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Therapy with mesenchymal stromal cells (MSCs) has shown promise in inflammatory bowel disease—leveraging their immunosuppressive and regenerative properties. However, the potential immunogenic complications of allogenic MSCs sourced from different tissues raise concern. Thus, we assessed the fitness and functionality of autologous intestinal MSCs as a potential platform for cellular therapy. Mucosal biopsy-derived MSCs from Crohn’s disease (n = 11), ulcerative colitis (n = 12), and controls (n = 14) were analyzed by microscopy and flow cytometry for doubling-time, morphology, differentiation potential, and immunophenotype. Gene expression, cell-subtype composition, along with surface marker and secretome changes after IFN-γ priming were measured by bulk and single-cell RNA sequencing coupled with a 30-plex Luminex panel. MSCs expanded ex vivo demonstrate canonical MSC markers, similar growth kinetics, and tripotency regardless of the patient phenotype. Global transcription patterns were similar at baseline though inflammatory bowel disease (IBD) rectal MSCs showed changes in select immunomodulatory genes. IFN-γ priming resulted in upregulation of shared immunoregulatory genes (particularly in PD-1 signaling) and overrode the transcriptional differences observed at baseline. Furthermore, MSCs secrete key immunomodulatory molecules at baseline and in response to IFN-γ including CXCL10, CXCL9, and MCP-1. Overall, MSCs from IBD patients have normal transcriptional and immunomodulatory properties with therapeutic potential and can be sufficiently expanded.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) causes ventricular arrhythmias (VAs) and sudden cardiac death (SCD). In 2019, a risk prediction model that estimates the 5-year risk of incident VAs in ARVC was developed (ARVCrisk.com). This study aimed to externally validate this prediction model in a large international multicentre cohort and to compare its performance with the risk factor approach recommended for implantable cardioverter-defibrillator (ICD) use by published guidelines and expert consensus. In a retrospective cohort of 429 individuals from 29 centres in North America and Europe, 103 (24%) experienced sustained VA during a median follow-up of 5.02 (2.05-7.90) years following diagnosis of ARVC. External validation yielded good discrimination [C-index of 0.70 (95% confidence interval-CI 0.65-0.75)] and calibration slope of 1.01 (95% CI 0.99-1.03). Compared with the three published consensus-based decision algorithms for ICD use in ARVC (Heart Rhythm Society consensus on arrhythmogenic cardiomyopathy, International Task Force consensus statement on the treatment of ARVC, and American Heart Association guidelines for VA and SCD), the risk calculator performed better with a superior net clinical benefit below risk threshold of 35%. Using a large independent cohort of patients, this study shows that the ARVC risk model provides good prognostic information and outperforms other published decision algorithms for ICD use. These findings support the use of the model to facilitate shared decision making regarding ICD implantation in the primary prevention of SCD in ARVC. P.J. is supported by the Daniel Bravo Foundation grant and Spanish Society of Cardiology Magda Heras mobility grant, A.G. by the Wilton W. Webster Fellow of Heart Rhythm Society, The Johns Hopkins ARVD/C Programme by the Leonie-Wild Foundation, Leyla Erkan Family Fund for ARVD Research, The Hugh Calkins, Marvin H. Weiner, and Jacqueline J. Bernstein Cardiac Arrhythmia Center, Dr Francis P. Chiramonte Private Foundation, Dr Satish, Rupal, and Robin Shah ARVD Fund at Johns Hopkins, Bogle Foundation, Campanella Family, Patrick J. Harrison Family, Peter French Memorial Foundation, and Wilmerding Endowments, H.K.J. by grants from Novo Nordisk Foundation, Denmark (NNF18OC0031258 and NNF20OC0065151), S.A.L. by NIH grant 1R01HL139731 and American Heart Association 18SFRN34250007, R.T. by the Canada Research Chairs programme, J.C.T. by the Philippa and Marvin Carsley Cardiology Research Chair. S.G.P. receives support from Ricerca Corrente funding scheme of the Italian Ministry of Health and Italian Ministry of Research and University Dipartimenti di Eccellenza 2018 to 2022 grant to the Molecular Medicine Department (University of Pavia). Sí

Motivation There is widespread interest in identifying genetic variants that exhibit parent-of-origin effects (POEs) wherein the effect of an allele on phenotype expression depends on its parental origin. POEs can arise from different phenomena including genomic imprinting and have been documented for many complex traits. Traditional tests for POEs require family data to determine parental origins of transmitted alleles. As most genome-wide association studies (GWAS) sample unrelated individuals (where allelic parental origin is unknown), the study of POEs in such datasets requires sophisticated statistical methods that exploit genetic patterns we anticipate observing when POEs exist. We propose a method to improve discovery of POE variants in large-scale GWAS samples that leverages potential pleiotropy among multiple correlated traits often collected in such studies. Our method compares the phenotypic covariance matrix of heterozygotes to homozygotes based on a Robust Omnibus Test. We refer to our method as the Parent of Origin Inference using Robust Omnibus Test (POIROT) of multiple quantitative traits. Results Through simulation studies, we compared POIROT to a competing univariate variance-based method which considers separate analysis of each phenotype. We observed POIROT to be well-calibrated with improved power to detect POEs compared to univariate methods. POIROT is robust to non-normality of phenotypes and can adjust for population stratification and other confounders. Finally, we applied POIROT to GWAS data from the UK Biobank using BMI and two cholesterol phenotypes. We identified 338 genome-wide significant loci for follow-up investigation. Availability and implementation The code for this method is available at https://github.com/staylorhead/POIROT-POE.

