Correlation between automated DNA ploidy measurements of Hürthle-cell tumors and their histopathologic and clinical features

The treatment of Hürthle-cell tumors of the thyroid is controversial because of their rarity and the inconsistent histopathologic criteria for their diagnosis. In order to obtain more objective criteria for the management of Hürthle-cell tumors, the nuclear DNA content of cells from 20 cases was measured with the MicroTICAS system and the correlation between the DNA distribution patterns and the clinical and histopathologic findings was evaluated. Three main DNA patterns were found: euploid, polyploid and aneuploid. The euploid or polyploid Hürthle-cell tumors came from patients who did not develop distant metastases or recurrence whereas the aneuploid variants came from patients who died of their disease and/or developed distant metastases and recurrence. Various correlation analyses were performed between DNA ploidy and age, sex, size of tumor, growth pattern, pleomorphism, invasion and metastases. Our data suggests that an aneuploid DNA pattern or one with a large percentage of aneuploid nuclei with DNA content exceeding 5N may predict eventual metastases or recurrence from Hürthle-cell tumor.     

High-resolution color video cytophotometry

Comparison was made between cytophotometric measurements obtained using two data acquisition systems, one a microphotometer and the other a rapid video camera system, to ascertain whether the degradation of data with the faster video acquisition system still results in recorded images of sufficient quality to permit computer discrimination between cells of very similar appearance. Normal-appearing intermediate cells from cases with normal cytology and those from patients with dysplasia or malignant disease, as well as the subvisual markers within these cells that have rendered them capable of cytophotometric discrimination, were used for the study. Comparison of the data recorded by the two systems indicates that the diagnostic information is preserved in the change-over to a full-field, video-rate scanning system, with differences in the data caused primarily by differences in the spectral response of the two systems. This was reflected in the substantial differences observed in the color-related features and the lesser differences seen in the textural features, while the morphometric features (outline and shape) were virtually unaffected. The differences were primarily expressed on a cell-to-cell basis; in sets of about 300 cells, which would be used in patient-to-patient comparisons, the feature values showed remarkable consistency between the two systems.     

Transport and transformation of hexavalent chromium through soils and into ground water

A detailed characterization of the underlying and adjacent soils of a chrome‐plating shop was performed to provide information on the extent of soil and aquifer contamination at the site and on the potential for off‐site migration and environmental impact. Intact, moist cores were obtained from more than 40 different locations, resulting in more than 200 discrete samples for total metal analysis, selective extraction tests, and adsorption‐reduction experiments, to assess the chemical speciation and distribution of chromium on the contaminated soils and its leaching potential. Surface analytical techniques were also used to determine chemical speciation and to further elucidate mineral fractions responsible for retention of the chromium on the soils and sediments. Adsorption and reduction capacities of the saturated aquifer sediments were variable and low, while the unsaturated soils’ reduction capacities were much greater and were correlated with depth (decreasing capacity with increasing depth). The soils’ adsorption and reduction capacities were eventually overwhelmed, however, and permitted the passage of Cr(VI) into the underlying ground water. Adsorption capacity differences were primarily related to clay content and pH, and less so to the presence of amorphous iron oxide coatings on matrix minerals as operationally defined by the selective extraction methods used in the study. Reduction of Cr(VI) to Cr(III) and subsequent precipitation as (Fe, Cr)(OH)₃ is proposed as the primary attenuation mechanism in the unsaturated soils immediately beneath the shop, based on extraction and surface analyses results.     

PRFS-Based MR Thermometry Versus an Alternative T1 Magnitude Method – Comparative Performance Predicting Thermally Induced Necrosis in Hepatic Tumor Ablation

Objective

To compare the accuracy of a semi-quantitative proton resonance frequency shift (PRFS) thermal mapping interface and an alternative qualitative T1 thermometry model in predicting tissue necrosis in an established routine setting of MRI-guided laser ablation in the human liver.

Materials and Methods

34 cases of PRFS-guided (GRE) laser ablation were retrospectively matched with 34 cases from an earlier patient population of 73 individuals being monitored through T1 magnitude image evaluation (FLASH 2D). The model-specific real-time estimation of necrotizing thermal impact (above 54 °C zone and T1 signal loss, respectively) was correlated in size with the resulting necrosis as shown by lack of enhancement on the first-day contrast exam (T1). Matched groups were compared using the Mann-Whitney test.

Results

Online PRFS guidance was available in 33 of 34 cases. Positive size correlation between calculated impact zone and contrast defect at first day was evident in both groups (p < 0.0004). The predictive error estimating necrosis was median 21 % (range 1 % - 52 %) in the PRFS group and 61 % (range 22 - 84 %) in the T1 magnitude group. Differences in estimating lethal impact were significant (p = 0.004), whereas the real extent of therapy-induced necrosis showed no significant difference (p > 0.28) between the two groups.

Conclusion

PRFS thermometry is feasible in a clinical setting of thermal hepatic tumor ablation. As an interference-free MR-tool for online therapy monitoring its accuracy to predict tissue necrosis is superior to a competing model of thermally induced alteration of the T1 magnitude signal.     

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.     

Differences in the Direction of Change of Cerebral Function Parameters Are Evident over Three Years in HIV-Infected Individuals Electively Commencing Initial cART

BackgroundChanges in cerebral metabolite ratios (CMR) measured on ¹H-MRS and changes in cognitive function (CF) are described in subjects commencing combination antiretroviral therapy (cART), although the dynamics of such changes are poorly understood.MethodsNeuroasymptomatic, HIV-infected subjects electively commencing cART were eligible. CMR were assessed in three anatomical voxels and CF assessed at baseline, week 48 and week 144. Overall differences in absolute change in CMRs and CF parameters between 0–48 and 48–144 weeks were assessed.ResultsTwenty-two subjects completed study procedures. Plasma HIV-RNA was <50 copies/mL in all at week 48 and in all, but two subjects at week 144. In general, between weeks 0–48 a rise in N-acetyl-aspartate(NAA)/Creatine(Cr) ratio and a decline in myo-Inositol(mI)/Cr ratio were observed. Between weeks 48–144, small rises in NAA/Cr ratio were observed in two anatomical voxels, whereas a rise in mI/Cr ratio was observed in all anatomical locations (0.31 (0.66) and -0.27 (1.35) between weeks 0–48 and 0.13 (0.91) and 1.13 (1.71) between weeks 48–144 for absolute changes in NAA/Cr and mI/Cr (SD) in frontal-grey voxel, respectively). Global CF score improved between weeks 0–48 and then declined between weeks 48–144 (0.63 (1.16) and -0.63 (0.1.41) for mean absolute change (SD) between weeks 0–48 and weeks 48–144, respectively).ConclusionsThe direction of change of cerebral function parameters differs over time in HIV-infected subjects commencing cART, highlighting the need for long-term follow-up in such studies. The changes we have observed between weeks 48–144 may represent the initial development of cerebral toxicities from cART.     

Novel Biocatalysts by Identification and Design

Enzymes produced from bacteria and eukaryotic organisms are presently being used for a large variety of different biotechnological applications. The rapidly increasing demand for enzymes which are active towards novel and often non-natural substrates has triggered the development of novel molecular biological methods of enzyme isolation and design. The metagenome approach is a cultivation-independent method which allows the direct cloning and expression of environmental DNA thereby providing access to a wealth of so-far unknown biocatalysts. Additionally, newly identified or existing biocatalysts can be further optimized by different methods of directed evolution. Here, the principle of the metagenome approach is outlined and a strategy is presented for the optimization of a bacterial lipase using a combination of rational design and directed evolution.     

Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.     

Deacetylation of xylans by acetyl esterases of Trichoderma reesei

Summary Two previously purified esterases of Trichoderma reesei were used to study the deacetylation of polymeric, oligomeric and dimeric acetylated xylan fragments. For the first time nearly complete enzymatic deacetylation of polymeric xylan with purified acetyl xylan esterase was demonstrated, resulting in precipitation of the remaining polymer structure. The esterases had very different substrate specifities, one having a preference for high molecular weight substrates and the other showing high activity only towards acetyl xylobiose. The latter enzyme was also regioselective, cleaving off the acetyl substituent only from the C-3 position of the xylopyranose ring. The highest xylose yield from acetylated xylan was obtained by the synergistic action of xylanase, \-xylosidase and acetyl xylan esterase. Offprint requests to: M. Sundberg     

A regulatory role for CD37 in T cell proliferation

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.