Module-detection approaches for the integration of multilevel omics data highlight the comprehensive response of Aspergillus fumigatus to caspofungin.

BACKGROUND: Omics data provide deep insights into overall biological processes of organisms. However, integration of data from different molecular levels such as transcriptomics and proteomics, still remains challenging. Analyzing lists of differentially abundant molecules from diverse molecular levels often results in a small overlap mainly due to different regulatory mechanisms, temporal scales, and/or inherent properties of measurement methods. Module-detecting algorithms identifying sets of closely related proteins from protein-protein interaction networks (PPINs) are promising approaches for a better data integration. RESULTS: Here, we made use of transcriptome, proteome and secretome data from the human pathogenic fungus Aspergillus fumigatus challenged with the antifungal drug caspofungin. Caspofungin targets the fungal cell wall which leads to a compensatory stress response. We analyzed the omics data using two different approaches: First, we applied a simple, classical approach by comparing lists of differentially expressed genes (DEGs), differentially synthesized proteins (DSyPs) and differentially secreted proteins (DSePs); second, we used a recently published module-detecting approach, ModuleDiscoverer, to identify regulatory modules from PPINs in conjunction with the experimental data. Our results demonstrate that regulatory modules show a notably higher overlap between the different molecular levels and time points than the classical approach. The additional structural information provided by regulatory modules allows for topological analyses. As a result, we detected a significant association of omics data with distinct biological processes such as regulation of kinase activity, transport mechanisms or amino acid metabolism. We also found a previously unreported increased production of the secondary metabolite fumagillin by A. fumigatus upon exposure to caspofungin. Furthermore, a topology-based analysis of potential key factors contributing to drug-caused side effects identified the highly conserved protein polyubiquitin as a central regulator. Interestingly, polyubiquitin UbiD neither belonged to the groups of DEGs, DSyPs nor DSePs but most likely strongly influenced their levels. CONCLUSION: Module-detecting approaches support the effective integration of multilevel omics data and provide a deep insight into complex biological relationships connecting these levels. They facilitate the identification of potential key players in the organism's stress response which cannot be detected by commonly used approaches comparing lists of differentially abundant molecules.

Application of fuzzy inference to European patients to predict cervical lymph node metastasis in carcinoma of the tongue.

In head and neck cancers, the presence of cervical lymph node metastasis is an important determinant of outcome. Many attempts have been made to predict cervical lymph node metastasis, but the accuracy of currently available techniques remains inadequate. We used fuzzy inference to predict cervical lymph node metastasis retrospectively in 75 patients with squamous cell carcinoma of the tongue and prospectively in 23 patients. Our model was based on three variables: tumor size, keratinization, and mode of invasion. The accuracy of fuzzy inference for the prediction of cervical lymph node metastasis in the 75 patients studied retrospectively was 86.7%, the sensitivity was 70.8%, and the specificity was 94.1%. In the 23 patients studied prospectively, the accuracy was 91.3%, the sensitivity was 50.0%, and the specificity was 95.2%. The accuracy obtained in this European series of patients was similar to that previously obtained in Japanese patients. We conclude that fuzzy inference may be a useful method for predicting cervical lymph node metastasis. Its high specificity is likely to reduce the number of unnecessary neck dissections. However, the current level sensitivity is inadequate for routine clinical use. Therefore, other predictors of lymph node metastasis should be identified to refine the current model.

FH-Freiburg: a novel missense mutation (C317Y) in growth factor repeat A of the low density lipoprotein receptor gene in a German patient with homozygous familial hypercholesterolemia.

We describe the characterization of a novel mutation in the low density lipoprotein receptor (LDL-R) gene in a patient with true homozygous familial hypercholesterolemia (FH). The combined use of denaturing gradient gel electrophoresis (DGGE) and sequencing of genomic DNA revealed a guanine to adenine base substitution at nucleotide position 1013 of the LDL-R cDNA. This point mutation results in a change from cysteine to tyrosine at amino acid residue 317 of repeat A of the epidermal growth factor (EGF) precursor homology domain. Binding, uptake and degradation of iodinated LDL in skin fibroblasts from the homozygous patient were less than 10% of normal. In contrast, binding, uptake and degradation of iodinated VLDL was reduced by only 60, 30, and 38%, respectively. Incubation of the patient's fibroblasts in the presence of cholesterol diminished the residual binding of VLDL by 50%, suggesting that the loss of the highly conserved cysteine at position 317 results in a LDL-R that fails to bind LDL, but retains some ability to bind VLDL by interacting with the apolipoprotein E. Both parents were heterozygous for the C317Y mutation. Interestingly, however, the father presented with markedly elevated levels of triglycerides and VLDL cholesterol, whereas his LDL cholesterol was unexpectedly low. The mother of the index patient had only slightly elevated LDL cholesterol. These observations testify to the biological complexity of genotype-environment interactions in individuals carrying mutations at the LDL-R locus and indicate that genetic analysis importantly complements the clinical and biochemical diagnosis of patients with hyperlipidemia.

Effects of organometals on cellular signaling. II. Inhibition of reincorporation of free arachidonic acid and influence on paf-acether synthesis by triethyllead.

Organometal compounds affect many enzymes, especially those containing SH-groups as acyl- and acetyltransferases involved in lysophospholipid reacylation. In HL-60 cells, organotin and -lead compounds stimulate phospholipase A2 activity, contributing thus to increase the level of lysophospholipids. In the present study, we have tested whether paf-acether (paf) biosynthesis was affected by treatment with triethyllead (Et3PbCl) in HL-60 cells. Et3PbCl inhibits the incorporation of exogenous arachidonic acid in the presence of high (> or = 50 microM) but not low concentrations (< or = 1 microM). High concentrations of the lead compound are unable to induce paf formation by itself, however, lower concentrations (< or = 10 microM) acted synergistically with TPA or fMLP to stimulate paf formation. Whereas unstimulated cells produced 0.4 pmole paf/2 x 10(6) cells, the stimulation with low fMLP (0.1 microM) resulted in the synthesis of 1.7 pmole and with low TPA (2 ng/ml) in 0.5 pmole paf. Preincubation of the cells with 10 microM Et3PbCl for 20 to 30 min increased the amount of paf formed by these cells to 3.3 pmole after treatment with 0.1 microM fMLP and 1.5 pmole after TPA. Furthermore, the results showed an inhibition of acetyltransferase (the key enzyme of paf synthesis) by the high and not by low concentrations of the lead compound. We conclude that low concentrations of Et3PbCl (< or = 10 microM) may act as a synergistic inducer of paf synthesis initiated via a receptor-coupled stimulation.

Preparation of functional group analogs of unsaturated fatty acids and their effects on the circadian rhythm of a fatty-acid-deficient mutant of Neurospora crassa.

Functional group analogs of oleic, linoleic and linolenic acids were prepared by coverting their double bonds to dibromo, cyclopropyl, epoxy, methoxy, and, in the case of oleic acid, hydroxy groups. These compounds were supplemented to the bd csp cel strain of the mold Neurospora crassa. The cel mutation confers a partial requirement for saturated fatty acids and, also, perturbs the circadian rhythm of spore formation. For example, the period of bd csp cel's rhythm is dramatically lengthened upon supplementation by natural cis-unsaturated fatty acids. Of the analogs tested, only the monoepoxy, monomethoxy, dibromo, and hexabromo stearic acids gave significant period lengthening. Other analogs, which should have comparable abilities to disrupt lipid bilayer packing, gave no rhythm effect. Further, the inactive di- and tri-methoxystearic acid analogs were incorporated to a greater extent than the active mono-methoxystearic acid. The results do not, therefore, support a direct alteration in membrane "fluidity' as the mode of action of the period-lengthening fatty acids.

Comparison of fuzzy inference, logistic regression, and classification trees (CART). Prediction of cervical lymph node metastasis in carcinoma of the tongue.