Background The gut and oral microbiome have independently been shown to be associated with inflammatory bowel disease (IBD). However, it is not known to what extent gut and oral microbial disease markers converge in terms of their composition in IBD. Further, the spatial and temporal variation within the oral microenvironments of IBD remain to be elucidated. Patients and Methods We used a prospectively recruited cohort of patients with IBD (n = 47) and unrelated healthy control patients (n = 18) to examine the spatial and temporal distribution of microbiota within the various oral microenvironments, represented by saliva, tongue, buccal mucosa, and plaque, and compared them with stool. Microbiome characterization was performed using 16S rRNA gene sequencing. Results The oral microbiome displayed IBD-associated dysbiosis, in a site- and taxa-specific manner. Plaque samples depicted a relatively severe degree of dysbiosis, and the disease-associated dysbiotic bacterial groups were predominantly the members of the phylum Firmicutes. Our 16S rRNA gene analyses show that oral microbiota can distinguish patients with IBD from healthy control patients, with salivary microbiota performing the best, closely matched by stool and other oral sites. Longitudinal profiles of microbial composition suggest that some taxa are more consistently perturbed than others, preferentially in a site-dependent fashion. Conclusions Collectively, these data indicate the potential of using oral microbial profiles in screening and monitoring patients with IBD. Furthermore, these results support the importance of spatial and longitudinal microbiome sampling to interpret disease-associated dysbiotic states and eventually to gain insights into disease pathogenesis.

Aims The aim was to determine the survival and persistence of Escherichia coli in soil amended with compost from different manure sources. Method and results Complex interactions of abiotic and biotic factors on E. coli survival were characterized in field experiment plots receiving randomly assigned compost treatments: dairy windrow, dairy vermicompost, poultry windrow or no compost. Biomass, activity and function of indigenous microbial communities in the composts and soils were measured concurrently to determine whether mechanisms of compost were driven by biotic or abiotic properties. E. coli persisted in compost containing poultry amendments but not in composts containing dairy or no amendments. Poultry compost contained more NH4 -N and a distinct microbial community compared to dairy and no compost treatments. A laboratory experiment performed on compost extracts suggested that E. coli survived better in extracts devoid of indigenous microbes as long as bioavailable nutrients were plentiful. Conclusions Dairy-based composts are less likely to support E. coli survival than poultry-based composts. Significance and impact of the study Results aid in risk assessment of the use of different types of manure-based compost and soil amendments in fruit and vegetable production by elucidating the roles of nutrient and microbial community composition on survival of E. coli in amended field soils.

Author(s): Denson, Lee A; Jurickova, Ingrid; Karns, Rebekah; Shaw, Kelly A; Cutler, David J; Okou, David; Valencia, C Alexander; Dodd, Anne; Mondal, Kajari; Aronow, Bruce J; Haberman, Yael; Linn, Aaron; Price, Adam; Bezold, Ramona; Lake, Kathleen; Jackson, Kimberly; Walters, Thomas D; Griffiths, Anne; Baldassano, Robert N; Noe, Joshua D; Hyams, Jeffrey S; Crandall, Wallace V; Kirschner, Barbara S; Heyman, Melvin B; Snapper, Scott; Guthery, Stephen L; Dubinsky, Marla C; Leleiko, Neal S; Otley, Anthony R; Xavier, Ramnik J; Stevens, Christine; Daly, Mark J; Zwick, Michael E; Kugathasan, Subra | Abstract: BACKGROUND:Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling. METHODS:Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined. RESULTS:We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohn's disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P l 0.0001). CONCLUSIONS:Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD.

The evolutionary theories of mutation accumulation (MA) and disposable soma (DS) provide possible explanations for the existence of human aging. To better understand the relative importance of these theories, we devised a test to identify MA- and DS-consistent sites across the genome using familial DNA methylation data. Two key characteristics of DNA methylation allowed us to do so. First, DNA methylation exhibits distinct and widespread changes with age, with numerous age-differentially-methylated sites observed across the genome. Second, many sites show heritable DNA methylation patterns within families. We extended heritability predictions of MA and DS to DNA methylation, predicting that MA-consistent age-differentially-methylated sites will show increasing heritability with age, while DS-consistent sites will show the opposite. Variance components models were used to test for changing heritability of methylation with age at 48,601 age-differentially-methylated sites across the genome in 610 individuals from 176 families. Of these, 102 sites showed significant MA-consistent increases in heritability with age, while 2266 showed significant DS-consistent decreases in heritability. These results suggest that both MA and DS play a role in explaining aging and aging-related changes, and that while the majority of DNA methylation changes observed in aging are consistent with epigenetic drift, targeted changes exist and may mediate effects of aging-related genes.

