Determining Protease Substrates Within a Complex Protein Background Using the PROtein TOpography and Migration Analysis Platform (PROTOMAP).

The PROtein TOpography and Migration Analysis Platform (PROTOMAP) approach is a degradomics technique used to determine protease substrates within complex protein backgrounds. The method involves protein separation according to protein relative mobility, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel lanes are then sliced into horizontal sections, and in-gel trypsin digestion performed for each gel slice. Extracted peptides and corresponding proteins are identified using liquid chromatography-tandem mass spectrometry and bioinformatics. Results are compiled in silico to generate a peptograph for every identified protein, being a pictorial representation of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins shown by their peptograph to have migrated further through the gel (i.e., to a lower gel slice) in the lane containing the active protease(s) of interest, as compared to the control, are deemed putative protease substrates. PROTOMAP has broad applicability to a range of experimental conditions and protein pools. Coupling this with its simple and robust methodology, the PROTOMAP approach has emerged as a valuable tool with which to determine protease substrates in complex systems.

Larval morphology of Metaphycus flavus and its role in host attachment and larval cannibalism.

Metaphycus flavus (Howard) (Hymenoptera: Encyrtidae) is a facultatively gregarious endoparasitoid of soft scales (Hemiptera: Coccidae). When it develops in superparasitised hosts, the larvae often attack and consume brood mates six or more days post oviposition. Under our laboratory conditions (25±1°C and 14 hours of light followed by 18±1°C and ten hours of darkness in 50-70% R.H.), M. flavus eggs hatched three days after oviposition. Measurements of the mandibles and tentorium indicate there are four larval instars, and M. flavus reaches the fourth instar by day six post oviposition, and pupates on day eight. Thus, cannibalism among M. flavus larvae occurs during the fourth instar. During this instar, M. flavus larvae separate from their attachment to the scale cuticle, to which they were tethered by a respiratory structure during the previous three larval instars. Once detached, they are free to move within the scale, which increases the probability of larval encounters and aggressive behaviours. Moreover, the mandibles of the fourth instar are better adapted for fighting than are those of the first three larval instars, since they are larger and more sclerotized. The cranium and mouthparts of M. flavus have four different types of sensory organs, some of which are almost certainly olfactory, an unexpected function for a larva that presumably is surrounded by an aqueous medium where gustatory sensilla would seem to be more appropriate. The cranium also bears two pairs of what appear to be secretory pores.

Regulatory T cells in peripheral blood, lymph node, and thyroid tissue in patients with medullary thyroid carcinoma.

BACKGROUND: Immunological response of the human body is controlled by the suppressive characteristics of regulatory T cells (Tregs). In various diseases a change in the number of Tregs is evident. For example, whereas Tregs are reduced in auto-immunological processes, an increase of Tregs is found with various malignant tumors. Regarding medullary thyroid carcinoma (MTC) no such studies have been performed to date. METHODS: Expression of CD4 and CD25 in CD45+ leukocytes from blood and lymph nodes was studied by flow cytometry in patients with MTC and patients with benign goiter. We also examined the marker forkhead box P3 (FoxP3), an intracellular transcription factor, which is supposed to be the most specific marker for Tregs. Immunohistochemical staining for FoxP3 was performed on lymph node and thyroid tissue. RESULTS: The number of FoxP3+ lymphocytes in peripheral blood was significantly higher in patients with MTC than in controls (p = 0.02). This result was confirmed immunohistochemically in lymph node and thyroid tissue, as well as in carcinoma tissue. No difference in CD4+CD25+ lymphocytes was observed between the two groups. After clinical staging (International Union against Cancer-UICC-stages) of MTC patients, triplication of FoxP3+ lymphocytes could be observed from MTC < UICC II to MTC > UICC II. CONCLUSIONS: An increase of FoxP3+ lymphocytes could be shown in peripheral blood of patients with MTC but not in patients with benign goiter; this increase also correlates with findings in lymph nodes and thyroid gland. The number of FoxP3+ cells correlated with the patients' prognosis. Therefore, FoxP3+ lymphocytes are a good diagnostic criterion for malignancy in patients with medullary thyroid carcinoma, and their presence at staging may influence therapeutic decisions.

A modular approach to linear uncertainty analysis.

This paper introduces a methodology to simplify the uncertainty analysis of large-scale problems where many outputs and/or inputs are of interest. The modular uncertainty technique presented here can be utilized to analyze the results spanning a wide range of engineering problems with constant sensitivities within parameter uncertainty bounds. The proposed modular approach provides the same results as the traditional propagation of errors methodology with fewer conceptual steps allowing for a relatively straightforward implementation of a comprehensive uncertainty analysis effort. The structure of the modular technique allows easy integration into most experimental/modeling programs or data acquisition systems. The proposed methodology also provides correlation information between all outputs, thus providing information not easily obtained using the traditional uncertainty process based on analyzing one data reduction equation (DRE)/model at a time. Finally, the paper presents a straightforward methodology to obtain the covariance matrix for the input variables using uncorrelated elemental sources of systematic uncertainties along with uncorrelated sources corresponding to random uncertainties.