OBJECTIVES: In this paper three statistical methods [logistic regression, classification and regression tree (CART), and fuzzy inference] for the prediction of lymph node metastasis in carcinoma of the tongue are compared. METHODS: A retrospective collection of data in 75 patients treated for tongue cancer was carried out at the Clinic and Policlinic for Oral and Maxillo-facial Surgery at the University Hospital of Freiburg in Germany between January 1990 and December 1999; biopsy material was used for laboratory evaluations. Statistical methods for the prediction of lymph node metastasis were compared using ROC curves and accuracy rates. RESULTS: All three methods show similar results for the prediction of lymph node metastasis with slightly superior results for fuzzy inference and CART. A great overlap is apparent in the ROC curves. The best result observed for fuzzy inference and CART was a sensitivity of 79.2% [95% confidence interval: (57.8%; 92.9%)] and a specificity of 86.3% (73.7%; 94.3%); the best result for predictions based on the logistic regression was a sensitivity of 66.7% (44.7%; 84.4%) and a specificity of 80.4% (66.9%; 90.2%). Accuracy rates of fuzzy method and CART were higher [accuracy rate for fuzzy method and CART: 84% (73.7%; 91.4%), for logistic regression method: 73.3%, 95%-CI: (61.9%; 82.9%)]. CONCLUSIONS: From a clinical point of view, the predictive ability of the three methods is not sufficiently large to justify use of these methods in daily practice. Other factors probably on the molecular level are needed for the prediction of lymph node metastasis.

[Diagnostic and therapeutic management of a big cyst at the floor of the mouth--a case report]. / Diagnostisch-therapeutisches Management einer grossen Epidermoidzyste des Mundbodens--Ein Fallbericht.

BACKGROUND: The growth of dysontogenetic cysts at the floor of the mouth is considered as a rare condition. Cysts of the floor of the mouth may be differentiated depending on location or on histopathologic-embryological criteria into dermoids, epidermoids and teratomas. PATIENTS AND METHODS: We report a case of a large cyst at the floor of the mouth and present the results of clinical examination, preoperative diagnostics and surgical approach. RESULTS: The tumour was resected by an enoral operation. Histological findings confirmed a large sublingual epidermoid cyst. There were no complication or relaps during postoperative course. The case is discussed within the framework of scientific literature. CONCLUSIONS: Dysontogenetic cysts should be considered in differential diagnosis of tumours of the floor of the mouth. Diagnostic and surgical management depends first of all on size and location of dysontogenetic cysts.

Tissue defect at the gunshot entrance wound: what happens to the skin?

To investigate the question what happens to the tissue lost at the entrance wound, experimental studies were performed on composite models consisting of dyed pig skin and gelatin blocks. For the test shots to the skin-gelatin preparations, cartridges calibre .38 spec. with different bullet types (round nose, hollow point, flat nose, truncated cone) were used. In all shots, a multitude of coloured skin particles were macroscopically discernible along the bullet tracks. In addition, small cell aggregations could be demonstrated microscopically even in those sections of the bullet paths which did not show skin fragments visible to the naked eye. The distribution of the skin particles showed certain peculiarities depending on the type of projectile.

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant.

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.

Molecular evolution of the small subunit ribosomal DNA in woodlice (Crustacea, Isopoda, Oniscidea) and implications for Oniscidean phylogeny.

The small subunit ribosomal DNA (ssu rDNA) of 13 isopods was sequenced. The entire length of the ribosomal gene is unusually long, resulting from the presence of five expansion elements accounting for more than 40% of the gene. We found that in terrestrial isopods the length of the ssu rDNA ranges from 2414 bp (Ligidium hypnorum) to 3537 bp (Cubaris murina). This is the longest metazoan ssu rDNA reported to date. The conserved regions are highly informative for analysis of the early nodes of the tree, whereas the variable expansion elements are better suited to reconstruction of the branching pattern between closely related taxa. The suggested relationship among Synochaeta, Crinochaeta, and Diplochaeta based on the conserved regions confirms that based on previous morphological analyses. In contrast, the phylogeny within the Crinochaeta based on the entire ssu rDNA including the variable domains is in conflict with that based on most of the morphological analyses. The phylogenetic analyses of the ssu rDNA support a repeated independent evolution of the three different types of pleopodal lungs in the Crinochaeta.

