Unique Changes in Mitochondrial Genomes Associated with Reversions of S-Type Cytoplasmic Male Sterility in Maizemar

Cytoplasmic male sterility (CMS) in plants is usually associated with the expression of specific chimeric regions within rearranged mitochondrial genomes. Maize CMS-S plants express high amounts of a 1.6-kb mitochondrial RNA during microspore maturation, which is associated with the observed pollen abortion. This transcript carries two chimeric open reading frames, orf355 and orf77, both unique to CMS-S. CMS-S mitochondria also contain free linear DNA plasmids bearing terminal inverted repeats (TIRs). These TIRs recombine with TIR-homologous sequences that precede orf355/orf77 within the main mitochondrial genome to produce linear ends. Transcription of the 1.6-kb RNA is initiated from a promoter within the TIRs only when they are at linear ends. Reversions of CMS-S to fertility occur in certain nuclear backgrounds and are usually associated with loss of the S plasmids and/or the sterility-associated region. We describe an unusual set of independently recovered revertants from a single maternal lineage that retain both the S plasmids and an intact orf355/orf77 region but which do not produce the 1.6-kb RNA. A 7.3-kb inversion resulting from illegitmate recombination between 14-bp microrepeats has separated the genomic TIR sequences from the CMS-associated region. Although RNAs containing orf355/orf77 can still be detected in the revertants, they are not highly expressed during pollen development and they are no longer initiated from the TIR promoter at a protein-stabilized linear end. They appear instead to be co-transcribed with cytochrome oxidase subunit 2. The 7.3-kb inversion was not detected in CMS-S or in other fertile revertants. Therefore, this inversion appears to be a de novo mutation that has continued to sort out within a single maternal lineage, giving rise to fertile progeny in successive generations

Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man

Stain-Free Quantification of Chromosomes in Live Cells Using Regularized Tomographic Phase Microscopy

Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells.National Institute of Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1

Between-day reliability of electromechanical delay of selected neck muscles during performance of maximal isometric efforts

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to assess the between-day reliability of the electromechanical delay (EMD) of selected neck muscles during the performance of maximal isometric contractions in five different directions.</p> <p>Methods</p> <p>Twenty-one physically active males participated in two testing sessions separated by seven to eight days. Using a custom-made fixed frame dynamometer, cervical force and surface electromyography (EMG) were recorded bilaterally from the splenius capitis, upper trapezius and sternocleidomastoid muscles during the performance of efforts in extension, flexion, left and right lateral bending, and protraction. The EMD was extracted using the Teager-Kaiser Energy Operator. Reliability indices calculated for each muscle in each testing direction were: the difference in scores between the two testing sessions and corresponding 95% confidence intervals, the standard error of measurement (SEM) and intra-class correlation coefficients (ICC).</p> <p>Results</p> <p>EMD values showed no evidence of systematic difference between the two testing sessions across all muscles and testing directions. The SEM for extension, flexion and lateral bending efforts ranged between 2.5 ms to 4.8 ms, indicating a good level of measurement precision. For protraction, SEM values were higher and considered to be imprecise for research and clinical purposes. ICC values for all muscles across all testing directions ranged from 0.23 to 0.79.</p> <p>Conclusion</p> <p>EMD of selected neck muscles can be measured with sufficient precision for the assessment of neck muscle function in an athletic population in the majority of directions tested.</p

The Finnish Cardiovascular Study (FINCAVAS): characterising patients with high risk of cardiovascular morbidity and mortality

BACKGROUND: The purpose of the Finnish Cardiovascular Study (FINCAVAS) is to construct a risk profile – using genetic, haemodynamic and electrocardiographic (ECG) markers – of individuals at high risk of cardiovascular diseases, events and deaths. METHODS AND DESIGN: All patients scheduled for an exercise stress test at Tampere University Hospital and willing to participate have been and will be recruited between October 2001 and December 2007. The final number of participants is estimated to reach 5,000. Technically successful data on exercise tests using a bicycle ergometer have been collected of 2,212 patients (1,400 men and 812 women) by the end of 2004. In addition to repeated measurement of heart rate and blood pressure, digital high-resolution ECG at 500 Hz is recorded continuously during the entire exercise test, including the resting and recovery phases. About 20% of the patients are examined with coronary angiography. Genetic variations known or suspected to alter cardiovascular function or pathophysiology are analysed to elucidate the effects and interactions of these candidate genes, exercise and commonly used cardiovascular medications. DISCUSSION: FINCAVAS compiles an extensive set of data on patient history, genetic variation, cardiovascular parameters, ECG markers as well as follow-up data on clinical events, hospitalisations and deaths. The data enables the development of new diagnostic and prognostic tools as well as assessments of the importance of existing markers

Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor

High blood pressure in school children: prevalence and risk factors

BACKGROUND: The purpose of this study was to determine the prevalence of high blood pressure (HBP) and associated risk factors in school children 8 to 13 years of age. METHODS: Elementary school children (n = 1,066) were examined. Associations between HBP, body mass index (BMI), gender, ethnicity, and acanthosis nigricans (AN) were investigated using a school based cross-sectional study. Blood pressure was measured and the 95(th )percentile was used to determine HBP. Comparisons between children with and without HBP were utilized. The crude and multiple logistic regression adjusted odds ratios were used as measures of association. RESULTS: Females, Hispanics, overweight children, and children with AN had an increased likelihood of HBP. Overweight children (BMI ≥ 85(th )percentile) and those with AN were at least twice as likely to present with HBP after controlling for confounding factors. CONCLUSION: Twenty one percent of school children had HBP, especially the prevalence was higher among the overweight and Hispanic group. The association identified here can be used as independent markers for increased likelihood of HBP in children

Constitutive Activation of PrfA Tilts the Balance of Listeria monocytogenes Fitness Towards Life within the Host versus Environmental Survival

PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells