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[Image: see text] 1,N(6)-ethenoadenine (εA) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises εA in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/α-ketoglutarate-dependent ALKBH2 enzyme repairs εA by direct conversion of εA to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145–147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on εA lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of εA at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of εA and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded εA lesions, but only AAG was capable of full repair of any εA in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

MOTIVATION: We undertook large citizen science surveys of bird distributions (atlases) in Britain and Ireland, aimed at quantifying breeding bird distributions on a 20‐year cycle and wintering bird distributions on a c. 30‐year cycle. We use these to generate spatially referenced information on apparent changes in bird distributions over c. 40 years. MAIN TYPE OF VARIABLE CONTAINED: Detection of breeding and wintering bird species in grid squares during five periods, and changes in detection between periods. The combined distribution dataset contains 1,410,938 records detailing detections of 465 bird species in 3,880 grid cells in different periods. The combined distribution change dataset contains 1,297,791 records describing stability, apparent colonization or apparent extinction of individual species in grid squares between pairs of atlases spanning up to c. 40 years. SPATIAL LOCATION AND GRAIN: Grid squares (10 km × 10 km) containing land throughout Britain, Ireland, the Isle of Man and the Channel Islands. The majority of data are at 10‐km resolution, but data for rare species are summarized at 20‐ or 50‐km resolution to protect sensitive locations. TIME PERIOD: The data represent summarized detection information derived from fieldwork during five periods: the breeding seasons 1968–1972, 1988–1991 and 2008–2011, and the winters 1981/1982–1983/1984 and 2007/2008–2010/2011. MAJOR TAXA STUDIED AND LEVEL OF MEASUREMENT: Birds; their distribution derived from citizen science surveys. SOFTWARE FORMAT: Data are supplied as comma‐separated text files.

Monoterpenes contribute to the characteristic aroma of several hop varieties and may occur as nonvolatile glycosides. Upon hydrolysis, the volatile terpenes are released from the glycoside precursors. Little is known, however, about the glycoside composition of hops. Seven pentose-hexose monoterpene alcohol glycosides from dried Humulus lupulus L. cv. Citra cones were isolated using high performance liquid chromatography separation and fractionation on a reverse phase phenyl-hexyl column. Further evaluation of each isolated fraction through HPLC qTOF MS with porous graphitic carbon (PGC) showed that the seven isolated monoterpenyl glycoside fractions could be further resolved into 20 isomers. Isolation on phenyl-hexyl followed by separation on PGC was needed to distinguish each isomer present. Additionally, the hop cones were screened for potential aroma glycosides. Using the PGC column combined with a database of over 900 potential glycosides, the identification of 21 additional monoterpene-polyol, norisoprenoid, volatile phenol, and aliphatic alcohol glycosides is reported.

A recently discovered post-translational modification of histone proteins is the irreversible proteolytic clipping of the histone N-terminal tail domains. This modification is involved in the regulation of various biological processes, including the DNA damage response. In this work, we used chemical footprinting to characterize the structural alterations to nucleosome core particles (NCPs) that result from a lack of a histone H2B or H3 tail. We also examine the influence of these histone tails on excision of the mutagenic lesion 1,N6-ethenoadenine (eA) by the repair enzyme alkyladenine DNA glycosylase. We found that the absence of the H2B or H3 tail results in altered DNA periodicity relative to that of native NCPs. We correlated these structural alterations to eA excision by utilizing a global analysis of 21 eA sites in NCPs and unincorporated duplex DNA. In comparison to native NCPs, there is enhanced excision of eA in tailless H2B NCPs in regions that undergo DNA unwrapping. This enhanced excision is not observed for tailless H3 NCPs; rather, excision is inhibited in more static areas of the NCP not prone to unwrapping. Our results support in vivo observations of alkylation damage profiles and the potential role of tail clipping as a mechanism for overcoming physical obstructions caused by packaging in NCPs but also reveal the potential inhibition of repair by tail clipping in some locations. Taken together, these results further our understanding of how base excision repair can be facilitated or diminished by histone tail removal and contribute to our understanding of the underlying mechanism that leads to mutational hot spots.

The increased use of Holstein genetic material in the Irish dairy herd has consequences for beef production. In all, 42 spring-born steers [14 Holsteins (HO), 14 Friesian (FR) and 14 Charolais × Holstein-Friesian (CH)] were reared to slaughter at between 26 and 37 mo of age. Carcass weight was higher and the lipid concentration of m. longissimus thoracis et lumborum was lower (P < 0.05) for CH than the dairy breeds. Overall acceptability tended to be lower (P = 0.055) while tenderness, texture and chewiness were lower (P < 0.05) for CH compared with the dairy breeds. The proportion of C16:1 in the total lipid tended to be lower (P = 0.055) for CH than the dairy breeds. Replacing male offspring of traditional “Irish” Friesian bulls with offspring from a genetically superior (from a dairy perspective) strain of Holstein bull had no commercially important impact on beef nutritional or eating quality.

Ultra-high-performance liquid chromatography (UHPLC) accurate mass tandem mass spectrometry is a powerful tool for identifying and profiling plant metabolites. Here, we describe an approach to characterize glycosidically bound precursors of monoterpenoids, norisoprenoids, volatile phenols, aliphatic alcohols, and sesquiterpenoids in grapes. Chromatographic separation of glycosylated compounds was evaluated using phenyl-hexyl (reverse phase), glycan/hydrophilic interaction, and porous graphitic carbon (PGC) stationary phases. PGC provided the best UHPLC separation for 102 tentatively identified aroma precursors in

The double-beta decay of 82Se to the 0 1 + excited state of 82Kr has been studied with the NEMO-3 detector using 0.93 kg of enriched 82Se measured for 4.75 y, corresponding to an exposure of 4.42 kg⋅y. A dedicated analysis to reconstruct the γ-rays has been performed to search for events in the 2e2γ channel. No evidence of a 2 ν β β decay to the 0 1 + state has been observed and a limit of T 1 / 2 2 ν ( Se 82 , 0 g s + → 0 1 + ) > 1.3 × 10 21 y at 90% CL has been set. Concerning the 0 ν β β decay to the 0 1 + state, a limit for this decay has been obtained with T 1 / 2 0 ν ( Se 82 , 0 g s + → 0 1 + ) > 2.3 × 10 22 y at 90% CL, independently from the 2 ν β β decay process. These results are obtained for the first time with a tracko-calo detector, reconstructing every particle in the final state.

The V27A mutation confers adamantane resistance on the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to determine structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to that of the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics simulation of the M2(22-46) V27A-spiro-adamantyl amine complex predicts with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2(22-46) V27A complex. Copyright © 2020 American Chemical Society.

The base excision repair (BER) pathway removes modified nucleobases that can be deleterious to an organism. BER is initiated by a glycosylase, which finds and removes these modified nucleobases. Most of the characterization of glycosylase activity has been conducted in the context of DNA oligomer substrates. However, DNA within eukaryotic organisms exists in a packaged environment with the basic unit of organization being the nucleosome core particle (NCP). The NCP is a complex substrate for repair in which a variety of factors can influence glycosylase activity. In this Review, we focus on the geometric positioning of modified nucleobases in an NCP and the consequences on glycosylase activity and initiating BER.

DNA is comprised of chemically reactive nucleobases that exist under a constant barrage from damaging agents. Failure to repair chemical modifications to these nucleobases can result in mutations that can cause various diseases, including cancer. Fortunately, the base excision repair (BER) pathway can repair modified nucleobases and prevent these deleterious mutations. However, this pathway can be hindered through several mechanisms. For instance, mutations to the enzymes in the BER pathway have been identified in cancers. Biochemical characterisation of these mutants has elucidated various mechanisms that inhibit their activity. Furthermore, the packaging of DNA into chromatin poses another obstacle to the ability of BER enzymes to function properly. Investigations of BER in the base unit of chromatin, the nucleosome core particle (NCP), have revealed that the NCP acts as a complex substrate for BER enzymes. The constituent proteins of the NCP, the histones, also have variants that can further impact the structure of the NCP and may modulate access of enzymes to the packaged DNA. These histone variants have also displayed significant clinical effects both in carcinogenesis and patient prognosis. This review focuses on the underlying molecular mechanisms that present obstacles to BER and the relationship of these obstacles to cancer. In addition, several chemotherapeutics induce DNA damage that can be repaired by the BER pathway and understanding obstacles to BER can inform how resistance and/or sensitivity to these therapies may occur. With the understanding of these molecular mechanisms, current chemotherapeutic treatment regiments may be improved, and future therapies developed.

