Gel Bands Research Articles

STUDY OF GENETIC DIVERSITY AND ANALYSIS OF THE DEGREE OF SIMILARITY IN SOME APPLE AND PLUM VARIETIES USING RAPD MARKERS

Molecular evaluation of germplasm is an important step in breeding programs, and the application of molecular biological techniques has led to important results in terms of both within- and between-species variability of traits. The RAPD technique has been successfully used to reveal allelic polymorphism as well as to measure genetic similarity. In this study, the genetic diversity of 25 genotypes and cultivars for apple species and 26 genotypes and cultivars for plum species was assessed with six RAPD markers. All these cultivars belong to the ex situ collection of apple and the ex situ collection of plum at the Research Institute for Fruit Growing Pitesti. The average number of amplified bands was 19.2 for apple and 17.66 for plum. Statistical analysis of intraspecific allelic polymorphism was expressed using the PIC (Polymorphic Information Content) index, which takes into account the allelic frequency. Two statistical indices were used to quantify genetic diversity: the Shannon index and the Simpson index. The degree of similarity between varieties was analyzed using the NTSYSpc version 2.1. Following RAPD analyses, the allele sizes of the analyzed varieties were within the range quoted in the literature, the genetic profiles of the studied varieties suggesting a medium to high genetic diversity, except for markers OPBC-04 and OPBB-05 for plum species, which expressed a high genetic diversity. Genetic distances calculated based on polymorphism of migrated bands in agarose gel confirmed the known genealogies of the apple and plum varieties studied. Thus, the smallest genetic distance for apple species was found between 'Jonagold' and 'Golden Delicious', 'Pionier' and 'Rustic', 'Jonathan' and 'Idared', 'Wagener Premiat' and 'Granny Smith', 'Remar' and 'Aura', 'Romus 3' and 'Rome Beauty', and the largest between Malus floribunda and the other genotypes studied. In plum, the smallest genetic distance was found between 'Dani' and 'Tita', 'Roman' and 'Tuleu gras', 'Dara' and 'Haganta', 'Romanța' and 'Stanley', 'Anna Spath' and 'Renclod Violet', and the largest between 'Lama', 'Black Diamond' and the other genotypes studied.

Production of 7,10-dihydroxy-8(E)-octadecenoic acid using cell-free supernatant of Pseudomonas aeruginosa

Cell-free synthesis has been adopted in the bioconversion process due to its known advantages, such as fast production rate, high product content, and no substrate/product inhibition effect. In this study, the cell-free supernatant of Pseudomonas aeruginosa was used to improve the production of 7,10‐dihydroxy‐8(E)‐octadecenoic acid (DOD) from oleic acid. DOD production using cell-free supernatant demonstrated reduction in bioconversion duration and higher product concentration than conventional method using whole cell culture. The maximum DOD concentration (6.41 g/L) was obtained after 36 h of biotransformation using 1 % v/v oleic acid as a substrate with a productivity of 0.178 g/L/h and a yield of 74.8 %. DOD concentration, productivity, and yield using cell-free supernatant were 2.12, 7.12, and 2.22 times higher, respectively, than using the conventional whole cell culture method. Of the carbon and nitrogen sources used in pre-culture, galactose and sodium glutamate along with diammonium phosphate were found to be the most effective for DOD production. An incubation temperature of 27 °C and pH 8.0 were found to be most favorable for DOD production. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis demonstrated the presence of enzymes related to DOD production in the cell-free supernatant, which was substantiated by performing DOD production experiment using the supernatant enzymes extracted from protein gel bands with oleic acid as a substrate. To the best of our knowledge, this is the first report on DOD production using a cell-free supernatant and verifying the existence of the relevant enzymes in the cell-free supernatant. Compared to whole cell process, cell-free DOD production holds several advantages, including higher DOD productivity which could be beneficial for large-scale production.

