Impact of Recombinant VSV-HIV Prime, DNA-Boost Vaccine Candidates on Immunogenicity and Viremia on SHIV-Infected Rhesus Macaques.

Currently, no effective vaccine to prevent human immunodeficiency virus (HIV) infection is available, and various platforms are being examined. The vesicular stomatitis virus (VSV) vaccine vehicle can induce robust humoral and cell-mediated immune responses, making it a suitable candidate for the development of an HIV vaccine. Here, we analyze the protective immunological impacts of recombinant VSV vaccine vectors that express chimeric HIV Envelope proteins (Env) in rhesus macaques.

Insight into the Binding Mechanisms of Quartz-Selective Peptides: Toward Greener Flotation Processes.

Mining practices, chiefly froth flotation, are being critically reassessed to replace their use of biohazardous chemical reagents in favor of biofriendly alternatives as a path toward green processes. In this regard, this study aimed at evaluating the interactions of peptides, as potential floatation collectors, with quartz using phage display and molecular dynamics (MD) simulations. Quartz-selective peptide sequences were initially identified by phage display at pH = 9 and further modeled by a robust simulation scheme combining classical MD, replica exchange MD, and steered MD calculations.

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30 µm).

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer.

Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant.

Background: Unlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt.

Optimisation of Cytochrome P450 BM3 Assisted by Consensus-Guided Evolution.

Cytochrome P450 enzymes have attracted much interest over the years given their ability to insert oxygen into saturated carbon-hydrogen bonds, a difficult feat to accomplish by traditional chemistry. Much of the activity in this field has centered on the bacterial enzyme CYP102A1, or BM3, from Bacillus megaterium, as it has shown itself capable of hydroxylating/acting upon a wide range of substrates, thereby producing industrially relevant pharmaceuticals, fine chemicals, and hormones. In addition, unlike most cytochromes, BM3 is both soluble and fused to its natural redox partner, thus facilitating its use.

Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures.

Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives.

α-2-Macroglobulin induces the shedding of microvesicles from cutaneous wound myofibroblasts.

Microvesicles (MVs) are recognized as an important class of cell-to-cell messengers. Although the properties of MVs are increasingly documented, the mechanisms regulating MV biogenesis remain debated. Myofibroblasts are a key cellular component of wound healing and have been shown to produce MVs upon stimulation with serum.

Dynamics of Endothelial Cell Responses to Laminar Shear Stress on Surfaces Functionalized with Fibronectin-Derived Peptides.

Surface endothelialization could improve the long-term performance of vascular grafts and stents. We previously demonstrated that aerosol-generated fibronectin-derived peptide micropatterns consisting of GRGDS spots over a WQPPRARI background increase endothelial cell yields in static cultures. We developed a novel fluorophore-tagged RGD peptide (RGD-TAMRA) to visualize cell-surface interactions in real-time.

Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models.

Monitoring of adherent live cells morphology using the undecimated wavelet transform multivariate image analysis (UWT-MIA).

Cell morphology is an important macroscopic indicator of cellular physiology and is increasingly used as a mean of probing culture state in vitro. Phase contrast microscopy (PCM) is a valuable tool for observing live cells morphology over long periods of time with minimal culture artifact. Two general approaches are commonly used to analyze images: individual object segmentation and characterization by pattern recognition.

Hutchinson-Gilford progeria syndrome as a model for vascular aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a de novo genetic mutation that leads to the accumulation of a splicing isoform of lamin A termed progerin. Progerin expression alters the organization of the nuclear lamina and chromatin. The life expectancy of HGPS patients is severely reduced due to critical cardiovascular defects.

Analytical solution of Luedeking-Piret equation for a batch fermentation obeying Monod growth kinetics.

Not so many fermentation mathematical models allow analytical solutions of batch process dynamics. The most widely used is the combination of the logistic microbial growth kinetics with Luedeking-Piret bioproduct synthesis relation. However, the logistic equation is principally based on formalistic similarities and only fits a limited range of fermentation types.

A fluorophore-tagged RGD peptide to control endothelial cell adhesion to micropatterned surfaces.