The full data set of the NEMO-3 experiment has been used to measure the half-life of the two-neutrino double beta decay of $^{100}$Mo to the ground state of $^{100}$Ru, $T_{1/2} = \left[ 6.81 \pm 0.01\,\left(\mbox{stat}\right) ^{+0.38}_{-0.40}\,\left(\mbox{syst}\right) \right] \times10^{18}$ y. The two-electron energy sum, single electron energy spectra and distribution of the angle between the electrons are presented with an unprecedented statistics of $5\times10^5$ events and a signal-to-background ratio of ~80. Clear evidence for the Single State Dominance model is found for this nuclear transition. Limits on Majoron emitting neutrinoless double beta decay modes with spectral indices of n=2,3,7, as well as constraints on Lorentz invariance violation and on the bosonic neutrino contribution to the two-neutrino double beta decay mode are obtained., 11 pages, 9 figures

Using data from the NEMO-3 experiment, we have measured the two-neutrino double beta decay ( $2\nu \beta \beta $ ) half-life of$^{82}$ Se as $T_{\smash {1/2}}^{2\nu } \!=\! \left[ 9.39 \pm 0.17\left( \text{ stat }\right) \pm 0.58\left( \text{ syst }\right) \right] \times 10^{19}$ y under the single-state dominance hypothesis for this nuclear transition. The corresponding nuclear matrix element is $\left| M^{2\nu }\right| = 0.0498 \pm 0.0016$ . In addition, a search for neutrinoless double beta decay ( $0\nu \beta \beta $ ) using 0.93 kg of$^{82}$ Se observed for a total of 5.25 y has been conducted and no evidence for a signal has been found. The resulting half-life limit of $T_{1/2}^{0\nu } > 2.5 \times 10^{23} \,\text{ y } \,(90\%\,\text{ C.L. })$ for the light neutrino exchange mechanism leads to a constraint on the effective Majorana neutrino mass of $\langle m_{\nu } \rangle < \left( 1.2{-}3.0\right) \,\text{ eV }$ , where the range reflects $0\nu \beta \beta $ nuclear matrix element values from different calculations. Furthermore, constraints on lepton number violating parameters for other $0\nu \beta \beta $ mechanisms, such as right-handed currents, majoron emission and R-parity violating supersymmetry modes have been set.

We have designed and tested software for the acquisition and analysis of high-resolution gamma-ray spectra during on-site inspections under the Comprehensive Nuclear-Test-Ban Treaty (CTBT). The On-Site Inspection RadioIsotopic Spectroscopy—OSIRIS—software filters the spectral data to display only radioisotopic information relevant to CTBT on-site inspections, e.g., 131 I. A set of over 100 fission-product spectra was employed for OSIRIS testing. These spectra were measured where possible, or generated by modeling. The test spectral compositions include non-nuclear-explosion scenarios, e.g., a severe nuclear reactor accident, and nuclear-explosion scenarios such as a vented underground nuclear test. Comparing its computer-based analyses to expert visual analyses of the test spectra, O SIRIS correctly identifies CTBT-relevant fission product isotopes at the 95% level or better.

The Portable Isotopic Neutron Spectroscopy (PINS) is a commercialized system developed by Idaho National Laboratory to examine chemical compounds in munitions and containers non-destructively, utilizing the Prompt Gamma Neutron Activation Analysis (PGNAA) technique. The PINS system takes advantage of a germanium detector’s high energy resolution, and gamma-ray peak analysis provides input to its chemical identification logic using a probabilistic decision tree. Multivariate analysis (MVA) techniques were contemplated with the expectation that they could supplement the current PINS algorithm. Principal Component Analysis (PCA) was selected to project gamma-ray spectra into the principal component domain. A PCA-based chemical identification algorithm was tested, and the results are presented in this study.

We report the results of a first experimental search for lepton number violation by four units in the neutrinoless quadruple-β decay of ^{150}Nd using a total exposure of 0.19 kg yr recorded with the NEMO-3 detector at the Modane Underground Laboratory. We find no evidence of this decay and set lower limits on the half-life in the range T_{1/2}(1.1-3.2)×10^{21} yr at the 90% C.L., depending on the model used for the kinematic distributions of the emitted electrons.

