Electron paramagnetic resonance imaging of tumor hypoxia: enhanced spatial and temporal resolution for in vivo pO2 determination.

The time-domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen-dependent linewidth (LW) to generate pO2 maps with minimal errors. A methodology for noninvasive pO2 imaging based on the application of TD-EPR using oxygen-induced LW broadening of a triarylmethyl (TAM)-based radical is presented. The decay of pixel intensities in an image is used to estimate T2*, which is inversely proportional to pO2. Factors affecting T2* in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line-broadening. Suitable experimental and image-processing parameters were obtained to produce pO2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor-bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of approximately 1.0 mm and a temporal resolution of approximately 9 s for 2D pO2 mapping, with a reliable oxygen resolution of approximately 1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution.     

DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620–1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of ^Gγ-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 × 10⁻⁴²). Together, common SNPs at the BCL11A, HBS1L-MYB, and β-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.     

Deoxyribonucleic acid synthesis-dependent casein gene expression: species differences

Mammary gland explants from mature virgin mice, rats, and rabbits exhibit an increased rate of both DNA and casein syntheses when cultured in the presence of specific combinations of insulin, hydrocortisone, and PRL. If cytosine arabinoside, a potent inhibitor of DNA synthesis, is added to the culture medium, casein synthesis is inhibited in explants from mice but not in those from rats or rabbits. This inhibition is at the level of accumulation of casein mRNA; an 89% reduction of stimulated levels was observed. The nature of this block was investigated further by examining the general response of mature virgin mouse mammary explants to hydrocortisone and PRL, hormones considered essential for casein gene expression in this species. Cytosine arabinoside did not prevent an increment in either hydrocortisone-induced NADH-cytochrome c-reductase or PRL-induced total RNA synthesis. Previous work has shown that certain insulin-induced responses are also unaffected. Taken collectively, these results suggest that the lesion induced by cytosine arabinoside inhibition of DNA synthesis is distal to the receptor for one or more of these hormones. The necessity for coupling DNA synthesis with overt differentiation in the mature virgin mouse, but not in the rat or rabbit, is one of numerous examples of species variation in regard to the interaction of hormones with the mammary gland.     

Specific factors influencing histotypic aggregation of chick embryo hepatocytes.

Conditions are described for the reproducible assay of substances affecting the in vitro rate of aggregation of isolated chick embryo hepatocytes. Two low molecular weight (less than 1000) fractions--one that promotes hepatocyte aggregation (HAP) and the other that inhibits this stimulation (HAI)--have been isolated and partially purified from adult chicken liver. One major active component of HAP was identified as taurine (2-aminoethanesulfonate). The presence of HAP during the entire time of assay was required for largest aggregate formation. HAP had no effect on aggregation of chick embryo neural retina, kidney, or heart cells. Our results and the fact that puromycin completely inhibits aggregate formation suggest that HAP and HAI influence the specific synthesis and interaction of membrane macromolecules involved in the aggregation process.     

Heat-stable low molecular weight form of phosphodiesterases from bovine pineal gland.

The 105,000 X g supernatant fraction of bovine pineal gland contains a phosphodiesterase activity that hydrolyzes both cyclic AMP and cyclic GMP. The rate of hydrolysis is 4-5 times greater with cyclic GMP as substrate than with cyclic AMP. Chromatography of supernatant fraction on Sephadex G-150 resolves phosphodiesterase activity into two fractions designated PDE I and PDE II. These are distinguishable on the basis of their molecular size, substrate specificity, and kinetic parameters. PDE I hydrolyzes cyclic GMP at a faster rate than cyclic AMP and has a molecular weight of 163,000. PDE II appears to be a smaller protein with a molecular weight of 24,400 and is specific for cyclic AMP. PDE I has apparent Km values of 83 and 53 micron for cyclic AMP and cyclic GMP, respectively, whereas PDE II exhibits an apparent Km value of 330 micron for cyclic AMP. With subsaturating concentrations of cyclic AMP as substrate, the phosphodiesterase activity of PDE I is inhibited by the addition of cyclic GMP. However, PDE II activity remains unaffected by cyclic GMP even at concentrations up to 125 micron. PDE II appears to be thermostable, losing only 20% of its activity on heating at 80 degrees for 2 min. Similar treatment completely abolishes the enzyme activity of PDE I.     

加拿大新生儿重症监护室院内感染及其变化趋势对胎龄<33周早产儿预后的影响

败血症是新生儿重症监护室(NICU)的危重病症及造成新生儿死亡的重要原因之一,根据其发病时间,可分为早发(生后2 d 内)和晚发(生后2 d 后)。其中晚发的新生儿败血症,一般考虑为院内感染(NI)。胎龄越小,出生体重越低,NI 的发生率越高。NI 的发生率在加拿大各 NICU 之间差别较大,与各中心医护人员的相关诊疗行为密切相关。在国家卫生研究院"基于循证医学的医疗质量改进项目" 的推动下,2003~2009 年间加拿大全国 NICU 总的NI 率明显下降,但不同中心各自感染率的变化趋势仍极为不同。本研究纳入了加拿大全国共24 家 NICU 在2008~2012 年间所有收入院的胎龄<33 周的早产儿共18 961 名,探讨NI 率的变化趋势以及其与早产儿预后的关系。     

Symptomatic and asymptomatic Cryptosporidium infections in children in a semi-urban slum community in southern India

Cryptosporidium is a leading cause of childhood diarrhea in developing countries. We investigated symptomatic and asymptomatic cryptosporidiosis in 20 children less than two years of age in a semi-urban slum in southern India. All surveillance (conducted every two weeks) and diarrheal samples from 20 children (n = 1,036) with cryptosporidial diarrhea previously identified by stool microscopy were tested by polymerase chain reaction-restriction fragment length polymorphism for species and subgenotype determination. Thirty-five episodes of cryptosporidiosis were identified in 20 children, of which 25 were diarrheal. Fifteen episodes were associated with prolonged oocyst shedding. Multiple episodes of cryptosporidiosis occurred in 40% of the children. Most infections were with C. hominis, subtype Ia. Children with multiple infections had significantly lower weight-for-age and height-for-age Z scores at 24 months but had scores comparable with children with a single episode by 36 months. Multiple symptomatic Cryptosporidium infections associated with prolonged oocyst shedding occur frequently in this disease-endemic area and may contribute to the long-term effects of cryptosporidiosis on physical growth in these children.