Search

OBJECTIVE: To explore family factors that influence sexual behaviors among a group of Black and Hispanic adolescent males. METHODS: One hundred seventy-eight subjects were selected from a convenience sample of 431 adolescents who participated in a 1996 Youth Survey conducted in San Bernardino County, California. RESULTS: Having a sibling who was a teen parent was significantly associated with engaging in sexual intercourse among Hispanics. Among Blacks, having a father who was a teen dad was significantly associated with sexual intercourse. CONCLUSIONS: Findings showed relationships between family characteristics and sexual attitudes and behaviors among participants. [ABSTRACT FROM AUTHOR]

Abstract Background Glomerular filtration rate (GFR) and urinary albumin excretion (UAE) are markers of kidney function that are known to be heritable. Many endocrine conditions have strong familial components. We tested for association between the Affymetrix GeneChip Human Mapping 100K single nucleotide polymorphism (SNP) set and measures of kidney function and endocrine traits. Methods Genotype information on the Affymetrix GeneChip Human Mapping 100K SNP set was available on 1345 participants. Serum creatinine and cystatin-C (cysC; n = 981) were measured at the seventh examination cycle (1998–2001); GFR (n = 1010) was estimated via the Modification of Diet in Renal Disease (MDRD) equation; UAE was measured on spot urine samples during the sixth examination cycle (1995–1998) and was indexed to urinary creatinine (n = 822). Thyroid stimulating hormone (TSH) was measured at the third and fourth examination cycles (1981–1984; 1984–1987) and mean value of the measurements were used (n = 810). Age-sex-adjusted and multivariable-adjusted residuals for these measurements were used in association with genotype data using generalized estimating equations (GEE) and family-based association tests (FBAT) models. We presented the results for association tests using additive allele model. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg Equilibrium p-value ≥ 0.001, and call rates of at least 80%. Results The top SNPs associated with these traits using the GEE method were rs2839235 with GFR (p-value 1.6*10-05), rs1158167 with cysC (p-value 8.5*10-09), rs1712790 with UAE (p-value 1.9*10-06), and rs6977660 with TSH (p-value 3.7*10-06), respectively. The top SNPs associated with these traits using the FBAT method were rs6434804 with GFR(p-value 2.4*10-5), rs563754 with cysC (p-value 4.7*10-5), rs1243400 with UAE (p-value 4.8*10-6), and rs4128956 with TSH (p-value 3.6*10-5), respectively. Detailed association test results can be found at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. Four SNPs in or near the CST3 gene were highly associated with cysC levels (p-value 8.5*10-09 to 0.007). Conclusion Kidney function traits and TSH are associated with SNPs on the Affymetrix GeneChip Human Mapping 100K SNP set. These data will serve as a valuable resource for replication as more SNPs associated with kidney function and endocrine traits are identified.

Abstract Introduction Subclinical atherosclerosis (SCA) measures in multiple arterial beds are heritable phenotypes that are associated with increased incidence of cardiovascular disease. We conducted a genome-wide association study (GWAS) for SCA measurements in the community-based Framingham Heart Study. Methods Over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1345 subjects from 310 families. We calculated sex-specific age-adjusted and multivariable-adjusted residuals in subjects tested for quantitative SCA phenotypes, including ankle-brachial index, coronary artery calcification and abdominal aortic calcification using multi-detector computed tomography, and carotid intimal medial thickness (IMT) using carotid ultrasonography. We evaluated associations of these phenotypes with 70,987 autosomal SNPs with minor allele frequency ≥ 0.10, call rate ≥ 80%, and Hardy-Weinberg p-value ≥ 0.001 in samples ranging from 673 to 984 subjects, using linear regression with generalized estimating equations (GEE) methodology and family-based association testing (FBAT). Variance components LOD scores were also calculated. Results There was no association result meeting criteria for genome-wide significance, but our methods identified 11 SNPs with p < 10-5 by GEE and five SNPs with p < 10-5 by FBAT for multivariable-adjusted phenotypes. Among the associated variants were SNPs in or near genes that may be considered candidates for further study, such as rs1376877 (GEE p < 0.000001, located in ABI2) for maximum internal carotid artery IMT and rs4814615 (FBAT p = 0.000003, located in PCSK2) for maximum common carotid artery IMT. Modest significant associations were noted with various SCA phenotypes for variants in previously reported atherosclerosis candidate genes, including NOS3 and ESR1. Associations were also noted of a region on chromosome 9p21 with CAC phenotypes that confirm associations with coronary heart disease and CAC in two recently reported genome-wide association studies. In linkage analyses, several regions of genome-wide linkage were noted, confirming previously reported linkage of internal carotid artery IMT on chromosome 12. All GEE, FBAT and linkage results are provided as an open-access results resource at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. Conclusion The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis.

Abstract Background Obesity is related to multiple cardiovascular disease (CVD) risk factors as well as CVD and has a strong familial component. We tested for association between SNPs on the Affymetrix 100K SNP GeneChip and measures of adiposity in the Framingham Heart Study. Methods A total of 1341 Framingham Heart Study participants in 310 families genotyped with the Affymetrix 100K SNP GeneChip had adiposity traits measured over 30 years of follow up. Body mass index (BMI), waist circumference (WC), weight change, height, and radiographic measures of adiposity (subcutaneous adipose tissue, visceral adipose tissue, waist circumference, sagittal height) were measured at multiple examination cycles. Multivariable-adjusted residuals, adjusting for age, age-squared, sex, smoking, and menopausal status, were evaluated in association with the genotype data using additive Generalized Estimating Equations (GEE) and Family Based Association Test (FBAT) models. We prioritized mean BMI over offspring examinations (1–7) and cohort examinations (10, 16, 18, 20, 22, 24, 26) and mean WC over offspring examinations (4–7) for presentation. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg equilibrium p ≥ 0.001, and call rates of at least 80%. Results The top SNPs to be associated with mean BMI and mean WC by GEE were rs110683 (p-value 1.22*10-7) and rs4471028 (p-values 1.96*10-7). Please see http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007 for the complete set of results. We were able to validate SNPs in known genes that have been related to BMI or other adiposity traits, including the ESR1 Xba1 SNP, PPARG, and ADIPOQ. Conclusion Adiposity traits are associated with SNPs on the Affymetrix 100K SNP GeneChip. Replication of these initial findings is necessary. These data will serve as a resource for replication as more genes become identified with BMI and WC.

