Sodium Dodecyl Sulfate Polyacrylamide Research Articles

Characterization of a Bacterial Keratinolytic Protease for Effective Degradation of Chicken Feather Waste into Feather Protein Hydrolysates.

Chicken feathers contribute to large quantities of keratinaceous wastes that pose serious environmental problems and must be catered to properly. Chicken feathers are also a potential source of vital proteins, peptides, and amino acids, which could be used as low-cost animal feeds. Therefore, there has been increasing interest in keratinase-producing microbes for reprocessing and using keratinous biomaterials. Among the five isolated keratinolytic microorganisms, one microbe, Bacillus XT 01, produced a significant amount of enzyme activity, which was partially characterized. The potential of this protease-producing microbe was investigated for converting feather keratin waste to valuable protein hydrolysate. Maximum keratinase production was observed after 5 days of incubating Bacillus XT 01 at an optimum temperature of 45 °C and pH 8.5. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and zymogram of ammonium sulfate precipitated culture supernatant showed the presence of several proteolytic enzymes with molecular weights between 30 and 60 kDa. The Bacillus strain caused almost complete feather degradation (98%) after 7 days of incubation at 45 °C in a shake culture medium. Antioxidant and reducing activities of the feather protein hydrolysate (FPH) elevated with increased cultivation time. Investigation of the effect of feather protein hydrolysate on plants indicated improved plant growth regarding the agronomic parameters, such as plant height, number of trifoliate leaves, number of pods, pod length, number of seeds per pod, and root length, which increased by 30.84%, 49.32%, 70.90%, 53.27%, 60.03%, and 54.71%, respectively. The prospective of Bacillus XT 01 for degrading feather waste keratin to highly valued hydrolyzed feather protein offers effectiveness in the poultry industry and ultimately decreases environmental pollution hazards.

Relative Protein Profile of Philippine Cinnamomum (Schaeffer) Using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Philippine Cinnamomum are trees with aromatic leaves and bark. It has several economic and medicinal uses, but concerns about protein profile are limited. Thus, this study was conducted to determine the relative protein component of the species by Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). A total of 27 cinnamon species were run in Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-Page) with LSB (Laemli Sample Buffer) and Tris HCI (1:1) as sample buffer. Determining protein profiles is a tool for the systematic classification of the species. Results showed that Philippine Cinnamomum leaf samples have the same component proteins. The SDS-PAGE provided a similar protein banding pattern, although the same bands' intensity varies. Pronounced protein bands were observed corresponding to 50 to 75 kDa and 25 kDa, with additional minor bands indicating a higher protein content. The results in SDS-PAGE showed that the component proteins of Cinnamomum leaves were comparable regardless of geographical source and different stages of growth of the samples. However, the concentration of some component proteins may vary, as indicated by the difference in intensity of protein bands. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) is a tool in the biochemical method of determining the relative protein profile of Cinnamomum species. It can be used to classify plants up to the species level. Based on the study results, Philippine Cinnamomum has a higher protein content that can be used in culinary, medicinal, and pharmaceuticals.

Human buccal epithelial cells as a model system for molecular analysis of DNA, RNA and protein

We report use of human buccal epithelial cells as an easy model system for isolation and molecular analysis of genomic DNA, RNA, and protein to study any gene of interest by Polymerase Chain Reaction (PCR), RNA expression by Reverse Transcription-PCR (RT-PCR), protein-profiling, and expression by western blot as well as DNA-methylation by Msp I/Hpa II-restriction digestion. We used simple methods to isolate genomic DNA, RNA and protein from human buccal cells collected by oral swab and cultured them in-vitro. The microscopic observation of haematoxylin and eosin (EA-50) stained cells, genomic PCR of house-keeping genes (GAPDH and β-actin), RT-PCR of GAPDH and β-actin mRNAs and whole cell protein-profiling by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) were carried out. Expression of β-actin protein in supernatant and pellet fractions of the cells was determined by western blot analysis. MTT-assay was carried out to assess the cell viability and cell growth. Green Fluorescence Protein (GFP)-DNA was expressed in these cells by transient transfection. DNA-methylation in the genome was analyzed by Msp I/ Hpa II restriction digestion and agarose gel electrophoresis. Thus these methods can be used for molecular analysis of DNA, RNA and protein from the human buccal epithelial cells for studying and monitoring health, disease, population genetics/genomics and epidemiology under different conditions.

Plant proteases and anti-bacterial substances in Allium sativum L. varieties

Allium sativum L. protease still remains largely understudied although new varieties of garlic appear quite often, e.g., lanang garlic. This study tested the antibacterial effect of garlic and the effectiveness of various A. sativum proteases as meat tenderizers. 
 The research involved powder extracts of four varieties of A. sativum: kating, lanang, black garlic, and sin-chung. The degradation kinetics was defined based on the Lineweaver-Burk equation. The degradation zones were measured using sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). Scan electron microscopy served to test the changes in meat connective tissue.
 Lanang demonstrated the largest inhibition zones against Escherichia coli (9.75 ± 0.15 mm) and Staphylococcus aureus (1.04 mm). Sin-chung protease degraded beef protein with the highest Vmax of 0.1818 μg/μL/min at 10–22 KDa (small peptide, troponin C, and troponin I), 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin), and 100–140 KDa (protein C). The same garlic variety degraded mutton meat protein at 10–17 KDa (small peptide) and 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin) with Vmax of 0.1135 μg/μL/min.
 All four A. sativum proteases proved to be quite effective meat tenderizers.

Innovative Wound Dressing Coated with Drug-Loaded Adipose Mesenchymal Stem Cells to Promote Wound Healing in Diabetes

Impaired wound healing is associated with hyperglycaemia in patients with diabetes. Hyperglycaemia induces protein glycation and the formation of Advanced Glycation End-Products (AGEs). The accumulation of AGEs in the body results in the structural and functional modification of tissue proteins. This study was conducted to evaluate compounds with antiglycation activities (S-Ally1 Cysteine (SAC), N-Acetylcysteine (NAC) and the mimic compound A). The extent of glycation in the presence and absence of several inhibitors was assessed via several methods including fluorescence, Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)- silver stain, Western blotting, and Enzyme-Linked Immunosorbent Assays (ELISA). Additionally, this research aimed to evaluate and quantify the potential of Human Adipose Mesenchymal Stem Cells (hADMSCs) to uptake and release these drugs as potential therapeutics. To achieve this, hADMSCs were primed with a combination of SAC/NAC and mimic compound A and their concentrations were analysed using High-Performance Liquid Chromatography (HPLC). The SAC/NAC and mimic compound A prohibit the formation of AGEs while the Conditioned Medium (CM) from SAC/NACand compound A-loaded hADMSCs induced cell migration and tube formation in BAECs. hADMSCs provide a unique opportunity for the development of an innovative targeting and drug-delivery system which could effectively deliver therapeutics to specific regions of wounds or other damaged tissues. The data provided demonstrate the potential of hADMSCs as a drug delivery method with the potential to improve wound healing, and it may offer potential therapeutic targeting for the development of diabetic complications.

Plasmid and Total Protein Analyses of Extended Spectrum Beta Lactamase Producing Bacteria from Periplaneta americana (Coackroaches)

Background: The importance of plasmid-encoded Extended Spectrum Beta Lactamase (ESBL) organisms in the dissemination of multi-drug resistance and complicated infections are contributory factors to treatment failure. Despite these, there is a paucity of information regarding plasmid-borne associated infection from primary sources including cockroaches while studies correlating total protein and plasmid-encoded ESBL are also limited especially in cockroaches. The objective of this study was therefore aimed at determining the presence of plasmid in the ESBL organisms from cockroaches as well as their total protein profiles.
 Materials and Methods: Bacterial isolation, characterization and molecular identification were carried out in our previous work following standard recommended techniques. These molecularly identified organisms which were delineated into twenty-two (22) were further subjected to plasmid and total protein analyses using alkaline lysis and Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) respectively.
 Results: Results obtained identifies 18(81.8%) of the ESBL isolates to be harbouring plasmids of different molecular weights (2-21.5kbp). The total protein profile of the plasmid-encoded ESBL and the non-plasmid encoded ESBL reveals differential protein expression patterns except for two non- plasmid encoded ESBL isolates that have similar patterns as that of plasmid-encoded ESBL organisms (isolates 2 and 3).
 Conclusion: There is a need to monitor the primary sources of infection in the epidemiological distribution of resistance. Improving environmental sanitation through proper disinfection of carrier insects may be a means to curtailing such spread.

