Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer.

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

Effect of temperature on biogas yield increase and formation of refractory COD during thermal hydrolysis of waste activated sludge.

Thermal hydrolysis (TH) increases the anaerobic biodegradability of waste activated sludge (WAS), but also refractory organic and nutrient return load to a wastewater treatment plant (WWTP). This could lead to an increase in effluent chemical oxygen demand (COD) of the WWTP. The aim of this study was to investigate the trade-off between increase in biogas production through TH and anaerobic digestion and increase in refractory COD in dewatered sludge liquors at different temperatures of TH in lab-scale. WAS was thermally hydrolyzed in temperature range of 130-170 °C for 30 min to determine its biomethane potential (BMP). After BMP test, sludge was dewatered and sludge liquor was aerated in Zahn-Wellens test to determine its non-biodegradable soluble COD known as refractory soluble COD (sCODref). With increasing temperature in the range of 130-170 °C, BMP of WAS increased by 17-27%, while sCODref increased by 3.9-8.4%. Dewaterability was also enhanced through relative increase in cake solids by 12-30%. A conversion factor was defined through mass balance to relate sCODref to volatile solids of raw WAS. Based on the conversion factor, expected increase in effluent CODs of six WWTPs in Berlin were predicted to be in the range of 2-15 mg/L after implementation of TH at different temperatures. It was concluded that with a slight decrease in temperature, formation of sCODref could be significantly reduced, while still benefiting from a substantial increase in biogas production and dewaterability improvement.

Reductive transformation of carbamazepine by abiotic and biotic processes.

The antiepileptic drug carbamazepine (CBZ) is ubiquitously present in the anthropogenic water cycle and is therefore of concern regarding the potable water supply. Despite of its persistent behavior in the aquatic environment, a redox dependent removal at bank filtration sites with anaerobic aquifer passage was reported repeatedly but not elucidated in detail yet. The reductive transformation of CBZ was studied, using abiotic systems (catalytic hydrogenation, electrochemistry) as well as biologically active systems (column systems, batch degradation tests). In catalytic hydrogenation CBZ is gradually hydrogenated and nine transformation products (TPs) were detected by liquid chromatography high-resolution mass spectrometry. 10,11-Dihydro-CBZ ((2H)-CBZ) was the major stable product in these abiotic, surface catalyzed reduction processes and turned out to be not a precursor of the more hydrogenated TPs. In the biotic reduction processes the formation of (2H)-CBZ alone could not explain the observed CBZ decline. There, also traces of (6H)-CBZ and (8H)-CBZ were formed by microbes under anaerobic conditions and four phase-II metabolites of reduced CBZ could be detected and tentatively identified. Thus, the spectrum of reduction products of CBZ is more diverse than previously thought. In environmental samples CBZ removal along an anaerobic soil passage was confirmed and (2H)-CBZ was determined at one of the sites.

Transcriptional regulation of the GLAST/EAAT-1 gene in rat and man.

Various acute and chronic brain diseases result in disturbed expression of the glial glutamate transporters, GLAST/EAAT-1 and GLT-1/EAAT-2, and subsequent secondary neuronal cell death. The idea that glutamate-induced brain damage can be prevented by restoring glutamate homeostasis in the injured brain, focussed previous efforts on identifying the network controlling astrocytic glutamate transport. Since most of this work was performed with rat astrocytes, we now sought to compare the transcriptional regulation of the GLAST/EAAT-1 gene in rat and man. Reporter gene assay demonstrated that the human GLAST/EAAT-1 promoter comprises the 2.3 kb region immediately flanking the 5'-end of the human GLAST/EAAT-1 gene. Cloning of the previously unknown promoter of rat GLAST/EAAT-1 gene demonstrated maximal reporter gene activity with a sequence comprising the 1.5 kb region flanking the 5'-end of the gene as well as non-coding exon 1, and intron 1-2. Although the promoter regions from both species lacked sequence homology, they contained numerous identical consensus motifs. In human promoter constructs, dbcAMP, PACAP, EGF, and TGFα, which represent potent stimulators of endogenous GLAST/EAAT-1 expression, only further increased reporter gene activity in the presence of the GLAST/EAAT-1 3'-UTR. By contrast, the rat GLAST/EAAT-1 3'-UTR only mediated the stimulatory increases of dbcAMP. Moreover, the GLAST/EAAT-1 3'-UTR repressed constitutive GLAST/EAAT-1 expression in man, but enhanced GLAST/EAAT-1 transcription in rat. Together, our findings suggest the existence of close functional similarities of the GLAST/EAAT-1 promoter regions in man and rat and further point to a species-specific function of the GLAST/EAAT-1 3'-UTR in constitutive and regulated GLAST/EAAT-1 expression.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex.

