Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.
We utilized molecular dynamics (MD) simulations and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) free energy calculations to investigate the specificity of two oligonucleotide probes, namely probe B and probe D, in detecting single-stranded DNA (ssDNA) within three bacteria families: Enterobacteriaceae, Pasteurellaceae, and Vibrionaceae. Due to the limited understanding of molecular mechanisms in the previous research, we have extended the discussion to focus specifically on investigating the binding process of bacteria-probe DNA duplexes, with an emphasis on analyzing the binding free energy. The role of electrostatic contributions in the specificity between the oligonucleotide probes and the bacterial ssDNAs was investigated and found to be crucial. Our calculations yielded results that were highly consistent with the experimental data. Through our study, we have successfully exhibited the benefits of utilizing in-silico approaches as a powerful virtual-screening tool, particularly in research areas that demand a thorough comprehension of molecular interactions.
This study aimed to evaluate the two-stage and one-stage anaerobic co-digestion of vinasse and spent brewer yeast cells (SBY) for biohydrogen and methane production. Optimization of the vinasse-to-SBY ratio and fly ash concentration of the two-stage and one-stage production processes was investigated. In the two-stage process, the vinasse-to-SBY ratio and fly ash concentration were optimized, and the leftover effluent was used for methane production. The optimum conditions for biohydrogen production were a vinasse-to-SBY ratio of 7:3% v/w and fly ash concentration of 0.4% w/v, in which the maximum hydrogen yield was 43.7 ml-H2/g-VSadded. In contrast, a vinasse-to-SBY ratio of 10:0% v/w and fly ash concentration of 0.2% w/v were considered optimal for methane production, and resulted in a maximum methane yield of 214.6 ml-CH4/g-VSadded. For the one-stage process, a vinasse-to-SBY ratio of 10:0% v/w and fly ash concentration of 0.1% w/v were considered optimal, and resulted in a maximum methane yield of 243.6 ml-CH4/g-VSadded. In the two-stage process, the energy yield from hydrogen (0.05-0.47 kJ/g-VSadded) was 0.62%-11.78%, and the major fraction was approximately 88.22%-99.38% gain from methane (3.19-7.73 kJ/g-VSadded). For the one-stage process, the total energy yield distribution ranged from 4.20 to 8.77 kJ/g-VSadded.
Seaweeds are photosynthetic marine macroalgae known for their rapid biomass growth and their significant contributions to global food and feed production. Seaweeds play a crucial role in mitigating various environmental issues, including greenhouse gases, ocean acidification, hypoxia, and eutrophication. Tropical seaweeds are typically found in tropical and subtropical coastal zones with warmer water temperatures and abundant sunlight. These tropical seaweeds are rich sources of proteins, vitamins, minerals, fibers, polysaccharides, and bioactive compounds, contributing to their health-promoting properties and their diverse applications across a range of industries. The productivity, cultivability, nutritional quality, and edibility of tropical seaweeds have been well-documented. This review article begins with an introduction to the growth conditions of selected tropical seaweeds. Subsequently, the multifunctional properties of tropical seaweeds including antioxidant and anti-inflammatory, anti-coagulant, anti-carcinogenic and anti-proliferative, anti-viral, therapeutic and preventive properties were comprehensively evaluated. The potential application of tropical seaweeds as functional foods and feeds, as well as their contributions to sustainable cosmetics, bioenergy, and biofertilizer production were also highlighted. This review serves as a valuable resource for researchers involved in seaweed farming as it provides current knowledge and insights into the cultivation and utilization of seaweeds.
Fusarium oxysporum f. sp. cubense is one of the most severe and threatening pathogens of bananas, causing "Panama wilt" worldwide. Confrontation assay of Foc antagonistic bacterial endophyte, Bacillus velezensis YEBBR6, with the Foc and GC-MS profiling of excised agar from the zone of inhibition, led to the unveiling of secondary metabolites produced by the endophyte. To refine the probable antifungal compounds among the numerous biomolecules formed during their di-trophic interaction with the pathogen, fungal protein targets were modeled, and docking studies (AutoDock Vina module of the PyRx 0.8 server) were done with all the compounds. Triamcinolone acetonide exhibited the most excellent affinity for the protein targets among the compounds studied. It had a maximum binding affinity of 11.2 kcal/mol for XRN2 (5' → 3'). Further, the protein-ligand complex formation kinetics was done through Molecular Dynamic Simulation studies. Graphs for the RMSD, RMSF, Rg, potential energy, and SASA were generated, and the values during the simulation period suggested the stability of the biomolecule as a complex with the protein. This indicated Triamcinolone acetonide's potential ability to act as a functional disrupter of the target protein and likely an antifungal molecule. Further, the biomolecule was tested for its activity against Foc by screening in the wet lab through the poisoned plate technique, and it was found to be fully inhibitory to the growth of the pathogen at 1000 ppm.
