High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.     

Informatics-guided procurement of patient samples for biomarker discovery projects in cancer research

Modern cancer research for biomarker discovery program requires solving several tasks that are directly involved with patient sample procurement. One requirement is to construct a highly efficient workflow on the clinical side for the procurement to generate a consistent supply of high quality samples for research. This undertaking needs a network of interdepartmental collaborations and participations at various levels, including physical human interactions, information technology implementations and a bioinformatics tool that is highly effective and user-friendly to busy clinicians and researchers associated with the sample procurement. Collegial participation that is sequential but continual from one department to another demands dedicated bioinformatics software coordinating between the institutional clinic and the tissue repository facility. Participants in the process include admissions, consenting process, phlebotomy, surgery center and pathology. During this multiple step procedures, clinical data are collected for detailed analytical endpoints to supplement logistics of defining and validating the discovery of biomarkers.

Fusogenic potential of sperm membrane lipids: nature's wisdom to accomplish targeted gene delivery

The membrane-membrane fusion during fertilization of oocyte by spermatozoa is believed to be mainly mediated by so called "fusion proteins". In the present study we have tried to demonstrate that beside the proteins, lipid components of membrane may play an important role in fusion of oocyte with spermatozoa. Conventional membrane-membrane fusion assays were used as means to demonstrate fusogenic potential of human sperm membrane lipids. The liposomes (spermatosomes) made of the lipids isolated from sperm membrane were found to undergo strong membrane-membrane fusion as evident from fluorescence dequenching and resonance energy transfer assays. Furthermore, the fusion of these liposomes with living cells (J774 A.1 macrophage cell line) was demonstrated to result in an effective transfer of a water-soluble fluorescent probe (calcein) to cytosol of the target cell. Lastly, the liposomes were demonstrated to behave like efficient vehicles for the in vivo cytosolic delivery of the antigens to target cells resulting in elicitation of antigen specific CD8(+) T cell responses.     

Signaling pathways involving adenosine A<Subscript>2A</Subscript> and A<Subscript>2B</Subscript> receptors in wound healing and fibrosis

Collagen and matrix deposition by fibroblasts is an essential part of wound healing but also contributes to pathologic remodeling of organs leading to substantial morbidity and mortality. Adenosine, a small molecule generated extracellularly from adenine nucleotides as a result of direct stimulation, hypoxia, or injury, acts via a family of classical seven-pass G protein-coupled protein receptors, A_2A and A_2B, leading to generation of cAMP and activation of downstream targets such as PKA and Epac. These effectors, in turn, lead to fibroblast activation and collagen synthesis. The regulatory actions of these receptors likely involve multiple interconnected pathways, and one of the more interesting aspects of this regulation is opposing effects at different levels of cAMP generated. Additionally, adenosine signaling contributes to fibrosis in organ-specific ways and may have opposite effects in different organs. The development of drugs that selectively target these receptors and their signaling pathways will disrupt the pathogenesis of fibrosis and slow or arrest the progression of the important diseases they underlie.     

Determination of gentamicin in bovine milk using liquid chromatography with post-column derivatization and fluorescence detection

A method capable of separating and quantifying the three major and one minor components of gentamicin in milk has been developed. The method is capable of detecting 15 ng/ml gentamicin, based on a total of the four components. Milk samples are centrifuged at 4°C, the fat layer removed, and the samples deproteinated with 30% trichloracetic acid. After a second centrifugation, the supernatant is passed through a C₁₈ solid-phase extraction column. The column is washed with water, water-methanol (50:50) and methanol. Ammonium hydroxide (16%) in methanol is used to elute the gentamicin. The eluent is evaporated to near dryness and taken up with water. An aliquot of the sample is then mixed with an ion-pairing reagent for chromatography. Separation is achieved using pentanesulfonic acid in a water-methanol mobile phase on a C₁₈ reversed-phase column. The o-phthalaldehyde fluorescence derivatives of gentamicin are formed post-column and are detected with excitation at 340 nm and emission at 430 nm. The percent recovery of gentamicin averaged 72, 78 and 88% from milk samples fortified at 15, 30 and 60 ng/ml, respectively.     

