Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer’s disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer’s disease and multiple sclerosis in particular. Inflammatory processes are involved in the pathogenesis of a variety of degenerative diseases, such as Alzheimer’s disease (AD), multiple sclerosis (MS) and rheumatoid arthritis (RA). More recent studies have established that acetylcholine (ACh), the classical neurotransmitter in the central and peripheral nervous systems, acts as a suppressor of inflammatory responses of lymphocytes, mediated by binding to α7-nicotinic ACh receptors (α7-nAChRs) However, there are still unresolved questions regarding this hypothesis. In particular that (i) ACh must be able to diffuse considerable distances from the cholinergic nerve terminals and (ii) resist the action of two extremely efficient ACh-degrading enzymes, acetyl- (AChE) and butyryl-cholinesterase (BuChE), which are abundant in extracellular fluids such as plasma and cerebrospinal fluids (CSF). In addition, the immune-suppressive activity requires that ACh has to be present at certain extrasynaptic levels to exert its putative role on immune cells by way of activating α7-nAChR ion-channels.Additional questions arise from our recent demonstration in patients with AD that beta-amyloid (Aβ) peptides, the main component of senile plaques in the AD brain, together with high ApoE protein interact physically with BuChE and AChEThus, an abnormal formation and accumulation of BAβACs at synapses or within the brain parenchyma provides a plausible explanation for the main characteristic features of AD, namely the selective deficit in the cholinergic signaling However, it is difficult to conceive how hyperactivation of two enzymes with high intrinsic ACh-hydrolyzing capacity may have a meaningful pathophysiological impact on extrasynaptic ACh levels. In order to balance the actions of highly abundant and efficient cholinesterases, we hypothesized that ACh-synthesizing machinery is also present in extracellular fluids, whose function is to uphold steady-state equilibrium of ACh levels. If so, nerve activity dependent release of Aβ would result in a transitory ACh-hydrolytic burst through hyperactivation of BAβACs, with rapid lowering of ACh levels. The subsequent re-uptake of Aβ peptides would terminate this regulatory signaling so that ACh equilibrium can be re-established.The main ACh-synthesizing enzyme is choline acetyltransferase (ChAT), which was discovered about 70 years ago Several splice variants of ChAT  are generated from the coding gene region at the “cholinergic gene locus”. In human, the M-type mRNA transcript may produce both a larger (82 kDa) and a smaller (69 kDa) molecular isoform Both the well-established cholinergic-antiinflammatory hypothesis and our model where extracellular hydrolyzing activity is balanced by capacity to regenerate ACh are challenged by lack of convincing evidence for extracellular ChAT, with several conflicting reportsThe current study was hence initiated as a proof-of-concept, with the main objective of a thorough investigation if ChAT protein and activity is present in extracellular fluids of the peripheral and CNS compartments. We used human plasma and CSF as surrogates of the extracellular fluids in these compartments. We also used mouse lymphocytes and human-brain primary astrocytes and human embryonic stem cells to investigate the probable source of ChAT in the extracellular fluids.We here demonstrate conclusive evidence for the presence of high levels of functional ChAT in human plasma and CSF. We show that CSF ChAT maintains a steady state level of ACh in the presence of physiological levels of fully active cholinesterases. We also show that ChAT is actively secreted by different non-neuronal cells, and that the levels of ChAT in the extracellular fluids are physiologically balanced to the levels of the counteracting ACh-degrading enzymes. We show that ChAT level is elevated in patients with MS compared to controls, and this strongly correlates to CSF levels of the complement factors of innate immune system., Blots I & II), and pooled brain homogenates (BH) from controls and AD patients , we here used an analogous terminology for the detected molecular forms of ChAT in CSF  in the peripheral circulation was 11 198±234 nmol/min/ml, whereas the corresponding value in CSF was just 25±1 nmol/min/ml Fig. 2b In order to test if ChAT protein present in CSF and plasma is functionally intact a simple sensitive colorimetric assay was applied. First, ChAT activity was directly measured in the samples using a microtiter plate Fig. 2g.Both the functional ELISA assay and the overall direct assay yielded essentially similar levels of ChAT activity Fig. 2i.p<0.0013).All of these ACh-related enzymes are simultaneously present in their active forms. Therefore, it is imperative to determine if ChAT is capable of maintaining certain levels of ACh in extracellular fluids, such as plasma and CSF, in the presence of fully functional degrading enzymes Fig. 3aExperiments were also performed without adding choline to the samples, since endogenous choline concentration was sufficiently high (31±2 µM). A similar steady-state level of ACh was observed at both temperatures (data not shown).We next addressed whether or not ChAT is a secreted protein. This was tested first by using human embryonic stem (hES) cells, where the culture medium was assayed by sandwich ELISA. These cells secreted substantial amounts of ChAT into the medium Fig. 2c.Lymphocytes also possess the essential components of the cholinergic system, including ChAT and both muscarinic and nicotinic ACh receptors Very few cells displayed positive ChAT immunostaining under resting conditions, and no significant change occurred upon incubation for up to 48 hours . The sedimentation analysis revealed that the difference was most likely related to the amount of the G2 variant of ChAT in BCHE-Wt homozygotes and the G1 and G4 variants in the heterozygotes compared to the BCHE-K homozygotes  and a matching control group consisting of patients with non-inflammatory neurological/psychiatric conditions.Consistent with the observation in AD, we found that CSF levels of BuChE, but not AChE 6c–d, hiThe ratio of ChAT to cholinesterases (ChAT/ChE-ratio) may be indicative of the extrasynaptic ACh equilibrium status, since a high ChAT/ChE ratio plausibly will lead to more generated than degraded ACh. This ratio was significantly higher in the CSF of patients with MS compared to patients with other non-inflammatory neurological conditions (OND) .The complement system constitutes an important part of the innate immune system. Upstream complement components are expressed by cholinoceptive cells in the CNS, such as microglia and astrocytes. ACh equilibrium in the brain interstitial fluid may therefore influence expression of complement components. We have recently found that TNF-α mediated increase in expression of complement C3 in cultured rat, mice and human astrocytes is attenuated by addition of ACh to the culture medium . The relevance of this question has been increased by the relatively recent observation that ACh plays an important immune regulatory effect outside of neuronal synapses, where ACh is assumed to diffuse over longer distances in the presence of abundant ACh-degrading enzymes. The finding here that ChAT is abundant in extracellular compartments, such as CSF and plasma, makes it possible to reconcile this model with ACh’s short half-life Fig. 9. We previously hypothesized that ApoE and Aβ attenuate cholinergic transmission through formation, gradual accumulation, and ultra-activation of BAβACs, in turn causing low-degree systemic inflammation in AD Another fundamentally important question concerns a native physiological function for Aβ peptides. It is difficult to conceive that such sophisticated and complex enzymatic machinery has been evolved for the universal production and release of Aβ peptides in the brain  activity or protein levels. As a relative index for the extracellular ACh equilibrium state, we calculated the ratio of ChAT to overall ChE activity in the CSF. A high ratio should indicate a low CSF ACh-hydrolyzing status and/or a high ACh-synthesizing status. Such a status would indicate a high extracellular ACh concentration. We found that this ratio to be two-fold higher in the CSF of the MS group than in the OND group. This observation suggests that increased ACh is a counter-regulatory response to inflammation, which may be driven by an aberrant T-cell response with autoimmune neuroinflammation. In the CNS compartment, immune cells are greatly outnumbered by CNS inherent neurons and glia. It is likely that a large proportion of the ChAT activity detected in the CSF stems from the latter. Regardless of the source, the findings of increased ChAT and BuChE indicate that this pathway is malfunctioning in patients with MS.The current study also report an integrated simple, reliable and specific assay for simultaneous measurement of both activity and protein levels of ChAT in practically any biological fluid. We used undiluted or diluted human CSF and plasma, rather than concentrating the samples by centrifugation and filtration, which has been done in previous studies n) subunits. We here used the same nomenclature, where the monomeric G1 subunit of CSF ChAT had a Mw between 50–70 kDa, in close agreement with earlier reports 2 molecular form of ChAT had an Mw of 100–140 kDa, likely representing the assembly of two G1 ChAT subunits. It was the most abundant molecular complex of ChAT in CSF. The heavier G4 molecular form of CSF ChAT had an Mw ranging between 200–300 kDa, which fits well with assembly of four G1 subunits.The common view is that ChAT in the brain exists as a 68 kDa and a 80 kDa protein A much smaller ∼25 kDa subunit ChAT protein was detected in CSF, plasma and brain homogenates, both with immunoblotting and sucrose density gradient analysis. This may represent a partially degraded enzyme. However, reports indicate that the human gene for ChAT generates two alternatively spliced transcripts, which produce two protein variants of ChAT: a 68 kDa protein and a 27 kDa protein 2 form, was associated with genetic risk factors of AD, APOE4 and BCHE-K genotypes Interestingly, the level of CSF ChAT, in particular the GThe findings reported here warrant further studies of the various molecular forms of ChAT not resolved here. For instance, do the subunits of ChAT assemble through disulfide bridges in a similar manner as the subunits of AChE and BuChE? Is there any membrane-anchored extracellular ChAT present on astrocytes or lymphocytes in similar fashion