Membrane distillation (MD) has lower operating temperature and potential to recycle waste heat for desalination which catches much attention of the researchers in the recent years. However, the biofouling is still a challenging hurdle to be overcome for such applications. The microbial growth rate, secretion and biofilm formation are sensitive to heat. Membrane distillation is a thermally driven separation, so the increase of temperature in the seawater feed could influence the extent of biofouling on the unit parts. In this review, we present the effect of temperature on algal growth, the range of temperature the microbes, marine algae and planktons able to survive and the changes to those planktons once exceed the critical temperature. Thermal effect on the biofilm, its composition and properties are discussed as well, with association of the biofilm secreting microbes, but the study related to membrane distillation unit seems to be lacking and MD biofouling factors are not fully understood. Characterization of the algae, biofilm and EPS that govern biofouling are discussed. This information not only will help in designing future studies to fill up the knowledge gaps in biofouling of membrane distillation, but also to some extent, assist in pointing out possible fouling factors and predicting the degree of biofouling in the membrane distillation unit.
The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 °C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.
Suppression subtractive hybridization (SSH) is an effective method to identify different genes with different expression levels involved in a variety of biological processes. This method has often been used to study molecular mechanisms of plants in complex relationships with different pathogens and a variety of biotic stresses. Compared to other techniques used in gene expression profiling, SSH needs relatively smaller amounts of the initial materials, with lower costs, and fewer false positives present within the results. Extraction of total RNA from plant species rich in phenolic compounds, carbohydrates, and polysaccharides that easily bind to nucleic acids through cellular mechanisms is difficult and needs to be considered. Remarkable advancement has been achieved in the next-generation sequencing (NGS) field. As a result of progress within fields related to molecular chemistry and biology as well as specialized engineering, parallelization in the sequencing reaction has exceptionally enhanced the overall read number of generated sequences per run. Currently available sequencing platforms support an earlier unparalleled view directly into complex mixes associated with RNA in addition to DNA samples. NGS technology has demonstrated the ability to sequence DNA with remarkable swiftness, therefore allowing previously unthinkable scientific accomplishments along with novel biological purposes. However, the massive amounts of data generated by NGS impose a substantial challenge with regard to data safe-keeping and analysis. This review examines some simple but vital points involved in preparing the initial material for SSH and introduces this method as well as its associated applications to detect different novel genes from different plant species. This review evaluates general concepts, basic applications, plus the probable results of NGS technology in genomics, with unique mention of feasible potential tools as well as bioinformatics.
The synergistic effects of antimicrobial nanostructures with antibiotics present a promising solution for overcoming resistance in methicillin-resistant Staphylococcus aureus (MRSA). Previous studies have introduced iron as a novel coating for silver nanoparticles (AgNPs) to enhance both economic efficiency and potency against S. aureus. However, there are currently no available data on the potential of these novel nanostructures to reverse MRSA resistance. To address this gap, a population study was conducted within the MRSA community, collecting a total of 48 S. aureus isolates from skin lesions. Among these, 21 isolates (43.75%) exhibited cefoxitin resistance as determined by agar disk diffusion assay. Subsequently, a PCR test confirmed the presence of the mecA gene in 20 isolates, verifying them as MRSA. These results highlight the cefoxitin disk diffusion susceptibility test as an accurate screening method for predicting mecA-mediated resistance in MRSA. Synergy tests were performed on cefoxitin, serving as a marker antibiotic, and iron-coated AgNPs (Fe@AgNPs) in a combination study using the checkerboard assay. The average minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) of cefoxitin were calculated as 11.55 mg/mL and 3.61 mg/mL, respectively. The findings indicated a synergistic effect (FIC index
Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.
Biomolecules produced by living organisms can perform vast array of functions and play an important role in the cell. Important biomolecules such as lysozyme, bovine serum albumin (BSA), and bromelain are often studied by researchers due to their beneficial properties. The application of reverse micelles is an effective tool for protein separation from their sources due to the special system structure. Mechanisms of transferring biomolecules and factors that influence the extraction of biomolecules are reviewed in this paper. The enhancement of biomolecule extraction could be achieved depending on the properties of reverse micelles. This paper provides an overall review on lysozyme, BSA, and bromelain extraction by reverse micelle for various applications.
Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.
The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69-0.73 mg/g of fresh weight, with A(260)/A(280) ratio of 1.79-1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.
Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.
