Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Germline ATM variants predispose to melanoma: a joint analysis across the GenoMEL and MelaNostrum consortia.

PURPOSE: Ataxia-Telangiectasia Mutated (ATM) has been implicated in the risk of several cancers, but establishing a causal relationship is often challenging. Although ATM single-nucleotide polymorphisms have been linked to melanoma, few functional alleles have been identified. Therefore, ATM impact on melanoma predisposition is unclear. METHODS: From 22 American, Australian, and European sites, we collected 2,104 familial, multiple primary (MPM), and sporadic melanoma cases who underwent ATM genotyping via panel, exome, or genome sequencing, and compared the allele frequency (AF) of selected ATM variants classified as loss-of-function (LOF) and variants of uncertain significance (VUS) between this cohort and the gnomAD non-Finnish European (NFE) data set. RESULTS: LOF variants were more represented in our study cohort than in gnomAD NFE, both in all (AF = 0.005 and 0.002, OR = 2.6, 95% CI = 1.56-4.11, p < 0.01), and familial + MPM cases (AF = 0.0054 and 0.002, OR = 2.97, p < 0.01). Similarly, VUS were enriched in all (AF = 0.046 and 0.033, OR = 1.41, 95% CI = 1.6-5.09, p < 0.01) and familial + MPM cases (AF = 0.053 and 0.033, OR = 1.63, p < 0.01). In a case-control comparison of two centers that provided 1,446 controls, LOF and VUS were enriched in familial + MPM cases (p = 0.027, p = 0.018). CONCLUSION: This study, describing the largest multicenter melanoma cohort investigated for ATM germline variants, supports the role of ATM as a melanoma predisposition gene, with LOF variants suggesting a moderate-risk.

POT1 germline mutations but not TERT promoter mutations are implicated in melanoma susceptibility in a large cohort of Spanish melanoma families.

BACKGROUND: Germline mutations in telomere-related genes such as POT1 and TERT predispose individuals to familial melanoma. OBJECTIVES: To evaluate the prevalence of germline mutations in POT1 and TERT in a large cohort of Spanish melanoma-prone families (at least two affected first- or second-degree relatives). METHODS: Overall, 228 CDKN2A wild-type melanoma-prone families were included in the study. Screening of POT1 was performed in one affected person from each family and TERT was sequenced in one affected patient from 202 families (26 families were excluded owing to DNA exhaustion/degradation). TERT promoter sequencing was extended to an additional 30 families with CDKN2A mutation and 70 patients with sporadic multiple primary melanoma (MPM) with a family history of other cancers. RESULTS: We identified four families with potentially pathogenic POT1 germline mutations: a missense variant c.233T>C (p.Ile78Thr); a nonsense variant c.1030G>T (p.Glu344*); and two other variants, c.255G>A (r.125_255del) and c.1792G>A (r.1791_1792insAGTA, p.Asp598Serfs*22), which we confirmed disrupted POT1 mRNA splicing. A TERT promoter variant of unknown significance (c.-125C>A) was detected in a patient with MPM, but no germline mutations were detected in TERT promoter in cases of familial melanoma. CONCLUSIONS: Overall, 1·7% of our CDKN2A/CDK4-wild type Spanish melanoma-prone families carry probably damaging mutations in POT1. The frequency of TERT promoter germline mutations in families with melanoma in our population is extremely rare.

A magnetic topological semimetal Sr1-yMn1-zSb2 (y, z < 0.1).

Weyl (WSMs) evolve from Dirac semimetals in the presence of broken time-reversal symmetry (TRS) or space-inversion symmetry. The WSM phases in TaAs-class materials and photonic crystals are due to the loss of space-inversion symmetry. For TRS-breaking WSMs, despite numerous theoretical and experimental efforts, few examples have been reported. In this Article, we report a new type of magnetic semimetal Sr1-yMn1-zSb2 (y, z < 0.1) with nearly massless relativistic fermion behaviour (m∗ =  0.04 - 0.05m0, where m0 is the free-electron mass). This material exhibits a ferromagnetic order for 304 K  <  T  <  565 K, but a canted antiferromagnetic order with a ferromagnetic component for T  <  304 K. The combination of relativistic fermion behaviour and ferromagnetism in Sr1-yMn1-zSb2 offers a rare opportunity to investigate the interplay between relativistic fermions and spontaneous TRS breaking.

Focusing of a Rydberg Positronium Beam with an Ellipsoidal Electrostatic Mirror.

