A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO, due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

Dolutegravir-containing HIV therapy reversibly alters mitochondrial health and morphology in cultured human fibroblasts and peripheral blood mononuclear cells.

OBJECTIVES: Given the success of combination antiretroviral therapy (cART) in treating HIV viremia, drug toxicity remains an area of interest in HIV research. Despite newer integrase strand transfer inhibitors (InSTIs), such as dolutegravir (DTG) and raltegravir (RAL), having excellent clinical tolerance, there is emerging evidence of off-target effects and toxicities. Although limited in number, recent reports have highlighted the vulnerability of mitochondria to these toxicities. The aim of the present study is to quantify changes in cellular and mitochondrial health following exposure to current cART regimens at pharmacological concentrations. A secondary objective is to determine whether any cART-associated toxicities would be modulated by human telomerase reverse transcriptase (hTERT). METHODS: We longitudinally evaluated markers of cellular (cell count, apoptosis), and mitochondrial health [mitochondrial reactive oxygen species (mtROS), membrane potential (MMP) and mass (mtMass)] by flow cytometry in WI-38 human fibroblast with differing hTERT expression/localization and peripheral blood mononuclear cells. This was done after 9 days of exposure to, and 6 days following the removal of, seven current cART regimens, including three that contained DTG. Mitochondrial morphology was assessed by florescence microscopy and quantified using a recently developed deep learning-based pipeline. RESULTS: Exposure to DTG-containing regimens increased apoptosis, mtROS, mtMass, induced fragmented mitochondrial networks compared with non-DTG-containing regimens, including a RAL-based combination. These effects were unmodulated by telomerase expression. All effects were fully reversible following removal of drug pressure. CONCLUSION: Taken together, our observations indicate that DTG-containing regimens negatively impact cellular and mitochondrial health and may be more toxic to mitochondria, even among the generally well tolerated InSTI-based cART.

Untapped potential of nature-based activities for mental health: need for further research.

In April 2022, the Intergovernmental Panel on Climate Change stated clearly that without immediate and deep reductions in greenhouse gas emissions, it would be impossible to limit global temperature rise to 1.5 °C above pre-industrial levels. A growing body of research shows an increasing reaction to witnessing or experiencing current climate impacts and concern regarding apparent inaction in response, manifests as negative cognitive, emotional, and behavioural responses, including eco-anxiety. This commentary contends that nature-based activities (NBAs) have a potentially important, but currently neglected, role to play in supporting those experiencing eco-anxiety. However, existing research limitations hinder their adoption in the United Kingdom's social prescribing agenda and they remain a largely untapped therapeutic resource that need to be mainstreamed into clinical and social care provision. The paper calls for investment in research that advances our understanding of the individual and community benefits of NBAs, the longevity of their impact, and their associated, comparative and opportunity costs.

Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq.

Guanine-rich RNAs can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), and participate in a wide range of biological processes. Here we describe in detail a G4-RNA-specific precipitation (G4RP) protocol, which enables the transcriptomic profiling of G4-RNAs. The G4RP protocol consists of a chemical cross-linking step, followed by affinity capture with a G4-specific probe, BioTASQ. G4RP can be coupled with sequencing to capture a comprehensive global snapshot of folded G4-RNAs. This method can also be used to profile induced changes (i.e., through G4 ligand treatments) within the G4-RNA transcriptome. The entire protocol can be completed in 1-2 weeks and can be scaled up or down depending on the specific experimental goals. In addition to the protocol details, we also provide here a guide for optimization in different laboratory setups.

G-quadruplexes mark alternative lengthening of telomeres.