The present study focused on preparing and characterizing magnetite-polyvinyl alcohol (PVA) hybrid nanoparticles using Acanthophora spicifera marine algae extract as a reducing agent. Various analytical techniques, including UV-Visible spectrometry, Fourier-transform infrared (FTIR) analysis, energy-dispersive X-ray (EDX), scanning electron microscopy (SEM), and X-ray diffraction (XRD) analysis, were used to characterize the nanoparticles. The results showed the successful synthesis of nanoparticles with a characteristic color change and absorption peak at 400 nm in UV-Visible spectrometry. FTIR analysis indicated an interaction between the carboxyl group and magnetite-polyvinyl alcohol hybrid ions. SEM analysis revealed spherical nanoparticles with sizes ranging from 20 to 100 nm. EDX analysis confirmed the presence of strong magnetite peaks in Acanthophora spicifera, validating successful preparation. XRD analysis indicated the crystalline nature of the nanoparticles. Furthermore, the antimicrobial potential of As-PVA-MNPs was evaluated, demonstrating a significant zone of inhibition against tested bacterial and fungal samples at a concentration of 100 µg. These findings suggest the promising antimicrobial activity of the synthesized nanoparticles for potential applications in combating pathogenic microorganisms.
Pasteurella multocida is the main cause of haemorrhagic septicaemia (HS) outbreak in livestock, such as cattle and buffaloes. Conventional vaccines such as alum-precipitated or oil-adjuvant broth bacterins were injected subcutaneously to provide protection against HS. However, the immunity developed is only for short term and needed to be administered frequently. In our previous study, a short gene fragment from Pasteurella multocida serotype B was obtained via shotgun cloning technique and later was cloned into bacterial expression system. pQE32-ABA392 was found to possess immunogenic activity towards HS when tested in vivo in rat model. In this study, the targeted gene fragment of ABA392 was sub-cloned into a DNA expression vector pVAX1 and named as pVAX1-ABA392. The new recombinant vaccine was stable and expressed on mammalian cell lines. Serum sample collected from a group of vaccinated rats for ELISA test shows that the antibody in immunized rats was present at high titer and can be tested as a vaccine candidate with challenge in further studies. This successful recombinant vaccine is immunogenic and potentially could be used as vaccine in future against HS.
The world of pharmaceutical research has been increasingly turning its gaze toward the treasure trove of natural products in search of novel drugs and therapeutic agents. Amidst the vast array of medicinal plants that dot our planet, the Asclepiadaceae family unexplored species have piqued the interest of researchers. Both medicinal plants are indigenous to specific regions and have been integral to traditional medicine systems for centuries. This systematic review aims to provide a comprehensive summary of the current knowledge regarding the phytochemical profile of these plants and their potential implications in the pharmaceutical industry. These plants are rich in phytochemical constituents such as alkaloids, flavonoids, terpenoids, phenolic compounds, glycosides, and saponins. These constituents have been found to exhibit a range of pharmacological activities. They have antimicrobial properties, providing a defense against various microorganisms. They also show anti-inflammatory properties, helping to reduce inflammation in the body. In addition, these plants have antioxidant properties, which help protect cells from damage by harmful free radicals. They have shown anticancer activity, offering potential for cancer treatment. Their neuroprotective properties could be beneficial in treating neurological disorders. The analgesic properties of these plants could be harnessed for pain relief. Furthermore, they have antidiabetic properties, offering potential for diabetes management. The hope is that this review will stimulate further research into these fascinating plants and contribute to discovering new drugs from natural herbs.
EgENOD93 was first identified in a cDNA microarray study of oil palm tissue culture where it was highly expressed in leaf explants with embryogenic potential. Functional characterization via an RNA interference study of its orthologue in Medicago truncatula demonstrated a significant role of this gene in somatic embryo formation. In this study, EgENOD93 was overexpressed in the important model plant Arabidopsis thaliana to investigate the embryogenic potential of EgENOD93 transgenic Arabidopsis explants compared to explants from control plants (pMDC140 and WT). Experiments using leaf explants revealed higher numbers of regenerated shoots at day 27 in all the homozygous transgenic Arabidopsis cultures (Tg01, Tg02 and Tg03) compared to controls. The expression level of EgENOD93 in Arabidopsis cultures was quantified using reverse transcription quantitative real-time PCR (RT-qPCR). The results supported the overexpression of this gene in transgenic Arabidopsis cultures, with 6 and 10 times higher expression of EgENOD93 in callus at Day 9 and Day 20, respectively. Overall, the results support the role of EgENOD93 in the enhancement of shoot regeneration in transgenic Arabidopsis. This together with the previous results observed in oil palm and Medicago truncatula suggests that ENOD93 plays a key role in the induction of somatic embryogenesis. A similarity to early nodulation-like ontogeny is possible.
Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.