Pedigree-based estimation of human mobile element retrotransposition rates.

Germline mutation rates in humans have been estimated for a variety of mutation types, including single-nucleotide and large structural variants. Here, we directly measure the germline retrotransposition rate for the three active retrotransposon elements: L1, Alu, and SVA. We used three tools for calling mobile element insertions (MEIs) (MELT, RUFUS, and TranSurVeyor) on blood-derived whole-genome sequence (WGS) data from 599 CEPH individuals, comprising 33 three-generation pedigrees. We identified 26 de novo MEIs in 437 births. The retrotransposition rate estimates for Alu elements, one in 40 births, is roughly half the rate estimated using phylogenetic analyses, a difference in magnitude similar to that observed for single-nucleotide variants. The L1 retrotransposition rate is one in 63 births and is within range of previous estimates (1:20-1:200 births). The SVA retrotransposition rate, one in 63 births, is much higher than the previous estimate of one in 900 births. Our large, three-generation pedigrees allowed us to assess parent-of-origin effects and the timing of insertion events in either gametogenesis or early embryonic development. We find a statistically significant paternal bias in Alu retrotransposition. Our study represents the first in-depth analysis of the rate and dynamics of human retrotransposition from WGS data in three-generation human pedigrees.

Polyamorphism Mirrors Polymorphism in the Liquid-Liquid Transition of a Molecular Liquid.

Liquid-liquid transitions between two amorphous phases in a single-component liquid have courted controversy. All known examples of liquid-liquid transitions in molecular liquids have been observed in the supercooled state, suggesting an intimate connection with vitrification and locally favored structures inhibiting crystallization. However, there is precious little information about the local molecular packing in supercooled liquids, meaning that the order parameter of the transition is still unknown. Here, we investigate the liquid-liquid transition in triphenyl phosphite and show that it is caused by the competition between liquid structures that mirror two crystal polymorphs. The liquid-liquid transition is found to be between a geometrically frustrated liquid and a dynamically frustrated glass. These results indicate a general link between polymorphism and polyamorphism and will lead to a much greater understanding of the physical basis of liquid-liquid transitions and allow the systematic discovery of other examples.

Experimental observation of nanophase segregation in aqueous salt solutions around the predicted liquid-liquid transition in water.

The liquid-liquid transition in supercooled liquid water, predicted to occur around 220 K, is controversial due to the difficulty of studying it caused by competition from ice crystallization (the so-called "no man's land"). In aqueous solutions, it has been predicted to give rise to phase separation on a nanometer scale between a solute-rich high-density phase and a water-rich low-density phase. Here we report direct experimental evidence for the formation of a nanosegregated phase in eutectic aqueous solutions of LiCl and LiSCN where the presence of crystalline water can be experimentally excluded. Femtosecond infrared and Raman spectroscopies are used to determine the temperature-dependent structuring of water, the solvation of the SCN- anion, and the size of the phase segregated domains.

Phenotype of CM-AVM2 caused by variants in EPHB4: how much overlap with hereditary hemorrhagic telangiectasia (HHT)?

PURPOSE: EPHB4 variants were recently reported to cause capillary malformation-arteriovenous malformation 2 (CM-AVM2). CM-AVM2 mimics RASA1-related CM-AVM1 and hereditary hemorrhagic telangiectasia (HHT), as clinical features include capillary malformations (CMs), telangiectasia, and arteriovenous malformations (AVMs). Epistaxis, another clinical feature that overlaps with HHT, was reported in several cases. Based on the clinical overlap of CM-AVM2 and HHT, we hypothesized that patients considered clinically suspicious for HHT with no variant detected in an HHT gene (ENG, ACVRL1, or SMAD4) may have an EPHB4 variant. METHODS: Exome sequencing or a next-generation sequencing panel including EPHB4 was performed on individuals with previously negative molecular genetic testing for the HHT genes and/or RASA1. RESULTS: An EPHB4 variant was identified in ten unrelated cases. Seven cases had a pathogenic EPHB4 variant, including one with mosaicism. Three cases had an EPHB4 variant of uncertain significance. The majority had epistaxis (6/10 cases) and telangiectasia (8/10 cases), as well as CMs. Two of ten cases had a central nervous system AVM. CONCLUSIONS: Our results emphasize the importance of considering CM-AVM2 as part of the clinical differential for HHT and other vascular malformation syndromes. Yet, these cases highlight significant differences in the cutaneous presentations of CM-AVM2 versus HHT.

