An Analysis of the Radiosensitiser Applications in the Biomedical Field.

BACKGROUND: Various types of radiosensitisers have been introduced from the past until the present day for applications in the biomedical field. However, there is a lack of understanding and comparison between the various parameters introduced in addition to a lack of consensus among researchers on the optimal radiosensitiser for applications in the biomedical field. OBJECTIVE: This review aimed to investigate the usage of radiosensitisers in the biomedical field, determine their important parameters, and suggest radiosensitisers with potential among the analysed radiosensitisers. RESULTS AND CONCLUSION: This review has discussed several parameters for radiosensitisers, including median lethal dose, cell survival, tumour size, cell viability, Dose Enhancement Factor (DEF), Reactive Oxygen Species (ROS) concentration, radiosensitiser production complexity, radiosensitiser administration technique, and radiosensitiser toxicity. General trends regarding the development of radiosensitisers, including the types, effectiveness, and their production complexity, have also been discussed within this review article.

Protein expression, purification, crystallization and crystallographic studies of BPSL0741 from Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of the lethal disease melioidosis. This bacterium infects animals and humans and is increasingly resistant to multiple antibiotics. Recently, genes associated with survival of the bacterium in the infected host have been identified. One of these genes, bpsl0741, is annotated as a hypothetical protein of 185 amino acids. Here, recombinant BPSL0741 (rBPSL0741) protein was expressed, purified, verified by mass spectrometry, crystallized and analyzed by X-ray diffraction. rBPSL0741 was crystallized by vapor diffusion using a reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30% PEG 4000. The crystals diffracted to 2.1 Šresolution using an in-house X-ray diffractometer and belonged to an orthorhombic space group, with unit-cell parameters a = 62.92, b = 64.57, c = 89.16 Å. The Matthews coefficient (VM) was calculated to be 2.18 Å3 Da-1, suggesting the presence of two molecules per asymmetric unit and an estimated solvent content of 43.5%. The crystal was deemed to be suitable for further structural studies, which are currently ongoing.

Functional and structural characterization of a thermostable flavin reductase from Geobacillus mahadii Geo-05.

Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2-12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.

Bacterial biofilm growth and perturbation by serine protease from Bacillus sp.

In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.

Biochemical and in silico structural characterization of a cold-active arginase from the psychrophilic yeast, Glaciozyma antarctica PI12.

Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.

Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family.

Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified ßαßßαßα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.

X-ray crystallography of mutant GDSL esterase S12A of Photobacterium marinum J15.

GDSL esterase is designated as a member of Family II of lipolytic enzymes known to catalyse the synthesis and hydrolysis of ester bonds. The enzyme possesses a highly conserved motif Ser-Gly-Asn-His in the four conserved blocks I, II, III and V respectively. The enzyme characteristics, such as region-, chemo-, and enantioselectivity, help in resolving the racemic mixture of single-isomer chiral drugs. Recently, crystal structure of GDSL esterase from Photobacterium J15 has been reported (PDB ID: 5XTU) but not in complex with substrate. Therefore, GDSL in complex with substrate could provide insights into the binding mode of substrate towards inactive form of GDSL esterase (S12A) and identify the hot spot residues for the designing of a better binding pocket. Insight into molecular mechanisms is limited due to the lack of crystal structure of GDSL esterase-substrate complex. In this paper, the crystallization of mutant GDSL esterase (S12A) (PDB ID: 8HWO) and its complex with butyric acid (PDB ID: 8HWP) are reported. The optimized structure would be vital in determining hot spot residue for GDSL esterase. This preliminary study provides an understanding of the interactions between enzymes and hydrolysed p-nitro-phenyl butyrate. The information could guide in the rational design of GDSL esterase in overcoming the medical limitations associated with racemic mixture.

Spatial, Temporal, and Demographic Patterns in the Prevalence of Hemorrhagic Septicemia in 41 Countries in 2005-2019: A Systematic Analysis with Special Focus on the Potential Development of a New-Generation Vaccine.

Hemorrhagic septicemia (HS) caused by Pasteurella multocida B:2 and E:2 is among the fatal bacterial diseases in cattle and buffaloes that are economically valuable in Asian and African countries. The current work aims to study the prevalence of HS among buffaloes, cattle, sheep, and goats in 41 countries in 2005-2019. The data analysis revealed that 74.4% of the total infection rate in the world was distributed among cattle, followed by buffaloes (13.1%). The mortality of HS among cattle and buffaloes increased in 2017-2019 compared to the period between 2014 and 2016. The best measure to control the disease is through vaccination programs. Current commercial vaccines, including live-attenuated vaccines and inactivated vaccines, have some shortcomings and undesirable effects. Virus-like particles (VLPs) have more potential as a vaccine platform due to their unique properties to enhance immune response and the ability to use them as a platform for foreign antigens against infectious diseases. VLPs-based vaccines are among the new-generation subunit vaccine approaches that have been licensed for the human and veterinary fields. However, most studies are still in the late stages of vaccine evaluation.

Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from Plasmodium knowlesi for the Development of Anti-Malarial Drugs.

Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl ß-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% ß strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.

