Applications In Molecular Medicine Research Articles

SpotClean adjusts for spot swapping in spatial transcriptomics data

Spatial transcriptomics is a powerful and widely used approach for profiling the gene expression landscape across a tissue with emerging applications in molecular medicine and tumor diagnostics. Recent spatial transcriptomics experiments utilize slides containing thousands of spots with spot-specific barcodes that bind RNA. Ideally, unique molecular identifiers (UMIs) at a spot measure spot-specific expression, but this is often not the case in practice due to bleed from nearby spots, an artifact we refer to as spot swapping. To improve the power and precision of downstream analyses in spatial transcriptomics experiments, we propose SpotClean, a probabilistic model that adjusts for spot swapping to provide more accurate estimates of gene-specific UMI counts. SpotClean provides substantial improvements in marker gene analyses and in clustering, especially when tissue regions are not easily separated. As demonstrated in multiple studies of cancer, SpotClean improves tumor versus normal tissue delineation and improves tumor burden estimation thus increasing the potential for clinical and diagnostic applications of spatial transcriptomics technologies.

TissueSpace: a web tool for rank-based transcriptome representation and its applications in molecular medicine.

Cross-platform or cross-experiment transcriptome data is hard to compare as the original gene expression values from different platforms cannot be compared directly. The inherent gene expression ranking information is rarely utilized. Use of reduced vector to represent transcriptome data independent of platforms. Thus, we turned the expression profile into a rank vector, where a higher expression has a higher rank value, then applied Latent semantic analysis (LSA) to get compact and continuous 100-dimensional vector representations for samples. Results showed that the reconstructed vector has a precision of 96.7% in recovering tissue labels from an independent dataset. A user-friendly tool TissueSpace was developed, which provides users the following functionalities: (1) convert different gene ID types to Ensembl gene IDs; (2) project any human transcriptome profile to get vector representation for downstream analysis; (3) functional enrichment for each of the 100-dimensional vector features. Case studies for its applications in human common diseases indicate its usefulness. TissueSpace could be used to generate testable hypotheses for translational medicine. The TissueSpace tool is available at http://bioinformatics.fafu.edu.cn/tissuespace/ .

Biología molecular del carcinoma de tiroides de estirpe folicular (II). Aplicaciones clínicas

El notable avance que ha experimentado la medicina molecular en los últimos años proporciona una visión tremendamente atractiva sobre las nuevas posibilidades de abordaje diagnóstico y terapéutico del carcinoma de tiroides, a la vez que nos ayuda a comprender su comportamiento biológico. 
 
 La aplicación clínica de dichos hallazgos comienza a ser una realidad. Este conocimiento puede proporcionar información para esclarecer un diagnóstico dudoso, distinguir las células malignas de las benignas, predecir la agresividad de un tumor o indicar su origen. Del mismo modo puede resultar útil para conocer las posibilidades de respuesta al tratamiento convencional más adecuado, o las implicaciones que tendrá el emergente campo de la farmacogenética. Del mismo modo las aplicaciones de la medicina molecular deben ser tenidas en cuenta en los estudios de orientación pronóstica tanto en los tumores espontáneos como en las neoplasias de origen familiar. Dicha información puede mejorar de forma importante las limitaciones actuales de los métodos de pronóstico basados en criterios clinico-patológicos.

Treatments Principles in Malignant Homeopathies

Aim of this study is to present the latest researches in the field of molecular medicine, in terms of treatments in malignant hemopathies, emerged from the P53 gene deletion in human lymphoma genome. Method: In recent years proved that the best techniques in the investigation of malignant lymphocytes are the Flow Cytometry, Elisa, ICT and Fluorescence in situ hybridization (FISH). This method is used as an alternative to chromosomal banding, a conventional application in molecular medicine. Discussion: Recent, endogenous somatic gene therapy research is a basic of trial clinical and therapeutic trial. The DNA is used to treat a disease arising as a result of mutations in chromosomal regions. In the past few years, this method has been included in the treatment of CLL, acute lymphocytic leukemia, [ALL], or multiple myeloma [MM]. Conclusion: The frequencies of P53 gene mutations and deletion in CLL can be categorized as individual biomarkers in proteomic and genomic profile for this type of leukemia that can be implemented in targeted patient treatment of personalized medicine.

