Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Late Quaternary Environmental and Human Impacts on the Mitochondrial DNA Diversity of Four Commensal Rodents in Myanmar

Journal of Mammalian Evolution - We addressed the spatiotemporal characteristics of four commensal rodent species occurring in Myanmar in comparison with other areas of the Indo-Malayan region. We...     

Modification of plant bait technique for direct diagnosis for Rhizoctonia rice pathogens from Myanmar paddy field soils

A modified baiting technique was conducted for selective isolation, fungal DNA diagnosis and fungal cell lipid assay derived from Myanmar isolates of Rhizoctonia spp., causal agents of rice sheath diseases by trapping selective plant stem segments. Bait plant materials of rice, mat rush and cotton were successfully used to isolate R. solani AG1-IA, R. oryzae and R. oryzae-sativae. Moreover, the three plant materials were also effectively used to detect genomic DNA derived from all Rhizoctonia spp. obtained from Myanmar. Rice segment was the most successful materials for detection of fungal cell lipids including palmitic, stearic and linoleic acids. The results of this experiment demonstrate that bait plant materials of rice, mat rush and cotton were the best useful tools for not only direct isolation, but also fungal DNA diagnosis and cell lipid assay of Myanmar soil environmental conditions.     

Single Low Dose Primaquine (0.25mg/kg) Does Not Cause Clinically Significant Haemolysis in G6PD Deficient Subjects

Background

Primaquine is the only drug consistently effective against mature gametocytes of Plasmodium falciparum. The transmission blocking dose of primaquine previously recommended was 0.75mg/kg (adult dose 45mg) but its deployment was limited because of concerns over haemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is an inherited X-linked enzymatic defect that affects an estimated 400 million people around the world with high frequencies (15–20%) in populations living in malarious areas. To reduce transmission in low transmission settings and facilitate elimination of P. falciparum, the World Health Organization now recommends adding a single dose of 0.25mg/kg (adult dose 15mg) to Artemisinin-based Combination Therapies (ACTs) without G6PD testing. Direct evidence of the safety of this low dose is lacking. Adverse events and haemoglobin variations after this treatment were assessed in both G6PD normal and deficient subjects in the context of targeted malaria elimination in a malaria endemic area on the North-Western Myanmar-Thailand border where prevalence of G6PD deficiency (Mahidol variant) approximates 15%.

Methods and Findings

The tolerability and safety of primaquine (single dose 0.25 mg base/kg) combined with dihydroartemisinin-piperaquine (DHA-PPQ) given three times at monthly intervals was assessed in 819 subjects. Haemoglobin concentrations were estimated over the six months preceding the ACT + primaquine rounds of mass drug administration. G6PD deficiency was assessed with a phenotypic test and genotyping was performed in male subjects with deficient phenotypes and in all females. Fractional haemoglobin changes in relation to G6PD phenotype and genotype and primaquine round were assessed using linear mixed-effects models. No adverse events related to primaquine were reported during the trial. Mean fractional haemoglobin changes after each primaquine treatment in G6PD deficient subjects (-5.0%, -4.2% and -4.7%) were greater than in G6PD normal subjects (0.3%, -0.8 and -1.7%) but were clinically insignificant. Fractional drops in haemoglobin concentration larger than 25% following single dose primaquine were observed in 1.8% of the population but were asymptomatic.

Conclusions

The single low dose (0.25mg/kg) of primaquine is clinically well tolerated and can be used safely without prior G6PD testing in populations with high prevalence of G6PD deficiency. The present evidence supports a broader use of low dose primaquine without G6PD testing for the treatment and elimination of falciparum malaria.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01872702","term_id":"NCT01872702"}}NCT01872702     

Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo

The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1—which binds Myo2—its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo.     

Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway

Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK.     

The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome

Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself.     

Age Related Macular Degeneration and Total Hip Replacement Due to Osteoarthritis or Fracture: Melbourne Collaborative Cohort Study

Osteoarthritis is the leading cause of total hip replacement, accounting for more than 80% of all total hip replacements. Emerging evidence suggests that osteoarthritis has a chronic inflammatory component to its pathogenesis similar to age-related macular degeneration. We evaluated the association between age-related macular degeneration and total hip replacement as proxy for severe osteoarthritis or fractured neck of femur in the Melbourne Collaborative Cohort Study. 20,744 participants had complete data on both age-related macular degeneration assessed from colour fundus photographs taken during 2003–2007 and total hip replacement. Total hip replacements due to hip osteoarthritis and fractured neck of femur during 2001–2011 were identified by linking the cohort records to the Australian Orthopedic Association National Joint Replacement Registry. Logistic regression was used to examine the association between age-related macular degeneration and risk of total hip replacement due to osteoarthritis and fracture separately, adjusted for confounders. There were 791 cases of total hip replacement for osteoarthritis and 102 cases of total hip replacement due to fractured neck of femur. After adjustment for age, sex, body mass index, smoking, and grouped country of birth, intermediate age-related macular degeneration was directly associated with total hip replacement for osteoarthritis (odds ratio 1.22, 95% CI 1.00–1.49). Late age-related macular degeneration was directly associated with total hip replacement due to fractured neck of femur (odds ratio 5.21, 95% CI2.25–12.02). The association between intermediate age-related macular degeneration and an increased 10-year incidence of total hip replacement due to osteoarthritis suggests the possibility of similar inflammatory processes underlying both chronic diseases. The association of late age-related macular degeneration with an increased 10-year incidence of total hip replacement due to fractured neck of femur may be due to an increased prevalence of fractures in those with poor central vision associated with the late complications of age-related macular degeneration.     

Site-specific sonoporation of human melanoma cells at the cellular level using high lateral-resolution ultrasonic micro-transducer arrays

We developed a new instrumental method by which human melanoma cells (LU1205) are sonoporated via radiation pressures exerted by highly-confined ultrasonic waves produced by high lateral-resolution ultrasonic micro-transducer arrays (UMTAs). The method enables cellular-level site-specific sonoporation within the cell monolayer due to UMTAs and can be applicable in the delivery of drugs and gene products in cellular assays. In this method, cells are seeded on the biochip that employs UMTAs for high spatial resolution and specificity. UMTAs are driven by 30-MHz sinusoidal signals and the resulting radiation pressures induce sonoporation in the targeted cells. The sonoporation degree and the effective lateral resolution of UMTAs are determined by performing fluorescent microscopy and analysis of carboxylic-acid-derivatized CdSe/ZnS quantum dots passively transported into the cells. Models representing the transducer-generated ultrasound radiation pressure, the ultrasound-inflicted cell membrane wound, and the transmembrane transport through the wound are developed to determine the ultrasound-pressure-dependent wound size and enhanced cellular uptake of nanoparticles. Model-based calculations show that the effective wound size and cellular uptake of nanoparticles increase linearly with increasing ultrasound pressure (i.e., at applied radiation pressures of 0.21, 0.29, and 0.40 MPa, the ultrasound-induced initial effective wound radii are 150, 460, and 650 nm, respectively, and the post-sonoporation intracellular quantum-dot concentrations are 7.8, 22.8, and 29.9 nM, respectively) and the threshold pressure required to induce sonoporation in LU1205 cells is ～0.12 MPa.