Measles virus spread initiated at international mass gatherings in Europe, 2011.

Three parallel transmission chains of measles virus (MV) variant 'D8-Villupuram' (D8-V) originated from two coinciding international mass gathering (MG) events in Rimini, Italy, in June 2011. MV D8-V was independently introduced into Germany by two unvaccinated persons, and into Slovenia by one unvaccinated person who had attended these events. Secondary spread of D8-V was restricted to two generations of transmission in Slovenia as well as in Germany where the virus was further disseminated at another MG. Serological and epidemiological investigation of the D8-V-associated German and Slovenian cases revealed different antibody responses and age distributions. Primary infected young persons between 11 and 27 years-old were affected in Germany, whereas the group of Slovenian cases comprised adults aged from 28 to 47 years and a high proportion (9/14; 64%) of patients with secondary vaccine failure (SVF). Our study demonstrates that monitoring of MV transmission chains in an international context and adequate serological investigation of cases with remote vaccination can contribute to identify susceptibility gaps.

Rise in the number of notified human hantavirus infections since October 2011 in Baden-Wurttemberg, Germany.

From October 2011 to April 2012, 852 human hantavirus infections were notified in Germany, of which 580 (68%) were in Baden-Württemberg. Case numbers started to rise earlier than they did before the previous outbreaks in 2007 and 2010, and are the largest ever reported in this state during October to April of any year. The early rise could be due to a beech mast year in 2011, followed by an early and massive reproduction of the reservoir bank vole populations during winter 2011 and spring 2012.

Intrinsic promoter nucleosome stability/dynamics variations support a novel targeting mechanism.

Genomic processes like transcription initiation typically involve the alteration of nucleosome structure, to expose DNA elements for regulatory factor binding. Nucleosome altering/modifying complexes have been identified, but precisely how these complexes find their specific targets remains unclear. We have shown that nucleosomes can exhibit significant DNA sequence-dependent stability and dynamics variations and have suggested that these inherent variations could facilitate nucleosome recognition and thus aid in specific targeting. Here, we confirm an important prediction of the model, namely, that stability and DNA dynamics features can correlate with the transcriptional involvement of specific promoter nucleosomes. A transcriptionally inert Mouse Mammary Tumor Virus promoter-region nucleosome (MMTV-D) has greater inherent stability than and reduced dynamics compared to a nearby nucleosome (MMTV-B) that is the initial target of transcription activation-associated processes on this promoter. MMTV-D stability could help direct activation-associated events to the less stable and more dynamic target, MMTV-B.

Yeast chromatin subunit structure.

Micrococcal nuclease digestion of in situ (intranuclear) and in vitro yeast chromatin produces distributions of DNA molecules of discrete sizes. In both cases, these molecules appear to be integral multiples of the smallest size on polyacrylamide gel electrophoresis. This result implies a widespread generic occurrence of the periodic organization of chromatin seen in mammalian systems.

Volatilization of high molecular weight DNA by pulsed laser ablation of frozen aqueous solutions.

DNA has been volatilized by pulsed laser ablation of a thin film of a frozen aqueous DNA solution. The target film was irradiated in vacuum by a pulsed laser at power densities sufficient to ablate the ice matrix. The expanding ablated water vapor entrained embedded DNA molecules and expelled them into the gas phase. Ejection of DNA molecules as large as 410 kilodaltons was verified by collection of the ablation products and subsequent mass analysis by polyacrylamide gel electrophoresis with autoradiographic detection.

Design of a novel antisymmetric coil array for parallel transmit cardiac MRI in pigs at 7 T.

The design, simulation, assembly and testing of a novel dedicated antisymmetric transmit/receive (Tx/Rx) coil array to demonstrate the feasibility of cardiac magnetic resonance imaging (cMRI) in pigs at 7 T was described. The novel antisymmetric array is composed of eight elements based on mirrored and reversed loop orientations to generate varying B1+ field harmonics for RF shimming. The central four loop elements formed together a pair of antisymmetric L-shaped channels to allow good decoupling between all neighboring elements of the entire array. The antisymmetric array was compared to a standard symmetric rectilinear loop array with an identical housing dimension. Both arrays were driven in the parallel transmit (pTx) mode forming an 8-channel transmit and 16-channel receive (8Tx/16Rx) coil array, where the same posterior array was combined with both anterior arrays. The hardware and imaging performance of the dedicated cardiac arrays were validated and compared by means of electromagnetic (EM) simulations, bench-top measurements, phantom, and ex-vivo MRI experiments with 46 kg female pig. Combined signal-to-noise ratio (SNR), geometry factor (g-factor), noise correlation maps, and high resolution ex-vivo cardiac images were acquired with an in-plane resolution of 0.3 mm × 0.3 mm using both arrays. The novel antisymmetric array enhanced the SNR within the heart by about two times and demonstrated good decoupling and improved control of the B1+ field distributions for RF shimming compared to the standard coil array. Parallel imaging with acceleration factor (R) up to 4 was possible using the novel antisymmetric coil array while maintaining the mean g-factor within the heart region of 1.13.

