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1.
Int J Mol Sci ; 23(9)2022 May 06.
Article in English | MEDLINE | ID: mdl-35563580

ABSTRACT

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins' molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings.


Subject(s)
Proteome , Ribosomal Proteins , Ascorbate Peroxidases , Keratins , Mechanistic Target of Rapamycin Complex 1 , Proteome/metabolism
2.
Biology (Basel) ; 11(2)2022 Feb 11.
Article in English | MEDLINE | ID: mdl-35205152

ABSTRACT

TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes.

3.
JCI Insight ; 6(21)2021 11 08.
Article in English | MEDLINE | ID: mdl-34554930

ABSTRACT

IL-33, a nuclear alarmin released during cell death, exerts context-specific effects on adaptive and innate immune cells, eliciting potent inflammatory responses. We screened blood, skin, and kidney tissues from patients with systemic lupus erythematosus (SLE), a systemic autoimmune disease driven by unabated type I IFN production, and found increased amounts of extracellular IL-33 complexed with neutrophil extracellular traps (NETs), correlating with severe, active disease. Using a combination of molecular, imaging, and proteomic approaches, we show that SLE neutrophils, activated by disease immunocomplexes, release IL-33-decorated NETs that stimulate robust IFN-α synthesis by plasmacytoid DCs in a manner dependent on the IL-33 receptor ST2L. IL33-silenced neutrophil-like cells cultured under lupus-inducing conditions generated NETs with diminished interferogenic effect. Importantly, NETs derived from patients with SLE are enriched in mature bioactive isoforms of IL-33 processed by the neutrophil proteases elastase and cathepsin G. Pharmacological inhibition of these proteases neutralized IL-33-dependent IFN-α production elicited by NETs. We believe these data demonstrate a novel role for cleaved IL-33 alarmin decorating NETs in human SLE, linking neutrophil activation, type I IFN production, and end-organ inflammation, with skin pathology mirroring that observed in the kidneys.


Subject(s)
Dendritic Cells/metabolism , Extracellular Traps/immunology , Interferon-alpha/immunology , Interleukin-33/metabolism , Lupus Erythematosus, Systemic/immunology , Case-Control Studies , Humans
4.
Sci Rep ; 10(1): 4815, 2020 03 16.
Article in English | MEDLINE | ID: mdl-32179759

ABSTRACT

Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epithelium-derived factor), AMBP (Protein AMBP), LYZ (Lysozyme C), HBB (Hemoglobin subunit beta) and IGHA1 (Immunoglobulin heavy constant alpha 1)], as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho = 0.5-0.9) with results from respective ELISA. Five proteins [AMBP, B2M, LYZ, HBB and SERPINF1] were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p < 0.0001) compared to individual markers. Collectively, our study describes a multiplex assay for absolute quantification and verification analysis of previously described putative CKD prognostic markers, laying the groundwork for further use in prospective validation studies.


Subject(s)
Alpha-Globulins , Complement C1 Inhibitor Protein , Mass Spectrometry/methods , Muramidase/blood , Renal Insufficiency, Chronic/diagnosis , beta 2-Microglobulin/blood , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Cohort Studies , Disease Progression , Female , Follow-Up Studies , Glomerular Filtration Rate , Hemoglobin Subunits , Humans , Longitudinal Studies , Male , Middle Aged , Prognosis
5.
Oncol Rep ; 42(4): 1441-1450, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31524267

