As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method ...for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding
enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of
enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.
The probability of contamination of non-transgenic varieties with genetically modified (GM) products increase as a result of global expansion of areas sown with transgenic crops. DNA-based methods as ...accurate, efficient and reliable methods are preferable for detection of GM material in raw or highly processed foods. Isolation of high quality DNA with a suitable and efficient DNA extraction protocol is crucial for getting precise results in DNA amplification. In this study, we performed modifications of previously known Sodium dodecyl sulfate (SDS)-based DNA extraction method regarding the incubation period, DNA pellet washing and addition of organic solvent extraction, to improve DNA quality and to reduce costs. Raw corn kernels and roasted soybean seed were used as samples. DNA was extracted following three protocols, modifications of Edwards protocol. The type of detergent used in raw corn sample did not cause significant effects on extracted DNA yield and purity, while in roasted soybean samples the 2% (w/v) SDS lysis buffer gave the highest DNA yield. The additional incubation step raised the DNA yield from raw corn for 121%, while the purest DNA from soybean sample was obtained using organic solvent extraction. Electrophoretic determination of DNA integrity showed varying degree of DNA smearing from roasted soybean. Contrary, all extraction protocols used on raw corn kernels produced a high molecular weight DNA. Thus, our in-house DNA extraction protocol is as efficient but more cost effective compared to commercial kits and can be used for raw corn, while the protocol for roasted soybean needs further improvement.
Exposure to sublethal heat stress activates a complex cascade of signaling events, such as activators (NO), signal molecules (PKCɛ), and mediators (HSP70 and COX-2), leading to implementation of heat ...preconditioning, an adaptive mechanism which makes the organism more tolerant to additional stress. We investigated the time frame in which these chemical signals are triggered after heat stress (41 ± 0.5°C/45 min), single or repeated (24 or 72 h after the first one) in heart tissue of male Wistar rats. The animals were allowed to recover 24, 48 or 72 h at room temperature. Single heat stress caused a significant increase of the concentration of HSP70, NO, and PKC level and decrease of COX-2 level 24 h after the heat stress, which in the next course of recovery gradually normalized. The second heat stress, 24 h after the first one, caused a significant reduction of the HSP70 levels, concentration of NO and PKCɛ, and significant increase of COX-2 concentration. The second exposure, 72 h after the first heat stress, caused more expressive changes of HSP70 and NO in the 24 h-recovery groups. The level of PKCɛ was not significantly changed, but there was significantly increased COX-2 concentration during recovery. Serum activity of AST, ALT, and CK was reduced after single exposure and increased after repeated exposure to heat stress, in both time intervals. In conclusion, a longer period of recovery (72 h) between two consecutive sessions of heat stress is necessary to achieve more expressive changes in mediators (HSP70) and triggers (NO) of heat preconditioning.
Screening and confirmatory methods for Aflatoxin M1 (AFM1) analysis were optimized and studied regarding the European Decision 2002/657/EC requirements. The recoveries of AFM1 from the spiked samples ...at levels between 0.025 and 0.075 µg kg-1 were 99.6-110.3 % with obtained precision 10.5-13.4 %, confirming the immunochemical method precision and reliability for intensive surveillance studies. According to the regulatory requirements, for confirmation of the obtained positives from the screening, the reference high-performance liquid chromatographic method was evaluated in reference to the performance criteria. The obtained between-day recovery was in the range 67.2-72.8 %, and the precision range 5.4-9.1 %. 205 positives from the screening were subjected to confirmatory analysis. The comparison of the data obtained from the immunoassay and chromatographic method (R2=0.764) revealed a slight overestimation of the screening method, but no cases of false-negative determinations occurred.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This work presents the first insight on the aflatoxin M1 (AFM1) occurrence in raw milk collected from Macedonia, during the mycotoxin incidence in 2013. The total tested samples number was 3635, ...collected from February 2013 to January 2014. They were analyzed for AFM1 with ELISA as screening, and HPLC – fluorescence detection as confirmatory method. The AFM1 concentrations exceeded the maximum permitted levels in 2.9% of the samples, and the highest detected concentration was 408.1 ng/kg. The follow-up actions including feed sampling revealed aflatoxin B1 contamination in 31.8% of the samples tested, applying the HPLC-FD method. We estimated the carry-over rate between 0.22 and 3.47%. The experience obtained from the coordinated milk-feed aflatoxin survey could contribute towards increasing awareness for establishing continuous monitoring systems.
