Acromelic frontonasal dysostosis and ZSWIM6 mutation: phenotypic spectrum and mosaicism.

Acromelic frontonasal dysostosis (AFND) is a distinctive and rare frontonasal malformation that presents in combination with brain and limb abnormalities. A single recurrent heterozygous missense substitution in ZSWIM6, encoding a protein of unknown function, was previously shown to underlie this disorder in four unrelated cases. Here we describe four additional individuals from three families, comprising two sporadic subjects (one of whom had no limb malformation) and a mildly affected female with a severely affected son. In the latter family we demonstrate parental mosaicism through deep sequencing of DNA isolated from a variety of tissues, which each contain different levels of mutation. This has important implications for genetic counselling.

A temperature-sensitive disorder in basal transcription and DNA repair in humans.

The xeroderma pigmentosum group D (XPD) helicase subunit of TFIIH functions in DNA repair and transcription initiation. Different mutations in XPD give rise to three ultraviolet-sensitive syndromes: the skin cancer-prone disorder xeroderma pigmentosum (XP), in which repair of ultraviolet damage is affected; and the severe neurodevelopmental conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD). In the latter two, the basal transcription function of TFIIH is also presumed to be affected. Here we report four unusual TTD patients with fever-dependent reversible deterioration of TTD features such as brittle hair. Cells from these patients show an in vivo temperature-sensitive defect of transcription and DNA repair due to thermo-instability of TFIIH. Our findings reveal the clinical consequences of impaired basal transcription and mutations in very fundamental processes in humans, which previously were only known in lower organisms.

Rhizobium radiobacter as an opportunistic pathogen in central venous catheter-associated bloodstream infection: case report and review.

Rhizobium radiobacter is an uncommon opportunistic pathogen present in soil. It has been particularly associated with indwelling intravascular devices in immunocompromised patients. In this report, we summarise the case of a patient with multiple myeloma who developed R. radiobacter bacteraemia during autologous stem cell leucopheresis. Retrospective investigation revealed exposure to soil after central venous catheter placement for chemotherapy and leucopheresis access. This is the first reported case of R. radiobacter bacteraemia following probable colonisation of the catheter from soil exposure. We further review the existing literature to delineate prevention and treatment recommendations for line-associated R. radiobacter infections.

Akathisia--seen from the patients' perspective.

Akathisia is a side-effect that continues to affect a large percentage of psychiatric patients though the focus over recent years has moved away from extrapyramidal side-effects to metabolic problems. As cognitive behavioural therapy has become an integrated part of the management plan for psychotic patients an increased understanding of patients' interpretation of their symptoms and side-effects are encouraged. This case series illustrates different views that patients take on medication induced side-effects.

Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth.

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

Two subgroups of antipsychotic-naive, first-episode schizophrenia patients identified with a Gaussian mixture model on cognition and electrophysiology.

Deficits in information processing and cognition are among the most robust findings in schizophrenia patients. Previous efforts to translate group-level deficits into clinically relevant and individualized information have, however, been non-successful, which is possibly explained by biologically different disease subgroups. We applied machine learning algorithms on measures of electrophysiology and cognition to identify potential subgroups of schizophrenia. Next, we explored subgroup differences regarding treatment response. Sixty-six antipsychotic-naive first-episode schizophrenia patients and sixty-five healthy controls underwent extensive electrophysiological and neurocognitive test batteries. Patients were assessed on the Positive and Negative Syndrome Scale (PANSS) before and after 6 weeks of monotherapy with the relatively selective D2 receptor antagonist, amisulpride (280.3±159 mg per day). A reduced principal component space based on 19 electrophysiological variables and 26 cognitive variables was used as input for a Gaussian mixture model to identify subgroups of patients. With support vector machines, we explored the relation between PANSS subscores and the identified subgroups. We identified two statistically distinct subgroups of patients. We found no significant baseline psychopathological differences between these subgroups, but the effect of treatment in the groups was predicted with an accuracy of 74.3% (P=0.003). In conclusion, electrophysiology and cognition data may be used to classify subgroups of schizophrenia patients. The two distinct subgroups, which we identified, were psychopathologically inseparable before treatment, yet their response to dopaminergic blockade was predicted with significant accuracy. This proof of principle encourages further endeavors to apply data-driven, multivariate and multimodal models to facilitate progress from symptom-based psychiatry toward individualized treatment regimens.

