Biological Sciences

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Dancing to Another Tune—Adhesive Moonlighting Proteins in Bacteria

Author(s):Veera Kainulainen -- Timo K. Korhonen
Journal: Biology
Publisher:
Abstract
| Pages: 178-204
Biological moonlighting refers to proteins which express more than one function. Moonlighting proteins occur in pathogenic and commensal as well as in Gram-positive and Gram-negative bacteria. The canonical functions of moonlighting proteins are in essential cellular processes, i.e., glycolysis, protein synthesis, chaperone activity, and nucleic acid stability, and their moonlighting functions include binding to host epithelial and phagocytic cells, subepithelia, cytoskeleton as well as to mucins and circulating proteins of the immune and hemostatic systems. Sequences of the moonlighting proteins do not contain known motifs for surface export or anchoring, and it has remained open whether bacterial moonlighting proteins are actively secreted to the cell wall or whether they are released from traumatized cells and then rebind onto the bacteria. In lactobacilli, ionic interactions with lipoteichoic acids and with cell division sites are important for surface localization of the proteins. Moonlighting proteins represent an abundant class of bacterial adhesins that are part of bacterial interactions with the environment and in responses to environmental changes. Multifunctionality in bacterial surface proteins appears common: the canonical adhesion proteins fimbriae express also nonadhesive functions, whereas the mobility organelles flagella as well as surface proteases express adhesive functions.

Automated Sample Preparation Platform for Mass Spectrometry-Based Plasma Proteomics and Biomarker Discovery

Author(s):Vilém Guryča -- Daniel Roeder -- Paolo Piraino -- Jens Lamerz -- Axel Ducret -- Hanno Langen -- Paul Cutler
Journal: Biology
Publisher:
Abstract
| Pages: 205-219
The identification of novel biomarkers from human plasma remains a critical need in order to develop and monitor drug therapies for nearly all disease areas. The discovery of novel plasma biomarkers is, however, significantly hampered by the complexity and dynamic range of proteins within plasma, as well as the inherent variability in composition from patient to patient. In addition, it is widely accepted that most soluble plasma biomarkers for diseases such as cancer will be represented by tissue leakage products, circulating in plasma at low levels. It is therefore necessary to find approaches with the prerequisite level of sensitivity in such a complex biological matrix. Strategies for fractionating the plasma proteome have been suggested, but improvements in sensitivity are often negated by the resultant process variability. Here we describe an approach using multidimensional chromatography and on-line protein derivatization, which allows for higher sensitivity, whilst minimizing the process variability. In order to evaluate this automated process fully, we demonstrate three levels of processing and compare sensitivity, throughput and reproducibility. We demonstrate that high sensitivity analysis of the human plasma proteome is possible down to the low ng/mL or even high pg/mL level with a high degree of technical reproducibility.

Local Similarity Search to Find Gene Indicators in Mitochondrial Genomes

Author(s):Ruby L. V. Moritz -- Matthias Bernt -- Martin Middendorf
Journal: Biology
Publisher:
Abstract
| Pages: 220-242
Given a set of nucleotide sequences we consider the problem of identifying conserved substrings occurring in homologous genes in a large number of sequences. The problem is solved by identifying certain nodes in a suffix tree containing all substrings occurring in the given nucleotide sequences. Due to the large size of the targeted data set, our approach employs a truncated version of suffix trees. Two methods for this task are introduced: (1) The annotation guided marker detection method uses gene annotations which might contain a moderate number of errors; (2) The probability based marker detection method determines sequences that appear significantly more often than expected. The approach is successfully applied to the mitochondrial nucleotide sequences, and the corresponding annotations that are available in RefSeq for 2989 metazoan species. We demonstrate that the approach finds appropriate substrings.

Conjugates of Small Molecule Drugs with Antibodies and Other Proteins

Author(s):Yang Feng -- Zhongyu Zhu -- Weizao Chen -- Ponraj Prabakaran -- Kedan Lin -- Dimiter S. Dimitrov
Journal: Biomedicines
Publisher:
Abstract
| Pages: 1-13
Conjugates of small molecule drugs with antibodies (ADCs) and with other proteins (protein-drug conjugates, PDC) are used as a new class of targeted therapeutics combining the specificity of monoclonal antibodies (mAbs) and other proteins with potent cytotoxic activity of small molecule drugs for the treatment of cancer and other diseases. A(P)DCs have three major components, antibody (targeting protein), linker and payload, the cytotoxic drug. Recently, advances in identifying targets, selecting highly specific mAbs of preferred isotypes, optimizing linker technology and improving chemical methods for conjugation have led to the approval of two ADCs by Food and Drug Administration (FDA) and more than 30 ADCs in advanced clinical development. However, the complex and heterogeneous nature of A(P)DCs often cause poor solubility, instability, aggregation and eventually unwanted toxicity. This article reviews the main components of A(P)DCs, and discusses the choices for drugs, linkers and conjugation methods currently used. Future work will need to focus on developments and strategies for overcoming such major problems associated with the A(P)DCs.