•We have shortened the abstract.•We have revised results and discussion section.•We have introduced new subtitle in the results and discussion section.•We have reduced the number of references.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
A different, reliable, and cost-effective strategy for the analysis of aflatoxins, ochratoxin A, and zearalenone in corn-based foods was proposed, including one multi-toxin immunoaffinity column ...(IAC) sample preparation and three different high-performance liquid chromatography fluorescence detection methods. The analytical procedures were tested and verified, keeping in mind their occurrence at trace levels in corn-based foods. With the validation of the proposed multi-toxin IAC methodology and comparison of the performance characteristics with methods using a single-toxin IAC, we confirmed the reliability of the multi-toxin IAC procedure versus the single-toxin IAC. The methods were validated by revealing satisfactory performance characteristics; for example, the obtained values of limit of detection were significantly lower than the maximum limits for all mycotoxins of concern. In addition, the recovery values were between 70.9 % and 106.1 % for all mycotoxins of interest, with precision values lower than 10.5 %.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A different, reliable, and cost-effective strategy for the analysis of aflatoxins, ochratoxin A, and zearalenone in corn-based foods was proposed, including one multi-toxin immunoaffinity column ...(IAC) sample preparation and three different high-performance liquid chromatography fluorescence detection methods. The analytical procedures were tested and verified, keeping in mind their occurrence at trace levels in corn-based foods. With the validation of the proposed multi-toxin IAC methodology and comparison of the performance characteristics with methods using a single-toxin IAC, we confirmed the reliability of the multi-toxin IAC procedure versus the single-toxin IAC. The methods were validated by revealing satisfactory performance characteristics; for example, the obtained values of limit of detection were significantly lower than the maximum limits for all mycotoxins of concern. In addition, the recovery values were between 70.9 % and 106.1 % for all mycotoxins of interest, with precision values lower than 10.5 %.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The Macedonian folk calendar is a rich treasury of cultural and spiritual wealth, connected with many beliefs and ritual activities. People organized life and activities according to their folk ...calendar, which is a complex system of behavioral rules. The folk calendar explained the cyclical changes in nature and natural phenomena. The year was perceived as a closed circle that has its own beginning and end but also has the quality of rebirth, i.e., renewal. The beginning of a new annual cycle represented a re-actualization of the cosmogony, which implies a repetition of time from its beginning, whatever it was in the moments of its creation. By establishing the system of holidays throughout the year, people realized the periodic re-actualization of the original memories. Therefore, the holiday is a nonworking day, it is not an empty day, but on the contrary, it is densely filled with many ritual-magical activities, through which the participants manage to perform another type of work. The importance and significance of all holidays, as well as the St. George's Day holiday and the festive ritual in it, have a crucial significance for the cultures that have the holiday system as a form of regulation of social relations in their system, as it is in the folk culture of the Macedonian people.
Mammary tumors are among the most common neoplasms in intact female dogs. They have a complex morphology, usually affecting middle age and older bitches. Almost 50% of the mammary tumors in dogs are ...malignant neoplasms. Prognosis is based on several factors: stage, age, tumor size, metastasis, histopathology, ovariectomy status and hormone-receptor activity. Immunohistochemical (IHC) measurement has become increasingly an important diagnostic and prognostic parameter, with the development of monoclonal antibodies against nuclear estrogen and progestin receptors. The aim of this study was to detect the presence of ER receptors in malignant canine mammary tumors and to identify their association with the clinical course of the tumor. Mammary tumor samples have been obtained by mastectomy from dogs presented at our clinic. Detailed clinical examination, CBC and basic serum biochemical profile were performed in all patients. Surgery was the only treatment. Histopathological examination and immunohistochemical detection of estrogen α receptors (ERα) was performed on 8 formalin-fixed, paraffin-embedded tissue samples, using the PT LINK immunoperoxidase technique. Histopathological examination of the mammary tumor samples (n=11) revealed tubular adenocarcinoma (n=6,54.5%) and ductal adenocarcinoma (n=3, 27.3%), one patient with benign adenoma and one with mastitis. Patients with positive ER tumors are alive, without remission, while 3 of the patients that were ER negative died due to lung metastases. According to our results, it can be concluded that the appearance and development of canine mammary tumors is highly connected with ovarian steroid hormones and that immunostaining of the tumors may be used as a good prognostic parameter in these patients.
Prognostic value of immunohistochemical expression of HER-2/neu in patients with lung carcinoma Background. The amplification and the overexpression of the Her-2/neu gene have been shown in certain ...human tumours and are postulated to be important in human carcinogenesis. In this study we evaluated the expression of HER-2/neu gene in patients with lung carcinoma (LC) and assessed its prognostic significance. Patients and methods. HER-2/neu expression was determined in 127 LC patients using immunohistochemistry (IHC) performed on paraffin-embedded section - Hercep Test(TM) (DAKO). Results. The overall HER-2/neu expression was seen in 36 (28.35%) of 127 LC patients. According to the histological type, HER-2/neu overexpression was detected in 12 patients with adenocarcinomas (60%), in 19 patients with squamous cell carcinomas (31.14%), in 4 patients with small cell-lung carcinomas (10%) and in 1 patient with other carcinomas (16.66%). Only in patients with small cell-lung carcinomas HER-2/neu overexpression was in correlation with the stage of the disease (p<0.001). The patients with HER-2/neu positive expression were associated with a significantly shorter survival compared with those who were HER-2/neu negative (log rank, p<0.002). Conclusions. These observations suggest that HER-2/neu positivity may serve as a prognostic indicator in patients with LC. HER-2/neu plays a role in identifying patients at risk for the shortened survival, who may benefit from a more aggressive therapy.
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