Induction of hepatocyte proliferation and liver hyperplasia by the targeted expression of cyclin E and skp2.

Cells in culture become competent to replicate in the absence of growth factor after progressing beyond the late G1 restriction point, suggesting that a set of genes expressed during G1 phase is sufficient to trigger completion of the cell cycle. However, this has not been demonstrated in an in vivo system. In this study, we examined whether transfection of genes associated with the G1/S transition could trigger hepatocyte replication. Co-transfection of cyclin E and skp2 synergistically promoted cell cycle progression in cultured primary hepatocytes in the absence of mitogen or in the presence of growth inhibitors. Furthermore, transfection of hepatocytes in vivo with cyclin E and skp2 promoted abundant hepatocyte replication and hyperplasia of the liver. These studies confirm that transfection with a small number of genes can trigger proliferation of quiescent hepatocytes in vivo, and suggest that therapies to enhance liver regeneration by targeting cell cycle control genes may be feasible.

Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70 kDa family of heat-shock proteins.

This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H-II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer-generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins.

The crystal structure of tris-inhibited phospholipase C from Bacillus cereus at 1.9 A resolution. The nature of the metal ion in site 2.

We report here the crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and the widely used biochemical buffer tris (hydroxymethyl)-methylamine (Tris). The structure has been determined at 1.9 A resolution and refined to R = 20.3%. Tris has metal-binding properties, especially to Zn2+, and has been reported to reduce the activity of PLC. The amine nitrogen atom in Tris is co-ordinated to one of the three Zn2+ ions in the active site of the enzyme, thus confirming its chelating properties and the involvement of the metal ions in the catalytic process. The occupancy of the Zn2+ ion in site 2 in native PLC is 0.6 which could imply the presence of Ca2+ rather than Zn2+. The fact that Tris binds to this metal ion, the nature of the site 2 co-ordination shell and comparison with several homologous Zn-metalloenzymes indicate that PLC is a 3-Zn metalloenzyme. This study is one of a series which explores the active site of PLC by complexing the enzyme with inhibitors and substrate analogues.

Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue.

The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.

Crystallization and preliminary X-ray crystallographic studies of CO-hemoglobin from the North Atlantic salmon (Salmo salar).

Purification of hemoglobin from North Atlantic salmon (Salmo salar) gave three different types. The CO-complexes of types I and III have been crystallized by the batch method at 4 degrees C from solutions 18% (w/v) in polyethylene glycol 2000, 50 mg/ml in hemoglobin and 0.05 M in phosphate buffer (pH 8.3). Orthorhombic crystals, space group P2(1)2(1)2(1), were obtained for both, with cell dimensions a = 53.9 A, b = 80.4 A, c = 132.4 A, and a = 58.7 A, b = 95.0 A, c = 107.4 A, for types I and III, respectively.

Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar).

Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.

Genetic basis of familial neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant (adFNDI) or X-linked recessive (xrFNDI) disorder characterized by the development in early childhood of an irreversible deficiency of arginine vasopressin (AVP) secretion. Autopsy data in adFNDI reveal selective destruction of the posterior pituitary magnocellular neurons that normally produce the hormone. These abnormalities are due to a variety of mutations in the gene that encodes the AVP-neurophysin II precursor. Each one predicts a change in the primary structure of the preprohormone, and all but one are of a type known or reasonably presumed to impair the folding and cellular trafficking of the preprohormone. This pattern and the uniform clinical characteristics of adFNDI suggest that the disease is due to the production of a mutant precursor that is toxic for magnocellular neurons, because it cannot be folded, processed, or otherwise disposed of efficiently. Although the gene responsible for xrFNDI has not yet been cloned, the striking clinical similarities between adFNDI and xrFNDI suggest that similar pathophysiologic mechanisms may be involved.

