Correlation of two-photon in vivo imaging and FIB/SEM microscopy.

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two-photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long-term two-photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three-dimensional high-resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.

Whole-genome copy number variation analysis in anophthalmia and microphthalmia.

Anophthalmia/microphthalmia (A/M) represent severe developmental ocular malformations. Currently, mutations in known genes explain less than 40% of A/M cases. We performed whole-genome copy number variation analysis in 60 patients affected with isolated or syndromic A/M. Pathogenic deletions of 3q26 (SOX2) were identified in four independent patients with syndromic microphthalmia. Other variants of interest included regions with a known role in human disease (likely pathogenic) as well as novel rearrangements (uncertain significance). A 2.2-Mb duplication of 3q29 in a patient with non-syndromic anophthalmia and an 877-kb duplication of 11p13 (PAX6) and a 1.4-Mb deletion of 17q11.2 (NF1) in two independent probands with syndromic microphthalmia and other ocular defects were identified; while ocular anomalies have been previously associated with 3q29 duplications, PAX6 duplications, and NF1 mutations in some cases, the ocular phenotypes observed here are more severe than previously reported. Three novel regions of possible interest included a 2q14.2 duplication which cosegregated with microphthalmia/microcornea and congenital cataracts in one family, and 2q21 and 15q26 duplications in two additional cases; each of these regions contains genes that are active during vertebrate ocular development. Overall, this study identified causative copy number mutations and regions with a possible role in ocular disease in 17% of A/M cases.

Natural bond-bond polarizability: a Hückel-like electronic delocalization index.

We show how the bond-bond polarizability index, as originally introduced by Coulson and Longuet-Higgins in the Hückel-theoretic context, can be generalized in the natural bond orbital (NBO) framework to ab initio molecular orbital and density functional theory levels. We demonstrate that such a "natural bond-bond polarizability" (NBBP) index provides a flexible and quantitative descriptor for a broad spectrum of delocalization effects ranging from strong π aromaticity to weak intra- and intermolecular hyperconjugative phenomena. Illustrative applications are presented for representative delocalization effects in saturated and unsaturated species, chemical reactions, and hydrogen-bonding interactions.

A review of full-body radiography in nontraumatic emergency medicine.

This paper reports on the application of full-body radiography to nontraumatic emergency situations. The Lodox Statscan is an X-ray machine capable of imaging the entire body in 13 seconds using linear slit scanning radiography (LSSR). Nontraumatic emergency applications in ventriculoperitoneal (VP) shunt visualisation, emergency room arteriography (ERA), detection of foreign bodies, and paediatric emergency imaging are presented. Reports show that the fast, full-body, and low-dose scanning capabilities of the Lodox system make it well suited to these applications, with the same or better image quality, faster processing times, and lower dose to patients. In particular, the large format scans allowing visualisation of a greater area of anatomy make it well suited to VP shunt monitoring, ERA, and the detection of foreign bodies. Whilst more studies are required, it can be concluded that the Lodox Statscan has the potential for widespread use in these and other nontraumatic emergency radiology applications.

Variability in the preoperative management of infants with hypoplastic left heart syndrome.

Infants with hypoplastic left heart syndrome (HLHS) commonly undergo initial surgical palliation during the first week of life. Few data exist on optimal preoperative management strategies; therefore, the management of these infants prior to surgery is anecdotal and variable. To more fully define this variability in preoperative care of infants with HLHS, a survey was designed to describe current preoperative management practices in the infant with HLHS. The questionnaire explored management styles as well as preoperative monitoring techniques and characteristics of the respondent's health care institution. The responses were compiled and are reported. A striking lack of consistency in preoperative management techniques for infants with HLHS is apparent. The impact of these preoperative strategies is unknown. Despite challenges in anatomic and hemodynamic variability at presentation, a prospective randomized controlled trial comparing ventilatory management techniques, enteral feeding strategies, and the utility of various monitoring tools on short- and long-term outcome is needed.

