Evaluating the cost-effectiveness of polygenic risk score-stratified screening for abdominal aortic aneurysm.

As the heritability of abdominal aortic aneurysm (AAA) is high and AAA partially shares genetic architecture with other cardiovascular diseases, genetic information could help inform AAA screening strategies. Exploiting pleiotropy and meta-analysing summary data from large studies, we construct a polygenic risk score (PRS) for AAA. Leveraging related traits improves PRS performance (R2) by 22.7%, relative to using AAA alone. Compared with the low PRS tertile, intermediate and high tertiles have hazard ratios for AAA of 2.13 (95%CI 1.61, 2.82) and 3.70 (95%CI 2.86, 4.80) respectively, adjusted for clinical risk factors. Using simulation modelling, we compare PRS- and smoking-stratified screening with inviting men at age 65 and not inviting women (current UK strategy). In a futuristic scenario where genomic information is available, our modelling suggests inviting male current smokers with high PRS earlier than 65 and screening female smokers with high/intermediate PRS at 65 and 70 respectively, may improve cost-effectiveness.

Deformable nature of various damaged DNA duplexes estimated by an electrochemical analysis on electrodes.

We report bending flexibility of damaged duplexes possessing an apurinic/apyrimidinic (AP) site analogue, a cyclobutane pyrimidine dimer (CPD), and a pyrimidine(6-4)pyrimidone photoproduct (6-4PP). Based on the electrochemical evaluation on electrodes, the duplex flexibilities of the lesions increased in the following order: CPD < AP < 6-4PP. We discussed the possibility that the emerging local flexibility might be a good sign for UV-damaged DNA-binding proteins on duplexes.

A genome-wide association study suggests that a locus within the ataxin 2 binding protein 1 gene is associated with hand osteoarthritis: the Treat-OA consortium.

To identify the susceptibility gene in hand osteoarthritis (OA) the authors used a two-stage approach genome-wide association study using two discovery samples (the TwinsUK cohort and the Rotterdam discovery subset; a total of 1804 subjects) and four replication samples (the Chingford Study, the Chuvasha Skeletal Aging Study, the Rotterdam replication subset and the Genetics, Arthrosis, and Progression (GARP) Study; a total of 3266 people). Five single-nucleotide polymorphisms (SNPs) had a likelihood of association with hand OA in the discovery stage and one of them (rs716508), was successfully confirmed in the replication stage (meta-analysis p = 1.81x10(-5)). The C allele conferred a reduced risk of 33% to 41% using a case-control definition. The SNP is located in intron 1 of the A2BP1 gene. This study also found that the same allele of the SNP significantly reduced bone density at both the hip and spine (p<0.01), suggesting the potential mechanism of the gene in hand OA might be via effects on subchondral bone. The authors' findings provide a potential new insight into genetic mechanisms in the development of hand OA.

Bone mineral density, osteoporosis, and osteoporotic fractures: a genome-wide association study.

BACKGROUND: Osteoporosis is diagnosed by the measurement of bone mineral density, which is a highly heritable and multifactorial trait. We aimed to identify genetic loci that are associated with bone mineral density. METHODS: In this genome-wide association study, we identified the most promising of 314 075 single nucleotide polymorphisms (SNPs) in 2094 women in a UK study. We then tested these SNPs for replication in 6463 people from three other cohorts in western Europe. We also investigated allelic expression in lymphoblast cell lines. We tested the association between the replicated SNPs and osteoporotic fractures with data from two studies. FINDINGS: We identified genome-wide evidence for an association between bone mineral density and two SNPs (p<5x10(-8)). The SNPs were rs4355801, on chromosome 8, near to the TNFRSF11B (osteoprotegerin) gene, and rs3736228, on chromosome 11 in the LRP5 (lipoprotein-receptor-related protein) gene. A non-synonymous SNP in the LRP5 gene was associated with decreased bone mineral density (rs3736228, p=6.3x10(-12) for lumbar spine and p=1.9x10(-4) for femoral neck) and an increased risk of both osteoporotic fractures (odds ratio [OR] 1.3, 95% CI 1.09-1.52, p=0.002) and osteoporosis (OR 1.3, 1.08-1.63, p=0.008). Three SNPs near the TNFRSF11B gene were associated with decreased bone mineral density (top SNP, rs4355801: p=7.6x10(-10) for lumbar spine and p=3.3x10(-8) for femoral neck) and increased risk of osteoporosis (OR 1.2, 95% CI 1.01-1.42, p=0.038). For carriers of the risk allele at rs4355801, expression of TNFRSF11B in lymphoblast cell lines was halved (p=3.0x10(-6)). 1883 (22%) of 8557 people were at least heterozygous for these risk alleles, and these alleles had a cumulative association with bone mineral density (trend p=2.3x10(-17)). The presence of both risk alleles increased the risk of osteoporotic fractures (OR 1.3, 1.08-1.63, p=0.006) and this effect was independent of bone mineral density. INTERPRETATION: Two gene variants of key biological proteins increase the risk of osteoporosis and osteoporotic fracture. The combined effect of these risk alleles on fractures is similar to that of most well-replicated environmental risk factors, and they are present in more than one in five white people, suggesting a potential role in screening.

Retrons, msDNA, and the bacterial genome.

Retrons are distinct DNA sequences that code for a reverse transcriptase (RT) similar to the RTs produced by retroviruses and other types of retroelements. Retron DNAs are commonly associated with prophage DNA and are found in the genomes of a wide variety of different bacteria. The retron RT is used to synthesize a strange satellite DNA known as msDNA. msDNA is actually a complex of DNA, RNA, and probably protein. It is composed of a small, single-stranded DNA, linked to a small, single-stranded RNA molecule. The 5' end of the DNA molecule is joined to an internal guanosine residue of the RNA molecule by a unique 2'-5' phosphodiester bond. msDNA is produced in many hundreds of copies per cell, but its function remains unknown. Although retrons are absent from the genome of most members of a population of related bacteria, retrons may not be entirely benign DNAs. Evidence is beginning to suggest that retron elements may produce small but potentially significant effects on the host cell. This includes the generation of repeated copies of the msDNA sequence in the genome, and increasing the frequency of spontaneous mutations. Because these events involve the retron RT, this may represent a source of reverse transcription in the bacterial cell. Thus, the process of reverse transcription, a force that has profoundly affected the content and structure of most eukaryotic genomes, may likewise be responsible for changes in some prokaryotic genomes.

Relationship of the fistulas to the rectum and genitourinary tract in mouse fetuses with high anorectal malformations induced by all-trans retinoic acid.

Since high anorectal malformations with fistulae in human embryos and fetuses of successive developmental stages have not been reported, the embryologic relationship between the rectal fistula (RF) and the genitourinary tract (GUT) in high anorectal agenesis (ARA) remains to be elucidated. This study investigates the developmental relationship between the RF and the GUT in male and female fetuses with high ARA using our established model for high ARA with fistula in mice. Pregnant mice received all-trans retinoic acid suspended in corn oil (5 mg/ml) 100 mg/kg i.p. on day 9 of pregnancy. All fetuses were removed from the uterus on a single day from days 12 to 18 of pregnancy. The caudal regions were analyzed histologically with hematoxylin and eosin staining. All fetuses examined had high ARA with fistula. On day 12 of pregnancy, an anomalous communication was seen between the urogenital sinus (UGS) and the rectum. In the affected female fetuses, on day 14 of pregnancy the paramesonephric (müllerian) ducts and müllerian tubercle were located above the rectocloacal fistula (RCF), and on day 18 of pregnancy the uterovaginal canal was located between the cloaca and the RCF. In the male fetuses, on day 14 of pregnancy the junction between the mesonephric (wolffian) duct and the UGS was located away from the junction between the rectum and the UGS. On day 18 of pregnancy the ejaculatory duct was located between the urinary bladder and the rectourethral fistula. The results of our experiment clearly show the embryologic relationship between the RF and the GUT with high ARA. The anomalous communication between the UGS and the rectum may interfere with normal caudal migration along the dorsal wall of the UGS at the junction between the UGS and the mesonephric or paramesonephric duct.

Neurogenesis of heterotopic gray matter in the brain of the microcephalic mouse.

Neurogenesis of heterotopic gray matter in the brain of the microcephalic mouse prenatally exposed to X-rays at embryonic day 13 (E13) was studied immunohistochemically. Bromodeoxyuridine (BrdU) as a marker to label the migrating position of neuroblasts generated at various embryonic stages showed that no "inside-out" pattern of neuronal migration occurred in the heterotopic cell mass similar to that seen in the laminated cortex. Further results in which midkind (MK) immunoreactive radial glial fibers did not appear in the heterotopic cell mass demonstrated that heterotopia formed in the absence of radial glia system. Different types of cells (pyramidal and non-pyramidal neurons) in the heterotopic cell mass were identified with immunoreactivity for anti-parvalbumin and anti-calbindin D-28K antibodies in addition to current histological methods. Two major types of neurons were mixed together with random distribution in the heterotopic cell mass. This finding indicates that irradiation might have no selective effects on the precursors of pyramidal and non-pyramidal neurons. Moreover, anti-glial fibrillary acidic protein (GFAP) immunostaining showed that numerous astrocytes were present in the heterotopic cell mass. The fact that astrocytes appeared in the heterotopia without the transition from classic radial glial cells to astrocytes suggests that astrocytes might be generated directly from a separate astroglial precursor.

Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones.

Several proteases require propeptides for the correct folding of their own protease domain. We have recently found that the propeptide from a thermostable subtilisin homolog aqualysin I can refold subtilisin BPN' when added in trans. Here, we constructed chimeric genes with subtilisin E and aqualysin I to attempt the in cis folding of subtilisin E by means of the propeptide of aqualysin I. Our results indicate that the propeptide of aqualysin I can to some extent chaperone the intramolecular folding of the denatured subtilisin E. These results suggest that propeptides in the subtilisin family, despite their sequence diversity, have similar functions. Further, some enzymatic properties of some chimeras in which the subtilisin mature domain is partly swapped with that of aqualysin I were shown to be more similar to those of aqualysin I.

Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin.

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.

Predicting outcomes of patients in Japan after first acute stroke using a simple model.

OBJECTIVE: Prediction of patient outcome can be useful as an aid to clinical decision making. Many studies, including my own, have constructed predictive multivariate models for outcome following stroke rehabilitation therapy, but these have often required several minutes work with a pocket calculator. The aim is to develop a simple, easy-to-use model that has strong predictive power. METHODS: Four hundred sixty-four consecutive patients with first stroke who were admitted to a rehabilitation hospital during a period of 19 mo were enrolled in the study. Sex, age, the stroke type, Functional Independence Measure total score on admission (X), onset to admission interval (number of days from stroke onset to rehabilitation admission), and length of rehabilitation hospital stay (number of days from hospital admission to discharge) were the independent variables. Functional Independence Measure total score at discharge (Y) was the dependent variable. RESULTS: Stepwise multiple regression analysis resulted in the model containing age (P < 0.0001), X (P < 0.0001), and onset to admission interval (P < 0.0001). The equation was: Y = 68.6 - 0.32 (age) + 0.80X - 0.13 (onset to admission interval), a multiple correlation coefficient (R) = 0.82, and a multiple correlation coefficient squared (R2) = 0.68. Simple regression analysis revealed the relation between Xand Y: Y = 0.85X + 37.36, and R = 0.80 R2 = 0.64. In fact, plots of X vs. Ywere nonlinear, but seemed to be able to be linearized by some form of equation. It was found that there is a linear relation between logX and Y. The equation is Y = 106.88x - 95.35, where x = logX, R = 0.84, and R2 = 0.70. The correlation is improved by a regression analysis of a natural logarithmic transformation of X (R = 0.84 vs. R = 0.82). CONCLUSION: The results in this study confirm that the simple regression model using a logarithmic transformation of X (R = 0.84) has predictive power over the simple regression model (R = 0.80). This model is well validated and clinically useful.

Influence of initial status on functional gain for Japanese patients with first cerebral hemorrhage.

It is important to identify in advance patients who will achieve the greatest functional gains from rehabilitation therapy, as specialist rehabilitation resources are still scarce in Japan. The purpose of this study was to determine whether functional score at admission influences the functional change (functional score at discharge minus functional score at admission) after inpatient rehabilitation for first cerebral hemorrhage. One hundred and ninety-three patients with cerebral hemorrhage were enrolled in this study. They were assessed using the Functional Independence Measure (FIM) at admission and discharge and underwent inpatient rehabilitation treatment. Patients were stratified into 3 groups according to their FIM total scores on admission as follows: (1) < or = 36 (severely affected patient group); (2) 37-72 (moderately affected patient group); and (3) >73 (mildly affected patient group). Scheffe's multiple comparison test showed that patients in group 1 were significantly older (mean +/- SD = 63 +/- 10 years) than those in groups 2 (56 +/- 10 years) or 3 (53 +/- 12 years). Patients in group 2 showed significantly greater FIM gain (37 +/- 17) compared with patients in groups 3 (23 +/- 12) or 1 (27 +/- 23). The results suggest that moderately affected patients at admission will show significantly higher functional gain compared with severely or mildly affected patients. Mildly affected patients at admission had a significantly shorter length of hospital stay for rehabilitation than the other groups. There was no significant difference in onset to admission interval between the 3 groups. The functional levels of affected patients on admission, as stratified by the FIM scale, roughly predict the degree of functional gain following rehabilitation in patients with first cerebral hemorrhage. Moderately affected patients will benefit from intensive rehabilitation. This study may be useful in determining how best to prioritize rehabilitation therapy.

The msDNAs of bacteria.

msDNAs are small, structurally unique satellite DNAs found in a number of Gram-negative bacteria. Composed of hundreds of copies of single-stranded DNA--hence the name multicopy single-stranded DNA--msDNA is actually a complex of DNA, RNA, and probably protein. These peculiar molecules are synthesized by a reverse transcription mechanism catalyzed by a reverse transcriptase (RT) that is evolutionarily related to the polymerase found in the HIV virus. The genes, including the RT gene, responsible for the synthesis of msDNA are encoded in a retron, a genetic element that is carried on the bacterial chromosome. The retron is, in fact, the first such retroelement to be discovered in prokaryotic cells. This report is a comprehensive review of the many interesting questions raised by this unique DNA and the fascinating answers it has revealed. We have learned a great deal about the structure of msDNA: how it is synthesized, the structure and functions of the RT protein required to make it, its effects on the host cell, the retron element that encodes it, its possible origins and evolution, and even its potential usefulness as a practical genetic tool. Despite the impressive gains in our understanding of the msDNAs, however, the simple, fundamental question of its natural function remains an enduring mystery. Thus, we have much more to learn about the msDNAs of bacteria.

Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies.

The dynamics of the natively unfolded form of the pro-peptide of subtilisin (PPS) have been characterized at two different pHs (6.0 and 3.0) by 15N relaxation experiments. 15N relaxation data is obtained at multiple field strengths and a detailed comparison of spectral density mapping, the model free approach and the recently proposed Cole-Cole model free (CC-MF) analysis is presented. The CC-MF analysis provides a better fit to the observed magnetic field dependence of 15N relaxation data of unfolded PPS than conventional model free approaches and shows that fluctuations in R2 may be accounted for by a distribution of correlation times on the nanosecond timescale. A new parameter epsilon derives from the analysis and represents the width of the distribution function and the heterogeneity of the dynamics on the nanosecond timescale at a particular site. Particularly interesting is the observation that epsilon is sensitive to pH changes and that PPS samples a wider distribution of nanosecond time scale motions at less acidic pHs than at more acidic pHs. These results suggest that PPS experiences a higher degree of correlated motion at pH 6.0 and that electrostatic interactions may be important for inducing correlated motions on the nanosecond timescale in unfolded PPS.

Predicting models of outcome stratified by age after first stroke rehabilitation in Japan.

OBJECTIVE: A multivariate model predicting the function at discharge following inpatient rehabilitation has been previously produced. The aim of this study is to determine predictors of function at discharge for stroke outcome and examine their accuracy of prediction. DESIGN: Four hundred sixty-four stroke patients were enrolled. Sex, the nature of the stroke, age, onset to rehabilitation admission interval and length of rehabilitation hospital stay were obtained from their medical records. Patients were divided into the following five groups according to age: < or = 49, 50-59, 60-69, 70-79, and > or = 80 yr. Disability was assessed on admission and at discharge by the FIM. Stepwise multiple regression analysis was performed in each group. RESULTS: The model for patients aged 60-69 yr was best for accuracy of prediction and explained 76% of variation for discharge FIM total score. The equation: (expected discharge FIM total score) = 111.88 + 0.08 x (the type of stroke) - 0.11 x (age) + 0.81 x (admission FIM total score) - 0.12 x (onset to rehabilitation admission interval), R = 0.87, R2 = 0.76, P < 0.0001. The type of stroke = 1 for cerebral infarction and 0 otherwise. Length of rehabilitation stay is not selected as a predictor. CONCLUSION: The stratification of patients by age is useful to determine predictors of function at discharge for stroke outcome and to improve their accuracy of prediction.

Nonsense mutations in cspA cause ribosome trapping leading to complete growth inhibition and cell death at low temperature in Escherichia coli.

CspA, the major cold shock protein of Escherichia coli, is dramatically induced immediately after cold shock. CspA production is transient and reduces to a low basal level when cells become adapted. Here we show that expression from multicopy plasmids of mutant cspA mRNAs bearing nonsense mutations in the coding region caused sustained high levels of the mutant mRNAs at low temperature, resulting in complete inhibition of cell growth ultimately leading to cell death. We demonstrate that the observed growth inhibition was caused by largely exclusive occupation of cellular ribosomes by the mutant cspA mRNAs. Such sequestration of ribosomes even occurs without a single peptide bond formation, implying that the robust translatability of the cspA mRNA is determined at the step of initiation. Further analysis demonstrated that the downstream box of the cspA mRNA was dispensable for the effect, whereas the upstream box of the mRNA was essential. Our system may offer a novel means to study sequence or structural elements involved in the translation of the cspA mRNA and may also be utilized to regulate bacterial growth at low temperature.

An essential GTPase, der, containing double GTP-binding domains from Escherichia coli and Thermotoga maritima.

A gene encoding a putative GTPase containing two tandemly repeated GTP-binding domains from a hyperthermophilic bacterium, Thermotoga maritima, was cloned and expressed in Escherichia coli. The gene (TM1446) termed der is highly conserved in Eubacteria including E. coli. The purified der product (Tm-Der) has GTPase activity but no ATPase activity. GTP, GDP, and dGTP but not GMP, ATP, CTP, and UTP compete for GTP binding to Tm-Der. An optimal condition for the GTPase assay was determined to be pH 7.5 in 400 mm KCl and 5 mm MgCl(2) at 70 degrees C, where K(m), V(max), and k(cat) values were determined to be 110 microm, 3.46 microm/min, and 0.87 min(-1), respectively. A der deletion strain of E. coli was constructed by replacing the der gene (originally annotated yfgK) with a kanamycin resistance gene. The deletion strain was found to form colonies only if the cells harbored a plasmid containing der, indicating that der is essential for E. coli growth.

Antibacterial activity of 4,5-dihydroxy-2-cyclopentan-1-one (DHCP) and cloning of a gene conferring DHCP resistance in Escherichia coli.

In the present study we report that 4,5-dihydroxy-2-cyclopentan-1-one (DHCP), which is derived from heat-treatment of uronic acid or its derivatives, has antibacterial activity against Escherichia coli. The compound causes complete growth inhibition at 350 microM concentration. We have cloned a gene from E. coli, which confers DHCP resistance when present in multicopy. The putative protein encoded by this gene (dep- DHCP efflux protein) is a transmembrane efflux protein with a high homology to other antibiotic-efflux proteins including those for chloramphenicol, bicyclomycin and tetracycline. However, the Dep protein does not confer cross-resistance to any of the antibiotics tested.

Histological characteristics of the pelage skin of rough fur mice (C3H/HeJ- ruf/ruf).

Pelage skin of C3H/HeJ mice homozygous at an autosomal recessive mutant locus, rough fur (ruf) which is located on chromosome 9, was histologically analyzed. Sebaceous glands synthesizing lipids were larger in the mutant mice than in controls in an examination by Sudan IV staining. Electron microscopic analysis of the sebaceous gland showed that lipid droplets were denser in mutant mice than in control mice, and that they were irregular in shape in ruf mice while those of controls were round. Our results suggested that rough fur (ruf) mice might be an animal model for hyperlipogenesis of the pelage skin.

Expression of ZAKI-4 messenger ribonucleic acid in the brain during rat development and the effect of hypothyroidism.

We identified ZAKI-4 (also designated as DSCR1L1) as a thyroid hormone responsive gene in cultured human skin fibroblasts. Recently it has been reported that ZAKI-4 belongs to an evolutionary conserved family of proteins that function as calcineurin inhibitor. In human, ZAKI-4 and calcineurin are highly expressed in brain, where thyroid hormones play essential roles in the development during fetal and neonatal periods. In the present study, we examined the temporal and spatial expression patterns of ZAKI-4 messenger RNA (mRNA) in control and hypothyroid rat brains. Northern blot analysis revealed that ZAKI-4 mRNA was detected in both cerebral cortex and cerebellum as early as embryonic day (E)18. In the cerebral cortex, the expression level gradually increased with age, reaching a plateau at postnatal day (P)7 and remained constant thereafter until P30. A similar pattern of increase with age was also observed in hypothyroid rats; however, the magnitude of the increase was significantly reduced. In control rats, the fold increase in ZAKI-4 mRNA level from E18 to P17 was 10.8; whereas in hypothyroid rats, it was 7.4. In cerebellum the expression level did not change with age or by thyroid status. In situ hybridization revealed that ZAKI-4 mRNA is widely expressed in neurons throughout the brain. It is noteworthy that the expression in the neurons of layer VI of the cerebral cortex was more evident in control rats than that in hypothyroid rats from P17 to P30. Though not influenced by hypothyroidism, there were several regions of the brain in which ZAKI-4 mRNA was strongly expressed. These regions were the mitral cell layer of the olfactory bulb, the substantia nigra, and the hippocampus, where calcineurin is also abundantly expressed. Therefore, it may be hypothesized that ZAKI-4 plays an important role in the development and function of the brain by modulating calcineurin function; and decrease in ZAKI-4 mRNA expression in the specific brain areas may explain, in some parts, the mechanism of abnormal brain development by hypothyroidism.

Induction of CspA, an E. coli major cold-shock protein, upon nutritional upshift at 37 degrees C.

BACKGROUND: The synthesis of CspA, the major cold-shock protein of Escherichia coli, is dramatically induced upon cold shock. It was recently reported that there is massive presence of CspA under nonstress conditions, and it is thus claimed that CspA as the cold-shock protein is a misnomer. RESULTS: Here, we re-examined and confirmed that CspA is induced upon culture dilution at 37 degrees C. However, its induction level is one-sixth of the cold-shock-induced level, clearly indicating that the major stress that induces CspA is cold shock. It was further found that CspA induction can be achieved not only by culture dilution but also by the simple addition of nutrients, and that it was almost completely abolished in the presence of rifampicin or nalidixic acid. Nutritional upshift causes the induction of only CspA but not other cold-shock-inducible CspA homologues. The amount of cspA mRNA rapidly and transiently increased by culture dilution, but its stability was not significantly changed. CONCLUSIONS: These results suggest that CspA is a nutritional-upshift stress protein as well as a cold-shock stress protein, and that CspA induction following nutritional upshift may be due to transcriptional activation.