Circadian rhythms in Neurospora crassa: effects of saturated fatty acids.

To assess their effects on the conidiation rhythm in Neurospora, 14 saturated fatty acids from 6 to 24 carbons long were used to supplement the bd csp and bd csp cel strains. Both strains express a circadian spore-forming rhythm when grown on solid media; the cel mutation confers a partial fatty acid requirement. Fatty acid supplements from 8 to 13 carbons long lengthened the free-running period of bd csp cel compared with the control value of 21 h; the maximal effect (33 h) was obtained with nonanoic acid (9:0) at a concentration of 5 x 10(-4) M. In contrast, the period of bd csp remained unchanged under all experimental conditions. The short-chain fatty acids (<14 carbons) reduced the rate of advance of the growth front in both strains, compared with unsupplemented controls. However, this inhibition did not appear to be responsible for the lengthened periods in bd csp cel. Nor was direct incorporation of the short-chain (period-lengthening) fatty acids into mycelial total lipids responsible, since such incorporation was not observed. In fact, extensive metabolic conversion of these supplements by both strains was indicated by the disappearance of short-chain fatty acids from the agar media coupled with their absence in mycelial lipids, and by the liberation of (14)CO(2) from cultures supplemented with [1-(14)C]lauric acid (12:0).

9(10)-alkoxystearic acids lengthen the circadian period of a fatty acid auxotroph of Neurospora crassa.

9- and 10-Alkoxystearic acids with alkoxy groups of varying size were synthesized by solvomercuration of methyl oleate. These "knobbed" fatty acids were used to supplement a Neurospora strain carrying the cel mutation, which confers a partial fatty acid deficiency. Supplemental unsaturated or short-chain fatty acids are known to lengthen dramatically the circadian period of spore formation (conidiation) in cel strains. A similar dramatic lengthening was obtained with the alkoxy supplements. The change in period, however, did not correlate positively with the size of the knob. Smaller knobbed analogs gave somewhat greater period lengthening and were removed from the medium faster. It may be that the knobbed analogs cause period lengthening by an indirect effect on Neurospora membranes rather than, or in addition to, a direct perturbation.

[Effect of mode of delivery on early morbidity and mortality of premature infants (< or = 34th week of pregnancy)]. / Der Einfluss des Entbindungsmodus auf Frühmorbidität und Mortalität von Frühgeborenen (< or = 34. SSW).

In a retrospective analysis (1985-1994) the influence of the mode of delivery on mortality and early morbidity of low birth weight infants (< or = 2500 gm and < or = 34 weeks of gestation) was assessed (n = 450). We only included cases that offered a choice between abdominal and vaginal delivery (cephalic and breech presentation, premature rupture of membranes and preterm labour). We found significantly higher survival rates in infants with birth weights of 1500 gm or less in case of breech and cephalic presentation after cesarean births. In infants with cephalic presentation and birth weights of 1500 gm or less no parameter of early morbidity was changed by the mode of delivery. Infants with breech presentation and birth weights of 1500 gm or less have less severe cerebral hemorrhages, cord pH values < or = 7.15 and have a lower incidence of primary intubation after abdominal delivery. Infants with birth weights of 1501-2500 gm and cephalic presentation are better off after a vaginal delivery, because there is the same survival rate and some parameters of early morbidity (1' and 5' Apgar score, incidence of primary intubation) are better after spontaneous births. In breech infants with birth weights of 1501-2500 gm there are no differences in mortality and early morbidity after vaginal and abdominal birth. A logistic regression analysis showed, that the risk of dying was significantly higher in infants with cerebral hemorrhages (I degree-IV degrees) and in infants born before 1990. In infants with birth weights of 1500 gm or less the mortality rate was higher after vaginal delivery and in case of an 10' Apgar score <8. Regarding infants with birth weights of 1501-2500 gm the risk of dying was higher in infants that suffered from a respiratory distress syndrome I degree-IV degrees and in case of birth weights of 2000 gm or less.

