Presence of Chlamydiales DNA in ticks and fleas suggests that ticks are carriers of Chlamydiae.

The Chlamydiales order includes the Chlamydiaceae, Parachlamydiaceae, Waddliaceae, Simkaniaceae, Criblamydiaceae, Rhabdochlamydiaceae, Clavichlamydiaceae, and Piscichlamydiaceae families. Members of the Chlamydiales order are obligate intracellular bacteria that replicate within eukaryotic cells of different origins including humans, animals, and amoebae. Many of these bacteria are pathogens or emerging pathogens of both humans and animals, but their true diversity is largely underestimated, and their ecology remains to be investigated. Considering their potential threat on human health, it is important to expand our knowledge on the diversity of Chlamydiae, but also to define the host range colonized by these bacteria. Thus, using a new pan-Chlamydiales PCR, we analyzed the prevalence of Chlamydiales DNA in ticks and fleas, which are important vectors of several viral and bacterial infectious diseases. To conduct this study, 1340 Ixodes ricinus ticks prepared in 192 pools were collected in Switzerland and 55 other ticks belonging to different tick species and 97 fleas belonging to different flea species were harvested in Algeria. In Switzerland, the prevalence of Chlamydiales DNA in the 192 pools was equal to 28.1% (54/192) which represents an estimated prevalence in the 1340 individual ticks of between 4.0% and 28.4%. The pan-Chlamydiales qPCR was positive for 45% (25/55) of tick samples collected in Algeria. The sequencing of the positive qPCR amplicons revealed a high diversity of Chlamydiales species. Most of them belonged to the Rhabdochlamydiaceae and Parachlamydiaceae families. Thus, ticks may carry Chlamydiales and should thus be considered as possible vectors for Chlamydiales propagation to both humans and animals.

Chlamydiaceae and Chlamydia-like organisms in free-living small mammals in Europe and Afghanistan.

Few data are available on the occurrence of chlamydial infections in wild small mammals. We investigated the significance of free-living small mammals as reservoirs or transmission hosts for microorganisms of the phylum/class Chlamydiae. We obtained 3,664 tissue samples from 911 animals in Switzerland, Germany, Austria, the Czech Republic, and Afghanistan. Samples included internal organs (n = 3,652) and feces (n = 12) from 679 rodents (order Rodentia) and 232 insectivores (order Soricomorpha) and were tested by three TaqMan® real-time PCRs specific for members of the family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia spp. and Waddlia spp. Only one of 911 (0.11%) animals exhibited a questionable positive result by Chlamydiaceae-specific real-time PCR. Five of 911 animals were positive by specific real-time PCR for Parachlamydia spp. but could not be confirmed by quantitative PCR targeting the Parachlamydia acanthamoebae secY gene (secY qPCR). One of 746 animals (0.13%) was positive by real-time PCR for Waddlia chondrophila. This result was confirmed by Waddlia secY qPCR. This is the first detection of Chlamydia-like organisms in small wildlife in Switzerland. Considering previous negative results for Chlamydiaceae in wild ruminant species from Switzerland, these data suggest that wild small mammals are unlikely to be important carriers or transport hosts for Chamydiaceae and Chlamydia-like organisms.

Retinal ischemia-induced apoptosis is associated with alteration in Bax and Bcl-x(L) expression rather than modifications in Bak and Bcl-2.

