Enzymatic Digestion Method Coupled with Artificial Intelligence Techniques in Forensic Drowning Diatom Detection / 中山大学学报(医学科学版)

ObjectiveArtificial intelligence （AI） full smear automated diatom detection technology can perform forensic pathology drowning diatom detection more quickly and efficiently than human experts.However， this technique was only used in conjunction with the strong acid digestion method， which has a low extraction rate of diatoms. In this study， we propose to use the more efficient proteinase K tissue digestion method （hereinafter referred to as enzyme digestion method） as a diatom extraction method to investigate the generalization ability and feasibility of this technique in other diatom extraction methods. MethodsLung tissues from 6 drowned cadavers were collected for proteinase K ablation and made into smears， and the smears were digitized using the digital image matrix cutting method and a diatom and background database was established accordingly.The data set was divided into training set， validation set and test set in the ratio of 3：1：1， and the convolutional neural network （CNN） models were trained， internally validated， and externally tested on the basis of ImageNet pre-training. ResultsThe results showed that the accuracy rate of the external test of the best model was 97.65 %， and the area where the model features were extracted was the area where the diatoms were located. The best CNN model in practice had a precision of more than 80 % for diatom detection of drowned corpses. ConclusionIt is shown that the AI automated diatom detection technique based on CNN model and enzymatic digestion method in combination can efficiently identify diatoms and can be used as an auxiliary method for diatom detection in drowning identification.

The role of enzymes in the angiogenic activity of Hancornia speciosa latex

The Hancornia speciosa latex has shown angiogenic activity. Angiogenesis plays a major role in wound healing, and materials that stimulate this process could be used to develop drugs. This study aimed to explain the role of proteins in the H. speciosa serum fraction latex in angiogenesis. Hence, this material was treated with proteinase K and the proteins were inactivated. After protein inactivation, angiogenic activity was assessed with the chick chorioallantoic membrane assay. The result showed that the proteins in the serum fraction are responsible for angiogenic activity. Then, the total protein content in the serum fraction and its enzymatic activity were investigated. The low protein content observed in the H. speciosa serum fraction latex suggests that this biomaterial could be used to develop new drugs with a hypoallergenic response. Despite the low protein content, there was a significant enzymatic activity of at least three enzymes in the serum fraction latex: ß-1,3 glucanase, ß-glucosidase, and proteases. These enzymes seem to influence the healing process, assisting debridement, extracellular matrix remodeling, and collagen deposition, and decreasing the chances of contamination by microorganisms. In conclusion, the enzymes in the H. speciosa serum latex are associated with the angiogenic activity of this biomaterial and may be used to assist the wound healing process.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

Evaluación de un método de extracción y purificación de DNA a partir de tejido muscular fijado en formaldehido de cadáveres no identificados / Evaluation of a method of extracting and purifying DNA from muscle tissue fixed in formaldehyde unidentified bodies / Avaliação de um método de extracção e purificação de ADN a partir de tecido muscular fixado em formaldeído corpos não identificados

La obtención de DNA humano en buena cantidad y alta pureza a partir de tejido muscular fijado en Formaldehido no tamponado es de suma importancia en la amplificación por la Reacción en Cadena de la Polimerasa (PCR) para su aplicación en estudios de identificación y filiación genética. En este estudio se evaluó la eficiencia del Kit QIAampR DNA FFPE TISSUE y una modificación del mismo basado en lavados con PBS y el tiempo de digestión con proteinasa K, frente a la cantidad y calidad de este ácido nucléico. Las diferencias fueron significativas entre los tiempos de acción con la proteinasa K (PK) en relación a la cantidad y en la pureza producto de los lavados del tejido muscular previa extracción. Estos resultados proporcionan una pauta para el diseño de experimentos de acuerdo con el efecto de la fijación, optimizando recursos humanos e insumos.

Obtaining human DNA in large quantity and high purity from muscle tissue fixed in formaldehyde unbuffered is of utmost importance in amplification by Chain Reaction (PCR) for application in identification studies and genetic affiliation. In this study, the efficiency of DNA FFPE TISSUE QIAampR Kit and a modification thereof based washes with PBS and the time of digestion with proteinase K, compared to the amount and quality of the nucleic acid were evaluated. Differences were significant between the exposure times with proteinase K (PK) in relation to the quantity and purity of the product prior washings muscle tissue extraction. These results provide a guideline for the design of experiments according to the effect of fixing, optimizing human resources and inputs.

