Changes in Nuclear Orientation Patterns of Chromosome 11 during Mouse Plasmacytoma Development

Studying changes in nuclear architecture is a unique approach toward the understanding of nuclear remodeling during tumor development. One aspect of nuclear architecture is the orientation of chromosomes in the three-dimensional nuclear space. We studied mouse chromosome 11 in lymphocytes of [T38HxBALB/c]N mice with a reciprocal translocation between chromosome X and 11 (T38HT(X;11)) exhibiting a long chromosome T(11;X) and a short chromosome T(X;11) and in fast-onset plasmacytomas (PCTs) induced in the same strain. We determined the three-dimensional orientation of chromosome 11 using a mouse chromosome 11 specific multicolor banding probe. We also examined the nuclear position of the small translocation chromosome T(X;11) which contains cytoband 11E2 and parts of E1. Chromosomes can point either with their centromeric or with their telomeric end toward the nuclear center or periphery, or their position is found in parallel to the nuclear border. In T38HT(X;11) nuclei, the most frequently observed orientation pattern was with both chromosomes 11 in parallel to the nuclear border ("PP"). PCT cells showed nuclei with two or more copies of chromosome 11. In PCTs, the most frequent orientation pattern was with one chromosome in parallel and the other pointing with its centromeric end toward the nuclear periphery ("CP"). There is a significant difference between the orientation patterns observed in T38HT(X;11) and in PCT nuclei (P < .0001)

Telomere Dysfunction Is Associated with Altered {DNA} Organization in Trichoplein/Tchp/Mitostatin ({TpMs}) Depleted Cells

Abstract: Recently, we highlighted a novel role for the protein Trichoplein/TCHP/Mitostatin (TpMs), both as mitotic checkpoint regulator and guardian of chromosomal stability. TpMs-depleted cells show numerical and structural chromosome alterations that lead to genomic instability. This condition is a major driving force in malignant transformation as it allows for the cells acquiring new functional capabilities to proliferate and disseminate. Here, the effect of TpMs depletion was investigated in different TpMs-depleted cell lines by means of 3D imaging and 3D Structured illumination Microscopy. We show that TpMs depletion causes alterations in the 3D architecture of telomeres in colon cancer HCT116 cells. These findings are consistent with chromosome alterations that lead to genomic instability. Furthermore, TpMs depletion changes the spatial arrangement of chromosomes and other nuclear components. Modified nuclear architecture and organization potentially induce variations that precede the onset of genomic instability and are considered as markers of malignant transformation. Our present observations connect the tumor suppression ability of TpMs with its novel functions in maintaining the proper chromosomal segregation as well as the proper telomere and nuclear architecture. Further investigations will investigate the connection between alterations in telomeres and nuclear architecture with the progression of human tumors with the aim of developing personalized therapeutic interventions

Risk Stratification and Treatment in Smoldering Multiple Myeloma.

Smoldering multiple myeloma is a heterogeneous asymptomatic precursor to multiple myeloma. Since its identification in 1980, risk stratification models have been developed using two main stratification methods: clinical measurement-based and genetics-based. Clinical measurement models can be subdivided in three types: baseline measurements (performed at diagnosis), evolving measurements (performed over time during follow-up appointments), and imaging (for example, magnetic resonance imaging). Genetic approaches include gene expression profiling, DNA/RNA sequencing, and cytogenetics. It is important to accurately distinguish patients with indolent disease from those with aggressive disease, as clinical trials have shown that patients designated as "high-risk of progression" have improved outcomes when treated early. The risk stratification models, and clinical trials are discussed in this review

Combination of whole genome sequencing and supervised machine learning provides unambiguous identification of eae-positive Shiga toxin-producing Escherichia coli

Introduction: The objective of this study was to develop, using a genome wide machine learning approach, an unambiguous model to predict the presence of highly pathogenic STEC in E. coli reads assemblies derived from complex samples containing potentially multiple E. coli strains. Our approach has taken into account the high genomic plasticity of E. coli and utilized the stratification of STEC and E. coli pathogroups classification based on the serotype and virulence factors to identify specific combinations of biomarkers for improved characterization of eae-positive STEC (also named EHEC for enterohemorrhagic E.coli) which are associated with bloody diarrhea and hemolytic uremic syndrome (HUS) in human. Methods: The Machine Learning (ML) approach was used in this study on a large curated dataset composed of 1,493 E. coli genome sequences and 1,178 Coding Sequences (CDS). Feature selection has been performed using eight classification algorithms, resulting in a reduction of the number of CDS to six. From this reduced dataset, the eight ML models were trained with hyper-parameter tuning and cross-validation steps. Results and discussion: It is remarkable that only using these six genes, EHEC can be clearly identified from E. coli read assemblies obtained from in silico mixtures and complex samples such as milk metagenomes. These various combinations of discriminative biomarkers can be implemented as novel marker genes for the unambiguous EHEC characterization from different E. coli strains mixtures as well as from raw milk metagenomesPeer Reviewe

Granzyme B (GraB) Autonomously Crosses the Cell Membrane and Perforin Initiates Apoptosis and GraB Nuclear Localization

Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4°C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus

Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

<p>Abstract</p> <p>Background</p> <p>Expression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts <it>in vivo</it>. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.</p> <p>Results</p> <p>The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system.</p> <p>Conclusion</p> <p>The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.</p

Genital invasion or perigenital spread may pose a risk of marginal misses for Intensity Modulated Radiotherapy (IMRT) in anal cancer

Background: While intensity modulated radiotherapy (IMRT) in anal cancer is feasible and improves high-dose conformality, the current RTOG/AGITG contouring atlas and planning guidelines lack specific instructions on how to proceed with external genitalia. Meanwhile, the RTOG-Protocol 0529 explicitly recommends genital sparing on the basis of specific genital dose constraints. Recent pattern-of-relapse studies based on conventional techniques suggest that marginal miss might be a potential consequence of genital sparing. Our goal is to outline the potential scope and increase the awareness for this clinical issue. Methods: We present and discuss four patients with perigenital spread in anal cancer in both early and advanced stages (three at time of first diagnosis and one in form of relapse). Genital/perigenital spread was observed once as direct genital infiltration and thrice in form of perigenital lymphatic spread. Results: We review the available data regarding the potential consequences of genital sparing in anal cancer. Pattern-of-relapse studies in anal cancer after conventional radiotherapy and the current use of IMRT in anal cancer are equivocal but suggest that genital sparing may occasionally result in marginal miss. An obvious hypothesis suggested by our report is that perigenital lymphovascular invasion might be associated with manifest inguinal N+ disease. Conclusions: Local failure has low salvage rates in recent anal cancer treatment series. Perigenital spread may pose a risk of marginal misses in IMRT in anal cancer. To prevent marginal misses, meticulous pattern-of-relapse analyses of controlled IMRT-series are warranted. Until their publication, genital sparing should be applied with caution, PET/CT should be used when possible and meeting genital dose constraints should not be prioritized over CTV coverage, especially (but not only) in stage T3/4 and N+ disease