Update on liver transplantation using cyclosporine.

After the introduction of cyclosporine into liver transplantation in 1983, 1-year patient survival more than doubled. Later, with the improved microemulsified formulation of cyclosporine (Neoral) more stable pharmacokinetics were achieved. Today, C(2) monitoring of cyclosporine blood levels allows a more accurate estimation of the area under the concentration-versus-time curve as the single best indicator of cyclosporine exposure. As a consequence, with better control of side effects as well as desired effects the results of cyclosporine in liver transplantation have been further improved. The introduction of mycophenolate mofetil and basiliximab/daclizumab combination therapy has provided new options for the prevention of allograft rejection. The safety profile of individual immunosuppressive regimens comes more into focus since acute allograft rejection may be controlled successfully with competing strategies. As the focus in liver transplantation is shifting toward greatly improved long-term results, late posttransplant mortality with a functioning graft is a major concern. Prevention of long-term complications associated with highly effective immunosuppressants--posttransplant lymphoproliferative disease, cytomegalovirus infection, diabetes, hypertension, and hyperlipidemia-gains importance. Technical advances in living-related and cadaveric split-liver transplantation have lead to increasing use of segmental liver transplantation with the need to consider the effects of immunosuppression on liver regeneration and metabolism. The individualized orchestration of immunosuppression taking into account the underlying liver disease as well as other individual predispositions remains a future challenge.

A simple numerical procedure for estimating nonlinear uncertainty propagation.

Accurately determining the effect of the propagation of uncertainty in nonlinear applications can be awkward and difficult. The Monte Carlo approach requires statistically significant numbers of function evaluations (typically 10(5) or more) and analytical methods are intractable for all but the simplest cases. This paper derives and demonstrates a method to estimate the propagation of uncertainty in nonlinear cases by representing the function in a piecewise fashion with straight line segments. The probability density function of the result can be calculated from the transformation of the line segments. The mean and confidence intervals of the result can then be calculated from the probability density function. For the special case of a normal distribution in the independent variable, calculation of the mean and confidence intervals requires evaluation of only the error function (erf). A simple example is presented to demonstrate the technique. Variations on the basic approach are presented and discussed.

Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps.

The intracellular bacterium Wolbachia is one of the most common symbionts in arthropods and, because of its manipulative effects on host reproduction, is assumed to be an important factor in several evolutionary processes. These bacteria are mainly vertically transmitted from mother to daughter through the egg cytoplasm, and horizontal transmission is generally assumed to be rare. Here, we show natural inter- and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia between parasitoid wasps of the genus Trichogramma. Horizontal transfer was observed when infected and uninfected larvae shared the same host egg. This is the first report, to our knowledge, on interspecific horizontal transfer of Wolbachia between closely related sympatric species. Some originally uninfected immature wasps acquired Wolbachia while inside the host egg, but not all of these newly infected females exhibited the parthenogenesis phenotype. In general, intraspecific horizontal transfer was more successful than interspecific transfer. Wolbachia underwent vertical transmission in the new species but the infection tended to be lost within several generations. Our results have important implications for understanding the evolution of Wolbachia-host associations.

[Surgical treatment for hilar cholangiocarcinoma (Klatskin's tumor)]. / Chirurgische Therapie hilärer Gallengangskarzinome (Klatskin-Tumoren).

Surgical treatment of hilar bile duct carcinoma remains difficult, which is due to the inadequate possibilities in assessing tumor extent during the preoperative diagnostic procedure as well as intraoperatively. Radical resection with negative histologic margins offers the best chance for long-term survival. The decision regarding the appropriate surgical approach is challenging due to the complexity of tumor localization and neighboring vascular structures. Aggressive resection demands extended liver resection, which is associated with the risk of postoperative liver failure. However, even limited surgery such as hilar resection can be curative and leads to long-term survival in individual cases. The principles of surgical oncology have led to more aggressive procedures, including the combination of liver transplantation and multivisceral resection, and can be performed with calculable morbidity and mortality. Nevertheless, the high risk of tumor recurrence under long-term immunosuppression, the limited availability of donor organs and the excellent results of liver transplantation in non-malignant diseases do not justify this procedure at present. Neoadjuvant radiochemotherapy has failed to demonstrate major benefit. In patients with irresectable tumor or distant metastases palliative measures are aimed at restoring an unobstructed bile flow with endoscopic placement of metal stents. Palliative treatment with additional radio- or photodynamic therapy may be considered in individual cases.

[Living kidney transplantation. A comparison of Scandinavian countries and Germany]. / Lebendnierenspende. Ein Vergleich zwischen den skandinavischen Ländern und Deutschland.

The discussion of compensating for shortages of cadaveric donation with increased living donation often reveals differences between the Scandinavian countries and Germany. Possible adoption of Scandinavian structures to improve the rate of living donations in Germany warrants analysis of the actual differences between these two regions. Close examination reveals that significantly higher rates of living donation are achieved only in Sweden and Norway. In Norway, a frequently postulated negative effect on cadaveric donation due to very high rates of living donation could not be confirmed. In contrast to Germany and as a consequence of Norwegian geography, kidney transplantation has been regarded in Norway as the first-line therapy for endstage renal disease for more than 35 years. Living donation has since been actively pursued and is traditionally the transplantation of first choice. In Germany, living donation is still regarded as the second choice after cadaveric donation, due to legal regulations. Significant improvements in living donation frequencies could be achieved there by adopting the active Norwegian approach to living donor identification.

Successful outcome of acute graft-versus-host disease in a liver allograft recipient by withdrawal of immunosuppression.

BACKGROUND: Graft-versus-host disease (GVHD) after liver transplantation is uncommon, and the outcome is almost always fatal. Since 1987, about 30 cases have been described, and patient survival is mostly exceptional. METHODS: A 29-year-old man underwent retransplantation due to chronic cholestatic syndrome, 5 years after his first liver transplantation. Indication for the first liver transplantation was acute liver failure caused by exsiccosis. After the second transplantation, the patient had an initially uneventful course, developing thrombocytopenia at day 21 followed by skin rash and septic complications. Diagnosis of acute GVHD was made by using serological techniques for HLA-A and HLA-DRB and subsequently by fluorogenic sequence-specific primed polymerase chain reaction. In addition, donor lymphocytes were marked by immunohistochemical methods via biopsies of the skin. Immunosuppressive therapy was withdrawn to allow the patient's own immune system to eliminate donor cells. RESULTS: By withdrawing the immunosuppressive therapy, clinical and morphological signs of GVHD vanished. The patient is doing well without recurrence 13 months after transplantation. CONCLUSION: Withdrawal of immunosuppressive therapy is a promising approach in the treatment of acute GVHD to allow the patient's immune system to reconstitute itself, reject offending lymphocytes, and avoid lethal septic complications.

meso-1,2-bis(methylazo)-1,2-diphenylethane.

The title compound, meso-1,2-bis(methyldiazenyl)-1,2-diphenylethane, C16H18N4, is arranged in a disordered manner around an inversion point. The N-N atom distances in the azo group of 1.192 (8) and 1.195 (8) A, and the C-C atom distances in the ethylene moiety at 1.512 (8) and 1.503 (8) A in the two models [refined to 51.7 (6) and 48.3 (6)% occupancies] were not significantly different.

Synthesis and characterization of ReCl(H(2))(AsMePh(2))(4), a classical hydride complex; reexamination of ReCl(H(2))(PMePh(2))(4) and theoretical calculations on model compounds.

The synthesis of ReCl(H(2))(AsMePh(2))(4) is reported and verified on the basis of (1)H NMR and FAB-MS. The minimum T(1) time for the metal-bonded hydrogen atoms was determined to be 44 ms (-78 degrees C) and 89 (-48 degrees C) ms at 200 and 400 MHz, respectively. From this minimum T(1) time, and, allowing for contributions from other nuclei to the relaxation, a metal-bonded H to H atom distance of 1.57 A can be assessed. The complex is reversibly oxidized at a potential E(1/2)(ox) = +0.07 V. The J(HD) coupling in the related ReCl(HD)(AsMePh(2))(4) compound was at 3.3 Hz. These data for ReCl(H(2))(AsMePh(2))(4) suggest that this molecule contains classical hydrides, and interpretations on T(1) and X-ray data published previously for ReCl(H(2))(PMePh(2))(4) suggest that this molecule contains a very long metal-bonded H-H interaction of 1.39 A. Theoretical calculations on model compounds ReCl(H(2))(XR(3))(4) (X = P, As; R = H, Me) give virtually identical average d(H-H) values of 1.5 (XH(3)) and 1.63 (XMe(3)) A.

Selfish element maintains sex in natural populations of a parasitoid wasp.