Abstract Background Prior studies reported conflicting findings on the association between metabolic syndrome and inflammatory biomarkers. We tested the cross-sectional associations between metabolic syndrome and nine inflammatory markers. Methods We measured C-reactive protein, CD40 ligand, interleukin-6, intercellular adhesion molecule-1, monocyte chemoattractant protein-1, osteoprotegerin, P-selectin, tumor necrosis factor-alpha, and tumor necrosis factor receptor-2 in 2570 Framingham Offspring Study participants free of diabetes and cardiovascular disease at examination 7. Metabolic syndrome was defined by National Cholesterol Education Program criteria. We performed multivariable linear regressions for each biomarker with metabolic syndrome as the exposure adjusting for age, sex, smoking, aspirin use, and hormone replacement. We subsequently added to the models components of the metabolic syndrome as continuous traits plus lipid lowering and hypertension treatments. We considered P Results Metabolic syndrome was present in 984 (38%) participants and was statistically significantly associated with each biomarker (all P < 0.02) except osteoprotegerin. After adjusting for its component variables, the metabolic syndrome was associated only with P-selectin (1.06 fold higher in metabolic syndrome, 95% CI 1.02, 1.10, p = 0.005). Conclusions Metabolic syndrome was associated with multiple inflammatory biomarkers. However, adjusting for each of its components eliminated the association with most inflammatory markers, except P-selectin. Our results suggest that the relation between metabolic syndrome and inflammation is largely accounted for by its components.

Abstract Background Animal studies suggest that local adipocyte-mediated activity of the renin-angiotensin-aldosterone system (RAAS) contributes to circulating levels, and may promote the development of obesity-related hypertension in rodents. Methods We examined relations of systemic RAAS activity, as assessed by circulating plasma renin activity (PRA), serum aldosterone level, and aldosterone:renin ratio (ARR), with specific regional adiposity measures in a large, community-based sample. Third Generation Framingham Heart Study participants underwent multidetector computed tomography assessment of SAT and VAT volumes during Exam 1 (2002 and 2005). PRA and serum aldosterone were measured after approximately 10 minutes of supine rest; results were log-transformed for analysis. Correlation coefficients between log-transformed RAAS measures and adiposity measurements were calculated, adjusted for age and sex. Partial correlations between log-transformed RAAS measures and adiposity measurements were also calculated, adjusted for standard CVD risk factors. Results Overall, 992 women and 897 men were analyzed (mean age 40 years; 7% hypertension; 3% diabetes). No associations were observed with SAT (renin r = 0.04, p = 0.1; aldosterone r = -0.01, p = 0.6) or VAT (renin r = 0.03, p = 0.2; aldosterone r = -0.03, p = 0.2). Similar results were observed for ARR, in sex-stratified analyses, and for BMI and waist circumference. Non-significant partial correlations were also observed in models adjusted for standard cardiovascular risk factors. Conclusions Regional adiposity measures were not associated with circulating measures of RAAS activity in this large population-based study. Further studies are required to determine whether adipocyte-derived RAAS components contribute to systemic RAAS activity in humans.

Abstract Background Ectopic fat accumulation in the renal sinus is associated with chronic kidney disease and hypertension. The genetic contributions to renal sinus fat accumulation in humans have not been well characterized. Methods The present analysis consists of participants from the Framingham Offspring and Third Generation who underwent computed tomography; renal sinus fat and visceral adipose tissue (VAT) were quantified. Renal sinus fat was natural log transformed and sex- and cohort-specific residuals were created, adjusted for (1) age, (2) age and body mass index (BMI), and (3) age and VAT. Residuals were pooled and used to calculate heritability using variance-components analysis in SOLAR. A genome-wide association study (GWAS) for renal sinus fat was performed using an additive model with approximately 2.5 million imputed single nucleotide polymorphisms (SNPs). Finally, we identified the associations of renal sinus fat in our GWAS results with validated SNPs for renal function (n = 16), BMI (n = 32), and waist-to-hip ratio (WHR, n = 14), and applied a multi-SNP genetic risk score method to determine if the SNPs for each renal and obesity trait were in aggregate associated with renal sinus fat. Results The heritability of renal sinus fat was 39% (p < 0.0001); results were not materially different after adjustment for BMI (39%) or VAT (40%). No SNPs reached genome-wide significance in our GWAS. In our candidate gene analysis, we observed nominal, direction consistent associations with renal sinus fat for one SNP associated with renal function (p = 0.01), two associated with BMI (p < 0.03), and two associated with WHR (p < 0.03); however, none remained significant after accounting for multiple testing. Finally, we observed that in aggregate, the 32 SNPs associated with BMI were nominally associated with renal sinus fat (multi-SNP genetic risk score p = 0.03). Conclusions Renal sinus fat is a heritable trait, even after accounting for generalized and abdominal adiposity. This provides support for further research into the genetic determinants of renal sinus fat. While our study was underpowered to detect genome-wide significant loci, our candidate gene BMI risk score results suggest that variability in renal sinus fat may be associated with SNPs previously known to be associated with generalized adiposity.