Genome sequencing reveals a deep intronic splicing ACVRL1 mutation hotspot in Hereditary Haemorrhagic Telangiectasia.

INTRODUCTION: Hereditary haemorrhagic telangiectasia (HHT) is a genetically heterogeneous disorder caused by mutations in the genes ENG, ACVRL1, and SMAD4. Yet the genetic cause remains unknown for some families even after exhaustive exome analysis. We hypothesised that non-coding regions of the known HHT genes may harbour variants that disrupt splicing in these cases. METHODS: DNA from 35 individuals with clinical findings of HHT and 2 healthy controls from 13 families underwent whole genome sequencing. Additionally, 87 unrelated cases suspected to have HHT were evaluated using a custom designed next-generation sequencing panel to capture the coding and non-coding regions of ENG, ACVRL1 and SMAD4. Individuals from both groups had tested negative previously for a mutation in the coding region of known HHT genes. Samples were sequenced on a HiSeq2500 instrument and data were analysed to identify novel and rare variants. RESULTS: Eight cases had a novel non-coding ACVRL1 variant that disrupted splicing. One family had an ACVRL1intron 9:chromosome 3 translocation, the first reported case of a translocation causing HHT. The other seven cases had a variant located within a ~300 bp CT-rich 'hotspot' region of ACVRL1intron 9 that disrupted splicing. CONCLUSIONS: Despite the difficulty of interpreting deep intronic variants, our study highlights the importance of non-coding regions in the disease mechanism of HHT, particularly the CT-rich hotspot region of ACVRL1intron 9. The addition of this region to HHT molecular diagnostic testing algorithms will improve clinical sensitivity.

Rock Climber Self-Rescue Skills.

INTRODUCTION: Rock climbing involves some inherent danger, and rock climbers should be able to carry out basic rescue techniques for their own safety. This study seeks to assess such abilities by examining self-rescue skills in a cohort of rock climbers. METHODS: Climbers who participate in multipitch sport or traditional climbing styles were recruited via posters at a local climbing gym and on social media. Participants completed a survey assessing climbing history and confidence in their rescue skills and then were evaluated on 3 rescue scenarios in an indoor, standardized setting. Scenario pass rates were calculated and compared with rescue skill confidence on the survey. RESULTS: Twenty-five climbers participated in the study. Mean confidence in rescue skills varied from 4 to 4.5 (on a 7-point scale). The pass rates for the 3 scenarios were 28%, 68%, and 52%. Only 24% of climbers passed all 3 scenarios. Surveyed confidence in rescue skills and pass rate statistically correlated in only 1 scenario. CONCLUSIONS: Self-rescue skills were generally lacking in our study population. Climber confidence, experience, training, and climbing frequency did not appear to be associated with a higher level of rescue skills. Self-rescue skills should be emphasized in climbing instruction and courses to increase overall safety.

Remote Collection of Patient-Reported Outcomes Following Outpatient Hand Surgery: A Randomized Trial of Telephone, Mail, and E-Mail.

PURPOSE: Obtaining remote patient-reported outcomes (PRO) is limited by low patient response rates and resource-intensive collection methods. We hypothesized that an e-mail-delivered Web-based data collection tool would outperform the traditional methods of telephone and standard mail for collecting long-term Boston Carpal Tunnel Questionnaire (BCTQ) scores at a minimum of 1 year following carpal tunnel release (CTR). METHODS: We conducted a randomized trial of 969 patients who underwent CTR at a tertiary medical center within the past 5 years. Participants were randomized to the PRO collection methods of mail, telephone, and e-mail. The primary outcome was survey response rate at 1 year after surgery. Secondary analyses included data completeness and the effect of time from surgery, mode effects, and patient modality preference. RESULTS: At 1 year from surgery, the response rates were 64% for telephone and 42% for both mail and e-mail. Ninety-nine percent of telephone surveys were complete compared with 88% and 83% for mail and e-mail, respectively. There was no significant difference in the overall response rate at 1 or 5 years after surgery, nor in the BCTQ score between the modalities. CONCLUSIONS: A higher response rate and increased survey completeness was achieved by telephone contact methods compared with standard mailings or Web-based methods for PRO collection after CTR 1 to 5 years after surgery. A Web-based method demonstrated response rates equivalent to those of standard mail, was the most preferred modality, and offered logistical advantages such as automation and immediate integration with outcome databases. CLINICAL RELEVANCE: Obtaining PRO routinely after treatment may increase in importance. A Web-based interface may assist clinicians in decreasing the resource utilization typically associated with more traditional methods used to obtain outcome data.

