Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A2 (iPLA2-VIA). Induction of stress with acidic pH, C2-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA2-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA2 (cPLA2α). The results demonstrate that iPLA2-VIA provides fatty acids for TAG synthesis while cPLA2α allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A2 (cPLA2α). Knocking down cPLA2α expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA2α fusion protein (EGFP-cPLA2) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA2α at Ser505. Transfection of a S505A mutant cPLA2α showed that phosphorylation at Ser505 is key for enzyme activity and LD formation. cPLA2α contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA2α inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA2α in the formation of nascent LD from the endoplasmic reticulum

Stand-Alone Artificial Intelligence for Breast Cancer Detection in Mammography: Comparison With 101 Radiologists.

BACKGROUND: Artificial intelligence (AI) systems performing at radiologist-like levels in the evaluation of digital mammography (DM) would improve breast cancer screening accuracy and efficiency. We aimed to compare the stand-alone performance of an AI system to that of radiologists in detecting breast cancer in DM. METHODS: Nine multi-reader, multi-case study datasets previously used for different research purposes in seven countries were collected. Each dataset consisted of DM exams acquired with systems from four different vendors, multiple radiologists' assessments per exam, and ground truth verified by histopathological analysis or follow-up, yielding a total of 2652 exams (653 malignant) and interpretations by 101 radiologists (28 296 independent interpretations). An AI system analyzed these exams yielding a level of suspicion of cancer present between 1 and 10. The detection performance between the radiologists and the AI system was compared using a noninferiority null hypothesis at a margin of 0.05. RESULTS: The performance of the AI system was statistically noninferior to that of the average of the 101 radiologists. The AI system had a 0.840 (95% confidence interval [CI] = 0.820 to 0.860) area under the ROC curve and the average of the radiologists was 0.814 (95% CI = 0.787 to 0.841) (difference 95% CI = -0.003 to 0.055). The AI system had an AUC higher than 61.4% of the radiologists. CONCLUSIONS: The evaluated AI system achieved a cancer detection accuracy comparable to an average breast radiologist in this retrospective setting. Although promising, the performance and impact of such a system in a screening setting needs further investigation

Cell cycle control and HIV-1 susceptibility are linked by CDK6-Dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain-containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67+) primary CD4+ T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability. © 2014 by The American Association of Immunologists, Inc

Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection

3D chromatin remodelling in the germ line modulates genome evolutionary plasticity

Chromosome folding has profound impacts on gene regulation, whose evolutionary consequences are far from being understood. Here we explore the relationship between 3D chromatin remodelling in mouse germ cells and evolutionary changes in genome structure. Using a comprehensive integrative computational analysis, we (i) reconstruct seven ancestral rodent genomes analysing whole-genome sequences of 14 species representatives of the major phylogroups, (ii) detect lineage-specific chromosome rearrangements and (iii) identify the dynamics of the structural and epigenetic properties of evolutionary breakpoint regions(EBRs) throughout mouse spermatogenesis. Our results show that EBRs are devoid of programmed meiotic DNA double-strand breaks (DSBs) and meiotic cohesins in primary spermatocytes, but are associated in post-meiotic cells with sites of DNA damage and functional long-range interaction regions that recapitulate ancestral chromosomal configurations. Overall, we propose a model that integrates evolutionary genome reshuffling with DNA damage response mechanisms and the dynamic spatial genome organisation of germ cell

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of “emerging” LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storag