Abstract Background Renal sinus fat may mediate obesity-related vascular disease, although this fat depot has not been assessed in a community-based sample. We sought to develop a protocol to quantify renal sinus fat accumulation using multi-detector computed tomography (MDCT). Methods Protocol development was performed in participants in the Framingham Offspring cohort who underwent MDCT. Volumetric renal sinus fat was measured separately within the right and left kidneys, and renal sinus fat area within a single MDCT scan slice was measured in the right kidney. Due to the high correlation of volumetric and single-slice renal sinus fat in the right kidney (Pearson correlation [r] = 0.85, p < 0.0001), we optimized a single-slice protocol to capture renal sinus fat in the right kidney alone. Pearson correlation coefficients were used to compare to assess the correlation of volumetric and single-slice renal sinus fat in the right kidney with other measures of adiposity. Inter- and intra-reader reproducibility was assessed using intra-class correlation coefficients. Results Single-slice measurements were obtained in 92 participants (mean age 60 years, 49% women, median renal sinus fat 0.43 cm2). Intra- and inter-reader intra-class correlation coefficients were 0.93 and 0.86, respectively. Single-slice renal sinus fat was correlated with body mass index (r = 0.35, p = 0.0006), waist circumference (r = 0.31, p = 0.003), and abdominal visceral fat (r = 0.48, p < 0.0001). Similar correlations were observed for volumetric renal sinus fat in the right kidney. Conclusions Measuring renal sinus fat is feasible and reproducible using MDCT scans in a community-based sample.

Abstract Background A common variant of the UMOD gene was linked with prevalent chronic kidney disease (CKD) in large, genomics consortia. One community-based study found that urine concentrations of the uromodulin protein forecast risk of incident CKD. This study within persons with known coronary artery disease (CAD) evaluated whether uromodulin concentrations could distinguish CKD risk. Methods In the Heart and Soul Study, the UMOD snp (12917707) was genotyped in 879 individuals with baseline creatinine clearance (CrCl) measured from a 24-hour urine collection. Uromodulin protein was measured from stored urine specimens among a subset of 120 participants, balanced by genotype. Incident CKD cases (N = 102) were defined by an initial CrCl > 70 ml/min and a 5-year follow-up CrCl Results Among 527 self-described White participants with DNA, 373 (71%) were homozygous for the dominant allele (G/G), 133 (25%) were heterozygous (G/T) and only 21 (4%) were homozygous for the minor allele (T/T). The T/T genotype had an approximately 11 ml/min higher CrCl than the other 2 groups, but this difference did not reach statistical significance (p = 0.20). The T/T genotype had significantly lower uromodulin levels than the common G/G genotype, and the G/T genotype had intermediate levels. However, uromodulin concentrations were similar between cases and controls (44 vs. 48 mg/dL, p = 0.88). Conclusions This study among a cohort of persons with established CAD found no association between urine uromodulin and incident CKD, although UMOD genotype was associated with urine uromodulin concentrations.

Abstract Background Genome-wide association (GWA) studies that use population-based association approaches may identify spurious associations in the presence of population admixture. In this paper, we propose a novel three-stage approach that is computationally efficient and robust to population admixture and more powerful than the family-based association test (FBAT) for GWA studies with family data. We propose a three-stage approach for GWA studies with family data. The first stage is to perform linear regression ignoring phenotypic correlations among family members. SNPs with a first stage p-value below a liberal cut-off (e.g. 0.1) are then analyzed in the second stage that employs a linear mixed effects (LME) model that accounts for within family correlations. Next, SNPs that reach genome-wide significance (e.g. 10-6 for 34,625 genotyped SNPs in this paper) are analyzed in the third stage using FBAT, with correction of multiple testing only for SNPs that enter the third stage. Simulations are performed to evaluate type I error and power of the proposed method compared to LME adjusting for 10 principal components (PC) of the genotype data. We also apply the three-stage approach to the GWA analyses of uric acid in Framingham Heart Study's SNP Health Association Resource (SHARe) project. Results Our simulations show that whether or not population admixture is present, the three-stage approach has no inflated type I error. In terms of power, using LME adjusting PC is only slightly more powerful than the three-stage approach. When applied to the GWA analyses of uric acid in the SHARe project of FHS, the three-stage approach successfully identified and confirmed three SNPs previously reported as genome-wide significant signals. Conclusions For GWA analyses of quantitative traits with family data, our three-stage approach provides another appealing solution to population admixture, in addition to LME adjusting for genetic PC.

Abstract Background African Americans have an increased incidence and worse prognosis with chronic kidney disease (CKD - estimated glomerular filtration rate [eGFR] 2) than their counterparts of European-descent. Inflammation has been related to renal disease in non-Hispanic whites, but there are limited data on the role of inflammation in renal dysfunction in African Americans in the community. Methods We examined the cross-sectional relation of log transformed C-reactive protein (CRP) to renal function (eGFR by Modification of Diet and Renal Disease equation) in African American participants of the community-based Jackson Heart Study's first examination (2000 to 2004). We conducted multivariable linear regression relating CRP to eGFR adjusting for age, sex, body mass index, systolic and diastolic blood pressure, diabetes, total/HDL cholesterol, triglycerides, smoking, antihypertensive therapy, lipid lowering therapy, hormone replacement therapy, and prevalent cardiovascular disease events. In a secondary analysis we assessed the association of CRP with albuminuria (defined as albumin-to-creatinine ratio > 30 mg/g). Results Participants (n = 4320, 63.2% women) had a mean age ± SD of 54.0 ± 12.8 years. The prevalence of CKD was 5.2% (n = 228 cases). In multivariable regression, CRP concentrations were higher in those with CKD compared to those without CKD (mean CRP 3.2 ± 1.1 mg/L vs. 2.4 ± 1.0 mg/L, respectively p < 0.0001). CRP was significantly associated with albuminuria in sex and age adjusted model however not in the multivariable adjusted model (p > 0.05). Conclusion CRP was associated with CKD however not albuminuria in multivariable-adjusted analyses. The study of inflammation in the progression of renal disease in African Americans merits further investigation.

Abstract Background Studies suggest end-stage renal disease incidence and all-cause mortality rates among patients with chronic kidney disease (CKD) differ by age. The association of diabetes mellitus and hypertension with CKD across the adult lifespan is not well established. Methods Data from NHANES 1999–2004 were used to determine the association of risk factors for stage 3 or 4 CKD (n = 12,518) and albuminuria (n = 12,778) by age grouping (20 to 49, 50 to 69, and ≥70 years). Stage 3 or 4 CKD was defined as an estimated glomerular filtration rate of 15 to 59 ml/min/1.73 m2 and albuminuria as an albumin to creatinine ratio ≥30 mg/g. Results For adults 20 to 49, 50 to 69 and ≥70 years of age, the prevalence ratios (95% confidence interval) of stage 3 or 4 CKD associated with hypertension were 1.94 (0.86 – 4.35), 1.51 (1.09 – 2.07), 1.31 (1.15 – 1.49), respectively (p-trend = 0.038). The analogous prevalence ratios (95% confidence interval) were 3.01 (1.35 – 6.74), 1.61 (1.15 – 2.25), 1.40 (1.15 – 1.69), respectively, for diagnosed diabetes mellitus (p-trend = 0.067); and 2.67 (0.53 – 13.4), 1.35 (0.69 – 2.63), 1.08 (0.78 – 1.51), respectively, for undiagnosed diabetes mellitus (p-trend = 0.369). The prevalence ratios of albuminuria associated with hypertension and diagnosed and undiagnosed diabetes mellitus were lower at older age (each p < 0.05). Conclusion Among US adults, diabetes mellitus and hypertension are associated with CKD and albuminuria regardless of age. However, the associations were stronger at younger ages.

Abstract Background The Framingham Heart Study (FHS) recently obtained initial results from the first genome-wide association scan for renal traits. The study of 70,987 single nucleotide polymorphisms (SNPs) in 1,010 FHS participants provides a list of SNPs showing the strongest associations with renal traits which need to be verified in independent study samples. Methods Sixteen SNPs were selected for replication based on the most promising associations with chronic kidney disease (CKD), estimated glomerular filtration rate (eGFR), and serum cystatin C in FHS. These SNPs were genotyped in 15,747 participants of the Atherosclerosis in Communities (ARIC) Study and evaluated for association using multivariable adjusted regression analyses. Primary outcomes in ARIC were CKD and eGFR. Secondary prospective analyses were conducted for association with kidney disease progression using multivariable adjusted Cox proportional hazards regression. The definition of the outcomes, all covariates, and the use of an additive genetic model was consistent with the original analyses in FHS. Results The intronic SNP rs6495446 in the gene MTHFS was significantly associated with CKD among white ARIC participants at visit 4: the odds ratio per each C allele was 1.24 (95% CI 1.09–1.41, p = 0.001). Borderline significant associations of rs6495446 were observed with CKD at study visit 1 (p = 0.024), eGFR at study visits 1 (p = 0.073) and 4 (lower mean eGFR per C allele by 0.6 ml/min/1.73 m2, p = 0.043) and kidney disease progression (hazard ratio 1.13 per each C allele, 95% CI 1.00–1.26, p = 0.041). Another SNP, rs3779748 in EYA1, was significantly associated with CKD at ARIC visit 1 (odds ratio per each T allele 1.22, p = 0.01), but only with eGFR and cystatin C in FHS. Conclusion This genome-wide association study provides unbiased information implicating MTHFS as a candidate gene for kidney disease. Our findings highlight the importance of replication to identify common SNPs associated with renal traits.

Abstract Background Previously, we reported significant linkage of body mass index (BMI) to chromosomes 6 and 11 across six examinations, covering 28 years, of the Framingham Heart Study. These results were on all individuals available at each exam, thus the sample size varied from exam to exam. To remove any effect of sample size variation we have now constructed six subsets; for each exam individuals were only included if they were measured at every exam, i.e. for each exam, included individuals comprise the intersection of the original six exams. This strategy preferentially removed older individuals who died before reaching the sixth exam, thus the intersection datasets are smaller (n = 1114) and significantly younger than the full datasets. We performed variance components linkage analysis on these intersection datasets and on their sex-specific subsets. Results Results from the sex-specific genome scans revealed 11 regions in which a sex-specific maximum lodscore was at least 2.0 for at least one dataset. Randomization tests indicated that all 11 regions had significant (p < 0.05) differences in sex-specific maximum lodscores for at least three datasets. The strongest sex-specific linkage was for men on chromosome 16 with maximum lodscores 2.70, 3.00, 3.42, 3.61, 2.56 and 1.93 for datasets 1–6 respectively. Results from the full genome scans revealed that linked regions on chromosomes 6 and 11 remained significantly and consistently linked in the intersection datasets. Surprisingly, the maximum lodscore on chromosome 10 for dataset 1 increased from 0.97 in the older original dataset to 4.23 in the younger smaller intersection dataset. This difference in maximum lodscores was highly significant (p < 0.0001), implying that the effect of this chromosome may vary with age. Age effects may also exist for the linked regions on chromosomes 6 and 11. Conclusion Sex specific effects of chromosomal regions on BMI are common in the Framingham study. Some evidence also exists for age-specific effects of chromosomal regions.

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hoggard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metacognitive biases have been repeatedly associated with transdiagnostic psychiatric dimensions of 'anxious-depression' and 'compulsivity and intrusive thought', cross-sectionally. To progress our understanding of the underlying neurocognitive mechanisms, new methods are required to measure metacognition remotely, within individuals over time. We developed a gamified smartphone task designed to measure visuo-perceptual metacognitive (confidence) bias and investigated its psychometric properties across two studies (N = 3410 unpaid citizen scientists, N = 52 paid participants). We assessed convergent validity, split-half and test-retest reliability, and identified the minimum number of trials required to capture its clinical correlates. Convergent validity of metacognitive bias was moderate (r(50) = 0.64, p < 0.001) and it demonstrated excellent split-half reliability (r(50) = 0.91, p < 0.001). Anxious-depression was associated with decreased confidence (β =  - 0.23, SE = 0.02, p < 0.001), while compulsivity and intrusive thought was associated with greater confidence (β = 0.07, SE = 0.02, p < 0.001). The associations between metacognitive biases and transdiagnostic psychiatry dimensions are evident in as few as 40 trials. Metacognitive biases in decision-making are stable within and across sessions, exhibiting very high test-retest reliability for the 100-trial (ICC = 0.86, N = 110) and 40-trial (ICC = 0.86, N = 120) versions of Meta Mind. Hybrid 'self-report cognition' tasks may be one way to bridge the recently discussed reliability gap in computational psychiatry., (© 2024. The Author(s).)

Background and Aims: Subjective confidence plays an important role in guiding behaviour, especially when objective feedback is unavailable. Systematic misjudgements in confidence can foster maladaptive behaviours and have been linked to various psychiatric disorders. In this study, we adopted a transdiagnostic approach to examine confidence biases in problem gamblers across three levels: local decision confidence, global task performance confidence, and overall self-esteem. The importance of taking a transdiagnostic perspective is increasingly recognised, as it captures the dimensional nature of psychiatric symptoms that often cut across diagnostic boundaries. Accordingly, we investigated if any observed confidence biases could be explained by transdiagnostic symptom dimensions of Anxiety-Depression and Compulsive Behaviour and Intrusive Thought. This approach allows us to gain a more comprehensive understanding of the role of metacognitive processes in problem gambling, beyond the constraints of traditional diagnostic categories., Methods: Thirty-eight problem gamblers and 38 demographically matched control participants engaged in a gamified metacognition task and completed self-report questionnaires assessing transdiagnostic symptom dimensions., Results: Compared to controls, problem gamblers displayed significantly elevated confidence at the local decision and global task levels, independent of their actual task performance. This elevated confidence was observed even after controlling for the heightened symptom levels of Anxiety-Depression and Compulsive Behaviour and Intrusive Thought among the problem gamblers., Discussion: The results reveal a notable disparity in confidence levels between problem gamblers and control participants, not fully accounted for by the symptom dimensions Anxiety-Depression and Compulsive Behaviour and Intrusive Thought. This suggests the contribution of other factors, perhaps linked to gambling-specific cognitive distortions, to the observed confidence biases., Conclusion: The findings highlight the intricate link between metacognitive confidence and psychiatric symptoms in the context of problem gambling. It underscores the need for further research into metacognitive biases, which could enhance therapeutic approaches for individuals with psychiatric conditions.

Recently, Mn-doped semiconductor nanocrystals (NCs) with high brightness, long lifetimes, and low-energy excitation are emerging for time-resolved luminescence biosensing/imaging. Following our previous work on Mn-doped NCs, in this work we developed poly(styrene-co-maleic anhydride) (PSMA)-encapsulated Mn-doped AgZnInS/ZnS NCs as signal transducers for immunoassay of capsular polysaccharide (CPS), a surface antigen and also a biomarker of Burkholderia pseudomallei which causes a fatal disease called melioidosis. To enhance the assay sensitivity, a surface treatment for PSMA-encapsulated NCs (NC-probes) was performed to promote the presence of carboxyl groups that help conjugate more anti-CPS antibodies to the surface of NC-probes and thus enhance bioassay signals. Meanwhile, time-resolved reading on the luminescence of NC-probes was adopted to minimize the assay background autofluorescence. Both strategies essentially enhance the assay signal-to-background ratio (or equivalently the assay sensitivity) by increasing the signal and decreasing the background, respectively. Through performing and comparing immunoassays with different NC-probes (with and without surface treatment) and different signal reading methods (time-resolved reading and non-time-resolved reading), it was proven that the immunoassay adopting surface-treated NC-probes and time-resolved reading achieved a lower limit-of-detection (LOD) than the ones adopting non-surface-treated NC-probes or non-time-resolved reading. Moreover, the achieved LOD is comparable to the LOD of immunoassay using enzyme horseradish peroxidase as a signal transducer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Prior studies have found metacognitive biases are linked to a transdiagnostic dimension of anxious-depression, manifesting as reduced confidence in performance. However, previous work has been cross-sectional and so it is unclear if under-confidence is a trait-like marker of anxious-depression vulnerability, or if it resolves when anxious-depression improves. Data were collected as part of a large-scale transdiagnostic, four-week observational study of individuals initiating internet-based cognitive behavioural therapy (iCBT) or antidepressant medication. Self-reported clinical questionnaires and perceptual task performance were gathered to assess anxious-depression and metacognitive bias at baseline and 4-week follow-up. Primary analyses were conducted for individuals who received iCBT (n=649), with comparisons between smaller samples that received antidepressant medication (n=82) and a control group receiving no intervention (n=88). Prior to receiving treatment, anxious-depression severity was associated with under-confidence in performance in the iCBT arm, replicating previous work. From baseline to follow-up, levels of anxious-depression were significantly reduced, and this was accompanied by a significant increase in metacognitive confidence in the iCBT arm ( β =0.17, SE=0.02, p<0.001). These changes were correlated (r(647)=-0.12, p=0.002); those with the greatest reductions in anxious-depression levels had the largest increase in confidence. While the three-way interaction effect of group and time on confidence was not significant (F(2, 1632)=0.60, p=0.550), confidence increased in the antidepressant group ( β =0.31, SE = 0.08, p<0.001), but not among controls ( β =0.11, SE = 0.07, p=0.103). Metacognitive biases in anxious-depression are state-dependent; when symptoms improve with treatment, so does confidence in performance. Our results suggest this is not specific to the type of intervention., Competing Interests: CF, AH, TS, KL, VO, CG No competing interests declared, CL The PhD studentship of Chi Tak Lee is co-funded by SilverCloud Health and the Irish Research Council, SH Siobhán Harty is a current employees of SilverCloud Health, DR Derek Richards is a current employees of SilverCloud Health, JP Jorge Palacios is a current employees of SilverCloud Health, KS Klaas Enno Stephan acknowledges support by the René and Susanne Braginsky Foundation and the ETH Foundation, (© 2023, Fox et al.)

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1-183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60-80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle's lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin B as the hydrophobic bioactive agent.

Cardiolipin (CL) is a unique phospholipid that is fundamental to the structure and function of the highly curved cristae membranes of mitochondria. Given its distinctive cone-shaped molecular architecture, CL induces negative membrane curvature in a bilayer setting. Another key feature of CL is its intrinsic ability to interact with various ligands, including cytochrome c, the anti-neoplastic anthracycline, doxorubicin, and the divalent cation, calcium. Although these, and other, binding interactions exert profound effects on mitochondrial and cellular function, they are difficult to study in intact mitochondria. Whereas liposomes provide a potential model membrane system, their relatively large size, limited ability to accommodate CL and the presence of an inaccessible interior bilayer leaflet, make these structures suboptimal. The discovery that CL can be formulated into aqueous soluble, reconstituted high density lipoprotein particles, termed nanodisks (ND), provides an alternative model membrane system. Comprised solely of CL and an apolipoprotein scaffold, CL-ND exist as a disk-shaped phospholipid bilayer whose perimeter is stabilized by contact with the scaffold protein. In these nanoscale particles, both leaflets of the bilayer are solvent accessible, an advantage for studies of ligand interactions. Recent experiments employing CL-ND have yielded novel insight into apoptosis, cardiotoxicity and CL-dependent bilayer to non-bilayer transitions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Doxorubicin (DOX) is an aqueous soluble anthracycline therapeutic widely used in cancer treatment. Although DOX anti-cancer activity is dose-dependent, increased dosage enhances the risk of cardiotoxicity. Despite intensive investigation, the molecular basis of this undesirable side effect has yet to be established. In addition to serving as a DNA intercalation agent, DOX is known to bind to the signature mitochondrial phospholipid, cardiolipin (CL). Consistent with this, DOX associates with aqueous soluble nanoparticles, termed nanodisks (ND), comprised solely of CL and an apolipoprotein scaffold. Fluorescence microscopy analysis revealed that DOX uptake, and targeting to the nucleus of cultured hepatocarcinoma (HepG2) or breast cancer (MCF7) cells, was unaffected by its association with CL-ND. Subsequent studies revealed that free DOX and DOX-CL-ND were equivalent in terms of growth inhibition activity in both cell lines. By contrast, in studies with H9C2 cardiomyocytes, DOX-CL-ND induced a lesser concentration-dependent decline in cell viability than free DOX. Whereas incubation of H9C2 cardiomyocytes with free DOX caused a steep decline in maximal oxygen consumption rate, DOX-CL-ND treated cells were largely unaffected. The data indicate that association of DOX with CL-ND does not diminish its cancer cell growth inhibition activity yet confers protection to cardiomyocytes from DOX-induced effects on aerobic respiration. This study illustrates that interaction with CL plays a role in DOX-induced mitochondrial dysfunction and suggests CL-ND provide a tool for investigating the mechanistic basis of DOX-induced cardiotoxicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability., (© 2022 Regan-Mochrie et al.; Published by Cold Spring Harbor Laboratory Press.)