PURPOSE: Apoptosis is known to play a key role in cell death after retinal ischemia. However, little is known about the kinetics of the signaling pathways involved and their contribution to this process. The aim of this study was to determine whether changes in the expression of molecules in the mitochondrial apoptotic pathway might explain the progression of retinal damage following ischemia/reperfusion. METHODS: Retinal ischemia was induced by elevating intraocular pressure in the vitreous cavity to 150 mmHg for a period of 60 min. At time 0, 3 h (early phase), and 24 h (late phase) after reperfusion, the retinas were harvested and modifications in the expression of Bax, Bak, Bcl-2, and Bcl-x(L) as well as caspase-3 and -7, were examined by qPCR and, in some cases, by western blot. RESULTS: qPCR analysis performed at the early phase after ischemia revealed a time dependent decrease in Bax, Bak, and Bcl-x(L) and no alteration in Bcl-2 mRNA expression in response to retinal ischemia. At the protein level, proapoptotic Bax and Bak were not modulated while Bcl-2 and Bcl-x(L) were significantly upregulated. At this stage, the Bax per Bcl-2 and Bax:Bcl-x(L) ratios were not modified. At the late phase of recovery, Bax and Bcl-x(L) mRNAs were downregulated while Bak was increased. Increased Bax:Bcl-2 and Bax:Bcl-x(L) ratios at both the mRNA and protein levels were observed 24 h after the ischemic insult. Analysis of caspases associated with mitochondria-mediated apoptosis revealed a specific increase in the expression of caspase-3 in the ischemic retinas 24 h after reperfusion, and a decrease in the expression of caspase-7. CONCLUSIONS: This study revealed that Bcl-2-related family members were differently regulated in the early and late phases after an ischemic insult. We showed that the Bax:Bcl-2 and Bax:Bcl-x(L) balances were not affected in the initial phases, but the Bax:Bcl-x(L) ratio shifted toward apoptosis during the late phase of recovery. This shift was reinforced by caspase-3 upregulation.

Altered expression of the transcription factor Mef2c during retinal degeneration in Rpe65-/- mice.

Purpose. To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed. Methods. Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR) and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells. Results. Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2 fold at 2 and 4 months and by 3.5 fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas, from post-natal day (P)13 onward, was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression, as well as those of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin and M-opsin genes. Conclusions. These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They further indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

A new blood-based screening test for colorectal cancer: a pilot study

Background: Colorectal cancer (CRC) can be cured when diagnosed in its early or precancerous (adenoma) stages. Mostly due to poor compliance towards invasive screening procedures, detection rates for adenoma and early CRCs are still low. Available non-invasive screening tests have unfortunately low sensitivity and specificity performances. Therefore, there is a large unmet need calling for a cost-effective, reliable and non-invasive test to screen for early neoplastic and pre-neoplastic lesions. Objective: To develop a routine screening test based on a nucleic acids multi-gene assay performed on peripheral blood mononuclear cells (PBMCs) that can detect early CRCs and adenomas. Methods: 116 patients (mean age: 55 years; range: 18 to 74 years; female/male ration 0.98) were included in this pilot, nonblinded, colonoscopy-controlled study. Colonoscopy revealed 21 patients with CRC, 30 patients with adenoma bigger than 1 cm, 24 patients with inflammatory bowel disease (IBD) and 41 patients had no neoplastic or inflammatory lesions. Blood samples were taken from each patient the day of the colonoscopy and PBMCs were purified. Total RNA was extracted following standard procedures. Multiplex RT-qPCR was applied on 92 different candidate biomarkers. Different univariate and multivariate statistical methods were applied on these candidates, and among them, 57 biomarkers with significant p values (<0.01, Wilcoxon test) were selected, including ADAMTS1, MMP9, CXCL10, CXCR4, VEGFA and CDH1. Two distinct biomarker signatures are used to separate patients without neoplastic lesion from those with cancer (named COLOX 1 test), respectively from those with adenoma (named COLOX 2 test). Result: COLOX 1 and 2 tests have successfully separated patients without neoplastic lesion from those with CRC (sensitivity 70%, specificity 90%, AUC 0.88), respectively from those with adenoma bigger than 1cm (sensitivity 61%, specificity 80%, AUC 0.80). 6/24 patients in the IBD group have a positive COLOX 1 test. Conclusion: These two COLOX tests demonstrated an acceptable sensitivity and a high specificity to detect the presence of CRCs and adenomas bigger than 1 cm. The false positives COLOX 1 test in IBD patients could possibly be due to the chronic inflammatory state. A prospective, multicenter, pivotal study is underway in order to confirm these promising results in a larger cohort.

Homeostatic and circadian contribution to EEG and molecular state variables of sleep regulation.