About 10-15% of all human cancer cells employ a telomerase-independent recombination-based telomere maintenance method, known as alternative lengthening of telomere (ALT), of which the full mechanism remains incompletely understood. While implicated in previous studies as the initiating signals for ALT telomere repair, the prevalence of non-canonical nucleic acid structures in ALT cancers remains unclear. Extending earlier reports, we observe higher levels of DNA/RNA hybrids (R-loops) in ALT-positive (ALT+) compared to telomerase-positive (TERT+) cells. Strikingly, we observe even more pronounced differences for an associated four-stranded nucleic acid structure, G-quadruplex (G4). G4 signals are found at the telomere and are broadly associated with telomere length and accompanied by DNA damage markers. We establish an interdependent relationship between ALT-associated G4s and R-loops and confirm that these two structures can be spatially linked into unique structures, G-loops, at the telomere. Additionally, stabilization of G4s and R-loops cooperatively enhances ALT-activity. However, co-stabilization at higher doses resulted in cytotoxicity in a synergistic manner. Nuclear G4 signals are significantly and reproducibly different between ALT+ and TERT+ low-grade glioma tumours. Together, we present G4 as a novel hallmark of ALT cancers with potential future applications as a convenient biomarker for identifying ALT+ tumours and as therapeutic targets.

Hepatitis C virus screening of high-risk patients in a community hospital emergency department: Retrospective review of patient characteristics and future implications.

BACKGROUND: Chronic hepatitis C virus infection (HCV) is a common infectious disease that affects more than 2.7 million people in the US. Because the emergency department (ED) can present an ideal opportunity to screen patients who may not otherwise get routine screening, we implemented a risk-based screening program for ED patients and established a system to facilitate linkage to care. METHODS AND FINDINGS: A risk-based screening algorithm for HCV was programmed to trigger an alert in Epic electronic medical record system. Patients identified between August 2018 and April 2020 in the ED were tested for HCV antibody reflex to HCV RNA. Patients with a positive screening test were contacted for the confirmatory test result and to establish medical care for HCV treatment. Patient characteristics including age, sex, self-awareness of HCV infection, history of previous HCV treatment, history of opioids use, history of tobacco use, and types of insurance were obtained. A total of 4,525 patients underwent a screening test, of whom 131 patients (2.90%) were HCV antibody positive and 43 patients (0.95%) were HCV RNA positive, indicating that only 33% of patients with positive screening test had chronic HCV infection. The rate of chronic infection was higher in males as compared to females (1.34% vs 0.60%, p = 0.01). Patients with history of opioid use or history of tobacco use were found to have a lower rate of spontaneous clearance than patients without each history (opioids: 48.6% vs 72.0%, p = 0.02; tobacco: 56.6% vs 80.5%, p = 0.01). Among 43 patients who were diagnosed with chronic hepatitis C, 26 were linked to a clinical setting that can address chronic HCV infection, with linkage to care rate of 60.5%. The most common barrier to this was inability to contact patients after discharge from the ED. CONCLUSIONS: A streamlined EMR system for HCV screening and subsequent linkage to care from the ED can be successfully implemented. A retrospective review suggests that male sex is related to chronic HCV infection, and history of opioid use or history of tobacco use is related to lower HCV spontaneous clearance.

Mild catalytic defects of tert rs61748181 polymorphism affect the clinical presentation of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a disorder of accelerated lung aging. Multiple pieces of evidence support that the aging biomarker short telomeres, which can be caused by mutations in telomerase reverse transcriptase (TERT), contribute to COPD pathogenesis. We hypothesized that short telomere risk-associated single nucleotide polymorphisms (SNPs) in TERT, while not able to drive COPD development, nonetheless modify the disease presentation. We set out to test the SNP carrying status in a longitudinal study of smokers with COPD and found that rapid decline of FEV1 in lung function was associated with the minor allele of rs61748181 (adjusted odds ratio 2.49, p = 0.038). Biochemical evaluation of ex vivo engineered human cell models revealed that primary cells expressing the minor allele of rs61748181 had suboptimal telomere length maintenance due to reduced telomerase catalytic activity, despite having comparable cell growth kinetics as WT-TERT expressing cells. This ex vivo observation translated clinically in that shorter telomeres were found in minor allele carriers in a sub-population of COPD patients with non-declining lung function, over the 5-year period of the longitudinal study. Collectively, our data suggest that functional TERT SNPs with mild catalytic defects are nonetheless implicated in the clinical presentation of COPD.

Telomerase Biogenesis and Activities from the Perspective of Its Direct Interacting Partners.

Telomerase reverse transcriptase (TERT)-the catalytic subunit of telomerase-is reactivated in up to 90% of all human cancers. TERT is observed in heterogenous populations of protein complexes, which are dynamically regulated in a cell type- and cell cycle-specific manner. Over the past two decades, in vitro protein-protein interaction detection methods have discovered a number of endogenous TERT binding partners in human cells that are responsible for the biogenesis and functionalization of the telomerase holoenzyme, including the processes of TERT trafficking between subcellular compartments, assembly into telomerase, and catalytic action at telomeres. Additionally, TERT have been found to interact with protein species with no known telomeric functions, suggesting that these complexes may contribute to non-canonical activities of TERT. Here, we survey TERT direct binding partners and discuss their contributions to TERT biogenesis and functions. The goal is to review the comprehensive spectrum of TERT pro-malignant activities, both telomeric and non-telomeric, which may explain the prevalence of its upregulation in cancer.

Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance.

Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.

Small-molecule affinity capture of DNA/RNA quadruplexes and their identification in vitro and in vivo through the G4RP protocol.

Guanine-rich DNA and RNA sequences can fold into higher-order structures known as G-quadruplexes (or G4-DNA and G4-RNA, respectively). The prevalence of the G4 landscapes in the human genome, transcriptome and ncRNAome (non-coding RNA), collectively known as G4ome, is strongly suggestive of biological relevance at multiple levels (gene expression, replication). Small-molecules can be used to track G4s in living cells for the functional characterization of G4s in both normal and disease-associated changes in cell biology. Here, we describe biotinylated biomimetic ligands referred to as BioTASQ and their use as molecular tools that allow for isolating G4s through affinity pull-down protocols. We demonstrate the general applicability of the method by purifying biologically relevant G4s from nucleic acid mixtures in vitro and from human cells through the G4RP-RT-qPCR protocol. Overall, the results presented here represent a step towards the optimization of G4-RNAs identification, a key step in studying G4s in cell biology and human diseases.

Pharmacokinetics and investigation of optimal dose ertapenem in intermittent hemodialysis patients.

BACKGROUND: Previous pharmacokinetic studies demonstrated an increase in serum ertapenem concentrations with decreasing kidney function, including patients receiving renal replacement therapy. This study evaluated the pharmacokinetic parameters of ertapenem in patients receiving hemodialysis. METHODS: This prospective, single-center, open-label study examined the pharmacokinetics of a single intravenous (IV) dose of ertapenem 1 g in seven hospitalized noninfected patients undergoing hemodialysis. Blood samples were collected prior to ertapenem administration and at 0.5, 1, 2, 6, 12 and 48 hours (h) after administration. Ertapenem concentrations were determined by validated liquid chromatography mass spectrometry assay. RESULTS: Following an IV bolus of 1 g ertapenem, plasma concentrations declined relatively slowly with a mean ±standard deviation (SD) elimination half-life of 19.3 ±6.6 h. Plasma concentrations were similar in all subjects, with maximum mean plasma concentration observed of 343±48 µg/mL postdose. The mean ±SD values for systemic plasma clearance (CL) and volume of distribution at steady state (Vss) were 2±0.5 mL/min and 3295±1187 mL, respectively. The area under the curve for 0 h-∞ (AUCinf) was 7494 ±1424 h•µg/mL. No gender effect was observed and no serious adverse events were reported. CONCLUSIONS: Ertapenem half-life was prolonged in hemodialysis patients. Considering the nonrenal clearance and the expected 70% removal with high-efficacy hemodialysis, the dose of 1 g ertapenem, three times weekly, after hemodialysis may produce pharmacodynamically sufficient exposure for potential antimicrobial efficacy. Further studies are warranted to assess the clinical efficacy and safety of this dose with prolonged duration of therapy.

