Potential for colonization of O111:H25 atypical enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis>

Using clonal phylogenetic methods, it has been demonstrated that O111:H25 atypical enteropathogenic E. coli (aEPEC) strains belong to distinct clones, suggesting the possibility that their ability to interact with different hosts and abiotic surfaces can vary from one clone to another. Accordingly, the ability of O111:H25 aEPEC strains derived from human, cat and dogs to adhere to epithelial cells has been investigated, along with their ability to interact with macrophages and to form biofilms on polystyrene, a polymer used to make biomedical devices. The results demonstrated that all the strains analyzed were able to adhere to, and to form pedestals on, epithelial cells, mechanisms used by E. coli to become strongly attached to the host. The strains also show a Localized-Adherence-Like (LAL) pattern of adhesion on HEp-2 cells, a behavior associated with acute infantile diarrhea. In addition, the O111:H25 aEPEC strains derived either from human or domestic animals were able to form long filaments, a phenomenon used by some bacteria to avoid phagocytosis. O111:H25 aEPEC strains were also encountered inside vacuoles, a characteristic described for several bacterial strains as a way of protecting themselves against the environment. They were also able to induce TNF-α release via two routes, one dependent on TLR-4 and the other dependent on binding of Type I fimbriae. These O111:H25 strains were also able to form biofilms on polystyrene. In summary the results suggest that, regardless of their source (i.e. linked to human origin or otherwise), O111:H25 aEPEC strains carry the potential to cause human disease.     

An efficient route to bispecific antibody production using single-reactor mammalian co-culture

Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho β that is being developed for type 2 diabetes and other obesity-linked disorders.     

Environmental Mapping of Paracoccidioides spp. in Brazil Reveals New Clues into Genetic Diversity,Biogeography and Wild Host Association

BackgroundParacoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques.Conclusions/SignificanceData may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.     

Oral insulin delivery by means of solid lipid nanoparticles

The aim of this work was to produce and characterize cetyl palmitate-based solid lipid nanoparticles (SLN) containing insulin, and to evaluate the potential of these colloidal carriers for oral administration. SLN were prepared by a modified solvent emulsification-evaporation method based on a w/o/w double emulsion. The particle size, zeta potential and association efficiency of unloaded and insulin-loaded SLN were determined and were found to be around 350 nm, negatively charged and the insulin association efficiency was over 43%. After oral administration of insulin-loaded SLN to diabetic rats, a considerable hypoglycemic effect was observed during 24 hours. These results demonstrated that SLN promote the oral absorption of insulin.     

Chemical Characterization,Antioxidant and Cytotoxic Activities of the Edible Fruits of Brosimun gaudichaudii Trécul,a Native Plant of the Cerrado Biome

In Brazil, there is a large diversity of species of small edible fruits that are considered sources of nutrients and functional properties. They present a high innovation domain for the pharmaceutical, cosmetic and food industries due to their health-promoting properties. Edible fruits from Brosimum gaudichaudii (Moraceae) are widely consumed and used in folk medicine and in feed by the population of the Brazilian Cerrado. Nevertheless, detailed information on the chemical fingerprint, antiradical activity and safety aspects of these fruits is still unknown. Thus, the aim of this work was to investigate the bioactive compounds of hydroethanolic extracts of fruits from Brosimum gaudichaudii using high-performance liquid chromatography combined with mass spectrometry using electrospray ionization (HPLC ESI-MS). Eighteen different compounds, including flavonoids, coumarins, arylbenzofurans, terpenoids, stilbenes, xanthones and esters, were detected. Moreover, the study indicated that the hydroethanolic extract of fruits from B. gaudichaudii presented low scavenging activity against 2,2-diphenyl-1-picrylhydrazyl radicals (IC₅₀>800 μg mL⁻¹) and was cytotoxic (IC₅₀<30 μg mL⁻¹) in Chinese hamster ovary cells (CHO−K1) by an in vitro assay. This is the first report of the chemical profile, antioxidant activity and cytotoxic properties of the hydroethanolic extract of fruits from B. gaudichaudii.     

Identification and distribution of chondroitin sulfate in the three electric organs of the electric eel, Electrophorus electricus (L.)

The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.     

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971–974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h^–1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7×10⁵ cells ml^–1) and a high variability of aggregate size (70–400 m after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4×10⁴ cells ml^–1) and the cells thus obtained are able to differentiate into neurons.     

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.     

Expansion of mouse embryonic stem cells on microcarriers

Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large-scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling-up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage-dependent cells, a proper support is necessary. Cytodex-3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5-3.9 x 10(6) cells/mL. Improved expansion was achieved using an inoculum of 5 x 10(4) cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real-time PCR and immunostaining analysis.