Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division.

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3' untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3' end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.

Polymorphisms in MIR27A Associated with Early-Onset Toxicity in Fluoropyrimidine-Based Chemotherapy

PURPOSE The microRNA miR-27a was recently shown to directly regulate dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. A common polymorphism (rs895819A>G) in the miR-27a genomic region (MIR27A) was associated with reduced DPD activity in healthy volunteers, but the clinical relevance of this effect is still unknown. Here, we assessed the association of MIR27A germline variants with early-onset fluoropyrimidine toxicity. EXPERIMENTAL DESIGN MIR27A was sequenced in 514 patients with cancer receiving fluoropyrimidine-based chemotherapy. Associations of MIR27A polymorphisms with early-onset (cycles 1-2) fluoropyrimidine toxicity were assessed in the context of known risk variants in the DPD gene (DPYD) and additional covariates associated with toxicity. RESULTS The association of rs895819A>G with early-onset fluoropyrimidine toxicity was strongly dependent on DPYD risk variant carrier status (Pinteraction = 0.0025). In patients carrying DPYD risk variants, rs895819G was associated with a strongly increased toxicity risk [OR, 7.6; 95% confidence interval (CI), 1.7-34.7; P = 0.0085]. Overall, 71% (12/17) of patients who carried both rs895819G and a DPYD risk variant experienced severe toxicity. In patients without DPYD risk variants, rs895819G was associated with a modest decrease in toxicity risk (OR, 0.62; 95% CI, 0.43-0.9; P = 0.012). CONCLUSIONS These results indicate that miR-27a and rs895819A>G may be clinically relevant for further toxicity risk stratification in carriers of DPYD risk variants. Our data suggest that direct suppression of DPD by miR-27a is primarily relevant in the context of fluoropyrimidine toxicity in patients with reduced DPD activity. However, miR-27a regulation of additional targets may outweigh its effect on DPD in patients without DPYD risk variants.

Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Comparative effectiveness of immediate antiretroviral therapy versus CD4-based initiation in HIV-positive individuals in high-income countries: observational cohort study.

BACKGROUND Recommendations have differed nationally and internationally with respect to the best time to start antiretroviral therapy (ART). We compared effectiveness of three strategies for initiation of ART in high-income countries for HIV-positive individuals who do not have AIDS: immediate initiation, initiation at a CD4 count less than 500 cells per μL, and initiation at a CD4 count less than 350 cells per μL. METHODS We used data from the HIV-CAUSAL Collaboration of cohort studies in Europe and the USA. We included 55 826 individuals aged 18 years or older who were diagnosed with HIV-1 infection between January, 2000, and September, 2013, had not started ART, did not have AIDS, and had CD4 count and HIV-RNA viral load measurements within 6 months of HIV diagnosis. We estimated relative risks of death and of death or AIDS-defining illness, mean survival time, the proportion of individuals in need of ART, and the proportion of individuals with HIV-RNA viral load less than 50 copies per mL, as would have been recorded under each ART initiation strategy after 7 years of HIV diagnosis. We used the parametric g-formula to adjust for baseline and time-varying confounders. FINDINGS Median CD4 count at diagnosis of HIV infection was 376 cells per μL (IQR 222-551). Compared with immediate initiation, the estimated relative risk of death was 1·02 (95% CI 1·01-1·02) when ART was started at a CD4 count less than 500 cells per μL, and 1·06 (1·04-1·08) with initiation at a CD4 count less than 350 cells per μL. Corresponding estimates for death or AIDS-defining illness were 1·06 (1·06-1·07) and 1·20 (1·17-1·23), respectively. Compared with immediate initiation, the mean survival time at 7 years with a strategy of initiation at a CD4 count less than 500 cells per μL was 2 days shorter (95% CI 1-2) and at a CD4 count less than 350 cells per μL was 5 days shorter (4-6). 7 years after diagnosis of HIV, 100%, 98·7% (95% CI 98·6-98·7), and 92·6% (92·2-92·9) of individuals would have been in need of ART with immediate initiation, initiation at a CD4 count less than 500 cells per μL, and initiation at a CD4 count less than 350 cells per μL, respectively. Corresponding proportions of individuals with HIV-RNA viral load less than 50 copies per mL at 7 years were 87·3% (87·3-88·6), 87·4% (87·4-88·6), and 83·8% (83·6-84·9). INTERPRETATION The benefits of immediate initiation of ART, such as prolonged survival and AIDS-free survival and increased virological suppression, were small in this high-income setting with relatively low CD4 count at HIV diagnosis. The estimated beneficial effect on AIDS is less than in recently reported randomised trials. Increasing rates of HIV testing might be as important as a policy of early initiation of ART. FUNDING National Institutes of Health.

Dihydropyrimidinase and β-ureidopropionase gene variation and severe fluoropyrimidine-related toxicity

AIMS To assess the association of DPYS and UPB1 genetic variation, encoding the catabolic enzymes downstream of dihydropyrimidine dehydrogenase, with early-onset toxicity from fluoropyrimidine-based chemotherapy. PATIENTS & METHODS The coding and exon-flanking regions of both genes were sequenced in a discovery subset (164 patients). Candidate variants were genotyped in the full cohort of 514 patients. RESULTS & CONCLUSIONS Novel rare deleterious variants in DPYS (c.253C > T and c.1217G > A) were detected once each in toxicity cases and may explain the occurrence of severe toxicity in individual patients, and associations of common variants in DPYS (c.1-1T > C: padjusted = 0.003; OR = 2.53; 95% CI: 1.39-4.62, and c.265-58T > C: padjusted = 0.039; OR = 0.61; 95% CI: 0.38-0.97) with 5-fluorouracil toxicity were replicated.

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H+-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.