Multiscale modelling of the extracellular matrix.

The extracellular matrix is a complex three-dimensional network of molecules that provides cells with a complex microenvironment. The major constituents of the extracellular matrix such as collagen, elastin and associated proteins form supramolecular assemblies contributing to its physicochemical properties and organization. The structure of proteins and their supramolecular assemblies such as fibrils have been studied at the atomic level (e.g., by X-ray crystallography, Nuclear Magnetic Resonance and cryo-Electron Microscopy) or at the microscopic scale. However, many protein complexes are too large to be studied at the atomic level and too small to be studied by microscopy. Most extracellular matrix components fall into this intermediate scale, so-called the mesoscopic scale, preventing their detailed characterization. Simulation and modelling are some of the few powerful and promising approaches that can deepen our understanding of mesoscale systems. We have developed a set of modelling tools to study the self-organization of the extracellular matrix and large motion of macromolecules at the mesoscale level by taking advantage of the dynamics of articulated rigid bodies as a mean to study a larger range of motions at the cost of atomic resolution.

Electronic signature and attestation in conveyancing practice: A Malaysian legal perspective.

Background The Corona Virus Disease 2019 (COVID-19) pandemic brought about an unprecedented disruption to global business activities. Physical face-to-face activities must be restricted due to movement control order (MCO). The clients are required to sign the documents physically in the presence of the solicitor who must subsequently attest to the signature of the clients. The issue arises whether electronic signature (e-signature) and attestation are permissible under the laws of Malaysia. The aim of this research was to study the legality of e-signature and attestation in conveyancing practice in Malaysia and subsequently to propose recommendations to overcome these issues. Methods This is qualitative study and not an empirical study. The data was collected by library-based research from various primary and secondary data sources, including case law in Malaysia, written statutes, publication of journal and article. Results The Digital Signatures Act 1997 (DSA) and the Electronic Commerce Act 2006 (ECA) have legalised e-signatures. The DSA is the law that governs the digital signatures in Malaysia. ECA has listed a few documents which are not legally accepted if signed electronically, namely Power of Attorney, the Wills and codicils, the Trusts, and negotiable instruments. However, with regards to the issue of attestation of these documents, there are no clear laws which govern the attestation. The legal issue arises when the lawyers who have attested these documents are liable to be called as witness under the Evidence Act 1950 to testify their signature if these documents are tendered as evidence in any court proceedings. Conclusion Thus, it is suggested that there is a need for unique legal framework for e-signature and attestation in Malaysia due to the lack of specific laws which govern the issues of electronic signature and attestation.

Effects of changes in glycan composition on glycoprotein dynamics: example of N-glycans on insulin receptor.

Glycosylation is among the most common post-translational modifications in proteins, although it is observed in only about 10% of all the protein structures in protein data bank (PDB). Modifications of sugar composition in glycoproteins profoundly impact the overall physiology of the organism. One such example is the development of insulin resistance, which has been attributed to the removal of sialic acid residues from N-glycans of insulin receptor (IR) from various experimental studies. How such modifications affect the glycan-glycoprotein dynamics, and ultimately their function is not clearly understood to date. In this study, we performed molecular dynamics simulations of glycans in different environments. We studied the effects of removal of sialic acid on the glycan, as well as on the dynamics of leucine-rich repeat L1 domain of the IR ectodomain. We observed perturbations in L1 domain dynamics as a result of the removal of sialic acid. The perturbations include an increase in the flexibility of insulin-binding residues, which may affect insulin binding with IR. These changes are accompanied by perturbations in glycan-protein interactions and perturbation of long-range allosteric dynamics. Our observations will further aid in understanding the role of sugars in maintaining homeostasis and how changes in glycan composition may lead to perturbations in homeostasis, ultimately leading to conditions such as insulin resistance.

Mesoscopic Rigid Body Modelling of the Extracellular Matrix Self-Assembly.

The extracellular matrix (ECM) plays an important role in supporting tissues and organs. It even has a functional role in morphogenesis and differentiation by acting as a source of active molecules (matrikines). Many diseases are linked to dysfunction of ECM components and fragments or changes in their structures. As such it is a prime target for drugs. Because of technological limitations for observations at mesoscopic scales, the precise structural organisation of the ECM is not well-known, with sparse or fuzzy experimental observables. Based on the Unity3D game and physics engines, along with rigid body dynamics, we propose a virtual sandbox to model large biological molecules as dynamic chains of rigid bodies interacting together to gain insight into ECM components behaviour in the mesoscopic range. We have preliminary results showing how parameters such as fibre flexibility or the nature and number of interactions between molecules can induce different structures in the basement membrane. Using the Unity3D game engine and virtual reality headset coupled with haptic controllers, we immerse the user inside the corresponding simulation. Untrained users are able to navigate a complex virtual sandbox crowded with large biomolecules models in a matter of seconds.

Chromatin fiber allostery and the epigenetic code.

The notion of allostery introduced for proteins about fifty years ago has been extended since then to DNA allostery, where a locally triggered DNA structural transition remotely controls other DNA-binding events. We further extend this notion and propose that chromatin fiber allosteric transitions, induced by histone-tail covalent modifications, may play a key role in transcriptional regulation. We present an integrated scenario articulating allosteric mechanisms at different scales: allosteric transitions of the condensed chromatin fiber induced by histone-tail acetylation modify the mechanical constraints experienced by the embedded DNA, thus possibly controlling DNA-binding of allosteric transcription factors or further allosteric mechanisms at the linker DNA level. At a higher scale, different epigenetic constraints delineate different statistically dominant subsets of accessible chromatin fiber conformations, which each favors the assembly of dedicated regulatory complexes, as detailed on the emblematic example of the mouse Igf2-H19 gene locus and its parental imprinting. This physical view offers a mechanistic and spatially structured explanation of the observed correlation between transcriptional activity and histone modifications. The evolutionary origin of allosteric control supports to speak of an 'epigenetic code', by which events involved in transcriptional regulation are encoded in histone modifications in a context-dependent way.

DNA topology in chromosomes: a quantitative survey and its physiological implications.

Using a simple geometric model, we propose a general method for computing the linking number of the DNA embedded in chromatin fibers. The relevance of the method is reviewed through the single molecule experiments that have been performed in vitro with magnetic tweezers. We compute the linking number of the DNA in the manifold conformational states of the nucleosome which have been evidenced in these experiments and discuss the functional dynamics of chromosomes in the light of these manifold states.

Cytotoxic activity of kenaf (Hibiscus cannabinus L.) seed extract and oil against human cancer cell lines

Objective: To examine the cytotoxic properties of both the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cervical cancer, human breast cancer, human colon cancer and human lung cancer cell lines.Methods:kenaf seed oil on human cancer cell lines was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sulforhodamine B assays. Cell morphological changes were observed by using an inverted light microscope.Results:The in vitro cytotoxic activity of the kenaf (Hibiscus cannabinus L.) seed extract and cancer cell lines. Morphological alterations in the cell lines after KSE and KSO treatment were observed. KSE and KSO possessed effective cytotoxic activities against all the cell lines been selected.Conclusions:KSE and KSO could be potential sources of natural anti-cancer agents. Further The kenaf seed extract (KSE) exhibited a lower IC50 than kenaf seed oil (KSO) in all of the investigations on using kenaf seeds for anti-proliferative properties are warranted.

How to build a yeast nucleus.

Biological functions including gene expression and DNA repair are affected by the 3D architecture of the genome, but the underlying mechanisms are still unknown. Notably, it remains unclear to what extent nuclear architecture is driven by generic physical properties of polymers or by specific factors such as proteins binding particular DNA sequences. The budding yeast nucleus has been intensely studied by imaging and biochemical techniques, resulting in a large quantitative data set on locus positions and DNA contact frequencies. We recently described a quantitative model of the interphase yeast nucleus in which chromosomes are represented as passively moving polymer chains. This model ignores the DNA sequence information except for specific constraints at the centromeres, telomeres, and the ribosomal DNA (rDNA). Despite its simplicity, the model accounts for a large majority of experimental data, including absolute and relative locus positions and contact frequency patterns at chromosomal and subchromosomal scales. Here, we also illustrate the model's ability to reproduce observed features of chromatin movements. Our results strongly suggest that the dynamic large-scale architecture of the yeast nucleus is dominated by statistical properties of randomly moving polymers with a few sequence-specific constraints, rather than by a large number of DNA-specific factors or epigenetic modifications. In addition, we show that our model accounts for recently measured variations in homologous recombination efficiency, illustrating its potential for quantitatively understanding functional consequences of nuclear architecture.

