Stearoyl-CoA desaturase 1 deficiency increases CTP:choline cytidylyltransferase translocation into the membrane and enhances phosphatidylcholine synthesis in liver

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.     

Membrane topology of mouse stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.     

Restoration of the CD4 T cell compartment after long-term highly active antiretroviral therapy without phenotypical signs of accelerated immunological aging

It remains uncertain whether full T cell reconstitution can be established in HIV-infected children and adults with long-term sustained virological control by highly active antiretroviral therapy (HAART). In this study, we comprehensively analyzed various phenotypical markers of CD4 T cell recovery. In addition to measuring T cell activation and proliferation markers, CD4 T cell generation and aging of the CD4 T cell compartment were assessed by measuring TCR excision circles and the fraction of CD31-expressing naive CD4 T cells. In all children and in adults with relatively high CD4 T cell counts at start of therapy (>200 cells/microl), total CD4 T cell numbers normalized within 1 year of therapy. After long-term HAART (4.4-9.6 years), naive CD4 T cell counts had normalized in both groups. Although in adults with low baseline CD4 T cell counts (<200 cells/microl) total CD4 T cell numbers normalized eventually after at least 7 years of HAART, naive CD4 T cell counts had still not recovered. TCR excision circle data showed that thymic T cell production contributed to naive T cell recovery at all ages. The fraction of CD31-expressing naive CD4 T cells was found to be normal, suggesting that the CD4 T cell repertoire was diverse after long-term HAART. Hence, under sustained viral suppression during long-term HAART, the T cell compartment has the potential to fully recover by generating new naive T cells both in children and in adults with high baseline CD4 T cells counts. Irrespective of baseline CD4 T cell counts, reconstitution occurred without a significant effect on T cell aging as reflected by markers for replicative history.     

Autophagy activity is associated with membranous sodium iodide symporter expression and clinical response to radioiodine therapy in non-medullary thyroid cancer

Although non-medullary thyroid cancer (NMTC) generally has a good prognosis, 30–40% of patients with distant metastases develop resistance to radioactive iodine (RAI) therapy due to tumor dedifferentiation. For these patients, treatment options are limited and prognosis is poor. In the present study, expression and activity of autophagy was assessed in large sets of normal, benign and malignant tissues and was correlated with pathology, SLC5A5/hNIS (solute carrier family 5 member 5) protein expression, and with clinical response to RAI ablation therapy in NMTC patients. Fluorescent immunostaining for the autophagy marker LC3 was performed on 100 benign and 80 malignant thyroid tissues. Semiquantitative scoring was generated for both diffuse LC3-I intensity and number of LC3-II-positive puncta and was correlated with SLC5A5 protein expression and clinical parameters. Degree of diffuse LC3-I intensity and number of LC3-II-positive puncta scoring were not discriminative for benign vs. malignant thyroid lesions. Interestingly, however, in NMTC patients significant associations were observed between diffuse LC3-I intensity and LC3-II-positive puncta scoring on the one hand and clinical response to RAI therapy on the other hand (odds ratio [OR] = 3.13, 95% confidence interval [CI] =1.91–5.12, P = 0.01; OR = 5.68, 95%CI = 3.02–10.05, P = 0.002, respectively). Mechanistically, the number of LC3-II-positive puncta correlated with membranous SLC5A5 expression (OR = 7.71, 95%CI = 4.15–11.75, P<0.001), number of RAI treatments required to reach remission (P = 0.014), cumulative RAI dose (P = 0.026) and with overall remission and recurrence rates (P = 0.031). In conclusion, autophagy activity strongly correlates with clinical response of NMTC patients to RAI therapy, potentially by its capacity to maintain tumor cell differentiation and to preserve functional iodide uptake.     

Reconciling Longitudinal Naive T-Cell and TREC Dynamics during HIV-1 Infection

Naive T cells in untreated HIV-1 infected individuals have a reduced T-cell receptor excision circle (TREC) content. Previous mathematical models have suggested that this is due to increased naive T-cell division. It remains unclear, however, how reduced naive TREC contents can be reconciled with a gradual loss of naive T cells in HIV-1 infection. We performed longitudinal analyses in humans before and after HIV-1 seroconversion, and used a mathematical model to investigate which processes could explain the observed changes in naive T-cell numbers and TRECs during untreated HIV-1 disease progression. Both CD4⁺ and CD8⁺ naive T-cell TREC contents declined biphasically, with a rapid loss during the first year and a much slower loss during the chronic phase of infection. While naive CD8⁺ T-cell numbers hardly changed during follow-up, naive CD4⁺ T-cell counts continually declined. We show that a fine balance between increased T-cell division and loss in the peripheral naive T-cell pool can explain the observed short- and long-term changes in TRECs and naive T-cell numbers, especially if T-cell turnover during the acute phase is more increased than during the chronic phase of infection. Loss of thymic output, on the other hand, does not help to explain the biphasic loss of TRECs in HIV infection. The observed longitudinal changes in TRECs and naive T-cell numbers in HIV-infected individuals are most likely explained by a tight balance between increased T-cell division and death, suggesting that these changes are intrinsically linked in HIV infection.     

Focus on Quality: Investigating Residents’ Learning Climate Perceptions

Background

A department’s learning climate is known to contribute to the quality of postgraduate medical education and, as such, to the quality of patient care provided by residents. However, it is unclear how the learning climate is perceived over time.

Objectives

This study investigated whether the learning climate perceptions of residents changed over time.

Methods

The context for this study was residency training in the Netherlands. Between January 2012 and December 2014, residents from 223 training programs in 39 hospitals filled out the web-based Dutch Residency Educational Climate Test (D-RECT) to evaluate their clinical department’s learning climate. Residents had to fill out 35 validated questions using a five point Likert-scale. We analyzed data using generalized linear mixed (growth) models.

Results

Overall, 3982 D-RECT evaluations were available to investigate our aim. The overall mean D-RECT score was 3.9 (SD = 0.3). The growth model showed an increase in D-RECT scores over time (b = 0.03; 95% CI: 0.01–0.06; p < 0.05).

Conclusions

The observed increase in D-RECT scores implied that residents perceived an improvement in the learning climate over time. Future research could focus on factors that facilitate or hinder learning climate improvement, and investigate the roles that hospital governing committees play in safeguarding and improving the learning climate.     

The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Phi = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.     

Optimization of human immunodeficiency virus type 1 envelope glycoproteins with V1/V2 deleted, using virus evolution

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.