Enzymological characteristics of pepsinogens and pepsins purified from lizardfish (Saurida micropectoralis) stomach

One major pepsinogen, PG-I, and two minor pepsinogens, PG-II and PG-III were purified from lizardfish stomach by ammonium sulfate precipitation and two chromatographic columns. The three purified PGs migrated as single bands in native-PAGE gels with molecular weights (MW) ranging from 36 to 38 kDa. Each PG was converted to pepsin (P) at pH 2.0, and the MW were determined as 32 kDa (for P-I), 31 kDa (for P-II) and 30 kDa (for P-III). The optimum pH and temperature of pepsins were 2.0–3.5, and 40–50 °C. All 3 pepsins were strongly inhibited by pepstatin A. Divalent cations slightly stimulated the pepsin activities, but ATP had no effect on the pepsins. Purified pepsins were effective in the hydrolysis of various proteins. Km and kcat of the three pepsins for hemoglobin hydrolysis were 107.64–276.61 µM and 18.30–32.68 s−1, respectively. The new pepsins have potential for use in protein food procession and modification.

Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.

Allergenicity assessment and allergen profile analysis of different Chinese wheat cultivars

BackgroundsAs one of the most important cereals, wheat (Triticum aestivum) can cause severe allergic reactions, such as baker's asthma, allergic rhinitis, and atopic dermatitis. A growing number of people are developing allergies to Chinese wheat; however, only a few wheat cultivars have been screened on allergenicity in China. ObjectiveThe aim of the present study was to assess the allergenicity of different Chinese wheat cultivars and characterize wheat allergen profiles of patients with allergic rhinitis. MethodsWe determined protein (soluble protein, gliadin, and glutenin) composition in Chinese wheat by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the immunoglobulin E (IgE) binding capacity by enzyme-linked immunosorbent assay (ELISA) and Western blot using 10 positive sera from wheat allergy patients. We identified 5 gel bands with significant IgE binding capacity using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsSoluble protein, albumin, and globulin, showed the highest allergenicity, followed by gliadin, while glutenin only had slight allergenicity. In soluble protein, 5 protein bands with molecular weights of 27, 28, 53, 58, and 62 kDa showed very significant allergenicity. Meanwhile, the relative abundances of 28 kDa-protein and 58 kDa-protein were significantly positively correlated with the IgE-binding capacity of Chinese wheat cultivars, which were identified as rRNA N-glycosidase and β-amylase, respectively, among other proteins in those highly complex gel bands. Conclusion and clinical relevance28 kDa-protein (rRNA N-glycosidase) and 58 kDa-protein (β-amylase) were speculated to be the main allergens of Chinese wheat causing baker's asthma and allergic rhinitis. These results provide new insights into the prevention and treatment of wheat allergy and the development of hypoallergenic wheat products, whose clinical significance is worth further evaluation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor.

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle

BackgroundDNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples.ResultsThe present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards.ConclusionsThe present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

Detection of simultaneous production of kurstakin, fengycin and surfactin lipopeptides in Bacillus mojavensis using a novel gel-based method and MALDI-TOF spectrometry.

Bacterial lipopeptides have become a research focus of many studies owing to their industrial and pharmaceutical importance. Although such studies focused on researching purification procedures and qualitative analysis, much remains to be explored and developed to improve the current methods. To enable thorough studies of lipopeptides, this paper describes a new method for purification and characterization of in-gel anionic lipopeptides. Specifically, lipopeptides attributed to the anti-staphylococcal activity of Bacillus mojavensis HF were separated using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and subsequently characterized using mass spectrometry. Lipopeptide band obtained by gel electrophoresis was first visualized using three different staining methods. Next, the lipopeptide isomers were efficiently recovered from the gel band and structural characterization of the extracted lipopeptides was carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MS analysis revealed that Bacillus mojavensis HF produced three types of lipopeptides including surfactin, fengycin, and kurstakin. 14 clusters of ion peaks were identified as fengycin A with fatty acid of C15-C17, fengycin B (C16, C17), surfactin (C13-C16), and kurstakin (C9-C12). Moreover, tandem mass spectrometric analysis (MS/MS) revealed the sequences of fengycin A and surfactin. In this study, we identified a high variety and number of surfactin and fengycin isomers, which previous reports lacked. To the best of our knowledge, we are the first to report the presence of kurstakin in Bacillus mojavensis species. Finally, we demonstrated that our gel-based study of lipopeptides allowed for a precise and reproducible investigation of these molecules.