ABSTRACT

Cervical cancer remains the fourth most common and most lethal type of cancer in women, despite the applied regular screening and prevention strategies, while the available treatment schemes still pose a threat to fertility. Substantial understanding of the underlying mechanisms and development of novel diagnostic, prognostic and therapeutic approaches are critical steps for improving cervical cancer management. Towards this goal, a comparative proteomic analysis was conducted between three cervical cancer cell lines (HeLa: HPV18+, SiHa: HPV16+, C33A: HPV­) and normal cervical keratinocytes (HCK1T). The total cell extract of each cell line was analyzed by liquid chromatography coupled to tandem mass spectrometry (LC­MS/MS). Differential expression analysis revealed 919, 826 and 1,370 deregulated proteins in the comparisons of HeLa, SiHa and C33A with HCK1T cell lines, respectively. Pathway enrichment analysis of the differentially expressed proteins highlighted common cancer characteristics such as high metabolic demands and increased cell turnover, confirming the validity of the proteomic results. Extensive literature mining of the consistently differentially expressed proteins that resulted from the three comparisons was performed leading to a shortlist of 21 proteins that are potentially involved in cervical malignancy. The criteria for this shortlisting were the association of the proteins with various types of cancer, while there is no study as yet associating their expression to cervical cancer. Moreover, the expression trend of two of the shortlisted proteins was validated using western blot analysis. The proteomic datasets generated in this study can be utilized to enrich the current knowledge on cervical cancer pathology and unveil key molecular mechanisms of carcinogenesis. In conclusion, the shortlist of consistently deregulated proteins between cervical cancer cell lines and normal cervical keratinocytes can be used for validation in clinical samples and in functional investigation experiments that could ultimately lead to the discovery of novel disease biomarkers and drug targets.

6.
Expert Rev Mol Diagn ; 19(6): 499-515, 2019 06.
Article in English | MEDLINE | ID: mdl-31057016

ABSTRACT

Introduction: Multiple (or selected) reaction monitoring-mass spectrometry (MRM/SRM) is a targeted proteomic method that can be used for relative and absolute quantification. Multiple reports exist supporting the potential of the approach in proteomic biomarker validation. Areas covered: To get an overview of the applications of MRM in protein quantification in plasma, a search in MedLine/PubMed was performed using the keywords: 'MRM/SRM plasma proteomic/proteomics/proteome'. The retrieved studies were further filtered to focus on disease biomarkers and the main results are summarized. Expert opinion: MRM is increasingly employed for the quantification of both well-established but also newly discovered putative biomarkers and occasionally their post-translationally modified forms in plasma. Fractionation is regularly required for the detection of low abundance proteins. Standardized procedures to facilitate assay establishment and marker quantification have been proposed and, in few cases, implemented. Nevertheless, in most cases, absolute quantification is not performed. To advance, multiple technical issues including the regular use of standard labeled peptides and appropriate quality controls to monitor assay performance should be considered. Additionally, clinical aspects involving careful study design to address biomarker clinical use should also be considered.


Subject(s)
Biomarkers/blood , Proteome , Proteomics , Disease Susceptibility , Humans , Molecular Diagnostic Techniques , Proteomics/methods
7.
Methods Mol Biol ; 1959: 205-223, 2019.
Article in English | MEDLINE | ID: mdl-30852825

ABSTRACT

Selected/multiple reaction monitoring-mass spectrometry (SRM/MRM) is an analytical method that is frequently combined to the use of stable isotope-labeled standard (SIS) peptides for absolute protein quantification. The application of SRM/MRM is a relatively recent development in the proteomics field for analysis of biological samples (plasma, urine, cell/tissue lysates) targeting to a large extent biomarker validation. Although MRM generally by-passes the use of antibodies (being linked to sub-optimal assay specificity in many cases), bioanalytical validation of MRM protocols has not been robustly applied because of sensitivity issues, in contrary to antibody-based methods. In this chapter, we will discuss the points that should be addressed for MRM method development in clinical proteomics applications.


