THE PERIODIC AND DYNAMIC STRUCTURE OF CHROMATIN

The organisation of chromatin is non-random and shows a broad diversity across cell types, developmental stages, and cell cycle stages. During G0 and G1 phase of interphase, chromatin displays a bivalent status. The condensed chromatin (heterochromatin) at the nuclear periphery is mostly associated with low levels of gene expression, while the loosened chromatin (euchromatin) towards the interior of the nucleus is associated with higher gene expression. This quiescent picture of interphase radically changes when the cell cycle progresses toward cell division. Firstly, during S phase, DNA is replicated, and chromatin progressively condenses. This is followed by the G2 phase that shows a compact heterochromatin recruited towards the centre of the nucleus. At the beginning of mitosis, the chromosomes condense with a significant topological change in their organisation and are segregated during the next stages of the cell division. Meiotic chromosomes are also highly condensed as mitotic chromosomes but show a particular functional structure, which prepares germ cells to exchange DNA sequences between their homologous chromosomes to generate diversity. To summarise, chromatin experiences dramatic organisational changes during mitosis and meiosis. These changes in chromatin organisation during the lifetime of a cell show that chromatin is not a static entity but highly dynamic in nature. For a variety of reasons, conventional light and electron microscopy have not been able to fully capture the finer details of chromatin organisation and dynamics. For a long time, description of the interphase nucleus was limited to delineate the euchromatin-heterochromatin dichotomy or describe some specific nuclear elements such as the nucleolus. Advancements in molecular biology during the last thirty years have brought an immense amount of information about how chromatin is organised and genes are regulated. As a classical example, the globin gene has been shown to display a highly constrained shape forced by chromatin looping that brings the regulatory regions to the promoter of the gene. Nowadays, genomic studies can acquire an immense amount of information regarding chromatin organisation and gene regulation, leaving one with the expectation that structure of individual genes could potentially be described visually if sufficient specificity and resolution were reached. With the advent of various super-resolution methods, in particular, single molecule localisation microscopy (SMLM) based methods and recently developed strategies for labelling DNA, it is now possible to study chromatin organisation and underlying gene regulatory mechanisms at the nanoscale. During my PhD, I have analysed a broad range of nuclear phenotypes using SMLM. My analyses contribute to the description of a periodic and dynamic structure of chromatin. Moreover, I have described several elementary chromatin structures that I call chromatin domains, both in interphase and meiosis, that are potentially associated with a local function such as gene activation or silencing. Firstly with colleagues, I established an experimental setup to study chromatin organisation with single molecule localisation microscopy. I investigated how UV-induced photo-conversion of conventional DNA dyes allows increasing sufficiently the labelling density such that it is possible to study various organisational aspects of chromatin in basal interphase. An adequate imaging protocol has been established to bring DNA minor groove binding dyes such as Hoechst 33258, Hoechst 33342 and DAPI (4’,6-diamidino-2-phenylindole) into an efficient blinking state necessary to record single molecule locations with high precision. This method was applied to several cell types to investigate the chromatin organisation during different stages of the cell cycle at the highest resolution currently achievable with light microscopy. The results show that the method can capture several hierarchical levels of chromatin organisation. In reverse hierarchical order, I could describe previously known chromatin territories of 1000 nm, subchromosomal domains of 500 nm, chromatin domains of 100 to 400 nm (and further sub-categories of active or repressed domains) and chromatin fibres below 100 nm, mostly between 30 to 60 nm. Individual nucleosomal domains are also described, which tend to cluster in batches of 10-15 nucleosomes, a number close to one found in genomic studies upstream to promoter regions. Next, with colleagues, I studied the dynamics of chromatin using stress as a model system. It was found that short-term oxygen and nutrient deprivation provokes chromatin to shrink to a hollow, condensed ring and rod-like configuration, which reverses back to the initial structure when the stress conditions cease. The condensed network of rods and rings interspersed with large, chromatin-sparse nuclear voids were 40-700 nm in dimension, capturing another level of chromatin organisation not described before. Finally, I explored the unique properties of chromatin during meiosis, which has escaped analysis at the single-molecule level until now. Single molecule analysis revealed unexpected highly recognisable periodic patterns of chromatin. Firstly, I observed that meiotic chromatin show unique clusters of 250 nm diameter along the synaptonemal complex, extended laterally by chromatin fibres forming loops. These clusters show a remarkable periodicity of 500 nm, a pattern possible to spot because of the highly deterministic nature of pachytene chromosomes and the resolution of the experimental setup. Furthermore, guided by genomic data, I selected histone modifications associated with different chromatin states to dissect the morphology of meiotic chromosomes. I could examine the morphology of these chromosomes into three spatially distinct nanoscale sub-compartments. Histone mark H3K4me3 associated with active chromatin was found in a lateral position, potentially located at the places of \textit{de novo} double-strand breaks. Repressive histone mark H3K27me3 was shown to display a surprising medial symmetrical and periodic pattern, putatively associated with recombination. Finally, centromeric histone mark H3K9me3 locates at one of meiotic chromosome ends and is potentially associated with repression of repeated regions and pairing of homologous chromosomes at early stages. I summarise these findings in a comprehensive final model. Overall, I have used new information brought by super-resolution technologies to show the dynamics of chromatin in various processes and novel orders of chromatin compaction, which were not reported previously. Among these new levels of chromatin compaction are the interphase hierarchical chromatin domains, the stress pattern of cells upon oxygen and nutrients deprivation and the novel epigenetic domains found at pachytene stage of meiosis. These architectures show that the organisation of chromatin is more complex than thought before, dynamic in nature and shows a high order of periodicity. Further investigation is, therefore, necessary to understand how chromatin transits from a ’beads-on-string’ model to the intermediary chromatin domains and finally to the commonly observed X-shaped chromosomes

Foetal kidney length as a parameter for determination of gestational age in pregnancy by ultrasonography

Background: Establishing the gestational age of the fetus, especially in late trimester is a challenge to aptly treat the pregnant woman. Ultrasound parameters like BPD, HC, AC and FL in second and third trimesters are not very reliable for dating the pregnancy. Fetal kidney length has been studied and shown to strongly correlate with the gestational age in late trimesters even in IUGR fetuses.Methods: This cross section hospital based study was conducted in the Department of Obstetrics and Gynecology, P.B.M. and Associated Group of Hospitals, attached to Sardar Patel Medical College, Bikaner during study period of one year from 2015 to 2016. 100 pregnant women with known dates of different parity and ages were included in this study.Results: According to the observations, the mean deviation from the gestational age at all the weeks is least for KL. The result indicates that the kidney length in the present study correlated well with the assigned gestational age and found almost same as all the ultrasound biometric parameters put together.Conclusions: Kidney length can be used as an individual parameter in estimating gestational age, especially in later trimesters, where biometric indices may not be much reliable

Comparative Compositional Analysis and Pesticidal Efficacy of Essential Oils from Leaves of Skimmia Aanquetilia N.P. Taylor and Airy Shaw

The objective of the current study was to re-examine the chemical components of the essential oil (EO) from the aerial parts of Skimmia anquetilia N.P. Taylor & Airy Shaw in two different seasons designated as Skimmia anquetilia rainy season essential oil (SKREO) and Skimmia anquetilia winter season essential oil (SKWEO). The GC-MS analysis of SKREO and SKWEO resulted in the identification of 42 and 48 constituents, comprising of 95.3 % and 95.4 % of the total composition respectively. Both SKREO and SKWEO varied in their chemical composition in terms of quantity viz: linalyl acetate (15.8% - 17.6%), linalool (13.2% - 13.9%), geijerene (11.6% - 11.7%), α-thujene (11.3% - 11.1%), α-terpineol (6.1% - 6.1%), geranyl acetate (5.0% - 5.1%), α-terpinyl acetate (3.3% - 3.1%), myrcene (3.0% - 3.1%), geraniol (2.6% - 1.9%), α-pinene (2.1% - 2.2%), trans-β-ocimene (2.1% - 2.3%), cis-β-ocimene (2.0% - 2.2%) and neryl acetate (2.3% - 2.4%). Besides qualitative differences SKREO and SKWEO, both were studied for their pesticidal activities. The study exhibited potent antifeedant activity against Spodoptera litura and nematicidal activity against Meloidogyne incognita. Based on the present observations, it was found that besides its academic importance, shrub Skimmia anquetilia can be a good source of phytochemicals like linalyl acetate, linalool, geijerene, thujene and can be used for the development of herbal source for antifeedant and nematicidal activity after proper clinical trials

Super-resolution structured illumination microscopy: past, present and future.