In the 1930s, the Japanese army used forest management in its effort to transform the puppet Manchu Nation (Manchukuo) it had created in Northeast China into the cornerstone of a pan-Asian ...

The NEMO-3 experiment at the Modane Underground Laboratory has investigated the double-$\beta$ decay of $^{48}{\rm Ca}$. Using $5.25$ yr of data recorded with a $6.99\,{\rm g}$ sample of $^{48}{\rm Ca}$, approximately $150$ double-$\beta$ decay candidate events have been selected with a signal-to-background ratio greater than $3$. The half-life for the two-neutrino double-$\beta$ decay of $^{48}{\rm Ca}$ has been measured to be $T^{2\nu}_{1/2}\,=\,[6.4\, ^{+0.7}_{-0.6}{\rm (stat.)} \, ^{+1.2}_{-0.9}{\rm (syst.)}] \times 10^{19}\,{\rm yr}$. A search for neutrinoless double-$\beta$ decay of $^{48}{\rm Ca}$ yields a null result and a corresponding lower limit on the half-life is found to be $T^{0\nu}_{1/2} > 2.0 \times 10^{22}\,{\rm yr}$ at $90\%$ confidence level, translating into an upper limit on the effective Majorana neutrino mass of $ < 6.0 - 26$ ${\rm eV}$, with the range reflecting different nuclear matrix element calculations. Limits are also set on models involving Majoron emission and right-handed currents.

Epidemiologic data suggest an association between cadmium exposure and cardiovascular disease though the underlying mechanisms remain unclear. This study explored the hypothesis that cadmium exposure is associated with elevated C-reactive protein (CRP) and fibrinogen, two risk factors for developing cardiovascular disease.The current study investigated associations between urinary cadmium and the prevalence of elevated blood CRP (or = 2.2 mg/L) and fibrinogen (or = 10.35 micromol/L) using data from a sample of 6497 participants aged 40-79 in the Third National Health and Nutrition Examination Survey. Logistic regression model was used to investigate existing associations while adjusting for confounding variables.Both simple and covariate-adjusted models indicated that urinary cadmium was associated with elevated CRP and fibrinogen in a dose-dependent fashion (p(trend)0.05 for both). Adjusted odds ratios for increased CRP and fibrinogen comparing highest with lowest quartiles of urinary cadmium were 1.62 (95% confidence interval [CI], 1.24-2.12) and 2.12 (1.43-3.14), respectively.This analysis shows that cadmium exposure is associated with high prevalence of elevated CRP and fibrinogen. Additional study will be required to determine whether the increased risk derives from cadmium per se or from the environmental circumstances responsible for acquiring the contamination, e.g., cigarette smoking.

In 32 Florida lakes, Secchi depth (SD), light attenuation coefficient measured with a light meter, plant and sediment type, and slope were examined with respect to the maximum depth of plant colonization (MDC). The MDC was shown to be significantly related to light through measurements taken by SD (R2 = 0.46; p < 0.05) and a light meter (R2 = 0.41; p < 0.05). While both light measurements can be used to estimate MDC, SD accounted for more variance in MDC than light attenuation coefficients. Plant type, sediment type and slope did not account for more variance in MDC than light measurements for these Florida lakes. Additional unpublished data from 187 Florida Lakes (Florida LAKEWATCH, 279 lake-years of data) also showed a significant positive relationship between SD and MDC (R2 = 0.68; p < 0.05). The best fit MDC-SD regression line in meters was: log (MDC) = 0.66 log (SD) + 0.31. A maximum MDC line relating MDC to SD in meters was also calculated and was found to be equal to: log (max MDC) = 0.53 ...

The NEMO-3 detector, which had been operating in the Modane Underground Laboratory from 2003 to 2010, was designed to search for neutrinoless double $\beta$ ($0\nu\beta\beta$) decay. We report final results of a search for $0\nu\beta\beta$ decays with $6.914$ kg of $^{100}$Mo using the entire NEMO-3 data set with a detector live time of $4.96$ yr, which corresponds to an exposure of 34.3 kg$\cdot$yr. We perform a detailed study of the expected background in the $0\nu\beta\beta$ signal region and find no evidence of $0\nu\beta\beta$ decays in the data. The level of observed background in the $0\nu\beta\beta$ signal region $[2.8-3.2]$ MeV is $0.44 \pm 0.13$ counts/yr/kg, and no events are observed in the interval $[3.2-10]$ MeV. We therefore derive a lower limit on the half-life of $0\nu\beta\beta$ decays in $^{100}$Mo of $T_{1/2}(0\nu\beta\beta)> 1.1 \times 10^{24}$ yr at the $90\%$ Confidence Level, under the hypothesis of light Majorana neutrino exchange. Depending on the model used for calculating nuclear matrix elements, the limit for the effective Majorana neutrino mass lies in the range $\langle m_{\nu} \rangle < 0.33$--$0.62$ eV. We also report constraints on other lepton-number violating mechanisms for $0\nu\beta\beta$ decays.

