Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice

Background and Aims: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate‐limiting step in this pathway. Na+‐coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+‐dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo‐ and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. Approach and Results: Utilizing AnxA6 knockout mice (AnxA6−/−), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6−/− mice liver proliferation and energetic metabolism. Most strikingly, AnxA6−/− mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6−/− mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6−/− mice was the consequence of an impaired alanine‐dependent GNG in AnxA6−/− hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6−/− hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. Conclusions: We conclude that the lack of AnxA6 compromises alanine‐dependent GNG and liver regeneration in mice

Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation

Overview of the instrumentation for the Dark Energy Spectroscopic Instrument

The Dark Energy Spectroscopic Instrument (DESI) embarked on an ambitious 5 yr survey in 2021 May to explore the nature of dark energy with spectroscopic measurements of 40 million galaxies and quasars. DESI will determine precise redshifts and employ the baryon acoustic oscillation method to measure distances from the nearby universe to beyond redshift z > 3.5, and employ redshift space distortions to measure the growth of structure and probe potential modifications to general relativity. We describe the significant instrumentation we developed to conduct the DESI survey. This includes: a wide-field, 3.°2 diameter prime-focus corrector; a focal plane system with 5020 fiber positioners on the 0.812 m diameter, aspheric focal surface; 10 continuous, high-efficiency fiber cable bundles that connect the focal plane to the spectrographs; and 10 identical spectrographs. Each spectrograph employs a pair of dichroics to split the light into three channels that together record the light from 360–980 nm with a spectral resolution that ranges from 2000–5000. We describe the science requirements, their connection to the technical requirements, the management of the project, and interfaces between subsystems. DESI was installed at the 4 m Mayall Telescope at Kitt Peak National Observatory and has achieved all of its performance goals. Some performance highlights include an rms positioner accuracy of better than 0.″1 and a median signal-to-noise ratio of 7 of the [O ii] doublet at 8 × 10−17 erg s−1 cm−2 in 1000 s for galaxies at z = 1.4–1.6. We conclude with additional highlights from the on-sky validation and commissioning, key successes, and lessons learned

The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils

A 10 gene classifier for indeterminate thyroid nodules: development and multicenter accuracy study

Background: In most of the world, diagnostic surgery remains the most frequent approach for indeterminate thyroid cytology. Although several molecular tests are available for testing in centralized commercial laboratories in the United States, there are no available kits for local laboratory testing. The aim of this study was to develop a prototype in vitro diagnostic (IVD) gene classifier for the further characterization of nodules with an indeterminate thyroid cytology. Methods: In a first stage, the expression of 18 genes was determined by quantitative polymerase chain reaction (qPCR) in a broad histopathological spectrum of 114 fresh-tissue biopsies. Expression data were used to train several classifiers by supervised machine learning approaches. Classifiers were tested in an independent set of 139 samples. In a second stage, the best classifier was chosen as a model to develop a multiplexed-qPCR IVD prototype assay, which was tested in a prospective multicenter cohort of fine-needle aspiration biopsies. Results: In tissue biopsies, the best classifier, using only 10 genes, reached an optimal and consistent performance in the ninefold cross-validated testing set (sensitivity 93% and specificity 81%). In the multicenter cohort of fine-needle aspiration biopsy samples, the 10-gene signature, built into a multiplexed-qPCR IVD prototype, showed an area under the curve of 0.97, a positive predictive value of 78%, and a negative predictive value of 98%. By Bayes' theorem, the IVD prototype is expected to achieve a positive predictive value of 64-82% and a negative predictive value of 97-99% in patients with a cancer prevalence range of 20-40%. Conclusions: A new multiplexed-qPCR IVD prototype is reported that accurately classifies thyroid nodules and may provide a future solution suitable for local reference laboratory testing.Biomedical Research Consortium 13CTI-21526P2 CORFO 14IEAT-2867