Sequences of the non-coding RNA, NTAB, are contained within the 3'-UTR of human and rat EAAT2/GLT-1 transcripts and act as transcriptional enhancers.

In the CNS, extracellular glutamate is predominantly cleared by astroglial cells through the high-affinity glutamate transporter subtype, EAAT2/GLT-1. Expression of EAAT2/GLT-1 is perturbed in various acute and chronic brain diseases eventually allowing for the onset of neurotoxic extracellular glutamate concentrations and subsequent excitotoxic neuronal cell death. The idea that glutamate-induced brain damage could be prevented by restoring glutamate homeostasis in the injured brain, spurred considerable interest in identifying the mechanisms controlling EAAT2/GLT-1 expression. Since to date most of this study was done with rat astrocytes, an emerging issue is to whether these findings would also apply to humans. While so far it is known that the promoter region of the EAAT2/GLT-1 gene is strikingly similar in rat and man, little information is available on the function of the EAAT2/GLT-1 3'-UTR in the control of EAAT2/GLT-1 expression in general as well as across both species. We now report on the presence of a homologous sequence within the 3'-UTR of the human and rat EAAT2/GLT-1 gene which we identified as a partial sequence of the putative non-coding RNA, Ntab. We further demonstrate that fragments of Ntab act as enhancers of EAAT2/GLT-1 transcription. Finally, we unravel that partial Ntab sequences are selectively present in the vicinity of the EAAT2/GLT-1 gene in several other mammalians, implying a conserved function of this sequence in the vertebrate CNS.

The genome of the stick insect Medauroidea extradentata is strongly methylated within genes and repetitive DNA.

BACKGROUND: Cytosine DNA methylation has been detected in many eukaryotic organisms and has been shown to play an important role in development and disease of vertebrates including humans. Molecularly, DNA methylation appears to be involved in the suppression of initiation or of elongation of transcription. Resulting organismal functions are suggested to be the regulation of gene silencing, the suppression of transposon activity and the suppression of initiation of transcription within genes. However, some data concerning the distribution of methylcytosine in insect species appear to contradict such roles. PRINCIPAL FINDINGS: By comparison of MspI and HpaII restriction patterns in genomic DNA of several insects we show that stick insects (Phasmatodea) have highly methylated genomes. We isolated methylated DNA fragments from the Vietnamese Walking Stick Medauroidea extradentata (formerly known as Baculum extradentatum) and demonstrated that most of the corresponding sequences are repetitive. Bisulfite sequencing of one of these fragments and of parts of conserved protein-coding genes revealed a methylcytosine content of 12.6%, mostly found at CpG, but also at CpT and CpA dinucleotides. Corresponding depletions of CpG and enrichments of TpG and CpA dinucleotides in some highly conserved protein-coding genes of Medauroidea reach a similar degree as in vertebrates and show that CpG methylation has occurred in the germline of these insects. CONCLUSIONS: Using four different methods, we demonstrate that the genome of Medauroidea extradentata is strongly methylated. Both repetitive DNA and coding genes appear to contain high levels of methylcytosines. These results argue for similar functions of DNA methylation in stick insects as those already known for vertebrates.

Mitochondrial genomes reveal an explosive radiation of extinct and extant bears near the Miocene-Pliocene boundary.

BACKGROUND: Despite being one of the most studied families within the Carnivora, the phylogenetic relationships among the members of the bear family (Ursidae) have long remained unclear. Widely divergent topologies have been suggested based on various data sets and methods. RESULTS: We present a fully resolved phylogeny for ursids based on ten complete mitochondrial genome sequences from all eight living and two recently extinct bear species, the European cave bear (Ursus spelaeus) and the American giant short-faced bear (Arctodus simus). The mitogenomic data yield a well-resolved topology for ursids, with the sloth bear at the basal position within the genus Ursus. The sun bear is the sister taxon to both the American and Asian black bears, and this clade is the sister clade of cave bear, brown bear and polar bear confirming a recent study on bear mitochondrial genomes. CONCLUSION: Sequences from extinct bears represent the third and fourth Pleistocene species for which complete mitochondrial genomes have been sequenced. Moreover, the cave bear specimen demonstrates that mitogenomic studies can be applied to Pleistocene fossils that have not been preserved in permafrost, and therefore have a broad application within ancient DNA research. Molecular dating of the mtDNA divergence times suggests a rapid radiation of bears in both the Old and New Worlds around 5 million years ago, at the Miocene-Pliocene boundary. This coincides with major global changes, such as the Messinian crisis and the first opening of the Bering Strait, and suggests a global influence of such events on species radiations.