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.

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.

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.

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.

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.

Visualization of RNA-Quadruplexes in Live Cells.

Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

The accumulation and not the specific activity of telomerase ribonucleoprotein determines telomere maintenance deficiency in X-linked dyskeratosis congenita.

X-linked dyskeratosis congenita (X-DC) is caused by mutations in the housekeeping nucleolar protein dyskerin. Amino acid changes associated with X-DC are remarkably heterogeneous. Peripheral mononuclear blood cells and fibroblasts isolated from X-DC patients harbor lower steady-state telomerase RNA (TER) levels and shorter telomeres than healthy age-matched controls. Previously, we showed that retroviral expression of recombinant TER, together with expression of recombinant telomerase reverse transcriptase, restored telomere maintenance and proliferative capacity in X-DC patient cells. Using rare X-DC isoforms (ΔL37 and A386T dyskerin), we showed that telomere maintenance defects observed in X-DC are solely due to decreased steady-state levels of TER. Disease-associated reductions in steady-state TER levels cause deficiencies in telomere maintenance. Here, we confirm these findings in other primary X-DC patient cell lines coding for the most common (A353V dyskerin) and more clinically severe (K314R and A353V dyskerin) X-DC isoforms. Using cell lines derived from these patients, we also examined the steady-state levels of other hinge-ACA motif RNAs and did not find differences in their in vivo accumulations. We show, for the first time, that purified telomerase holoenzyme complexes from different X-DC cells have normal catalytic activity. Our data confirm that dyskerin promotes TER stability in vivo, endorsing the development of TER supplementation strategies for the treatment of X-DC.

Severity of X-linked dyskeratosis congenita (DKCX) cellular defects is not directly related to dyskerin (DKC1) activity in ribosomal RNA biogenesis or mRNA translation.

Dyskerin (encoded by the DKC1 locus) is the pseudouridine synthase responsible for the modification of noncoding RNA. Dyskerin is also an obligate member of the telomerase enzyme, and participates in the biogenesis of telomerase. Genetic lesions at the DKC1 locus are associated with X-linked dyskeratosis congenita (X-DC) and the Hoyeraal-Hreidarsson Syndrome (HHS). Both syndromes have been linked to deficient telomere maintenance, but little is known about the RNA modification activities of dyskerin in X-DC and HHS cells. To evaluate whether X-DC-associated dyskerin mutations affect the modification or function of ribosomal RNA, we studied five telomerase-rescued X-DC cells (X-DC(T) ). Our data revealed a small reproducible loss of pseudouridines in mature rRNA in two X-DC variants. However, we found no difference in protein synthesis between telomerized wild-type (WT(T) ) and X-DC(T) cells, with an internal ribosomal entry site translation assay, or by measuring total protein synthesis in live cells. X-DC(T) cells and WT(T) cells also exhibited similar tolerances to ionizing radiation and endoplasmic reticulum stress. Despite the loss in rRNA pseudouridine modification, functional perturbations from these changes are secondary to the telomere maintenance defects of X-DC. Our data show that telomere dysfunction is the primary and unifying etiology of X-DC.

A cohort study of the risk of seizures in a pediatric population treated with atomoxetine or stimulant medications.

PURPOSE: Stimulant medications used for treating attention deficit hyperactivity disorder (ADHD) can be associated with an increased risk of seizures. Atomoxetine is a non-stimulant medication approved for treating ADHD. This retrospective cohort analysis evaluated risk of seizures among pediatric patients naïve to ADHD medication therapy, with exposure to atomoxetine relative to stimulant medications. METHODS: Among members of a large US health plan from 1/1/2003 to 12/31/2006, aged 6-17 years, we identified initiators of atomoxetine or stimulants with no evidence of prior study drug use. We created study cohorts using propensity score matching within 6-month calendar blocks. The outcome was a seizure event in the 6-month follow-up period verified through medical record review. Relative risks (RR) based on current use of each study drug adjusted for baseline covariates were calculated using Poisson regression. We estimated hazard ratios from Cox proportional hazards models for the comparison of atomoxetine to stimulants based on initial cohort assignment. RESULTS: We matched 13,398 initiators of atomoxetine to 13,322 initiators of stimulants. We identified 97 seizure events. After adjustment, current atomoxetine therapy was associated with a non-statistically significant 28% lower risk of seizure compared to current stimulant therapy (RR 0.72; 95%CI 0.37, 1.38). The adjusted RR of seizure with atomoxetine compared to stimulants based on initial cohort assignment was 0.90 (95%CI 0.54, 1.49). CONCLUSIONS: These results do not support an increase in the risk of seizure with atomoxetine therapy. The risk of seizure was not significantly different between pediatric patients taking atomoxetine compared with those taking stimulants.

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.

Electrophysiological properties of heteromeric TRPV4-C1 channels.