Electronic structures and reactivity aspects of ruthenium-nitrosyls

Nitrosyl complexes with {Ru-NO}⁶ (4(ClO₄)₃) and {Ru-NO}⁷ (4(ClO₄)₂) configurations have been isolated in the selective molecular framework of [Ru(tpm)(pap)(NO)]ⁿ⁺ (tpm = tris(1-pyrazolyl)methane and pap = 2-phenylazopyridine). The DFT optimized structures of [Ru^II(tpm)(pap)(NO⁺)]³⁺ (4³⁺) and [Ru^II(tpm)(pap)(NO)]²⁺ (4²⁺) predict that the Ru-N-O groups in the complexes are in almost linear and bent geometries, respectively. In agreement with largely NO centered reduction a sizeable shift in ν(NO) frequency of 324 cm⁻¹ has been observed on moving from {Ru^II-NO⁺} state in 4³⁺ to {Ru^II-NO) state in 4²⁺. The DFT proposed NO centered spin in {Ru^II-NO) (4²⁺) (Mulliken spin-densities: 0.860 (NO) and 0.087 (Ru)) has been evidenced by its free radical EPR spectrum with g = 1.989. The strongly electrophilic {Ru^II-NO⁺} state in 4³⁺ (ν(NO): 1962 cm⁻¹) can be transformed to the corresponding complex (3⁺) in the presence of nucleophile, OH⁻ with k = 2.03 × 10⁻¹ M⁻¹ s⁻¹ at 298 K in CH₃CN. On irradiation with light the acetonitrile solution of [Ru^II(tpm)(pap)(NO⁺)]³⁺ (4³⁺) undergoes facile photorelease of NO (k_NO, s⁻¹ = 0.1 × 10⁻¹ and t_1/2, s = 69.3) with the concomitant formation of the solvate [Ru^II(tpm)(pap)(CH₃CN)]²⁺ (2²⁺). The photoreleased NO can be trapped as an Mb-NO adduct.     

Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects. TRIAL REGISTRATION: Clinicaltrials.gov NCT01155492.     

Long circulating lectin conjugated paclitaxel loaded magnetic nanoparticles: a new theranostic avenue for leukemia therapy

Amongst all leukemias, Bcr-Abl positive chronic myelogenous leukemia (CML) confers resistance to native drug due to multi drug resistance and also resistance to p53 and fas ligand pathways. In the present study, we have investigated the efficacy of microtubule stabilizing paclitaxel loaded magnetic nanoparticles (pac-MNPs) to ascertain its cytotoxic effect on Bcr-Abl positive K562 cells. For active targeted therapy, pac-MNPs were functionalized with lectin glycoprotein which resulted in higher cellular uptake and lower IC(50) value suggesting the efficacy of targeted delivery of paclitaxel. Both pac-MNPs and lectin conjugated pac-MNPs have a prolonged circulation time in serum suggesting increased bioavailability and therapeutics index of paclitaxel in vivo. Further, the molecular mechanism pertaining to pac-induced cytotoxicity was analyzed by studying the involvement of different apoptotic pathway proteins by immunoblotting and quantitative PCR. Our study revealed simultaneous activation of JNK pathway leading to Bcr-Abl instability and the extrinsic apoptotic pathway after pac-MNPs treatment in two Bcr-Abl positive cell lines. In addition, the MRI data suggested the potential application of MNPs as imaging agent. Thus our in vitro and in vivo results strongly suggested the pac-MNPs as a future prospective theranostic tool for leukemia therapy.     

VD3 and LXR agonist (T0901317) combination demonstrated greater potency in inhibiting cholesterol accumulation and inducing apoptosis via ABCA1-CHOP-BCL-2 cascade in MCF-7 breast cancer cells

Molecular Biology Reports - Obesity is associated with hypercholesterolemia and is a global epidemic. Epidemiological and animal studies revealed cholesterol is an essential regulator of estrogen...