that the G2 AChE is anchored to the outer membrane of blood cells? Does the 25-kDa ChAT splice variant bind to the full-length ∼65 kDa variant, and has such a binding a regulatory role of the ChAT activity? There are also other critical issues such as the source of acetyl-Co enzyme A in extracellular fluids, which is required by ChAT for the synthesis of ACh. For instance, it is possible that this cofactor is released simultaneously by the cells together with ChAT.All studies involving human subjects and animals had been approved by the Regional Ethical Review Board in Stockholm or the Research Ethics Committee of the South, Huddinge University Hospital. Written informed consent was obtained from each patient or the responsible caregivers prior to collection and storage of samples for research purposes in Biobank of the Karolinska University Hospital, Huddinge. This study was conducted according to the Declaration of Helsinki and subsequent revisions.C57BL/6 mice were housed under standard conditions at the Department of Microbiology, Tumor and Cell Biology at both Karolinska Institutet and Karolinska University Hospital Huddinge, Stockholm, Sweden. All procedures were performed in conformance with both institutional and national guidelines.Cortical brain homogenates (BH) from parietal cortex of AD patients  and parietal cortex of non-AD controls  were prepared as previously described A (APOEε44/BCHEWW); B (APOEε44/BCHEKK); C (APOEε44/BCHEWK); D (APOEε43/BCHEWW); E (APOEε43/BCHEKK); F (APOEε43/BCHEWK); G (APOEε33/BCHEWW); H (APOEε33/BCHEKK); I (APOEε33/BCHEWK)  Fig. 1a.All pooled brain extracts and CSF/plasma samples were kept at −80°C until the assay.The pooled human CSF and plasma samples, as well as the pooled homogenates from the control and the AD brains, were used  and dot-blotted on nitrocellulose membranes . The analysis was done according to standards procedures. The anti-ChAT antibodies were either a mouse monoclonal  or a rabbit polyclonal antibody . The corresponding HRP-conjugated secondary antibody was a goat anti-rabbit or a goat anti-mouse antibody . Finally, the membranes were incubated for five min in 3 mL/blot of ECL plus reagents (Amersham Life Science). The blots were then photographed with a digital camera , according to standard procedures.Neat and diluted pooled human samples  were used. ChAT was detected by immunoblotting after resolution by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, as described before To ensure the identity of the proteins detected by the dot-blot and Western-blotting analyses, we designed sandwich ELISA assays using a combination of three different anti-ChAT antibodies and purified recombinant human ChAT protein  as a standard.0.05% .Briefly, the in-house constructed sandwich ELISA was set up as follows: High-binding plates  were coated with the anti-ChAT capturing antibodies [PAB14536 (1/2000) or MAB 3447 (1/250)], diluted in sodium carbonate buffer (pH 9.5). The plate was then blocked for two hours with the carbonate buffer, containing 5% w/v bovine serum albumin. All washing steps were done by 3 times of 5 min incubation with TBST0.05% buffer, containing 5% BSA and 0.01% NaN3. The reporter system was alkaline phosphatase (AP)-conjugated agent appropriate for the detecting antibody .All samples and standards were diluted in TBS buffer, containing 0.05% Triton-X100; 0.1% BSA and 1 mM EDTA. All samples or standards  were added in triplicate. The plate was incubated ON at 4°C under gentle orbital shaking. The plate was then incubated with one of the following detecting anti-ChAT antibodies: MAB 3447 (1/250), PAB 14536 (1/2000), or a biotin-labeled goat polyclonal anti-ChAT antibody  in TBSTp-Nitrophenyl phosphate, Cat no 73724, Sigma-Aldrich) in 1.0 M diethanolamine buffer, pH 9.8, containing 1 M MgCl, 0.01% NaN3. The reaction was monitored at 405 nm wavelength using an Infinite® M1000 Tecan microplate reader.Finally, the plate was incubated with the substrate solution . The pooled CSF (0.5 mL) or brain homogenate (0.3 mL) samples were applied on top of the gradients. Approximately 50 fractions were collected from the bottom of each tube. Concentration of ChAT protein in the collected fractions was then determined by sandwich ELISA as outlined above. Enzymes of known sedimentation coefficient, bovine liver catalase  and calf intestinal alkaline phosphatase , were used in the gradients to estimate the sedimentation coefficient of different molecular forms of ChAT.Molecular forms of ChAT were also separated by sucrose-gradient sedimentation technique and ultracentrifugation at 165000×g in a continuous sucrose gradient (5–20% w/v) for 18 hours at 4°C in a Beckman rotor (SW 41 Ti) as described before 0.05% .Neat or diluted human samples  were used for this assay. In part of the analysis, individual CSF (n = 10) and plasma (n = 10) samples were used instead of the pooled samples, described in the aforementioned section. These samples were also from patients with mild-to-moderate AD with the following demographic data . All samples were collected prior to treatment with cholinesterase inhibitors. When diluted sample was used, the dilution buffer was TBS-TrThe ChAT assay presented here is in principle based on the chemiluminescence assay for ChAT described by Menzel et al.,1988 Cocktail-A were added to each well. The Cocktail-A was prepared in the TBS-Tr0.05% buffer and contained 62.5 µM of lithium salt of acetyl coenzyme-A ; 1.25 U/ml of phosphotransacetylase ; 8.75 mM of lithium potassium acetyl-phosphate ; 6.25 µM choline chloride and 0.75 mM of eserine hemisulfate . The function of phosphotransacetylase and acetyl phosphate was to regenerate back the ACoA in the reaction wells Cocktail-A was not added to the choline standards, which quantified the amount of choline remaining in the wells at the end of the ChAT reaction. The plate was then sealed and incubated in a humid chamber for one hour at 38°C, under constant, gentle linear shaking. Immediately, after the incubation, 25 µl/well of Cocktail B  was added to all the wells, including the choline standards. Reaction absorbance was then monitored using a microplate spectrophotometer reader  at 500 nm wavelength.Then 40 µL of ChAT activity, in which Cd and Cn are the measured choline concentrations (in pmol) in the denatured and native samples, respectively, t is the incubation time (in minutes) at 38°C, and Vs is the volume (mL) of the samples.ChAT activity (nmol/min/mL samples) was calculated according to the following formula: This should be noted that at the end of this step the plate can be sealed and incubated ON at 4°C. Then the plate is washed as before, and incubated with the detecting antibody, PAB 14536 (1/2000) and the subsequent steps in the sandwich ELISA described in the previous section. This procedure will with obvious advantages integrate the ChAT protein determination to the ChAT activity assay.CSF samples from 10 patients with multiple sclerosis (MS) and 11 patients with other non-inflammatory neurological conditions (OND) were used. The MS and OND CSF samples were obtained from patients attending the Neurology Clinic, Karolinska University Hospital, Solna. Clinical examinations were performed by specialists in neurology, and all MS patients fulfilled the McDonald criteria The OND group consisted of patients with non-inflammatory neurological/psychiatric conditions with normal MRI scans and without signs of inflammatory activity in CSF in terms of pleocytosis or intrathecal IgG production. There were no significant concomitant diseases, such as infections. Corticosteroids had not been given within three months of sampling.The CSF levels of C3, C3a, C1q, C4, and H components of the complement system were measured using a sandwich ELISA, essentially as previously described AChE and BuChE activities were determined as described 6 cells/mL), which were from C57BL/6 mice, were stimulated with lipopolysaccharides  or with anti-CD3 (clone 2C11 at 5 µg/mL). The cell cultures were run in parallel in duplicates in RPMI1640 (HyClone Cat no SH3009601), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 g/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol.Spleen cells . The cells were fixed and prepared for the subsequent confocal microscopy as described 5 cells/well), and left in medium overnight to recover. Following this, the cells were stimulated with ACh or tumour necrosis factor-α (TNF-α) alone, ACh+TNF-α, or left unstimulated as control. ACh concentration varied between 1 µM to 10 mM and TNF-α concentration was kept constant at 20 ng/ml. The astrocytes were stimulated for 24 hours, after which the cells were lysed for RNA extraction and subsequent reverse transcriptase-PCR expressional analysis. The cultured mediums were saved for measuring AChE and BuChE activity and protein levels. All conditions were in triplicate. The pH of the medium was lowered to 7.1 to increase the stability of ACh. Recombinant human TNF-α was obtained from R&D systems (Cat no 210-TA-010). Acetylcholine chloride was obtained from Sigma (Cat no. A2661).Primary human astrocytes were obtained from ScienCell Research Laboratories (Cat no 1800). Cells were cultured in astrocyte medium (Sciencell Cat no 1801) in poly-L-lysine-coated T-175 flasks, and handled according to manufacturer’s protocols. Confluent cells were then detached, and seeded in a 48-well plate (3×10www.ncbi.nlm.nih.gov/tools/primer-blast), and checked for specificity with melt-curve analysis. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize the mRNA expression levels of the studied transcripts. Normalized expression levels were calculated with Bio-Rad CFX manager.Cells were lysed in RLT buffer  for total RNA preparation. Total RNA was extracted and purified. Column DNase I was treated using an RNeasy Mini kit (Qiagen) and RNase-Free DNase Set (Qiagen), according to the manufacturer’s protocols. The RNA was synthesized to cDNA by incubation with 5x iScript reaction mix (Bio-Rad) for five min at 25°C, 30 min at 42°C, and five min at 85°C. All steps were performed under RNase-free conditions. Real-time PCR was conducted using a three-step PCR protocol and Bio-Rad CFX manager software . Samples were run in triplicate. All primers and probes were designed with primer blast (GAPDH: Forward: 5′-AGGGCTGCT TTTAACTCTGGTAAA-3′; Reverse: 5′-CATATTGGAACATGTAAACCATGTAGT TG-3′. CHAT: Forward: 5′-ATGGGCTCTTCTCCTCCTAC-3′Reverse: 5′-GTGGAGTCTTTCACGAGGAC-3′.The primers sequences were as follows:


IGF-2 transcript expression and subsequent competence for embryonic development.The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid ; 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF  or 3) tissue culture medium  and then subsequently in vitro fertilized and cultured for 9 days.Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control .Immunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development.GM-CSF enhanced cumulus cell expansion of  The granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein with several molecular-weight species ranging from 18 to 30 kDa . Upon biBoth GM-CSF and its receptor have been highly characterized in the hematopoietic cell line, as well as in other cell types including fibroblasts, oligodendrocytes, trophoblast, endothelial and neoplastic -10. In rIn vitro studies have shown that cumulus cells are able to uptake and metabolize glucose allowing transport of glycolytic products such as pyruvate and lactate through gap junctions into the oocyte . Two additional groups were incorporated in the experimental design: SOF alone (n = 75) and a positive control maturation media consisted of tissue culture medium (n = 95)  supplemented with 10% FBS , 0.2 μM Pyruvate, 5 μg/ml LH , 40 mg/ml FSH  and 50 μg/ml gentamicin.After follicular aspiration, COC were classified into five groups based on the morphology of their surrounding cumulus cells . Group Ain vitro maturation (IVM) in 50-μl droplets of treatment media in Petri dishes under mineral oil for 22 h in humidified atmosphere consisting in 5% CO2 at 38.5°C.Groups of 10–15 COC were allocated for After 22 h of IVM, oocytes were collected and evaluated according to the cumulus expansion and then nuclear maturation. Cumulus expansion was determined using three different methods: 1) higher and a lower diameter for each COC were measured using a micrometric rule previously calibrated using a 0.1 mm objective (Nikon); 2) oocytes were microphotographed and higher and lower diameters were measured using a Fluoview software ; and 3) a subjective scale was used  to estimAfter cumulus expansion evaluation, cumulus cells were removed mechanically by vortex in PBS-0.1% BSA and washed twice in the same solution. Oocytes were placed between glass and cover slides with silicone and fixed with a mixture of acetic acid and ethanol (1:3) overnight at room temperature. Oocytes were then stained using 1% aceto-orcein for 1 h and destained using a mixture of acetic acid, glycerol and distilled water (1:1:3). Stained oocytes were examined under a phase contrast microscope for intact nucleus with germinal vesicle (GV), germinal vesicle breakdown (GVBD) or metaphase II (MII)-arrested.in vitro maturation media: SOF alone, SOF supplemented with GM-CSF at a concentration of 1, 10 or 100 ng/ml of GM-CSF or TCM 199 as described above. Groups of 10–15 COC were allocated for in vitro maturation (IVM) in 50-μl droplets of treatment media in Petri dishes under mineral oil for 22 h in humidified atmosphere consisting of 5% CO2 at 38.5°C. An additional sample of COC (n = 40-45/per group) was in vitro matured in SOF medium alone or supplemented with 10 and 100 μM of LY294002 a PI 3-kinase inhibitor or DMSO (DMSO was used as a diluent control). Cumulus cells were removed mechanically by vortex in PBS-0.1% BSA at 22 h. A 50 μl aliquot of cell suspension was mixed with 5 μl of Trypan Blue for cell viability using a Neubauer chamber.Cumulus oocyte complexes (n = 52-60/per group) were randomly assigned to the following in vitro maturation media: 1- SOF without GM-CSF supplementation (n = 123), 2- SOF supplemented with 100 ng/ml of GM-CSF or 3- TCM 199 as described above (n = 159).Cumulus oocyte complexes were randomly assigned to the following Immunohistochemical staining for cortical granules was also performed for evaluation of oocyte cytoplasm maturation. The type of cortical granules  was evaluated as previously described . BrieflyOocytes were examined and evaluated under epifluorescence inverted microscope .IGF-2 gene transcript in bovine cumulus cells and oocytes were determined in COC (n = 30 per group) in vitro matured in TCM, SOF alone or supplemented with 100 ng/ml of GM-CSF. Total RNA was extracted from lysed cells using the RNAeasy extraction mini kit . All subsequent RNA purification steps were carried out according to the manufacturer’s instructions. cDNA was synthesized using the oligo-dT method  with 1 μg of total RNA as a template in a reaction volume of 20 μl. Sequences of forward and reverse bovine IGF-2 were: ATCCAGCCGCATAAACCG and GGACGGTACAGGGATTTCAG. A reaction mixture containing a volume of 50 μl was prepared . All the reagents were acquired from Promega. The reaction was heated on a Stratagene Thermo Cycler  to 95°C for 7 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final extension step of 72°C for 10 min. As a normalization control for RNA loading, parallel reactions in the same multiwell plate were performed using GAPDH as a target. Quantification of gene amplification was made following quantitative PCR by determining the threshold cycle (CT) number for SYBR fluorescence within the geometric region of the semilog plot generated during PCR. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. The relative quantification of the target gene expression across treatment was evaluated using the comparative ∆∆CT method. The CT value was determined by subtracting the GAPDH CT value from the target CT value of the sample. Calculation of ∆∆ CT involved using target gene expression on immature control  as an arbitrary constant to subtract from all other CT sample values. Relative target mRNA expression was calculated as fold changes in relation to immature control sample and expressed as 2-∆∆CT value.Relative expression of in vitro maturation media consisting of: 1) SOF alone ; 2) SOF supplemented with 100 ng/ml of GM-CSF  or 3) Tissue Culture Medium  and then subsequently in vitro fertilized and cultured for 9 days.A sample of COC was randomly assigned to in vitro maturation, fertilization and culture [6 sperm/ml of heparin-containing (1 μg/ml) TALP-IVF medium . The sperm suspension was pippeted into 35 mm-petri dishes in 50 μl microdrops and covered with mineral oil. Thereafter, 10–12 matured COC per drop were added and incubated in 5% CO2 and 5% O2 in humidified air at 38.5°C. After 18–20 h, the presumptive zygotes were vortexed in PBS-0.1% BSA medium to remove the cumulus cells. Denuded zygotes were cultures in 30 μl of bicarbonate-buffered SOF medium for 7 days in a humid chamber under an atmosphere containing 5% CO2, 5% O2 and 90% N2.Embryos were produced using standard protocols for  culture -39. Frozin vitro fertilization (Day 0 = in vitro fertilization) and blastocyst formation were recorded on Days 7 and 9 of in vitro culture. Blastocysts from days 9 of in vitro culture (n = 30/per group) were used for cell number determination. Embryos were placed on a slide with dye bisbenzimide  for 5 min at 39°C. Hoechst dye was removed, and cover lips were mounted with wax and then firmly pushed onto the slide to spread the embryo. Staining nuclei was visualized with an epifluorescence microscope .Early cleavage was evaluated on Day 2 after IGF-2 mRNA levels were estimated using one-way analysis of variance (ANOVA). Tukey’s multiple comparison was used as a post-hoc test when a significant difference was detected. Data from cumulus diameter was normalized to a logarithmic scale in order to accomplish homocedasticity. CEI and number of cells values were compared among treatments using non-parametric Kruskal-Wallis and multicomparison tests. Data from cellular viability were arcsin-transformed and analyzed using one-way ANOVA and Tukey’s test. All statistical analyses were performed using the Statistica 7.0  software package.Single point measurements such as the difference among treatments for cumulus expansion, nuclear and cytoplasmic maturation, and Immunofluorescence analyses were performed to detect expression of α and β receptors of GM-CSF in whole-mounted bovine cumulus cells Figure A-F and oA dose–response experiment was performed to estimate the effect of GM-CSF on nuclear maturation. The maturation state was evaluated using aceto-orcein staining. Data showed that the proportion of oocytes undergoing metaphase II after treatment with 1, 10 or 100 ng/ml of GM-CSF was not significantly different compared to the untreated controls  in cumulus diameter was observed in COC treated with 10 and 100 ng/ml of GM-CSF (329 ± 68 and 400 ± 88 μm) compared with control COC  in cumulus cells and oocytes after in vitro maturation compared to the immature state. GM-CSF induced no significant effect over IGF-2 expression neither in cumulus cells or oocytes  in cumulus cells and oocytes after maturation in TCM (2.81 and 2.64 fold compared to the immature control).Furthermore, we evaluated the effect of GM-CSF during d P < 0.0 in cumulin vitro maturation on subsequent embryonic development we utilized COC matured in TCM, SOF and SOF + 100 ng/ml of GM-CSF for in vitro fertilization. Results showed that COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) or embryonic nuclei count (98 vs. 96) compared to untreated controls -kinase prevented the GM-CSF effect, suggesting that the 3PI-kinase pathway is associated with glucose uptake, mediated by the activity of GM-CSF.Immunofluorescence analyses demonstrated a wide distribution of α- and β- subunits of the GM-CSF receptor in bovine oocytes and cumulus cells. Immunolabeling associated to both α and β receptors appeared to be located in the cytoplasm of cumulus cells. Oocytes collected from antral follicles were stripped from cumulus cells and processed for immunofluorescence analyses. Confocal microscopy showed a pattern of immunoreactivity for the α receptor in the cytoplasm in proximity to the plasmatic membrane. In contrast, the β subunit was homogeneously distributed in the cytoplasm. A previous report indicated the expression of the α-subunit but not the β- subunit in mouse COC by RT-PCR . HoweverAddition of 100 ng/ml to cumulus cell culture resulted in higher percentage of total cells (45.4% higher) compared to untreated controls. The proportion of nonviable cells remained similar to controls indicating that a higher proportion of live cells accounted for the total cells. These data suggest that GM-CSF induced proliferation instead of survival of cumulus cells. The proliferative effect of GM-CSF was blocked after addition Ly294002 indicating that PI3- kinase activity mediated the GM-CSF effect. The intracellular signaling that intermediates proliferative and survival effects of GM-CSF have been previously characterized . The proIGF-2 in bovine cumulus and oocytes after IVM. Our results showed that IGF-2 expression in cumulus cells and oocytes was not affected by GM-CSF treatment during in vitro maturation. However, we found