Signification of Protein P-53 Isoforms in CLL

In the past few years has used the technique for analyzing deletions of genes, its rearrangements, cross-reactivity or multiplications in human genome affected of a variety genetic diseases. Was proved, the best techniques in the investigation of malignant lymphocytes are the Flow Cytometry, Elisa, ICT and Fluorescence in situ hybridization (FISH). Last method, FISH is used as an alternative to chromosomal banding, a conventional application in molecular medicine and can detect the chromosomal rearrangements and complexes of different genes in malignant diseases, like chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia, (ALL), or multiple myeloma (MM). Identification of P53 gene deletions and mutations in regions of chromosome 17 in hematological malignancies is important because these mutations have an impact on the clinical management of patients.

New Principles of Treatments in Malignant Hematological Diseases, as Acute and Chronic Lymphocytic Leukemia or Multiple Myeloma

Aim of this study is to present the latest researches in the field of molecular medicine, in terms of treatments in malignant hemopathies, emerged from the P53 gene deletion in human lymphoma genome. Method: In recent years proved that the best techniques in the investigation of malignant lymphocytes are the Flow Cytometry, Elisa, ICT and Fluorescence in Situ Hybridization (FISH). This method is used as an alternative to chromosomal banding, a conventional application in molecular medicine. Discussion: Recent, endogenous somatic gene therapy research is a basic of trial clinical and therapeutic trial. The DNA is used to treat a disease arising as a result of mutations in chromosomal regions. In the past few years, this method has been included in the treatment of CLL, acute lymphocytic leukemia, [ALL], or multiple myeloma [MM]. Conclusion: The frequencies of P53 gene mutations and deletion in CLL can be categorized as individual biomarkers in proteomic and genomic profile for this type of leukemia that can be implemented in targeted patient treatment of personalized medicine.

A biodistribution study of two differently shaped plant virus nanoparticles reveals new peculiar traits

Self-assembling plant virus nanoparticles (pVNPs) have started to be explored as nanometre-sized objects for biomedical applications, such as vaccine or drug delivery and imaging. Plant VNPs may be ideal tools in terms of biocompatibility and biodegradability endowed with a wide diversity of symmetries and dimensions, easy chemical/biological engineering, and rapid production in plants. Recently, we defined that icosahedral Tomato bushy stunt virus (TBSV) and filamentous Potato virus X (PVX) are neither toxic nor teratogenic. We report here the results of an interdisciplinary study aimed to define for the first time the biodistribution of unlabelled, unpegylated, underivatized TBSV and PVX by proved detecting antibodies. These data add new insights on the in vivo behaviour of these nano-objects and demonstrate that the pVNPs under scrutiny are each intrinsically endowed with peculiar properties foreshadowing different applications in molecular medicine.

14. Engineering and Characterization of a Cloned Adenoviral-Library to Explore Natural Virus Diversity

Large double-stranded DNA (dsDNA) viruses such as adenovirus (Ad), herpesvirus, poxvirus and their recombinant counterparts were well explored in basic virology and biomedical research. Especially for the development of novel vaccines and gene therapies, Ad gained special attention and represents the most widely explored vector worldwide. Although ~70 types of human Ad and numerous nonhuman Ad (>200) have been identified so far a system for efficient Ad genome cloning and manipulation was lacking and therefore the majority of recombinant adenoviral vectors (AdVs) are based on a small fraction of Ad types. Here we report the generation and characterization of an engineered human adenoviral-library allowing exploration and system studies of the natural Ad diversity. Towards that end we first established that adenoviral genomes can be cloned and tagged in a high-throughput manner utilizing advanced homologous recombination techniques. Wild type Ads from clinical isolates including around half of the currently known adenovirus types that represent all seven human adenovirus species were propagated and direct high-throughput cloning (HTC) applied. The integrity of cloned Ad genomes was confirmed by DNA restriction enzyme pattern and virus reconstitution was conducted using optimized conditions. Most importantly next-generation sequencing (NGS) and phylogenetic analysis was performed. As a further step, half of all cloned adenoviruses representing each species were tagged with a 2A peptide-mediated multicistronic expression cassette providing a Turbo Green fluorescent protein as in vitro marker, a NanoLuc luciferase for in vivo studies and kanamycin/neomycin as selection marker. For HTC of tagged viruses the reporter cassette was inserted into the adenovirus E3 region using different orientations because the orientation of the transgene was essential for reconstitution efficiencies. After successful reconstitution these double reporters-labeled AdVs were evaluated in cultured cell lines and mouse models. In vitro characterization revealed distinct tropisms for tested viruses. Among the currently evaluated cell lines, specie B viruses demonstrated high transduction efficiencies in epithelial (Hela- and A549 cells) and endothelial cells, while Ad5 still displayed highest transduction rates in other human and murine cell types (hepatocytes, lymphocytes, neuroblastoma cells and myoblasts). For the further characterization of unknown receptor usage, we injected recombinant viruses into DSG2 or CD46 transgenic mice for in vivo bio-distribution analyses on genome level by quantitative PCR and on protein level by immunohistology analyses. We anticipate that our engineered adenoviral-library will provide a spacious novel view to the adenovirus field. As a broader perspective it will bring AdV and also other large dsDNA viruses from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology.

FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.

Chemical biology of nucleic acids.

he XXI International Round Table (IRT) on Chemical Biology of Nucleic Acids was held in PoznanPoland, August 24�28, 2014. It attracted 400 scientists from 32 countries. Various aspects of chemistry and biology of nucleic acids and their components were discussed. There were 23 invited talks and 33 oral presentations covering topics ranging from chemical synthesis of nucleic acids to their application as drugs and tools. The meeting highlighted recent advances in DNA and RNA chemistry, biology, biochemistry, and biophysics and identified emerging concepts of their application in molecular medicine. Many talks presented at the meeting illustrated ways to approach biological properties to answer both general and detailed questions in chemical biology of nucleosides, nucleotides, and nucleic acids. More than 250 posters were presented. The poster session offered an opportunity for undergraduate Ph.D. students, postdoctoral fellows, and early career researchers to present their results and interact together and with senior scientists.To acknowledge outstanding scientific results of these young scientists, several IS3NA, A. HolyIRT Poster Awards, sponsored by Gilead Sciences were distributed in honor of Professor Antoni

48. High-Throughput Cloning and Tagging of an Engineered Adenovirus-Library Empowers Vector Diversity for Broad Applications

Adenoviral vectors are the most widely used vectors in gene therapy, largely owing to their high efficacy in gene delivery and the ability to transduce a broad variety of cell types. Although more than 60 human adenoviruses types have been identified, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types (Ad2 and 5 from species C) and their genetic modification. Predefined tissue tropism and preexisting immunity considerably limit the application of currently used vectors, making the developing of alternative AdV s mandatory. However, generation of AdVs other than species C was hampered by the lack of convenient cloning methods, for traditional approaches were limited by the low efficiency and complicate procedure.We believe that the engineered adenovirus-library is going to provide spacious novel view to the adenovirus field. As a toolkit, it will bring the AdVs from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology studies.Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here we established a seamless recombineering pipeline for high-throughput cloning and tagging of adenoviral genomes. We amplified and purified wild type adenoviruses that represent all seven human adenovirus species. Using the recombineering pipeline we generated an engineered adenovirus-library including around half of the currently available adenovirus types. To prove integrity of the cloned adenovirus genomes we performed rescue experiments in respective cell lines permissive for adenovirus infection and we optimized rescue condition by testing different molecular forms of the adenoviral DNA (linear, circular, precise release of the DNA molecule). A limited number of selected types from each species will be tagged with a P2A peptide-mediated bicistronic-expression cassette providing a Turbo Green fluorescent protein as in vitro marker and a NanoLuc luciferase for in vivo studies. These tagged adenoviral vectors will be evaluated in cultured cell lines and mouse model. A summary of the human adenoviral vector-library which can be used for further genetic modification and tagging based on the recombineering technology is shown in figure 1.We believe that the engineered adenovirus-library is going to provide spacious novel view to the adenovirus field. As a toolkit, it will bring the AdVs from mono- to multitypes and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology studies. View Large Image | Download PowerPoint Slide

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine.

BackgroundIn molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells.ResultsThe parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization.ConclusionThis methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Advances in Lipid-Based Platforms for RNAi Therapeutics

Sequence-specific gene silencing, known as RNA interference (RNAi), is a natural process that can be exploited for knocking-down specific genes involved in the insurgence/development of pathological processes. In 2001 the discovery that small interfering RNA (siRNA) can induce gene silencing without immunoresponse turned RNAi into a promising technique for the control of post-transcriptional gene expression. Nowadays, the major challenge remains infusion in vivo. Therefore, vehicles providing protection and selective transport are to be developed for efficient systemic delivery. The most used vectors are lipid-based, offering a wide range of biocompatible formulations. Here their application in molecular medicine is discussed, especially with regard to recent clinical trials where conventional therapies have failed. The role played by extended physicochemical characterization for the success of RNAi therapeutics is also evidenced.

Evolutionary molecular medicine

Evolution has long provided a foundation for population genetics, but some major advances in evolutionary biology from the twentieth century that provide foundations for evolutionary medicine are only now being applied in molecular medicine. They include the need for both proximate and evolutionary explanations, kin selection, evolutionary models for cooperation, competition between alleles, co-evolution, and new strategies for tracing phylogenies and identifying signals of selection. Recent advances in genomics are transforming evolutionary biology in ways that create even more opportunities for progress at its interfaces with genetics, medicine, and public health. This article reviews 15 evolutionary principles and their applications in molecular medicine in hopes that readers will use them and related principles to speed the development of evolutionary molecular medicine.

Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine

Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.

RNAi methods for management of insects and their pathogens.

Abstract RNA interference (RNAi), a post-transcriptional gene silencing method discovered in 1998, has been used in basic research in almost every area of biology to silence genes in order to learn about their function. This technology also has many potential applications in molecular medicine, in agriculture and many other fields. RNAi has been extensively used to understand the molecular basis of physiological, developmental, behavioural and reproductive processes in insects. This technology also has a potential to assist in developing target-specific management methods for insect pests and pathogens that cause diseases in beneficial insects. Recent literature on RNAi methods and their applications for controlling insect pests and pathogens that cause diseases in beneficial insects will be reviewed.

Molecular diagnosis of muscular dystrophies, focused on limb girdle muscular dystrophies

Muscular dystrophies include a spectrum of muscle disorders, some of which are phenotypically well characterized. The identification of dystrophin as the causative factor in Duchenne muscular dystrophy has led to the development of molecular genetics and has facilitated the division of muscular dystrophies into distinct groups, among which are the 'limb girdle muscular dystrophies'. This article reviews the methodology to be used in the diagnosis of muscular dystrophies, focused on the groups of limb girdle muscular dystrophies, and the development of new strategies to reach a final molecular diagnosis. A literature review (Medline) from 1985 to the present. Immunohistochemistry and western blotting analyses of the proteins involved in the various forms of muscular dystrophies have permitted a refined pathological approach necessary to conduct genetic studies and to offer appropriate genetic counseling. The application of molecular medicine in genetic muscular dystrophies also brings great hope to the therapeutic management of these patients.

Mass transfer model for drug delivery in tissue cells with reversible electroporation

Reversible electroporation is the temporary permeabilization of the cell membrane through the formation of nano-scale pores that are transient defects in the membrane. These pores are caused by short electrical pulses, typically on the order of a few to several hundred microseconds that are delivered by electroporation electrodes inserted around the treated tissue. Reversible electroporation has become an important technique in molecular medicine. It is used to introduce macromolecules such as genes or anticancer drugs, to which the cell membrane is normally not permeable, into the cytosol. For optimal application of molecular medicine, it is important to be able to predict precisely the mass transfer in tissue during reversible electroporation. In this study, we introduce a first attempt at developing a macroscopic mathematical model for analyzing the mass transfer into cells during reversible electroporation of tissue. The model combines a macroscopic model of the electrical fields around electroporation electrodes with a new cells-scale model of electroporation-driven mass transfer and with a macroscopic mass transfer model in tissue. The model is illustrated for a situation typical to that in electrochemotherapy in which cancer is treated with reversible electroporation and a non cell membrane permeant drug such as bleomycin.

Modification-Specific Proteomics of Plasma Membrane Proteins: Identification and Characterization of Glycosylphosphatidylinositol-Anchored Proteins Released upon Phospholipase D Treatment

Plasma membrane proteins are displayed through diverse mechanisms, including anchoring in the extracellular leaflet via glycosylphosphatidylinositol (GPI) molecules. GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family, and their importance is well-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural heterogeneity of the GPI moiety, making PLD a generally useful reagent for proteomic investigations of GPI-anchored proteins in a variety of cells, tissues, and organisms. A total of 11 human GPI-APs and 35 Arabidopsis thaliana GPI-APs were identified, representing a significant addition to the number of experimentally detected GPI-APs in both species. Computational GPI-AP sequence analysis tools were investigated for the characterization of the identified GPI-APs, and these demonstrated that there is some discrepancy in their efficiency in classification of GPI-APs and the exact assignment of omega-sites. This study highlights the efficiency of an integrative proteomics approach that combines experimental and computational methods to provide the selectivity, specificity, and sensitivity required for characterization of post-translationally modified membrane proteins.

Molecular Approaches in Pediatric Oncology

The use of molecular approaches has become part of the standard of care in the management of pediatric cancer patients. Molecular approaches are now included in the initial diagnosis, definition of prognostically distinct patient subgroups, selection of patients for specific therapies, prediction of risk for toxicities to therapy, and monitoring of patients receiving both conventional and novel targeted therapies. This clinical application of molecular medicine has been based on a growing molecular understanding of cancer biology. Studies of pediatric cancers have contributed to this understanding in many ways. We present a model for understanding cancer biology, using specific examples taken from pediatric oncology, and then discuss the application of molecular techniques to the clinical management of pediatric cancer patients.