Zein based-nanoparticles loaded botanical pesticides in pest control: An enzyme stimuli-responsive approach aiming sustainable agriculture

Nanoencapsulation of biopesticides is an important strategy to increase the efficiency of these compounds, reducing losses and adverse effects on non-target organisms. This study describes the preparation and characterisation of zein nanoparticles containing the botanical compounds limonene and carvacrol, responsive to proteolytic enzymes present in the insects guts. The spherical nanoparticles, prepared by the anti-solvent precipitation method, presented in the nanoparticle tracking analysis (NTA) a concentration of 4.7 × 1012 ± 1.3 × 1011 particles.mL−1 and an average size of 125 ± 2 nm. The formulations showed stability over time, in addition to not being phytotoxic to Phaseolus vulgaris plants. In vivo tests demonstrated that formulations of zein nanoparticles containing botanical compounds showed higher mortality to Spodoptera frugiperda larvae. In addition, the FTIC probe (fluorescein isothiocyanate) showed wide distribution in the larvae midgut, as well as being identified in the feces. The trypsin enzyme, as well as the enzymatic extract from insects midgut, was effective in the degradation of nanoparticles containing the mixture of botanical compounds, significantly reducing the concentration of nanoparticles and the changes in size distribution. The zein degradation was confirmed by the disappearance of the protein band in the electrophoresis gel, by the formation of the lower molecular weight fragments and also by the greater release of FTIC after enzymes incubation. In this context, the synthesis of responsive nanoparticles has great potential for application in pest management, increasing the selectivity and specificity of the system and contributing to a more sustainable agriculture.

Formation of light-harvesting complex II aggregates from LHCII–PSI–LHCI complexes in rice plants under high light

During low light- (LL) induced state transitions in dark-adapted rice (Oryza sativa) leaves, light-harvesting complex (LHC) II become phosphorylated and associate with PSI complexes to form LHCII-PSI-LHCI supercomplexes. When the leaves are subsequently transferred to high light (HL) conditions, phosphorylated LHCII complexes are no longer phosphorylated. Under the HL-induced transition in LHC phosphorylation status, we observed a new green band in the stacking gel of native green-PAGE, which was determined to be LHCII aggregates by immunoblotting and 77K chlorophyll fluorescence analysis. Knockout mutants of protein phosphatase 1 (PPH1) which dephosphorylates LHCII failed to form these LHCII aggregates. In addition, the ability to develop non-photochemical quenching in the PPH1 mutant under HL was less than for wild-type plants. As determined by immunoblotting analysis, LHCII proteins present in LHCII-PSI-LHCI supercomplexes included the Lhcb1 and Lhcb2 proteins. In this study, we provide evidence suggesting that LHCII in the LHCII-PSI-LHCI supercomplexes are dephosphorylated and subsequently form aggregates to dissipate excess light energy under HL conditions. We propose that this LHCII aggregation, involving LHCII L-trimers, is a newly observed photoprotective light-quenching process operating in the early stage of acclimation to HL in rice plants.

