The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.     

Biosorption of Cr(VI) by Ceratocystis paradoxa MSR2 Using Isotherm Modelling,Kinetic Study and Optimization of Batch Parameters Using Response Surface Methodology

This study is focused on the possible use of Ceratocystis paradoxa MSR2 native biomass for Cr(VI) biosorption. The influence of experimental parameters such as initial pH, temperature, biomass dosage, initial Cr(VI) concentration and contact time were optimized using batch systems as well as response surface methodology (RSM). Maximum Cr(VI) removal of 68.72% was achieved, at an optimal condition of biomass dosage 2g L⁻¹, initial Cr(VI) concentration of 62.5 mg L⁻¹ and contact time of 60 min. The closeness of the experimental and the predicted values exhibit the success of RSM. The biosorption mechanism of MSR2 biosorbent was well described by Langmuir isotherm and a pseudo second order kinetic model, with a high regression coefficient. The thermodynamic study also revealed the spontaneity and exothermic nature of the process. The surface characterization using FT-IR analysis revealed the involvement of amine, carbonyl and carboxyl groups in the biosorption process. Additionally, desorption efficiency of 92% was found with 0.1 M HNO₃. The Cr(VI) removal efficiency, increased with increase in metal ion concentration, biomass concentration, temperature but with a decrease in pH. The size of the MSR2 biosorbent material was found to be 80 μm using particle size analyzer. Atomic force microscopy (AFM) visualizes the distribution of Cr(VI) on the biosorbent binding sites with alterations in the MSR2 surface structure. The SEM-EDAX analysis was also used to evaluate the binding characteristics of MSR2 strain with Cr(VI) metals. The mechanism of Cr(VI) removal of MSR2 biomass has also been proposed.     

Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin

Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a null mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.     

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage,Senescence and Apoptosis in Colorectal Cancer Cells

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC₅₀ of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.     

Subgroups of Paediatric Acute Lymphoblastic Leukaemia Might Differ Significantly in Genetic Predisposition to Asparaginase Hypersensitivity

L-asparaginase (ASP) is a key element in the treatment of paediatric acute lymphoblastic leukaemia (ALL). However, hypersensitivity reactions (HSRs) to ASP are major challenges in paediatric patients. Our aim was to investigate genetic variants that may influence the risk to Escherichia coli-derived ASP hypersensitivity. Sample and clinical data collection was carried out from 576 paediatric ALL patients who were treated according to protocols from the Berlin—Frankfurt—Münster Study Group. A total of 20 single nucleotide polymorphisms (SNPs) in GRIA1 and GALNT10 genes were genotyped. Patients with GRIA1 rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-cell ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04), while no such association was found in pre-B-cell ALL. In the medium risk group two SNPs of GRIA1 (rs2055083 and rs707176) were associated significantly with the occurrence of ASP hypersensitivity (OR = 0.21 (0.09–0.53); p = 8.48E-04 and OR = 3.02 (1.36–6.73); p = 6.76E-03, respectively). Evaluating the genders separately, however, the association of rs707176 with ASP HSRs was confined only to females. Our results suggest that genetic variants of GRIA1 might influence the risk to ASP hypersensitivity, but subgroups of patients can differ significantly in this respect.     

The Angiopoietin-1 Variant COMP-Ang1 Enhances BMP2-Induced Bone Regeneration with Recruiting Pericytes in Critical Sized Calvarial Defects

Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.