N(pro) fusion technology: On-column complementation to improve efficiency in biopharmaceutical production.

N(pro) fusion technology, a highly efficient system for overexpression of proteins and peptides in Escherichia coli, was further developed by splitting the autoprotease N(pro) into two fragments to generate a functional complementation system. The size of the expression tag is thus reduced from 168 to 58 amino acids, so by 66%. Upon complementation of the fragments auto-proteolytic activity is restored. This process has been shown for three model proteins of different size, a short 16 aa-peptide, MCP-1, and lysozyme. Moreover, the complementation was still functional after immobilization of the N-terminal fragment to a solid support which enables recycling of the immobilized fragment. This strategy enhances overall productivity of N(pro) Fusion Technology and thus allows more efficient production of recombinant proteins with reduced costs and in higher yields. Overall, the N(pro) complementation system has, depending on the size of the target molecule, potential to increase the productivity up to 4 fold for batch refolding and even more for on-column refolding strategies by the proven possibility of regeneration of the immobilized fragment.

ß-catenin-independent WNT signaling in basal-like breast cancer and brain metastasis.

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of ß-catenin remained uninfluenced. Consistently, ß-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. ß-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

Diffusion-weighted MRI for tumour volume delineation: comparison with morphological MRI.

Diffusion-weighted magnetic resonance imaging (dwMRI) is sensitive to tissue microstructure on the cellular level and may therefore help to define biological tumour subvolumes and add complementary information to morphology-based cancer treatment protocols and therapy monitoring. The purpose of this study was therefore to evaluate the potential of dwMRI as compared with morphological MRI (mMRI) for tumour volume delineation using a nude rat human tumour xenograft model. Sixteen tumour-bearing rats (10 H1299, six FaDu) were examined with mMRI (T2-weighted true fast imaging with steady precession (TrueFISP), T1-weighted fast low angle shot (FLASH), T2-weighted dual echo steady state (DESS)) and echo-planar dwMRI in a clinical scanner at 1.5 T. For each method, we compared tumour volume and intra- and inter-observer variability of tumour outer edge delineation (disregarding intra-tumoural structure) as well as tumour signal-to-noise ratio (SNR) and tumour-to-muscle contrast-to-noise ratio (CNR). Tumours were visualised with significantly higher SNR and CNR in dwMRI. Median tumour volumes as measured by dwMRI (3.5 cm(3)) and mMRI (TrueFISP: 3.3 cm(3); FLASH: 3.3 cm(3); DESS: 3.2 cm(3)) were not significantly different and significantly correlated. Related to partial volume effects, the intra- and inter-observer variability of dwMRI (intra/inter: 12%/12%) was larger than for mMRI (TrueFISP: 4%/4%; FLASH: 5%/5%; DESS: 5%/5%). In conclusion, dwMRI allows tumour delineation with overall volume estimation comparable with mMRI approaches but slightly higher observer variability. Thus, besides tumour outline, it may potentially supplement morphology-based therapy planning and monitoring with additional biological information.

Tightening elongated ACL grafts by application of bipolar electromagnetic energy (ligament shrinkage).

In ACL transplants showing elongation with functional deficit but no interruption of structure (Marburg Arthroscopic Score type III) there is the possibility of reinsertion, augmentation, or revision ACL reconstruction to stabilize the joint. Bipolar electromagnetic energy can be used to induce the tightening of elongated ACL transplants. We examined 14 patients with a secondary instability after ACL transplantation (mean 23.6 months). During arthroscopy a type III transplant without signs of nonisometric position was found. The elongated transplants were treated by bipolar application probe. All knees where protected by an unlimited orthosis over a period of 12 weeks. Postoperative follow-up took 9.4 months. There was a significant improvement in subjective discomforts (35.9 to 88.5 points on the Lysholm score), and the Rollimeter test showed a reduction of the ventral tibial translation (13.1 to 2.6 mm). There was a correlation between the degree of chondral damage and a Lysholm score but not between subjective discomforts and the degree of instability. Our findings confirm that elongated ACL transplants can be tightened by applying bipolar electromagnetic energy during rearthroscopy, thus avoiding revision ACL reconstruction in some cases.

[Significance of minimal residual disease for the estimation of the prognosis and for therapeutic decisions in solid tumors]. / Der Stellenwert der minimalen Resterkrankung für die Einschätzung der Prognose und für Therapieentscheidungen bei soliden Tumoren.

The detection of disseminated tumor cells in bone marrow and blood is increasingly used for staging and therapeutic decisions in breast cancer and other solid tumors. Molecular biological methods improve the diagnostic accuracy. Limitations of the approach relate to the lack of disease-specific marker genes. The detection of tumor cells in the bone marrow after primary therapy is a negative prognostic parameter in many solid tumours. Axillary lymph node dissection and histopathology remain the standard staging procedure in breast cancer, but nodal negative patients exhibiting tumor cells in the bone marrow have an inferior outcome and may benefit from adjuvant therapy. The immunohistochemical and molecular detection of tumour cells in lymph nodes reduces the number of truly nodal-negative patients considerably. Tumour cells in bone marrow and blood may be used to directly monitor therapeutic responses.

Diagnostic challenges in aspiration cytology of the salivary glands.

The main goal of fine-needle aspiration (FNA) of salivary gland lesions is to assist the clinician in the management of patients who present with a mass lesion. Cytologic examination aims to determine, if a process is inflammatory and/or reactive, benign or malignant neoplasm and if possible renders a specific diagnosis. It has been argued that in the area of salivary gland tumors, surgical management relies less heavily on a specific preoperative diagnosis, because almost all neoplastic salivary gland lesions will undergo surgical excision. However, knowing beforehand if a lesion is malignant or benign, will aid in planning surgery and may prompt or postpone decisions for surgical intervention. The salivary glands are unique in their histologic complexity and morphological variability of tumors, which is reflected in the cytologic material. In addition to the overlapping morphologic patterns of salivary gland tumors, they also represent relatively rare lesions, thus making it more difficult to acquire diagnostic expertise in FNA. Other than approaching salivary gland tumors by a description of single entities in their benign and malignant categories, we favor a more practical approach to diagnosis based on the key morphologic features noted in FNAs. This article addresses differential diagnoses according to the predominant cytologic presentation with attention to the cell type and size, nature of the cytoplasm, and the smear background.

Syntheses and structural characterization of dinuclear and tetranuclear iron(III) complexes with dinucleating ligands and their reactions with hydrogen peroxide.

The iron(III) complexes [Fe(2)(HPTB)(mu-OH)(NO(3))(2)](NO(3))(2).CH(3)OH.2H(2)O (1), [Fe(2)(HPTB)(mu-OCH(3))(NO(3))(2)](NO(3))(2).4.5CH(3)OH (2), [Fe(2)(HPTB)(mu-OH)(OBz)(2)](ClO(4))(2).4.5H(2)O (3), [Fe(2)(N-EtOH-HPTB)(mu-OH)(NO(3))(2)](ClO(4))(NO(3)).3CH(3)OH.1.5H(2)O (4), [Fe(2)(5,6-Me(2)-HPTB)(mu-OH)(NO(3))(2)](ClO(4))(NO(3)).3.5CH(3)OH.C(2)H(5)OC(2)H(5).0.5H(2)O (5), and [Fe(4)(HPTB)(2)(mu-F)(2)(OH)(4)](ClO(4))(4).CH(3)CN.C(2)H(5)OC(2)H(5).H(2)O (6) were synthesized (HPTB = N,N,N',N'-tetrakis(2-benzimidazolylmethyl)-2-hydroxo-1,3-diaminopropane, N-EtOH-HPTB = N,N,N',N'-tetrakis(N' '-(2-hydroxoethyl)-2-benzimidazolylmethyl)-2-hydroxo-1,3-diaminopropane, 5,6-Me(2)-HPTB = N,N,N',N'-tetrakis(5,6-dimethyl-2-benzimidazolylmethyl)-2-hydroxo-1,3-diaminopropane). The molecular structures of 2-6 were established by single-crystal X-ray crystallography. Iron(II) complexes with ligands similar to the dinucleating ligands described herein have been used previously as model compounds for the dioxygen uptake at the active sites of non-heme iron enzymes. The same metastable (mu-peroxo)diiron(III) adducts were observed during these studies. They can be prepared by adding hydrogen peroxide to the iron(III) compounds 1-6. Using stopped-flow techniques these reactions were kinetically investigated in different solvents and a mechanism was postulated.

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis.

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.

Follow-up of atypical glandular cells in cervical-endocervical smears.

Atypical glandular cells of undetermined significance (AGUS) is a diagnostic category of the Bethesda system encompassing glandular-type cells that show either endometrial or endocervical differentiation and display greater atypia than expected for a reactive process but do not meet the criteria for invasive adenocarcinoma. We investigated AGUS in a follow-up study of cervical-endocervical smears with either histology or repeat cytology follow-up. From the cytology files at Northwestern Memorial Hospital over a 4-year period, 136 cervical-endocervical smears were diagnosed with AGUS, which were further subdivided into atypical glandular cells, unqualified (AGC-U); atypical glandular cells, favor reactive (AGC-FR); or atypical glandular cells, favor neoplasia (AGC-FN). Of 96 cases with either histologic or cytologic (cervical-endocervical smear) follow-up, 39 cases of AGC-U had a variety of diagnoses on follow-up, with mostly benign entities in 72% and squamous intraepithelial lesions in 28%. Follow-up of the 36 cases of AGC-FR also demonstrated mostly benign entities (82%) and five cases of squamous intraepithelial lesions. The largest number of premalignant and malignant diagnoses (48%) was found during follow-up of patients with an initial diagnosis of AGC-FN, including the only two cases of adenocarcinoma in situ in our study. In conclusion, our study confirms that AGUS encompasses a wide spectrum of diagnoses, most of which prove to be benign. Subclassification of these cases into "favor reactive" and "favor neoplasia" was found to be helpful in predicting the follow-up status of these patients. However, the small but distinctive percentage of preneoplastic and neoplastic diagnoses seen on follow-up warrant further diagnostic procedures and/or close monitoring in patients with this diagnosis.

[Adenosarcoma and other uterine sarcomas]. / Adénosarcomes et autres sarcomes utérins.

Sarcomas are tumors of conjunctive origin, rarely relating to the gynaecological sphere; among these, the adenosarcoma only represent one particular aspect. From one clinical case and from literature, the authors attempt to sum up the situation on the different clinical, para-clinical and therapeutic aspects of those bad-know neoplasia.

The effects of four commonly used drugs on platelet function.

A number of commonly used drugs have been reportd to inter