A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness

Nuclear chromosome locations dictate segregation error frequencies

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis [1, 2, 3, 4, 5]. Selective pressures then shape distinct aneuploidy and rearrangement patterns—for example, in cancer [6, 7] —but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing [8] after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes [9] and aneuploidies in early development [10] occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development. 1. van Jaarsveld, R. H. &amp; Kops, G. J. P. L. Difference makers: chromosomal instability versus aneuploidy in cancer. Trends Cancer 2, 561–571 (2016). 2. Compton, D. A. Mechanisms of aneuploidy. Curr. Opin. Cell Biol. 23, 109–113 (2011). 3. Zhang, C. Z. et al. Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184 (2015). 4. Ly, P. et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat. Genet. 51, 705–715 (2019). 5. Shoshani, O. et al. Chromothripsis drives the evolution of gene amplification in cancer. Nature 591, 137–141 (2021). 6. Davoli, T. et al. Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962 (2013). 7. Knouse, K. A., Davoli, T., Elledge, S. J. &amp; Amon, A. Aneuploidy in cancer: seq-ing answers to old questions. Annu. Rev. Cancer Biol. 1, 335–354 (2017). 8. Bolhaqueiro, A. C. F. et al. Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nat. Genet. 51, 824–834 (2019). 9. Cortés-Ciriano, I. et al. Comprehensive analysis of chromothripsis in 2, 658 human cancers using whole-genome sequencing. Nat. Genet. 52, 331–341 (2020). 10. McCoy, R. C. et al. Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development. PLoS Genet. 348, 235–238 (2015)

The relation between big data and informational privacy in the context of the healthcare

Big data is a broad term that is related to the collection, storage and analysis of large volumes of data. The term big data is often associated with the popular 3V’s model, which defined that data is growing significantly in the characteristics volume, variety and velocity. In this research we defined big data as: the collection, storage and transformation of structured and unstructured data from multiple sources into useful information (or knowledge) to improve decision-making within organizations. The significant growth of data is also occurring in the health care sector. A lot of these scattered data sources, possessing large volumes of personal health data of patients, are present in the health care. Big data have shown potential to support health care, by combining and transforming health data. Big data can be used to support medical and healthcare functions, including among others clinical decision support, disease surveillance, and population health (Raghupathi & Raghupathi, 2014). The increasing availability of large data sets from various sources in combination with the development of more advanced analytical tools for big data makes it more and more difficult to ensure privacy. Big data in its current form is still relatively new, and the knowledge on the implications on the security and privacy issues that it brings is still limited. This study explores the relation between big data and privacy in the health care. The research objective of this study is to gather knowledge on how big data affects privacy in the health care. In order to reach this objective, semi-structured interviews have been conducted with eight experts in either big data, health care or privacy in the Netherlands. In this research, a conceptual model of privacy has been created based on existing theories of privacy (e.g. nonintrusion theory, seclusion theory, control theory and restricted access theory). The conceptual model of privacy defines privacy in the elements: natural privacy, normative privacy, control aspect of privacy and the condition of privacy and has been used as a structure to analyze the relation between big data and privacy.Management of TechnologyICTTechnology, Policy and Managemen

The geology of the Rouchel Brook - Davis Creek District, N.S.W.

Bachelor Honours - Bachelor of Science (Honours)An area of approximately fifty square miles of the Carboniferous System has been mapped, north-east of Muswellbrook, N.S.W.. The earliest deposition outcropping in the area is a sequence of shallow-water, marine deposits (Waverley Formation) consisting mainly of fossiliferous lutites, arenites and minor limestones. The Waverley Formation contains fauna characteristic of the <i>Schellwienella cf. burlingtonensis</i> and <i>Pustula gracilis</i> assemblage zones. This sequence is followed by the non-marine Isismurra Formation, a sequence of coarse, lithic, zeolithic arenites with thin interbedded volcanics (mainly ignimbrites). Only the lowermost ignimbrite member (Curra Keith Ignimbrite Member) can as yet be correlated outside the area, namely with the Greenland Ignimbrite Member (Hansen, 1968). The Isismurra Formation intertoungues extensively with the Woolooma Formation, a sequence of marine lutites and arenites which lenses out to the west in the Stoney Creek area and which thickens in the east until it directly overlies the Waverley Formation. The Woolooma Formation contains fauna characteristic of <i>Thomasaria voiseyi</i>, <i>Werriea australis</i> and <i>Delepinea aspinosa</i> assemblage zones. An attempt was made to correlate the sequence in the area mapped, to the north (Manser, 1968), as well as to the south (Blayden, 1964; Rudd, 1967) and to use nomenclature originating in both areas. In late Palaeozoic times, the are was subjected to the compressive and shearing forces of the Hunter-Bowen Crogeny which produced extensive folding and faulting

Acitretin for lichen amyloidosus

We present two cases of lichen amyloidosus treated with retinoids. A 57-year-old Vietnamese woman has had extensive generalized recalcitrant lichen amyloidosus for 23 years. Treatment with oral etretinate (25 mg/day) for 3 years, and later oral acitretin (10 mg/day) for the past 10 years, has controlled the pruritus and flattened the hyperkeratotic papules. Whenever the acitretin was ceased her symptoms flared within weeks. On each occasion reintroduction of acitretin was effective within 1-2 months. The second case is that of a 51-year-old Australian Aboriginal woman who had a 2-year history of lichen amyloidosus affecting her lower legs. A 2-month course of oral acitretin (25 mg b.d.) produced a marked improvement in both the pruritus and hyperkeratotic papules. She was then lost to follow up for 2 years, during which time her symptoms recurred

Validity of the standard cross-correlation test for model structure validation.

Abstract In the standard prediction error framework of system identification, statistical properties of estimated models are typically derived under the assumption that the true system is in the model class. The standard model structure validation test for plant models is the sample cross-correlation test between the residuals of the model and the input. It turns out that the standard test itself is valid only under exactly those assumptions it is meant to verify, i.e. system is in the model class. It is shown that for reliable results of the validation test a vector-valued test is required and that accurate noise modelling is indispensable for reliable model structure validation. This shows the limitation of separate validation of plant and noise model structures. Improvements of the test are presented, while it is motivated that reserving data only to be used for model validation is not efficient