Slow atoms in Rydberg states can exhibit specular reflection from a cylindrical surface upon which an azimuthally periodic potential is imposed. We have constructed a concave mirror of this type, in the shape of a truncated oblate ellipsoid of revolution, which has a focal length of (1.50±0.01) m measured optically. When placed near the center of a long vacuum pipe, this structure brings a beam of n=32 positronium (Ps) atoms to a focus on a position sensitive detector at a distance of (6.03±0.03) m from the Ps source. The intensity at the focus implies an overall reflection efficiency of ∼30%. The focal spot diameter (32±1) mm full width at half maximum is independent of the atoms' flight times from 20 to 60 µs, thus indicating that the mirror is achromatic to a good approximation. Mirrors based on this principle would be of use in a variety of experiments, allowing for improved collection efficiency and tailored transport or imaging of beams of slow Rydberg atoms and molecules.

Newborn screening for X-linked adrenoleukodystrophy in New York State: diagnostic protocol, surveillance protocol and treatment guidelines.

PURPOSE: To describe a diagnostic protocol, surveillance and treatment guidelines, genetic counseling considerations and long-term follow-up data elements developed in preparation for X-linked adrenoleukodystrophy (X-ALD) newborn screening in New York State. METHODS: A group including the director from each regional NYS inherited metabolic disorder center, personnel from the NYS Newborn Screening Program, and others prepared a follow-up plan for X-ALD NBS. Over the months preceding the start of screening, a series of conference calls took place to develop and refine a complete newborn screening system from initial positive screen results to long-term follow-up. RESULTS: A diagnostic protocol was developed to determine for each newborn with a positive screen whether the final diagnosis is X-ALD, carrier of X-ALD, Zellweger spectrum disorder, acyl CoA oxidase deficiency or D-bifunctional protein deficiency. For asymptomatic males with X-ALD, surveillance protocols were developed for use at the time of diagnosis, during childhood and during adulthood. Considerations for timing of treatment of adrenal and cerebral disease were developed. CONCLUSION: Because New York was the first newborn screening laboratory to include X-ALD on its panel, and symptoms may not develop for years, long-term follow-up is needed to evaluate the presented guidelines.

Response of knee fibrocartilage to joint destabilization.

OBJECTIVE: A major challenge to understanding osteoarthritis (OA) pathology is identifying the cellular events that precede the onset of cartilage damage. The objective of this study is to determine the effect of joint destabilization on early changes to fibrocartilage in the joint. DESIGN/METHODS: The anterior cruciate ligament was transected in collagen reporter mice (Col1CFP and ColXRFP). Mineralization labels were given every 2 weeks to measure new mineralized cartilage apposition. Novel fluorescent histology of mineralized tissue was used to characterize the changes in fibrocartilage at 2 and 4 weeks post-injury. RESULTS: Changes in fibrocartilaginous structures of the joint occur as early as 2 weeks after injury and are well developed by 4 weeks. The alterations are seen in multiple entheses and in the medial surface of the femoral and tibial condyles. In the responding entheses, mineral apposition towards the ligament midsubstance results in thickening of the mineralize fibrocartilage. These changes are associated with increases in ColX-RFP, Col1-CFP reporter activity and alkaline phosphatase enzyme activity. Mineral apposition also occurs in the fibrocartilage of the non-articular regions of the medial condyles by 2 weeks and develops into osteophytes by 4 weeks post-injury. An unexpected observation is punctate expression of tartrate resistant acid phosphatase activity in unmineralized fibrochondrocytes adjacent to active appositional mineralization. DISCUSSION: These observations suggest that fibrocartilage activates prior to degradation of the articular cartilage. Thus clinical and histological imaging of fibrocartilage may be an earlier indicator of disease initiation and may indicate a more appropriate time to start preventative treatment.

Effect of daily lithium chloride administration on bone mass and strength in growing broiler chickens.

The objective was to determine the effects of oral lithium chloride supplementation on bone strength and mass in broiler chickens. Ninety-six broilers were assigned to 1 of 2 treatment groups (lithium chloride or control; n=48/treatment). Beginning at 1 or 3 wk of age, chickens were administered lithium chloride (20 mg/kg body weight) or water daily by oral gavage. At 6 wk of age, chickens were euthanized and bone and muscle samples were collected. A 24 h lithium chloride (20 mg/kg body weight) challenge determined that serum lithium chloride increased within 2 h and cleared the system within 24 h, demonstrating the effective delivery of lithium chloride. Treatment did not influence body weight (P≥0.20) or feed intake (P≥0.81), demonstrating that lithium chloride did not negatively affect broiler growth. To determine bone strength, 3-point bending was performed on the femora and tibiae obtained from control and lithium chloride-treated birds in the 1 wk group. Lithium chloride-treated birds had a 22% reduction in stiffness compared with control in the femora (P=0.02) without a corresponding reduction in elastic modulus. No differences were observed in yield or ultimate load and in the corresponding calculations of stresses (P≥0.26). The toughness of tibiae was not altered in lithium chloride compared with control (P=0.11). Bone length and micro-CT imaging were performed on the tibiae of control and lithium chloride groups. No differences (P≥0.52) in bone length, cortical or trabecular bone volume, trabecular thickness, number, or spacing were observed. Lithium chloride treatment did not affect pectoralis muscle color or lipid oxidation (P>0.05). In conclusion, lithium chloride treatment in broilers did not negatively affect growth or meat quality. A reduction in bone stiffness of the femur with lithium chloride treatment was observed, however unlike the mouse model, the dosages of lithium chloride used in the current study did not result in anabolic effects on broiler long bones.