Systematic characterization of the conformation and dynamics of budding yeast chromosome XII.

Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ~100 kb along the chromosome, and investigated their motion over broad time scales (0.2-400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.

A predictive computational model of the dynamic 3D interphase yeast nucleus.

BACKGROUND: Despite the absence of internal membranes, the nucleus of eukaryotic cells is spatially organized, with chromosomes and individual loci occupying dynamic, but nonrandom, spatial positions relative to nuclear landmarks and to each other. These positional preferences correlate with gene expression and DNA repair, recombination, and replication. Yet the principles that govern nuclear organization remain poorly understood and detailed predictive models are lacking. RESULTS: We present a computational model of dynamic chromosome configurations in the interphase yeast nucleus that is based on first principles and is able to statistically predict the positioning of any locus in nuclear space. Despite its simplicity, the model agrees with extensive previous and new measurements on locus positioning and with genome-wide DNA contact frequencies. Notably, our model recapitulates the position and morphology of the nucleolus, the observed variations in locus positions, and variations in contact frequencies within and across chromosomes, as well as subchromosomal contact features. The model is also able to correctly predict nuclear reorganization accompanying a reduction in ribosomal DNA transcription, and sites of chromosomal rearrangements tend to occur where the model predicted high contact frequencies. CONCLUSIONS: Our results suggest that large-scale yeast nuclear architecture can be largely understood as a consequence of generic properties of crowded polymers rather than of specific DNA-binding factors and that configurations of chromosomes and DNA contacts are dictated mainly by genomic location and chromosome lengths. Our model provides a quantitative framework to understand and predict large-scale spatial genome organization and its interplay with functional processes.

On the topology of chromatin fibres.

The ability of cells to pack, use and duplicate DNA remains one of the most fascinating questions in biology. To understand DNA organization and dynamics, it is important to consider the physical and topological constraints acting on it. In the eukaryotic cell nucleus, DNA is organized by proteins acting as spools on which DNA can be wrapped. These proteins can subsequently interact and form a structure called the chromatin fibre. Using a simple geometric model, we propose a general method for computing topological properties (twist, writhe and linking number) of the DNA embedded in those fibres. The relevance of the method is reviewed through the analysis of magnetic tweezers single molecule experiments that revealed unexpected properties of the chromatin fibre. Possible biological implications of these results are discussed.

A molecular model of chromatin organisation and transcription: how a multi-RNA polymerase II machine transcribes and remodels the beta-globin locus during development.

We present a molecular model of eukaryotic gene transcription. For the beta-globin locus, we hypothesise that a transcription machine composed of multiple RNA polymerase II (PolII) assembles using the locus control region as a foundation. Transcription and locus remodelling can be achieved by pulling DNA through this multi-PolII 'reading head'. Once a transcription complex is formed, it may engage an active gene in several rounds of transcription. Observed intergenic sense and antisense transcripts may be the result of PolII pulling the DNA through the reading head whilst searching for the promoter of a gene. Support for this hypothesis is provided using various data from the literature. In the model, DNA is packed in a 30-nm chromatin fibre, thus gene regulatory regions separated by kilobases are close in space. This, and the need to store transcription-induced supercoiling, may explain why functionally interacting regions are often separated by many kilobases.

An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length.

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.

Nucleosome chiral transition under positive torsional stress in single chromatin fibers.

Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the [H3-H4](2) tetramer bound with approximately 50 bp of DNA) obtained by depletion of H2A-H2B dimers suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented right-handed tetrasome. In view of its low energy, <8 kT, we propose that this transition is physiologically relevant and serves to break the docking of the dimers on the tetramer that in the absence of other factors exerts a strong block against elongation of transcription by the main RNA polymerase.