Structural and functional analysis of Hydra Actinoporin-Like Toxin 1 (HALT-1).

Actinoporins are a family of α-pore-forming toxins (α-PFTs) that have been identified in sea anemones. Recently, a freshwater Hydra Actinoporin-Like Toxin (HALT) gene family was found in Hydra magnipapillata. Unlike sea anemone actinoporins that use sphingomyelin as their main recognition target, the HALTs proteins may recognise alternative lipid molecules as their target. To unveil the structural insights into lipid preference of HALTs protein as compared to sea anemone actinoporins, we have determined the first crystal structure of actinoporin-like toxin, HALT-1 at 1.43 Å resolution with an acetylated lysine residue K76. Despite the overall structure of HALT-1 sharing a high structural similarity to sea anemone actinoporins, the atomic resolution structure revealed several unique structural features of HALT-1 that may influence the lipid preference and oligomerisation interface. The HALT-1 contains a RAG motif in place of the highly conserved RGD motif found in sea anemone actinoporins. The RAG motif contributed to a sharper ß9-ß10 turn, which may sway its oligomerisation interface in comparison to sea anemone actinoporins. In the lipid-binding region, the HALT-1 contains a shorter α2 helix and a longer α2-ß9 loop due to deletion and subsequently an insertion of five amino acid residues in comparison to the sea anemone actinoporins. Structure comparison and molecular docking analysis further revealed that the HALT-1 lipid-binding site may favour sphingolipids with sulfate or phosphate head group more than the sphingomyelin. The structure of HALT-1 reported here provides a new insight for a better understanding of the evolution and lipid recognition mechanism of actinoporin.

Recombinant Production and Characterization of an Extracellular Subtilisin-Like Serine Protease from Acinetobacter baumannii of Fermented Food Origin.

Acinetobacter baumannii is a ubiquitous bacteria that is increasingly becoming a formidable nosocomial pathogen. Due to its clinical relevance, studies on the bacteria's secretory molecules especially extracellular proteases are of interest primarily in relation to the enzyme's role in virulence. Besides, favorable properties that extracellular proteases possess may be exploited for commercial use thus there is a need to investigate extracellular proteases from Acinetobacter baumannii to gain insights into their catalytic properties. In this study, an extracellular subtilisin-like serine protease from Acinetobacter baumannii designated as SPSFQ that was isolated from fermented food was recombinantly expressed and characterized. The mature catalytically active form of SPSFQ shared a high percentage sequence identity of 99% to extracellular proteases from clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae as well as a moderately high percentage identity to other bacterial proteases with known keratinolytic and collagenolytic activity. The homology model of mature SPSFQ revealed its structure is composed of 10 ß-strands, 8 α-helices, and connecting loops resembling a typical architecture of subtilisin-like α/ß motif. SPSFQ is catalytically active at an optimum temperature of 40 °C and pH 9. Its activity is stimulated in the presence of Ca2+ and severely inhibited in the presence of PMSF. SPSFQ also displayed the ability to degrade several tissue-associated protein substrates such as keratin, collagen, and fibrin. Accordingly, our study shed light on the catalytic properties of a previously uncharacterized extracellular serine protease from Acinetobacter baumannii that warrants further investigations into its potential role as a virulence factor in pathogenicity and commercial applications.

Crystallization and structure elucidation of GDSL esterase of Photobacterium sp. J15.

GDSL esterase J15 (EstJ15) is a member of Family II of lipolytic enzyme. The enzyme was further classified in subgroup SGNH hydrolase due to the presence of highly conserve motif, Ser-Gly-Asn-His in four conserved blocks I, II, III, and V, respectively. X-ray quality crystal of EstJ15 was obtained from optimized formulation containing 0.10 M ammonium sulphate, 0.15 M sodium cacodylate trihydrate pH 6.5, and 20% PEG 8000. The crystal structure of EstJ15 was solved at 1.38 Šwith one molecule per asymmetric unit. The structure exhibits α/ß hydrolase fold and shared low amino acid sequence identity of 23% with the passenger domain of the autotransporter EstA of Pseudomonas aeruginosa. The active site is located at the centre of the structure, formed a narrow tunnel that hinder long substrates to be catalysed which was proven by the protein-ligand docking analysis. This study facilitates the understanding of high substrate specificity of EstJ15 and provide insights on its catalytic mechanism.

Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli: Is there an optimal signal peptide design?

Recombinant protein fused to an N-terminal signal peptide can be translocated to the periplasm and, eventually, to the extracellular medium of Escherichia coli under specific conditions. In this communication, we described the use and optimization of a heterologous signal peptide (G1 signal peptide) from a Bacillus sp for improved recombinant protein secretion and cell viability in E. coli. Significant advantages in maintaining high cell viability and high specificity of target protein secretion were achieved by using G1 signal peptide compared to the well-known PelB signal peptide. Signal peptide sequence analysis and site-directed mutagenesis of G1 signal peptide demonstrated that an 'MKK' sequence in n-region and the presence of a helix-breaking residue at the centre of h-region are important elements for the design of an optimal signal peptide.

Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli.

A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.