Technical validation of a new microfluidic device for enrichment of CTCs from large volumes of blood by using buffy coats to mimic diagnostic leukapheresis products.

Diagnostic leukapheresis (DLA) enables to sample larger blood volumes and increases the detection of circulating tumor cells (CTC) significantly. Nevertheless, the high excess of white blood cells (WBC) of DLA products remains a major challenge for further downstream CTC enrichment and detection. To address this problem, we tested the performance of two label-free CTC technologies for processing DLA products. For the testing purposes, we established ficollized buffy coats (BC) with a WBC composition similar to patient-derived DLA products. The mimicking-DLA samples (with up to 400 × 106 WBCs) were spiked with three different tumor cell lines and processed with two versions of a spiral microfluidic chip for label-free CTC enrichment: the commercially available ClearCell FR1 biochip and a customized DLA biochip based on a similar enrichment principle, but designed for higher throughput of cells. While the samples processed with FR1 chip displayed with increasing cell load significantly higher WBC backgrounds and decreasing cell recovery, the recovery rates of the customized DLA chip were stable, even if challenged with up to 400 × 106 WBCs (corresponding to around 120 mL peripheral blood or 10% of a DLA product). These results indicate that the further up-scalable DLA biochip has potential to process complete DLA products from 2.5 L of peripheral blood in an affordable way to enable high-volume CTC-based liquid biopsies.

Analysis of DNA methylation in single circulating tumor cells.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells.

Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability-based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array-based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch(®) system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM-negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as "liquid biopsies."

Dermatophytosis in patients with human immunodeficiency virus infection: clinical aspects and etiologic agents.

Dermatophytosis in individuals with human immunodeficiency virus infection seems to manifest with atypical, multiple, or extensive lesions more frequently. In addition, there are reports of presentations with little inflammation, called anergics. Less common etiologic agents have been isolated in these individuals, such as Microsporum species. To describe clinical aspects and etiologic agents of dermatophytosis in individuals with human immunodeficiency virus (HIV) infection. Patients with clinical diagnosis of dermatophytosis underwent scarification for mycological diagnosis through direct microscopic examination and fungal isolation in culture on Sabouraud dextrose agar. Sixty individuals had a clinical hypothesis of dermatophytosis. In 20 (33.3%) of the 60 patients, dermatophytosis was confirmed through a mycological study. Tinea corporis, diagnosed in 14 patients, was the most frequent clinical form, followed by tinea unguium in 7, tinea cruris in 5, and tinea pedis in 1 patient. Most of the lesions of tinea corporis were anergic. Five patients with tinea unguium had involvement of multiple nails, with onychodystrophy as the predominant subtype. Multiple cutaneous lesions occurred in 3 patients and extensive cutaneous lesions in 4. Regarding the agent, Trichophyton rubrum was the most commonly isolated. The high occurrence of anergic skin lesions and involvement of multiple nails, especially as onychodystrophy, corroborates the hypothesis that atypical, disseminated, and more severe presentations are common in individuals with HIV infection. However, no Microsporum species was isolated even in atypical, extensive, or disseminated cases, in disagreement with previous reports. Therefore, the approach of squamous lesions in HIV-positive patients must include a mycological study, in view of the possibility of anergic dermatophytosis, to promote the introduction of a suitable therapeutic agent.

Antimicrobial activity of ß-lapachone encapsulated into liposomes against meticillin-resistant Staphylococcus aureus and Cryptococcus neoformans clinical strains.

The aim of this study was to determine whether encapsulation of ß-lapachone (ß-lap) into liposomes interferes with its in vitro antimicrobial activity against meticillin-resistant Staphylococcus aureus (MRSA) and Cryptococcus neoformans clinical strains. Liposomes (ß-lap:lipo or ß-lap:HPß-CD-lipo) were prepared using the hydration of thin lipid film method followed by sonication. The in vitro antimicrobial activities of ß-lap-loaded liposomes against MRSA and C. neoformans were evaluated using the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). The liposomes presented a mean particle size ranging from 88.7±1.5nm to 112.4±1.9nm with a polydispersity index ranging from 0.255 to 0.340, zeta potential from -0.26±0.01mV to +0.25±0.05mV and drug encapsulation efficiency from 97.4±0.3% to 98.9±0.4%. ß-Lap and ß-lap:HPß-CD had minimum inhibitory concentrations (MICs) ranging from 2mg/L to 4mg/L, whereas the MICs of ß-lap-lipo or ß-lap:HPß-CD-lipo ranged from 4mg/L to 16mg/L for the MRSA strains tested. ß-Lap and ß-lap:HPß-CD were able to inhibit fungal growth [MIC=2-8mg/L and minimum fungicidal concentration (MFC)=4-8mg/L]. However, ß-lap-lipo and ß-lap:HPß-CD-lipo were more efficient, with MICs and MFCs of <4mg/L. These findings suggest that the liposomal formulations tested do not interfere significantly with ß-lap antibacterial activity against MRSA and improve its antifungal properties against C. neoformans.

Protein-ligand docking in the new millennium--a retrospective of 10 years in the field.

Protein-ligand docking is currently an important tool in drug discovery efforts and an active area of research that has been the subject of important developments over the last decade. These are well portrayed in the rising number of available protein-ligand docking software programs, increasing level of sophistication of its most recent applications, and growing number of users. While starting by summarizing the key concepts in protein-ligand docking, this article presents an analysis of the evolution of this important field of research over the past decade. Particular attention is given to the massive range of alternatives, in terms of protein-ligand docking software programs currently available. The emerging trends in this field are the subject of special attention, while old established docking alternatives are critically revisited. Current challenges in the field of protein-ligand docking such as the treatment of protein flexibility, the presence of structural water molecules and its effect in docking, and the entropy of binding are dissected and discussed, trying to anticipate the next years in the field.

Isolation of cellulolytic fungi from waste of castor (Ricinus communis L.).

This is the first report of isolation of fungi present in fatty and defatted castor bean meal as well as the first of crop's selection to test the cellulolytic potential, in order to verify the diversity and potential of cellulolytic fungi in castor bean waste (Ricinus communis L.). For the screening on solid medium, it was used carboxymethylcellulose (CMC) as the sole carbon source. The microcrystalline cellulose (Avicel) was used as a substrate for submerged fermentation for production of cellobiohydrolase (FPase) and the CMC to produce endoglucanases (CMCase) and ß-glycosidases (BG). 189 cultures of fungi were isolated, including 40 species of filamentous fungi and three yeasts. The Aspergillus was the most frequent found genus. Regarding the distribution of isolated species from defatted castor bean meal, the A. niger was the most frequent one; and within the fatty castor bean meal, the Emericela variecolor prevailed among other species. Among the 67 fungal cultures tested in the initial screening on solid media to assess the cellulolytic potential, 54 disclosed Cellulolytic Index (CI) ranging from 1.04 to 6.00 mm. The isolates were selected for enzyme production in liquid medium with values above 2.0 CI. They were obtained with A. japonicus URM5620 FPase activity (4.99 U/ml) and BG (0.05 U/ml), and Rhodotorula glutinis URM5724 activity of CMCase 3.58 U/ml. These cases occurred after 168 h of submersion for both species of fungi. In our study, we could conclude that the castor bean is a promising source of fungi capable of producing cellulolytic enzymes.

Ciclopirox olamine: an antifungal alternative against cryptococcosis.

AIMS: The in vitro activity of ciclopirox olamine was evaluated against Cryptococcus spp. obtained from the cerebrospinal fluid (CSF) of immunocompromised patients. METHODS AND RESULTS: The antifungal activity of ciclopirox olamine was tested against Cryptococcus spp. obtained from the CSF of immunocompromised patients, using amphotericin B and fluconazole as controls. The minimal inhibitory concentration was determined following the microdilution method indicated by the Clinical and Laboratory Standards Institute. The minimal fungicide concentration was determined by the absence of growth on Sabouraud dextrose agar. The data obtained showed that antifungal activity of ciclopirox olamine ranged from 0·25 to 1 µg ml(-1) . CONCLUSIONS: This paper underscores the importance of the antifungal potential of ciclopirox olamine against Cryptococcus spp. as an alternative treatment against systemic cryptococosis. In vivo experiments are essential for future medical use. SIGNIFICANCE AND IMPACT OF THE STUDY: This was the first time that ciclopirox olamine was tested against Cryptococcus spp. using the reference method. The antifungal activity of this drug against this species suggests an applicable potential for systemic cryptococcosis therapy.

Antifungal activity of the naphthoquinone beta-lapachone against disseminated infection with Cryptococcus neoformans var. neoformans in dexamethasone-immunosuppressed Swiss mice

The in vivo antifungal activity of the naphthoquinone beta-lapachone against disseminated infection by Cryptococcus neoformans was investigated. Swiss mice were immunosuppressed daily with dexamethasone (0.5 mg per mouse) intraperitoneally for 3 days, the procedure was repeated 4 days later, and the animals were then challenged intravenously with C. neoformans (10(6) CFU/mL) 1 week later. Seven days after infection, the mice were divided into groups and treated daily with beta-lapachone (10 mg/kg, iv) for 7 (N = 6) and 14 days (N = 10). Amphotericin B (0.5 mg/kg) was used as comparator drug and an additional group received PBS. Treatment with beta-lapachone cleared the yeast from the spleen and liver, and the fungal burden decreased approximately 10(4) times in the lungs and brain 14 days after infection when compared to the PBS group (P < 0.05). This result was similar to that of the amphotericin B-treated group. Protection was suggestively due to in vivo antifungal activity of this drug and apparently not influenced by activation of the immune response, due to similar leukocyte cell counts among all groups. This study highlights the prospective use of beta-lapachone for treatment of disseminated cryptococcosis.

Antifungal activity of the naphthoquinone beta-lapachone against disseminated infection with Cryptococcus neoformans var. neoformans in dexamethasone-immunosuppressed Swiss mice.

The in vivo antifungal activity of the naphthoquinone beta-lapachone against disseminated infection by Cryptococcus neoformans was investigated. Swiss mice were immunosuppressed daily with dexamethasone (0.5 mg per mouse) intraperitoneally for 3 days, the procedure was repeated 4 days later, and the animals were then challenged intravenously with C. neoformans (10(6) CFU/mL) 1 week later. Seven days after infection, the mice were divided into groups and treated daily with beta-lapachone (10 mg/kg, iv) for 7 (N = 6) and 14 days (N = 10). Amphotericin B (0.5 mg/kg) was used as comparator drug and an additional group received PBS. Treatment with beta-lapachone cleared the yeast from the spleen and liver, and the fungal burden decreased approximately 10(4) times in the lungs and brain 14 days after infection when compared to the PBS group (P < 0.05). This result was similar to that of the amphotericin B-treated group. Protection was suggestively due to in vivo antifungal activity of this drug and apparently not influenced by activation of the immune response, due to similar leukocyte cell counts among all groups. This study highlights the prospective use of beta-lapachone for treatment of disseminated cryptococcosis.

Chromium(VI) induced alterations in mouse spleen cells: a short-term assay.

Cr(VI), the highest oxidation state for chromium, is a carcinogenic and mutagenic agent. In vivo and in vitro Cr(VI) toxic effects are related to its intracellular fate. Once inside the cell it is reduced to stable Cr(III) by cysteine, glutathione and ascorbic acid. Additionally, as Cr(V) and/or Cr(IV) intermediates have been reported in Cr(VI) reactions with biological reductants, chromium damage is thought to originate from these chemical species. This work investigated the morphology of splenic cells after short-term exposure to Cr(VI). A dose of 30 mg of K2CrO4/kg body weight was administered to mice and the effects were studied 24 and 48 h after the injections. Histological results revealed a time-dependency effect of Cr(VI) on splenic cells. Changes included enlargement of the capsule and depletion of the red pulp cells, accompanied by an increase in macrophages, 24 h after injection. Partial restoration of red pulp was noted after 48 h.

Relationship of mercury with aluminum, iron and manganese oxy-hydroxides in sediments from the Alto Pantanal, Brazil.

Sediments from nine floodplain lakes in Pantanal were analyzed for a large-scale (300 km) survey of mercury (Hg) load in sediments and soils of the Alto Pantanal and to study the relationship between Hg and reactive aluminum, iron, and manganese oxy-hydroxides. The results were compared with the Hg content in river and stream sediments from the Poconé gold mining area, where Hg has been extensively used and still is in use. The results indicate that the Hg concentrations were elevated in river sediment close to the mining area in Bento Gomes river basin (average in the < 74-microm fraction 88.9 ng Hg g(-1) dry wt.; interquartile range 50.3-119.5), but there was no clear indication that the local Hg emissions have contaminated the remote floodplain lakes, where concentrations were surprisingly low (average in the < 74-microm fraction 33.2 ng Hg g(-1) dry wt. sediment; interquartile range 18.4-46.8), in particular when considering geochemical characteristics of the sediment. The sediment from the floodplain lakes contained less Hg-tot and more reactive iron oxy-hydroxides than soils from the Tapajós area in the Amazon basin. This resulted in a mass ratio between Hg and amorphous oxy-hydroxides of only 5 x 10(-6) for Hg-tot/Fe-oxa (interquartile range 3-7 x 10(-6).