Quantification of cellular properties from external fields and resulting induced velocity: cellular hydrodynamic diameter.

An experimental technique is discussed in which the size distribution of a population of cells is determined by calculating each cell's settling velocity. The settling velocity is determined from microscopically obtained images which were recorded on SVHS tape. These images are then computer imaged and processed, and the cell's location and velocity are determined using a computer algorithm referred to as cell tracking velocimetry (CTV). Experimental data is presented comparing the distribution of human lymphocytes and a human breast cancer cell line, MCF-7, determined using a Coulter counter and the CTV approach.     

Quantification of cellular properties from external fields and resulting induced velocity: magnetic susceptibility.

An experimental technique is discussed in which the magnetic susceptibility of immunomagnetically labeled cells can be determined on a cell-by-cell basis. This technique is based on determining the magnetically induced velocity that an immunomagnetically labeled cell has in a well-defined magnetic energy gradient. This velocity is determined through the use of video recordings of microscopic images of cells moving in the magnetic energy gradient. These video images are then computer digitized and processed using a computer algorithm, cell tracking velocimetry, which allows larger numbers (>10(3)) of cells to be analyzed.     

Synthesis of aminated polysorbate 80 for polyplex‐mediated gene transfection

To develop novel gene delivery carriers, aminated polysorbate 80 (P80‐NH₂) was synthesized with strong positively charged properties through the introduction of three amine groups. The resulting P80‐NH₂ and DNA polyplex exhibited superb condensation abilities due to the high densities of positively charged amines groups. Size and surface charge of polyplex were shown to be well suited for cellular internalization. In addition, the P80‐NH₂/DNA polyplex demonstrated acceptable transfection efficiency in HeLa cells and was nontoxic relative to the conventional 25‐kDa polyethyleneimine system. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Novel hyaluronic acid (HA) coated drug carriers (HCDCs) for human breast cancer treatment

Hyaluronic acid (HA) coated drug carriers (HCDCs) were successfully synthesized by chemical conjugation method for targeted delivery of doxorubicin (DOX) as a prototype anticancer drug to CD44 expressed human breast cancer cell. From XPS analysis, the HCDCs by conjugation methods demonstrated the superior HA fixation amount and colloidal stability compared with the nanoparticles by nanoprecipitation. The cytotoxicity of the HCDCs formulation accessed by the MTT assay against the higher CD44 expressed cell line (MDA-MB-231) and lower CD44 expressed cell line (ZR-75-1) human breast cancer cell lines demonstrated that the HCDCs formulation exhibited excellent tumoricidal effect and their affinity to cancer cells was predominant. The in vitro drug release profile of the HCDCs showed sustained release behavior and after 14 days, 80% of the encapsulated DOX was released due to a high release rate of DOX from HCDCs. We synthesized that HCDCs have therapeutic potentials of cancer as a target specific fashion by increasing the tumoricidal efficacy of targeted cancer cells while reducing their cytotoxicity of non-targeted cells to minimize the side effect.     

Enhancement of monascus pigment production by the culture of Monascus sp. J101 at low temperature

In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 degrees C. In a batch culture at 30 degrees C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 degrees C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 degrees C was 10 times greater than at 30 degrees C.     

Continuous flow magnetic cell fractionation based on antigen expression level

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.     

Expressed sequence tags from a wheat-rye translocation line (2BS/2RL) infested by larvae of Hessian fly [<Emphasis Type="Italic">Mayetiola destructor</Emphasis> (Say)]

Of the 16 known biotypes of the Hessian fly [Mayetiola destructor (Say)], biotype L is recognized as being the most virulent. We have previously reported the development of near-isogenic lines (NILs) (BC₃F_3:4) by backcross introgression (Coker797*4/Hamlet) that differed by the presence or absence of the H21 gene on 2RL chromatin. Florescence in situ hybridization analysis revealed introgressed 2RLs in NILs possessing the H21 gene, but no signal was detected in NILs lacking 2RL. As part of an approach to elucidate molecular interactions between plants and the Hessian fly, a cDNA library from NILs with H21 infested by larvae of biotype L of the Hessian fly was constructed for expressed sequence tag (EST) analysis. Of 1,056 sequenced reactions attempted, 919 ESTs produced some lengths of readable sequences. Based on their putative identification, 730 ESTs that showed significant similarity with amino acid sequences registered in the gene bank were divided into 13 functional categories. Defense- and stress-related genes represented about 16.1%, including protease inhibition, oxidative burst, lignin synthesis, and phenylpropanoid metabolism. EST clones obtained from the cDNA library may provide a clue to the molecular interactions between plant and larva of the Hessian fly larval infestation.Abbreviations ESTs Expressed sequence tags - FISH Florescence in situ hybridization - NILs Near-isogenic linesCommunicated by P. PuigdoménechAll of the EST sequence data reported will appear in the dbEST and GenBank database (accession numbers CB307016 to CB307934)     

Long-term depletion of cereblon induces mitochondrial dysfunction in cancer cells

Cereblon (CRBN) is a multi-functional protein that acts as a sub-strate receptor of the E3 ligase complex and a molecular chaperone. While CRBN is proposed to function in mitochondria, its specific roles are yet to be established. Here, we showed that knockdown of CRBN triggers oxidative stress and calcium overload in mitochondria, leading to disruption of mitochondrial membrane potential. Notably, long-term CRBN depletion using PROteolysis TArgeting Chimera (PROTAC) induced irreversible mitochondrial dysfunction, resulting in cell death. Our collective findings indicate that CRBN is required for mitochondrial homeostasis in cells.     

Preparation of peptide-loaded polymer microparticles using supercritical carbon dioxide

In this study, peptide-loaded microparticles were prepared using an aerosol solvent extraction system (ASES) by employing supercritical carbon dioxide as an antisolvent. The effects of the molecular weight of poly(Llactide) (PLLA), poly(ethylene glycol) (PEG), the block length of methoxy poly(ethylene glycol)-b-poly(L-lactide) (mPEG-PLLA), the blending of PLLA and PEG, and the drug-to-polymer feed ratio on the formation of leuprolide acetate (LA)-loaded microparticles and their release characteristics were investigated. Scanning electron microscope observations showed that the LA-loaded polymer particles had a spherical morphology with a smooth surface. The entrapment efficiency of LA in the ASES-processed microparticles was found to be extremely high (about 99%), whereas the initial release rate of the LA-loaded microparticles was very low for PLLA. The release rate of LA was observed to increase as the PEG block length of mPEG-PLLA and/or the drug content in the microparticles increased. When PLLA was blended with PEG, the release rate of LA from the PLLA/PEG microparticles was significantly faster compared with the corresponding mPEG-PLLA copolymer.