A obtenção de DNA humano em grande quantidade e de alta pureza a partir de tecido muscular fixado em formol não tamponado é de extrema importância para a amplificação por Reacção em Cadeia (PCR), para sua aplicação em estudos de identificação e filiação genética. Neste estudo, a eficácia do kit QIAampR DNA FFPE TISSUE e uma modificação do processo por lavagens com PBS e o tempo de digestão com proteinase K, em comparação com a quantidade e a qualidade do ácido nucléico desta base. As diferenças foram significativas entre os tempos de exposição com proteinase K em relação à quantidade e pureza produto das lavagens do tecido muscular extraída previamente. Estes resultados fornecem uma diretriz para o desenho de experimentos de acordo com o efeito de fixação, otimizando recursos humanos e insumos.

The Analysis of Reclaiming Ratio for 3 Diatom Species from Experimentally Drowned Animal Organs / 대한법의학회지

Drowning is one of the most common causes accidental death worldwide, but its diagnosis remains a challenging task in forensic pathology. Several authors have suggested that diatom analysis be conducted via an enzymatic digestion method that uses proteinase K to provide objective evidence for drowning; we employed this method in our study because of its superior applicability as compared to the conventional disorganization methods. The purpose of this study was to examine the reclaiming ratio of diatoms from experimentally drowned animal organs, which could be influenced by diatom morphology. The authors injected 3 diatoms species (Cyclotella striata, Navicula incerta, and Pleurosigma angulatum) into a rat's airway and compared the detection rate to investigate the factors that influence the sensitivity of diatom analysis. The results are as follows: (1) Average reclaiming ratio in the lungs was 81.07 for Navicula incerta, 48.26 for Cyclotella striata, and 5.35 for Pleurosigma angulatum. (2) The detection rates from the closed organs in 15 experimental animals were highest in the kidney (73%, 11/15), followed by the heart (67%, 10/15), brain (60%, 9/15), and liver (53%, 8/15). (3) Two Cyclotella striata was detected in the kidney of postmortem control group which suggest the possibility of contamination during laboratory procedure. In conclusion, the authors propose that diatom size could be a significant influencing factor for diatom extraction from the organs of drowned bodies; therefore, the results of diatom analysis must be interpreted after considering the diatom population of the drowning medium at the scene and the possibility of contamination during the laboratory procedure.

The Comparison of Plankton Detection by Two Analysis Methods in the Seawater of Gageo Island / 대한법의학회지

The acid digestion method for extracting diatoms has been widely used to confirm death by drowning, but its reliability is still disputed because some diatoms can be destroyed during the extraction process due to treatment with strong acid and heat. There is a need to develop an efficient and reliable digestive method to overcome the limitation of the present analytical procedure. In this study, the reliability and efficacy of quantitative and qualitative diatom analysis from seawater by an enzymatic digestion method was evaluated. We confirmed the merit of the enzymatic method that used proteinase K instead of nitric acid in the conventional method. As a result, the enzymatic method showed a higher recovery ratio and better preservation of the diatom structure, which is essential for quantitative (diatom density) and qualitative (species) interpretation of diatom analysis. This result indicates that the enzymatic method can replace the conventional acid digestion method to confirm cases of death by drowning since it is more reliable and yields conclusive results.

Identification of the Proteinase K-resistant Antigen of Orientia tsutsugamushi by Monoclonal Antibodies

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we characterized two monoclonal antibodies, NT19 and WT14, against the proteinase K-resistant antigen of O. tsutsugamushi. Western blot analysis showed that MAb NT19 and WT14 strongly recognized two antigenic bands with molecular masses of 20 kDa and 24 kDa, which were resistant to proteinase K digestion. We suggest that the proteinase-resistant antigen might be polysaccharide. One patient serum reacted with a 24 kDa band that was similar to a band observed by WT14, suggesting the possibility of the role of this proteinase-resistant antigen as an antigenic molecule in human infection.

Rapid Extraction of DNA using Ion Exchange Resin for Early Detection ofMycobacterium tuberculosis by the Polymerase Chain Reaction / 결핵

BACKGROUND: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(Polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. METHODS: We used the InstaGene(TM) DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1, 245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2, 536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. RESULTS: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value.. CONCLUSION: Even though both methods had lower possibility of cross contamination, shorter time requrirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usfulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.