Forward genetic screening identifies a small molecule that blocks Toxoplasma gondii growth by inhibiting both host- and parasite-encoded kinases.

The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.

Analysis of archived residual newborn screening blood spots after whole genome amplification.

BACKGROUND: Deidentified newborn screening bloodspot samples (NBS) represent a valuable potential resource for genomic research if impediments to whole exome sequencing of NBS deoxyribonucleic acid (DNA), including the small amount of genomic DNA in NBS material, can be overcome. For instance, genomic analysis of NBS could be used to define allele frequencies of disease-associated variants in local populations, or to conduct prospective or retrospective studies relating genomic variation to disease emergence in pediatric populations over time. In this study, we compared the recovery of variant calls from exome sequences of amplified NBS genomic DNA to variant calls from exome sequencing of non-amplified NBS DNA from the same individuals. RESULTS: Using a standard alignment-based Genome Analysis Toolkit (GATK), we find 62,000-76,000 additional variants in amplified samples. After application of a unique kmer enumeration and variant detection method (RUFUS), only 38,000-47,000 additional variants are observed in amplified gDNA. This result suggests that roughly half of the amplification-introduced variants identified using GATK may be the result of mapping errors and read misalignment. CONCLUSIONS: Our results show that it is possible to obtain informative, high-quality data from exome analysis of whole genome amplified NBS with the important caveat that different data generation and analysis methods can affect variant detection accuracy, and the concordance of variant calls in whole-genome amplified and non-amplified exomes.

A randomized trial of the disabilities of the arm, shoulder, and hand administration: tablet computer versus paper and pencil.

PURPOSE: To compare the Disabilities of the Arm, Shoulder, and Hand (DASH) patient-reported outcome measure as administered by tablet computer to the traditional paper format. METHODS: In a prospective, randomized study design, 223 consecutive adult patients who presented to the clinic of a single hand surgeon at a tertiary medical center were randomized by visit time to receive the DASH by either paper or tablet computer. Test completeness, time to completion, DASH score, and diagnostic and demographic data were collected and compared between the two cohorts. In total, 120 participants took the DASH using the tablet and 103 using paper. RESULTS: 43% of the paper surveys had at least one question that was omitted, compared with 13% in the tablet group; 14% of the paper surveys were not scoreable (< 27 questions answered) compared with 4% of the tablet surveys. The mean time to complete was 3.1 minutes for the paper version of the DASH and 4.3 minutes for the tablet version. Among our study population, there was no influence of age, sex, or diagnosis category on the time required to complete either version of the test. The mean DASH score was 45 for the paper version and 32 for the tablet version. CONCLUSIONS: The use of digital data entry methods in the arena of health care outcomes research is increasing. Administration of the DASH via a tablet computer resulted in more complete data, slightly increased responder burden, and a lower DASH score. This finding may have important implications for the use of this metric in an electronic format in future research endeavors. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic II.

Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii.

BACKGROUND: Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages. RESULTS: By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively). CONCLUSIONS: The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.

Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains.

BACKGROUND: Toxoplasma gondii has a largely clonal population in North America and Europe, with types I, II and III clonal lineages accounting for the majority of strains isolated from patients. RH, a particular type I strain, is most frequently used to characterize Toxoplasma biology. However, compared to other type I strains, RH has unique characteristics such as faster growth, increased extracellular survival rate and inability to form orally infectious cysts. Thus, to identify candidate genes that could account for these parasite phenotypic differences, we determined genetic differences and differential parasite gene expression between RH and another type I strain, GT1. Moreover, as differences in host cell modulation could affect Toxoplasma replication in the host, we determined differentially modulated host processes among the type I strains through host transcriptional profiling. RESULTS: Through whole genome sequencing, we identified 1,394 single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) between RH and GT1. These SNPs/indels together with parasite gene expression differences between RH and GT1 were used to identify candidate genes that could account for type I phenotypic differences. A polymorphism in dense granule protein, GRA2, determined RH and GT1 differences in the evasion of the interferon gamma response. In addition, host transcriptional profiling identified that genes regulated by NF-ĸB, such as interleukin (IL)-12p40, were differentially modulated by the different type I strains. We subsequently showed that this difference in NF-ĸB activation was due to polymorphisms in GRA15. Furthermore, we observed that RH, but not other type I strains, recruited phosphorylated IĸBα (a component of the NF-ĸB complex) to the parasitophorous vacuole membrane and this recruitment of p- IĸBα was partially dependent on GRA2. CONCLUSIONS: We identified candidate parasite genes that could be responsible for phenotypic variation among the type I strains through comparative genomics and transcriptomics. We also identified differentially modulated host pathways among the type I strains, and these can serve as a guideline for future studies in examining the phenotypic differences among type I strains.