The goal of this study was to identify the contribution of common genetic variants to Down syndrome−associated atrioventricular septal defect, a severe heart abnormality. Compared with the euploid population, infants with Down syndrome, or trisomy 21, have a 2000-fold increased risk of presenting with atrioventricular septal defects. The cause of this increased risk remains elusive. Here we present data from the largest heart study conducted to date on a trisomic background by using a carefully characterized collection of individuals from extreme ends of the phenotypic spectrum. We performed a genome-wide association study using logistic regression analysis on 452 individuals with Down syndrome, consisting of 210 cases with complete atrioventricular septal defects and 242 controls with structurally normal hearts. No individual variant achieved genome-wide significance. We identified four disomic regions (1p36.3, 5p15.31, 8q22.3, and 17q22) and two trisomic regions on chromosome 21 (around PDXK and KCNJ6 genes) that merit further investigation in large replication studies. Our data show that a few common genetic variants of large effect size (odds ratio >2.0) do not account for the elevated risk of Down syndrome−associated atrioventricular septal defects. Instead, multiple variants of low-to-moderate effect sizes may contribute to this elevated risk, highlighting the complex genetic architecture of atrioventricular septal defects even in the highly susceptible Down syndrome population.

Autism spectrum disorder (ASD) is a heterogeneous disorder with substantial heritability, most of which is unexplained. ASD has a population prevalence of one percent and affects four times as many males as females. Patients with fragile X E (FRAXE) intellectual disability, which is caused by a silencing of the X-linked gene AFF2 ,d isplay an umber of ASD-like phenotypes. Duplications and deletions at theAFF2 locus have also been reported in cases with moderate intellectual disability and ASD. We hypothesized that other rare X-linked sequence variants at the AFF2 locus might contribute to ASD. We sequenced the AFF2 genomic region in 202 male ASD probands and found that 2.5% of males sequenced had missense mutations at highly conserved evolutionary sites. When compared with the frequency of missense mutations in 5545 X chromosomes from unaffected controls, we saw a statistically significant enrichment in patients with ASD (OR: 4.9; P < 0.014). In addition, we identified rare AFF2 3 " UTR variants at conserved sites which alter gene expression in a luciferase assay. These data suggest that rare variation in AFF2 may be a previously unrecognized ASD susceptibility locus and may help explain some of the male excess of ASD.

Seed development ends with a maturation phase that imparts desiccation tolerance, nutrient reserves, and dormancy degree. Here, we report the functional analysis of an Arabidopsis thaliana C2H2 zinc finger protein INDETERMINATE DOMAIN1 (IDD1)/ENHYDROUS (ENY). Ectopic expression of IDD1/ENY (2x35S:ENY) disrupted seed development, delaying endosperm depletion and testa senescence, resulting in an abbreviated maturation program. Consequently, mature 2x35S:ENY seeds had increased endosperm-specific fatty acids, starch retention, and defective mucilage extrusion. Using RAB18 promoter ENY lines (RAB18:ENY) to confine expression to maturation, when native ENY expression increased and peaked, resulted in mature seed with lower abscisic acid (ABA) content and decreased germination sensitivity to applied ABA. Furthermore, results of far-red and red light treatments of 2x35S:ENY and RAB18:ENY germinating seeds, and of artificial microRNA knockdown lines, suggest that ENY acts to promote germination. After using RAB18:ENY seedlings to induce ENY during ABA application, key genes in gibberellin (GA) metabolism and signaling were differentially regulated in a manner suggesting negative feedback regulation. Furthermore, GA treatment resulted in a skotomorphogenic-like phenotype in light-grown 2x35S:ENY and RAB18:ENY seedlings. The physical interaction of ENY with DELLAs and an ENY-triggered accumulation of DELLA transcripts during maturation support the conclusion that ENY mediates GA effects to balance ABA-promoted maturation during late seed development.

A common goal in the discovery of rare functional DNA variants via medical resequencing is to incur a relatively lower proportion of false positive base-calls. We developed a novel statistical method for resequencing arrays (SRMA, sequence robust multi-array analysis) to increase the accuracy of detecting rare variants and reduce the costs in subsequent sequence verifications required in medical applications. SRMA includes single and multi-array analysis and accounts for technical variables as well as the possibility of both low- and high-frequency genomic variation. The confidence of each base-call was ranked using two quality measures. In comparison to Sanger capillary sequencing, we achieved a false discovery rate of 2% (false positive rate 1.2 × 10⁻⁵, false negative rate 5%), which is similar to automated second-generation sequencing technologies. Applied to the analysis of 39 nuclear candidate genes in disorders of mitochondrial DNA (mtDNA) maintenance, we confirmed mutations in the DNA polymerase gamma POLG in positive control cases, and identified novel rare variants in previously undiagnosed cases in the mitochondrial topoisomerase TOP1MT, the mismatch repair enzyme MUTYH, and the apurinic-apyrimidinic endonuclease APEX2. Some patients carried rare heterozygous variants in several functionally interacting genes, which could indicate synergistic genetic effects in these clinically similar disorders.

Borrelial relapsing fever was once a major worldwide epidemic disease that made a significant impact on Livingstone during his epic travels through Africa and throughout Europe. Indeed, the term 'relapsing fever' was first used to describe clinical cases of this disease in Edinburgh. During the last century, we have witnessed the demise of the louse-borne infection, largely through improving standards of living resulting in a reduction in body lice, the vector for Borrelia recurrentis [louse-borne relapsing fever (LBRF)]. The tick-borne zoonotic form of the disease persists in endemic foci around the world [tick-borne relapsing fever (TBRF)]. Indeed, TBRF is reportedly the most common bacterial infection from Senegal and listed within the top ten causes of mortality in children under five in Tanzania. In Ethiopia, LBRF is again within the top ten causes of hospital admission, associated with significant morbidity and mortality. Despite these figures, many now regard relapsing fever as an unusual tropical disease. Certainly, recent cases have been imported following travel from endemic zones. More surprisingly, cases have been reported following family reunions in Colorado, USA. A further case was reported from the Mt Wilson observatory in Los Angeles, USA. In many regions, the infection is zoonotic with natural reservoirs in several vertebrate species. In West Africa, infection is again primarily zoonotic. Whether those species found predominantly in East Africa are zoonoses or are infections of humans alone is still debated, however, the life cycle may be determined by the feeding preferences of their arthropod vectors.

Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5′ proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5′ deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT–PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5′ deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms.

Almost 2000 drought-responsive genes were identified in Arabidopsis thaliana under progressive soil drought stress using whole-genome oligonucleotide microarrays. Most of the drought-regulated genes recovered to normal expression levels by 3 h after rewatering. It has previously been shown that the abscisic acid (ABA) analogue (+)-8′-acetylene-ABA (PBI425) hyperinduces many ABA-like changes in gene expression to reveal a more complete list of ABA-regulated genes, and it is demonstrated here that PBI425 produced a correspondingly increased drought tolerance. About two-thirds of drought-responsive genes (1310 out of 1969) were regulated by ABA and/or the ABA analogue PBI425. Analysis of promoter motifs suggests that many of the remaining drought-responsive genes may be affected by ABA signalling. Concentrations of endogenous ABA and its catabolites significantly increased under drought stress and either completely (ABA) or partially (ABA catabolites) recovered to normal levels by 3 h after rehydration. Detailed analyses of drought transcript profiles and in silico comparisons with other studies revealed that the ABA-dependent pathways are predominant in the drought stress responses. These comparisons also showed that other plant hormones including jasmonic acid, auxin, cytokinin, ethylene, brassinosteroids, and gibberellins also affected drought-related gene expression, of which the most significant was jasmonic acid. There is also extensive cross-talk between responses to drought and other environmental factors including light and biotic stresses. These analyses demonstrate that ABA-related stress responses are modulated by other environmental and developmental factors.

Cystic fibrosis (CF), the most common lethal single gene disorder in Caucasians, is due to mutations in the CFTR gene. Twin and sibling analysis indicates that modifier genes, rather than allelic variation in CFTR, are responsible for most of the variability in severity of lung disease, the major cause of mortality in CF patients. We used a family-based approach to test for association between lung function and two functional SNPs (rs1800469, '-509' and rs1982073, 'codon 10') in the 5' region of transforming growth factor-beta1 (TGFB1), a putative CF modifier gene. Quantitative transmission disequilibrium testing of 472 CF patient-parent-parent trios revealed that both TGFB1 SNPs showed significant transmission distortion when patients were stratified by CFTR genotype. Although lung function and nutritional status are correlated in CF patients, there was no evidence of association between the TGFB1 SNPs and variation in nutritional status. Additional tagging SNPs (rs8179181, rs2278422, rs8110090, rs4803455 and rs1982072) that capture most of the diversity in TGFB1 were also typed but none showed association with variation in lung function. However, a haplotype composed of the -509 C and codon 10 T alleles along with the C allele of the 3' SNP rs8179181 was highly associated with increased lung function in patients grouped by CFTR genotype. These results demonstrate that TGFB1 is a modifier of CF lung disease and reveal a previously unrecognized beneficial effect of TGFB1 variants upon the pulmonary phenotype.

We have functionally characterized an Arabidopsis (Arabidopsis thaliana) gene AtHSD1 (At5g50600) that encodes a protein with homology to animal 11-beta-hydroxysteroid dehydrogenase (HSD). Transgenic Arabidopsis plants overexpressing AtHSD1 (designated AOHSD plants) under the control of the cauliflower mosaic virus 35S promoter showed increased growth and seed yield as well as increased tolerance of saline stress and reduced seed dormancy. In canola (Brassica napus), transgenic plants overexpressing AtHSD1 also outgrew wild-type plants. AOHSD phenotypes were similar to those of plants that overproduced brassinosteroids (BRs) or overexpressed the BR receptor gene BRI1. A loss-of-function hsd mutant produced by RNA interference displayed a semi-dwarfed phenotype with reduced sensitivity to BRs. In contrast, AOHSD plants were hypersensitive to BRs and exhibited increased catabolism of abscisic acid (ABA). Germination of AOHSD seeds was less sensitive to ABA, while hsd seed was more sensitive to ABA during germination. AtHSD transcription was rapidly induced by BR treatment in wild type and was expressed widely in aerial plant parts, especially vascular tissues. This study demonstrates that AtHSD1 is involved in regulating growth and development in plants and is likely to promote or mediate BR effects. The gene has significant potential for improving growth and yield of canola and other agricultural crops.

Purpose: We present the concept of the Green House, articulated by William Thomas as a radically changed, ‘‘deinstitutionalized’’ nursing home well before its first implementation, and we describe and discuss implications from the first Green Houses in Tupelo, Mississippi. Design and Methods: Green Houses are small, selfcontained houses for 10 or fewer elders, each with private rooms and full bathrooms and sharing family-style communal space, including hearth, dining area, and full kitchen. Line staff at the level of certified nursing assistants, called Shahbazim, are ‘‘universal workers,’’ who cook meals, do laundry, provide personal care, assist with habilitation, and promote the elders’ quality of life. Nurses, doctors, and other professionals comprise a visiting clinical support team for the residents and Shahbazim. Multiple Green Houses comprise a nursing home, meeting all nursing facility regulations and working within state-reimbursement levels. In 2003, four Green Houses were built on the campus of a retirement community; in June of that year, 40 residents relocated from the 140-bed nursing home to the Green Houses, including 20 residents previously living in the locked dementia unit. Results: Experiences to date are positive for residents, family, and staff. The sponsor is converting the entire facility to Green Houses, and other providers around the country plan to implement Green House variants. Implications: Because nursing home stock is aging, many physical plants are or soon will be slated for major rebuilding, thereby providing sponsors with an opportunity to consider Green Houses. Early experience suggests that Green Houses are feasible and that outcomes are likely to be positive, and it also suggests that there are some potential issues to overcome in such a dramatic reengineering of nursing homes.

Background: Crohn's disease (CD) is highly heritable. NOD2 has emerged as the main susceptibility gene among individuals of European ancestry; however, NOD2 does not appear to contribute to CD susceptibility among many non-European populations. Today's African American (AA) population represents an admixture of West African (80%) and European (20%) ancestry. Since genotype-based tools are becoming increasingly available for CD, it is important that we validate the risk variants in different populations, such as admixed AAs. Methods: We analyzed the NOD2 variants among admixed AAs (n = 321, 240 with CD and 111 healthy controls [HCs]) and nonadmixed West Africans (n = 40) by genotyping four known disease-causing NOD variants. We extracted the publicly available 1000 Genomes data on NOD2 variants from 500 subjects of West African origin. Association with disease was evaluated by logistic regression. Results: An association with CD was found for the classical single nucleotide polymorphism (SNP) 1007fs (2.6% CD, 0% HC, P = 0.012); there was no association when the genotypic and allelic frequencies of the risk alleles were compared for SNPs R702W and G908R. No known NOD2 risk alleles were seen in either the West African cohort or in subjects of African ancestry from the 1000 Genomes project. Conclusions: The NOD2 gene is a risk for CD in AAs, although the allele frequencies and the attributable risk are much lower compared with Caucasians. The risk alleles are not seen in the West African population, suggesting that the risk for CD contributed by NOD2 among AAs is due exclusively to recent European admixture. (Inflamm Bowel Dis 2012;)

Background. Quality of life (QOL) is a goal for nursing home residents, but measures are needed to tap this phenomenon. Methods. In-person QOL interviews were attempted for 1988 residents, stratified by cognitive functioning, from 40 nursing homes in five states. Likert-type response options were used with reversion to dichotomous responses when necessary; z-score transformations were used to combine the formats. Tests of internal consistency and confirmatory factor analysis were performed; cluster analysis was used to shorten the scales. Correlations between domain scores were examined, and tests of convergent validity performed. Analyses were repeated for subgroups based on cognitive functioning levels. Results. Long QOL scales were constructed for 1316 of the 1988 residents, including many with substantial cognitive impairment. Confirmatory factor analysis confirmed 10 QOL domains. Cronbach alphas ranged from .76 to .52. The majority (93%) of the 45 possible interscale correlations among domains were below .l4 and the rest were between .4 and .5. QOL scales were correlated with, but distinct from, residents’ emotions ratings and overall satisfaction, and each was correlated with a corresponding summary rating for the domain. Conclusions. QOL can be feasibly measured from resident self-report for much of the nursing home population, including cognitively impaired residents. Additional research is suggested on the measures, but the approach has promise for regulation, continuous quality improvement, and public information.

Previous analyses of the alpha-esterase cluster of Drosophila melanogaster revealed 10 active genes and the DmalphaE4a-Psi pseudogene. Here, we reconstruct the evolution of the pseudogene from the sequences of 12 alleles from widely scattered D. melanogaster populations and single alleles from Drosophila simulans and Drosophila yakuba. All of the DmalphaE4a-Psi alleles contain numerous inactivating mutations, suggesting that pseudogene alleles are fixed in natural populations. Several lines of evidence also suggest that DmalphaE4a is now evolving without selective constraint in the D. melanogaster lineage. There are three polymorphic indels which result in frameshifts; a key nucleotide of the intron splice acceptor is polymorphic; the neutral mutation parameter is the same for replacement and silent sites; one of the nonsilent polymorphisms results in a stop codon; only 1 of the 13 replacement polymorphisms is biochemically conservative; residues that are conserved among active esterases have different states in DmalphaE4a-Psi; and there are about half as many transitional polymorphisms as transversional ones. In contrast, the D. simulans and D. yakuba orthologs DsalphaE4a and DyalphaE4a do not have the inactivating mutations of DmalphaE4a-Psi and appear to be evolving under the purifying selection typical of protein- encoding genes. For instance, there have been more substitutions in the introns than in the exons, and more in silent sites than in replacement sites. Furthermore, most of the amino acid substitutions that have occurred between DyalphaE4a and DsalphaE4a are located in sites that typically vary among active alpha-esterases rather than those that are usually conserved. We argue that the original alphaE4a gene had a function which it has lost since the divergence of the D. melanogaster and D. simulans lineages.

Rates of molecular evolution at some protein-encoding loci are more irregular than expected under a simple neutral model of molecular evolution. This pattern of excessive irregularity in protein substitutions is often called the “overdispersed molecular clock” and is characterized by an index of dispersion, R(T) > 1. Assuming infinite sites, no recombination model of the gene R(T) is given for a general stationary model of molecular evolution. R(T) is shown to be affected by only three things: fluctuations that occur on a very slow time scale, advantageous or deleterious mutations, and interactions between mutations. In the absence of interactions, advantageous mutations are shown to lower R(T); deleterious mutations are shown to raise it. Previously described models for the overdispersed molecular clock are analyzed in terms of this work as are a few very simple new models. A model of deleterious mutations is shown to be sufficient to explain the observed values of R(T). Our current best estimates of R(T) suggest that either most mutations are deleterious or some key population parameter changes on a very slow time scale. No other interpretations seem plausible. Finally, a comment is made on how R(T) might be used to distinguish selective sweeps from background selection.

Objectives. To determine if the curvilinear relationship between age and voluntary association memberships is due to compositional differences between age groups. Methods. Data are drawn from 12 nationally representative surveys covering the period 1974‐1994 and including items on membership in 16 types of associations. Age is categorized into 14 groups ranging from 18‐24 through 85+. Patterns of age differences in number of association memberships are examined before and after controlling for several variables related both to age and to memberships. Results. At the bivariate level, number of memberships increases through ages 40‐44 and then declines fairly steadily. When controls for compositional differences are introduced, the curvilinear pattern of age differences is replaced by one showing an increasing number of memberships through ages 55‐59, a slight decrease in the 60‐64 age group, and then generally stable membership levels through the 85+ age group. Discussion. The lower levels of voluntary association memberships of older persons consistently identified in bivariate analyses appear to be largely due to differences in compositional characteristics between the older and other age groups.

Abscisic acid (ABA) 8′-hydroxylase catalyzes the first step in the oxidative degradation of (+)-ABA. The development of a robust in vitro assay has now permitted detailed examination and characterization of this enzyme. Although several factors (buffer, cofactor, and source tissue) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevich [1997] J Exp Bot 48: 1787–1795). (+)-ABA 8′-hydroxylase is an integral membrane protein that is localized to the microsomal fraction in suspension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, although there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)-ABA was progressively inhibited at O2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N2. (+)-ABA 8′-hydroxylase activity was inhibited by tetcyclacis (50% inhibition at 10−6 m), cytochrome c (oxidized form), and CO. The CO inhibition was reversible by light from several regions of the visible spectrum, but most efficiently by blue and amber light. These data strongly support the contention that (+)-ABA 8′-hydroxylase is a cytochrome P450 monooxygenase.

Microspore-derived embryos ofBrassica napus cv Reston were used to examine the effects of exogenous (+)-abscisic acid (ABA) and related compounds on the accumulation of very-long-chain monounsaturated fatty acids (VLCMFAs), VLCMFA elongase complex activity, and induction of the 3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing enzyme of the VLCMFA elongation system. Of the concentrations tested, (+)-ABA at 10 μm showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 μm (+)-ABA treatment, was 60% higher than that of the untreated embryos at 24 h. The transcript of the KCS gene was induced by 10 μm (+)-ABA within 1 h and further increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with (+)-ABA for 72 h. Also, (+)-8′-methylene ABA, which is metabolized more slowly than ABA, had a stronger ABA-like effect on the KCS gene transcription, elongase complex activity (28% higher), and level of VLCMFAs (25–30% higher) than ABA. After 24 h approximately 60% of the added (+)-[3H]ABA (10 μm) was metabolized, yielding labeled phaseic and dihydrophaseic acid. This study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via increased expression of the KCS gene and that reducing ABA catabolism would increase VLCMFAs in microspore-derived embryos.

In vivo measurements of the enzymatic hydroxylation of the phytohormone (+)-abscisic acid to hormonally inactive ( -)-phaseic acid in corn cell cultures revealed that (+)-abscisic acid 8' hydroxylase activity was induced by (+)-abscisic acid treatment. This induction was blocked by the protein synthesis inhibitor cycloheximide and by the transcription inhibitor cordycepin. Following an induction treatment with abscisic acid, the amount of induced enzyme was measured by addition of both cycloheximide, to prevent further induction, and fresh abscisic acid as substrate for the induced enzyme. Phaseic acid production was related to the amount of enzyme induced. The experimental system was optimized for studying enzyme induction and the substrate specificity of the induced enzyme. The induced 8' hydroxylase was specific for (+)-abscisic acid, whereas the low basal activity also hydroxylated (-)-abscisic acid at the 7' position to form 7'-hydroxy abscisic acid. After induction, the 8' hydroxylase was rapidly degraded with a half-life of approximately 2 h. Phaseic and (-)-abscisic acid were weak inducers; however, the unstable oxidation product 8'-hydroxy abscisic acid exhibited 42% of the activity of (+)-abscisic acid. When corn cells were placed under water stress by increasing concentrations of mannitol in the culture medium, 8' hydroxylase induction was suppressed. The experimental system described will be useful for further studies of the physiological and hormonal factors that modulate abscisic acid metabolism and for testing potential enzyme inhibitors and hormone analogues.

We report a 55-year-old woman with acrodermatitis chronica atrophicans (ACA) and a peripheral sensory neuropathy. ACA is an uncommon late cutaneous manifestation of Lyme disease, which follows disseminated Borrelia burgdorferi infection. This is the second published case from the U.K. since serological diagnosis has been available. In this patient the diagnosis was confirmed by serology using a sensitive enzyme-linked immunosorbent assay and immunoblotting techniques. B. burgdorferi DNA was demonstrated in the affected skin using the polymerase chain reaction, although staining and cultures for the organism were negative. Recommended treatment of ACA is with oral doxycycline 100 mg twice daily for 28 days, but our patient did not respond well to this regimen. She was therefore treated with ceftriaxone intravenously for 21 days, which resulted in a rapid symptomatic and clinical response.

Nine patients with RA received two doses of 155 mg racemic hydroxychloroquine each, as a tablet and by i.v. infusion, in a randomized cross-over design study. Blood concentrations over the first 32 h following each dose were determined. Bioavailability was estimated using a sequential exponential least squares deconvolution method. The mean fraction absorbed from the tablet was 0.79 (range 0.39 to 1.27). The mean absorption lag-time was 1.3 h (range 0.5 to 3.7 h) and the mean time for 50% absorption was 4.3 h (range 1.9 to 10.3 h). Mean rate and extent of hydroxychloroquine absorption were not significantly different from that previously reported for healthy volunteers, although the interindividual variability in absorption parameters was greater in the patient group. Variability in the extent of absorption would lead to differences in steady-state hydroxychloroquine concentrations between patients, potentially contributing to the variability in response observed in clinical practice.

Interpretation of immunoblot results for Lyme borreliosis remains an area of controversy. This study was undertaken to devise a simple method which incorporated distinctions between specific and non-specific bands. A numerical value was assigned to bands reflecting their relative importance. This scheme was evaluated with sera from 61 forestry workers, which were previously tested by immunoblotting and interpreted subjectively. These results were reported as five separate categories representing levels of reactivity. A total of 72% (44) of sera fell into the same categories as they were originally reported. The method was also assessed with 40 sera routinely submitted for Lyme borreliosis serology. These were interpreted by two members of staff without previous experience of immunoblotting. Their results gave 80% and 72.5% agreement with those of an experienced person used to reading immunoblots for Lyme borreliosis.