Risk network structure in the early epidemic phase of HIV transmission in Colorado Springs.

This study describes the risk network structure of persons with HIV infection during its early epidemic phase in Colorado Springs, USA, using analysis of community-wide HIV/AIDS contact tracing records (sexual and injecting drug partners) from 1985 to 1999. Paired partner information from other STD/HIV programme records was used to augment network connections. Analyses were conducted with and without this supplemental information. The results suggest that a combined dendritic and cyclic structural network pattern is associated with low to moderate HIV propagation in Colorado Springs, and may account for the absence of intense propagation of the virus.

Energy distribution and redistribution and chemical reactivity. The generalized delta overlap-density method for ground state and electron transfer reactions: a new quantitative counterpart of electron-pushing.

A new approach to prediction of organic reactions and understanding of the electron flow involved in the reaction mechanisms is presented. The method developed permits comparison of electronic structures of species different in multiplicity, charge, and geometry based on use of spin- and charge-independent entities-"overlap corrected density matrices". The method utilizes the basis orbitals of one molecule A (e.g. reactant) in the computation of a second molecule, B, derived from the first by an approach to product. This then provides two Overlap-Density Matrices with a common set of basis (e.g. hybrid) orbitals. Subtraction of Overlap-Density Matrix B from Matrix A affords the Delta Overlap-Density Matrix. Each element of the Delta Overlap-Density matrix gives the change in electron population of a bond or of a single hybrid orbital. Molecule B may differ from A by the addition or loss of an electron, by stretching of a bond, by electronic excitation, or by some other perturbation. The Delta Overlap-Density matrices afford a detailed description of the reaction process and provide predictions of overall reactions including such subtleties as regiochemistry.

Excited- and ground-state versions of the tri-pi-methane rearrangement: mechanistic and exploratory organic photochemistry.

The di-pi-methane rearrangement with two pi-groups bonded to a single carbon leading to pi-substituted cyclopropanes is now well established. The present research had as its goal the exploration of molecular systems having three pi-moieties attached to an sp(3)-hybridized atom in a search for a tri-pi-methane rearrangement. Indeed, it was found that such systems do rearrange photochemically to afford cyclopentenes. However, it was also established that vinylcyclopropanes ring-expand to cyclopentenes on direct irradiation. Since both three-ring and five-ring photoproducts often are found to be produced, it was important to establish that the observed photochemistry was really the result of a true single-step tri-pi-methane rearrangement and not the consequence of two sequential rearrangements, first to form a vinyl cyclopropane which subsequently ring expanded to the cyclopentene. The general situation has three species-A, B, and C-corresponding to tri-pi-methane reactant A, vinylcyclopropane photoproduct B, and cyclopentene photoproduct C. Three rate constants are involved, k(1) for A --> B, k(2) for A --> C, and k(3) for B --> C. The kinetics were applied to two examples with provision to avoid differential light absorption; this utilized singlet sensitization. It was determined that direct formation of the cyclopentene photoproduct proceeds more rapidly than the ring-expansion route. In contrast to the di-pi-methane rearrangement, the tri-pi-methane reaction was found to be preferred by the singlet, while in these sterically congested systems, the triplet led to di-pi-methane reactivity. Finally, a ground-state counterpart of the reaction was obtained.

Management of drug-induced liver disease.

The treatment and prevention of drug-induced liver injury starts with the recognition of hepatotoxicity at the earliest possible time so that the suspected drug can be discontinued expeditiously. Both liver enzyme monitoring and vigilance for signs of hypersensitivity involving the liver are useful strategies for many agents known to cause hepatocellular necrosis leading to liver failure. Specific antidotes to prevent or limit hepatic damage exist for only a few drugs, the most important being N-acetylcysteine for the treatment of acetaminophen hepatotoxicity. Corticosteroids are of unproven benefit in the setting of fulminant failure. Ursodiol may be helpful in instances of cholestatic injury. For other agents, supportive measures and the increasing use of liver-assist devices as well as emergency liver transplantation are available when drug injury evolves into irreversible liver failure. It is hoped that a better understanding of hepatotoxicity mechanisms will lead to the development of more specific and effective forms of therapy in the near future.

The need to know: experiences of critically ill patients.

BACKGROUND: Critically ill patients vary in their memories of their experience in the intensive care unit. Some have little recall and need to learn about their critical illness. Others have more vivid memories of their experiences, some of which were extremely unpleasant. Patients' not knowing what was happening may have exacerbated the unpleasant experiences. OBJECTIVES: To elicit the experience of knowing for critically ill patients and to explore the differences in perceptions between patients who were intubated and those who were not intubated during the illness. METHODS: Grounded theory was used to explore the meaning of knowing and not knowing and the process by which knowing occurs. Unstructured interviews were done with 14 patients. RESULTS: Knowing had 2 phases: the need to know (1) during and (2) after the critical illness. The first phase had 3 facets: needing information, needing to be oriented, and having confusing perceptions. The second phase had 2 facets: needing information about what had happened and piecing together events. Many experiences with knowing during and after a critical illness were similar for both intubated and nonintubated patients. The main difference was the intensity of the experience in some categories. CONCLUSIONS: Critically ill patients have a strong need to know throughout and after their time in the intensive care unit. Nurses must address this need for constant reorientation to the past and present in these patients. In addition, adequate nursing staff must be available for these patients.

Drug-induced liver disease.

Drug-induced liver disease may account for between 10% and 50% of adult patients with elevated enzymes, especially in patients over age 50 years. It accounts for nearly 25% of patients with fulminant hepatic failure. Liver injury can be cytotoxic, cholestatic, or mixed. A variety of systemic manifestations can accompany drug-induced hepatotoxicity. Drug-induced liver disease can mimic autoimmune hepatitis or it can evolve to cirrhosis. It can also mimic veno-occulusive disorders. The plethora of herbal and traditional agents currently ingested by many people should always be considered in any patient with abnormal hepatic biochemistry.

Pharmacokinetics of estradiol valerate 2mg + dienogest 2mg (climodien® 2/2) after single and repeated oral administration in healthy postmenopausal women.

OBJECTIVE: To evaluate the pharmacokinetics and tolerability of estradiol valerate 2.0mg plus dienogest 2.0mg (Climodien® 2/2). DESIGN AND SETTING: This was an open single-and multiple-dose study. STUDY PARTICIPANTS: 16 healthy postmenopausal women. INTERVENTIONS: Pharmacokinetic parameters were determined in plasma after single and multiple daily intake of Climodien® 2/2 for 12 weeks. Accumulation during multiple administration was calculated from the area under the plasma concentration-time curve (AUC). Changes in plasma levels of other hormones and sex hormone-binding globulin (SHBG) were also measured. RESULTS: The observed accumulation of estradiol (accumulation ratio R(1) = 3.3) and free estrone (R(1) = 2.4) was higher than that predicted from single-dose data (R(theor) = 1.7 and 2.0 for estradiol and free estrone, respectively). This was thought to be due to high interindividual variability in estrogen parameters, or the degree of extrapolation required when calculating the half-life (t1/2). The observed accumulation of total estrone after multiple-drug administration was as predicted from single-dose results (R(1) and R(theor) = 1.5). The pharmacokinetics of dienogest were not time dependent, the observed accumulation (AUC(0-24h) 627 vs 483 µg/L · h) was as predicted from single-dose results (R(1) and R(theor) = 1.3). Reduced total plasma testosterone levels confirmed the antiandrogenic effect of dienogest.The main adverse events with Climodien® 2/2 (breast tension in five participants and irregular vaginal bleeding in four) reflected its hormonal content, and laboratory screening tests revealed no tolerability concerns. CONCLUSIONS: Estradiol may accumulate in plasma during multiple-drug administration with Climodien® 2/2 more than predicted from single-dose results. However, dienogest kinetics after multiple-drug administration were as predicted from single-dose results. Climodien® 2/2 demonstrated antiandrogenic effects and was well tolerated.

Variability of intracellular concentrations of basic fibroblast growth factor in cultured human gliomas.

BACKGROUND: Basic fibroblast growth factor (bFGF) is a small peptide with angiogenic and mitogenic properties that supports the growth and proliteration of human malignant glial tumors in vitro and in vivo in an autocrine fashion. The purpose of this study was to examine the potential relationship between intracellular bFGF concentrations and grade of glial malignancy using a monolayer cell culture system. MATERIALS AND METHODS: Samples from 13 histopathologically verified astrocytic brain tumors and two non-tumorous astrocyte specimens were grown in tissue culture and examined both early and late after explantation together with a bFGF-producing reference cell line. RESULTS: Elevated intracellular concentrations of bFGF were noted in the reference line as well as two of five other glioblastoma multiforme-derived cell cultules, three of five anaplastic glioma-derived cell cultures, and two of three astrocytoma-derived cell cultures. The cells derived from non-tumorous astrocyte specimens expressed low concentrations of bFGF. CONCLUSION: This study demonstrates that overlap exists between the grade ot glial malignancy and intracellular bFGF levels.

Drug-induced liver disease.

The past year has seen several additions to the list of drugs that cause hepatic injury. Many of these agents produce fulminant hepatic necrosis and, in some cases, were withdrawn from the market (eg, bromfenac). Other drugs had warnings placed in their labeling along with stringent monitoring guidelines to alert physicians and patients alike to the potential for severe hepatic injury (eg, troglitazone, tolcapone). New reports of hepatoxicity continued to appear for many older agents, in some cases expanding the hepatotoxic spectrum for the drugs. Vanishing bile duct syndrome has drawn increasing attention and is now associated with more than 30 drugs. Ibuprofen is among those drugs newly described as causing this syndrome. Hepatitis C virus infection was reported as a possible risk factor for ibuprofen hepatotoxicity, raising the issue of safe use of nonprescription as well as prescription drugs in patients with underlying liver disease. Reports have appeared about acetaminophen-induced hepatotoxicity in several dozen children from unintentional overdoses, in addition to cases of therapeutic misadventure in adults.

Drug- and chemical-induced cholestasis.

Cholestasis resulting from drugs is an increasingly recognized cause of liver disease. It produces a broad clinical-pathologic spectrum of injury that includes simple jaundice, cholestatic hepatitis, and bile duct injury that can mimic extrahepatic biliary obstruction, primary biliary cirrhosis, and sclerosing cholangitis. Although the risk of drug-induced cholestasis leading to a fatal outcome is quite rare, knowledge and recognition of the various forms of cholestatic injury assumes an importance whenever clinicians are confronted with jaundice or other manifestations of liver disease in patients receiving medicinal or chemical agents.

High-level expression of the human CB2 cannabinoid receptor using a baculovirus system.