Sequence-dependent variations associated with H2A/H2B depletion of nucleosomes.

Mechanisms that can alter nucleosome structure to enhance DNA accessibility are of great interest because of their potential involvement in genomic processes. One such mechanism is H2A/H2B release from nucleosomes; it occurs in vivo and is involved in the in vitro activities of several transcription-associated complexes. Using fluorescence approaches based on Förster resonance energy transfer, we previously detected sequence-dependent structure/stability variations between 5S and two types of promoter nucleosomes (from yeast GAL10 or mouse mammary tumor virus promoters). Those variations included differing responses when nucleosomes were diluted to concentrations (sub-nM) known to produce H2A/H2B loss. Here, we show that treatment of these same three types of nucleosomes with the histone chaperone yNAP-1, which causes H2A/H2B release from nucleosomes in vitro, produces the same differential Förster resonance energy transfer responses, again demonstrating sequence-dependent variations associated with conditions that produce H2A/H2B loss. Single-molecule population data indicate that DNA dynamics on the particles produced by diluting nucleosomes to sub-nM concentrations follow two-state behavior. Rate information (determined by fluorescence correlation spectroscopy) suggests that these dynamics are enhanced in MMTV-B or GAL10 compared to 5S particles. Taken together, the results indicate that H2A/H2B loss has differing effects on 5S compared to these two promoter nucleosomes and the differences reflect sequence-dependent structure/stability variations in the depleted particles.

Nucleosomal stability and dynamics vary significantly when viewed by internal versus terminal labels.

Nucleosomes are a major impediment to regulatory factor activities and therefore to the operation of genomic processes in eukaryotes. One suggested mechanism for overcoming in vivo nucleosomal repression is factor-mediated removal of H2A/H2B from nucleosomes. Using nucleosomes labeled internally with FRET fluorophores, we previously observed significant, DNA sequence-dependent variation in stability and dynamics under conditions (subnanomolar concentrations) reported to produce H2A/H2B release from nucleosomes. Here, the same analytical approaches are repeated using 5S and MMTV-B nucleosomes containing FRET labels that monitor the terminal regions. The results show that stability and dynamics vary significantly within the nucleosome; terminally labeled constructs report significantly reduced stability and enhanced DNA dynamics compared to internally labeled constructs. The data also strongly support previous suggestions (1) that subnanomolar concentrations cause H2A/H2B release from nucleosomes, including the 5S, and (2) that stabilities in the internal regions of 5S and two promoter-derived nucleosomes (MMTV-B, GAL10) differ. Sequence-dependent nucleosome stability/dynamics differences could produce inherent variations in the accessibility of histone-associated DNA in vivo. Such intrinsic variation could also provide a mechanism for producing enhanced effects on specific nucleosomes by processes affecting large chromatin regions, thus facilitating the localized targeting of alterations to nucleosomes on crucial regulatory sequences. The results demonstrate clearly the importance of studying physiologically relevant nucleosomes.

AFM imaging of protein movements: histone H2A-H2B release during nucleosome remodeling.

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi-Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi-Snf complexes.

Conformational transition in DNA on a cold surface.

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2 degrees C. The contraction is not unique to a specific sequence and does not occur in solution at 2 degrees C or on a surface at 25 degrees C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.

Yeast chromatin structure and regulation of GAL gene expression.

Yeast genomic DNA is covered by nucleosome cores spaced by short, discrete length linkers. The short linkers, reinforced by novel histone properties, create a number of unique and dynamic nucleosome structural features in vivo: permanent unpeeling of DNA from the ends of the core, an inability to bind even full 147 bp core DNA lengths, and facility to undergo a conformational transition that resembles the changes found in active chromatin. These features probably explain how yeast can maintain most of its genome in a transcribable state and avoid large-scale packaging away of inactive genes. The GAL genes provide a closely regulated system in which to study gene-specific chromatin structure. GAL structural genes are inactive without galactose but are highly transcribed in its presence; the expression patterns of the regulatory genes can account for many of the features of GAL structural gene control. In the inactive state, GAL genes demonstrate a characteristic promoter chromosomal organization; the major upstream activation sequence (UASG) elements lie in open, hypersensitive regions, whereas the TATA and transcription start sites are in nucleosomes. This organization helps implement gene regulation in this state and may benefit the organism. Induction of GAL expression triggers Gal4p-dependent upstream nucleosome disruption. Disruption is transient and can readily be reversed by a Gal80p-dependent nucleosome deposition process. Both are sensitive to the metabolic state of the cell. Induction triggers different kinds of nucleosome changes on the coding sequences, perhaps reflecting the differing roles of nucleosomes on coding versus promoter regions. GAL gene activation is a complex process involving multiple Gal4p activities, numerous positive and negative cofactors, and the histone tails. DNA bending and chromosomal architecture of the promoter regions may also play a role in GAL regulation. Regulator-mediated competition between nucleosomes and the TATA binding protein complex for the TATA region is probably a central aspect of GAL regulation and a focal point for the numerous factors and processes that contribute to it.