Locusta migratoria apolipophorin III (apoLp-III) possesses the ability to exist as a water soluble amphipathic α-helix bundle and a lipid surface seeking apolipoprotein. The intrinsic ability of apoLp-III to transform phospholipid vesicles into reconstituted discoidal high-density lipoproteins (rHDL) has led to myriad applications. To improve the yield of recombinant apoLp-III, studies were performed in a bioreactor. Induction of apoLp-III expression generated a protein product that is secreted from E. coli into the culture medium. Interaction of apoLp-III with gas and liquid components in media produced large quantities of thick foam. A continuous foam fractionation process yielded a foamate containing apoLp-III as the sole major protein component. The yield of recombinant apoLp-III was ~0.2 g / liter bacterial culture. Mass spectrometry analysis verified the identity of the target protein and indicated no modifications or changes to apoLp-III occurred as a result of foam fractionation. The functional ability of apoLp-III to induce rHDL formation was evaluated by incubating foam fractionated apoLp-III with phosphatidylcholine vesicles. FPLC size exclusion chromatography revealed a single major population of particles in the size range of rHDL. The results described offer a novel approach to bioreactor-based apoLp-III production that takes advantage of its intrinsic biosurfactant properties., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl 2 resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl 2 -induced effects. Centrifugation of CaCl 2 -treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl 2 at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl 2 under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca 2+ and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Studies of "pre β" high density lipoprotein (HDL) and reconstituted HDL (rHDL) have contributed to our understanding of the Reverse Cholesterol Transport pathway. The relative ease with which discoidal rHDL can be generated in vitro has led to novel applications including a) infusion of rHDL into patients to promote regression of atherosclerosis; b) use of rHDL as a miniature membrane for integration of transmembrane proteins in a native-like conformation and c) incorporation of hydrophobic bioactive molecules into rHDL, creating a delivery device. The present review is focused on bioactive agent containing rHDL. The broad array of hydrophobic bioactive molecules successfully incorporated into these particles is discussed, as well as the use of natural lipids and synthetic lipid analogs to confer distinctive binding activity. This technology remains in its infancy with the full potential of these simple, yet elegant, nanoparticles still to be discovered., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognitioncomplex (ORC). The ORC-DNA complex directs the formation of origins, the specific chromosomal regions where DNA synthesis initiates. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (Forkhead homolog) protein and its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. Several of the FHA-dependent origins examined required a distinct Fkh1 binding site located 5' of and proximal to their ORC sites (5'-FKH-T site). Genetic and molecular experiments provided evidence that the Fkh1-FHA domain promoted origin activity directly through Fkh1 binding to this 5' FKH-T site. Nucleotide substitutions within two relevant origins that enhanced their ORC-DNA affinity bypassed the requirement for their 5' FKH-T sites and for the Fkh1-FHA domain. Significantly, assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism was relevant at ∼25% of yeast origins. Thus, the FHA domain of the conserved cell-cycle transcription factor Fkh1 enhanced origin selection in yeast at the level of ORC-origin binding., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Novel solutions are needed for expediting margin assessment to guide basal cell carcinoma (BCC) surgeries. Ex vivo fluorescence confocal microscopy (FCM) is starting to be used in freshly excised surgical specimens to examine BCC margins in real time. Training and educational process are needed for this novel technology to be implemented into clinic., Objective: To test a training and reading process, and measure diagnostic accuracy of clinicians with varying expertise level in reading ex vivo FCM images., Methods: An international three-center study was designed for training and reading to assess BCC surgical margins and residual subtypes. Each center included a lead dermatologic/Mohs surgeon (clinical developer of FCM) and three additional readers (dermatologist, dermatopathologist, dermatologic/Mohs surgeon), who use confocal in clinical practice. Testing was conducted on 30 samples., Results: Overall, the readers achieved 90% average sensitivity, 78% average specificity in detecting residual BCC margins, showing high and consistent diagnostic reading accuracy. Those with expertise in dermatologic surgery and dermatopathology showed the strongest potential for learning to assess FCM images., Limitations: Small dataset, variability in mosaic quality between centers., Conclusion: Suggested process is feasible and effective. This process is proposed for wider implementation to facilitate wider adoption of FCM to potentially expedite BCC margin assessment to guide surgery in real time., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Electroconvulsive therapy (ECT) is a rapidly effective treatment for severe depression. Treatment with right unilateral (RUL) or bitemporal (BT) ECT may explain individual differences in speed of ECT effectiveness. There is limited evidence for demographic and clinical factors that predict speed of response and remission with ECT. We aimed to investigate differences in speed of improvement as well as achieving response and remission between twice-weekly brief-pulse high-dose (6 × seizure threshold) RUL ECT and moderate-dose (1.5 × seizure threshold) BT ECT. We also explored demographic and clinical characteristics that predict speed of response and remission., Methods: Weekly 24-item Hamilton Depression Rating Scale scores were assessed among patients with severe depression who participated in the EFFECT-Dep trial (ISRCTN23577151). Speed of improvement in patients randomised to RUL ECT (n = 69) or BT ECT (n = 69) was compared using independent sample t tests. Pearson's chi-square and Fisher's exact tests compared proportions of responders and remitters at each weekly assessment. Predictors of speed of response and remission were explored using Cox regression analyses., Results: There were no significant differences between RUL and BT ECT in speed of improvement, response or remission. Exploratory analyses indicated that speed of response and remission were not predicted by a wide variety of demographic and clinical characteristics., Conclusion: ECT electrode placement did not have predictive value when determining speed of improvement, response and remission with ECT. Other clinical factors, such as cognitive side-effects, may be more relevant when making the clinical choice between RUL and BT ECT., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In 2018, Cape Town, South Africa, nearly ran out of water. That this has not yet happened is in large part due to the water-saving efforts of its citizens. It is highly likely that this situation will be repeated in Cape Town and that similar situations will be experienced by major cities in other parts of the world. Efforts to save water should thus continue and the lessons learned in Cape Town should be shared. The functioning of Veterinary Services during a drought is affected in the same way as any business, in terms of running an office, but veterinary professionals face an increased risk of exposure to pathogens, compared to that of many occupations, and of veterinary officials becoming disease vectors. One component of Veterinary Services is veterinary laboratory services. Laboratory procedures rely heavily on water and, without advance planning, a laboratory's function can be severely limited by a restricted water supply. In many cases, innovative water-saving techniques can be used to reduce water use substantially without compromising the quality of the services offered. Here, the authors share their experiences and some lessons learned while working in Veterinary Services in the Western Cape province of South Africa.

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication., Competing Interests: The authors declare no competing interest.

Doxorubicin (DOX) is a potent anticancer agent that binds both DNA and cardiolipin (CL). To investigate DOX binding to CL versus DNA, aqueous soluble, CL-enriched nanoparticles, termed nanodisks (ND), were employed. Upon incubation with CL-ND, but not with phosphatidylcholine ND, DOX binding was detected. DOX binding to CL-ND was sensitive to buffer pH and ionic strength. To investigate if a DOX binding preference for DNA versus CL-ND exists, an agarose gel-based dye binding assay was developed. Under conditions wherein the commercial fluorescent dye, GelRed, detects a 636 bp DNA template following electrophoresis, DOX staining failed to visualize this DNA band. Incubation of the template DNA with DOX prior to electrophoresis resulted in a DOX concentration-dependent attenuation of GelRed staining intensity. When the template DNA was pre-incubated with equivalent amounts of free DOX or DOX-CL-ND, no differences in the extent of GelRed staining intensity attenuation were noted. When DOX was incubated with DNA alone, or a mixture of DNA and CL-ND, the extent of DOX-induced GelRed staining intensity attenuation was equivalent. Thus, DOX has a binding preference for DNA versus CL and, moreover, DOX-CL-ND offer a potential strategy to prevent DOX-induced cardiotoxicity while not affecting its affinity for DNA., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct tissue-specific developmental defects. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, MCM5, and DONSON) are mutated in individuals diagnosed with MGS. The essential role of ORC is to license origins during the G1 phase of the cell cycle, but ORC has also been implicated in several nonreplicative functions. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in genes encoding ORC lead to the tissue-specific defects associated with the disease. To begin to address these issues, we used Cas9-mediated genome engineering to generate a Drosophila melanogaster model of individuals carrying a specific Meier-Gorlin syndrome mutation in ORC4 along with control strains. Together these strains provide the first metazoan model for an MGS mutation in which the mutation was engineered at the endogenous locus along with precisely defined control strains. Flies homozygous for the engineered MGS allele reach adulthood, but with several tissue-specific defects. Genetic analysis revealed that this Orc4 allele was a hypomorph. Mutant females were sterile, and phenotypic analyses suggested that defects in DNA replication was an underlying cause. By leveraging the well-studied Drosophila system, we provide evidence that a disease-causing mutation in Orc4 disrupts DNA replication, and we propose that in individuals with MGS defects arise preferentially in tissues with a high-replication demand., (Copyright © 2020 by the Genetics Society of America.)

Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro , while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Miniature membranes comprised of tetramyristoylcardiolipin (CL) and apolipoprotein (apo) A-I, termed nanodisks (ND), are stable, aqueous soluble, reconstituted high density lipoproteins. When CL ND, but not dimyristoylphosphatidylcholine (PC) ND, were incubated with CaCl 2 , a concentration dependent increase in sample turbidity occurred, consistent with CL undergoing a bilayer to non-bilayer transition. To assess the cation specificity of this reaction, CL ND were incubated with various mono- and divalent cations. Whereas monovalent cations had no discernable effect, MgCl 2 and SrCl 2 induced a response similar to CaCl 2 . When ND were formulated using different weight ratios of CL and PC, those possessing 100% CL or 75% CL remained susceptible to CaCl 2 induced sample turbidity development while ND possessing 50% CL displayed reduced susceptibility. ND comprised of 25% CL and 75% PC were unaffected by CaCl 2 under these conditions. SDS PAGE analysis of insoluble material generated by incubation of CL ND with CaCl 2 revealed that nearly all apoA-I was recovered in the insoluble fraction along with CL. One h after addition of EDTA to CaCl 2 -treated CL ND, sample clarity was restored. Collectively, the data are consistent with a model wherein Ca 2+ forms a bidentate interaction with anionic phosphates in the polar head group of CL. As phosphate group repositioning occurs to maximize Ca 2+ binding, CL acyl chains reposition, accentuating the conical shape of CL to an extent that is incompatible with the ND bilayer structure., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biochemical, proteomic, and epigenetic studies of chromatin rely on the ability to efficiently isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae , which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state and is applicable for both parallel multisample spin-column purification and large-scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes and features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy. The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies and serve as a mild, acid- and detergent-free sample preparation method for MS analysis., (© 2018 Kuznetsov et al.)

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins., Competing Interests: The authors have declared that no competing interests exist.

Environmental managers in the United States and elsewhere are increasingly perceiving dam removal as a critical tool for river restoration and enhancing watershed resilience. In New England, over 125 dams have been dismantled for ecological and economic rationales. A surprising number of these removals, including many that are ongoing, have generated heated conflicts between restoration proponents and local communities who value their dammed landscapes. Using a comparative case study approach, we examine the environmental conflict around efforts to remove six dams in New England. Each of these removal efforts followed quite different paths and resultant outcomes: successful removal, stalled removal, and failure despite seemingly favorable institutional conditions. Lengthy conflicts often transpired in instances where removals occurred, but these were successfully arbitrated by paying attention to local historical-geographical conditions conducive to removal and by brokering effective compromises between dam owners and the various local actors and stakeholders involved in the removal process. Yet our results across all cases suggest that these are necessary, but not sufficient conditions for restoration through dam removal since a similar set of conditions typified cases where removals are continuously stalled or completely halted. Scholars examining the intersection between ecological restoration and environmental politics should remain vigilant in seeking patterns and generalities across cases of environmental conflict in order to promote important biophysical goals, but must also remain open to the ways in which those goals are thwarted and shaped by conflicts that are deeply contingent on historical-geographical conditions and broader institutional networks of power and influence.

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.

Background: Fathers are at risk for depression during a mother's gestation and postpartum. Assessment, detection, and treatment are hampered by the lack of consensus on this issue. The purpose of this study was to reach expert consensus through the Delphi method on the defining factors of depression in peripartum fathers., Methods: Purposive sampling resulted in the surveying of 14 international expert panelists. The study used a modified Delphi approach in which experts participated in two rounds of open-ended and scale questionnaires, followed by two rounds of opportunities to adjust their responses and/or comment on evolving data until consensus was achieved., Results: Experts responded to 10 questions on terminology, diagnostics, symptomology, risk/protective factors, biological factors, assessment tools/protocol, cost implications, and key stakeholders. Of these 10 questions presented for discussion, the analysis resulted in 197 coded themes. Consensus was met for 119 of the 197 coded responses (60.41%)., Limitations: Diversity of opinion within this Delphi Study was excluded for the sake of consensus. Regression to the mean may have occurred after continuous surveying and when evolving results were shared with panelists. Critics of Delphi methodologies have pointed to the issue of small expert samples typically used and the subjectivity of "expert.", Conclusion: Consensus identified diagnostic criteria and symptomology that differentiates the paternal experience of peripartum depression. Experts indicated the importance of a father's social context, biological risk factors, limitations of current assessment tools, key stakeholders, and potential financial costs. Stakeholders on this issue would benefit from translating consensus into assessment and treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.

The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements., (Copyright © 2016 Hoggard et al.)