The PINS Chemical Assay System, developed by Idaho National Laboratory (INL) is a field-portable Prompt Gamma-ray Neutron Activation Analysis (PGNAA) system. [1] For over 20 years, the U.S. military has used the PINS to nondestructively identify the fill chemical inside thousands of suspect chemical warfare munitions. First- and second-generation PINS systems use a 252Cf neutron source to induce capture reactions and inelastic-scattering reactions in the object under test. Currently, we are testing two third-generation PINS systems, using neutron generators in lieu of a 252Cf radioisotopic source, to simplify PINS shipping and storage logistics. We present here a comparison of the spectra collected during a series of blind tests performed with actual chemical warfare agents, using all three neutron source types. To our knowledge, this is the first test directly comparing the gamma-ray response of live-chemical warfare agents to neutrons from a 252Cf source, a DD neutron generator, and a DT neutron generator.

After analysis of 5797 h of data from the detector NEMO3, new limits on neutrinoless double beta decay of Mo-100 (T_{1/2} > 3.1 10^{23} y, 90% CL) and Se-82 (T_{1/2} > 1.4 10^{23} y, 90% CL) have been obtained. The corresponding limits on the effective majorana neutrino mass are: m 1.4 10^{22} y (90% CL) for Mo-100 and T_{1/2}> 1.2 10^{22} y (90%CL) for Se-82. Corresponding bounds on the Majoron-neutrino coupling constant are g < (0.5-0.9) 10^{-4} and < (0.7-1.6) 10^{-4}. Two-neutrino 2b-decay half-lives have been measured with a high accuracy, T_{1/2} Mo-100 = [7.68 +- 0.02(stat) +- 0.54(syst) ] 10^{18} y and T_{1/2} Se-82 = [10.3 +- 0.3(stat) +- 0.7(syst) ] 10^{19} y.

The objective of this study was to determine the impact of increasing the rate of carcass growth of steers grazing autumn grass by either supplementing with concentrates or by increasing grass supply on fatty acid composition of triacylglycerols within the M. longissmus thoracis . One hundred and ten steers (567 kg) were assigned to ten dietary treatments in a 3 (daily grass allowances: 6, 12 and 18 kg dry matter (DM)/animal) by 3 (daily concentrate allowances: 0, 2.5 and 5 kg/animal) factorial experiment with a control group offered concentrates ad libitum. Animals were slaughtered after 100 days and lipid extracted from samples of the M. longissmus thoracis for fatty acid analysis. There was an interaction ( P P P P P P P

The first comprehensive investigation of pike Esox lucius trophic ecology in a region (Ireland) where they have long been thought to be a non-native species is presented. Diet was investigated across habitat types (lake, river and canal) through the combined methods of stable-isotope and stomach content analyses. Variations in niche size, specialization and the timing of the ontogenetic dietary switch were examined, revealing pronounced opportunism and feeding plasticity in E. lucius, along with a high occurrence of invertivory (up to 60 cm fork length, LF ) and a concomitant delayed switch to piscivory. Furthermore, E. lucius were found to primarily prey upon the highly available non-native roach Rutilus rutilus, which may alleviate predation pressure on brown trout Salmo trutta, highlighting the complexity of dynamic systems and the essential role of research in informing effective management.

Most currently, viable double beta decay experiments require highly enriched isotopic sources. These sources must be extraordinarily free of radioactive contamination. The double beta decay experiment NEMO 3 will study 100Mo, for which physical and chemical purification techniques have been investigated. The success of the chemical purification process is discussed in the context of ultra-low background, high-purity germanium spectrometer measurements.

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) 3-phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375–13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P 5 ) 3-phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P 5 ( K m =7.1 μM) and PtdIns(3,4,5)P 3 ( K m =26 μM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P 5 levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P 5 to inositol 1,4,5,6-tetrakisphosphate is a cell signaling event.

