Saccharomyces cerevisiae biomass as a source of next-generation food preservatives: Evaluating potential proteins as a source of antimicrobial peptides.

Saccharomyces cerevisiae is the main biotechnological tool for the production of Baker's or Brewer's biomasses, largely applied in beverage and fermented-food production. Through its gene expression reprogramming and production of compounds that inactivate the growth of other microorganisms, S. cerevisiae is able to grow in adverse environments and in complex microbial consortia, as in fruit pulps and root flour fermentations. The distinct set of up-regulated genes throughout yeast biomass propagation includes those involved in sugar fermentation, ethanol metabolization, and in protective responses against abiotic stresses. These high abundant proteins are precursors of several peptides with promising health-beneficial activities such as antihypertensive, antioxidant, antimicrobial, immunomodulatory, anti-obesity, antidiabetes, and mitogenic properties. An in silico investigation of these S. cerevisiae derived peptides produced during yeast biomass propagation or induced by physicochemical treatments were performed using four algorithms to predict antimicrobial candidates encrypted in abundantly expressed stress-related proteins encoded by different genes like AHP1, TSA1, HSP26, SOD1, HSP10, and UTR2, or metabolic enzymes involved in carbon source utilization, like ENO1/2, TDH1/2/3, ADH1/2, FBA1, and PDC1. Glyceraldehyde-3-phosphate dehydrogenase and enolase II are noteworthy precursor proteins, since they exhibited the highest scores concerning the release of antimicrobial peptide candidates. Considering the set of genes upregulated during biomass propagation, we conclude that S. cerevisiae biomass, a food-grade product consumed and marketed worldwide, should be considered a safe and nonseasonal source for designing next-generation bioactive agents, especially protein encrypting antimicrobial peptides that display broad spectra activity and could reduce the emergence of microbial resistance while also avoiding cytotoxicity.

ImmunoPET Predicts Response to Met-targeted Radioligand Therapy in Models of Pancreatic Cancer Resistant to Met Kinase Inhibitors.

Background: Pancreatic ductal adenocarcinoma (PDAC) has limited standard of care therapeutic options. While initially received with enthusiasm, results from targeted therapy with small molecule tyrosine kinases inhibitors (TKIs) have been mixed, in part due to poor patient selection and compensatory changes in signaling networks upon blockade of one or more kinase of tumors. Here, we demonstrate that in PDACs otherwise resistant to rational kinase inhibition, Met-directed immuno-positron emission tomography (immunoPET) can identify targets for cell-signaling independent targeted radioligand therapy (RLT). In this study, we use Met-directed immunoPET and RLT in models of human pancreatic cancer that are resistant to Met- and MEK-selective TKIs, despite over-expression of Met and KRAS-pathway activation. Methods: We assessed cell membrane Met levels in human patient samples and pancreatic ductal adenocarcinoma (PDAC) cell lines (BxPC3, Capan2, Suit2, and MIA PaCa-2) using immunofluorescence, flow cytometry and cell-surface biotinylation assays. To determine whether Met expression levels correlate with sensitivity to Met inhibition by tyrosine kinase inhibitors (TKIs), we performed cell viability studies. A Met-directed imaging agent was engineered by labeling Met-specific onartuzumab with zirconium-89 (Zr-89) and its in vivo performance was evaluated in subcutaneous and orthotopic PDAC xenograft models. To assess whether the immunoPET agent would predict for targeted RLT response, onartuzumab was then labeled with lutetium (Lu-177) as the therapeutic radionuclide to generate our [177Lu]Lu-DTPA-onartuzumab RLT agent. [177Lu]Lu-DTPA-onartuzumab was administered at 9.25MBq (250µCi)/20µg in three fractions separated by three days in mice subcutaneously engrafted with BxPC3 (high cell-membrane Met) or MIA PaCa-2 (low cell-membrane Met). Primary endpoints were tumor response and overall survival. Results: Flow cytometry and cell-surface biotinylation studies showed that cell-membrane Met was significantly more abundant in BxPC3, Capan2, and Suit2 when compared with MIA PaCa-2 pancreatic tumor cells. Crizotinib and cabozantinib, TKIs with known activity against Met and other kinases, decreased PDAC cell line viability in vitro. The TKI with the lowest IC50 for Met, capmatinib, had no activity in PDAC lines. No additive effect was detected on cell viability when Met-inhibition was combined with MEK1/2 inhibition. We observed selective tumor uptake of [89Zr]Zr-DFO-onartuzumab in mice subcutaneously and orthotopically engrafted with PDAC lines containing high cell-surface levels of Met (BxPC3, Capan2, Suit2), but not in mice engrafted with low cell-surface levels of Met (MIA PaCa-2). Significant tumor growth delay and overall survival benefit were observed in both BxPC3 and MIA PaCa-2 engrafted animals treated with RLT when compared to controls, however, the benefit was more pronounced and more durable in the BxPC3 engrafted animals treated with [177Lu]Lu-DTPA-onartuzumab RLT. Conclusions: Our findings demonstrate that while over-expression of Met is not predictive of Met-directed TKI response, immunoPET can detect Met over-expression in vivo and predicts for therapeutic response to Met-selective RLT. This phenomenon can be exploited for other Met-overexpressing tumor types specifically, and to any differentially overexpressed surface molecule more broadly.