We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4-C1 channels [12]. In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4-C1 channels. 4α-Phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1. The activity of the channels was abrogated by a TRPC1-targeting blocking antibody T1E3. Conductance of the channels was ~95pS for outward currents and ~83pS for inward currents. The channels with similar conductance were also recorded in cells expressing TRPV4-C1 concatamers, in which assembled channels were expected to be mostly 2V4:2C1. Fluorescence Resonance Energy Transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2C1 stoichiometry while suggesting an unlikeliness of 3V4:1C1 or 1V4:3C1 stoichiometry. Monovalent cation permeability profiles were compared between heteromeric TRPV4-C1 and homomeric TRPV4 channels. For heteromeric TRPV4-C1 channels, their permeation profile was found to fit to Eisenman sequence VI, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence IV for a weak field strength binding site. Compared to homomeric TRPV4 channels, heteromeric TRPV4-C1 channels were slightly more permeable to Ca2+ and had a reduced sensitivity to extracellular Ca2+ inhibition. In summary, we found that, when TRPV4 and TRPC1 were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2C1. The heteromeric TRPV4-C1 channels display distinct electrophysiological properties different from those of homomeric TRPV4 channels.

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.

Subnuclear shuttling of human telomerase induced by transformation and DNA damage.

The telomerase ribonucleoprotein complex caps chromosome ends by adding telomeric repeats. Here we show that catalytically active human telomerase has a regulated intranuclear localization that is dependent on the cell-cycle stage, transformation and DNA damage. In primary cell lines, low expression of a fusion protein of green fluorescent protein and telomerase reverse transcriptase (GFP-hTERT) increases telomerase activity and stabilizes the maintenance of telomere length. Confocal microscopy shows that the release of telomerase to the nucleoplasm from sequestration at nucleolar sites is enhanced at the expected time of telomere replication. By contrast, in tumour and transformed cells, there is an almost complete dissociation of telomerase from nucleoli at all stages of the cell cycle. Transfection of the simian virus 40 genome into a primary cell line is sufficient to mobilize telomerase from nucleoli to the nucleoplasm. Conversely, ionizing radiation induces the reassociation of telomerase with nucleoli in both primary and transformed cells. These findings show that transformation and DNA damage have opposite effects on the cellular regulation of active telomerase, affecting the enzyme's access to both telomeric and nontelomeric substrates.

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.

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.

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.

Is antiquitin a mitochondrial Enzyme?

Antiquitin is an aldehyde dehydrogenase involved in the catabolism of lysine. Mutations of antiquitin have been linked with the disease pyridoxine-dependent seizures. While it is well established that lysine metabolism takes place in the mitochondrial matrix, evidence for the mitochondrial localization of antiquitin has been lacking. In the present study, the subcellular localization of antiquitin was investigated using human embryonic kidney HEK293 cells. Three different approaches were used. First, confocal microscopic analysis was carried out on cells transiently transfected with fusion constructs containing enhanced green fluorescent protein with different lengths of antiquitin based on the different potential start codons of translation. Second, immunofluorescence staining was used to detect the localization of antiquitin directly in the cells. Third, subcellular fractionation was carried out and the individual fraction was analyzed for the presence of antiquitin by Western blot and flow cytometric analyses. All the results showed that antiquitin was present not only in the cytosol but also in the mitochondria.

Adherence to recommended dosing and monitoring for mitoxantrone in patients with multiple sclerosis: a healthcare claims database study supplemented with medical records--the RETRO study.

PURPOSE: Mitoxantrone was approved for treatment of multiple sclerosis (MS) in October 2000. Monitoring and dosing guidelines in the product labeling accompanying this indication include blood counts, liver function, and pregnancy tests at each administration. Due to potential cardiotoxicity, left ventricular ejection fraction (LVEF) testing prior to initial infusion and all infusions at a cumulative dose >or=100 mg/m(2) was recommended until April 2005 when LVEF testing before all infusions was recommended in the approved labeling. We sought to estimate provider adherence to dosing and monitoring guidelines and the effect of changes in LVEF monitoring guidelines. METHODS: MS patients who received mitoxantrone between October 2000 and June 2006 were selected from the claims of a large US health insurer. Claims for infusions and for specified tests prior to an infusion determined adherence to guidelines, with medical records providing additional information for a subset. RESULTS: There were 1827 mitoxantrone infusions to 548 eligible patients; medical records were obtained for 261 patients (1096 infusions). Most mitoxantrone recipients were 30-59 years of age and 73% were female. Adherence to recommended dosing was higher than for recommended monitoring. Blood counts were conducted for most infusions (78-83%), while liver function tests (LFT) were performed less often (47-54% of infusions). Pregnancy tests were performed for 10% or fewer of the infusions administered to reproductive age women. Adherence with LVEF testing guidelines improved following labeling changes. CONCLUSIONS: Adherence to recommended monitoring was incomplete, but amenable to change. Automated assessment through insurance claims supplemented with medical record data provides a balanced means for studying adherence to recommendations.