/ABL obtained by ODK-1201 with those of WHO standard panel. To validate the results obtained by ODK-1201, we collected 267 peripheral blood samples from CML patients as a multicenter study, and 120 samples in these were measured by ODK-1201 and in the international reference laboratory (Adelaide Lab), respectively. In addition, we conducted another multicenter study to compare ODK-1201 with an Ipsogen BCR-ABL1 Mbcr IS-MMR DX Kit (QIAGEN) and a One-Step qRT-PCR BCR-ABL Kit (Molecular MD), in which we measured the samples from 201 CML patients, 25 patients with non-CML hematologic malignancies and 25 healthy volunteers by these methods. All of the samples were obtained after the written informed consent was given. Results: The CF of ODK-1201 determined by the WHO standard panel was 1.12. After conversion to the IS by this CF, %BCR-ABL/ABLIS determined by ODK-1201 ranged from 0.0007% to 144.6867% in CML patients. In contrast, all of the values were below 0.0007% in the samples from patients with non-CML hematologic malignancies and healthy volunteers. The overall correlation between ODK-1201 and Adelaide Lab system was very good (94% within 3-fold bias) and the evaluation of MMR was concordant in 84% of CML samples. As for 46 CML samples with %BCR-ABL/ABL IS between 0.0032% and 0.1%, IS values by ODK-1201 were similar to the ones by Adelaide Lab system (with MR4.5; 0.0032%) with 89% within 3-fold and high correlation coefficient (r=0.89). Also, the correlation between ODK-1201 and Ipsogen was r = 0.97 (n = 224) and that between ODK-1201 and Molecular MD kit was r = 0.97 (n = 221), indicating that ODK-1201 was as accurate as Ipsogen and Molecular MD kits. Together, these results indicate that ODK-1201 was capable of measuring at least MR4.5 with the lowest value detected as 0.0007% (calculated sensitivity of less than MR5.0) by the IS. Conclusions: We developed a major BCR-ABLmRNA kit, ODK-1201 with CF for the IS and confirmed its high sensitivity (0.0007%) and accuracy. The procedure is very simple and takes only 2.5 h, and it needs a small amount of blood because RT-qPCR is performed in a single tube. It will enable us to evaluate deeper molecular responses in any laboratories. Conflict of interest :received research fundinand and honoraria from Otsuka I.M. received honoraria from Otsuka during the conduct of the study. H.N. received research funding from Otsuka during the conduct of the study and received research funding and honoraria from otsuka outside the submitted work. C.Y. received research funding from Otsuka during the conduct of the study and honoraria from Novartis and Bristol Myers Squibb. S.B. received research funding and honoraria from Novartis, Bristol Myers Squibb, Otsuka and Ariad. L.F. has no conflict of interest. K.D. and T.S. are employee of Otsuka. Y.K. received honoraria from Otsuka during the conduct of the study. T.N. received grants and personal fees from Otsuka during the conduct of the study, and grants and personal fees from Otsuka outside the submitted work. Disclosures Matsumura: Otsuka: Consultancy, Honoraria. Nakamae:Otsuka : Research Funding. Yoshida:Otsuka: Research Funding. Branford:Otsuka: Research Funding. Koga:Otsuka: Employment. Sogabe:Otsuka: Employment. Kanakura:Otsuka: Consultancy. Naoe:Otsuka: Consultancy.

Abstract 3472: Mesodissection of slide mounted tissue: Applications including tumor tissue enrichment, expression analysis, and FISH on tissue fragments

Abstract We previously reported the development of a mill based instrument for dissection of slide-mounted tissue sections. This instrument employs a specialized disposable mill bit that simultaneously dispenses liquid, cuts tissue from the slide surface using rotary motion, and aspirates the liquid along with the displaced tissue fragments. An accompanying software package is capable of transferring digital annotations between images of serially cut tissue sections to guide dissection and generate an electronic record of the process. Since the original report, a new instrument and software package has been developed with a computer controlled stage for automatic dissection of tissue. Here we report three applications using this mesodissection system. The first application investigated the relationship between dissection resolution and percent tumor tissue recovered. Areas of high tumor content were indicated on digital images of lung tumor samples. The areas of highest concentration were dissected at 400 micron resolution, the remaining areas of tumor dissected at 800 micron resolution, and then surrounding areas that would typically be recovered using manual macrodissection were dissected at 2 mm resolution. The percent mutant DNA, determined using the Sequenom OncoCarta panel, was greater than 60% for the 400 micron resolution dissections, about 45% for the 800 micron resolution dissections, and less than 10% for the remaining regions. The second application investigated the effect of various dissection related parameters on expression analysis, as assayed by Reverse Transcription and Quantitative PCR (RT-qPCR). These parameters included placing freshly microtomed FFPE tissue sections directly on slides vs. first floating on water, dissection of deparaffinized vs. paraffinized tissue, the use of crude lysates vs. purified RNA for the reverse transcription reactions, and manual dissection vs. mesodissection of the same areas on serial sections. RT-qPCR results were robust and equivalent for all parameters except those derived from crude lysates, which were equivalent to the no template controls. The third application investigated if mesodissected tissue fragments could be re-adhered to glass slides and used for FISH analysis. This application has the potential to automate and increase the throughput of FISH on tissue sections while reducing sample consumption. The preliminary results demonstrate that tissue fragments can produce good FISH signals, but a more detailed comparison to undissected tissue sections is ongoing. A significant concern is the loss of context of these fragments. This concern can be somewhat minimized by adjusting dissection conditions to obtain fragments larger than 1 mm in diameter and by mapping the dissected area onto the corresponding area a serial H&amp;E stained tissue section using the mesodissection software. Citation Format: Nils Adey, Dale Emery, Derek Bosh, Robert Parry. Mesodissection of slide mounted tissue: Applications including tumor tissue enrichment, expression analysis, and FISH on tissue fragments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3472. doi:10.1158/1538-7445.AM2014-3472

Expression Analysis of Rare Cellular Subsets: Direct RT-PCR on Limited Cell Numbers Obtained by FACS or Soft Agar Assays

Since tissues and tumors are heterogenous populations containing different cell types, their transcriptomes are blends of multiple mRNA expression profiles. Although fluorescence-activated cell sorting (FACS) allows isolation of individual cell types, RNA isolation and quantification remain problematic from rare subsets, such as tissue stem cells. Likewise, identification of transcriptional changes relevant to the tumorigenic potential of mammalian cells while they are actively growing as colonies in soft agar is also hampered by limited amounts of starting material. Here we describe a convenient method that fills the gap between single cell and whole tissue mRNA analysis, enabling mRNA quantification for individual colonies picked from soft agar. Our method involves direct lysis, reverse transcription and quantitative PCR (RT-qPCR) on 500 sorted cells or a single soft agar colony, thus allowing evaluation of up to 20 transcripts in functionally distinct subpopulations without the need for RNA isolation or amplification.

Steroidogenesis in primary cultures of neonatal porcine Leydig cells from Duroc and Norwegian Landrace breeds

Breed differences in steroidogenic activity between primary Leydig cells derived from neonatal purebred Duroc and Norwegian Landrace boars were investigated in vitro. Concentrations of testosterone, estradiol, androstenone, cortisol and progesterone produced into the medium were determined. To explore underlying mechanisms the cellular expression of a suite of genes relevant in steroidogenesis was measured using reverse transcription and quantitative PCR (RT-qPCR). Basal steroid concentrations indicated a larger production capacity for steroids in unstimulated Duroc cells. Stimulation of the cells with LH increased steroid hormone secretion significantly in both breeds in a dose dependent manner. Testosterone and androstenone concentrations increased approximately 50- and 15-fold, respectively, whereas concentrations of estradiol, cortisol and progesterone increased to a lesser extent. At levels of maximal LH stimulation, absolute steroid concentrations were higher in Duroc. However, the relative increase in hormone concentrations was significantly lower in Duroc cells for estradiol, progesterone and cortisol when compared to basal levels. LH exposure was associated with a general up-regulation of mRNA levels for steroidogenic genes, stronger in Duroc than in Norwegian Landrace. This was in agreement with the higher absolute concentrations of steroid hormones measured in culture medium from the LH-stimulated Duroc Leydig cells, but did not concur with the fact that the relative increase in hormone production was lower in Duroc than in Norwegian Landrace Leydig cells for some hormones. It was concluded that breed differences in steroid hormone concentrations and gene expression between Norwegian Landrace and Duroc are complex and cannot be explained by a simple mechanism of action.

Transmission of plant-pathogenic bacteria by nonhost seeds without induction of an associated defense reaction at emergence.

An understanding of the mechanisms involved in the different steps of bacterial disease epidemiology is essential to develop new control strategies. Seeds are the passive carriers of a diversified microbial cohort likely to affect seedling physiology. Among seed-borne plant-pathogenic bacteria, seed carriage in compatible situations is well evidenced. The aims of our work are to determine the efficiency of pathogen transmission to seeds of a nonhost plant and to evaluate bacterial and plant behaviors at emergence. Bacterial transmission from flowers to seeds and from seeds to seedlings was measured for Xanthomonas campestris pv. campestris in incompatible interactions with bean. Transmissions from seeds to seedlings were compared for X. campestris pv. campestris, for Xanthomonas citri pv. phaseoli var. fuscans in compatible interactions with bean, and for Escherichia coli, a human pathogen, in null interactions with bean. The induction of defense responses was monitored by using reverse transcription and quantitative PCR (RT-qPCR) of genes representing the main signaling pathways and assaying defense-related enzymatic activities. Flower inoculations resulted in a high level of bean seed contamination by X. campestris pv. campestris, which transmitted efficiently to seedlings. Whatever the type of interaction tested, dynamics of bacterial population sizes were similar on seedlings, and no defense responses were induced evidencing bacterial colonization of seedlings without any associated defense response induction. Bacteria associated with the spermosphere multiply in this rich environment, suggesting that the colonization of seedlings relies mostly on commensalism. The transmission of plant-pathogenic bacteria to and by nonhost seeds suggests a probable role of seeds of nonhost plants as an inoculum source.

TGF-β Inhibits CYP17 Transcription in H295R Cells Acting via Activin Receptor-Like Kinase 5

Objective. Transforming growth factor β (TGF-β) is a potent inhibitor of 17α-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-β in adrenocortical cells. Methods. H295R cells were culture and incubated with TGF-β, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TβRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. Results. TGF-β significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-β on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-β on CYP17 expression. Conclusion. We concluded that the TGF-β-dependent decrease of 17α-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor.