The C-terminus of the phage λ Orf recombinase is involved in DNA binding

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.     

Estimating lifetime risk of diabetes in the Chinese population

In this Perspective, Fiona Bragg and Zhengming Chen discuss the burden of diabetes in the Chinese Population.

The worldwide epidemic of diabetes continues to grow [1]. In China, the rise in prevalence has been notably rapid; about 12% of the adult population has diabetes [2], accounting for almost one quarter of cases worldwide [1] and representing a 10-fold increase over the last 3 to 4 decades. It is appropriate, therefore, that diabetes—both prevention and management—is a major focus of current health policy initiatives in China [3,4], and their success depends on reliable quantification of the burden of diabetes. Commonly used measures such as prevalence and incidence fail to capture excess mortality risks or differences in life expectancy in diabetes [5]. Moreover, they may be less easily interpreted by policy makers and affected individuals. Estimates of lifetime risks and life years spent living with diabetes in an accompanying study by Luk and colleagues provide a valuable new perspective on the burden of diabetes in the Chinese population [6].The study used Hong Kong territory-wide electronic health records data for 2.6 million adults. Using a Markov chain model and Monte-Carlo simulations, Luk and colleagues estimated age- and sex-specific lifetime risks of diabetes (incorporating both clinically diagnosed and undiagnosed diabetes) and remaining life years spent with diabetes. Their findings showed a lifetime risk of 65.9% and 12.7 years of life living with diabetes for an average 20-year old with normoglycaemia. For an average 20-year old with prediabetes the corresponding estimates were 88.0% and 32.5 years, respectively. In other words, 6 out of 10 20-year olds with normoglycaemia and 9 out of 10 with prediabetes would be expected to develop diabetes in their lifetime. The estimated lifetime risks declined with increasing age and were higher among women than men at all ages, likely reflecting women’s higher life expectancy.These estimated lifetime risks are striking and concerning. Moreover, they are notably higher than western population estimates [7–10], including those considering both diagnosed and undiagnosed diabetes [9,10]. An Australian study estimated that 38% of 25-year olds would develop diabetes in their lifetime [10]. Another study in the Netherlands reported 31.3% and 74.0% probabilities of developing diabetes in the remaining lifetime for individuals aged 45 years without diabetes and with prediabetes, respectively [9]. Diabetes incidence and overall mortality influence population lifetime risks. Differences in the glycaemic indicators used to identify undiagnosed diabetes may have contributed to differences between studies in diabetes incidence. In the study by Luk and colleagues, a combination of fasting plasma glucose (FPG), HbA1c levels and oral glucose tolerance testing (OGTT) was used, while in the Australian [10] and the Netherlands [9] studies, they used FPG/OGTT and mainly FPG, respectively. However, it is unlikely these differences would fully account for the large disparities seen in lifetime risk. Similarly, differences between life expectancy in Hong Kong (84.8 years), Australia (83.4 years), and the Netherlands (82.2 years) are too small to explain the differences. Interestingly, the high lifetime risks observed in Hong Kong were more comparable to those in the Indian population, estimated at 55.5% and 64.6%, respectively, among 20-year-old men and women [11]. The typical type 2 diabetes (T2D) phenotype in these Asian populations may partly explain their higher estimated lifetime risks. More specifically, T2D in both Chinese and Indian populations is characterised by onset among younger and less adipose individuals than typically observed in western populations, exacerbated by rapid urbanisation and associated unhealthy lifestyles [12].However, aspects of Luk and colleagues’ study design may have overestimated lifetime diabetes risks. Chief among these is the data source used and associated selection bias. The Hong Kong Diabetes Surveillance Database includes only individuals who have ever had a plasma glucose or HbA1c measurement undertaken in a local health authority facility. Since measurement of glycaemic indicators is more likely among individuals at greater current or future risk of dysglycaemic states, this will have inflated estimates of lifetime risk and life years spent with diabetes. Although replication was undertaken by the study authors to address this bias in the smaller China Health and Retirement Longitudinal Survey (CHARLS) cohort, it does not fully allay these concerns, with modestly lower estimated lifetime diabetes risks in the CHARLS cohort, even after accounting for its higher mortality. A further limitation is their consideration of transition to dysglycaemic states as irreversible. Although data on long-term transition between glycaemic states are lacking, reversion from prediabetes (and less commonly diabetes) to normoglycaemia is well recognised, e.g., through lifestyle interventions [13].Large-scale population-based cohort studies could valuably address many of the limitations described [14]. Furthermore, lifetime risks are, by definition, population-based and represent the risk of an average person in the population, limiting their value for communicating long-term disease risks to specific individuals. However, the extensive phenotyping (e.g., adiposity) characteristic of many large contemporary cohorts [14] would facilitate incorporation of risk factors into lifetime risk estimates, enhancing their relevance to individuals. Previous studies have found greater lifetime risks of diabetes associated with adiposity [9,11], and this approach could be extended to incorporate other established, as well as more novel (e.g., genetic), risk factors. This is arguably of particular relevance to later-onset chronic conditions, such as T2D, in which changes in risk factors during middle age can influence lifetime risks. A valuable extension of Luk and colleagues’ study will be estimation of risk factor specific lifetime diabetes risks for the Chinese population.Importantly, the limitations described do not detract from the enormity and importance of the challenge diabetes poses for China, including Hong Kong, and the estimates presented by Luk and colleagues provide valuable impetus for action. The disease burden insights can inform treatment programmes and enhance understanding of current and future impacts of diabetes and associated complications on the healthcare system. Moreover, T2D is preventable, and arguably, the greatest value of these estimated lifetime risks is in highlighting the need for, and informing the planning and provision of, diabetes primary prevention programmes. This includes identification of high-risk individuals, such as those with prediabetes, who are most likely to benefit from prevention interventions. However, the magnitude of the estimated lifetime diabetes risks, including among the large proportion of the population in a normoglycaemic state, additionally demonstrates the need for population-level prevention approaches, including environmental, structural, and fiscal strategies. Without such actions, the individual and societal consequences of diabetes for present and future generations in Hong Kong, as well as mainland China, will be immense.     

A bacterial ecto-triphosphate diphosphohydrolase similar to human CD39 is essential for intracellular multiplication of Legionella pneumophila

As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.     

Genetic variation in Antarctic populations of the moss Sarconeurum glaciale

Sixty-six isolates of the moss Sarconeurum glaciale were collected from sites in continental Antarctica at Ross Island, southern Victoria Land and the Vestfold Hills. Genetic variation within and among the populations was estimated using isozymes and random amplified polymorphic DNA (RAPD) technology. Isozyme results only reproducibly showed variation between the populations with one enzyme; RAPDs indicated significantly higher levels of genetic variability within and among the Vestfold Hills samples than in the Ross Sea region samples. A dendrogram produced from the RAPD bands suggested that the Ross Island and southern Victoria Land samples form one population, and those from the Vestfold Hills form a separate and more variable population, possibly resulting from separate colonisation events on the continent. Received: 15 March 1996 / Accepted: 1 May 1997     

High‐Performance Liquid Chromatographic Resolution of Neutral and Cationic Hetero[6]Helicenes

Cationic hetero[6]helicenes 1⁺ , 2⁺ and 3⁺ have been recently disclosed. Herein we report on their enantiomeric separation using high‐performance liquid chromatography. Separation of the antipodes can be achieved in preparative scale on neutral adducts with Chiralcel OD‐I or Chiralpak ID CSP. Selectivity factors of 1.90, 1.67, and 1.96 were obtained for 1-H , 2-H , and 3-H , respectively. Separation can also be performed on the carbenium ions on regular Chiralpak IA CSP using water‐containing eluents, thus allowing for enantiomeric purity determinations in aqueous environments. Resolution of neutral and cationic helicenes is also achieved on more recently developed LARIHC columns. The versatility of the cyclofructan phases allows for baseline separations for both cases and their loading capabilities are demonstrated. Finally, the configurational stability of 1⁺ , 2⁺ , and 3⁺ was measured. For each replacement of an oxygen atom by an amino group, the racemization barrier increases significantly (ΔG^? = 29.8, 36.3 and >37 kcal mol^‐1 for 1⁺ , 2⁺ , and 3⁺ respectively). Chirality 28:282–289, 2016. © 2016 Wiley Periodicals, Inc.     

AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ～40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine     

Development and preliminary evaluation of a 90?K Axiom? SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa

Background

A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.

Results

About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.

Conclusions

The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1310-1) contains supplementary material, which is available to authorized users.