Halal Food Safety: PCR Based Detection of Porcine DNA in Imported Chocolate

Halal food and food products consumption is a major part of living in Muslim community. Pork meat or meat items are not considered halal in Muslim countries and consumers. Ensuring pork-free food items is a challenge for the food industry and exporters to Muslim nations. This study aims to detect porcine DNA in imported chocolate products in Bangladesh for halal safety assurance. The imported chocolate samples were collected from various multi shops in Dhaka. Polymerase chain reaction (PCR) method is used in our research to detect the porcine DNA. Two primer sets are used for the detection of porcine mitochondrial cyt-b (cytochrome-b) gene fragments in chocolate samples. To visualize the amplified DNA, agarose gel (1%) was used. After electrophoresis, DNA band in agarose gel indicated that the gene fragments are amplified properly. In our research, out of 42 chocolate samples, only 2 samples were found positive. The chocolate samples were branded as Wild Berry Flavor Chocolate and Cadbury Milk Tray Chocolate. In comparison with the positive pork sample, these two samples also containing the 165bp and 359bp fragment of the porcine cytochrome b gene. We reported that chocolate products contain the pork contamination were not labeled as halal. While other samples that did not have any halal logo originated from outside Bangladesh and imported also showed negative result. The present study established the DNA-based porcine detection system based on mitochondrial cyt-b that is viable in highly processed products. It can be used in the halal certification process to determine the pork items presence in food and halal safety. Our research also reported that imported chocolates should halal certify before release into the market.

Serum protein complex profiling reveals heterogeneity of Balanced constitutional population in traditional Chinese medicine through blue native polyacrylamide gel electrophoresis.

Pulse-taking is widely used for diagnosis and treatment in traditional Chinese medicine (TCM), and protein complexes in serum perform various biological functions. The Balanced constitution is one of the major constitutions in TCM, people with Balanced constitution can also share some common characteristics with unbalanced constitution types. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was applied to the serum of 25 people with balanced constitutions. The patterns of the protein complexes could be recognized according to the number, molecular weight, and intensity of the gel bands. All of the individual bands from these patterns were cut and in-gel-digested with trypsin, followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis for protein identification and biological function analysis. The protein complex patterns were roughly categorized as type A and B with high stability and reproducibility, and there were 15 and 16 gel bands in type A and type B, respectively. Among the 25 serum samples, 14 belonged to type A, and 11 belonged to type B. High-abundance proteins significantly decreased from 99% to 44% after BN-PAGE separation. The unique proteins in type A were mainly related to lipid metabolism, while the unique proteins in type B were involved in biological processes related to immune response and inflammatory regulation. The Qi-deficiency constitution converted score of type A was higher than that of type B, while the Damp-heat constitution converted score of type A was lower than that of type B. Our study provided an objective reference for diagnosis and prognosis, which might lay a foundation for establishing the characteristic protein complex spectra of all of the TCM constitutions.

Snake venom proteome of Protobothrops mucrosquamatus in Taiwan: Delaying venom-induced lethality in a rodent model by inhibition of phospholipase A2 activity with varespladib

Protobothrops mucrosquamatus, also known as the brown spotted pit viper or Taiwanese habu, is a medically significant venomous snake in Taiwan, especially in the northern area. To more fully understand the proteome profile of P. mucrosquamatus, we characterized its venom composition using a bottom-up proteomic approach. Whole venom components were fractionated by RP-HPLC and then analyzed by SDS-PAGE. Each protein band in gels was excised and subjected to protein identification by LC-MS/MS. A subsequent proteomic analysis revealed the presence of 61 distinct proteins belonging to 19 families in P. mucrosquamatus venom. Snake venom metalloproteinase (SVMP; 29.4%), C-type lectin (CLEC; 21.1%), snake venom serine protease (SVSP; 17.6%) and phospholipase A2 (PLA2; 15.9%) were the most abundant protein families, whereas several low-abundance proteins, categorized into eight protein families, were demonstrated in P. mucrosquamatus venom for the first time. Because PLA2 is known to make a major contribution to venom lethality, we evaluated whether the known PLA2 inhibitor, varespladib, was capable of preventing the toxic effects of P. mucrosquamatus venom. This small-molecule drug demonstrated the ability to inhibit PLA2 activity in vitro (IC50 = 101.3 nM). It also blunted lethality in vivo, prolonging survival following venom injection in a mouse model, but it showed limited potency against venom-induced local hemorrhage in this model. Our findings provide essential biological and pathophysiological insights into the composition of P. mucrosquamatus venom and suggest PLA2 inhibition as an adjunctive or alternative therapeutic strategy in the clinical management of P. mucrosquamatus envenoming in emergency medicine. SignificanceP. mucrosquamatus envenomation is a significant medical concern in Taiwan, especially in the northern region. Although antivenom is commonly used for rescuing P. mucrosquamatus envenoming, severe clinical events still occur, with more than 20% of cases requiring surgical intervention. Small-molecule therapy offers several advantages as a potential adjunctive, or even alternative, to antivenom treatment, such as heat stability, low antigenicity and ease of administration, among others. A deeper understanding of the venom proteome of P. mucrosquamatus would aid in the discovery of small-molecule drugs that could be repurposed to target specific venom proteins. Here, we applied a bottom-up proteomic approach to characterize the protein profile of P. mucrosquamatus venom. Varespladib, a small-molecule drug used to treat inflammatory disease, was repurposed to inhibit the toxicity of P. mucrosquamatus venom, and was shown to reduce the lethal effects of P. mucrosquamatus envenomation in a rodent model. Varespladib might be used as a first-aid therapeutic against P. mucrosquamatus envenoming in the pre-referral period and/or as an adjunctive agent administered together with anti-P. mucrosquamatus antivenom.

