Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications.

BACKGROUND: One of the most challenging aspects of nucleic acid amplification tests is the extraction of genomic DNA. However, achieving satisfactory quality and quantity of genomic DNA is not always easy, while the demand for rapid, low-cost and less laborious DNA isolation methods is ever-increasing. METHODS AND RESULTS: We have developed a rapid (⁓2 min) crude DNA extraction method leading to direct-PCR that requires minimum reagents and laboratory equipment. It was developed by eliminating the time-consuming purification steps of DNA extraction, by processing the sample in optimized amounts of Taq KCl PCR buffer and DNARelease Additive/Proteinase K in only two minutes and carrying out amplification using conventional Taq DNA polymerase. The DNA preparation method was validated on muscle tissue samples from 12 different species as well as 48 cooked meat samples. Its compatibility was also successfully tested with different types of PCR amplification platforms extensively used for genetic analysis, such as simplex PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), multiplex PCR, isothermal amplification, real-time PCR and DNA sequencing. CONCLUSIONS: The developed protocol provides sufficient amount of crude DNA from muscle tissues of different species for PCR amplifications to identify species-of-origin via different techniques coupled with PCR. The simplicity and robustness of this protocol make nucleic acid amplification assays more accessible and affordable to researchers and authorities for both laboratory and point-of-care tests.

Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.

BACKGROUND: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. METHODS AND RESULTS: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed - from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. CONCLUSIONS: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

Using deep learning for dermatologist-level detection of suspicious pigmented skin lesions from wide-field images.

A reported 96,480 people were diagnosed with melanoma in the United States in 2019, leading to 7230 reported deaths. Early-stage identification of suspicious pigmented lesions (SPLs) in primary care settings can lead to improved melanoma prognosis and a possible 20-fold reduction in treatment cost. Despite this clinical and economic value, efficient tools for SPL detection are mostly absent. To bridge this gap, we developed an SPL analysis system for wide-field images using deep convolutional neural networks (DCNNs) and applied it to a 38,283 dermatological dataset collected from 133 patients and publicly available images. These images were obtained from a variety of consumer-grade cameras (15,244 nondermoscopy) and classified by three board-certified dermatologists. Our system achieved more than 90.3% sensitivity (95% confidence interval, 90 to 90.6) and 89.9% specificity (89.6 to 90.2%) in distinguishing SPLs from nonsuspicious lesions, skin, and complex backgrounds, avoiding the need for cumbersome individual lesion imaging. We also present a new method to extract intrapatient lesion saliency (ugly duckling criteria) on the basis of DCNN features from detected lesions. This saliency ranking was validated against three board-certified dermatologists using a set of 135 individual wide-field images from 68 dermatological patients not included in the DCNN training set, exhibiting 82.96% (67.88 to 88.26%) agreement with at least one of the top three lesions in the dermatological consensus ranking. This method could allow for rapid and accurate assessments of pigmented lesion suspiciousness within a primary care visit and could enable improved patient triaging, utilization of resources, and earlier treatment of melanoma.

Fatty acid profile and effect of fish fermented silage on digestive enzymes in Labeo rohita / Perfil de ácidos graxos e efeito de peixe fermentado em silagem de enzimas digestivas em Labeo rohita

Due to inconsistency in demand and supply of fishmeal there is immense need of alternate protein sources. Present project was therefore designed to replace costly fishmeal (FM) with low-priced fermented fish silage (FFS) in fish feed. Fermented fish silage was prepared by fermentation process using Lacto bacillus bacteria and its fatty acid profile and effect on digestive system of Labeo rohita was investigated. Lipid contents were isolated by Soxhlet apparatus and recorded as 6.23 ± 1.23 g/100g of fermented fish silage (FFS). Fatty acid profile of extracted Lipids was determined by gas liquid chromatography (GLC), sufficient amount of unsaturated fatty acids were found with pattern mono unsaturated fatty acids (MUFA) > saturated fatty acids (SFA) >poly unsaturated fatty acids (PUFA). Three treatment diets containing 100% silage (T1), 75% silage (T2) and 50% silage (T3) were prepared by mixing it with soybean meal (SBM) and rice bran as co-ingredients while fermented fish silage was replaced by fishmeal in control diet (T0). The experiment was conducted in glass aquaria in triplicate. Fish growth parameters were recorded fortnightly while physicochemical parameters of water were recorded on daily basis. After completion of feeding trial, three fish were randomly dissected to excise out their intestines and determine activity for protease, amylase and lipase enzymes. Non-significant differences (P<0.05) were recorded in growth parameters and enzymatic activity among all diets except lipase enzyme. Deceptively, it can be concluded that FFS has reasonable concentration of nutrients and unsaturated fatty acids so it can successfully replace fishmeal in fish diets.