Protease inhibitory activity profile of Indonesia wild swamp eel (Monopterus albus)

A protease inhibitor is a compound that potentially could inhibit the activity of a protease or some protease enzymes. It has a significant role in fish processing to prevent quality deterioration. The study aimed to investigate the inhibitory activity of wild swamp eel (Monopterus albus) plasma fractionated with ethanol to papain enzyme. The parameters analyzed were protein content, inhibitory activity to papain enzyme, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis to demonstrate the protein profile. The result showed that the amount of protein in the plasma fractionated with ethanol was decreased. The effectiveness in inhibiting papain enzyme was increased when compared to that of the crude plasma, from 65 to 128.56% per mg protein. Analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis demonstrated that the protein complex alpha-2- macroglobulin in wild swamp eel plasma consists of two different subunits of 120 kDa and 110 kDa. This study suggested that plasma fractionated with ethanol showed inhibitory activity to papain enzyme greater than that of the crude plasma.

Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

Characterization of somatic and metabolic antigens of Cysticercus cellulosae and determination of immunodominant proteins

The present study shows the characterization of four somatic antigens (Whole Cyst Antigen (WCA), Cystic Fluid Antigen (CFA), Scolex Antigen (SA) and Membrane-Body Antigen (MBA)) and one metabolic antigen (Excretory-Secretory Antigen (ESA)) prepared from Cysticercus cellulosae originated from a naturally infected pig. Immunodominat proteins were determined using hyper immune rabbit sera. The Sodium Dodecyl SulphatePolyacrylamide Gel Electrophoresis (SDS-PAGE) profile of the antigens revealed different numbers and patterns of protein bands in the range of 11.80 to 176.74 kDa. The electrophoretic pattern of ESA also revealed a number of closely placed indistinct bands in the range of 20-34 kDa.. The proteins in the lower molecular weight range (< 30 kDa) in the case of WCA, CFA, ESA, and the medium molecular weight range (30-60 kDa) in the case of SA and MBA were observed to be immunodominant in the Enzyme-Linked Immunoelectrotransfer Blot (EITB) assay. These immunodominant proteins could be further investigated as potential diagnostic antigens in the serodiagnosis of cysticercosis in pigs, and neurocysticercosis in human patients.

Evaluation of PLGA-Encapsulated Recombinant GroEL of S. typhi immune Responses Against Enterohaemorrhagic and Enteropathogenic Escherichia coli

Heat Shock Proteins (HSPs) elicit humoral and cellular immune responses. Due to their high sequence homology, they can be developed as a new immunogen for cross prophylactic and vaccination effects against infectious agents such as Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC). This study aimed to evaluate the immunogenicity and cross-protective efficacy of rGroEL of Salmonella typhi (S. typhi) encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles against EPEC and EHEC. Recombinant GroEL was expressed in Escherichia coli (E. coli) and purified using Ni-NTA affinity chromatography. The protein was encapsulated in PLGA by the double emulsion method, and the nanoparticles were characterized physicochemically. BALB/c mice were immunized, and the efficacy of the protein to elicit immune responses was assessed. Over-expression in E. coli led to corresponding 64.5 kDa protein bands in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Non-aggregated nanoparticles had a spherical shape with a mean diameter of 194.3±3 nm and encapsulation efficiency of 89.5±2.5%. Antibody isotyping revealed that GroEL immunization induced both IgG1 and IgG2a antibodies. Moreover, immunization of the mice with recombinant GroEL protein conferred 80 and 60% protection against lethal infections by EPEC and EHEC, respectively. Furthermore, organ burden studies revealed a significant reduction in infection in the immunized mice compared to the non-immunized ones. Passive immunization with anti-GroEL sera also protected 50% of the mice against the lethal doses of EHEC and EPEC strains. The findings indicated that immunization of the mice with recombinant GroEL of S. typhi elicited cross-protection against other bacterial infections. This represented the immense potential of GroEL to be developed as a single vaccine against multiple pathogens.

Label free detection of SARS CoV-2 Receptor Binding Domain (RBD) protein by fabrication of gold nanorods deposited on electrochemical immunosensor (GDEI)

Coronavirus Disease 2019 (COVID-19) pandemic has shown the need for early diagnosis to manage infectious disease outbreaks. Here, we report a label free electrochemical Fluorine-Doped Tin Oxide (FTO) Immunosensor coupled with gold nanorods (GNRs) as an electron carrier for ultrasensitive detection of the Receptor Binding Domain (RBD) of SARS CoV-2 Spike protein. The RBD gene was cloned, and expressed in-house with confirmed molecular weight of ∼31 kDa via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). RBD antibodies (Ab) were generated to be used as a bioreceptor for sensor fabrication, and characterized using SDS-PAGE, Western Blot, and Enzyme-Linked Immunosorbent Assay (ELISA). GNRs were fabricated on the electrode surface, followed by immobilization of RBD Ab. The conjugation steps were confirmed by UV–Vis Spectroscopy, Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Cyclic Voltammetry (CV), and Differential Pulse Voltammetry (DPV). The fabricated electrode was further optimized for maximum efficiency and output. The detection limit of the developed electrode was determined as 0.73 fM for RBD antigen (Ag). Furthermore, the patient nasopharyngeal samples were collected in Viral Transport Media (VTM), and tested on the sensor surface that resulted in detection of SARS CoV-2 within 30 s, which was further validated via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, the immunosensor showed good repeatability, storage stability, and minimal cross reactivity against Middle East Respiratory Syndrome (MERS) spike protein. Along with ease of fabrication, the electrodes show future miniaturization potential for extensive and rapid screening of populations for COVID-19.

Comparative analysis of different methods used for molecular characterization of Burkholderia cepacia complex isolated from noncystic fibrosis conditions

PurposeBurkholderia is a Gram-negative opportunistic bacterium capable of causing severe nosocomial infections. The aim of this study was to characterize Burkholderia cepacia complex and to compare different molecular methods used in its characterization. MethodsIn this study, 45 isolates of Burkholderia cepacia complex (Bcc) isolated from clinical cases were subjected to RAPD (Random amplified polymorphic DNA), recA-RFLP (Restriction fragment length polymorphism), 16SrDNA-RFLP, whole-cell protein analysis, recA DNA sequencing and biofilm assay. ResultsOf the 45 isolates tested, 97.7% were sensitive to ceftazidime, 82.2% were sensitive to Cotrimoxazole, 73.3% were sensitive to meropenem, 55.5% were sensitive to minocycline and 42.2% were sensitive to levofloxacin. Majority of the isolates harbored all the tested virulence genes except bpeA and cblA. The RAPD generated 11 groups (R1-R11), recA-RFLP 10 groups (A1-A10), 16SrRNA-RFLP 5 groups (S1–S5) and SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis) whole cell protein analysis revealed 12 groups (C1–C12). recA sequencing revealed that most of the isolates belonging to the genomovar III Burkholderia cenocepacia. Though all the methods are found to be efficient in differentiating Burkholderia spp., recA-RFLP was highly discriminatory at 96% similarity value. The study also identified a new strain Burkholderia pseudomultivorans for the first time in the country. Further, recA sequencing could identify the strains to species level. Majority of the multidrug-resistant strains also showed moderate to strong biofilm-forming ability, which further contributes to the virulence characteristics of the pathogens. ConclusionsThe study highlights the importance of combination of molecular methods to characterize Burkholderia cepacia complex. Molecular typing of these human pathogens yields important information for the clinicians in order to initiate the most appropriate therapy in the case of severe infections and to implement preventive measures for the effective control of transmission of Burkholderia spp.