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

Liposomal Taro Lectin Nanocapsules Control Human Glioblastoma and Mammary Adenocarcinoma Cell Proliferation.

The search for natural anticancer agents and nanocarrier uses are a part of the current strategies to overcome the side effects caused by chemotherapeutics. Liposomal nanocapsules loaded with purified tarin, a potential immunomodulatory and antitumoral lectin found in taro corms, were produced. Liposomes were composed by 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, cholesterylhemisuccinate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000 prepared by thin-film hydration. Small unilamellar vesicles were achieved by sonication and extrusion. Scanning electron microscopy evidenced round-shaped nanocapsules presenting a smooth surface, 150 nm diameter and polydispersity index <0.2, estimated by dynamic light scattering. Tarin entrapment rates were over 80% and leakage of ~3% under 40 days of storage at 4 °C. Entrapped tarin exhibited an 83% release after 6 h at pH 4.6⁻7.4 and 36 °C. Both free and encapsulated tarin exhibited no in vitro toxicity against healthy mice bone marrow and L929 cells but stimulated the production of fibroblast-like and large round-shaped cells. Encapsulated tarin resulted in inhibition of human glioblastoma (U-87 MG) and breast adenocarcinoma (MDA-MB-231) proliferation, with an IC50 of 39.36 and 71.38 µg/mL, respectively. The effectiveness of encapsulated tarin was similar to conventional chemotherapy drugs, such as cisplatin and temozolide. Tarin liposomal nanocapsules exhibited superior pharmacological activity compared to free tarin as a potential chemotherapy adjuvant.

Tarin stimulates granulocyte growth in bone marrow cell cultures and minimizes immunosuppression by cyclo-phosphamide in mice.

Chemotherapeutic drugs, such as cyclophosphamide, cause severe immunosuppression and patients become susceptible to infections. Based on this, the immunomodulatory potential of tarin, a lectin from Colocasia esculenta, was evaluated in bone marrow cell cultures and in cyclophosphamide-immunosuppressed mice. Tarin promoted maintenance of hematopoietic progenitors and repopulation of Gr1 cells in vitro which was supported by in vivo results. In immunosuppressed mice, tarin increased bone marrow cell numbers and altered cell profile distribution by enhancing the frequency of Gr1+ progenitors, including Ly6-CintLy6-Glo, and anticipating their proliferation/differentiation in mature cells, especially Ly6-CloLy6-Ghi. Bone marrow cells harvested from tarin-treated immunosuppressed mice proliferated in response to GM-CSF or G-CSF in vitro and, the low numbers of bone marrow cells in the G0 phase, combined with a high number cells undergoing apoptosis confirmed that tarin promoted a faster and intense proliferation/differentiation, even in the presence of CY-induced toxicity. As a result, tarin minimized leukopenia in immunosuppressed mice promoting a faster recovery of peripheral leucocytes and protected erythroid bone marrow cells from CY-cytotoxicity in a dose-dependent manner. Data suggest that tarin could be considered a potential adjuvant to decrease leukopenia and possibly ameliorate anemia, if carefully evaluated in human cancer cell lineages and in clinical trials.