Bone-specific overexpression of NPY modulates osteogenesis.

OBJECTIVES: Neuropeptide Y (NPY) is a peptide involved in the regulation of appetite and energy homeostasis. Genetic data indicates that NPY decreases bone formation via central and peripheral activities. NPY is produced by various cell types including osteocytes and osteoblasts and there is evidence suggesting that peripheral NPY is important for regulation of bone formation. We sought to investigate the role of bone-derived NPY in bone metabolism. METHODS: We generated a mouse where NPY was over-expressed specifically in mature osteoblasts and osteocytes (Col2.3NPY) and characterized the bone phenotype of these mice in vivo and in vitro. RESULTS: Trabecular and cortical bone volume was reduced in 3-month-old animals, however bone formation rate and osteoclast activity were not significantly changed. Calvarial osteoblast cultures from Col2.3NPY mice also showed reduced mineralization and expression of osteogenic marker genes. CONCLUSIONS: Our data suggest that osteoblast/osteocyte-derived NPY is capable of altering osteogenesis in vivo and in vitro and may represent an important source of NPY for regulation of bone formation. However, it is possible that other peripheral sources of NPY such as the sympathetic nervous system and vasculature also contribute to peripheral regulation of bone turnover.

Functional modulation of the human voltage-gated sodium channel NaV1.8 by auxiliary ß subunits.

The voltage-gated sodium channel Nav1.8 mediates the tetrodotoxin-resistant (TTX-R) Na+ current in nociceptive primary sensory neurons, which has an important role in the transmission of painful stimuli. Here, we describe the functional modulation of the human Nav1.8 α-subunit in Xenopus oocytes by auxiliary ß subunits. We found that the ß3 subunit down-regulated the maximal Na+ current amplitude and decelerated recovery from inactivation of hNav1.8, whereas the ß1 and ß2 subunits had no such effects. The specific regulation of Nav1.8 by the ß3 subunit constitutes a potential novel regulatory mechanism of the TTX-R Na+ current in primary sensory neurons with potential implications in chronic pain states. In particular, neuropathic pain states are characterized by a down-regulation of Nav1.8 accompanied by increased expression of the ß3 subunit. Our results suggest that these two phenomena may be correlated, and that increased levels of the ß3 subunit may directly contribute to the down-regulation of Nav1.8. To determine which domain of the ß3 subunit is responsible for the specific regulation of hNav1.8, we created chimeras of the ß1 and ß3 subunits and co-expressed them with the hNav1.8 α-subunit in Xenopus oocytes. The intracellular domain of the ß3 subunit was shown to be responsible for the down-regulation of maximal Nav1.8 current amplitudes. In contrast, the extracellular domain mediated the effect of the ß3 subunit on hNav1.8 recovery kinetics.

Long-term drying of Mars by sequestration of ocean-scale volumes of water in the crust.

Geological evidence shows that ancient Mars had large volumes of liquid water. Models of past hydrogen escape to space, calibrated with observations of the current escape rate, cannot explain the present-day deuterium-to-hydrogen isotope ratio (D/H). We simulated volcanic degassing, atmospheric escape, and crustal hydration on Mars, incorporating observational constraints from spacecraft, rovers, and meteorites. We found that ancient water volumes equivalent to a 100 to 1500 meter global layer are simultaneously compatible with the geological evidence, loss rate estimates, and D/H measurements. In our model, the volume of water participating in the hydrological cycle decreased by 40 to 95% over the Noachian period (~3.7 billion to 4.1 billion years ago), reaching present-day values by ~3.0 billion years ago. Between 30 and 99% of martian water was sequestered through crustal hydration, demonstrating that irreversible chemical weathering can increase the aridity of terrestrial planets.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Laser-driven fast electron collimation in targets with resistivity boundary.