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed.

Selection and tuning of a fast and simple phase-contrast microscopy image segmentation algorithm for measuring myoblast growth kinetics in an automated manner.

Acquiring and processing phase-contrast microscopy images in wide-field long-term live-cell imaging and high-throughput screening applications is still a challenge as the methodology and algorithms used must be fast, simple to use and tune, and as minimally intrusive as possible. In this paper, we developed a simple and fast algorithm to compute the cell-covered surface (degree of confluence) in phase-contrast microscopy images. This segmentation algorithm is based on a range filter of a specified size, a minimum range threshold, and a minimum object size threshold.

New developments in lentiviral vector design, production and purification.

Introduction: Lentiviruses are a very potent class of viral vectors for which there is presently a rapidly growing interest for a number of gene therapy. However, their construction, production and purification need to be performed according to state-of-the-art techniques in order to obtain sufficient quantities of high purity material of any usefulness and safety.

Areas Covered: The recent advances in the field of recombinant lentivirus vector design, production and purification will be reviewed with an eye toward its utilization for gene therapy.

Cell culture tracking by multivariate analysis of raw LCMS data.

Liquid chromatography mass spectrometry (LCMS) is a powerful technique that could serve to rapidly characterize cell culture protein expression profile and be used as a process monitoring and control tool. However, this application is often hampered by both the sample proteome and the LCMS signal complexities as well as the variability of this signal. To alleviate this problem, culture samples are usually extensively fractionated and pretreated before being analyzed by top-end instruments.

Single-cell level analysis of megakaryocyte growth and development.

Several fundamental questions regarding cell growth and development can be answered by recording and analyzing the history of cells and their progeny. Herein, long-term and large-field live cell imaging was used to study the process of megakaryopoiesis at the single cell level (n = 9300) from human CD34+ cord blood (CB) in the presence of thrombopoietin (TPO) or the cytokine cocktail BS1 with or without nicotinamide (NIC). Comparative analyses revealed that the cocktail BS1 increased the mitotic and proplatelet rate of diploid and polyploid cells, respectively.

Megakaryocyte and platelet production from human cord blood stem cells.

The cloning of thrombopoietin together with advances in the culture of hematopoietic stem cells have paved the way for the study of megakaryopoiesis, ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes, such as platelets. This chapter describes a 14-day culture protocol for the production of human megakaryocytes (MKs) and platelets, and assays that can be used to characterize the functional properties of the platelets produced ex vivo. CD34(+) cells isolated from cord blood cells are grown in a serum-free medium supplemented with newly developed cytokine cocktails optimized for MK differentiation, expansion, and maturation.

Overview of current scalable methods for purification of viral vectors.

As a result of the growing interest in the use of viruses for gene therapy and vaccines, many virus-based products are being developed. The manufacturing of viruses poses new challenges for process developers and regulating authorities that need to be addressed to ensure quality, efficacy, and safety of the final product. The design of suitable purification strategies will depend on a multitude of variables including the vector production system and the nature of the virus.

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34(+) cells and megakaryocyte progenitors in culture.

Background Aims: Expansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.

Methods: We measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34(+) cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.

Polyploid megakaryocytes can complete cytokinesis.

Megakaryocytes (MK) undergo polyploidization through endomitosis, a mitotic process that ends prematurely due to aborted cytokinesis. To better understand this and other events associated with MK differentiation, we performed long-term and large-field live cell imaging of human MKs derived in cord blood (CB) and bone marrow (BM) CD34(+) cell cultures. Polyploid level of imaged cells was evaluated using three complementary approaches; cell history, cell size and ploidy correlation and nuclei staining.

New protocol for lentiviral vector mass production.

Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography.

High-level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene-switch.

Fast and efficient production of recombinant proteins for structural and functional studies is a crucial issue for research and for industry. To this end, we have developed an efficient system to generate in less than 2 months, starting from the cDNA, pools of CHO cells stably expressing high-level of recombinant proteins. It is based on lentiviral vectors (LVs) for stable transduction coupled with the cumate gene-switch for inducible and efficient gene expression.

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins.

Background: Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.