The production of clonal and axenic cultures of microalgae using fluorescence-activated cell sorting

Unialgal cultures of the flagellate algae Cyanophora paradoxa, Haematococcus lacustris, Monomastix sp., Scherffelia dubia and Spermatozopsis similis which contained bacteria were sorted by flow cytometry to obtain axenic clonal cultures. The variables used for fluorescence-activated cell sorting (FACS) were chlorophyll autofluorescence, forward scatter and side scatter of the laser beam. To produce clonal cultures, a single cell was sorted into each culture flask. Depending on the species, about 20–30% of the sorted cultures grew successfully and at least 20% of these were axenic even if the numerical ratio betweeen bacteria and algae in the original cultures was as high as 300:1. FACS represents an effective and rapid method for the preparation of clonal and axenic cultures of microalgae.     

An optimized microarray platform for assaying genomic variation in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> field populations

We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination.     

Confocal laser endomicroscopy for diagnosis and histomorphologic imaging of brain tumors in vivo

Early detection and evaluation of brain tumors during surgery is crucial for accurate resection. Currently cryosections during surgery are regularly performed. Confocal laser endomicroscopy (CLE) is a novel technique permitting in vivo histologic imaging with miniaturized endoscopic probes at excellent resolution. Aim of the current study was to evaluate CLE for in vivo diagnosis in different types and models of intracranial neoplasia. In vivo histomorphology of healthy brains and two different C6 glioma cell line allografts was evaluated in rats. One cell line expressed EYFP, the other cell line was used for staining with fluorescent dyes (fluorescein, acriflavine, FITC-dextran and Indocyanine green). To evaluate future application in patients, fresh surgical resection specimen of human intracranial tumors (n = 15) were examined (glioblastoma multiforme, meningioma, craniopharyngioma, acoustic neurinoma, brain metastasis, medulloblastoma, epidermoid tumor). Healthy brain tissue adjacent to the samples served as control. CLE yielded high-quality histomorphology of normal brain tissue and tumors. Different fluorescent agents revealed distinct aspects of tissue and cell structure (nuclear pattern, axonal pathways, hemorrhages). CLE discrimination of neoplastic from healthy brain tissue was easy to perform based on tissue and cellular architecture and resemblance with histopathology was excellent. Confocal laser endomicroscopy allows immediate in vivo imaging of normal and neoplastic brain tissue at high resolution. The technology might be transferred to scientific and clinical application in neurosurgery and neuropathology. It may become helpful to screen for tumor free margins and to improve the surgical resection of malignant brain tumors, and opens the door to in vivo molecular imaging of tumors and other neurologic disorders.     

Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high-caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high-caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin-induced glucose uptake in 3T3-L1 adipocytes.     

Measles cervicitis. Report of a case with cytologic and molecular biologic analysis.

We report on a 20-year-old woman who, in her third month of pregnancy, presented with a morbilliform rash, fever and diarrhea. Many multinucleate superficial, intermediate and metaplastic cells were noted in the cervical cytologic preparation. The presence of the measles virus in cells from the cervix was confirmed by the amplification of measles complementary DNA (cDNA) by the polymerase chain reaction. The patient's cervical Papanicolaou smear was unremarkable on reexamination four months after the onset of the disease. To our knowledge, this is the first verified reported case of infection of the uterine cervix by the measles virus.     

An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Iron uptake and storage in the HAB dinoflagellate <Emphasis Type="Italic">Lingulodinium polyedrum</Emphasis>

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive–oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to ⁵⁷Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.