STUDY OBJECTIVES: Besides their well-established role in circadian rhythms, our findings that the forebrain expression of the clock-genes Per2 and Dbp increases and decreases, respectively, in relation to time spent awake suggest they also play a role in the homeostatic aspect of sleep regulation. Here, we determined whether time of day modulates the effects of elevated sleep pressure on clock-gene expression. Time of day effects were assessed also for recognized electrophysiological (EEG delta power) and molecular (Homer1a) markers of sleep homeostasis. DESIGN: EEG and qPCR data were obtained for baseline and recovery from 6-h sleep deprivation starting at ZT0, -6, -12, or -18. SETTING: Mouse sleep laboratory. PARTICIPANTS: Male mice. INTERVENTIONS: Sleep deprivation. RESULTS: The sleep-deprivation induced changes in Per2 and Dbp expression importantly varied with time of day, such that Per2 could even decrease during sleep deprivations occurring at the decreasing phase in baseline. Dbp showed similar, albeit opposite dynamics. These unexpected results could be reliably predicted assuming that these transcripts behave according to a driven damped harmonic oscillator. As expected, the sleep-wake distribution accounted for a large degree of the changes in EEG delta power and Homer1a. Nevertheless, the sleep deprivation-induced increase in delta power varied also with time of day with higher than expected levels when recovery sleep started at dark onset. CONCLUSIONS: Per2 and delta power are widely used as exclusive state variables of the circadian and homeostatic process, respectively. Our findings demonstrate a considerable cross-talk between these two processes. As Per2 in the brain responds to both sleep loss and time of day, this molecule is well positioned to keep track of and to anticipate homeostatic sleep need. CITATION: Curie T; Mongrain V; Dorsaz S; Mang GM; Emmenegger Y; Franken P. Homeostatic and circadian contribution to EEG and molecular state variables of sleep regulation. SLEEP 2013;36(3):311-323.

Acid-sensing channels in human bladder: expression, function and alterations during bladder pain syndrome.

Purpose: To examine the possible role of H+-activated acid-sensing ion channels (ASICs) in pain perception we characterized their expression in bladder dome biopsies of Bladder Pain Syndrome (BPS) patients and controls, in cultured human urothelium and in urothelial TEU-2 cells.Materials and Methods: Cold cut biopsies from the bladder dome were obtained in 8 asymptomatic controls and 28 patients with symptoms of BPS. ASIC expression was analyzed by QPCR and immunofluorescence. The channel function was measured by electrophysiology.Results: ASIC1a, ASIC2a and ASIC3 mRNAs were detected in human bladder. Similar amounts of ASIC1a and -3 were detected in detrusor smooth muscle, whereas in urothelium ASIC3 levels were higher than -1a. ASIC2a mRNA levels were lower than either -1a or -3 in both layers. ASIC currents were measured in TEU-2 cells and in primary cultures of human urothelium, and ASIC expression was confirmed by QPCR. Differentiation of TEU-2 cells caused an up-regulation of ASIC2a and ASIC3, and a down-regulation of ASIC1a mRNAs. BPS patients showed an up-regulation of ASIC2a and -3 mRNA, whereas ASIC1a remained unchanged. In contrast, the mRNA levels of TRPV1 were down-regulated during BPS. All differences were statistically significant (p<0.05)Conclusions: Several different ASIC subunits are expressed in human bladder and TEU-2 cells, where their levels are regulated during urothelial differentiation. An up-regulation of ASIC2a and -3 in BPS suggests their involvement in increased pain and hyperalgesia. A down-regulation of TRPV1 mRNA levels might indicate a different regulatory mechanism, controlling its expression in human bladder.

ON THE ROLE OF PERIOD-2 IN THE CIRCADIAN AND HOMEOSTATIC REGULATION OF SLEEP

Humans spend one third of their life sleeping, then we could raise the basic question: Why do we sleep? Despite the fact that we still don't fully understand its function, we made much progress in understanding at different levels how sleep is regulated. One model suggests that sleep is regulated by two processes: a homeostatic process that tracks the need for sleep and by a circadian rhythm that determines the preferred time-of-day sleep occurs. At the molecular level circadian rhythms are a property of interlocking transcriptional regula-tors referred to as clock genes. The heterodimeric transcription factors BMAL1::CLOCK/NPAS2 drive the transcription of many target genes including the clock genes Cryptochome1 (Cry1), Cry2, Period1 (Per1), and Per2. The encoded CRY/PER proteins are transcriptional inhibitors of BMAL1::CLOCK/NPAS2 thereby providing negative feedback to their own transcription. These genes seem, however, also involved in sleep homeostasis because the brain expression of clock genes, es-pecially that of Per2, increase as a function of time-spent-awake and because mice lacking clock genes display altered sleep homeostasis. The aim of first part of my doctoral work has been to advance our understanding the link that exists between sleep homeostasis and circadian rhythms investigating a possible mechanism by which sleep deprivation could alter clock gene expression by quantifying DNA-binding of the core-clock genes BMAL1, CLOCK and NPAS2 to their target chromatin loci including the E-box enhancers of the Per2 promoter. We made use of chromatin immunoprecipitation (ChIP) and quantitative poly-merase chain reaction (qPCR) to show that DNA-binding of CLOCK and BMAL1 to their target genes changes as a function of time-of-day in both liver and cerebral cortex. We then performed a 6h sleep deprivation (SD) and observed a significant decrease in DNA-binding of CLOCK and BMAL1 to Dbp. This is consistent with a decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was similarly decreased following SD. However, SD has been previously shown to in-crease Per2 expression in the cortex which seems paradoxical. Our results demonstrate that sleep-wake history can affect the molecular clock machinery directly at the level of the chromatin thereby altering the cortical expression of Dbp and Per2, and likely other targets. However, the precise dy-namic relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive. The second aim of my doctoral work has been to perform an in depth characterization of cir-cadian rhythmicity, sleep architecture, analyze the response to SD in full null-Per2 knock-out (Per2-/-) mice, and Per1-/- mice, as well as their double knock-out offspring (Per1,2-/-) and littermate wildtype (Wt) mice. The techniques used include locomotor activity recording by passive infrared (PIR) sen-sors, EEG/EMG surgery, recording, and analysis, and cerebral cortex extraction and quantification of mRNA levels by qPCR. Under standard LD12:12 conditions, we found that wakefulness onset, as well as the time courses of clock gene expression in the brain and corticosterone plasma levels were ad-vanced by about 2h in Per2-/- mice compared to Wt mice. When released under constant dark condi-tions almost all Per2-/- mice (97%) became arrhythmic immediately. From these observations, we conclude that while Per2-/- mice seem to be able to anticipate dark onset, this does not result from a self-sustained circadian clock. Our results suggest instead that the earlier onset of activity results from a labile, not-self sustained 22h rhythm linked to light onset suggesting the existence of a light-driven rhythm. Analyses of sleep under LD12:12 conditions revealed that in both Per2-/- and Per1,2-/- mice the same sleep phenotypes are observed compared to Wt mice: increased NREM sleep frag-mentation and inability to adequately compensate the loss of NREM sleep. That suggests a possible role of PER2 in sleep consolidation and recovery.

Inflammation And Autoimmune Responses Are Independent Of Peripheral Mhc Class Ii Expression Driven By Ciita Piv In Collagen Induced Arthritis

Background In rheumatoid arthritis (RA), non-professional antigen presenting cells (APCs) such as fi broblast-like synoviocytes (FLS) can express MHC class II (MHCII) molecules and function as non-professional APCs in vitro.Objective To examine the regulation of MHCII expression in FLS and to investigate the role of FLS as non-professional APCs in collagen-induced arthritis (CIA). Methods Expression of MHCII, CIITA and Ciita isoforms pI, pIII and pIV was examined by RT-qPCR, immunohistochemistry and fl ow cytometry in human synovial tissues, arthritic mouse joints and human as well as mouse FLS. CIA was induced in mice knockout for the isoform IV of Ciita (pIV-/-), in pIV-/- mice transgenic for CIITA in the thymus (pIV-/- K14 CIITA) and in control littermates in the DBA/1 background by immunising with bovine collagen type II (CII) in complete Freund's adjuvant.Results HLA-DRA, total CIITA and CIITA pIII mRNA levels were signifi cantly increased in the synovial tissues from RA compared to osteoarthritis patients. Human FLS expressed surface MHCII via CIITA pIII and pIV, while MHCII expression in murine FLS was entirely mediated by pIV. pIV-/- mice lacked both inducible MHCII expression on non-professional APCs including FLS, and in the thymic cortex. The thymic defect in pIV-/- mice impaired CD4+ positive selection, thus protecting pIV-/- mice from CIA by preventing CD4+ T cells immune responses against CII and blocking the release of IFN-γ and IL-17 in ex vivo stimulated lymph node cells. The production of T dependent, arthritogenic anti-CII antibodies was also impaired in pIV-/- mice. A normal thymic expression of MHCII and CD4+ T cell repertoire was obtained in pIV-/- K14 CIITA Tg mice. Immune responses against CII were restored in pIV-/- K14 CIITA Tg mice, as well as the arthritis incidence and clinical severity despite the lack of MHCII expression by mouse FLS. At histology, infl ammation andneutrophils infi ltration scores were not reduced in pIV-/- K14 CIITA Tg mice, while the bone erosion score was signifi cantly lower than in controls.Conclusion Over expression of MHCII is tightly correlated with CIITA pIII in the arthritic human synovium. MHCII is induced via CIITA pIII and pIV in human FLS. In the mouse, MHCII expression in the thymic cortex and in FLS is strictly dependent upon Ciita pIV. The lack of Ciita pIV in the periphery of pIV-/- K14 CIITA Tg mice lowered the bone erosion score but did not signifi cantly protect from infl ammation and autoimmune responses in CIA.

Differential expression and hypoxic regulation of adenosine receptors and TRPC channels in the developing heart.

Objectives: To characterize the modifications of gene expression of adenosine receptors (AR), TRPC channels, HIF-1α and iNOS during the early cardiogenesis in response to chronic hypoxia exposure. Methods: 4-day-old chick embryos were subjected in ovo to 6H, 12H and 24H of hypoxia (10% O2). The mRNA expression was quantified by RT-qPCR. Results: The targeted genes were found to be expressed at mRNA level with a differential expression pattern within the heart. Hypoxia has no significant effect on mRNA expression of ARs, TRPCs channels and iNOS within the heart. By contrast, HIF-1α mRNA expression shows a tendency to be down-regulated by hypoxia. Conclusion: These results suggest that an intrauterine oxygen lack does not significantly affect expression of genes involved in adenosine signaling and in calcium handling by store operated channels (TRPC).

Assessment of airborne virus contamination in wastewater treatment plants

INTRODUCTION: Occupational exposure to bioaerosols in wastewater treatment plants (WWTP) and its consequence on workers׳ health are well documented. Most studies were devoted to enumerating and identifying cultivable bacteria and fungi, as well as measuring concentrations of airborne endotoxins, as these are the main health-related factors found in WWTP. Surprisingly, very few studies have investigated the presence and concentrations of airborne virus in WWTP. However, many enteric viruses are present in wastewater and, due to their small size, they should become aerosolized. Two in particular, the norovirus and the adenovirus, are extremely widespread and are the major causes of infectious gastrointestinal diseases reported around the world. The third one, hepatitis E virus, has an emerging status. GOAL AND METHODS: This study׳s objectives were to detect and quantify the presence and concentrations of 3 different viruses (adenovirus, norovirus and the hepatitis E virus) in air samples from 31 WWTPs by using quantitative polymerase chain reaction (qPCR) during two different seasons and two consecutive years. RESULTS: Adenovirus was present in 100% of summer WWTP samples and 97% of winter samples. The highest airborne concentration measured was 2.27×10(6) genome equivalent/m(3) and, on average, these were higher in summer than in winter. Norovirus was detected in only 3 of the 123 air samples, and the hepatitis E virus was not detected. CONCLUSIONS: Concentrations of potentially pathogenic viral particles in WWTP air are non-negligible and could partly explain the work-related gastrointestinal symptoms often reported in employees in this sector.

Two high throughput technologies to detect segmental aneuploidies identify new Williams-Beuren syndrome patients with atypical deletions.

OBJECTIVE: To develop and compare two new technologies for diagnosing a contiguous gene syndrome, the Williams-Beuren syndrome (WBS). METHODS: The first proposed method, named paralogous sequence quantification (PSQ), is based on the use of paralogous sequences located on different chromosomes and quantification of specific mismatches present at these loci using pyrosequencing technology. The second exploits quantitative real time polymerase chain reaction (QPCR) to assess the relative quantity of an analysed locus. RESULTS: A correct and unambiguous diagnosis was obtained for 100% of the analysed samples with either technique (n = 165 and n = 155, respectively). These methods allowed the identification of two patients with atypical deletions in a cohort of 182 WBS patients. Both patients presented with mild facial anomalies, mild mental retardation with impaired visuospatial cognition, supravalvar aortic stenosis, and normal growth indices. These observations are consistent with the involvement of GTF2IRD1 or GTF2I in some of the WBS facial features. CONCLUSIONS: Both PSQ and QPCR are robust, easy to interpret, and simple to set up. They represent a competitive alternative for the diagnosis of segmental aneuploidies in clinical laboratories. They have advantages over fluorescence in situ hybridisation or microsatellites/SNP genotyping for detecting short segmental aneuploidies as the former is costly and labour intensive while the latter depends on the informativeness of the polymorphisms.

COLOX: a new blood-based test for colorectal cancer (CRC)screening

BACKGROUND: The objective is to develop a cost-effective, reliable and non invasive screening test able to detect early CRCs and adenomas. This is done on a nucleic acids multigene assay performed on peripheral blood mononuclear cells (PBMCs). METHODS: A colonoscopy-controlled study was conducted on 179 subjects. 92 subjects (21 CRC, 30 adenoma >1 cm and 41 controls) were used as training set to generate a signature. Other 48 subjects kept blinded (controls, CRC and polyps) were used as a test set. To determine organ and disease specificity 38 subjects were used: 24 with inflammatory bowel disease (IBD),14 with other cancers (OC). Blood samples were taken and PBMCs were purified. After the RNA extraction, multiplex RT-qPCR was applied on 92 different candidate biomarkers. After different univariate and multivariate analysis 60 biomarkers with significant p-values (<0.01) were selected. 2 distinct biomarker signatures are used to separate patients without lesion from those with CRC or with adenoma, named COLOX CRC and COLOX POL. COLOX performances were validated using random resampling method, bootstrap. RESULTS: COLOX CRC and POL tests successfully separate patients without lesions from those with CRC (Se 67%, Sp 93%, AUC 0.87), and from those with adenoma > 1cm (Se 63%, Sp 83%, AUC 0.77). 6/24 patients in the IBD group and 1/14 patients in the OC group have a positive COLOX CRC. CONCLUSION: The two COLOX tests demonstrated a high Se and Sp to detect the presence of CRCs and adenomas > 1 cm. A prospective, multicenter, pivotal study is underway in order to confirm these promising results in a larger cohort.

DNA extraction using the QIAsymphony: Evaluation of PCR inhibitor removal

The goal of this study was to compare the quantity and purity of DNA extracted from biological tracesusing the QIAsymphony robot with that of the manual QIAamp DNA mini kit currently in use in ourlaboratory. We found that the DNA yield of robot was 1.6-3.5 times lower than that of the manualprotocol. This resulted in a loss of 8% and 29% of the alleles correctly scored when analyzing 1/400 and 1/800 diluted saliva samples, respectively. Specific tests showed that the QIAsymphony was at least 2-16times more efficient at removing PCR inhibitors. The higher purity of the DNA may therefore partlycompensate for the lower DNA yield obtained. No case of cross-contamination was observed amongsamples. After purification with the robot, DNA extracts can be automatically transferred in 96-wellsplates, which is an ideal format for subsequent RT-qPCR quantification and DNA amplification. Lesshands-on time and reduced risk of operational errors represent additional advantages of the robotic platform.