Devido à diferença na procura e na oferta de farinha de peixe há imensa necessidade de qualquer membro suplente da fonte de proteína. Tão presente projeto foi projetada para substituir a dispendiosa farinha de peixe (FM) com barato peixe fermentado de ensilagem (FFS) em alimentos para peixes. FFS foi preparado pelo processo de fermentação usando Lactobacillus bactérias e seu perfil de ácidos graxos e efeito sobre o sistema digestivo de Labeo rohita foi investigado. Conteúdo lipídico foram isoladas pelo aparelho de Soxhlet e registadas como 6,23 ± 1,23 g/100g de FFS. Perfil de ácidos graxos extraídos de lipídios foi determinada por cromatografia líquida de gás (GLC). Quantidade suficiente de ácidos graxos insaturados foram encontrados com padrão MUFA > SFA > AGPI. Tratamento de três dietas contendo silagem de 100% (T1), 75% silagem (T2) e 50% silagem (T3) foram preparados misturando com farinha de soja (SBM) e farelo de arroz como co ingredientes enquanto FFS foi substituído pela FM na dieta controle (T0). O experimento foi conduzido em aquários de vidro em triplicado. O Crescimento dos peixes foram anotados os parâmetros quinzenal enquanto parâmetros físico-químicos de água foram registradas diariamente. Após a conclusão do teste de alimentação, três peixes foram aleatoriamente dissecada a impostos especiais de consumo os seus intestinos e determinar a atividade de protease, enzimas amilase e lipase. As variações não significativas (P<0,05) foi registrada em parâmetros de crescimento e atividade enzimática entre as dietas exceto enzima lipase mostrou diferença significativa entre as dietas de tratamento. Aparentemente, é possível concluir que a concentração razoável de FFS tem nutrientes e ácidos graxos insaturados de modo que ela possa substituir com êxito a farinha de peixe na dieta de peixes.

Comparison of the effects of levofloxacin on QT/QTc interval assessed in both healthy Japanese and Caucasian subjects (pages.

AIMS: There is no consensus as to what extent the results of thorough QT interval/corrected QT interval (QT/QTc) studies need to be bridged. METHODS: The results of two studies using levofloxacin in Japanese and Caucasian subjects were compared in a post hoc analysis to investigate the similarity of dose­effect responses. RESULTS: Concentration­response analysis based on the change of QT interval corrected using Fridericia's formula (QTcF) from time-matched placebo was planned and performed in the combined data sets. At the geometric maximum mean concentration for the two doses in the Caucasian study, a predicted effect on QTcF comparable to the effects observed was found. For the Japanese study, the predicted effect was lower, but the difference was not statistically significant. CONCLUSIONS: No statistically significant differences in QTc-prolonging effect between Japanese and Caucasian subjects were observed following levofloxacin dosing. However, a trend suggests that Caucasian subjects may be more sensitive. Age and sex did not have an impact.

Repeated supratherapeutic dosing of strontium ranelate over 15 days does not prolong QT(c) interval in healthy volunteers.

