Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Computational investigations of physicochemical, pharmacokinetic, toxicological properties and molecular docking of betulinic acid, a constituent of Corypha taliera (Roxb.) with Phospholipase A2 (PLA2).

BACKGROUND: Betulinic acid (BA) is a natural triterpenoid compound and exhibits a wide range of biological and medicinal properties including anti-inflammatory activity. Therefore, this theoretical investigation is performed to evaluate (a) physicochemical properties such as acid dissociation constant (pKa), distribution coefficient (logD), partition coefficient (logP), aqueous solubility (logS), solvation free energy, dipole moment, polarizability, hyperpolarizability and different reactivity descriptors, (b) pharmacokinetic properties like human intestinal absorption (HIA), cellular permeability, skin permeability (PSkin), plasma protein binding (PPB), penetration of the blood brain barrier (BBB), (c) toxicological properties including mutagenicity, carcinogenicity, risk of inhibition of hERG gene and (d) molecular mechanism of anti-inflammatory action which will aid the development of analytical method and the synthesis of BA derivatives. METHODS: The physicochemical properties were calculated using MarvinSketch 15.6.29 and Gaussian 09 software package. The pharmacokinetic and toxicological properties were calculated on online server PreADMET. Further, the molecular docking study was conducted on AutoDock vina in PyRx 0.8. RESULTS: The aqueous solubility increased with increasing pH due to the ionization of BA leading to decrease in distribution coefficient. The solvation energies in water, dimethyl sulfoxide (DMSO), acetonitrile, n-octanol, chloroform and carbon tetrachloride were - 41.74 kJ/mol, - 53.80 kJ/mol, - 66.27 kJ/mol, - 69.64 kJ/mol, - 65.96 kJ/mol and - 60.13 kJ/mol, respectively. From the results of polarizability and softness, it was clear that BA is less stable and hence, kinetically more reactive in water. BA demonstrated good human intestinal absorption (HIA) and moderate cellular permeability. Further, BA also exhibited positive CNS activity due to high permeability through BBB. The toxicological study revealed that BA was a mutagenic compound but noncarcinogenic in mice model. Moreover, molecular docking study of BA with PLA2 revealed that BA interacts with GLY22 & GLY29 through hydrogen bond formation and LEU2, PHE5, HIS6, ALA17, ALA18, HIS47 and TYR51 through different types of hydrophobic interactions. The binding affinity of BA was - 41.00 kJ/mol which is comparable to the binding affinity of potent inhibitor 6-Phenyl-4(R)-(7-Phenyl-heptanoylamino)-hexanoic acid (BR4) (- 33.89 kJ/mol). CONCLUSIONS: Our computed properties may assist the development of analytical method to assay BA or to develop BA derivatives with better pharmacokinetic and toxicological profile.

Molecular identification and characterisation of Mannheimia haemolytica.

Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica-specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease.

Actinobacillus pleuropneumoniae: The molecular determinants of virulence and pathogenesis.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is responsible for high economic losses in swine herds across the globe. Pleuropneumonia is characterized by severe respiratory distress and high mortality. The knowledge about the interaction between bacterium and host within the porcine respiratory tract has improved significantly in recent years. A. pleuropneumoniae expresses multiple virulence factors, which are required for colonization, immune clearance, and tissue damage. Although vaccines are used to protect swine herds against A. pleuropneumoniae infection, they do not offer complete coverage, and often only protect against the serovar, or serovars, used to prepare the vaccine. This review will summarize the role of individual A. pleuropneumoniae virulence factors that are required during key stages of pathogenesis and disease progression, and highlight progress made toward developing effective and broadly protective vaccines against an organism of great importance to global agriculture and food production.

Thalassemias in South Asia: clinical lessons learnt from Bangladesh.

Thalassemias are emerging as a global public health concern. Due to remarkable success in the reduction of childhood mortality by controlling infectious diseases in developing countries, thalassemias are likely to be a major public health concern in the coming decades in South Asia. Despite the fact that Bangladesh lies in the world's thalassemia belt, the information on different aspects (epidemiology, clinical course, mortality, complications and treatment outcomes) of thalassemias is lacking. In this comprehensive review, the aim is to to depict the epidemiological aspects of thalassemias, mutation profile and current treatment and management practices in the country by sharing the experience of dealing with 1178 cases over 2009-2014 time periods in a specialized thalassemia treatment centre. We have also discussed the preventative strategies of thalassemias from the context of Bangladesh which could be effective for other developing countries.