Genomic conflicts between heritable elements with different modes of inheritance are important in the maintenance of sex and in the evolution of sex ratio. Generally, we expect sexual populations to exhibit a 1:1 sex ratio. However, because of their biology, parasitoid wasps often exhibit a female-biased sex ratio. Sex-ratio distorters can further alter this optimum, sometimes leading to the complete loss of sexual reproduction. In the parasitoid wasp Trichogramma kaykai ca. 4-26% of females in field populations are infected with a bacterial sex-ratio distorter, Wolbachia, allowing virgin mothers to produce daughters. In some micro-Hymenoptera these infections have led to the complete loss of sex, but in field populations of T. kaykai the proportion of individuals infected remains relatively stable. We tested several hypotheses to explain this low infection level, including inefficient and horizontal transmission of Wolbachia, suppressor genes negating the effect of Wolbachia and the presence of male-biasing sex-ratio distorters. Here, a male-biasing sex-ratio distorter, a parasitic B chromosome, causing females to produce only sons, keeps the frequency of Wolbachia low. The male-biasing factor of T. kaykai is the second known case of a B chromosome manipulating the reproduction of a parasitoid wasp.

Infectious parthenogenesis.

Parthenogenesis-inducing Wolbachia bacteria are reproductive parasites that cause infected female wasps to produce daughters without mating. This manipulation of the host's reproduction enhances the transmission of Wolbachia to future generations because the bacteria are passed on vertically only from mothers to daughters. Males are dead ends for cytoplasmically inherited bacteria: they do not pass them on to their offspring. Vertical transmission of Wolbachia has been previously considered to be the main mode of transmission. Here we report frequent horizontal transmission from infected to uninfected wasp larvae sharing a common food source. The transferred Wolbachia are then vertically transmitted to the new host's offspring. This natural and unexpectedly frequent horizontal transfer of parthenogensis-inducing Wolbachia intraspecifically has important implications for the co-evolution of Wolbachia and their host.

[Perfusion parameters in MRI diagnosis of pancreas transplants]. / Perfusionsparameter in der MRT-Diagnostik von Pankreastransplantaten.

PURPOSE: Evaluation of the role of perfusion parameters in the detection of circulatory disturbance and chronic rejection in patients after pancreas transplantation. MATERIALS AND METHODS: 70 examinations of 39 patients after pancreas transplantation were performed. Using a dynamic gadolinium-enhanced Turbo-FLASH-sequence, we evaluated the perfusion parameters in a group of patients with chronic rejection, with circulatory disturbance, and in a control group with normal organ function. RESULTS: There were statistically significant differences of the perfusion parameters in patients with chronic rejection and circulatory disturbance compared to the control group. CONCLUSION: Dynamic MRI can help detect patients with chronic rejection and circulatory disturbance and should therefore be part of the routine follow-up in patients after pancreas transplantation.

Tris(1,10-phenanthroline-N,N')chromium(III) triperchlorate hydrate.

The structure of the cation in [Cr(C(12)H(8)N(2))(3)](ClO(4))(3).H(2)O consists of the Cr atom bonded to the N atoms of the three 1,10-phenanthroline (phen) ligands, resulting in a distorted octahedral arrangement with the six Cr-N distances ranging from 2.040 (4) to 2.055 (4) A. One of the perchlorate anions is disordered and is located around two special positions.

ConStruct: a tool for thermodynamic controlled prediction of conserved secondary structure.

A tool for prediction of conserved secondary structure of a set of homologous single-stranded RNAs is presented. For each RNA of the set the structure distribution is calculated and stored in a base pair probability matrix. Gaps, resulting from a multiple sequence alignment of the RNA set, are introduced into the individual probability matrices. These 'aligned' probability matrices are summed up to give a consensus probability matrix emphasizing the conserved structural elements of the RNA set. Because the multiple sequence alignment is independent of any structural constraints, such an alignment may result in introduction of gaps into the homologous probability matrices that disrupt a common consensus structure. By use of its graphical user interface the presented tool allows the removal of such misalignments, which are easily recognized, from the individual probability matrices by optimizing the sequence alignment with respect to a structural alignment. From the consensus probability matrix a consensus structure is extracted, which is viewable in three different graphical representations. The functionality of the tool is demonstrated using a small set of U7 RNAs, which are involved in 3'-end processing of histone mRNA precursors. Supplementary Material lists further results obtained. Advantages and drawbacks of the tool are discussed in comparison to several other algorithms.

CMV in kidney transplantation: a single center experience over 22 years.

Analysis of a historic renal transplant population for risks of developing CMV disease demonstrated a low mortality (0.2%) and morbidity. In our population of 1,959 patients, 411 (21%) developed subclinical CMV infection and 220 (11%) had CMV disease which was severe in 41 (2%). Important factors for infection were baseline immunosuppression, indicating that triple therapy with the proliferation inhibitors, azathioprine and MMF, had significantly higher infection numbers in comparison to dual, CsA-based immunosuppression. The cumulative dose of steroids correlated strongly with an increased number of CMV infections and disease, as did the addition of ALG/ATG or OKT3 for either steroid-resistant rejections or induction therapy. While CMV serology had an impact on infection in cases of seropositive donors to seronegative recipients, seropositive patients, in general, demonstrated increased infection rates most likely due to reactivation of the virus. Prophylaxis had no impact on the incidence of infection but reduced the severity.