ASSESSMENT OF NUTRITIONAL RELEVANCE OF DIPLAZIUM ESCULENTUM BY QUALITATIVE PHYTOCHEMICAL AND OVERALL PROTEIN PROFILING

Diplazium esculentum is one of the very well-liked edible ferns, a common pteridophyte. Very few reports are published about the plant from India. Pteridophytes are acquainted for their biological and medicinal properties for long time but very fewevidences are seen about wetland pteridophytes. The present work focuses on assessment of nutritional relevance of D. esculentum collected from Alappuzha District of Kerala in India, by qualitative phytochemical and overall protein profiling. Chloroform and methanolic extracts of leaves of D. esculentum are used for both assays. The results showed the presence of vital phytoconstituents on qualitative analyses that included Alkaloids, Saponins, Flavonoids, Steroids, Phenols, Cardiac glycosides and Carbohydrates. On protein profiling by Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS PAGE), 5 distinct bands corresponding to molecular weights ~250 kDa, ~130 kDa, ~100 KDa, ~55 KDa and ~15 KDa which showed nutritional importance of the fern, Diplazium esculentum. The future perspectives of study may include the quantitative estimation of phytoconstituents and proteins that paves the way for the incredible health benefits of the fern for humans.

Mass spectrometry determination of potential species-specific peptide markers in commercial seasoning cubes

In recent years, issues related to food product authenticity have been alarming, particularly in meat-based products. Proteomic techniques in food authentication and their applications in determining quality and safety issues of ingredients have been progressively developed by researchers. The complexity of matrices in commercial seasoning cubes is one of the main concerns in developing a robust authentication method. Hence, the present study focuses on the development of a protein extraction method for commercial seasoning cubes and to determine potential species-specific peptide markers in commercial seasoning cubes. The efficacy of solvent extraction was determined using Bradford assays, while the electrophoretic profiles of commercial seasoning cubes were visualized via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The determination of potential species-specific peptide markers in commercial seasoning cubes was carried out via Liquid Chromatography (LC)-Orbitrap Fusion-Mass spectrometry (MS/MS) analysis (Thermo Fisher Scientific). In this study, the urea-thiourea solvent was ideal in extracting protein from commercial seasoning cubes as it produces a homogenous solution upon mixing, suggesting the diffusibility of solutes that is essential for continuing analysis. Moreover, a bovine-specific peptide marker ‘VGGHAAEYGAEALER’ and two porcine-specific peptide markers; ‘VGGQAGAHGAEALER’ and ‘EAVLGLWGK’ were discovered. The methodology developed in this study was successful in identifying species-specific peptide markers in seasoning cubes and can be used as a reference for validating the authenticity of food products, especially commercial seasonings.

Isolation and Identification of Protein Spike (Protein-S) in Field Isolates of Infectious Bronchitis Virus

Infectious Bronchitis (IB) is disease causes great economic impact across nations. Spike protein (protein-S) is one of structural protein of Infectious Bronchitis Virus (IBV) located on enveloped of the virion. Molecular characterization of IBV could be conducted use Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to reveal the protein weight then confirmed through Immuno-Blotting assay. These methods were conducted to know the molecule weight of field isolate compared to seed vaccine. The results of this study indicate the weight of protein-S of field isolate I-147/11 were 168.21 kDa which was same to protein-S of seed vaccine Massachusset.

Technological and Safety Attributes of Lactic Acid Bacteria and Yeasts Isolated from Spontaneously Fermented Greek Wheat Sourdoughs.

The aim of the present study was to assess the technological and safety potential of 207 lactic acid bacteria (LAB) and 195 yeast strains isolated from spontaneously fermented Greek wheat sourdoughs. More accurately, the amylolytic, proteolytic, lipolytic, phytase and amino acid decarboxylase activities, along with the production of exopolysaccharides and antimicrobial compounds by the LAB and yeast isolates, were assessed. A well diffusion assay revealed seven proteolytic LAB and eight yeast strains; hydrolysis of tributyrin was evident only in 11 LAB strains. A further Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicated partial hydrolysis of gluten. Lipolysis kinetics over 21 days was applied, exhibiting that lipolytic activity ranged from 6.25 to 65.50 AU/mL. Thirteen LAB inhibited Penicillium olsonii and Aspergillus niger growth and 12 yeast strains inhibited Pe. chrysogenum growth. Twenty-one Lactiplantibacillus plantarum strains exhibited inhibitory activity against Listeria monocytogenes, as well as several sourdough-associated isolates. The structural gene encoding plantaricin 423 was detected in 19 Lcb. plantarum strains, while the structural genes encoding plantaricins NC8, PlnE/F, PlnJ/K, and S were detected in two Lcb. plantarum strains. None of the microbial strains tested exhibited exopolysaccharide (EPS) production, amino acid decarboxylase, amylolytic or phytase activity. The technological and safety potential of the Lcb. plantarum and Wickerhamomyces anomalus strains was highlighted, since some of them exhibited proteolytic, lipolytic, antibacterial and antimould activities.

The Assembly of Bacteriophage Functional Enzymatic Models in Association with E. coli Proteins’ Profiles

Introduction: The invasion of bacteriophage on the associated host bacterium depends on their receptors’ orientation that adsorb them to cell surface. During phage replication a valuable number of proteins acts as lytic enzymes for host puncher at the beginning of the infection and other for burst after lytic cycle compilation. Accordingly, the proteomic relationship among phage and bacterium proteins could easily be studied by their protein profiles analysis. Objective: To detect bacteriophages functional enzymes during lytic cycle. Methods: The isolation and identification of Escherichia coli and their parasitic T7 phage group was done using bacterial culture and common plaque assay techniques. The investigations and protein-protein interactions’ assays were inveterate by proteins profile of phage and bacterium using Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) to find out their molecular weights, where the scaled location of each mobile band was compared to the standards of identified proteins weights in the molecular ladder. Thereafter, Protein model’s assembly and bands migration was done by computer analytical software. Results: Mobilization of the phage’ proteins inside the Two Dimensions (2D) gel ranged between 60 and 12 kDa where a model of 4 main bands with molecular weights of (46, 35, 24 and 14 kDa) is corresponded to the host ones, where pure 9 bands with molecular weight ranged between 96-24 kDa. The computational model analysis showed common shared molecular masses of 47, 34 and 16 kDa on plot area of the phage and the bacterium. Model interpretation confirmed that proteins ranged from 47.7 to 34.3 kDa resembles 43.3% of whole phage’s proteins that assembled the capsid head and the coil, while the molecular weight mass of 22.5 formed the tail’s proteins. The lytic enzymes’ molecular weight was ranged between 18-14 kDa according to the function of the enzyme. The study revealed that the 34 kDa band has the common shared peak between T7 phage group and associated Escherichia coli host. Conclusion: Functional models of analysed proteins during phage assembly, ensures lytic enzymes are built in the capsid head and the lysozyme in the tail, they facilitate the enzymatic decay for bacterial host. This enzymatic function is related to the lytic cycle of the bacteriophages and their phenomenon in employing the bacterial DNA in proteins manufacturing during their replication inside host.