Circadian rhythms in Neurospora crassa: a mutation affecting temperature compensation.

The circadian rhythm of conidiation (spore formation) in Neurospora crassa is known to be temperature compensated, that is, the period is only slightly affected by the incubation temperature. Thus, the Q10 (the relative rate enhancement corresponding to a 10 degrees C rise in temperature) of the rhythm of the bd csp strain from 14 to 30 degrees C was 1.1, whereas the Q10 of the uncompensated growth rate in the same interval was 2.4. A mutation at the cel locus resulted in loss of the temperature-compensation property in cultures grown below 22 degrees C. The Q10 of the rhythm below 22 degrees C was 2.2, and periods of about 40 hr were observed. In contrast, the Q10 of the rhythm above 22 degrees C was 1.1, with circadian periods of 18-21 hr. Thus, cel displayed a threshold temperature or "break point" for the temperature compensation of its rhythm. Supplementation of cel strains, which require fatty acids, with unsaturated or short-chain fatty acids raised the threshold temperature to 26 degrees C, whereas supplementation with long-chain saturated fatty acids lowered it to 18 degrees C. These data suggest a role for fatty acids, as liquid components or as cellular metabolites, in the mechanism of temperature compensation.

A simplified method of obtaining umbilical cord blood for pH using a heparinized vacutainer versus heparinized syringe.

Two experimental methods for collecting cord blood for acid-base analysis using green top vacutainer tubes were compared with a standard method using heparinized syringes. Paired samples from 30 newborn infants were collected using one of the vacutainer methods and the standard heparinized syringe method. No significant differences in pH, pCO2, base deficit, or pO2 were noted when comparing the two vacutainer methods with the standard syringe method. Both vacutainer methods were substantially easier to use than the heparinized syringe method. A green top vacutainer may be used to obtain an accurate cord blood sample for assessment of an infant's acid-base status at birth, and to aid the neonatologist as an indicator of intrapartum asphyxia.

[Myelodysplastic syndromes (MDS). Aspects of hematopathologic diagnosis]. / Myelodysplastisches Syndrom (MDS). Aspekte der hämatopathologischen Diagnostik.

Myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders which generally occur in older adults but may also affect children. Primary MDS should be distinguished from secondary MDS associated with antineoplastic or immunosuppressive therapy (t-MDS), exposure to toxic compounds, or genetic disorders. The establishment of a neoplastic clone is reflected by dysplastic features and impaired function which may affect all three hematopoietic cell lineages. The ineffective hematopoiesis which causes bone marrow failure is accompanied by peripheral blood cytopenia and is considered to result from increased apoptosis, at least in the less advanced MDS stages. The elucidation of the molecular pathogenesis of MDS has provided evidence that chromosomal abnormalities are present in about 50% of patients with primary MDS. They include numerical aberrations such as monosomy 5 or 7, trisomy 8, loss of the Y-chromosome and structural abnormalities such as deletion of the long arm of chromosome 5 (5q-syndrome), 7, or 8. Based on the percentage of blasts (<5%, 5-20%, 20-30%) and the presence of >15% ringed sideroblasts for marrows with <5% blasts, the French-American-British (FAB) classifies MDS into 4 morphologic categories: refractory anemia (RA), refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-t), and refractory anemia with ringed sideroblasts. The fifth morphologic type is chronic myelomonocytic leukemia characterized by peripheral blood monocytosis (>1x10(9)/l). However, a modification of this classification will be proposed by the World Health Organization, with the intention of lowering the threshold for the diagnosis of AML from 30% to 20% blast cells. In patients presenting with cytopenias suggesting impaired hematopoiesis, the initial diagnosis depends mainly on the cytological evaluation of bone marrow and blood smears and the histological findings of trephine bone marrow biopsy. In a retrospective analysis we evaluated the occurrence of the distinct FAB-categories as percentage of the total number of MDS cases diagnosed at the Institute of Pathology of the University of Freiburg. A total of 63% fullfilled the criteria of RA/RARS, 17% of RAEB, 14% of RAEB-t, and 6% of CMML. A fibrotic variant of MDS was observed in 7.67% of all cases, ranging from 2.34% in RA up to 15. 42-15.84% in the categories which did not show significant differences with regard to myelofibrosis. The histologic evaluation of a trephine bone marrow biopsy is of critical importance for the evaluation of fibrotic or hypocellular MDS since these patterns are not reflected by the cytological examination. The combined cytological and histological diagnosis of bone marrow and peripheral blood is a reliable tool for the initial diagnosis of MDS. In addition, cytogenetic and molecular analysis should be performed. Presently, the risk of leukemic transformation is evaluated using the International Prognostic Scoring System for MDS, which is the sum of the scores of bone marrow blasts, karyotypes and cytopenia. In the context of clinical trials therapeutic modalities should be considerd according to the age and the general performance state and the prognostic scores of individual patients.