that IGF-2 was up-regulated in cumulus cells and oocytes after maturation. Moreover, IGF-2 expression increased in oocytes and cumulus cells after maturation in TCM compared to maturation in SOF or supplementation with GM-CSF. Previous studies have suggested that IGF-2 play an autocrine and paracrine role associated to survival activity in embryos cultured under suboptimal conditions [IGF-2 expression may be modulated by culture conditions but not by supplementation of GM-CSF.To estimate the effect of GM-CSF at the transcription level we quantified the expression of nditions . These din vitro system, oocytes treated with and without GM-CSF were fertilized with frozen/thawed semen and cultured in SOF for 9 days. We found no differences among treatments on cleavage rate, blastocyst development and embryonic nuclei count. These results indicate that the effect of GM-CSF on cumulus expansion during maturation was not sufficient to improve the subsequent embryonic development. Previous studies have showed that addition of GM-CSF to culture media improved development rates in bovine [We further tested the effect of GM-CSF on cumulus expansion and the potential effect on oocyte competence by evaluating the embryonic development of COC matured with GM-CSF. Cumulus expansion has been associated with several oocyte functions including ovulation, cleavage and embryonic development -26. Cumun bovine  and human bovine  embryos.n bovine  and human bovine  embryos in vitro matured bovine COC, the oocyte nuclear and cytoplasmic maturation, IGF-2 mRNA levels or subsequent competence for embryonic development was not affected by the GM-CSF treatment. Our data suggest that GM-CSF may play a role in cumulus cell expansion in vitro and increasing cell proliferation.In conclusion, both α- and β-subunits of the GM- CSF receptor are expressed in bovine cumulus cells and oocytes. Despite, GM-CSF enhanced cumulus cell expansion of The authors declare that they have not competing interest.DB participated in designing the study, acquisition, analysis and interpretation of data, and in writing and revising the manuscript. OAP, AF, MOB participated in the acquisition and interpretation of the data. PS and AR participated in analysis and interpretation of data and critical revision of the manuscript. As Principal Investigators, MR and IC participated in the intellectual and experimental design of the study, the acquisition, analysis and interpretation of data, as well as writing and revising the manuscript. All authors read and approved the final manuscript.


Cystodiscus axonis and C. australis are pathogens of native and exotic Australian frog species. The pathology and ecological outcomes of infection with these parasites were investigated in this study. Gliosis was correlated to Cystodiscus axonis plasmodia in the brains of (9/60) tadpoles and (3/9) adult endangered Green and golden bell frogs using ordinal regression. Severe host reactions to C. axonis  were observed in the brains of threatened Southern bell frogs (8/8), critically endangered Booroolong frogs (15/44) and Yellow spotted bell frogs (3/3). Severe brain lesions were associated with behavioural changes, neurological dysfunction, and spontaneous death. Both C. axonis and C. australis develop in the bile ducts of tadpoles, the plasmodia were significantly associated with biliary hyperplasia, inflammation and the loss of hepatocytes in (34/72) Green and golden bell frog tadpoles using ordinal regression. These lesions were so severe that in some cases 70% of the total liver was diseased. Normal liver function in tadpoles is necessary for metamorphosis, metabolism, and immune function. We postulate that this extensive liver damage would have significant host health impacts. Severe hepatic myxosporidiosis was more prevalent in tadpoles examined in autumn and winter (overwintered), suggestive of delayed metamorphosis in infected tadpoles, which would have serious flow-on effects in small populations. We compared the sensitivity of histopathology and species-specific PCR in the detection of C. australis and C. axonis. PCR was determined to be the most sensitive method . Histology, however, had the advantage of assessing the impact of the parasite on the host. It was concluded that these parasites have the potential for significant ecological impacts, because of their high prevalence of infection and their ability to cause disease in some frogs.Myxosporean parasites  Sorex araneus) Talpa europaea) Myxobolus cerebralis, a parasite of salmonid fish, has caused morbidity and mortality in captive raised and wild fish wherever the parasite has been introduced to susceptible species Chloromyxum truttae, in contrast, is a common parasite in the same salmonid range as M. cerebralis and yet causes no host reaction in wild fish Myxosporea are two host metazoan parasites, closely related to jellyfish (Cnidarian) Cystodiscus axonis and Cystodiscus australis, have recently been characterized in Australian frogs Cystodiscus axonis and C. australis have presporogonic stages in bile ducts that are followed by the development of spore producing plasmodia in the gallbladder. Cystodiscus axonis is unique in that it has a presporogonic stage that develops in the central nervous system. Lesions in the central nervous system often accompany infection with C. axonis and liver lesions are found with infection by both of these parasites in some species of frogs, but not others. The origin of these parasites is not known, but both appear to have emerged in Australian frogs in the mid 1960's Litoria aurea, Litoria peronii, Litoria raniformis, Litoria castanea, Litoria booroolongensis, Limnodynastes peronii, Rhinella marina), representing 3  out of 5 frog families in Australia L. aurea, L. raniformis, L. booroolongensis and L. castanea). The populations of each endangered frog in which these parasites have been identified began declining from the 1970's and 80's Cystodiscus species first emerged Two myxosporean species, Infectious diseases are known to be important drivers of population declines and extinctions in amphibians and other species of animals. Disease causing organisms however, are often found in healthy populations of animals and, proving that organisms are causing significant disease and are having deleterious impacts on a population may be difficult C. axonis and C. australis as agents of significant disease and their potential to impact the survivability of both tadpoles and adult frogs. We compared the sensitivity and specificity of different techniques (histology and PCR) that can be used for diagnosis in individual animals and for population surveillance. Lastly, we provide evidence that differing host responses may play a role in the outcome of infection in these two parasites and show how the host-pathogen-environment interactions may have important ecological implications.It is the purpose of this study to document and statistically evaluate Green and golden bell frogs, Striped marsh frogs, Peron's tree frogs and Cane toads were collected under Scientific license numbers 12686 and 12969 . Animals were euthanized with AQUI-S  Litoria aurea) tadpoles (n = 97), metamorphs (n = 1) and adults (n = 11); Striped marsh frog (Limnodynastes peronii) tadpoles (n = 61) and adults (n = 27) and Peron's tree frog (Litoria peronii) tadpoles (n = 18), metamorphs (n = 1) and adults (n = 7) were collected from a semi-captive breeding colony for Green and golden bell frogs in Riverstone, NSW  from May 2008 to December 2010  frogs (n = 44) were collected from two locations, Maragle Creek (n = 25) and Abercrombie (n = 19) in NSW, Australia between 2007 and 2010. These animals were kept at the Taronga Zoo, Sydney, Australia for variable lengths of time and were either found dead or were euthanased as part of a disease screening program or due to some abnormal behaviour (e.g. leg dragging). Eight Southern bell frogs (Litoria raniformis) were wild caught in southern NSW, Australia in 2006 and kept in captivity until 2009 when they were euthanased due to lethargy and lack of breeding. Fourteen Yellow spotted bell frogs (Litoria castanea) were wild caught from southern NSW in 2010 and held in a captive breeding colony. Three frogs (1 sub-adult and 2 adults) died spontaneously and were submitted for necropsy.Booroolong (Rhinella marina) tadpoles (n = 14) and adults (n = 31) were collected from areas around Lismore, NSW  in February 2009 and 2010, at the southern end of their distribution in eastern Australia.Cane toad  were stained with Congo red to determine the presence of amyloidosis. For PCR analysis, brain, brainstem or liver were separately removed from the unfixed half of the tadpoles and adult frogs, or unfixed half of the brain stem and a representative portion of the liver of adult frogs. Individual tissues were stored at −80°C until processing.Many of the tadpoles were very small. In these cases, it was not always possible to get tissue for both histopathology and the PCR assay and therefore, tissues were only examined by histopathology.Southern bell frogs, Booroolong frogs and Yellow spotted bell frogs were maintained at Taronga Zoo, Sydney, Australia. All animals were subject to zoo staff observations; behavioural abnormalities were noted in individual pathology and diagnostic reports within the Australian Registry of Wildlife Health.A summary of the species and tissues examined to build a syndrome description is provided within Cystodiscus axonis infection and the presence of gliosis and other lesions in sagittal sections of the brain and spinal cord was recorded from 9 Green and golden bell frog tadpoles. Sagittal sections through the brain and 3 transverse sections through the spinal cord were also examined in 6 Green and golden bell frog adults. When an infected tadpole or frog was found, the severity of infection was assessed using the area of the central nervous system where the parasites were in highest concentration. Once this area was identified, parasite load was graded into three classes depending on the number of organisms that could be seen on 10X magnification: mild (1–5 plasmodia), moderate (6–10 plasmodia) and severe (more than 10 plasmodia). The entire brain and spinal cord sections were examined for evidence of host response to the parasite. Gliosis was recorded as either present or absent and when applicable, lesions such as perivascular lymphoid cell cuffing, malacia or haemorrhage were recorded.Gliosis was defined as increased numbers of glial cells occurring focally or more diffusely within a section of the brain or spinal cord. Cystodiscus species infection and microscopic liver disease, 72 tadpoles, 1 metamorph , and 11 adult Green and golden bell frogs were examined. Each liver section was examined for the presence or absence of plasmodial stages within the bile ducts, biliary hyperplasia, lymphoplasmacytic inflammation, loss of hepatocytes and biliary fibrosis. Bile duct plasmodia were recorded as either present or absent. The severity of the liver lesions were classified as mild (10–30% of liver affected), moderate (30–60% of the liver affected) and severe (60% or more of the liver affected) when a representative section of the liver was viewed under 10X magnification. It has been previously shown that C. axonis and C. australis are morphologically indistinguishable in liver sections and both infections can occur simultaneously C. axonis, C. australis or a combined infection.To examine the possible relationship between Associations were initially investigated by creating contingency tables of the presence of infection with presence of lesions and lesion severity. Univariable binary logistic regression analyses were then conducted to determine association between infection, age and season and the binary outcome variables – presence or absence of lesions in the brain  and UniLogistic macro  were used for all analyses. Odds ratios are reported with 95% confidence intervals (CI) and 2-sided Cystodiscus plasmodia in Green and golden bell frog tadpole livers and C. axonis in brains was compared to other species of tadpoles (Striped marsh and Peron's tree frog tadpoles) that were collected from the same location  in the same season. The overall differences in prevalence were evaluated with χ2 tests or Fisher's exact test if the cell frequencies were low. If the overall differences were significant, pair-wise comparisons were made using Z-test for comparing proportions or Fisher's exact test, as appropriate.Prevalence data for Cystodiscus spp. infection of the liver and the degree of severity of associated lesions in the liver seasonally were examined in tadpoles only (n = 72). Similarly, the presence or absence of Cystodiscus axonis infection in the brain was examined in tadpoles only (n = 60) , winter , spring  and summer . The presence or absence of (n = 60) .Due to the small amount of tissue available in a tadpole, not all sagittal sections contained both brain and liver. Therefore the numbers of animals that were included in the liver and brain seasonality study were not always the same  and 2. ACystodiscus spp. was recorded. The liver specimens were all from tadpoles of the Green and golden bell frogs. The brain specimens were from the Green and golden bell frogs , Peron's tree frog  and Striped marsh frog .A minimum of twelve serial sections were obtained for a sub-sample of the frog specimens . Depending on the size of the tissue, the number of 5 µm sections ranged from 12 to 34 in liver (n = 17) and 12 to 48 in brain (n = 15). Each section was screened individually and presence of For a subset of specimens for which a tissue sample was available in −20°C or −80°C, we isolated DNA and used a multiplex species specific PCR according to a previously published protocol The sensitivity of single histological section to demonstrate presence of the parasites in tissue compared with serial section and PCR and vice versa was calculated using a standard equation . The true positives were considered those returning positive outcomes in histology or PCR.C. axonis and C. australis myxospores (in PBS counted by haemocytometer). The DNA isolation and PCR reactions were performed according to Hartigan et al. To determine the detection limit of the multiplex species specific PCR Cystodiscus axonis produce 5–25 µm multinucleated non-sporogonic producing plasmodia within axons Inflammatory changes were observed in 83% of infected animals (10/12); changes were characterised by increased numbers of glial cells (gliosis) and the response varied between focal to multifocal and locally extensive in different intensities . The gliC. axonis plasmodia  (Gliosis is a nonspecific reaction and can be caused by multiple infectious agents  = 42.5) B. Gliosi = 42.5) B.Cystodiscus axonis plasmodia were found in the nervous system of Green and golden bell frog tadpoles in the spring, summer and autumn , then spring  and was significantly higher  in the autumn (C. axonis were identified in winter (n = 25) (d autumn . The pre(n = 25) .2 p = 0.0004). None of the Striped marsh frog tadpoles were found to be infected (0% 0/42) (2 p = 0.008). The numbers of organisms found in the brains of the Peron's tree frog tadpoles was low (100% mild infection load) and while gliosis was seen in these tadpoles, the severity was less than that seen in infected Green and golden bell frog tadpoles. Haemorrhage within the nervous system was not present in any of the Peron's tree frog tadpoles.Overall chi-sq analysis for brain plasmodia presence in tadpoles was found to be significant (p = <0.001). Prevalence of infection in the Peron's tree frog tadpoles  was significantly higher than that observed in the Green and golden bell frog tadpoles  and Yellow spotted bell frogs . Moderate prevalences were seen in the Peron's tree frogs  and the Booroolong frogs . Low prevalences were observed in Striped marsh frogs  and Cane toads   .Varying degrees of gliosis were found within the spinal cord , brain  and nervA Green and golden bell frog metamorph was captured because it could not use its back legs. This animal had many organisms and associated gliosis in the dorsal root ganglia and spinal nerves . Three oC. axonis and C. australis develop within the bile ducts as multinucleated intraluminal spherical to tubular structures distributed uniformly across the liver The plasmodia of The presence of hepatic plasmodia was significantly associated with age (tadpole) (p = 0.006) and biliary inflammation (p = <0.001) in univariable logistic regression models C. The pretal. Plasmodia in the gallbladder were observed in 14% (10/74) of Green and golden bell frog tadpoles (confirmed by PCR). Organisms were not attached to the epithelium, and were vacuolated with numerous vegetative nuclei in the cytoplasm, as described by Hartigan Cystodiscus spp. plasmodia were found in the livers of Green and golden bell frog tadpoles all year round. The prevalence of Cystodiscus spp. liver plasmodia in Green and golden bell frog tadpoles was lowest in the spring , increased in summer  and winter . The highest prevalence in the autumn   , howeverCystodiscus spp. plasmodia, age and season of collection in a multivariable model  and winter  than spring  and summer  . There wle model D.2 p = 0.009) (2 p = 0.033) and only mild lesions were seen in the livers of these tadpoles . Prevalence of Peron's tree frog tadpoles with hepatic plasmodia  was significantly higher than seen in the Green and golden bell frog tadpoles  E. The matadpoles . Early dtadpoles .C. axonis and C. australis develop spore producing plasmodia in the gallbladder of adult frogs Both Cystodiscus spp. infection. Biliary plasmodia, biliary hyperplasia and associated loss of hepatocytes, lesions found in tadpole livers, were only found in the metamorph . Myxospores were found in the gallbladder of one adult frog with no biliary lesions were seen.Twelve Green and golden bell frogs (11 adults and 1 metamorph) were examined in this study for evidence of 2 p = 0.002). Five of the frogs  with liver lesions had spore producing plasmodia in their gallbladders.Eight (7 adults and 1 metamorph) Peron's tree frogs were examined. The single metamorph had biliary plasmodia, biliary hyperplasia and associated loss of hepatocytes. Surrounding the proliferating bile ducts within the interstitium were multifocal deposits of amyloid-like eosinophilic fibrillar to amorphous material . Immatur2 p = 0.027). The degree of biliary hyperplasia, however, was mild resulting in a two to three fold increase in bile duct cross sections and typically only affected scattered portal triads. A mild degree of peribiliary fibrosis was present in 60% (12/20) of infected frogs, but these changes rarely impacted the surrounding hepatocytes.Twenty seven adult Striped marsh frog adults were examined and 20  were found to be infected and producing spores. Mild degrees of biliary hyperplasia were found in 12 (60%) of the 20 infected frogs. The prevalence of lesions in the liver  was significantly different from those in the Green and golden bell frogs (χLiver lesions found in the 15 infected adult Booroolong frogs with brain stages included amyloid-like deposits in 20% (3/15), mild periportal fibrosis in 6.7% (1/15) and mild biliary hyperplasia in 20% (3/15). Presporogonic plasmodia were also present in bile ducts of the 3 adult Booroolong frogs  with bilLiver lesions were found in all 8 frogs. These lesions varied from mild biliary hyperplasia with a minimal degree of fibrosis , to locaTwo adults and a sub-adult were examined. The sub-adult had a moderate degree of biliary hyperplasia, with accompanying inflammation and associated loss of adjacent hepatocytes . The subSix of the nine infected adults examined had fibrosis associated with the portal tract. No biliary hyperplasia or inflammation was observed in any of the livers seen. Large numbers of myxospores were observed in the gallbladders of adults .Cystodiscus spp. To calculate sensitivity of this approach we compared single histological section outcome with (i) multiple serial histological sectioning and (ii) used a previously developed multiplex PCR outcome Throughout this study we used the microscopic examination of single brain and liver histological sections to determine presence or absence of The sensitivity of single vs. serial histological sections for both brain and liver samples was 100%, however within the liver samples, there were four overall positive samples that contained sections in which parasites were not found  . Importantly, observing all serial sections including the false negative sections revealed histopathological lesions characteristic of hepatic myxosporidiosis .Cystodiscus was detected histologically in 43% of brains, and 33% of livers which tested positive by multiplex PCR. PCR was 100% sensitive in detecting brains that were microscopically Cystodiscus was observed, but since it did not detect infection in 2/19 liver samples that were positive on histology, sensitivity of PCR in liver is 66%  was equivalent to 1 myxospore, as confirmed by three independent PCR reactions. An equivalent of 0.1 myxospore DNA did not return species specific PCR amplicon for either Cystodiscus spp.The limit of detection using 10-fold serial dilutions of Cystodiscus axonis and C. australis are emerging myxosporean parasites of multiple species of Australian