Subject(s)
Biomarkers , Mass Spectrometry , Proteomics , Chromatography, Liquid , Isotope Labeling , Mass Spectrometry/methods , Peptides , Proteomics/methods , Reproducibility of Results , Workflow
8.
Int J Oncol ; 53(5): 2111-2122, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30106135

ABSTRACT

The available therapeutic approaches for cervical cancer can seriously affect the fertility potential of patient; thus, there is a pressing requirement for less toxic and targeted therapies. The membrane proteome is a potential source of therapeutic targets; however, despite the significance of membrane proteins in cancer, proteomic analysis has been a challenging task due to their unique biochemical properties. The aim of the present study was to develop an efficient membrane protein enrichment protocol, and to the best of our knowledge, to compare for the first time the expression pattern of membrane proteins of one normal cell line, HCK1T, and three cervical cancer cell lines, C33A, a human papilloma virus (HPV)-negative cell line, and two HPV-positive cell lines, SiHa (HPV16+) and HeLa (HPV18+). The study aimed to identify the proteins that are involved in cervical carcinogenesis and may constitute novel drug targets. Membrane protein isolation, liquid chromatography coupled with tandem mass spectrometry proteomics and bioinformatics analysis were performed in the membrane fraction of the informative cervical cell lines following a novel enrichment protocol. The percentages of membrane and transmembrane proteins in the enrichment protocol were higher compared with those of the corresponding data derived from total cell extract analysis. Differentially expressed proteins were detected by the comparison of the cervical cancer cell lines with the normal cell line. These proteins constitute molecular features of cancer pathology and participate in biological pathways relevant to malignancy, including 'HIPPO signaling', 'PI3K/Akt signaling', 'cell cycle: G2/M DNA damage checkpoint regulation' and 'EIF2 signaling'. These unique membrane protein identifications offer insights on a previously inaccessible region of the cervical cancer proteome, and may represent putative diagnostic and prognostic markers, and eventually therapeutic targets.


Subject(s)
Membrane Proteins/analysis , Membrane Proteins/metabolism , Proteomics/methods , Uterine Cervical Neoplasms/pathology , Cell Line, Tumor , Chromatography, Liquid , Female , HeLa Cells , Humans , Membrane Proteins/isolation & purification , Protein Interaction Maps , Reproducibility of Results , Software , Tandem Mass Spectrometry , Uterine Cervical Neoplasms/metabolism
9.
Proteomics ; 18(14): e1700463, 2018 07.
Article in English | MEDLINE | ID: mdl-29785737

ABSTRACT

Clinical proteomics, the application of proteome analysis to serve a clinical purpose, represents a major field in the area of proteome research. Over 1000 manuscripts on this topic are published each year, with numbers continuously increasing. However, the anticipated outcome, the transformation of the reported findings into improvements in patient management, is not immediately evident. In this article, the value and validity of selected clinical proteomics findings are investigated, and it is assessed how far implementation has progressed. A main conclusion from this assessment is that to achieve implementation, well-powered clinical studies are required in the appropriate population, addressing a specific clinical need and with a clear context-of-use. Efforts toward implementation, to be feasible, must be supported by the key players in science: publishers and funders. The authors propose a change on objectives, from additional discovery studies toward studies aiming at validation of the plethora of potential biomarkers that have been described, to demonstrate practical value of clinical proteomics. All elements required, potential biomarkers, technologies, and bio-banked samples are available (based on today's literature), hence a change in focus from discovery toward validation and application is not only urgently necessary, but also possible based on resources available today.


Subject(s)
Biomarkers/analysis , Clinical Medicine , Drug Discovery , Mass Spectrometry/methods , Proteins/metabolism , Proteome/analysis , Humans
10.
Oncol Rep ; 39(4): 1547-1554, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29436691

ABSTRACT

Cervical cancer incidence is tightly linked to HPV infection, and particularly virus types 16 and 18 cause the majority of cases presenting with pre-cancerous stages of cervical intraepithelial neoplasia (CIN). Structural and functional information concerning HPV proteins can offer novel insight into the mechanism(s) of cancer progression in the cervical epithelium. Recently, novel structural determinants of the interactions of viral proteins with their targets in keratinocytes have been elucidated. These exciting findings open the way for the development of targeted anti-oncogenic therapies, and may eventually allow the introduction of novel approaches for a rational cervical cancer treatment.