A human CB2 recombinant baculovirus (AcNPV-hCB2) was generated by site-specific transposition and employed to express the human CB2 cannabinoid receptor. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-hCB2 revealed novel expression of a unique 2.3 kb transcript when probed with hCB2 cDNA. This transcript corresponded to the size expected for hCB2 generated from the recombinant virus construct. Western immunoblot analysis of whole cell homogenates of recombinant baculovirus-infected Sf9 cells, using affinity-purified antibody to a human CB2 carboxy terminal domain (anti-hCB2.CV), revealed the presence of novel immunoreactive protein. In addition, when anti-hCB2.CV was employed in immunofluorescence staining, an intense signal was observed within AcNPV-hCB2-infected cells but not within uninfected cells or cells infected with a control beta-galactosidase recombinant baculovirus. The pattern of immunofluorescence at early periods post-infection was in a perinuclear arrangement with a "signet-ring" appearance, suggestive of glycosylation of the expressed recombinant protein. Transmission electron microscopy revealed regions of intranuclear recombinant virus assembly and the presence of numerous intracytoplasmic proteinaceous vesicular inclusions consistent with hyperproduction of hCB2. Scatchard-Rosenthal analysis of [3H]-(-)3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxypro pyl]cyclohexan-1-ol ([3H]CP 55,940) receptor binding indicated a Kd of 2.24 nM and a Bmax equal to 5.24 pmol/mg of protein. The lack of [3H]CP 55,940 displacement with N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamidehydrochloride (SR 141716A), the CB1-selective antagonist, confirmed the identity of the receptor as CB2. These data indicate that AcNPV-hCB2 expresses high levels of the human CB2, which retains properties of the native receptor. Thus, this recombinant virus may prove suitable for hyperproduction of receptor for basic biochemical and biophysical characterization studies.

Do autopsies of critically ill patients reveal important findings that were clinically undetected?

OBJECTIVE: To determine if autopsies performed on patients who die in the medical intensive care unit (ICU) provide clinically important new information. DESIGN: Retrospective review. SETTING: A 16-bed medical-coronary ICU. PATIENTS: Patients who underwent autopsy during a 1-yr period. INTERVENTIONS: Pre mortem diagnoses were determined from the medical record. Autopsy results were obtained from the final pathology report. A panel of three physicians with certification of added qualifications in critical care medicine reviewed the findings. MEASUREMENTS AND MAIN RESULTS: These questions were asked: a) Is the primary clinical diagnosis confirmed? b) Are the clinical and pathologic causes of death the same? c) Are new active diagnoses revealed? and d) If the new findings had been known before death, would the clinical management have differed? Forty-one autopsies (31% of deaths) were done that showed: a) the same primary clinical diagnosis and post mortem diagnosis in 34 (83%) patients; b) the same clinical and pathologic cause of death in 27 (66%) patients; c) new active diagnoses in 37 (90%) patients; and d) findings that would have changed medical ICU therapy had the findings been known in 11 (27%) patients. CONCLUSIONS: Although the primary clinical diagnosis was accurate in most cases before death, the cause of death was frequently unknown. Almost all autopsies demonstrated new diagnoses, and knowledge of these new findings would have changed medical ICU therapy in many cases. In the critical care setting, autopsies continue to provide information that could be important for education and quality patient care.

Granulomas in the livers of humans and fischer rats associated with the ingestion of mineral hydrocarbons: A comparison

Ninety-day feeding studies were conducted in Fischer 344 rats using a series of highly refined mineral hydrocarbons which included mineral oils and waxes representative of those used in consumer products and food applications. The series included materials which had been refined by oleum or hydrogenation. The materials tested were representative of the range of carbon chain lengths, molecular weights, and viscosities which are currently in use. Findings revealed the presence of granulomatous lesions in the liver and histiocytosis in the lymph nodes. Some mineral hydrocarbons did not induce any lesions; others induced relatively minor effects; and a low melting point wax induced the largest lesions in both liver and mesenteric lymph nodes, with inflammation and areas of focal necrosis in the livers. The majority of lesions reported were associated with the highest dose levels used. These studies are in contrast to studies in Sprague-Dawley rats in which comparable doses did not induce similar lesions, indicating marked strain variability. Lipogranulomas associated with the ingestion of mineral oil have been reported in humans. The comparative morphology of the lesions seen in the Fischer rat study and those observed in the human are discussed and differences are highlighted. The lesions in the human are not believed to progress to lesions of clinical significance. The pathogenesis of the lesions induced in Fischer rats and in humans is discussed and it is concluded that the majority, if not all of the lesions, in the rats are of no significance for humans. The possibility that a small proportion of cases of granulomatous hepatitis in humans may represent an atypical response to mineral hydrocarbons may need further investigation. Copyright 1998 Academic Press.