frogs. In addition to characterizing the prevalence and pathology associated with C. axonis and C. australis infection in several amphibian populations, the question that we address in this paper is whether these parasites have an ecological impact. Several biological characteristics are necessary for an emerging pathogen to have a significant and persistent ecological impact. The pathogen must be able to infect a high percentage of the susceptible population, it must be able to cause disease in a significant number of the animals that it infects, the disease that it generates must be sufficiently severe that it will directly or indirectly interfere with recruitment of juvenile animals into the breeding population or kill mature breeding adults, and the pathogen needs to persist in the environment C. axonis and C. australis exhibit these characteristics in our study populations and thus have a significant potential for ecosystem impact.C. axonis and C. australis caused high prevalences of infection in multiple species of frogs including the endangered Green and golden bell frog where prevalences of infection in tadpoles were as high as 65%. They also produced significant disease that directly or indirectly would have impacted their host in the common and endangered species of frog examined in this study. Neurological dysfunction is associated with similar haemorrhagic lesions in fish infected with myxosporea in the brain  C. axonis described here and possibly the presence of the parasite within axons would be expected to significantly interfere with neurological function and this was indeed the case in some frogs that were observed prior to death. The ecological impact of the nervous system form of C. axonis may be increased by its ability to cause disease in both tadpoles and adult frogs. Significant brain lesions were seen in a high percentage in adults of both the Southern bell frog and the Yellow spotted bell frog. However, whether these types of lesions occur in the wild is unknown. In this study, all the adult frogs with C. axonis lesions in the nervous system where held in captivity for months to years before their death. The stress of captivity can exacerbate the impacts of pathogens and disease Our findings show that C. axonis and C. australis were associated with severe disease in the liver of tadpoles of susceptible species. A healthy tadpole liver is essential for the hormonal cascades that bring about metamorphosis Both Cystodiscus spp. predisposes tadpoles to overwinter or if overwintered tadpoles are just infected for longer and therefore have a severe (chronic) host response. Regardless, any negative impact on the ability to metamorphose exposes tadpoles to more risks and removes one reproductive season from the life history of the tadpole/frog.Overwintering usually occurs when tadpoles do not have the resources to metamorphose and provides the advantage of extra time to feed and grow before spring C. axonis and C. australis to have spread so widely in 50 years could suggest an ability to persist in the environment. This could be explained by the resilience of myxospores in the water column as described for other myxosporea species Batrachochytrium dendrobatidis), where Striped marsh frogs were shown to have a higher tolerance for infection than the Green and golden bell frogs The ability of C. axonis. The question then arises as to whether it is a necessary part of the parasites life cycle? Based on the myxosporea of fish, the nervous system can act as a route of infection and conduit to other tissues, as occurs with M. cerebralis in salmonid fish C. axonis for amplification is based on the finding that the nervous system was infected by C. axonis in all species of frogs examined, arguing that it was a targeted tissue. Additionally, finding the organisms in cranial and peripheral nerves suggests that C. axonis may use the nervous system as an entry way to the tadpole. Lastly, finding organisms in close association with vessels could explain how the parasite moved from the central nervous system to other tissues as has been suggested for other myxosporea species C. axonis does use the nervous system as a site for amplification within the host it is likely that in its natural host it causes minimal or no disease as was seen in the Cane toad and the Striped marsh frog. Adverse impacts, as seen in Green and golden bell frog tadpoles and Booroolong frogs, and adults of the Southern bell frog and Yellow spotted bell frogs appear to represent reaction of the host's immune system to the parasite, and would be those expected in non-parasite adapted species. Ultimately, to determine if the nervous system stages of C. axonis represent an important developmental stage controlled infection trials where the sequence of the development of C. axonis can be followed will be necessary.Presporogonic development was observed to be a common finding in the nervous system of tadpoles and adult frogs infected with Cystodiscus infections occur only in the tadpole or at any time during the tadpole and frog life cycles. Based on studies of myxosporean infections in fish, age of the host and seasonal factors would be expected to impact susceptibility to infection and disease From a conservation perspective, it would be valuable to know if Cystodiscus species raises questions about the timing of infection. The low rate of infection in adult Green and golden bell frogs, compared to tadpoles suggests that infected tadpoles are either capable of ridding themselves of infection, or infected tadpoles have reduced survival rates compared with non-infected animals, or that adults of this species may be resistant to infection. Whether this represents infection in adult animals or infection of tadpoles with delayed development will require controlled experiments to determine. If Cystodiscus spp. are only able to infect tadpoles or only tadpoles of specific ages this could be valuable information for establishing captive bred populations that are free of disease and strategies for translocation of tadpoles.The difference in prevalence between frog and tadpoles for both C. axonis was unexpected because these stages were never seen in adult frogs of any other species in this study. These animals were euthanized immediately after capture from the wild and thus could have been recently infected. Alternately, the liver could have been colonized by reactivation of a latent infection in the brain or in other tissue. Why the bile duct environment could support presporogonic plasmodia in this species and not in others remains unknown and would require controlled infection trials to determine.The presence of presporogonic plasmodia in a small number of adult Booroolong frogs infected with Cystodiscus infections and that a combination of both is optimum.Screening frogs and tadpoles for infectious diseases is necessary to prevent the introduction of disease into captive breeding programs, preventing disease release into new areas from captive-raised frogs, and determining the geographic distribution of the disease. This study shows that both PCR and histopathology have advantages as screening tools for B. dendrobatidisCystodiscus, and the added advantage of PCR is that it can be used with pooled samples. The only disadvantage of PCR would be is susceptibility to contamination. Proving that animals were actually infected and that samples were not contaminated by environmental organisms can be difficult as has been shown for other amphibian pathogens in aquatic environments The PCR assay used in this study amplified an rDNA sequence for which as many as 10,000 copies may be present in mxyosporeans Cystodiscus spp., and that it can determine if infection is associated with disease. Combining these screening techniques or using histopathology as a confirmatory tool for populations that have been shown to be PCR positive would provide the most diagnostic power. Whichever technique is used, screening tadpoles in summer or autumn is optimal to detect infected populations as this is when infection prevalence is highest. In addition, development of an in situ hybridisation protocol for these two species could prove extremely informative for future research in these parasites.Histology as a screening tool has the advantages that it can be used to detect an array of infectious agents, in addition to C. axonis in Australian frogs (tadpoles and adults) if they are exhibiting gliosis and infection with either C. axonis or C. australis or both in tadpoles with biliary hyperplasia, particularly if there is minimal associated inflammation. Differential diagnoses for the lesions caused by C. axonis in the nervous system would be those caused by the rare extension of systemic bacterial and fungal infections to the nervous system and these would typically be accompanied by a neutrophilic or granulomatous inflammation. Liver disease caused by infectious agents , however, is common in frogs and tadpoles Cystodiscus spp. by the level of necrosis observed, multiple organs affected and the physical presence of the pathogen, e.g. fungal hyphae or nematode larvae.This study shows that pathologists should consider Cystodiscus axonis and Cystodiscus australis have the potential to infect and cause disease in common and endangered Australian frog species. They were also associated with spontaneous deaths in semi captive and captive populations of frogs. If these infection dynamics occur in wild frogs then they would also be expected to significantly reduce tadpole recruitment and adult frog longevity, potentially making them a key threatening process. Additionally, this study also provides evidence of certain host developmental stages being more susceptible to infection, which may have significant implications for release of captive bred animals. Lastly, this study provides investigators the necessary tools for screening wild and captive populations of frogs for the presence of these parasites and these tools may prove instrumental in determining the distribution of these parasites in Australia and establishing specific pathogen free captive breeding colonies.This study shows that both Figure S1Detection limit for multiplex species specific PCR. Ten fold dilutions of quantified myxospore samples for Cystodiscus axonis and C. australis using Cystodiscus internal transcribed spacer rDNA specific primers. Reactions were run in triplicate and visualised on a 2% agarose gel stained with Gel Red .(TIF)Click here for additional data file.Table S1Cystodiscus axonis and Cystodiscus australis with lesions of disease.Summary of statistical analyses results for associating (DOC)Click here for additional data file.


