Sequence-specific modification of genomic DNA by small DNA fragments

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, beta-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.     

Validation of novel optical imaging technologies: the pathologists' view

Noninvasive optical imaging technology has the potential to improve the accuracy of disease detection and predict treatment response. Pathology provides the critical link between the biological basis of an image or spectral signature and clinical outcomes obtained through optical imaging. The validation of optical images and spectra requires both morphologic diagnosis from histopathology and parametric analysis of tissue features above and beyond the declared pathologic "diagnosis." Enhancement of optical imaging modalities with exogenously applied biomarkers also requires validation of the biological basis for molecular contrast. For an optical diagnostic or prognostic technology to be useful, it must be clinically important, independently informative, and of demonstrated beneficial value to patient care. Its usage must be standardized with regard to methods, interpretation, reproducibility, and reporting, in which the pathologist plays a key role. By providing insight into disease pathobiology, interpretive or quantitative analysis of tissue material, and expertise in molecular diagnosis, the pathologist should be an integral part of any team that is validating novel optical imaging modalities. This review will consider (1) the selection of validation biomarkers; (2) standardization in tissue processing, diagnosis, reporting, and quantitative analysis; (3) the role of the pathologist in study design; and (4) reference standards, controls, and interobserver variability.     

Syndromic craniosynostosis due to complex chromosome 5 rearrangement and MSX2 gene triplication

Craniosynostosis is a common birth defect ( approximately 1/3,000 births) resulting from chromosome imbalances, gene mutations or unknown causes. We report a 6-month-old female with multiple sutural synostosis and prenatal onset growth deficiency, developmental delay, facial dysmorphism, congenital heart defect, and inguinal hernia. An integrated approach of standard cytogenetics, mBAND, locus-specific FISH, and 75 kb resolution array-CGH disclosed a complex chromosome 5 rearrangement, resulting in 3 paracentric inversions, 2 between-arm insertions, and partial duplication of 5q35. An extra copy of the MSX2 gene, which maps within the duplicated segment and is mutated in Boston-type craniosynostosis, was confirmed by molecular cytogenetic studies. Our study confirms that early fusion of cranial sutures commonly observed in the dup(5q) syndrome is caused by triplication of the MSX2 gene and strongly supports the crucial role of this gene in the development of craniofacial structures.     

High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?

We used Affymetrix 6.0 GeneChip SNP arrays to characterize copy number variations (CNVs) in a cohort of 70 patients previously characterized on lower-density oligonucleotide arrays affected by idiopathic mental retardation and dysmorphic features. The SNP array platform includes ∼900 000 SNP probes and 900 000 non-SNP oligonucleotide probes at an average distance of 0.7 Kb, which facilitates coverage of the whole genome, including coding and noncoding regions. The high density of probes is critical for detecting small CNVs, but it can lead to data interpretation problems. To reduce the number of false positives, parameters were set to consider only imbalances >75 Kb encompassing at least 80 probe sets. The higher resolution of the SNP array platform confirmed the increased ability to detect small CNVs, although more than 80% of these CNVs overlapped to copy number ‘neutral'' polymorphism regions and 4.4% of them did not contain known genes. In our cohort of 70 patients, of the 51 previously evaluated as ‘normal'' on the Agilent 44K array, the SNP array platform disclosed six additional CNV changes, including three in three patients, which may be pathogenic. This suggests that about 6% of individuals classified as ‘normal'' using the lower-density oligonucleotide array could be found to be affected by a genomic disorder when evaluated with the higher-density microarray platforms.     

Fatty acid percentage in erythrocyte membranes of atrial flutter/fibrillation patients and controls

Purpose  

Several epidemiological published data support the protective role of omega-3 consumption in coronary artery disease, sudden cardiac death and ventricular arrhythmias, but interestingly, this is not the case for atrial arrhythmias. The purpose of this study is to evaluate different fatty acid profile between AF/AFL subjects and healthy controls.     

