Effect of pH extraction buffer on antioxidant enzymes activities in water lily’s leaves and petioles

Water lily (Nymphaea antares) is one of the most valuable aquatic ornamental plants which has bright potential in the floriculture industry. It may be useful as an urban ecosystem and as a source of medicinal compounds. Due to its potential to become a new value-added product in the food industry, water lily (N. antares) was investigated in this study. Therefore, the goal of this study was to determine the nutritional content and antioxidant activity in water lily leaves and petioles with different pH extraction buffers. Water lily extract was obtained using three different pH extraction buffers, Tris buffer at pH 6.8, pH 7.1 and pH 7.8. The heat capacity of the extract was analysed using differential scanning calorimetry (DSC) and different functional groups were identified using Fourier transform infrared spectroscopy (FTIR). Thermal denaturation of the leaves sample was detected at 81.84°C. The antioxidant enzymes activities including catalase (CAT), peroxidase (POX), polyphenol oxidase (PPO), and superoxide dismutase (SOD) were determined in the leaves and petioles of water lily. In leaves, PPO activity was found to be higher in samples with pH 7.1 of the extraction buffer while there were no significant differences for activities of CAT, POX and SOD in all samples. In petioles, PPO and POX activity were found to be higher in samples with pH 7.8 and 7.1 of extraction buffer, respectively. Thus, the study found that a pH range of 7 to 9 extraction buffers did not greatly affect most of the analysis performed.

Multiplex Lateral Flow Immunoassay for the Detection of Expanded-Spectrum Hydrolysis and CTX-M Enzymes

Background: Early detection of expanded-spectrum cephalosporinase (ESC) hydrolyzing ß-lactamases is essential for antibiotic stewardship. Here we have developed a multiplex lateral flow immunoassay (LFIA) that detects cefotaxime-hydrolyzing activity as well as the most prevalent ESC-hydrolyzing ß-lactamases: the CTX-M-like. Methods: The Rapid LFIA ESC test was evaluated retrospectively on 188 (139 Enterobacterales, 30 Pseudomonas spp. and 14 Acinetobacter spp.) agar-grown bacterial isolates with well-characterized ß-lactamase content. One single colony was resuspended in 150 µL extraction buffer containing cefotaxime, incubated at room temperature for 30 min prior to loading on the LFIA for reading within 10 min. Results: Out of the 188 isolates, all 17 that did not express a β-lactamase hydrolyzing cefotaxime gave negative results, and all 171 isolates expressing a β-lactamase known to hydrolyze cefotaxime, gave a positive test result. In addition, all 86 isolates expressing a CTX-M-variant belonging to one of the five CTX-M-subgroups were correctly identified. The sensitivity and specificity was 100% for both tests. Conclusions: The results showed that the multiplex LFIA was efficient, fast, low cost and easy to implement in routine laboratory work for the confirmation of ESC hydrolyzing activity and the presence of CTX-M enzymes.

EFFICIENT METHOD OF DNA ISOLATION FROM BULKING SAMPLES OF SEEDLINGS

Forage annual and perennial grasses are the difficult subject for molecular and genetic studies because of the problem with obtaining qualitative genomic DNA for PCR, due of high content of proteins, polysaccharides and polyphenols. The known methods of DNA extraction or the numerous commercial kits allow isolating purified nucleic acids from the leaf tissue, but characterized by low efficiency at seedlings using. The modified method of DNA isolation, based on the SDS-extraction buffer (sodium dodecil sulfate), is presented in this study. Significant modifications were introduced in the reagents compound and the steps of procedure accordingly to used type of plant tissue and the result was positive at usage on the bulking samples, as well as on the individual genotypes (the only seedling). Reliability of this method and the functionality of the obtained DNA samples were tested in PCR with different molecular markers (SSR, SRAP and PawS) in researches on revealing of forage legume grasses DNA polymorphism. The general advantages of the proposed method are simplicity and effectiveness, the possibility to isolate qualitative DNA without toxic reagents application, as well as relatively low cost and availability of reagents. This method can be useful for studying the genetic biodiversity and for decision the different tasks, required the rapid analysis of large plant populations.

Evaluation of patatin content in proteins of potato genotypes grown in Latvia

Potato proteins contains essential amino acids in considerably high concentration, therefore potatoes are considered to be one of the most valuable plant origin food for human consumption. Patatin forms one of the largest group of potato proteins with high potential to be used in food industry as a novel food. This study has been performed to approbate patatin determination method for evaluation of protein quality of potato genotypes, as well as evaluate patatin relative abundance (PRA) for breeding programmes to create in the future potato cultivars with higher value and potential to develop new products. The evaluation of patatin was performed in following steps– extraction proteins from potato, determination of patatin concentration and calculation of its relative abundance in proteins. Separation of patatin from potato tubers was made using extraction by SDS extraction buffer and determination of patatin in organically and conventionally (with differnt N suply) grown samples of 20 potato genotypes. The results of one-year study showed that patatin relative abundance of different cultivars varied from 1.65% to 50.2% and it was significantly different among genotypes. The nitrogen content of soil and maturity type of potato did not affect PRA significantly. Results provide impetus for further research.