Relationship between mitochondrial lipid peroxidation and alpha-tocopherol levels in the guinea-pig adrenal cortex.

Lipid peroxidation in mitochondria from the functionally distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea-pig adrenal cortex was investigated. Ferrous ion (Fe2+)-induced lipid peroxidation was far greater in inner than outer zone mitochondria. Ascorbic acid similarly initiated lipid peroxidation to a greater extent in inner zone mitochondrial preparations. Differences in the unsaturated fatty acid content of inner and outer zone mitochondria could not account for the regional differences in lipid peroxidation. Total fatty acid concentrations were greater in the outer than in the inner zone, and the relative amounts of each fatty acid were similar in the two zones. However, mitochondrial concentrations of alpha-tocopherol, an antioxidant known to inhibit lipid peroxidation, were approx. 5-times greater in the outer than inner zone. The results demonstrate that there are regional differences in mitochondrial lipid peroxidation in the adrenal cortex which may be attributable to differences in alpha-tocopherol content. Thus, alpha-tocopherol may serve to protect outer zone mitochondrial enzymes from the consequences of lipid peroxidation and thereby contribute to some of the functional differences between the zones of the adrenal cortex.

Effects of tocopherol depletion on the regional differences in adrenal microsomal lipid peroxidation and steroid metabolism.

Studies were done to assess the contribution of alpha-tocopherol to the regional differences in microsomal lipid peroxidation (LP) and steroid metabolism in the guinea pig adrenal cortex. In normal guinea pigs, ferrous ion (Fe2+)- and ascorbic acid-induced LP are far greater in microsomal preparations from the inner adrenal zone (zona reticularis) than in those from the outer zones (zona fasciculata plus zona glomerulosa). The amounts of unsaturated fatty acids, substrates for LP, are similar in the two zones, but alpha-tocopherol concentrations are 4-5 times greater in outer than inner zone microsomes. Tocopherol depletion by dietary deprivation had little effect on LP in vitro in inner zone microsomes, but substantially increased LP in outer zone preparations. As a result, tocopherol deficiency eliminated the zonal differences in microsomal LP. Unsaturated FFA concentrations were lower in tocopherol-deficient microsomal preparations than in those from tocopherol-sufficient animals, suggesting peroxidative losses in vivo. Tocopherol deficiency decreased steroid C-17,20 lyase activity in outer zone microsomes, but had no effect on activity in inner zone preparations, eliminating the normal zonal difference in activity (outer greater than inner). The results indicate that alpha-tocopherol is a major determinant of adrenal LP and is responsible for the regional differences in microsomal LP in guinea pig adrenal cortex; the effects of ascorbic acid on LP in each zone are also affected by alpha-tocopherol. alpha-Tocopherol may influence the functional zonation of the adrenal cortex by selectively protecting outer zone steroidogenic enzymes from oxidative degradation.

alpha-Tocopherol depletion eliminates the regional differences in adrenal mitochondrial lipid peroxidation.

Prior studies demonstrated far greater amounts of lipid peroxidation (LP) in mitochondria from the zona reticularis (inner zone) of the guinea pig adrenal cortex than in mitochondria from the outer zone (zona fasciculata + zona glomerulosa) of the gland. alpha-Tocopherol concentrations, by contrast, were greater in the outer zone. To determine if the differences in alpha-tocopherol content were responsible for the regional differences in LP, the effects of alpha-tocopherol deficiency on mitochondrial LP were investigated. Tocopherol deficiency had relatively little effect on ferrous ion- or ascorbic acid-induced LP in inner zone mitochondria. However, depletion of adrenal tocopherol substantially increased outer zone LP, eliminating the differences between the two zones. Fatty acid analyses revealed that mitochondria from tocopherol-deficient animals contained significantly less linoleic acid (C18:2) and arachidonic acid (C20:4) than those from controls, suggesting peroxidative losses in vivo. In mitochondria from control animals, subphysiological concentrations of ascorbic acid stimulated LP, but physiological levels did not. However, in tocopherol-depleted mitochondria, even physiological concentrations of ascorbic acid stimulated LP. The results indicate that the intra-adrenal distribution of alpha-tocopherol is responsible for the regional differences in mitochondrial LP and that alpha-tocopherol is a major determinant of ascorbic acid actions on adrenal LP. The data also provide evidence of adrenal LP in vivo in tocopherol-deficient animals.

Calcified renal masses.

A review of the literature and the University of Kentucky Medical Center/Lexington Veterans Administration Medical Center experience regarding calcification of renal masses was undertaken. Twenty per cent of calcified renal masses cannot be easily characterized by CT scan as malignant or benign and are indeterminate. These lesions must be followed closely with follow-up CT scanning or undergo surgical exploration, as 40 per cent may be malignant.