Aortic pulse wave velocity (PWV) is considered as the "gold standard" measurement of arterial stiffness and is commonly calculated as the ratio between the distance separating two locations along the artery and the transit time (Δt) needed for the pressure or velocity wave to cover this distance. PWV is increasingly assessed by means of cardiovascular magnetic resonance (CMR). Our goal was evaluate the efficiency of a novel method for Δt estimation, based on the principle of group delay (TT-GD method).Flow curves were estimated from phase contrast (PC) images of 30 patients. The TT-GD method operates in the frequency domain and models the ascending aortic waveform as an input passing through a discrete-component "filter", producing the observed descending aortic waveform, so that the group delay (GD) of that filter represents the average time-delay. This method was compared with two previously described time-domain methods: TT-point using the half-maximum of the curves and TT-wave using cross correlation. In order to study the effect of the temporal resolution on ΔT estimates, the original flow curves were downsampled of a factor of two, three and four.Mean Δts obtained with the three methods were comparable .The TT-GD method was the most robust to reduced temporal resolution Table .While the TT-GD as well as the TT-wave produced comparable results for velocity and flow waveforms , the TT-point resulted in significant shorter Δt values when calculated from velocity waveforms .The TT-GD method was the most reproducible, with an intra-observer variability of 3.38% and an inter-observer variability of 3.67%.Since the TT-GD method operates in the frequency domain, it was more robust to reduced temporal resolution than either of the time-domain methods. Moreover, it was more robust to the waveform type and more reproducible.This work was part of National Institutes of Health trial supported by the National Heart Lung and Blood Institute grant # 1 RO1 HL075592-01A1.


Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls. It is well known that the RT step, required to convert RNA to complementary DNA (cDNA), is imprecise and that different reverse transcriptase enzymes PCR is continuing to gain recognition in the field as an extremely precise and reproducible methodology offering the potential for accurate, robust and highly sensitive measurement without the need for a standard curve While this may be true, many studies have demonstrated that the variability inherent in the RT component of the process far outweighs that observed from the PCR step when performing qPCR In this study we investigated how this characteristic of the RT might affect cDNA production and ultimately influence the dPCR measurement by performing RNA analysis by RT-dPCR and assessing the repeatability, linearity and sensitivity of dPCR measurement. We prepared a Transcriptomic Calibration Material (TCM) and measured both synthetic and endogenous targets, comparing RT-dPCR analysis to UV, and evaluated how different assays and commercially available one-step RT-qPCR kits perform using both endogenous targets and synthetic process controls.Saccharomyces cerevisiae), Sigma] at 25 ng/reaction was used as carrier in this study. All samples were diluted using RNA Storage Solution, RSS (Life Technologies), unless otherwise stated.LoBind® tubes were employed throughout this study . Primer and probe sequences for dPCR were designed in-house using Primer Express, software version 3  and obtained from Sigma . Primers/assays were positioned across different RNA secondary structure motifs  RNA transcripts  were selected for investigation . For brevity, the ERCCs shall be subsequently identified without the preceding zeros. Concentrations of plasmid were assigned by the supplier using UV spectrophotometry and converted to copy number using published methods In vitro transcription (IVT) performed using MEGAscript® T7 Kit (Life Technologies): 37°C overnight incubation, with Turbo DNase treatment. IVT ERCC RNA concentrations and insert sizes were subsequently estimated using Nanodrop UV spectrophotometry  and 2100 Bioanalyzer , respectively. 2100 Bioanalyzer data may be found in ERCC RNA was prepared from the corresponding plasmid DNA, as described previously Three human cell lines were employed for production of complex background material for endogenous target selection; Hep-G2 , SaOS-2  and Hs 683 , . Culturing details given in Based on confluency and cell size, eight to fourteen flasks were prepared for each cell type, as outlined in ase I (Life Technologies), as per manufacturer’s protocol . These preparations were then purified using RNeasy midi kit (Qiagen), assessed for quantity , and subsequently pooled (per cell-type).Total RNA was extracted from cell lysates by following the standard TRIzol protocol (Invitrogen), details given in ase treatment and clean-up, pooled cell-line RNA samples were subjected to standard quality metrics for concentration and integrity . Neat samples  were then stored in aliquots at −80°C.Following total RNA extraction, DNPooled cell line RNA stocks were diluted in RSS to 250 ng/µL (Hep-G2 and Hs683) or 100 ng/µL (SaOS-2), and the complex background material prepared by mixing different proportions of each cell line RNA to a final concentration of 50 ng/µL . A mix containing all six ERCC transcripts was spiked into the mixed ratio cell line solution, at approximately 1.0E+06 copies/µL , to produce the Transcriptomic Calibration Material (TCM) for analysis. The TCM solution was aliquoted (150 µL) to generate 245 replicate units prior to storage at −80°C.ase inactivate/denature) 95°C for 15 min, (PCR) 40 cycles 95°C for 15 s and 60°C for 60 s. Analysis was performed utilising dPCR analysis software (Fluidigm), version 3.0.2. dPCR calculations are explained in further detail in the dPCR experiments were performed using the Fluidigm Biomark platform. Both 12.765 and 48.770 chip formats were utilised. Assays were first optimised using the Prism 7900 HT real-time PCR system (Life Technologies) before transfer to the Biomark. One-step RT-dPCR utilised AgPath-ID one-step RT-PCR reagents (Ambion). Master reactions comprised RT-PCR buffer/master mix (1×), RT enzyme (1×), GE sample loading reagent , sequence-specific gene assay . These mA count of partitions showing positive amplification can be made and an absolute target concentration elucidated. “Estimated copies” or “Copies per panel” refer to the number of targets on the panel following a Poisson correction, to account for the fact that some positive partitions will contain more than one molecule. As the number of positive partitions increases, so does the probability that some partitions will contain more than one target molecule. See ases are derived from Moloney murine leukemia virus (MMLV) RTase. Alternatively, the Qiagen (Omniscript and Sensiscript) RTases are derived from a unique source (undisclosed). The Qiagen RTases maintain RNase H activity, while the Ambion and Invitrogen RTases are claimed to have reduced RNase H activity.For one-step kit comparison, two further commercial kits were evaluated; Quantitect Probe one-step RT-PCR Kit (Qiagen) and Superscript III Platinum one-step RT-qPCR system w/ROX (Invitrogen). Both the Ambion (Multiscribe) and Invitrogen (Superscript III) RTInitially, quantification was assessed for two external (ERCC-25 and ERCC-99) targets in both uniplex and duplex formats, between the three commercial one-step RT-qPCR kits: AgPath ID (Ambion), Quantitect (Qiagen) and Superscript III (Invitrogen). RT-dPCR was performed using Fluidigm Biomark 12.765 dPCR chips, n = 1 panel, plus three replicate experiments. Sample was diluted to approximately 1896 copies per panel (or 2062 copies/µL added to master mix), based on UV estimates. Following this, ERCC-25 and ERCC-99, plus two endogenous (UBC and MMP1) targets were compared between the kits. These assays were analysed in duplex: ERCC-25 with ERCC-99 (duplex A), UBC with MMP1 (duplex B), and ERCC-25 with UBC (duplex C). Sample was diluted to approximately 1886 copies per panel , based on UV estimates. RT-dPCR was performed using Fluidigm Biomark 12.765 dPCR chips, n = 3 replicate panels, plus two replicate experiments.Measurement variability of six ERCC targets was tested using RT-dPCR evaluated as above . ERCC targets were spiked into cell line-derived total RNA at approximately 1.0E+06 copies/µL (estimated by UV), enabling evaluation of potential assay bias. Sample was diluted to approximately 200–400 copies per panel. RT-dPCR was performed using Fluidigm Biomark 48.770 dPCR chips, n = 3 replicate experiments. Assays were analysed in uniplex.An evaluation of RT-dPCR quantification sensitivity was performed using ERCC-25 and ERCC-99 assays. Based on UV estimated values, sample was diluted in 0.5% Tween 20 (Sigma) to approximately 500, 250, 100, 50, 25, 10 and 5 copies per panel . Volumetric dilutions were performed independently for each dilution, rather than sequentially, to avoid volumetric error propagation during dilution steps. RT-dPCR was performed using Fluidigm Biomark 48.770 dPCR chips, n = 6 panels per dilution, plus three replicate experiments. Assays were analysed in duplex.http://www.r-project.org/). All data sets incorporated ANOVA calculations.All statistical analyses were performed using MS Excel 2007 and the R statistical programming environment  = 4.3.Weighted regression was used to stabilise the different variance between groups. Standard uncertainty estimates were made to 3 significant figures and have 2 degrees of freedom, with k = 4.3 to convert to expanded uncertainties. Only dispersion due to plate-to-plate variation was included. dPCR plate-to-plate