Chromatin Structure and Differential Accessibility of Homologous Human Mitotic Metaphase Chromosomes

The human mitotic metaphase chromosome is a product of complex chromatin restructuring during interphase. Metaphase chromosomes exhibit considerable plasticity in condensation. This is evident as distinct regions of accessible and compact chromatin fiber or epigenetic differences in histone and non-histone proteins. Such differences in chromatin condensation have been extensively described along the length of individual mitotic chromosomes but have not been recognized between homologous loci during metaphase. This thesis characterizes localized differences in condensation of homologous metaphase chromosomes that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy (SC) DNA probes used in fluorescence in situ hybridization. To investigate the physical and structural organization of chromatin at locus-specific sites, we developed correlated atomic force and fluorescence microscopy imaging. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicated that CENP-B and α-satellite DNA were distributed distinctly from one another and relative to observed centromeric ridge topography. At non-centromeic locations, short DNA probes that did not exhibit DA showed greater accessibility to the accessible chromatin topography on both homologs. Localized differential accessibility between chromosome homologs in metaphase was non-random and reproducible but not unique to known imprinted regions or specific chromosomes. Second, non-random DA was shown to be heritable within a 2 generation family. Third, DNA probe volume and depth measurements of hybridized metaphase chromosomes showed internal differences in chromatin accessibility of homologous regions by super-resolution 3D-structured illumination microscopy. Finally, genomic regions with equivalent accessibility were enriched for epigenetic marks of open interphase chromatin to a greater extent than regions with DA, suggesting that observed structural differences in accessibility may arise during or preceding metaphase chromosome compaction. Inhibition of the topoisomerase IIα-DNA cleavage complex mitigated DA by decreasing DNA superhelicity and axial metaphase chromosome condensation. Inter-homolog probe intensity ratios, depth, and volume between chromosomes treated with a catalytic inhibitor of topoisomerase IIα, were equalized compared to untreated cells. These data altogether suggest that DA is a reflection of allelic differences in metaphase chromosome compaction, dictated by the catenation state of the chromosome

Acute pancreatitis in children: efficacy of computed tomography severity index in the assessment, management, and prediction of complications

Aim The aim of the study was to describe the assessment and management aspects and the role of computed tomography severity index (CTSI) in children with acute pancreatitis.Materials and methods All the children (≤14 years) admitted to the pediatric surgery unit of our institution with acute pancreatitis from 2003 to 2014 were included. This retrospective analysis studied the demographic, clinical, diagnostic, and treatment aspects and the role of CTSI.Results The male-to-female ratio out of a total of 45 patients studied was 4 : 1. The differences in mean leukocyte count, mean serum amylase, and mean serum lipase were not significant in children with different CTSI scores. The children with higher CTSI scores are more likely to have both early and late complications, need for intensive care, and overall longer hospital stay.Conclusion CTSI plays an important role in early determination of the clinical severity, guiding the need for intensive care and in predicting the occurrence of early and late complications in children with acute pancreatitis. Keywords: acute pancreatitis, children, computed tomography severity inde

Neurofibromatosis type 1: a rare cause of parotid swelling in a child

Von Recklinghausen disease, also known as neurofibromatosis type 1, is an autosomal dominant disorder that presents as neurocutaneous syndrome. These patients have increased chances of developing other tumors such as plexiform neurofibromas. Plexiform neurofibromas are a proliferation of Schwann cells in the nerve sheath. Affliction of the parotid gland in a young child is a rare presentation of these tumors. We present the management of one such case.Keywords: neurofibromatosis type 1, parotid swelling, plexiform neurofibrom

Context-based FISH localization of genomic rearrangements within chromosome 15q11.2q13 duplicons