Subject(s)
Human papillomavirus 16/chemistry , Human papillomavirus 18/chemistry , Uterine Cervical Neoplasms/genetics , Viral Proteins/chemistry , Epithelium/pathology , Epithelium/virology , Female , Host-Pathogen Interactions/genetics , Human papillomavirus 16/genetics , Human papillomavirus 16/pathogenicity , Human papillomavirus 18/genetics , Human papillomavirus 18/pathogenicity , Humans , Keratinocytes/chemistry , Keratinocytes/virology , Precancerous Conditions/genetics , Precancerous Conditions/pathology , Precancerous Conditions/virology , Structure-Activity Relationship , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology , Viral Proteins/genetics
11.
Cancer Genomics Proteomics ; 14(6): 507-521, 2017.
Article in English | MEDLINE | ID: mdl-29109100

ABSTRACT

BACKGROUND: Oncogenic infection by HPV, eventually leads to cervical carcinogenesis, associated by deregulation of specific pathways and protein expression at the intracellular and secretome level. Thus, secretome analysis can elucidate the biological mechanisms contributing to cervical cancer. In the present study we systematically analyzed its constitution in four cervical cell lines employing a highly sensitive proteomic technology coupled with bioinformatics analysis. MATERIALS AND METHODS: LC/MS-MS proteomics and bioinformatics analysis were performed in the secretome of four informative cervical cell lines SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-) and HCK1T (normal). RESULTS: The proteomic pattern of each cancer cell line compared to HCK1T was identified and a detailed bioinformatics analysis disclosed inhibition of matrix metalloproteases in cancer cell lines. This prediction was further confirmed via zymography for MMP-2 and MMP-9, western blot analysis for ADAM10 and by MRM for TIMP1. The differential expression of important secreted proteins such as CATD, FUCA1 and SOD2 was also confirmed by western blot analysis. MRM-targeted proteomics analysis confirmed the differential expression of CATD, CATB, SOD2, QPCT and NEU1. CONCLUSION: High resolution proteomics analysis of cervical cancer secretome revealed significantly deregulated biological processes and proteins implicated in cervical carcinogenesis.


Subject(s)
Chromatography, Liquid/methods , Mass Spectrometry/methods , Matrix Metalloproteinases/genetics , Peptide Hydrolases/genetics , Proteomics/methods , Uterine Cervical Neoplasms/genetics , Cell Line, Tumor , Female , Humans
12.
Sci Rep ; 7(1): 15160, 2017 11 09.
Article in English | MEDLINE | ID: mdl-29123184

ABSTRACT

Mechanisms underlying the onset and progression of nephropathy in diabetic patients are not fully elucidated. Deregulation of proteolytic systems is a known path leading to disease manifestation, therefore we hypothesized that proteases aberrantly expressed in diabetic nephropathy (DN) may be involved in the generation of DN-associated peptides in urine. We compared urinary peptide profiles of DN patients (macroalbuminuric, n = 121) to diabetic patients with no evidence of DN (normoalbuminuric, n = 118). 302 sequenced, differentially expressed peptides (adjusted p-value < 0.05) were analysed with the Proteasix tool predicting proteases potentially involved in their generation. Activity change was estimated based on the change in abundance of the investigated peptides. Predictions were correlated with transcriptomics (Nephroseq) and relevant protein expression data from the literature. This analysis yielded seventeen proteases, including multiple forms of MMPs, cathepsin D and K, kallikrein 4 and proprotein convertases. The activity of MMP-2 and MMP-9, predicted to be decreased in DN, was investigated using zymography in a DN mouse model confirming the predictions. Collectively, this proof-of-concept study links urine peptidomics to molecular changes at the tissue level, building hypotheses for further investigation in DN and providing a workflow with potential applications to other diseases.