Understanding the emergence of the boson peak in molecular glasses.

A common feature of glasses is the "boson peak", observed as an excess in the heat capacity over the crystal or as an additional peak in the terahertz vibrational spectrum. The microscopic origins of this peak are not well understood; the emergence of locally ordered structures has been put forward as a possible candidate. Here, we show that depolarised Raman scattering in liquids consisting of highly symmetric molecules can be used to isolate the boson peak, allowing its detailed observation from the liquid into the glass. The boson peak in the vibrational spectrum matches the excess heat capacity. As the boson peak intensifies on cooling, wide-angle x-ray scattering shows the simultaneous appearance of a pre-peak due to molecular clusters consisting of circa 20 molecules. Atomistic molecular dynamics simulations indicate that these are caused by over-coordinated molecules. These findings represent an essential step toward our understanding of the physics of vitrification.

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.

TSPAN6 is a suppressor of Ras-driven cancer.

Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced KrasG12D-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation. Moreover, we show that inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. Our results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer.

Rapid genome sequencing identifies a novel de novo SNAP25 variant for neonatal congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a group of 32 disorders involving genetic dysfunction at the neuromuscular junction resulting in skeletal muscle weakness that worsens with physical activity. Precise diagnosis and molecular subtype identification are critical for treatment as medication for one subtype may exacerbate disease in another (Engel et al., Lancet Neurol 14: 420 [2015]; Finsterer, Orphanet J Rare Dis 14: 57 [2019]; Prior and Ghosh, J Child Neurol 36: 610 [2021]). The SNAP25-related CMS subtype (congenital myasthenic syndrome 18, CMS18; MIM #616330) is a rare disorder characterized by muscle fatigability, delayed psychomotor development, and ataxia. Herein, we performed rapid whole-genome sequencing (rWGS) on a critically ill newborn leading to the discovery of an unreported pathogenic de novo SNAP25 c.529C > T; p.Gln177Ter variant. In this report, we present a novel case of CMS18 with complex neonatal consequence. This discovery offers unique insight into the extent of phenotypic severity in CMS18, expands the reported SNAP25 variant phenotype, and paves a foundation for personalized management for CMS18.

Comprehensive variant calling from whole-genome sequencing identifies a complex inversion that disrupts ZFPM2 in familial congenital diaphragmatic hernia.

BACKGROUND: Genetic disorders contribute to significant morbidity and mortality in critically ill newborns. Despite advances in genome sequencing technologies, a majority of neonatal cases remain unsolved. Complex structural variants (SVs) often elude conventional genome sequencing variant calling pipelines and will explain a portion of these unsolved cases. METHODS: As part of the Utah NeoSeq project, we used a research-based, rapid whole-genome sequencing (WGS) protocol to investigate the genomic etiology for a newborn with a left-sided congenital diaphragmatic hernia (CDH) and cardiac malformations, whose mother also had a history of CDH and atrial septal defect. RESULTS: Using both a novel, alignment-free and traditional alignment-based variant callers, we identified a maternally inherited complex SV on chromosome 8, consisting of an inversion flanked by deletions. This complex inversion, further confirmed using orthogonal molecular techniques, disrupts the ZFPM2 gene, which is associated with both CDH and various congenital heart defects. CONCLUSIONS: Our results demonstrate that complex structural events, which often are unidentifiable or not reported by clinically validated testing procedures, can be discovered and accurately characterized with conventional, short-read sequencing and underscore the utility of WGS as a first-line diagnostic tool.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells.

BACKGROUND: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. METHODS: Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. RESULTS: RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~116 µM, and this uptake was reduced in the presence of extracellular Ca²+ and Mg²+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. CONCLUSIONS: P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. GENERAL SIGNIFICANCE: RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

Chromatin structure following UV-induced DNA damage-repair or death?

In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury.