Functional analysis of structural variants in single cells using Strand-seq

Somatic structural variants (SVs) are widespread in cancer, but their impact on disease evolution is understudied due to a lack of methods to directly characterize their functional consequences. We present a computational method, scNOVA, which uses Strand-seq to perform haplotype-aware integration of SV discovery and molecular phenotyping in single cells by using nucleosome occupancy to infer gene expression as a readout. Application to leukemias and cell lines identifies local effects of copy-balanced rearrangements on gene deregulation, and consequences of SVs on aberrant signaling pathways in subclones. We discovered distinct SV subclones with dysregulated Wnt signaling in a chronic lymphocytic leukemia patient. We further uncovered the consequences of subclonal chromothripsis in T cell acute lymphoblastic leukemia, which revealed c-Myb activation, enrichment of a primitive cell state and informed successful targeting of the subclone in cell culture, using a Notch inhibitor. By directly linking SVs to their functional effects, scNOVA enables systematic single-cell multiomic studies of structural variation in heterogeneous cell populations

Tensile Strength of Geological Discontinuities Including Incipient Bedding, Rock Joints and Mineral Veins

Geological discontinuities have a controlling influence for many rock-engineering projects in terms of strength, deformability and permeability, but their characterisation is often very difficult. Whilst discontinuities are often modelled as lacking any strength, in many rock masses visible rock discontinuities are only incipient and have tensile strength that may approach and can even exceed that of the parent rock. This fact is of high importance for realistic rock mass characterisation but is generally ignored. It is argued that current ISRM and other standards for rock mass characterisation, as well as rock mass classification schemes such as RMR and Q, do not allow adequately for the incipient nature of many rock fractures or their geological variability and need to be revised, at least conceptually. This paper addresses the issue of the tensile strength of incipient discontinuities in rock and presents results from a laboratory test programme to quantify this parameter. Rock samples containing visible, natural incipient discontinuities including joints, bedding, and mineral veins have been tested in direct tension. It has been confirmed that such discontinuities can have high tensile strength, approaching that of the parent rock. Others are, of course, far weaker. The tested geological discontinuities all exhibited brittle failure at axial strain less than 0.5 % under direct tension conditions. Three factors contributing to the tensile strength of incipient rock discontinuities have been investigated and characterised. A distinction is made between sections of discontinuity that are only partially developed, sections of discontinuity that have been locally weathered leaving localised residual rock bridges and sections that have been ‘healed’ through secondary cementation. Tests on bedding surfaces within sandstone showed that tensile strength of adjacent incipient bedding can vary considerably. In this particular series of tests, values of tensile strength for bedding planes ranged from 32 to 88 % of the parent rock strength (intact without visible discontinuities), and this variability could be attributed to geological factors. Tests on incipient mineral veins also showed considerable scatter, the strength depending upon the geological nature of vein development as well as the presence of rock bridges. As might be anticipated, tensile strength of incipient rock joints decreases with degree of weathering as expressed in colour changes adjacent to rock bridges. Tensile strengths of rock bridges (lacking marked discolouration) were found to be similar to that of the parent rock. It is concluded that the degree of incipiency of rock discontinuities needs to be differentiated in the process of rock mass classification and engineering design and that this can best be done with reference to the tensile strength relative to that of the parent rock. It is argued that the science of rock mass characterisation may be advanced through better appreciation of geological history at a site thereby improving the process of prediction and extrapolating properties

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles

The isolation of penicillin G acylase from the periplasmic space of a mutant of Escherichia coli ATCC 11105 by various extraction methods

In order to establish a simple and economic purification scheme for penicillin G acylase (PGA) (EC 3.5.1.11) from a mutant strain of Escherichia coli 11105 with high yield, various protein extraction methods were employed including osmotic and chloroform shocks, and osmotic shock after lysozyme and cetyltrimethylammonium bromide (CTAB) pretreatments of E. coli cells. A purification factor of 7.82 and recovery of 53 % were obtained with osmotic shock when sucrose and EDTA were used at 20 % and 1.22 % concentrations, respectively. Various treatment times of osmotic shock and lysozyme and CTAB pretreatments did not affect the yield of released PGA. The yield of released PGA was found to be lower using chloroform shock than when osmotic shock was employed. Two steps purification procedure developed in this research was satisfactory to obtain the high specific activity required for the production of immobilized PGA

THERMOSTABILIZATION OF PENICILLIN-G ACYLASE OBTAINED FROM A MUTANT OF ESCHERICHIA-COLI ATCC-11105 BY BISIMIDOESTERS AS HOMOBIFUNCTIONAL CROSS-LINKING AGENTS

We investigated the effects of three different bisimidoesters as homobifunctional cross-linking agents on the thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105. Cross-linkers were dimethyladipimidate (DMA), dimethylsuberimidate (DMS), and dimethyl-3,3'-dithiobispropionimidate (DTBP). The thermal inactivation mechanisms of the native and cross-linked PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active, susceptible transient stares. The efficacy of the cross-linkers on the thermostabilization of PGA was estimated to be DMA > DMS > DTBP. Optimal concentrations of DMA, DMS, and DTBP for cross-linking of PGA were found to be 0.5, 0.4, and 0.3% (w/v), respectively. The greatest enhancement of the thermostabilities was observed during DMA cross-linking, as a nearly 15-fold increase at temperatures above 50 degrees C. Cross-linking by DMA did not cause much change in the parameters V-m, K-m, and the optimal temperature values of PGA, but the activation energy of the enzyme was slightly decreased and k(cat) value slightly increased after cross-linking

INSTAGRAM DATA SET

Data set here is for the quantitative part only