Subject(s)
Diabetic Nephropathies/pathology , Diabetic Nephropathies/physiopathology , Peptide Hydrolases/analysis , Proteome/analysis , Urine/chemistry , Gene Expression Profiling , Humans
13.
Cancer Genomics Proteomics ; 14(4): 253-266, 2017.
Article in English | MEDLINE | ID: mdl-28647699

ABSTRACT

BACKGROUND: Both HPV-positive and -negative cervical cancers are primarily associated with features of cell cycle and cytoskeletal disruption; however, the actual biological processes affected remain elusive. To this end, we systematically characterized the intracellular proteomic profiles of four distinct and informative cervical cell lines. MATERIALS AND METHODS: Cell extracts from a normal cervical (HCK1T) and three cervical cancer cell lines, one HPV-negative (C33A), and two HPV-positive, SiHa (HPV16+) and HeLa (HPV18+), were analyzed by 2-dimensional electrophoresis and differentially expressed proteins were identified by MALDI-TOF mass spectrometry, while differential expression was confirmed by western blot analysis. RESULTS: In total, 113 proteins were found differentially expressed between the normal and the cervical cancer lines. Bioinformatics analysis revealed the actin cytoskeleton signaling pathway to be significantly affected, while up-regulation of cofilin-1, an actin depolymerizing factor, was documented and further validated by western blotting. Furthermore, two-way comparisons among the four cell lines, revealed a set of 18 informative differentially expressed proteins. CONCLUSION: These novel identified proteins provide the impetus for further functional studies to dissect the mechanisms operating in the two distinct pathways of cervical carcinogenesis.


Subject(s)
Cytoskeletal Proteins/metabolism , Proteomics , Uterine Cervical Neoplasms/metabolism , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uterine Cervical Neoplasms/pathology
14.
Biomed Res Int ; 2017: 4180703, 2017.
Article in English | MEDLINE | ID: mdl-28261610

ABSTRACT

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.


Subject(s)
Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Neoplastic , NF-E2-Related Factor 2/metabolism , Peroxiredoxins/metabolism , Transforming Growth Factor beta/metabolism , Uterine Cervical Neoplasms/metabolism , Algorithms , Carcinogenesis , Cell Line, Tumor , Computational Biology , Electrophoresis, Gel, Two-Dimensional , Female , HeLa Cells , Human papillomavirus 16 , Humans , Papillomavirus Infections/complications , Peptides/chemistry , Proteomics , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry
15.
Expert Rev Proteomics ; 13(8): 731-45, 2016 08.
Article in English | MEDLINE | ID: mdl-27398979

ABSTRACT

INTRODUCTION: The HPV virus accounts for the majority of cervical cancer cases. Although a diagnostic tool (Pap Test) is widely available, cervical cancer incidence still remains high worldwide, and especially in developing countries, attributed to a large extent to suboptimal sensitivities of the Pap test and unavailability of the test in developing countries. AREAS COVERED: Proteomics approaches have been used in order to understand the HPV virus correlation to cervical cancer pathology, as well as to discover putative biomarkers for early cervical cancer diagnosis and drug mode of action. Expert commentary: The present review summarizes the latest in vitro and in vivo proteomic studies for the discovery of putative cervical cancer biomarkers and the evaluation of available drugs and treatments.


Subject(s)
Biomarkers, Tumor/genetics , Papillomavirus Infections/genetics , Proteomics , Uterine Cervical Neoplasms/genetics , Early Detection of Cancer , Female , Humans , Papillomaviridae , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/pathology
16.
Virus Res ; 175(1): 1-11, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23583684

ABSTRACT

Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudotyped lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6×10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy.


Subject(s)
Genetic Vectors/isolation & purification , Lentivirus/isolation & purification , Ultrafiltration/methods , Virology/methods , Animals , Genetic Therapy/methods , Genetic Vectors/chemistry , Hematopoietic Stem Cells/virology , Humans , Lentivirus/chemistry , Mice , Mice, Inbred C57BL , Peptide Mapping , Transduction, Genetic , Ultracentrifugation/methods
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