Nondestructive Chemical Analysis of the Iron-Containing Protein Ferritin Using Raman Microspectroscopy.

Ferritin is a ubiquitous intracellular iron storage protein of animals, plants, and bacteria. The cavity of this protein acts like a reaction chamber for natural formation and storage of nano-sized particles via biomineralization. Knowledge of the chemical composition and structure of the iron core is highly warranted in the fields of nano technologies as well as biomolecules and medicine. Here, we show that Raman microspectroscopy (RM) is a suitable nondestructive approach for an analysis of proteins containing such nano-sized iron oxides. Our approach addresses: (1) synthesis of suitable reference materials, i.e., ferrihydrite, maghemite and magnetite nanoparticles; (2) optimization of parameters for Raman spectroscopic analysis; (3) comparison of Raman spectra from ferritin with apoferritin and our reference minerals; and (4) validation of Raman analysis by X-ray diffraction and Mössbauer spectroscopy as two independent complementary approaches. Our results reveal that the iron core of natural ferritin is composed of the iron(III) hydroxide ferrihydrite (Fe2O3 ∙ 0.5 H2O).

Fluorescence detection, enumeration and characterization of single circulating cells in vivo: technology, applications and future prospects.

There are many diseases and biological processes that involve circulating cells in the bloodstream, such as cancer metastasis, immunology, reproductive medicine, and stem cell therapies. This has driven significant interest in new technologies for the study of circulating cells in small animal research models and clinically. Most currently used methods require drawing and enriching blood samples from the body, but these suffer from a number of limitations. In contrast, 'in vivo flow cytometry' (IVFC) refers to set of technologies that allow study of cells directly in the bloodstream of the organism in vivo. In recent years the IVFC field has grown significantly and new techniques have been developed, including fluorescence microscopy, multi-photon, photo-acoustic, and diffuse fluorescence IVFC. In this paper we review recent technical advances in IVFC, with emphasis on instrumentation, contrast mechanisms, and detection sensitivity. We also describe key applications in biomedical research, including cancer research and immunology. Last, we discuss future directions for IVFC, as well as prospects for broader adoption by the biomedical research community and translation to humans clinically.

Membranous CD24 drives the epithelial phenotype of pancreatic cancer.

Surface CD24 has previously been described, together with CD44 and ESA, for the characterization of putative cancer stem cells in pancreatic ductal adenocarcinoma (PDAC), the most fatal of all solid tumors. CD24 has a variety of biological functions including the regulation of invasiveness and cell proliferation, depending on the tumor entity and subcellular localization. Genetically engineered mouse models (GEMM) expressing oncogenic KrasG12D recapitulate the human disease and develop PDAC. In this study we investigate the function of CD24 using GEMM of endogenous PDAC and a model of cerulein-induced acute pancreatitis. We found that (i) CD24 expression was upregulated in murine and human PDAC and during acute pancreatitis (ii) CD24 was expressed exclusively in differentiated PDAC, whereas CD24 absence was associated with undifferentiated tumors and (iii) membranous CD24 expression determines tumor subpopulations with an epithelial phenotype in grafted models. In addition, we show that CD24 protein is stabilized in response to WNT activation and that overexpression of CD24 in pancreatic cancer cells upregulated ß-catenin expression augmenting an epithelial, non-metastatic signature. Our results support a positive feedback model according to which (i) WNT activation and subsequent ß-catenin dephosphorylation stabilize CD24 protein expression, and (ii) sustained CD24 expression upregulates ß-catenin expression. Eventually, membranous CD24 augments the epithelial phenotype of pancreatic tumors. Thus we link the WNT/ß-catenin pathway with the regulation of CD24 in the context of PDAC differentiation.

Successful prevention of scedosporiosis after lung transplantation in a cystic fibrosis patient by combined local and systemic triazole therapy.

A persistent colonization with Scedosporium apiospermum (S. apiospermum) often results in disseminated infection with a high mortality rate in immunosuppressed patients. We present the first case of successful prevention of scedosporiosis in an adolescent female cystic fibrosis patient post double lung transplant, with a combination of local and systemic voriconazole therapy and surgical intervention.

Oxaliplatin, irinotecan, and gemcitabine: a novel combination in the therapy of progressed, relapsed, or refractory tumors in children.

Therapeutic options for unresectable neuroendocrine carcinomas and relapsed or refractory solid tumors are still limited in pediatric patients. We present a retrospective review of 12 children (3 to 16 y) in a case series treated with a novel combination of oxaliplatin, irinotecan, and gemcitabine (triple therapy). We defined its feasibility in a mainly outpatient setting and assessed its toxicity and effectiveness. Three patients with unresectable neuroendocrine carcinomas received triple therapy as first-line treatment; 9 children with relapsed or refractory solid tumors of different entities were assigned after failure of standard treatment protocols. The treatment schedule comprised oxaliplatin (85 mg/m²), irinotecan (175 mg/m²), and gemcitabine (1,000 mg/m²), the latter to be repeated on day 8. A median of 7 cycles was applied. Nine of 12 patients showed hematotoxicity 0-III degrees. Gastrointestinal toxicity I-II degrees were handled satisfactorily by supportive drugs. Tumor response was defined as partial response in 1 of 12 children, stable disease in 8 of 12 children, and progressive disease in 3 of 12 children with a median time of disease control of 7 months. We regard triple therapy as a well-tolerated outpatient treatment option offering children a high quality of life and showing considerable effectiveness in delaying tumor progress.

Large BRCA1 gene deletions are found in 3% of German high-risk breast cancer families.

We have tested for large BRCA1 gene rearrangements in German high-risk breast and ovarian cancer families previously screened negative for point mutations by dHPLC and sequencing. Using the novel MLPA method, two deletions of exons 1A, 1B and 2 and exon 17, respectively, were detected in four out of 75 families investigated in Southern Germany. An identical exon 17 deletion with the same breakpoints and a deletion of exons 1A, 1B and 2 were found by fluorescent multiplex PCR in two out of 30 families investigated in Northern Germany. Combining both populations, genomic rearrangements were found in 6% of the mutation-negative families and 3% of all high-risk families and account for 8% of all BRCA1 mutations. Our data indicate that the exon 17 deletion may be a founder mutation in the German population. The prevalence of BRCA1 gene deletions or duplications in our patients is similar to previous reports from Germany and France. Genomic quantification by MLPA is a useful method for molecular diagnostics in high-risk breast cancer families.