Determination of PEGylation homogeneity of polyethylene glycol-modified canine uricase.

Polyethylene glycol-modified canine uricase (PEG-UHC) was prepared by modifying the ε-amino group of lysine residues on the canine uricase (UHC) protein to near-saturation with 5kDa monomethoxyl-polyethylene glycol succinimide (mPEG-SPA-5k). In order to accurately determine the PEGylation uniformity of PEG-UHC, CZE, 3-8% gradient gel SDS-PAGE, and imaging CIEF (iCIEF) analyses were compared. CZE could not effectively separate PEG-UHC proteins with different degrees of modification, 3-8% gradient gel SDS-PAGE could separate PEG-UHC into seven gel bands; however, most of the gel bands were smeared or blurred, and the separation of PEG-UHC samples by iCIEF was significantly better than that by 3-8% gradient gel SDS-PAGE. Under denatured conditions, iCIEF separated 12 pI peaks, and could also accurately quantify the relative monomer PEG-UHC content. More than 85% of the total monomeric PEG-UHC was conjugated with 7-12 PEG molecules; of this 85%, approximately 40% was conjugated with 9-10 PEG molecules. These results demonstrated that iCIEF exhibits good potential for determining the PEGylation homogeneity of PEGylated protein drugs.

Lab hands and knowing toxic substances in Uganda

How does one know a toxic substance? Perhaps paradoxically, through contact with it, as I show ethnographically from a molecular biology laboratory in Uganda, a site where people routinely handle a range of chemicals, including potentially hazardous toxicants. I analyse an everyday situation in which researchers worked with ethidium bromide, a substance regularly used to stain DNA bands in gels at this Ugandan lab but also in many comparable labs elsewhere. Ethidium bromide is one of the most fear‐inspiring chemicals at this lab; it is considered a potent mutagen and thus counts as highly carcinogenic. While the lab has clear safety procedures concerning how to handle this substance correctly, these are often not implemented in a hard‐to‐control lab setting marked by both material deprivation and a steady flow of unremunerated volunteers who seek to gain practical experience. I show how experienced Ugandan scientists have learned to mitigate their own risk of exposure to ethidium bromide by developing choreographed routines that prevent them from inadvertently touching it. I examine this as a way of ‘handling’ a dangerous substance, developing and habituating skilled movements of hands and bodies that then are passed on to other researchers. It is not an abstract knowledge of a substance's chemical properties that counts here; instead, knowing a toxic substance implies practical routines of handling.