Cembrene A and a congeneric ketone isolated from the paracloacal glands of the Chinese alligator (Alligator sinensis).

A diterpene hydrocarbon and diterpene ketone isolated from the paracloacal gland secretions of the Chinese alligator (alligator sinensis) were examined by GC-MS and 1H-NMR, 13C-NMR, and IR spectroscopy, and identified as 4,8,12-trimethyl-1-(1-methylethenyl)-3,7,11-cyclotetradecatrien e (cembrene A, 1) and its congeneric ketone, 4,8,12-trimethyl-1-(1-methylethenyl)-3,7-cyclotetradecadien- 10-one (2), respectively. This is the first report of cembrene A from a vertebrate; the ketone has not been described previously.

[Proliferative activity and p53 expression in transitional cell carcinoma of the urinary bladder]. / Proliferative Aktivität und Expression des p53-Proteins in Ubergangszellkarzinomen der Harnblase.

Mutations of the p53 gene are important mechanisms in malignant transformation and are associated with dysregulation of normal cell growth. In the present study the expression of mutated p53-protein and proliferating cell nuclear antigen (PCNA) was investigated in a series of 31 human transitional cell carcinomas (TCC) by immunohistochemistry (IHC). The number of PCNA-positive cells and the pattern of expression was distinct in normal urothelium being confined to the basal cell layer. In dysplastic urothelium and in Carcinoma in situ (CIS) PCNA-immunoreactive nuclei were irregularly distributed throughout all layers. In tumor cell complexes the pattern of PCNA-immunoreactivity was different in papillary and primary infiltrating TCCs. Densitometric quantification of the intensity of the PCNA-reactivity using image analysis revealed an increase from normal to dysplastic urothelium and from dysplastic urothelium to invasive tumors. 21/31 (68%) of the tumors and tumor-associated CIS showed overexpression of p53 varying in percentage, pattern and reaction intensity. The percentage of PCNA-positive cells was higher in tumors overexpressing p53. Double IHC showed colocalization of both molecules in a significant proportion of tumor cells suggesting a link of p53 overexpression and the abnormal proliferative activity. The present results show that p53 over-expression is found in a significant percentage of TCCs and indicate a close association with a defective growth regulation resulting in increased PCNA-levels and enhanced cellular proliferation.

Acquired pure megakaryocytic aplasia: a separate haematological disease entity or a syndrome with multiple causes?

We report the case of a patient with acquired pure megakaryocytic aplasia. Until today, less than 20 cases of acquired pure megakaryocytic aplasia have been reported and the disease aetiology still seems to be unclear. This report summarizes the published data concerning possible aetiologies, treatment options and outcome of patients with acquired pure megakaryocytic aplasia. Furthermore, this case report presents an example for a possible disease progression.