Comparison of voxel- and volume-of-interest-based analyses in FDG PET scans of HIV positive and healthy individuals.

UNLABELLED: Abnormal glucose metabolic patterns in the brain have been reported for HIV-1 seropositive (HIV+) patients when compared with seronegative healthy individuals. The metabolic covariance pattern obtained from voxel- or volume-of-interest (VOI)-based multivariate data analysis techniques can be used to characterize disease and potentially to detect and monitor disease progression in the early stage of AIDS dementia complex. However, the arbitrary smoothing typically applied to PET data during reconstruction and processing to reduce noise has an unknown effect on the data, especially for the voxel-based analysis. METHODS: To investigate the impact of smoothing on a discrimination task, we applied principal component analysis with scaled subprofile-model preprocessing (SSM/PCA) followed by Fisher discriminant analysis to FDG PET data that were reconstructed and processed with different degrees of smoothing. Receiver operating characteristic curves were used to compare the ability of derived metabolic covariance patterns to discriminate HIV+ patients from healthy volunteers. RESULTS: For the voxel-based analysis, we found that the spatial distribution of voxel weights from the SSM/PCA analysis suggested edge effects along major tissue and cerebrospinal fluid boundaries, indicative of a disease-specific pattern of cerebral atrophy for the HIV+ patients. In terms of its discrimination performance, this metabolic covariance pattern is stable and insensitive to a wide range of smoothing kernels, except for ramp reconstruction and Hanning reconstruction with 7 x 7 x 7 block smoothing. In these reconstructions, the discrimination performance decreased as a result of high image noise and excessive smoothing, respectively. Our results also indicated that if sufficient variance from the VOI measurements is included, the overall performance of a conventional VOI-based analysis can be similar to that of the voxel-based analysis for the same discrimination task. However, the VOI-based analysis performed poorly at low false-positive fraction and is less tolerant to noise in the metabolic covariance pattern than the voxel-based analysis. CONCLUSION: We have obtained a unique covariance pattern of brain glucose metabolism for HIV+ patients compared with healthy volunteers. Discrimination based on this covariance pattern was found to be insensitive to a wide range of image smoothness.

Thyroid function, morphology and autoimmunity in young patients with insulin-dependent diabetes mellitus.

OBJECTIVE: An association between insulin-dependent diabetes mellitus (IDDM) and autoimmune thyroid disease is well recognized. We have studied the prevalence of thyroid dysfunction, autoimmunity and morphological abnormalities by ultrasonography in young diabetics. SUBJECTS AND METHODS: Among young IDDM patients less than 18 years old and living in the county of Funen, Denmark, 105 of 116 eligible patients participated. They were compared with 105 healthy children matched for sex and age. Routine thyroid function parameters (thyroxine (T4), tri-iodothyronine (T3), T3 resin uptake and TSH) and thyroid autoantibodies (anti-thyroid peroxidase, TPOab, and thyroglobulin antibodies, Tgab) were measured. Thyroid size and morphology were determined by ultrasonography. RESULTS: Two of the diabetics had previously diagnosed hypothyroidism and three new cases of subclinical hypothyroidism were found. There were no significant differences in thyroid function variables or thyroid volume between diabetics and controls. Thyroid volume correlated significantly with age and weight in both groups. Among diabetics, 17 had thyroid autoantibodies (13 with TPOab, 14 with Tgab and 10 with both) compared with 2 children in the control group (P<0.001). Forty-four with IDDM as opposed to 11 of the controls (P<0.001) had morphological abnormalities at ultrasonography. Most of them had various degrees of hypoechogenicity thought to be a marker of thyroid autoimmunity. Among the 17 diabetics with autoantibodies, 10 had morphological abnormalities at ultrasonography. CONCLUSIONS: A high proportion of young IDDM patients without any clinical signs of thyroid disease have markers of thyroid autoimmunity. Many have thyroid autoantibodies, but even more have abnormalities by thyroid ultrasonography.