Synthesis of CuO samples by co-precipitation and green mediated combustion routes: Comparison of their structural, optical properties, photocatalytic, antibacterial, haemolytic and cytotoxic activities

The goal of this research is to prepare novel, inexpensive, environment friendly and efficient crystalline CuO samples by green mediated combustion and co-precipitation routes towards environmental remediation and biomedical applications. The influence of preparation routes and Aloe barbadensis miller (Aloe vera) gel on the morphology, size, crystallinity, band gap, defects, photocatalytic, antibacterial, haemolytic and anticancer behaviour of CuO samples were explored. The PXRD, XPS, SEM, TEM, HRTEM and FT-IR were performed to confirm the formation of CuO samples. FTIR studies showed all possible bands of Aloe barbadensis miller gel. FTIR displaying the occurrence of new peaks and peak shifts in the CuO synthesized by green mediated combustion route compared to co-precipitation route is an evidence of the effective interactions between CuO and Aloe vera gel. The PL and UV–Vis-DRS techniques measured the optical sensitivity and tuning of band gap of the CuO samples. The crystallinity and surface properties dependent photocatalytic activities for decolourization of MB and RhB under both UV and Sunlight irradiation were investigated. The results indicated that green mediated synthesized CuO sample displayed high photocatalytic activity compared with co-precipitation route synthesized CuO sample, which mainly resulted from the low crystallinity and crystallite size (6 nm), narrow band gap (1.73 eV) and lower recombination of charge carriers. These synthesized CuO samples also demonstrate excellent antibacterial activity against the bacteria Gram-positive (S. aureus) and Gram-negative (E. coli). The present study probes into the cytotoxicity of CuO samples employing root cells of Allium cepa. In addition, we report the haemolytic activity on goat and human blood along with the implementation of green synthesized CuO sample using an Aloe barbadensis miller extract for the evaluation of anticancer activity in human cervical cancer (HeLa) cells with IC50 value of 310.1 μg/ml. Our studies focus on developing biosynthesized nanomaterials for environmental remediation and biomedical applications.

Activity profile of innate immune-related enzymes and bactericidal of freshwater fish epidermal mucus extract at different pH

The epidermal mucus of fish performs diverse functions from prevention of mechanical abrasion to limit pathogen invasions. The current experiment was designed to extract skin mucus proteins of three freshwater fish, i.e. common carp (Cyprinus carpio), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita) with organic solvent (methanol) and dissolve in different pH of Tris-HCl buffers to examine the significance of pH in the solubilisation of skin mucus proteins. The protein profiles of different pH solubilised methanol fish skin mucus extracts were determined by SDS-PAGE. The non-specific immune enzymes, alkaline phosphatase, lysozyme and protease of fish skin mucus were compared and this present study demonstrated that these enzymes differed in their activity depending on pH buffers. The higher lysozyme and protease activity were observed at the pH of 8.0 and higher alkaline phosphatase activity in the pH 9.0 of C. mrigala fish skin mucus methanol extract. In addition, the bactericidal activity was evaluated against the pathogens Proteus vulgaris and Pseudomonas aeruginosa. The pH 8.0 of C. mrigala skin mucus extract revealed better bactericidal activity than other fish species mucus pH buffers against both P. vulgaris and P. aeruginosa. In the case of protein profile from SDS-PAGE, based on pH buffers and the solubilisation of proteins, differences in the resolution of bands were observed. The higher alkaline pH of 9.0 showed smeared gel bands in all the three fish skin mucus methanol extract. The present study suggests that methanol extracted C. mrigala fish skin mucus at pH 8.0 showed better innate immune enzymes and bactericidal activity. The additional examinations of C. mrigala skin mucus methanol extract in this pH aids in identifying novel bioactive molecules. This is the study of proteome of three fish species skin mucus in the effect of pH. Further analyses are required to evaluate proteins present in fish skin mucus extracted with methanol and the influence of pH on protein solubility. These findings could be helpful in exploring natural alternatives to antibiotics in aquaculture industry against infectious pathogens.

