Radiation effects on the structure of chromosomes

The study aims to investigate the effect of ionising radiation on the structure of the chromosomes after lose-dose X-ray irradiation. The first aim was to investigate the changes that occurred in the molecular environment of the chromosomes after radiation exposure using Fluorescence Lifetime Imaging Microscopy (FLIM). The study’s second aim was to measure the mass of the human chromosomes and produce ‘X-ray karyotypes’ of each spread, plotting the measured mass against the number of base pairs using the X-ray ptychography technique. The purpose was to observe the changes in the mass of the X-ray induced chromosomes. A complete set of 46 human metaphase chromosomes in one frame is known as ‘spread’. Exponentially growing cells were irradiated with X-ray doses at 0.1 Gy (Gray), 0.5 Gy and 1 Gy, followed by chromosome preparation from the irradiated as well as non-irradiated cells. DNA specific stain, 4’,6-diamidino-2-phenylindole (DAPI), was used to stain the chromosomes during the analysis. Using FLIM, a significant reduction in the DAPI lifetime values (2.8 ± 0.03 ns) was observed in the heterochromatin region of heteromorphic chromosomes 1, 9, 15, and 16 compared to the non-heterochromatin region of the chromosomes (2.88 ± 0.02 ns) present in the non-irradiated chromosome spreads. Furthermore, in the case of irradiated chromosomes, the heterochromatic regions of chromosomes showed statistically significant reduced lifetime values, with the difference ranging between ~260 picoseconds (ps) to ~290 ps compared to the non-irradiated chromosomes. This indicates that chromosomes might have undergone chemical perturbations. However, DAPI lifetime values were not significantly different between low-dose radiations. Therefore, low-dose irradiation is thought to induce an adaptive or hermetic response under certain conditions. However, the sensitivity of the FLIM could assist in the early detection of epigenetic variations that occur in the structure of chromosomes due to radiation/diseases. Key results were obtained from the Diamond Light Source (Harwell), I-13-coherence beamline by imaging metal-stained and unstained human chromosome spreads at room temperature to determine the gain or loss of genetic material upon low-level X-ray irradiation doses due to radiation damage. The obtained better resolution phase-contrast images of chromosome spreads gave the distribution of electron density which correlates with the mass of the chromosomes. The masses of non-irradiated and irradiated chromosomes were karyotyped to observe the difference in the masses. Therefore, the mass of the human genome can be determined using phase-contrast X-ray ptychography. The X-ray ptychography results are fascinating. The importance of this research lies in developing a new and improved technique to image and weigh individual chromosomes that will benefit medicine in diagnosing cancer and other health problems from patient samples

Combining multicolor FISH with fluorescence lifetime imaging for chromosomal identification and chromosomal sub structure investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes

Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus

From MDPI via Jisc Publications RouterHistory: accepted 2021-05-23, pub-electronic 2021-06-01Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/H022597/1Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented

Parthenogenesis and Somatic cell nuclear transfer in sheep oocytes using Polscope

550-558Parthenogenesis and Somatic cell nuclear transfer (SCNT) techniques, offer a unique approach to manipulate the genetic composition of derived human embryonic stem cells – an essential step if the full opportunities for disease modeling, drug discovery or individualized stem cell therapy are to be realized. The present study describes the use of sheep oocytes to acquire expertise and establish methods to reconstruct embryos for obtaining blastocysts before venturing into human SCNT where the oocytes are a very precious starting material. Maturation of sheep eggs in vitro for 20-24 hr resulted in 65% metaphase II (MII) eggs which were either parthenogenetically activated using calcium ionomycin or ethanol or subjected to SCNT using cumulus cell as somatic cell. Sixteen blastocysts were produced by parthenogenetic activation of 350 eggs whereas reconstructed embryos, after SCNT carried out in 139 eggs, progressed only up to morula stage. The procedure of parthenogenesis and SCNT will be useful to generate autologous ES cells using human eggs

X-ray ptychography imaging of human chromosomes after low-dose irradiation

Studies of the structural and functional role of chromosomes in cytogenetics have spanned more than 10 decades. In this work, we take advantage of the coherent X-rays available at the latest synchrotron sources to extract the individual masses of all 46 chromosomes of metaphase human B and T cells using hard X-ray ptychography. We have produced \u27X-ray karyotypes\u27 of both heavy metal-stained and unstained spreads to determine the gain or loss of genetic material upon low-level X-ray irradiation doses due to radiation damage. The experiments were performed at the I-13 beamline, Diamond Light Source, Didcot, UK, using the phase-sensitive X-ray ptychography method

Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented

Ocimum sanctum Linn. A reservoir plant for therapeutic applications: An overview

The medicinal plants are widely used by the traditional medicinal practitioners for curing various diseases in their day to day practice. In traditional system of medicine, different parts (leaves, stem, flower, root, seeds and even whole plant) of Ocimum sanctum Linn. have been recommended for the treatment of bronchitis, malaria, diarrhea, dysentery, skin disease, arthritis, eye diseases, insect bites and so on. The O. sanctum L. has also been suggested to possess anti-fertility, anticancer, antidiabetic, antifungal, antimicrobial, cardioprotective, analgesic, antispasmodic and adaptogenic actions. Eugenol (1-hydroxy-2-methoxy-4-allylbenzene), the active constituents present in O. sanctum L. have been found to be largely responsible for the therapeutic potentials. The pharmacological studies reported in the present review confirm the therapeutic value of O. sanctum L. The results of the above studies support the use of this plant for human and animal disease therapy and reinforce the importance of the ethno-botanical approach as a potential source of bioactive substances