Selective expression of enzymes that adjust the intensity of turnover of diphosphoinositolpolyphosphates may regulate vesicle trafficking and DNA repair. For example, the type 2 human diphosphoinositolpolyphosphate phosphohydrolases (hDIPP2alpha and 2beta) are distinguished by a solitary amino-acid residue; the type 2beta isoform contains Gln86 whereas the type 2alpha isoform does not, yet the latter has 2-5 fold more catalytic activity than its beta counterpart (J. Biol.Chem. (2000) 12730). We discovered that both alpha and beta-type mRNAs were co-expressed in clonal cell-lines. We sought a genetic explanation for this microheterogeneity. Two BACs containing distinct, but intronless, hDIPP2beta genes were cloned. Only one of these genes could potentially give rise to our previously characterized hDIPP2beta mRNA; the other gene has several sequence differences and, in any case, is likely a processed pseudogene. These BACS were mapped to 1q12-q21 and 1p12-p13 by FISH. No analogous intronless hDIPP2alpha gene was detected by analysis of 21 individual genomic DNAs. However, sequence analysis of a third hDIPP2 gene (at 12q21) places the Gln86 CAG codon within an AGCAG pentamer, offering adjacent, alternate intronic 3'-boundaries. Thus, 'intron boundary skidding' by spliceosomes provides a mechanism for yielding both hDIPP2alpha and hDIPP2beta mRNAs. Our studies expand the repertoire of molecular mechanisms regulating diphosphoinositolpolyphosphate metabolism and function.

The aim of this experiment was to quantify the relationship between autumn grass supply and concentrate supplementation level on grass intake and animal performance. One hundred and ten continental steers (567 kg) were assigned to 10 treatments. The experimental design was a three grass allowances (6, 12 and 18 kg dry matter (DM) per head daily) by three concentrate levels: (0, 2·5 and 5 kg per head daily) factorial with a positive control group offered concentrates ad libitum and no grass. Grass allowance was offered daily and concentrates were given individually. The experiment began on 22 August and all animals were slaughtered after a mean experimental period of 95 days. Grass intake was calculated using the n-alkane technique and diet digestibility using ytterbium acetate as an indigestible marker. There was an interaction (P < 0·05) between grass allowance and concentrate level for grass intake. At the low grass allowance there was no effect of offering animals supplementary concentrates on grass intake, at the medium and high grass allowances, supplementary concentrates reduced grass intake by 0·43 and 0·81 kg DM respectively per kg DM concentrate offered. Increasing grass allowance increased (P < 0·001) complete diet organic matter (OM) digestibility at all concentrate levels and supplementary concentrates increased (P < 0·001) complete diet OM digestibility only at the low grass allowance. Both offering animals supplementary concentrates (P < 0·001) and increasing daily grass allowance (P < 0·001) increased their carcass growth rate. Relative to the animals offered the low grass allowance and no concentrate, supplementing with concentrate increased carcass growth by 116 g/kg concentrate DM eaten whereas increasing the grass allowance, increased carcass growth by 38 g/kg DM grass eaten. As a strategy for increasing the performance of cattle grazing autumn grass, offering supplementary concentrates offers more scope than altering grass allowance.

The objective of this study was to determine the effects on animal performance and rumen digestion characteristics of supplementing cattle grazing autumn grass offered at two levels of allowance, with concentrates formulated from different carbohydrate sources. Seventy-two steers (494 (s.e. 38·4) kg) were assigned to one of six treatments which were arranged in a two grass allowances (5·5 or 11 kg grass dry matter (DM) per head per day) by three concentrate types (starch, starch + fibre or fibre-based diets) factorial design. The experiment commenced on 22 August and had a mean duration of 84 days. Concentrates were formulated to have similar metabolizable energy and crude protein and animals were offered 5 kg per head daily. Grass allowances were offered daily and concentrates were offered individually. Six rumen fistulated steers were used concurrently in a Latin square design to determine the effect of the diets on rumen digestion characteristics. Grass and concentrates degradabilities were measured using the nylon bag technique. The concentrate DM was degraded at 0·189, 0·102 and 0·094 per h for the starch, starch + fibre and fibre-based diets, respectively. Concentrate carbohydrate source and grass allowance did not affect (P> 0·05) degradation rate of grass DM or nitrogen in the rumen, rumen fluid pH, rumen fluid concentration of volatile fatty acids, total lactic acid or ammonia concentration or plasma concentration of glucose, urea or beta-hydroxybutyrate. Animals offered the low grass allowance had larger (P< 0·05) diurnal variation in the rumen fluid acetate: propionate ratio than those offered the high grass allowance. Increasing grass allowance increased (P< 0·01) carcass gain whereas concentrate type had no effect. There was no interaction between concentrate carbohydrate source and grass allowance on any of the production variables measured. It is concluded that the rumen environment in cattle grazing autumn grass buffered the effects of concentrate carbohydrate degradability such that similar productivity was achieved with each concentrate.