Effects of Various Extraction Factors on Protein Yield of Haliotis discus hannai (Abalone)

Abalone (Haliotis discus hannai) is a widely consumed seafood in Asian countries. Rich in protein, abalone is consumed for refreshment, pregnancy care, and vitality. Although many studies have found that abalone protein has beneficial effects, the efficiency of the protein extraction method for abalone has rarely been studied. Therefore, this study aimed to examine the effects of various factors of abalone protein extraction, including extraction buffer, sonication, salt (NaCl) concentration, surfactant, and heating. Phosphate buffer showed higher protein yield compared with Tris-HCl buffer. In addition, the highest protein yield for each factor was observed at 60 s of sonication (84.44 µg/mg dw), 0.6 M NaCl (141.9 µg/mg dw), and 16 mM sodium dodecyl sulfate (SDS) (253.15 µg/mg dw). However, a combined effect was not observed. Lower protein extraction efficiency was observed for sous vide-cooked abalone. The electrophoresis assay revealed myofibrillar proteins, including paramyosin, actin, and tropomyosin. Overall, our results demonstrate that various extract conditions affect the protein extraction of abalone.

Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals

DETERMINATION OF THE OPTIMUM CONDITIONS FOR UREASE INHIBITTION EXTRACTED FROM SOME LOCAL PLANTS

In the current study, four types of plants commonly used namely Soybean, chickpea, bean, pea were obtained and screened for urease activity, among this plants, chickpea was chosen with maximum enzymatic activity, and it had the highest productivity of urease enzyme (1243 U/mg protein). Also sodium acetate buffer (0.2 M, pH 5.0) was chosen as a best extraction buffer with specific activity 1460 U/mg protein. The optimum extraction ratio represented by 1:8 (w:v) after 15 min, it was given 1988 U/mg protein. As well as four types of plants include garlic, red onion, green onion and cabbage were used to select the optimum plant material that inhibited urease enzyme. Cabbage was chosen, it had the highest inhibition activity of the enzyme (41%). Also tris buffer (0.2 M, pH 9) was selected as a best extraction buffer of plants inhibitor with inhibition activity 80%. The optimum extraction ratio represented by 1:8 (w:v) after 60 min, it was given 86% enzyme inhibition activity.

Isolation of high-quality RNA from plant seeds

The apple (Malus domestica Borkh.) is one of the major fruit tree crops, but it hasn’t been well-studied as a breeding object for molecular investigations. It is important to develop reliable and rapid methods that allow the preparation of plant material for future research. We introduce a quick and simple method for isolating high-quality RNA from lipid-rich apple seeds (M. domestica cv. Golden Delicious). Our method does not employ highly toxic reagents, because we exclude phenol, 2-mercaptoethanol and others. The chemical composition of the extraction buffer is simple and has a minimum level of toxicity. We showed that, in chaotropic conditions (i.e., with lithium chloride-urea), silica (SiO2) can bind with the lipids and RNA will remain in the solution. The extracted RNA was of high quality and we successfully used it for synthesizing cDNA and RT-PCR. The protocol developed by us can be useful for researchers working with RNA extraction from plant seeds.

Isolation of High Quality RNA for High Throughput Applications From Secondary Metabolite Rich Crocus Sativus L

Isolating high quality RNA is a basic requirement while performing high throughput sequencing, microarray and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction that include CTAB, TriZol, SDS based methods, they invariably yield less and poor quality RNA. In the present study we made certain changes in the available protocols including modifications in the extraction buffer and procedure viz-a-viz solutions used for precipitation of RNA. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) greater than 7.5. The quality of the RNA was further assessed by qPCR based amplification of mature miRNAs such as Cs-MIR166c and Cs- MIR396a. In conclusion, the study describes an efficient method of RNA extraction that is highly ideal for high throughput sequencing of small RNAs.

A rapid method for identifying ploidy level in Isatis indigotica Fortune and Isatis tinctoria Linnaeus

The genus Isatis is widely distributed throughout the world. In this work, thirty-two Isatis indigotica Fortune germplasms, collected from different regions and geographical locations in China, were analyzed the ploidy levels by flow cytometry. I. indigotica Fort. and Isatis tinctoria Linnaeus distinguished with root tip chromosome compression staining and cell flow cytometry. Microscopic observation showed that the chromosome numbers of I. indigotica Fort. and I. tinctoria L. were 2n = 14 and 2n = 28, respectively. In order to establish a flow cytometric nuclear experiment system suitable for I. indigotica Fort., the leaves of I. indigotica Fort. were used to prepare nuclear suspension with LB01, OTTO, Tris·MgCl2, patent of Luochang and Galbraith extraction buffer. It was found that the histogram generated by LB01, OTTO, Tris·MgCl2 and Patent of Luochang dissociation solution has poor peak shapes and large CV values, and Galbraith dissociation solution extraction buffer was suitable for extracting nuclei from most germplasms. Flow cytometry proved to be a simple, rapid, and highly accurate method for identifying ploidy levels Isatis species.