Transcriptome-wide identification of transient RNA G-quadruplexes in human cells.

Guanine-rich RNA sequences can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), whose biological roles are poorly understood, and in vivo existence is debated. To profile biologically relevant G4-RNA in the human transcriptome, we report here on G4RP-seq, which combines G4-RNA-specific precipitation (G4RP) with sequencing. This protocol comprises a chemical crosslinking step, followed by affinity capture with the G4-specific small-molecule ligand/probe BioTASQ, and target identification by sequencing, allowing for capturing global snapshots of transiently folded G4-RNAs. We detect widespread G4-RNA targets within the transcriptome, indicative of transient G4 formation in living human cells. Using G4RP-seq, we also demonstrate that G4-stabilizing ligands (BRACO-19 and RHPS4) can change the G4 transcriptomic landscape, most notably in long non-coding RNAs. G4RP-seq thus provides a method for studying the G4-RNA landscape, as well as ways of considering the mechanisms underlying G4-RNA formation, and the activity of G4-stabilizing ligands.

Transient Telomerase Inhibition with Imetelstat Impacts DNA Damage Signals and Cell-Cycle Kinetics.

Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of telomeres at the ends of linear chromosomes and contributes to proper telomere-loop (T-loop) formation. Formation of the T-loop, an obligate step before cell division can proceed, requires the generation of a 3'-overhang on the G-rich strand of telomeric DNA via telomerase or C-strand specific nucleases. Here, it is discovered that telomerase activity is critical for efficient cell-cycle progression using transient chemical inhibition by the telomerase inhibitor, imetelstat. Telomerase inhibition changed cell cycle kinetics and increased the proportion of cells in G2-phase, suggesting delayed clearance through this checkpoint. Investigating the possible contribution of unstructured telomere ends to these cell-cycle distribution changes, it was observed that imetelstat treatment induced γH2AX DNA damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Chromatin-immunoprecipitation with γH2AX antibodies demonstrated imetelstat treatment-dependent enrichment of this DNA damage marker at telomeres. Notably, the effects of telomerase inhibition on cell cycle profile alterations were abrogated by pharmacological inhibition of the DNA-damage-repair transducer, ATM. Also, imetelstat potentiation of etoposide, a DNA-damaging drug that acts preferentially during S-G2 phases of the cell cycle, depends on functional ATM signaling. Thus, telomerase inhibition delays the removal of ATM-dependent DNA damage signals from telomeres in telomerase-positive cancer cells and interferes with cell cycle progression through G2Implications: This study demonstrates that telomerase activity directly facilitates the progression of the cell cycle through modulation of transient telomere dysfunction signals. Mol Cancer Res; 16(8); 1215-25. ©2018 AACR.

Quantification of Pseudouridine Levels in Cellular RNA Pools with a Modified HPLC-UV Assay.

Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. With the help of different guide RNAs, the dyskerin complex pseudouridylates ribosomal RNA, small nuclear RNA and selective messenger RNAs. To monitor Ψ levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total Ψ levels in different cellular RNA fractions. Our method was validated to be accurate and precise within the linear range of 0.06-15.36 pmol/µL and to have absolute Ψ quantification levels as low as 3.07 pmol. Using our optimized HPLC assay, we found that 1.20% and 1.94% of all ribonucleosides in nuclear-enriched RNA and small non-coding RNA pools from the HEK293 cell line, and 1.77% and 0.98% of ribonucleosides in 18S and 28S rRNA isolated from the HeLa cell line, were pseudouridylated. Upon knockdown of dyskerin expression, a consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization.