To be sensitive to an effective Majorana neutrino mass, 〈 m ν 〉, on the order of 0.1 eV, the NEMO 3 double beta (ββ(0ν)) decay experiment requires precise knowledge and control of the backgrounds. The effect of neutrons and γ-rays from the Frejus underground laboratory (LSM, Laboratoire Souterrain de Modane) has been studied during 10 700 h of data collection. The data were taken with a NEMO 2 prototype detector using different shield configurations. Monte Carlo calculations with GEANT/MICAP code and a new library of γ-rays from neutron captures are presented. The implied consequences for the NEMO 3 detector which is under construction are discussed. The neutrinoless double beta decay background induced by neutrons and γ-rays within the LSM will be suppressed to the very suitable level of 0.1 event in a live time of five years given the appropriate shield and magnetic field.

The NEMO-2 tracking detector located in the Frejus Underground Laboratory was designed as a prototype for the NEMO-3 detector and to study different modes of double beta decay. Measurements with 100 Mo, 116 Cd, 82 Se and 96 Zr were carried out. Presented here are the experimental half-life limits on double beta decays for new Majoron emission modes and limits on effective neutrino–Majoron coupling constants.

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6kDelta cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo. This is the first time a PP-InsP(4) synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6). Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.

The turnover of the “high energy” diphosphoinositol polyphosphates by Ca2+- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype humandiphosphoinositol-polyphosphatephosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2α and hDIPP2β, distinguished from each other solely by hDIPP2β possessing one additional amino acid (Gln86). Candidate DIPP2α and DIPP2β homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2α > hDIPP2β. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX 2GX 6G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3′-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.

The ARGRIII gene of Saccharomyces cerevisiae encodes a transcriptional regulator that also has inositol polyphosphate multikinase (ipmk) activity [Saiardi et al. (1999) Curr. Biol. 9, 1323-1326]. To investigate how inositol phosphates regulate gene expression, we disrupted the ARGRIII gene. This mutation impaired nuclear mRNA export, slowed cell growth, increased cellular [InsP(3)] 170-fold and decreased [InsP(6)] 100-fold, indicating reduced phosphorylation of InsP(3) to InsP(6). Levels of diphosphoinositol polyphosphates were decreased much less dramatically than was InsP(6). Low levels of InsP(6), and considerable quantities of Ins(1,3,4,5)P(4), were synthesized by an ipmk-independent route. Transcriptional control by ipmk reflects that it is a pivotal regulator of nuclear mRNA export via inositol phosphate metabolism.

Background: Transient hyperthyroidism of hyperemesis gravidarum (THHG) is a self-limiting hyperthyroidism occurring in the context of hyperemesis gravidarum. Methods: A literature search of MEDLINE was undertaken, and a case report of a woman with THHG in pregnancy is described. Results and Conclusions: Because thyroid function tests cannot distinguish Graves disease from THHG, the diagnosis of THHG rests largely on the concurrent development of hyperemesis and hyperthyroidism and the absence of signs and symptoms of hyperthyroidism before and during pregnancy. THHG might be responsible for 40% to 70% of thyroid function abnormalities in pregnancy. Both the thyroid function abnormalities and hyperemesis are related to elevated levels of human chorionic gonadotropin. THHG resolves by 18 weeks of pregnancy without sequelae. No treatment is required. Diagnosis of THHG by the primary care provider can prevent unnecessary treatment or referral for specialty care.