Mapping changes in cortical activity during sleep in the first 4 years of life

A coherent body of evidence supports the notion that sleep is a local and use‐dependent process. Significant changes in brain morphology and function occur in the first years of life, revealing a postero–anterior trajectory of cortical maturation. On this basis, a recent study demonstrated that regional cortical maturation between early childhood and late adolescence is reflected in regional changes of sleep slow wave activity (SWA) during non‐rapid eye movement (NREM) sleep. Our hypothesis is that changes of electroencephalogram (EEG) rhythms during sleep from birth to childhood are also mirrored by parallel regional changes in the EEG rhythms of sleep according to the assumption of a postero–anterior gradient in cortical maturation. We studied all‐night EEG of 39 healthy, full‐term, infants and children aged between 0 and 48 months, evaluating regional differences in NREM sleep. We confirmed the strictly local nature of sleep with frequency‐specific regional differences. Specifically, we found a general shift of maxima of the upper alpha activity from occipital to prefrontal regions, expressed mainly by the ~11 Hz frequency. This shift corresponds to a postero–anterior trajectory of the so‐called ‘slow spindles’. The theta and alpha EEG activity of the frontal cortex exhibits a clear, positive, correlation with age. We conclude that specific local differences during NREM sleep, parallel cortical maturation also in the first 4 years of life.     

Linezolid pharmacokinetic/pharmacodynamic profile in critically ill septic patients: intermittent versus continuous infusion

Pharmacokinetics and pharmacodynamics are significantly altered in critically ill septic patients and the risk of prolonged periods with concentrations below the minimum inhibitory concentration (MIC) and of low area under the serum concentration-time curve/MIC (AUC/MIC) ratios is of concern. We compared the pharmacokinetic/pharmacodynamic (PK/PD) profile of linezolid administered by intermittent or continuous infusion in critically ill septic patients. Patients were divided into two groups: intermittent infusion (Group I) (600mg/12h); or continuous infusion (Group C) (300mg intravenous loading dose +900mg continuous infusion on Day 1, followed by 1200mg/daily from Day 2). Linezolid serum levels were monitored for 72h and microbiological data were collected. The clinical outcome was monitored. Sixteen patients completed the study. MICs of susceptible pathogens were 2mg/L for 80% of the isolates. In Group I, linezolid trough serum levels (C(min)) varied widely and were below the susceptibility breakpoint (4mg/L) during the study period; in 50% of patients C(min) was <1mg/L. In Group C, mean linezolid serum levels were more stable and, starting from 6h, were significantly higher than C(min) levels observed in Group I and were always above the susceptibility breakpoint. Time that the free drug concentration was above the MIC (T(free)>MIC) of>85% was more frequent in Group C than in Group I (P<0.05). Finally, with continuous infusion it was possible to achieve AUC/MIC values of 80-120 more frequently than with intermittent infusion (P<0.05). According to PK/PD parameters, continuous infusion has theoretical advantages over intermittent infusion in this population of patients.     

Maternal total vascular resistance and concentric geometry: a key to identify uncomplicated gestational hypertension

OBJECTIVE: To evaluate the prognostic impact of elevated total vascular resistance (TVR) on the outcome of pregnancy in early mild gestational hypertension (EMGH). DESIGN: Prospective observational study. SETTING: Data collected from women with EMGH referred to the obstetrics outpatient clinic of Tor Vergata University from June 2003 to June 2005. POPULATION: A total of 268 women with EMGH (systolic and diastolic blood pressure [BP] 140-150 mmHg and 90-99 mmHg, respectively, without significant proteinuria). METHODS: Women had a maternal echocardiographic examination and BP examination within 24 hours of diagnosis. From this, the TVR was calculated and the geometric pattern of the left ventricle assessed. MAIN OUTCOME MEASURES: Fetal/maternal adverse outcomes (pre-eclampsia, preterm delivery, placental abruption, other maternal medical problems, fetal distress, neonatal low birthweight, admittance to neonatal intensive care unit and perinatal death). RESULTS: Ninety-two out of the 268 pregnancies showed adverse outcomes (34.3%). The best independent predictor for the composite of maternal and fetal complications was TVR (OR 64.4, 95% CI 25.9-160.1). The cutoff value was 1340 dyn seconds/cm(5) with a sensitivity and a specificity of 90 and 91%, respectively. Concentric geometry of the left ventricle was also an independent predictor (OR 4.72, 95% CI 1.85-12.04). CONCLUSIONS: Echocardiography could help in identifying women with EMGH who subsequently develop maternal and fetal complications, allowing a classification in high-risk (TVR > 1340 dyn seconds/cm(5), concentric geometry of the left ventricle) and low-risk women (TVR < 1340 dyn seconds/cm(5), nonconcentric geometry of the left ventricle) for adverse outcomes of pregnancy.