<p>Abstract</p> <p>Background</p> <p>Segmental duplicons (SDs) predispose to an increased frequency of chromosomal rearrangements. These rearrangements can cause a diverse range of phenotypes due to haploinsufficiency, in <it>cis </it>positional effects or gene interruption. Genomic microarray analysis has revealed gene dosage changes adjacent to duplicons, but the high degree of similarity between duplicon sequences has confounded unequivocal assignment of chromosome breakpoints within these intervals. In this study, we localize rearrangements within duplicon-enriched regions of Angelman/Prader-Willi (AS/PWS) syndrome chromosomal deletions with fluorescence <it>in situ </it>hybridization (FISH).</p> <p>Results</p> <p>Breakage intervals in AS deletions were localized recursively with short, coordinate-defined, single copy (SC) and low copy (LC) genomic FISH probes. These probes were initially coincident with duplicons and regions of previously reported breakage in AS/PWS. Subsequently, probes developed from adjacent genomic intervals more precisely delineated deletion breakage intervals involving genes, pseudogenes and duplicons in 15q11.2q13. The observed variability in the deletion boundaries within previously described Class I and Class II deletion AS samples is related to the local genomic architecture in this chromosomal region.</p> <p>Conclusions</p> <p>Chromosome 15 abnormalities associated with SDs were precisely delineated at a resolution equivalent to genomic Southern analysis. This context-dependent approach can define the boundaries of chromosome rearrangements for other genomic disorders associated with SDs.</p

Localized, Non-Random Differences in Chromatin Accessibility Between Homologous Metaphase Chromosomes

BACKGROUND: Condensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization. RESULTS: Fourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 genomic regions (RGS7, CACNA1B, GABRA5, SNRPN, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were not unique to known imprinted domains or specific chromosomes. DNA probes within CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and intergenic region 1p36.3 showed no DA (equivalent accessibility), while OPCML showed unbiased DA. To pinpoint probe locations, we performed 3D-structured illumination microscopy (3D-SIM). This showed that genomic regions with DA had 3.3-fold greater volumetric, integrated probe intensities and broad distributions of probe depths along axial and lateral axes of the 2 homologs, compared to a low copy probe target (NOMO1) with equivalent accessibility. Genomic regions with equivalent accessibility were also enriched for epigenetic marks of open interphase chromatin (DNase I HS, H3K27Ac, H3K4me1) to a greater extent than regions with DA. CONCLUSIONS: This study provides evidence that DA is non-random and reproducible; it is locus specific, but not unique to known imprinted regions or specific chromosomes. Non-random DA was also shown to be heritable within a 2 generation family. DNA probe volume and depth measurements of hybridized metaphase chromosomes further show locus-specific chromatin accessibility differences by super-resolution 3D-SIM. Based on these data and the analysis of interphase epigenetic marks of genomic intervals with DA, we conclude that there are localized differences in compaction of homologs during mitotic metaphase and that these differences may arise during or preceding metaphase chromosome compaction. Our results suggest new directions for locus-specific structural analysis of metaphase chromosomes, motivated by the potential relationship of these findings to underlying epigenetic changes established during interphase

Reversing Chromatin Accessibility Differences that Distinguish Homologous Mitotic Metaphase Chromosomes

BACKGROUND: Chromatin-modifying reagents that alter histone associating proteins, DNA conformation or its sequence are well established strategies for studying chromatin structure in interphase (G1, S, G2). Little is known about how these compounds act during metaphase. We assessed the effects of these reagents at genomic loci that show reproducible, non-random differences in accessibility to chromatin that distinguish homologous targets by single copy DNA probe fluorescence in situ hybridization (scFISH). By super-resolution 3-D structured illumination microscopy (3D-SIM) and other criteria, the differences correspond to \u27differential accessibility\u27 (DA) to these chromosomal regions. At these chromosomal loci, DA of the same homologous chromosome is stable and epigenetic hallmarks of less accessible interphase chromatin are present. RESULTS: To understand the basis for DA, we investigate the impact of epigenetic modifiers on these allelic differences in chromatin accessibility between metaphase homologs in lymphoblastoid cell lines. Allelic differences in metaphase chromosome accessibility represent a stable chromatin mark on mitotic metaphase chromosomes. Inhibition of the topoisomerase IIα-DNA cleavage complex reversed DA. Inter-homolog probe fluorescence intensity ratios between chromosomes treated with ICRF-193 were significantly lower than untreated controls. 3D-SIM demonstrated that differences in hybridized probe volume and depth between allelic targets were equalized by this treatment. By contrast, DA was impervious to chromosome decondensation treatments targeting histone modifying enzymes, cytosine methylation, as well as in cells with regulatory defects in chromatid cohesion. These data altogether suggest that DA is a reflection of allelic differences in metaphase chromosome compaction, dictated by the localized catenation state of the chromosome, rather than by other epigenetic marks. CONCLUSIONS: Inhibition of the topoisomerase IIα-DNA cleavage complex mitigated DA by decreasing DNA superhelicity and axial metaphase chromosome condensation. This has potential implications for the mechanism of preservation of cellular phenotypes that enables the same chromatin structure to be correctly reestablished in progeny cells of the same tissue or individual

Design of multi-valued quaternary based analog-to-digital converter

Problem statement: The design of multi-valued quaternary based Analog-to-Digital Converter (ADC) circuit was presented. The ADC generates multi-valued logic outputs rather than the conventional binary output system to overall reduction in circuit complexity and size. Approach: Design was implemented using pipeline ADC architecture and was simulated using model parameters based on standard 0.13 µm CMOS process. Results: Performance analysis of the design showed desirable performance parameters in terms of response, low power consumption, and a sampling rate of 10 MHz at a supply voltage of 1.3V was achieved. Conclusion/Recommendations: The ADC design was suitable for the needs of mixed-signal integrated circuit design and can be implemented as a conversion circuit for systems based on multiple-valued logic design

Piece-wise linear analog to digital (PLADC) converter process

Transducers are devices that transform energy from one form to another. Such transformation process may be applicable in the measurement of physical quantities, transfer of information and also in performing a certain control action. Transducers used as measuring devices are generally termed as sensors. Such transducers detect the changes in characteristics of a physical quantity and convert the change into a corresponding electrical signal. This is a common phenomenon when transducers are used to detect temperature, speed, force, liquid level or viscosity. On the other hand transducers, used to carry out control actions, are termed as actuators. These transducers usually convert an electrical signal into some form of physical control action such as heating or movement and are carried by the control devices. Various types of transducers exist meant for sensing and controlling different physical quantities. For example, a light dependent resistor (LOR) or a photodiode can be used to sense light intensity of an environment while lamps and LED displays can be used to control it. Likewise, a thermistor can be used to measure the temperature of an environment while a heater/fan can be used to control it and a tachometer can be used to measure the speed of a device while a stepper motor can be used to control it [1-3]. The focus of this chapter is on the problems and issues related to the interfacing of transducers when used as measuring devices or sensors in smart applications. Smart applications are also termed as tuned control for detecting changes in the parameter of interest which used to be ignorable in the traditional measurement and control system

The outcomes of dapagliflozin use in real-life clinical settings in endocrinology clinics of Islamabad, Pakistan

Introduction: Dapagliflozin is a member of a novel class of drugs (sodium-glucose cotransporter-2 inhibitors) used to treat type 2 diabetes mellitus and licensed in Pakistan in 2017. This retrospective observational study evaluated the effects of dapagliflozin on glycated hemoglobin (HbA1c) concentrations in patients treated at endocrinology clinics in Islamabad, Pakistan. The secondary objectives included assessing the effects of dapagliflozin on weight reduction and blood pressure control and to determining its safety.Methodology: Patients with type 2 diabetes who were treated with dapagliflozin were identified by screening the electronic medical records at tertiary care hospitals in Islamabad. Data were collected at the first visit and at follow-up. Categorical variables were recorded as frequencies and percentages and compared by McNemar’s tests, and continuous variables were recorded as means and standard deviations and compared by paired sample t-tests.Results: Mean HbA1C concentration was significantly lower at follow-up than at the first visit (7.57%±0.98% vs. 9.07%±2.07%, respectively; p\u3c0.001). Bodyweight (85.09±15.92 kg vs. 87.07±16.11 kg, respectively; p\u3c0.001) and diastolic blood pressure (80.34±7.12 mmHg vs. 82.34±9.61 mmHg, respectively; p\u3c0.001) were also significantly lower at follow-up than at the first visit, whereas systolic pressure showed a marginally significant reduction (123.5±16.57 mmHg vs. 126.83±19.97 mmHg, p=0.048).Conclusion: This first observational study of patients in Pakistan treated with dapagliflozin found that HbA1c concentration, weight, and blood pressure were reduced after initiation of dapagliflozin treatment