Design and Potential of Non-Integrating Lentiviral Vectors

Author(s):Aaron Shaw -- Kenneth Cornetta
Journal: Biomedicines
Publisher:
Abstract
| Pages: 14-35
Lentiviral vectors have demonstrated promising results in clinical trials that target cells of the hematopoietic system. For these applications, they are the vectors of choice since they provide stable integration into cells that will undergo extensive expansion in vivo. Unfortunately, integration can have unintended consequences including dysregulated cell growth. Therefore, lentiviral vectors that do not integrate are predicted to have a safer profile compared to integrating vectors and should be considered for applications where transient expression is required or for sustained episomal expression such as in quiescent cells. In this review, the system for generating lentiviral vectors will be described and used to illustrate how alterations in the viral integrase or vector Long Terminal Repeats have been used to generate vectors that lack the ability to integrate. In addition to their safety advantages, these non-integrating lentiviral vectors can be used when persistent expression would have adverse consequences. Vectors are currently in development for use in vaccinations, cancer therapy, site-directed gene insertions, gene disruption strategies, and cell reprogramming. Preclinical work will be described that illustrates the potential of this unique vector system in human gene therapy.

Oncolytic Adenoviruses in Cancer Treatment

Author(s):Ramon Alemany
Journal: Biomedicines
Publisher:
Abstract
| Pages: 36-49
The therapeutic use of viruses against cancer has been revived during the last two decades. Oncolytic viruses replicate and spread inside tumors, amplifying their cytotoxicity and simultaneously reversing the tumor immune suppression. Among different viruses, recombinant adenoviruses designed to replicate selectively in tumor cells have been clinically tested by intratumoral or systemic administration. Limited efficacy has been associated to poor tumor targeting, intratumoral spread, and virocentric immune responses. A deeper understanding of these three barriers will be required to design more effective oncolytic adenoviruses that, alone or combined with chemotherapy or immunotherapy, may become tools for oncologists.

Stem Cell Banking for Regenerative and Personalized Medicine

Author(s):David T. Harris
Journal: Biomedicines
Publisher:
Abstract
| Pages: 50-79
Regenerative medicine, tissue engineering and gene therapy offer the opportunity to treat and cure many of today’s intractable afflictions. These approaches to personalized medicine often utilize stem cells to accomplish these goals. However, stem cells can be negatively affected by donor variables such as age and health status at the time of collection, compromising their efficacy. Stem cell banking offers the opportunity to cryogenically preserve stem cells at their most potent state for later use in these applications. Practical stem cell sources include bone marrow, umbilical cord blood and tissue, and adipose tissue. Each of these sources contains stem cells that can be obtained from most individuals, without too much difficulty and in an economical fashion. This review will discuss the advantages and disadvantages of each stem cell source, factors to be considered when contemplating banking each stem cell source, the methodology required to bank each stem cell source, and finally, current and future clinical uses of each stem cell source.

Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment

Author(s):J. Fraser Wright
Journal: Biomedicines
Publisher:
Abstract
| Pages: 80-97
Adeno-associated virus (AAV)-based vectors expressing therapeutic genes continue to demonstrate great promise for the treatment of a wide variety of diseases and together with other gene transfer vectors represent an emerging new therapeutic paradigm comparable in potential impact on human health to that achieved by recombinant proteins and vaccines. A challenge for the current pipeline of AAV-based investigational products as they advance through clinical development is the identification, characterization and lot-to-lot control of the process- and product-related impurities present in even highly purified preparations. Especially challenging are AAV vector product-related impurities that closely resemble the vector itself and are, in some cases, without clear precedent in established biotherapeutic products. The determination of acceptable levels of these impurities in vectors prepared for human clinical product development, with the goal of new product licensure, requires careful risk and feasibility assessment. This review focuses primarily on the AAV product-related impurities that have been described in vectors prepared for clinical development.

Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1

Author(s):Teresa Flett -- Elizabeth J. Campbell -- Elisabeth Phillips -- Margreet C. M. Vissers -- Gabi U. Dachs
Journal: Biomedicines
Publisher:
Abstract
| Pages: 98-109
Humans are unable to synthesise ascorbate (Vitamin C) due to the lack of a functional gulonolactone oxidase (Gulo), the enzyme that catalyses the final step in the biosynthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including as a cofactor for metalloenzymes that regulate the transcription factor hypoxia-inducible factor-1 (HIF-1), which governs cell survival under hypoxia. In most animals, ascorbate is made in liver cells. This study aimed to restore ascorbate synthesis to human hepatocellular carcinoma HepG2 cells and determine the effect of internally produced ascorbate on HIF-1 activation. HepG2 cells were gene-modified with a plasmid encoding the mouse Gulo cDNA, tested for genomic incorporation by PCR and ascorbate synthesis by high performance liquid chromatography. Levels of HIF-1 protein were measured using Western blotting. Gulo-modified HepG2 cells showed increased adherence compared to control HepG2 cells. A PCR-positive clone synthesised ascorbate when the Gulo substrate, l-gulono-1,4-lactone, was supplied. Intracellular ascorbate concentrations reached 5% of saturation levels (6 nmol/106 cells). Addition of ascorbate or gulonolactone reduced HIF-1 accumulation in the Gulo clone, but also in parental HepG2 cells. Our data confirm the requirement for a number of factors in addition to Gulo in the ascorbate biosynthesis pathway in human cells.
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