The relationship of white blood cell count to other cardiovascular risk factors.

Baseline data for the 12,866 men from the Multiple Risk Factor Intervention Trial was used to study factors related to white blood cell (WBC) count. White blood cell count was significantly higher in smokers (7853 cells/mm3) and ex-smokers (7091 cells/mm3) who stopped smoking less than one year before than in ex-smokers who stopped more than one year before (6255 cells/mm3) and those who never smoked (6094 cells/mm3). In current cigarette smokers, white blood cell count was significantly related to number of cigarettes smoked, degree of inhalation, and duration of smoking (p less than 0.001 for each). In addition, white blood cell count was higher in non-cigarette smokers who smoked pipes, cigars, or cigarillos than among men who did not smoke tobacco (p less than 0.001). White blood cell count was lower in blacks (by 877 cells/mm3) and Orientals (by 634 cells/mm3) than in whites. Leukocyte count also showed a strong inverse association with high density lipoprotein (HDL)-cholesterol, a positive association with triglycerides independent of cigarette use, and a positive association with low density lipoprotein (LDL)-cholesterol in smokers only. Leukocyte counts were inversely related to total family income and alcohol consumption. We conclude that elevated leukocyte count is independently associated with other risk factors for coronary heart disease (CHD) such as amount and duration of smoking as well as an atherogenic profile, and these relationships should be considered when using white blood cell count as a predictor of coronary heart disease.

Hepatocyte adhesion, growth and differentiated function on RGD-containing proteins.

There is currently much interest in designing synthetic substrates incorporating the cell binding motif RGD for tissue engineering. In this paper, hepatocyte function was examined on two synthetic RGD substrates and compared to that on fibronectin (Fn). One is a 2.3 kD RGD peptide (P-2) containing a single RGD, a short spacer in the middle and an end basic sequence to enhance adsorption. On bacteriological plastic, P-2 induced a rounded cell shape, enhanced differentiated function, and inhibited DNA synthesis. The other, a 73 kD synthetic RGD protein Pronectin F (PnF), contains repeating RGD units interspersed with a structural peptide. PnF induced cell spreading, dedifferentiation, and enhanced DNA synthesis, similar to Fn. In addition, only P-2 showed distinct differences in cell shape and DNA synthesis when coated on bacteriological plastic, or on Immulon II plastic, both intrinsically non-adhesive to cells. On bacteriologic plates coated with P-2, cells were round and showed diminished DNA synthesis while on Immulon II plates, they were spread and showed enhanced DNA synthesis. These results demonstrate that synthetic RGD peptides can induce very different hepatocyte function depending on the context in which they are presented to cells. It is likely that the RGD peptide conformation determines the specificity of cellular response.

Low plasma fibronectin after drowning.

The plasma concentrations of fibronectin were zero for 8 days in a 53-year-old male who was submersed for 5 min, but conscious at the time of admission to hospital. The patient developed multiple organ failure, disseminated intravascular coagulation and finally died after 15 days. The low fibronectin values indicate prolonged severe reticulo-endothelial failure, and may be a prognostic sign in cases of drowning or asphyxia of other etiologies.

Modeling the haemodynamic response in fMRI using smooth FIR filters.

Modeling the haemodynamic response in functional magnetic resonance (fMRI) experiments is an important aspect of the analysis of functional neuroimages. This has been done in the past using parametric response function, from a limited family. In this contribution, we adopt a semi-parametric approach based on finite impulse response (FIR) filters. In order to cope with the increase in the number of degrees of freedom, we introduce a Gaussian process prior on the filter parameters. We show how to carry on the analysis by incorporating prior knowledge on the filters, optimizing hyper-parameters using the evidence framework, or sampling using a Markov Chain Monte Carlo (MCMC) approach. We present a comparison of our model with standard haemodynamic response kernels on simulated data, and perform a full analysis of data acquired during an experiment involving visual stimulation.