variability was estimated by pooling the data for all six ERCCs. The relative standard deviation was approximately 7.59%  with 12 degrees of freedom (18 data points minus the six estimated group means).A linear mixed model fit was used with experiment as random effect. Additionally, an ANOVA was applied removing experiment from the model and applying a classical fixed effect linear model fit (with only assay and dilution as factors).The analysis was split into four groups, one per assay. The square root of the copy numbers was needed in order to stabilise the difference in variance between groups. The Qiagen kit always resulted in zero positive partitions for MMP1, which was therefore removed from the data set.Three commercially available kits were compared for quantitative performance by RT-dPCR. The three kits were initially assessed using both uniplex and duplex formats for quantification of two synthetic RNA targets: ERCC-25 and ERCC-99 . The choConsistent ratios for ERCC-25:ERCC-99 between uniplex and duplex measurements were not maintained between kits suggesting an assay-dependent as well as a kit associated difference . The ERCTo investigate this disparity further, RT-dPCR measurements using the Ambion kit were compared when measuring a further four ERCC targets  . dPCR es2O 0.5% v/v Tween 20 as diluent, to generate samples equating to approximately 500, 250, 100, 50, 25, 10 or 5 copies/panel.An additional aim was to identify RT-dPCR sensitivity and linearity of measurement for low copy targets. This was performed utilising the Ambion kit alone, due to its superior capabilities throughout our initial analyses. A dilution series of two synthetic RNA targets, ERCC-25 and ERCC-99, were analysed in duplex . DilutioThere was a significant difference identified between the two targets agreement with UV values, p<0.0001 , which cIn order to investigate the applicability of our findings to real samples, the same three, one-step RT-qPCR kits were tested to compare measurement of endogenous targets alongside external controls in various duplex combinations in the TCM . Again fTo establish whether different plex pairings influenced RT-dPCR results, duplex reactions were performed pairing different targets (Duplex A: ERCC-25+ ERCC-99. Duplex B: MMP1+ UBC. Duplex C: ERCC-25+ UBC). As observed above, there was a significant difference between the kits, but no significant difference observed in dPCR values between ERCC-25 or UBC when assessed in different duplex reactions using the Ambion reagents (ABC), p = 0.061 and 0.92, respectively. Therefore, for these targets, assays did not influence the quantification result of their duplex partners.In this study we used a Transcriptomic Calibration Material (TCM) containing synthetic RNA transcripts in a complex background made of mixtures of human cell line total RNA. This was used to both evaluate dPCR measurement and demonstrate the applicability of the TCM for supporting accurate RNA enumeration by RT-dPCR.The findings from the one-step kit comparison by dPCR , Table 2The analysis method was shown to significantly affect the RNA quantification result. There may be a number of reasons explaining the significant difference observed between dPCR and UV methodologies. While dPCR makes an absolute count of specific amplified cDNA target molecules, UV cannot discriminate between nucleic acid species, non-target RNA and fragmented/degraded/non-amplifiable targets Our linearity and sensitivity data clearly show a pattern of increased variability with the increase of dilution factor below 50–100 estimated copies. We have previously demonstrated that when analysing DNA targets, dPCR is highly precise down to 16 copies/panel The magnitude of the quantification difference between kits was not consistent between different targets, both synthetic and endogenous, suggesting an additional assay specific and kit associated bias. There was a greater difference between kits when measuring endogenous targets than for synthetic targets. Furthermore, both Invitrogen (1 positive partition) and Qiagen (0 positive partitions) kits were unable to provide satisfactory quantification values for MMP1 despite being measured with six replicates totalling some 4590 reactions. However, as the Ambion kit only measured on average 112 MMP1 positive partitions, it would suggest that this transcript was below the limit of detection for the two former kits. Measurement, or specifically enzyme, efficiency/sensitivity is an important consideration when measuring low abundance RNA targets, in order to avoid false negative results and our data suggests that choice of kit is crucial for ensuring the most sensitive result when performing RT-dPCR. It should also be noted that while MMP1 target was present at low abundance in the dilutions tested, evaluation of a more concentrated sample may circumvent the sensitivity issues associated with the two kits. Therefore, this must also be considered when validating protocols, and where possible, low copy measurements should be avoided.One of the most striking findings of this study is the large inter assay and inter kit difference in the estimated copies for a given target. There are a number of potential causes for these observations. It is clear from our data that some, if not all, of the kits analysed during this study were not measuring all the RNA molecules that were present. There may be a number of different reasons for this. The assumption that DNA measurement by dPCR can be precise, reproducible, and absolute cannot be readily extrapolated to the measurement of RNA In our recent study, we documented a dPCR phenomenon termed molecular dropout Template secondary structure and position of the assay is known to impact on the RT-qPCR reaction The recommendation from the MIQE guidelines ase efficiency ase used in the Invitrogen kit is not significantly inhibited by such total RNA components. These considerations taken together may in part explain the disparity displayed between different one-step RT-qPCR kits.There may be other factors contributing to RT yields. For example, the samples used were sourced from cell line lysates. Co-extracted inhibitors may affect different reverse transcriptases to different degrees. Furthermore, components of total RNA, such as rRNA and tRNA may additionally inhibit RTase/kit and all experimental plans should therefore be validated appropriately before embarking upon studies analysing important samples.As may be seen from this comparison, despite the accuracy conferred by dPCR, analysis of RNA using RT-dPCR needs to be approached with caution. While for RNA measurement the precision of the RT-dPCR technique is high, it nonetheless introduces increased variability into the measurement value than dPCR alone For accurate RNA analysis by RT-dPCR it is possible that unknown measurements should be properly correlated to an appropriate measurement standard, with a well-defined value and uncertainty This study has shown that dPCR is capable of making precise measurements of synthetic and endogenous RNA molecules in a complex RNA background. RT-dPCR quantification of RNA targets was significantly lower than that derived from UV values suggesting a possible underestimation bias. Furthermore, absolute measurements differed between the three one-step kits assessed, with bias in detection sensitivity. Linearity and precision were sustained for duplex dPCR measurement of synthetic RNA using the Ambion kit, while sensitivities differed between RNA targets. dPCR is unencumbered by the restraints of calibration curve measurements, however, the employment of dPCR-specific calibrant materials (reference samples) would facilitate greater accuracy for absolute quantification. Furthermore, use of the TCM shows the applicability of RT-dPCR for the target-dependent selection of suitable RT enzymes. This study is novel in demonstrating application of RT-dPCR for absolute quantification of RNA endogenous and synthetic targets. Our findings give strong weight to the applicability of RT-dPCR to measurement fields including RNA diagnostics and RNA viral measurement.Figure S1RNA Secondary Structure Predictions from mFold. (A) MMP1, (B) UBC, (C) ERCC-13, (D) ERCC-25, (E) ERCC-42, (F) ERCC-99, (G) ERCC-113 and (H) ERCC-171. Green highlighted regions indicate amplicon. Folding predictions were performed at 45°C (temperature of RT step).(DOCX)Click here for additional data file.Figure S2Integrity assessment of Synthetic RNA Transcripts. 2100 Bioanalyzer quantification for all six synthetic targets was comparable to nanodrop concentration estimates .(DOCX)Click here for additional data file.Figure S3Typical dPCR output data from this study. Both amplification plots and heatmaps are shown. Amplification plots display ΔRN versus cycle number. Heatmaps are the corresponding schematic representations of positive partitions as detected by the Biomark instrument. Black = no amplification. Red = FAM amplification. Blue = HEX amplification. Threshold was adjusted to eliminate cross talk between the filters (FAM versus HEX). (A) One-Step RT-qPCR Kit Comparison by dPCR. (B) Endogenous versus Synthetic Targets.(DOCX)Click here for additional data file.Table S1Primer and probe sequences.(DOCX)Click here for additional data file.Table S2ERCC RNA concentration and copy number estimates.(DOCX)Click here for additional data file.Table S3Assay Positions.(DOCX)Click here for additional data file.Table S4MIQE checklist for authors, reviewers and editors.(DOCX)Click here for additional data file.Appendix S1Materials and Methods.(DOCX)Click here for additional data file.Appendix S2dPCR Calculations Explained.(DOCX)Click here for additional data file.