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'

Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes

Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes

Economic evaluation of implementing a rapid point-of-care screening test for the identification of hepatitis C virus under National Viral Hepatitis Control Programme in Tamil Nadu, South India

Introduction: Viral hepatitis is a crucial public health problem in India. Hepatitis C virus (HCV) elimination is a national priority and a key strategy has been adopted to strengthen the HCV diagnostics services to ensure early and accurate diagnosis. Methods: To conduct an economic evaluation of implementing a rapid point-of-care screening test for the identification of HCV among the selected key population under the National Viral Hepatitis Control Programme in Tamil Nadu, South India. Economic evaluation of a point-of-care screening test for HCV diagnosis among the key population attending the primary health care centers. A combination of decision tree and Markov model was developed to estimate cost-effectiveness of point-of-care screening test for HCV diagnosis at the primary health care centers. Total costs, quality-adjusted life years (QALYs) of the intervention and comparator, and incremental cost-effectiveness ratio (ICER) were calculated. The model parameter uncertainties which would influence the cost-effectiveness outcome has been evaluated by one-way sensitivity analysis and probabilistic sensitivity analysis. Results: When compared to the tertiary level diagnostic strategy for HCV, the point-of-care screening for selected key population at primary health care level results in a gain of 57 undiscounted QALYs and 38 discounted QALYs, four undiscounted life years and two discounted life years. The negative ICER of the new strategy indicates that it is less expensive and more effective compared with the current HCV diagnosis strategy. Conclusions: The proposed strategy for HCV diagnosis in the selected key population in Tamil Nadu is dominant and cost-saving compared to the current strategy

Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map.

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.The study was supported by the Paul G. Allen Foundation Distinguished Investigator Program (E.M.U. and D.H.R.), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (D.H.R., D.G. and G. C.), BRAIN initiative (1U01 MH105991 to D.G.) and National Institute of Health (1R01 MH109912 to D.G.; P01NS08351 to D.H.R.), National Institute of Health Research and the European Union Seventh Framework (to P.H.), NINDS Informatics Center for Neurogenetics and Neurogenomics (P30 NS062691 to G.C.), Wellcome Trust core support (M.H., O.A.B.), European Research Council (281961 to M.G.H.), Fonds Wetenschappelijk Onderzoek (G066715N and 1523014N to M.G.H.), Stichting Alzheimer Onderzoek (S#16025 to M.G.H.) and VIB Institutional Support and Tech Watch funding (to M.G.H.), Howard Hughes Medical Institute and the Wellcome Trust (to D.H.R.)

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| < 0.03 at 95% confidence level. [Figure not available: see fulltext.

Measurement of t(t)over-bar normalised multi-differential cross sections in pp collisions at root s=13 TeV, and simultaneous determination of the strong coupling strength, top quark pole mass, and parton distribution functions

Peer reviewe

An embedding technique to determine ττ backgrounds in proton-proton collision data

An embedding technique is presented to estimate standard model tau tau backgrounds from data with minimal simulation input. In the data, the muons are removed from reconstructed mu mu events and replaced with simulated tau leptons with the same kinematic properties. In this way, a set of hybrid events is obtained that does not rely on simulation except for the decay of the tau leptons. The challenges in describing the underlying event or the production of associated jets in the simulation are avoided. The technique described in this paper was developed for CMS. Its validation and the inherent uncertainties are also discussed. The demonstration of the performance of the technique is based on a sample of proton-proton collisions collected by CMS in 2017 at root s = 13 TeV corresponding to an integrated luminosity of 41.5 fb(-1).Peer reviewe