“Development and comparison of reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP), RT-PCR and real time PCR for detection of Potato spindle tuber viroid in potato”

Potato spindle tuber viroid (PSTVd) is an important pathogen of potato infecting leaves, tubers as well as true potato seeds. We developed and validated a reverse transcription-loop mediated isothermal amplification (RT-LAMP) platform for visual, rapid, specific, and sensitive detection of PSTVd. The optimization of RT-LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs, and WarmStart Bst 2.0 polymerase. The assay is very simple and rapid which provide detectable results in less than 50 min by incubating all the reagents at 65 °C. The detection of RT-LAMP results could be accomplished as ladder-like bands in an agarose gel or visualized by naked eyes with inclusion of a SYBR Green I dye. The target specificity of RT-LAMP primers was assessed with an introduction of a simple restriction digestion step after the RT-LAMP assay. The RT-LAMP assay demonstrated absence of any cross-reaction with other, potato viruses. The analytical sensitivity of the assay was greater than RT-PCR either the test template was plasmid or total RNA. The detection of RT-LAMP was validated in potato samples and compared with real time PCR and RT-PCR. The RT-LAMP assay developed in this study has the potential as a beneficial tool in detection of PSTVd in a low-cost laboratory due to its simplicity, rapidness, and application in large samples.

Investigation Study of Human Spermatozoa Proteome

The current study has been conducted on health human sperm to identify the different types of amino acidsand entire proteins of sperm. Two methods have been applied to characterise of human sperm, firstly extractlysate of sperm, then digest and run it in mass spectrometry, secondly, total sperm lysate was run in SDSpage gel and bands were stained with coomassie, then band was cut and digest to extract of whole aminoacids and also was run in mass spectrometry. Result revealed that sperm have several proteins, which wereinvolved with different binding proteins, soluble proteins, and glycoproteins, transmembrane proteins.Current result was displayed that mass spectrometry can be used to recognise data of proteins of spermand detected types of it. This technique was applied on whole lysate and gel bands of sperm and createddatabase of sperm proteins which can be investigated and consider more interesting for researchers. Thisstudy is concluded that human sperm have important transmembrane, enzymes, glycosylated proteins,glycoproteins and uncharacterised proteins which might be answered many theoretical aspects of biologicaland biochemical sperm migrations before and after fertilization.

The Role of Cell Wall Degrading Enzymes in Antagonistic Traits of Trichoderma virens Against Rhizoctonia solani.

Background: High antagonistic ability of different Trichoderma species against a diverse range of plant pathogenic fungi has led them to be used as a biological fungicide in agriculture. They can also promote plant growth, fertility, resistance to stress, and absorption of nutrients. They are also opportunistic and symbiotic pathogens, which can lead to the activation of plant defense mechanisms.Objectives: The aim of this present study was to investigate possible enhancement of lytic enzymes production and biocontrol activity of T. virens against Rhizoctonia solani through gamma radiation and to find the relationship between changes in lytic enzyme production and antagonistic activity of T. virens.Material and Methods: Dual culture conditions were used to evaluate the antagonistic effect of T. virens and its gamma mutants against R. solani. Then, their chitinase and cellulase activities were measured. For more detailed investigation of enzymes, densitometry pattern of the proteins was extracted from the T. virens wild-type and its mutants were obtained via SDS-polyacrylamide gel electrophoresis.Results: The mutant T.vi M8, T. virens wild-type and mutant T.vi M20 strains showed the maximum antagonistic effects against the pathogen, respectively. Data showed that the mutant T. vi M8 reduced the growth of R. solani by 58 %. The mutants revealed significantly different (p<0.05) protein contents, chitinase and cellulase production (mg.mL-1) and activity (U.mL-1) compared to the wild-type strain. The highest extracellular protein production in the supernatant of chitinase and cellulase TFM was observed for the T.vi M11 and T.vi M17 strains, respectively. The T.vi M12 and wild-type strains secreted chitinase and cellulase significantly more than other strains did. Densitometry of SDS-PAGE gel bands indicated that both the amount and diversity of chitinase related proteins in the selected mutant (T. vi M8) were far higher than those of the wild-type. The diversity of molecular weight of proteins extracted from the T. virens M8 (20 proteins or bands) was very high compared to the wild-type (10 proteins) and mutant T.vi M15 (2 proteins).Conclusions: Overall, there was a strong link between the diversity of various chitinase proteins and the antagonistic properties of the mutant M8.