Good intentions, bad outcomes: Impact of mixed-fruit loading on banana fruit protein expression, physiological responses and quality

Mixed-fruit storage and/or transport could have detrimental impact on quality, due to varying ethylene sensitivities. This study evaluated the effects of mixed-fruit loading on physicochemical and physiological changes as well as protein expression of bananas during storage. Bananas, apples (Smartfresh™-treated), and pears were stored together (simulating mixed-fruit loading) and individually (control) at 15 °C for 9 days. Post-storage ethylene production rates were significantly higher (p ≤ 0.05) for fruit under mixed-fruit loading. Bananas from mixed-fruit had higher respiration rate (44.4 ± 8.83 mL/kg h) compared to control on day 9. Chroma values increased, while hue angle declined significantly for bananas across all treatments during storage (p ≤ 0.05). One-dimensional sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel electrophoresis for protein extracted from banana peels confirmed overexpression of proteins within 25−37 kDa on day 9. Results obtained emphasize the need for correct management of fruit based on ethylene sensitivity in order to extend shelf life.

Genetic diversity in the natural population of threatened fruit- bearing plant species Monotheca buxifolia (Falc.) A.D.

Monotheca buxifolia is an ethnomedicinally and economically important threatened fruit bearing plant species in Malakand Division Pakistan. The genetic diversity among the 92 various genotypes of Monotheca buxifolia was carried out using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) method. A considerable amount of inter districts genetic diversity (66.70%) was observed among the genotypes of M. buxifolia. Protein profiling was conducted on 12% gel electrophoresis. A total of 6 protein bands were observed in M. buxifolia genotypes. SDS-PAGE practice is a convenient scheme for the examination of both genetic diversity and relationship. Particularly, L-4 and L-5 were monomorphic in the inter districts Monotheca buxifolia genotypes and was recognized as species specific. The remaining other loci were polymorphic. In this investigation, the high inter and intra- districts specific diversity was observed demonstrating SDS-PAGE is an authoritative procedure for categorizing the genetically diverse germplasms in M. buxifolia. The findings from this study could be useful in the identification and selection of suitable M. buxifolia genotypes for future conservation programmes. Today, there is still a need to examine the genetic diversity and protect genetic resources, in particular wild species, for possible benefits in plant conservation programmes. To the best of our knowledge, this is the first ever report that addresses genetic variability in M. buxifolia.

The Evaluation of Inhibition Effects of Antibiotics on the Sheep Stomach Carbonic Anhydrase I and II Isozymes

Carbonic anhydrases are vital class of enzymes that participate in so many essential physiological events in the organism and associated with many diseases. Inhibitors of carbonic anhydrase enzymes are used in pharmacological applications in many areas such as antiobesity, antiglaucoma, anticancer agents and anticonvulsants. So, this study focuses on the characterization of CA-I and CA-II isoenzymes purified from sheep stomach and investigating the inhibition effects of antibiotics on these enzymes. The findings show that the antibiotics studied strongly inhibit CA-I and CA-II isozymes. In our presented study, using Sepharose 4B-L-Tyrosine sulfanilamide affinity gel chromatography, carbonic anhydrase I and carbonic anhydrase II isoenzymes were purified from sheep stomach with a yield of 51.9%, 78%, respectively and specific activity of CA I and CA II are 4864.8 EU/mg and 5652.02 EU/mg, respectively. The overall purifications from sheep stomach CA I and CA II were approximately 529.4 and 615.2 fold respectively. To check the purify of the enzymes, it was conducted an electrophoretic method so-called Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and single bands were detected for both enzymes. Additionally, the effects of antibiotics on sheep stomach CA I and CA II isozymes activities, using both hydratase and esterase activity methods, were researched. IC50 values of enrofloxacin, tylosin and ampicillin antibiotics that inhibit CA I enzyme with hydratase activity were found as 1.41, 0.033 and 1.56 mM, respectively. IC50 values for CA II enzyme with hydratase activity of the same antibiotics were found as 2.47, 0.039 and 1.63 mM, respectively. Kanamycin and amikacin antibiotics inhibited both CA I and CA II enzymes with esterase activity. IC50 values for CA I and CA II enzymes with esterase activity of kanamycin antibiotic were determined as 0.0488 and 0.118 mM, respectively. IC50 values of amikacin for CA I and CA II enzymes with esterase activity were determined as 0.0163 and 0.036 mM, respectively.