Charakterisierung des genetischen Defektes der GM1-Gangliosidose beim Alaskan Husky

Das Ziel dieser Arbeit war die Charakterisierung des genetischen Defektes der GM1-Gangliosidose beim Alaskan Husky und die Etablierung eines molekularbiologischen Nachweisverfahrens zur frühzeitigen Detektion heterozygoter Anlageträger. Um den genetischen Defekt zu charakterisieren, wurden auf mRNS-Ebene eine PCR mit dscDNS von gesunden Kontrolltieren, heterozygoten Anlageträgern und einem homozygoten kranken Alaskan Husky durchgeführt. Dadurch wurde beim heterozygoten Anlageträger die Anwesenheit zweier mRNS-Populationen nachgewiesen: eine mit dem normalen und eine mit einem abnormalen Exon 15. Um weitere genetische Defekte bzw. deren Auswirkungen zu untersuchen, wurden mehrere kanine saueren beta-Galaktosidase-Primer in der \u27nested\u27-PCR eingesetzt. Unter Verwendung eines Primerpaares, das den ganzen kodierenden Bereich des kaninen sauren beta-Galaktosidase-Gens amplifiziert, wurde bei gesunden Kontrolltieren eine Bande in der erwarteten Größe von 2022bp beobachtet. Bei heterozygoten Anlageträgern und beim kranken Alaskan Husky konnten 2 Banden mit einem 251bp bzw. 270bp Unterschied beobachtet werden. Mittels Sequenzierung wurde deren Zugehörigkeit zur kaninen sauren beta-Galaktosidase mRNS nachgewiesen. Zur Eingrenzung des genetischen Defektes wurden die erhaltenen PCR-Produkte als Matrize in einer \u27nested\u27-PCR mit kaninen sauren beta-Galaktosidase spezifischen Primerpaaren, die den Bereich zwischen den Positionen 79 1881 amplifizieren, eingesetzt. Die daraus resultierenden PCR-Produkte wurden in Plasmidvektoren eingebaut und anschließend in E. coli transformiert. Durch Sequenzierung der erhaltenen Plasmide wurde festgestellt, dass alle PCR-Produkte den kodierenden Bereich des kaninen sauren beta-Galaktosidase-Gens enthalten. Es konnte ein Verlust von Exon 15 und die Anwesenheit einer 19 Basenpaaren langen Duplikation auf dem Exon 15 zwischen den Positionen 1688-1689 bei heterozygoten Anlageträgern und dem homozygot kranken Alaskan Husky detektiert werden. Da Mutationen auf dem Exon 15 des sauren ß-Galaktosidase-Gens, welche die GM1-Gangliosidose bei Hund und Mensch hervorrufen, bekannt sind, wurde eine PCR bei gesunden, heterozygoten Anlageträgern und dem homozygot kranken Hund mit einem Primerpaar durchgeführt, das einen Abschnitt des Exon 15 zwischen den Positionen 1528-1710 amplifiziert. Dadurch wurde auf DNS-Ebene die Anwesenheit eines \u27Wildtyp\u27 Exon 15 bei gesunden, eines abnormalen Exon 15 bei kranken und beider Exons bei heterozygoten Tieren nachgewiesen. Durch die gefundene 19 bp Duplikation in dem kaninen sauren beta-Galaktosidase-Gen kommt es bei heterozygoten Anlageträgern und dem homozygoten Alaskan Husky zu einer Leserasterverschiebung (\u27frame shift\u27). Hierdurch entsteht ein frühzeitiges Stoppkodon, das zur Translation eines abnormalen, 67 Aminosäuren kürzeren, Polypeptids führt, welches im Carboxy-Terminalen Bereich keine Homologie zur kaninen sauren beta-Galaktosidase aufweist. Die 19bp Duplikation enthält mehrere Cytosin-Wiederholungen. Dieses Cytosin/Adenin-Muster (….CCCA....CCCCA….) bildet zusammen mit den 5’- und 3’- benachbarten Nukleotidsequenzen einen \u27exonic splicing silencer\u27, welcher das alternative Spleißen bevorzugt, ohne die vollständige Abschaffung des konstitutiven Spleißens. Das alternative Spleißen verursacht den Verlust von Exon 15, bis auf 5 Nukleotide (5’-GGAAG-3’), welche die normalen Spleißstellen enthalten. Dieses Nukleotidfragment verursacht eine Leserasterverschiebung und die Entstehung eines frühzeitiges Stoppkodons, das auf dem letzten Exon des kaninen sauren beta-Galaktosidase-Gens lokalisiert ist. Nach der Translation der falsch gespleißten mRNS entsteht ein 151 Aminosäure-verkürztes Polypeptid ohne Homologie mit der kaninen sauren beta-Galaktosidase mRNS im Carboxy-Terminalen Bereich. In der vorliegenden Studie gelang zum erstenmal in der Veterinär- und Humanmedizin die Identifikation und Charakterisierung einer genetischen Modifikation als Ursache für die GM1-Gangliosidose, die direkt eine Leserasterverschiebung bewirkt, aber auch indirekt durch Beeinflussung des mRNS-Spleißens als \u27exon skipping enhancer\u27 Element agiert. Da die GM1-Gangliosidose beim Alaskan Husky klinisch, biochemisch und pathologisch (Müller et al., 1998, 2001) diagnostiziert wurde, ist die Hypothese aufzustellen, dass die Leserasterverschiebung und der frühzeitige Abbruch der Translation die Ausfallserscheinungen verursachen. Bei heterozygoten Anlageträgern hingegen wurde eine Reduzierung der sauren beta-Galaktosidase-Aktivität im Vergleich zu gesunden Tieren festgestellt, was auf die verminderte Menge an Wildtyp mRNS-Molekülen (\u27gene dosis\u27 Effekt) zurückzuführen ist.The aim of the present study was to characterize the underlying genetic defect, in GM1-Gangliosiosis of Alaskan Huskies, and to establish a molecular assay for the detection of heterozygous animals. To identify the genetic defect which causes the GM1-Gangliosidosis in the Alaskan Husky, PCR with dscDNA from healthy control dogs, heterozygous carrier, homozygous diseased Alaskan Husky and the exon 15 specific primer pair was performed. The presence of two RNA-populations containing the \u27wild type\u27 and the abnormal exon 15 (with a 19bp duplication between the positions 1688-1698) was observed in heterozygous individuals. In healthy control dog the \u27wild type\u27 Exon 15 and in homozygous diseased Alaskan Husky the abnormal exon 15 (with the 19bp duplication) was also observed. The performed PCR allowed us to investigate only the exon 15 of canine GLB1. To investigate the presence of other genetic modifications \u27nested\u27-PCR with primer pairs covering the entire GLB1 coding region was performed. For this purpose the SMARTTMRACE method was used, which allows to obtain full-length cDNA. At first, a gene specific primer pair, to amplify the entire coding region of canine acid ß-galactosidase gene was used. Sequencing of all obtained PCR products confirmed their identity with published canine acid beta-galactosidase mRNA. The amplified coding region of canine acid beta-galactosidase was used as a template, \u27nested\u27-PCR with gene specific primer was for amplifying cDNA regions between position 79-1881. The performed experiments permitted to postulate the existence of one mRNA population in healthy control dogs, three mRNA populations in heterozygous carrier (a normal one, the second carrying the abnormal exon 15 with the duplication and the last one without exon 15) and two mRNA populations (one carrying the abnormal exon 15 and a second one without the exon 15) in homozygous diseased Alaskan Huskies. PCR experiments using exon 15 specific primers and DNA from heterozygous carriers as well as diseased homozygous and healthy dogs were also carried out. In these experiments the presence of a \u27wild type\u27 exon 15 in healthy dogs and an abnormal exon 15 (with 19bp duplication) in a diseased homozygous Alaskan Husky was detected. In heterozygous animals both exons were observed. The 19 bp duplication can act also as \u27exon skipping enhancer\u27. Through loss exon 15 (exon skipping) a premature termination codon is generated, because 5 nucleotides (containing the normal 5’ and 3’ splice site of exon 15) remain attached to the mature mRNA after removing exon 15. The mRNA molecules of both abnormal mRNA populations (with the abnormal exon 15 and with the exon skipping) are translated because of the frame shift and premature termination codons into shorter abnormal polypeptides. These polypeptides show at the carboxy-terminus (encoded by the exon 15, after the duplication, and by the exon 16) no homology to wild type canine acid beta-galactosidase. Homozygous disesased animals have only the abnormal acid beta-galactosidase gene and can not form an enzymatic active protein. In contrast, in heterozygous Alaskan Huskies the active canine acid beta-galactosidase are produced from the wild type allele. Phenotypically the dogs are healthy, but the enzymatic activity is lower compared to normal because of the presence of the abnormal allele. In the present study was achieved, for the first time in the veterinary and human medicine, the identification and characterization of a genetic modification, causing the GM1-Gangliosidosis, that directly determines a frame shift, but also indirectly act as an \u27exon skipping enhancer\u27 element influencing the mRNA-splicing

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development

Exceptional fracture toughness of CrCoNi-based medium- and high-entropy alloys close to liquid helium temperatures

Medium- and high-entropy alloys based on the CrCoNi-system have been shown to display outstanding strength, tensile ductility and fracture toughness (damage-tolerance properties), especially at cryogenic temperatures. Here we examine the JIc and (back-calculated) KJIc fracture toughness values of the face-centered cubic, equiatomic CrCoNi and CrMnFeCoNi alloys at 20 K. At flow stress values of ~1.5 GPa, crack-initiation KJIc toughnesses were found to be exceptionally high, respectively 235 and 415 MPa(square-root)m for CrMnFeCoNi and CrCoNi, with the latter displaying a crack-growth toughness Kss exceeding 540 MPa(square-root)m after 2.25 mm of stable cracking, which to our knowledge is the highest such value ever reported. Characterization of the crack-tip regions in CrCoNi by scanning electron and transmission electron microscopy reveal deformation structures at 20 K that are quite distinct from those at higher temperatures and involve heterogeneous nucleation, but restricted growth, of stacking faults and fine nano-twins, together with transformation to the hexagonal closed-packed phase. The coherent interfaces of these features can promote both the arrest and transmission of dislocations to generate respectively strength and ductility which strongly contributes to sustained strain hardening. Indeed, we believe that these nominally single-phase, concentrated solid-solution alloys develop their fracture resistance through a progressive synergy of deformation mechanisms, including dislocation glide, stacking-fault formation, nano-twinning and eventually in situ phase transformation, all of which serve to extend continuous strain hardening which simultaneously elevates strength and ductility (by delaying plastic instability), leading to truly exceptional resistance to fracture.Comment: 31 pages, 10 figures, including Supplementary Informatio

Basic Exploratory Study of Bisphenol A (BPA) Dietary Administration to Istrian Pramenka Rams and Male Toxicity Investigation

Bisphenol A (BPA), an endocrine-disrupting chemical and environmental pollutant, has been reported by many researchers to induce male reproductive toxicity in different experimental models. In this study, we investigated whether long-term exposure for two months to 25 mu g/kg body weight (low dose) of BPA affects spermatogenesis or sperm quality in young Istrian Pramenka rams exposed via diet. We evaluated body and testicular weights, histopathology of testes and epididymides, and sperm analyses, and compared these parameters between the group of treated rams and the control group of rams. Although there were some differences between the two groups, these differences were not large or statistically significant. The only statistically significant difference was the lower epithelial height of seminiferous tubules in treated rams, compared to control rams. In addition to assessing toxicity, BPA concentrations in the blood plasma of treated rams were determined after the first administration, and the toxicokinetic parameters of total BPA were calculated. In this study, no major signs of altered reproduction in rams were detected

Are there any good digraph width measures?

Many width measures for directed graphs have been proposed in the last few years in pursuit of generalizing (the notion of) treewidth to directed graphs. However, none of these measures possesses, at the same time, the major properties of treewidth, namely, 1. being algorithmically useful , that is, admitting polynomial-time algorithms for a large class of problems on digraphs of bounded width (e.g. the problems definable in MSO1MSO1); 2. having nice structural properties such as being (at least nearly) monotone under taking subdigraphs and some form of arc contractions (property closely related to characterizability by particular cops-and-robber games). We investigate the question whether the search for directed treewidth counterparts has been unsuccessful by accident, or whether it has been doomed to fail from the beginning. Our main result states that any reasonable width measure for directed graphs which satisfies the two properties above must necessarily be similar to treewidth of the underlying undirected graph

Developmental changes in resting‐state functional networks among individuals with and without internalizing psychopathologies

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147755/1/da22864.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147755/2/da22864_am.pd

Andean Leishmaniasis in Ecuador caused by Infection with Leishmania Mexicana and L. Major-Like Parasites

Between 1986 and 1988, epidemiologic studies were carried out in a small rural community in an Andean region of Ecuador, where cutaneous leishmaniasis is highly endemic. A total of 25 human cases, positive for Leishmania parasites by culture and/or smear, were examined. Fourteen of the cases were in infants less than one year of age, suggesting intradomiciliary transmission of the disease. Clinically, many of these cases were similar to descriptions of "uta," a form of cutaneous leishmaniasis which occurs in Andean regions of Peru and is reportedly caused by L. peruviana. Of the 11 positive cultures obtained from human cases in the present study, eight were identified by molecular characterization as L. mexicana and three were identified as L. major-like. Two additional isolates of L. mexicana were also made from an infected dog and from a sand fly, Lutzomyia ayacuchensis, living in the region, thus implicating the latter species as possible reservoir and vector, respectively, of L. mexicana in this highland community. The significance and validity of recent isolates of L. major-like parasites from the New World are also discussed