High-resolution crystal structures of Colocasia esculenta tarin lectin.

Tarin, the Colocasia esculenta lectin from the superfamily of α-d-mannose-specific plant bulb lectins, is a tetramer of 47 kDa composed of two heterodimers. Each heterodimer possesses homologous monomers of ~11.9 (A chain) and ~12.7 (B chain) kDa. The structures of apo and carbohydrate-bound tarin were solved to 1.7 Å and 1.91 Å, respectively. Each tarin monomer forms a canonical ß-prism II fold, common to all members of Galanthus nivalis agglutinin (GNA) family, which is partially stabilized by a disulfide bond and a conserved hydrophobic core. The heterodimer is formed through domain swapping involving the C-terminal ß-strand and the ß-sheet on face I of the prism. The tetramer is assembled through the dimerization of the B chains from heterodimers involving face II of each prism. The 1.91 Å crystal structure of tarin bound to Manα(1,3)Manα(1,6)Man reveals an expanded carbohydrate-binding sequence (QxDxNxVxYx4/6WX) on face III of the ß-prism. Both monomers possess a similar fold, except for the length of the loop, which begins after the conserved tyrosine and creates the binding pocket for the α(1,6)-terminal mannose. This loop differs in size and amino-acid composition from 10 other ß-prism II domain proteins, and may confer carbohydrate-binding specificity among members of the GNA-related lectin family.

Structural analysis and binding properties of isoforms of tarin, the GNA-related lectin from Colocasia esculenta.

The lectins, a class of proteins that occur widely in animals, plants, fungi, lichens and microorganisms, are known for their ability to specifically bind to carbohydrates. Plant lectins can be classified into 12 families including the Galanthus nivalis agglutinin (GNA)-related lectin superfamily, which is widespread among monocotyledonous plants and binds specifically to mannose, a behavior that confers remarkable anti-tumor, anti-viral and insecticidal properties on these proteins. The present study characterized a mitogenic lectin from this family, called tarin, which was purified from the crude extract from taro (Colocasia esculenta). The results showed that tarin is a glycoprotein with 2-3% carbohydrate content, composed of least 10 isoforms with pIs ranging from 5.5 to 9.5. The intact protein is a heterotetramer of 47kDa composed of two non-identical and non-covalently associated polypeptides, with small subunits of 11.9kDa and large subunits of 12.6kDa. The tarin structure is stable and recovers or maintains its functional structure following treatments at different temperatures and pH. Tarin showed a complex carbohydrate specificity, binding with high affinity to high-mannose and complex N-glycans. Many of these ligands can be found in viruses, tumor cells and insects, as well as in hematopoietic progenitor cells. Chemical modifications confirmed that both conserved and non-conserved amino acids participate in this interaction. This study determined the structural and ligand binding characteristics of a GNA-related lectin that can be exploited for several different purposes, particularly as a proliferative therapeutic molecule that is able to enhance the immunological response.

Burden of isolation for multidrug-resistant organisms in a tertiary public hospital in Southern Brazil.

We aimed to describe the use of isolation beds between September 2011 and August 2013 at a tertiary hospital located in Southern Brazil. The main cause for isolation was gram-negative carbapenem-resistant bacteria. Huge costs were associated with isolation practices. Considering the high burden on the isolation ward, practice of surveillance cultures and contact isolation should be balanced with other infection control practices.

Primary pleomorphic liposarcoma of the orbit: a case report.

To our knowledge, pleomorphic liposarcoma (PL) of the orbit has only been reported in the literature four times. This rarity makes it more difficult to diagnose and to treat in this clinical setting. A 62-year-old female presented with pruritus, edema, proptosis and diplopia 5 months OS. Imaging revealed an intraorbital mass displacing the globe, with infiltration into the sinus. The tumor was removed and the histological examination revealed a highly cellular tumor with heterogenous histology, with a few vacuolated cells and many malignant features. Immunohistochemistry allowed for the differential diagnosis, resulting in a diagnosis of PL of the orbit. The cells were immuno-positive for S-100 and negative for all other relevant markers. According to the literature, prognosis for this neoplasm is quite poor, and exenteration represents the best treatment option. The patient refused exenteration and radiation therapy, however, at 2 year follow-up, she remained recurrence-free.