We demonstrate experimentally that the relativistic electron flow in a dense plasma can be efficiently confined and guided in targets exhibiting a high-resistivity-core-low-resistivity-cladding structure analogous to optical waveguides. The relativistic electron beam is shown to be confined to an area of the order of the core diameter (50 µm), which has the potential to substantially enhance the coupling efficiency of electrons to the compressed fusion fuel in the Fast Ignitor fusion in full-scale fusion experiments.

Maturation and sexual ontogeny in the spangled emperor Lethrinus nebulosus.

The reproductive development and sexual ontogeny of spangled emperor Lethrinus nebulosus populations in the Ningaloo Marine Park (NMP) were investigated to obtain an improved understanding of its evolved reproductive strategy and data for fisheries management. Evidence derived from (1) analyses of histological data and sampled sex ratios with size and age, (2) the identification of residual previtellogenic oocytes in immature and mature testes sampled during the spawning season and (3) observed changes in testis internal structure with increasing fish size and age, demonstrated a non-functional protogynous hermaphroditic strategy (or functional gonochorism). All the smallest and youngest fish sampled were female until they either changed sex to male at a mean 277.5 mm total length (L(T)) and 2.3 years old or remained female and matured at a larger mean L(T) (392.1 mm) and older age (3.5 years). Gonad masses were similar for males and females over the size range sampled and throughout long reproductive lives (up to a maximum estimated age of c. 31 years), which was another correlate of functional gonochorism. That the mean L(T) at sex change and female maturity were below the current minimum legal size (MLS) limit (410 mm) demonstrated that the current MLS limit is effective for preventing recreational fishers in the NMP retaining at least half of the juvenile males and females in their landed catches.

ZNF265--a novel spliceosomal protein able to induce alternative splicing.

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-beta1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF(35). Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

Gating currents associated with sodium and calcium currents in an Aplysia neuron.

In a voltage-clamped aplysia neuron (R15), depolarization beyond -30 millivolts produces an inward sodium current. Depolarization beyond -10 millivolts produces an additional inward calcium current with slower kinetics than the sodium current. When these ionic currents have been suppressed and capacitive currents subtracted out, a small outward displacement current can be seen with depolarizations beyond -30 millivolts. An additional, slower displacement current is seen with depolarizations beyond -10 millivolts. The currents have an exponential decay with an increase in rate per 10 degrees C increase in temperature of about 3 and are thought to be sodium and calcium gating currents.

Conotoxin modulation of voltage-gated sodium channels.

The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the microO-conotoxins which inhibit VGSC Na+ conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the microO-conotoxins, which may develop into potential drug leads for the treatment of pain states.

The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis.

The RASSF1A isoform of RASSF1 is frequently inactivated by epigenetic alterations in human cancers, but it remains unclear if and how it acts as a tumor suppressor. RASSF1A overexpression reduces in vitro colony formation and the tumorigenicity of cancer cell lines in vivo. Conversely, RASSF1A knockdown causes multiple mitotic defects that may promote genomic instability. Here, we have used a genetic approach to address the function of RASSF1A as a tumor suppressor in vivo by targeted deletion of Rassf1A in the mouse. Rassf1A null mice were viable and fertile and displayed no pathological abnormalities. Rassf1A null embryonic fibroblasts displayed an increased sensitivity to microtubule depolymerizing agents. No overtly altered cell cycle parameters or aberrations in centrosome number were detected in Rassf1A null fibroblasts. Rassf1A null fibroblasts did not show increased sensitivity to microtubule poisons or DNA-damaging agents and showed no evidence of gross genomic instability, suggesting that cellular responses to genotoxins were unaffected. Rassf1A null mice showed an increased incidence of spontaneous tumorigenesis and decreased survival rate compared with wild-type mice. Irradiated Rassf1A null mice also showed increased tumor susceptibility, particularly to tumors associated with the gastrointestinal tract, compared with wild-type mice. Thus, our results demonstrate that Rassf1A acts as a tumor suppressor gene.

Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter.

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Detector for positronium temperature measurements by two-photon angular correlation.

We report on the design and characterization of a modular γ-ray detector assembly developed for accurate and efficient detection of coincident 511 keV back-to-back γ-rays following electron-positron annihilation. Each modular detector consists of 16 narrow lutetium yttrium oxyorthosilicate scintillators coupled to a multi-anode Hamamatsu H12700B photomultiplier tube. We discuss the operation and optimization of 511 keV γ-ray detection resulting from testing various scintillators and detector arrangements concluding with an estimate of the coincident 511 keV detection efficiency for the intended experiment and a preliminary test representing one-quarter of the completed array.