Cellular Detection of G-Quadruplexes by Optical Imaging Methods.

G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Radiation dose of digital radiography (DR) versus micro-dose x-ray (EOS) on patients with adolescent idiopathic scoliosis: 2016 SOSORT- IRSSD "John Sevastic Award" Winner in Imaging Research.

BACKGROUND: Patients with adolescent idiopathic scoliosis (AIS) frequently receive x-ray imaging at diagnosis and subsequent follow monitoring. The ionizing radiation exposure has accumulated through their development stage and the effect of radiation to this young vulnerable group of patients is uncertain. To achieve the ALARA (as low as reasonably achievable) concept of radiation dose in medical imaging, a slot-scanning x-ray technique by the EOS system has been adopted and the radiation dose using micro-dose protocol was compared with the standard digital radiography on patients with AIS. METHODS: Ninety-nine participants with AIS underwent micro-dose EOS and 33 underwent standard digital radiography (DR) for imaging of the whole spine. Entrance-skin dose was measured using thermoluminescent dosimeters (TLD) at three regions (i.e. dorsal sites at the level of sternal notch, nipple line, symphysis pubis). Effective dose and organ dose were calculated by simulation using PCXMC 2.0. Data from two x-ray systems were compared using independent-samples t-test and significance level at 0.05. All TLD measurements were conducted on PA projection only. Image quality was also assessed by two raters using Cobb angle measurement and a set of imaging parameters for optimization purposes. RESULTS: Entrance-skin dose from micro-dose EOS system was 5.9-27.0 times lower at various regions compared with standard DR. The calculated effective dose was 2.6 ± 0.5 (µSv) and 67.5 ± 23.3 (µSv) from micro-dose and standard DR, respectively. The reduction in the micro-dose was approximately 26 times. Organ doses at thyroid, lung and gonad regions were significantly lower in micro-dose (p < 0.001). Data were further compared within the different gender groups. Females received significantly higher (p < 0.001) organ dose at ovaries compared to the testes in males. Patients with AIS received approximately 16-34 times lesser organ dose from micro-dose x-ray as compared with the standard DR. There was no significant difference in overall rating of imaging quality between EOS and DR. Micro-dose protocol provided enough quality to perform consistent measurement on Cobb angle. CONCLUSIONS: Entrance-skin dose, effective dose and organ dose were significantly reduced in micro-dose x-ray. The effective dose of a single micro-dose x-ray (2.6 µSv) was less than a day of background radiation. As AIS patients require periodic x-ray follow up for surveillance of curve progression, clinical use of micro-dose x-ray system is beneficial for these young patients to reduce the intake of ionizing radiation.

Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method.

Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities.

Investigation of chromosome X inactivation and clinical phenotypes in female carriers of DKC1 mutations.

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer susceptibility syndrome caused by germline mutations in telomere biology genes. Germline mutations in DKC1, which encodes the protein dyskerin, cause X-linked recessive DC. Because of skewed X-chromosome inactivation, female DKC1 mutation carriers do not typically develop clinical features of DC. This study evaluated female DKC1 mutation carriers with DC-associated phenotypes to elucidate the molecular features of their mutations, in comparison with unaffected carriers and mutation-negative female controls. All female DKC1 mutation carriers had normal leukocyte subset telomere lengths and similarly skewed X-inactivation in multiple tissue types, regardless of phenotype. We observed dyskerin expression, telomerase RNA accumulation, and pseudouridylation present in all mutation carriers at levels comparable to healthy wild-type controls. Our study suggests that mechanisms in addition to X chromosome inactivation, such as germline mosaicism or epigenetics, may contribute to DC-like phenotypes present in female DKC1 mutation carriers. Future studies are warranted to understand the molecular mechanisms associated with the phenotypic variability in female DKC1 mutation carriers, and to identify those at risk of disease. Am. J. Hematol. 91:1215-1220, 2016. © 2016 Wiley Periodicals, Inc.