Histopathological diagnosis of corneal button specimens: an epidemiological study.

PURPOSE: To determine the most common histopathological diagnosis of corneal specimens from penetrating keratoplasty (PKP). METHODS: The records of 500 corneal specimens submitted to biopsy at the Henry Witelson Ocular Pathology Laboratory, Montreal, Canada, from 1999 to 2004 were reviewed. Age, sex, clinical indications, and histopathological findings were analyzed. RESULTS: Chronic keratitis (45.6%) was the most common pathological diagnosis, followed by corneal edema (25.8%), dystrophy (12.8%), keratoconus (KC) (9.2%), acute keratitis (5.6%), and degeneration (1.0%). Among the specimens with chronic keratitis, regraft was the most common clinical indication (39.0%). In the group of acute keratitis, ulcerative condition was the leading cause (75,0%). Fuchs' endothelial dystrophy represented 79.7% of the clinical diagnoses in the group of corneal dystrophies. The median patient age was 70-79 years, and the gender distribution was nearly symmetric. CONCLUSION: The present study is important for determining the most common histopathological diagnoses of corneal button specimens and the correlation with the age, gender, and clinical indications of PKP.

Immunohistochemical expression of COX-2 and c-kit in metastatic uveal melanoma.

CASE REPORT: We report a case of choroidal melanoma metastatic to the liver diagnosed by fine-needle aspiration. The biopsy sample was immunostained for COX-2 and c-kit. COMMENTS: Accurate diagnosis and identification of potential therapeutic targets are important for subsequent therapy and can be achieved by radiologically guided fine-needle aspiration biopsy.

Expression of EpCAM in uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults, and nearly 40% of UM will develop metastasis that will ultimately lead to death. The Epithelial Cell Adhesion Molecule (EpCAM) is a type I transmembrane glycoprotein expressed by carcinomas of head and neck, ovary, colon, breast, kidney and lung. Recently, antibodies against EpCAM such as Edrecolomab and Catumaxomab were developed, and clinical trials with these antibodies have been used in several types of neoplasia. We studied the expression of EpCAM in UM. METHODS: 25 enucleated formalin-fixed, paraffin-embedded UM specimens were immunostained for EpCAM. Histopathological analysis of the specimens with regards to prognostic factors such as cell type, largest (linear) tumor dimension, number of mitotic figures, scleral invasion and tumor infiltrating lymphocytes were done. RESULTS: None of them was positive for this EpCAM. CONCLUSION: In our report, UM did not express EpCAM. Therefore, it is not a helpful immunohistochemical marker to predict the behavior of UM. Further studies are needed to verify if EpCAM could also be related with prognosis and treatment of UM.

Orbital metastasis of urinary bladder carcinoma: a clinicopathologic report and review of the literature.

A 53-year-old male presented with a progressive mass of the left orbit. His medical history included an invasive carcinoma of the bladder diagnosed three weeks earlier. An orbital biopsy was performed and the diagnosis was that of an orbital metastasis of urinary bladder carcinoma. The patient developed widespread metastatic disease and unfortunately died one month after the diagnosis of orbital metastasis. Orbital metastasis of urinary bladder carcinoma is associated with a poor prognosis and is more frequently observed in older people. In addition, it is five times more prevalent in men than in women.

Corneal endothelial deposits associated with rifabutin use.

PURPOSE: The aim of this study was to report on the possible development of corneal endothelial deposits resulting from the use of rifabutin. METHODS: Case series consisting of 3 patients treated with rifabutin were retrospectively studied. Two of the patients were infected with human immunodeficiency virus. A corneal and external disease specialist performed a complete ophthalmologic exam and obtained medical histories of the patients. RESULTS: All cases developed corneal endothelial deposits after previous use of rifabutin. The deposits were bilateral, yellow-white colored, stellate, and mainly peripheral. CONCLUSIONS: In these 3 cases, the unique positive ocular finding was corneal endothelial deposits, which may be related to the use of rifabutin.