An experiment was carried out to compare the digestibility of five concentrate ingredients (barley, beet pulp, citrus pulp, maize gluten feed and grain screenings) in cattle or sheep, fed at ≈1.2 maintenance. Four wether sheep and four steers were fed each concentrate, which comprised ≈800 g/kg of the dietary dry matter (DM). The remaining 200 g/kg was a hay/soyabean meal mixture whose digestibility was determined simultaneously in another four sheep and cattle. Following diet introduction and adaptation, faeces were collected for a 10-day period. Across diets, there was no difference (p >0.05) between sheep and cattle in digestibility of organic matter (OM, 0.784 and 0.78, respectively, standard error of the difference (s.e.d.), 0.0049), crude protein (0.586 and 0.577, respectively, s.e.d. 0.0192), or neutral detergent fibre (0.632 and 0.628, respectively, s.e.d. 0.0116). However, cattle digested the OM (0.749 vs. 0.724, p 0.05) between sheep and cattle with any of the other four feeds. These results do not support previous conclusions that sheep digest concentrates better than cattle, but they do suggest that there are differences within specific feeds.

We have derived the full-length sequences of the human and rat forms of the multiple inositol polyphosphate phosphatase (MIPP); their structural and functional comparison with a chick histidine acid phosphatase (HiPER1) has revealed new information: (1) MIPP is approximately 50% identical to HiPER1, but the ER-targeting domains are divergent; (2) MIPP appears to share the catalytic requirement of histidine acid phosphatases, namely, a C-terminal His residue remote from the RHGxRxP catalytic motif; (3) rat MIPP mRNA is up-regulated during chondrocyte hypertrophy. The latter observation provides a context for proposing that MIPP may aid bone mineralization and salvage the inositol moiety prior to apoptosis.

Summary The distribution of acetyl-CoA carboxylase between the epidermal and mesophyll layers of leek leaves was determined. The specific activity of acetyl-CoA carboxylase was approximately three-fold higher in extracts of the epidermis than in those from the mesophyll layer. Four prominent biotinylated polypeptides of approximate molecular masses 240, 85, 37, and 35 ku (kDa) were detected in leaf extracts. The biotincontaining subunit of the homomeric acetyl-CoA carboxylase, the 240-ku biotinylated polypeptide, accumulates exclusively in the epidermis of leek leaves. Three of the subunits of the heteromeric acetyl-CoA carboxylase (the biotin carboxyl carrier, the biotin carboxylase, and s-subunit of the carboxyltransferase) were equally abundant in leek mesophyll and epidermal tissues. These data indicate that leek leaves contain two structurally distinct isozymes of acetyl-CoA carboxylase, one of which, the homomeric form, is restricted to the epidermis.

Stable isotope and gut content analyses, in conjunction with backcalculated length-at-age estimates of growth, were employed to examine the relationship between trophic ecology and growth rate of a successful invader, Rutilus rutilus, in eight lakes in Ireland. The data revealed that R. rutilus was a trophic generalist in Irish lakes. It utilized a greater proportion of pelagic resources in mesotrophic lakes than in eutrophic lakes, potentially due to a greater density of benthic macroinvertebrates in eutrophic systems. The species was characterized by a large dietary and isotopic niche width and high temporal and spatial variations in diet. Growth rates were typical of those found in the native range of the species and were unrelated to either lake productivity or fish's diet. A generalist trophic ecology confers significant advantages on an invasive species, allowing it to exploit a variety of novel resources and fluctuations in prey availability.

Initial infection with an attenuated form of human immunodeficiency virus type 1 (HIV-1) may give rise to some of the rare asymptomatic infections that have been observed. Recently, data have been presented suggesting that a persistent mutation in the essential activation domain of the HIV-1 Rev regulatory protein might have contributed to the maintenance of the asymptomatic state in one individual. Here, we have used a range of assays for in vivo Rev function to examine whether natural sequence variation in the normally highly conserved Rev activation domain can indeed affect Rev function. Analysis of five distinct natural sequence variants of the Rev domain demonstrated that each produced a two- to fourfold drop in Rev function when compared to the consensus activation domain sequence. A sixth sequence, reported for the MN isolate of HIV-1, proved entirely inactive. However, resequencing of this region of the MN genome revealed that this isolate actually encodes a consensus Rev activation domain. Overall, these data reveal that even natural sequence variation in the essential Rev activation domain can result in significantly reduced Rev function and suggest that isolates containing such sequence variation are likely to replicate less effectively.