Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.

Water-soluble precursors of beef flavour. Part II: effect of post-mortem conditioning

Changes in glycolytic metabolites, nucleotide degradation products, free amino acids and other amino compounds were monitored in beef muscle (M. longissimus lumborum), stored for 21 days at 4 degrees C, in order to evaluate how post-mortem conditioning may affect flavour formation in beef. The major effects observed in sugar-related substances were the dephosphorylation of the phosphates of glucose, fructose and mannose, to yield their free sugars, as well as the breakdown of inosine 5'-monophosphate, to give a sixfold increase in ribose. Total reducing sugars increased by only 15% during conditioning, while glycogen levels remained unchanged from 2 days post-slaughter. Free amino acids increased during conditioning, particularly between days 7 and 14. Phenylalanine, methionine, lysine, leucine and isoleucine were the amino acids showing the greatest increase with conditioning time, with methionine, in particular, showing a sevenfold increase during the conditioning period. The effects of these precursor changes on cooked beef flavour are discussed. (c) 2007 Elsevier Ltd. All rights reserved.

Reverências à vida terrena e post-mortem (Caicó-RN, século XIX)

ARAÚJO, M. M. ; Olivia M. de Medeiros Neta ; FIGUEIREDO, Franselma. Reverências à vida terrena e post-mortem (Caicó-RN, século XIX). Revista HISTEDBR On-line, v. 33, p. 179-193, mar. 2009

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

POST-MORTEM LESIONS of UROLITHIASIS IN A LESSER SEED FINCH (Sporophila angolensis).

Urolithiasis is a disease that despite being a commonly observed problem in veterinary practice is uncommon in birds. Such disease was not reported in passeriforms to date. Accordingly, the aim of the present article is to describe a case of urolithiasis in an adult female lesser seed finch (Sporophila angolensis) pet bird which presented abdominal distension, respiratory distress, and apathy prior to death. The bird had history of being fed with a diet rich in protein. After the bird death, a necropsy was conducted in order to determine the cause of death. At necropsy, accentuated ascites, hydropericardium, and ureteral stones in the left ureter could be grossly observed. Additional tests related with viral and bacterial microbiological testing and with the determination of calculi composition could not be performed since the owner did not consent with the procedures because of the cost. Since the bird was fed on a high protein diet, a relationship between the ureteroliths and dietary imbalance was suggested with participation of protein in calculi development by providing the organic nuclei. Additionally, we conclude that the presence of calculi in the ureter resulted in urinary flow blockage, ascites, and consequent acute respiratory failure due to filling of air sacs with liquid.

Ocorrência de lesões hepáticas provocadas por larvas de Strongylus spp diagnosticadas no exame post mortem de eqüídeos abatidos em Apucarana, Paraná

The objective of this study was to determinate the occurrence of hepatic lesions caused by migratory large Strongyle larvaes in horses slaughtered in city of Apucarana in state of Parana. The lesions were diagnostic in post mortem exam by macro and microscopic analysis. From April 2003 to April 2004,38,363 animals, coming from different regions of Brazil, were examined. The occurrence of granulomas in liver was observed in 14,443 (37.64%), with adhesions and spot in 6,645 (17,32%), and 17,275 (45.03%) without macroscopic lesions. Macroscopic analysis revealed the presence of livers with calcified nodules, the presence of whitish spots, and adhesions in the format of "lines" over the hepatic capsule. No larvae were found in the livers. Hepatic fragments were histologically processed and revealed, by optical microscopy, inflammatory cells with predominance of eosinophils around the granulomas with a moderate amount of macrophages and the presence of fibroblasts.

A standard protocol for obtaining fish chromosomes under post-mortem conditions

In order to improve cytogenetical analyses on fish, especially focusing on delicate and rare species, we have adopted a new in vitro methodology using dead animals. The results obtained from 24 neotropical freshwater and marine fish species demonstrate that chromosomes can be obtained under post-mortem conditions. Significantly, the samples analyzed provided reliable cytogenetical data in nearly all cases. Other advantages of this new methodology are also discussed. (c) 2006 Elsevier Ltd. All rights reserved.

Efeito do resfriamento sobre a textura post-mortem da carne do peixe matrinxã Brycon cephalus

Os mecanismos que causam o amolecimento e a perda na textura post-mortem da carne de matrinxã foram determinados por meio das mudanças na microestrutura do músculo, imediatamente após a morte e depois de 12 horas de estocagem a -3°C. As observações na microestrutura, realizadas com microscópio eletrônico de transmissão, foram semelhantes aos resultados obtidos na força de ruptura do músculo medidos com o texturômetro. Os valores da força da ruptura foram menores para a carne após o resfriamento. Observou-se que as fibras do colágeno do tecido conectivo pericelular se desintegraram e que as do colágeno do tecido conectivo do miocommata conservaram sua arquitetura e integridade. Houve pouca degradação da linha Z. Isso sugere que o amolecimento post-mortem da carne de mantrinxã, durante a estocagem a -3°C, é causado pela degradação do tecido conectivo pericelular.

Post mortem, citologia, histopatologia e bacteriologia no diagnóstico da tuberculose bovina - matadouro-frigorífico da Região Norte do Paraná

Pós-graduação em Medicina Veterinária - FMVZ

Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

Minimally invasive post-mortem CT-angiography in a case involving a gunshot wound

Non-contrast post-mortem computed tomography (pm-CT) is useful in the evaluation of bony pathologies, whereas minimally invasive pm-CT-angiography allows for the detection of subtle vascular lesions. We present a case of an accidentally self-inflicted fatal bullet wound to the chest where pm-CT-angiography revealed a small laceration of the anterior interventricular branch of the left coronary artery and a tiny disruption of the right ventricle with pericardial and pleural effusion. Subsequent autopsy confirmed our radiological findings. Post-mortem CT-angiography has a great potential for the detection of vascular lesions and can be considered equivalent to autopsy for selected cases in forensic medicine.

Virtobot--a multi-functional robotic system for 3D surface scanning and automatic post mortem biopsy

The Virtopsy project, a multi-disciplinary project that involves forensic science, diagnostic imaging, computer science, automation technology, telematics and biomechanics, aims to develop new techniques to improve the outcome of forensic investigations. This paper presents a new approach in the field of minimally invasive virtual autopsy for a versatile robotic system that is able to perform three-dimensional (3D) surface scans as well as post mortem image-guided soft tissue biopsies.