This paper presents results of a logit regression analysis of the correlates of senior center participation for a national sample of 13,737 elders from the Supplement on Aging of the 1984 National Health Interview Survey. Significant characteristics were higher levels of social interaction, decreasing income, living alone, fewer ADL-IADL difficulties, being female, and living in suburbs and rural nonfarm areas. Curvilinear relationships between center use and age and education were found. Race, self-reported health status, and residence in urban and rural farm areas were nonsignificant when the other variables were controlled.

Agings is commonly assumed to be associated with the adoption of increasingly conservative social and political attitudes. Empirical tests of this assumption have provided little support, but most analyses have focused on attitude domains where public opinion was shifting in a liberal direction. Using three items about law and order, a domain marked by conservative trends, this paper evaluates the aging-conservatism hypothesis with data drawn from 18 national surveys covering the period 1959-85. Attitude changes in four cohorts are analyzed with Taylor's hierarchical goodness-of-fit procedures for evaluating trends in public opinion. The findings show that each of the cohorts has participated in the shift toward more conservative opinions on law and order issues, and that either constant or zero differences characterize the trends in percentage differences between the oldest and the other cohorts. In the absence of differential rates of change, we conclude that older cohorts are no more likely than younger cohorts to adopt conservative law and order attitudes and that period effects are influencing each of the cohorts equally. Language: en

The percentage of soybean cell culture protoplasts permeabilized by electroporation was dependent on the voltage and the number of successive pulses that were applied. Best results were obtained with two 50 milliseconds, 400 volts per centimeter pulses after which 78% of the surviving protoplasts had been permeabilized to the fluorescent dye calcein. Quantitation of the volume of extracellular fluid taken up was performed using radioactive inulin (molecular weight 5000-5500). Typically between 20 and 40 nanoliters of fluid was taken up by 10(6) protoplasts. Electroporation and hypotonic shock treatments (M Saleem, AJ Cutler 1986 J Plant Physiol 124: 11-21) were compared with respect to the volume of fluid taken up under optimum conditions. Electroporation produced 10 times more uptake than hypotonic shock treatment. In all experiments there was a direct relationship between the number of protoplasts lysed and both the amount of fluid taken up and the percentage of surviving protoplasts that were permeabilized.

A critical analysis of historically used characters, based on a large series of specimens, is presented. Based on a comparative morphological analysis of type specimens, the 19 species of Sipunculus and four of Xenosiphon are reviewed. The genera and subgenera are redefined so that, after appropriate revisions arc made, Sipunculus (Sipunculus) has eight, Sipunculus (Contraporus) new subgenus has two, and Xenosiphon has only one remaining valid species. A zoogeographical note and a key to the species are included.

Nitrogen-14 and nitrogen-15 nuclear magnetic resonance (NMR) spectra were recorded for freshly dissected buds of Picea glauca and for buds grown for 3, 6 and 9 weeks on shoot-forming medium. Resonances for Glu (and other αNH2 groups), Pro, Ala, and the side chain groups in Gln, Arg, Orn, and γ-aminobutyric acid could be detected in in vivo15N NMR spectra. Peaks for α-amino groups, Pro, NO3− and NH4+ could also be identified in 14N NMR spectra. Perfusion experiments performed for up to 20 hours in the NMR spectrometer showed that 15N-labeled NH4+ and NO3− are first incorporated into the amide group of Gln and then in the αNH2 pool. Subsequently, it also emerges in Ala and Arg. These data suggest that the glutamine synthetase/ glutamate synthase pathway functions under these conditions. The assimilation of NH4+ is much faster than that of NO3−. Consequently after 10 days of growth more than 70% of the newly synthesized internal free amino acid pool derives its nitrogen from NH4+ rather than NO3−. If NH4+ is omitted from the medium, no NO3− is taken up during 9 weeks and the buds support limited growth by utilizing their endogenous amino acid pools. It is concluded that NH4+ and NO3− are both required for the induction of nitrate- and nitrite reductase.

The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline.

Several trends are noted which may lead future cohorts of older persons to belong to voluntary associations to a greater extent than current cohorts. Because the best previously available data which could be used to monitor changing membership levels are not two decades old, this study presents detailed age profiles of membership in 16 types of associations. Based on data from the 1974 and 1975 NORC General Social Surveys of the adult population of the USA (N = 2,974), various patterns of age differences in belonging to associations are identified and discussed for both the total sample and by sex. A rank order of the types of associations to which older persons are most likely to belong in 1974-1975 is compared with a similar rank order based on a 1955 survey. The paper concludes by specifying additional research questions which replicate studies should address.