AIMS: The study was performed to assess the safety of strontium ranelate in accordance with the ICH, E14 guidelines for QT/QT(c) studies. Its primary objective was to compare supratherapeutic repeated dosing of strontium ranelate (4 g day⁻¹ for 15 days) with placebo on the largest time-matched mean QT(c) variation, from baseline to under treatment values, in healthy subjects. METHODS: Ninety-six healthy male and female subjects (27.7 ± 7.5 years) were included to receive 1 day of placebo followed by 15 days of supratherapeutic repeated dosing of strontium ranelate (4 g day⁻¹), in a 4 month, randomized, placebo (16 days) and positive-controlled (single dose of moxifloxacin 400 mg preceded by 15 days of placebo), double-blind, double dummy, crossover design. Measurement of QT interval was performed automatically on the ECGs with subsequent manual onscreen over-reading by cardiologists using electronic callipers. RESULTS: The largest time-matched difference in QT(c) I (individual QT correction for heart rate) between moxifloxacin 400 mg and placebo was observed at 2 h post dose (mean [95% CI] 10.62 [7.90, 13.35] ms). For strontium ranelate (4 g day⁻¹) the largest time-matched difference in QT(c) I compared with placebo was observed at 1 h post dose (mean [90% CI] 7.54 [5.17, 9.90] ms). No subject had a QT(c) greater than 480 ms during the study. Both moxifloxacin and strontium ranelate were well tolerated in healthy subjects. CONCLUSIONS: The findings of this study demonstrate that the administration of supratherapeutic repeated oral doses of strontium ranelate (4 g day⁻¹ for 15 days) does not lead to a prolongation of the QT/QT(c) interval above the threshold of regulatory concern.

Shortening of the QT interval after food can be used to demonstrate assay sensitivity in thorough QT studies.

The effect of food was investigated under conditions of a thorough QT (TQT) study and with confirmation of assay sensitivity by the use of a positive control (400 mg of moxifloxacin). Fifty-five healthy subjects were randomized to treatment and a sequence of fasted and fed baseline electrocardiography days. Subjects received standard breakfast 30 to 10 minutes prior to dosing. Measurement of QT interval was performed automatically with subsequent manual onscreen overreading using electronic calipers. A profound increase in heart rate of 9.4 bpm was observed in the fed condition compared with the fasted condition at 1.5 hours after dose with a corresponding shortening of QT (27 milliseconds); (baseline data). When corrected, QTcF interval was shortened significantly with the maximal effect observed at 2 hours after dose of 8.2 (95% confidence interval, 6-10) milliseconds. A concurrent shortening of the PR interval with a maximum value of 5.6 milliseconds was also observed. The findings of this study demonstrate that food alters the QT-RR relationship and shortens QTc and PR for at least 4 hours after a carbohydrate-rich meal. The findings are of regulatory interest as this study shows that normal physiological causes can shorten QTc significantly and that it could be used as a method to demonstrate assay sensitivity.

Levofloxacin can be used effectively as a positive control in thorough QT/QTc studies in healthy volunteers.

AIMS: To characterize the effects of levofloxacin on QT interval in healthy subjects and the most appropriate oral positive control treatments for International Conference on Harmonization (ICH) E14 QT/QTc studies. METHODS: Healthy subjects received a single dose of levofloxacin (1000 or 1500 mg), moxifloxacin (400 mg) or placebo in a four-period crossover design. Digital 12-lead ECGs were recorded in triplicate. Measurement of QT interval was performed automatically with subsequent manual onscreen over-reading using electronic callipers. Blood samples were taken for determination of levofloxacin and moxifloxacin concentrations. RESULTS: Mean QTcI (QT interval corrected for heart rate using a correction factor that is applicable to each individual) was prolonged in subjects receiving moxifloxacin 400 mg compared with placebo. The largest time-matched difference in QTcI for moxifloxacin compared with placebo was observed to be 13.19 ms (95% confidence interval 11.21, 15.17) at 3.5 h post dose. Prolonged mean QTcI was also observed in subjects receiving levofloxacin 1000 mg and 1500 mg compared with placebo. The largest time-matched difference in QTcI compared with placebo was observed at 3.5 h post dose for both 1000 mg and 1500 mg of levofloxacin [mean (95%) 4.42 ms (2.44, 6.39) in 1000 mg and 7.44 ms (5.47, 9.42) in 1500 mg]. A small increase in heart rate was observed with levofloxacin during the course of the study. However, moxifloxacin showed a greater increase compared with levofloxacin. CONCLUSIONS: Both levofloxacin and moxifloxacin can fulfil the criteria for a positive comparator. The ICH E14 guidelines recommend a threshold of around 5 ms for a positive QT/QTc study. The largest time-matched difference in QTc for levofloxacin suggests the potential for use in more rigorous QT/QTc studies. This study has demonstrated the utility of levofloxacin on the assay in measuring mean QTc changes around 5 ms.