Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.     

Impact of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO)

The use of saline water as secondary quality water in urban environments for sanitation is a promising alternative towards mitigating fresh water scarcity. However, this alternative will increase the salinity in the wastewater generated that may affect the biological wastewater treatment processes, such as biological phosphorus removal. In addition to the production of saline wastewater by the direct use of saline water in urban environments, saline wastewater is also generated by some industries. Intrusion of saline water into the sewers is another source of salinity entering the wastewater treatment plant. In this study, the short-term effects of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) were investigated to assess the impact of salinity on enhanced biological phosphorus removal. Hereto, PAO and GAO cultures enriched at a relatively low salinity level (0.02 % W/V) were exposed to salinity concentrations of up to 6 % (as NaCl) in anaerobic batch tests. It was demonstrated that both PAO and GAO are affected by higher salinity levels, with PAO being the more sensitive organisms to the increasing salinity. The maximum acetate uptake rate of PAO decreased by 71 % when the salinity increased from 0 to 1 %, while that of GAO decreased by 41 % for the same salinity increase. Regarding the stoichiometry of PAO, a decrease in the P-release/HAc uptake ratio accompanied with an increase in the glycogen consumption/HAc uptake ratio was observed for PAO when the salinity increased from 0 to 2 % salinity, indicating a metabolic shift from a poly-P-dependent to a glycogen-dependent metabolism. The anaerobic maintenance requirements of PAO and GAO increased as the salinity concentrations risen up to 4 % salinity.     

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

Background

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Results

Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺ concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.

Conclusion

This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.     

Canopy structure measurement by gap fraction analysis using commercial instrumentation

In the past few years, a number of systems for making indirectcanopy structural estimates have become commercially available.These include linear sensors, such as the DEMON, line quantumsensors, and hemispherical sensors, such as the LAI-2000, theLeaf Laser, and the Cl-100. The performance of these instrumentsas reported in the literature is reviewed for forests, row crops,and individual trees. Key words: Leaf area index, LAI-2000, DEMON, SunLink, SunScan, Ceptometer, AccuPAR, Cl-100, gap fraction, sunflecks, hemispherical photography     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.