Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Herein, we show that dual deficiency for hematopoietic RXRα and RXRß induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRα and RXRß maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRα;RXRß-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRα;RXRß-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.

Ferrostatin-1 modulates dysregulated kidney lipids in acute kidney injury.

Ferroptosis, a form of regulated necrosis characterized by peroxidation of lipids such as arachidonic acid-containing phosphatidylethanolamine (PE), contributes to the pathogenesis of acute kidney injury (AKI). We have characterized the kidney lipidome in an experimental nephrotoxic AKI induced in mice using folic acid and assessed the impact of the ferroptosis inhibitor Ferrostatin-1. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to assess kidney lipidomics and it discriminated between glomeruli, medulla, and cortex in control kidneys, AKI kidneys, and AKI + Ferrostatin-1 kidneys. Out of 139 lipid species from 16 classes identified, 29 (20.5%) showed significant differences between control and AKI at 48 h. Total PE and lyso-sulfatide species decreased, while phosphatidylinositol (PI) species increased in AKI. Dysregulated mRNA levels for Pemt, Pgs1, Cdipt, and Tamm41, relevant to lipid metabolism, were in line with the lipid changes observed. Ferrostatin-1 prevented AKI and some AKI-associated changes in lipid levels, such as the decrease in PE and lyso-sulfatide species, without changing the gene expression of lipid metabolism enzymes. In conclusion, changes in the kidney lipid composition during nephrotoxic AKI are associated with differential gene expression of lipid metabolism enzymes and are partially prevented by Ferrostatin-1. © 2022 The Pathological Society of Great Britain and Ireland.

DNGR-1 limits Flt3L-mediated antitumor immunity by restraining tumor-infiltrating type I conventional dendritic cells.

BACKGROUND: Conventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a dead cell-sensing receptor highly restricted to cDC1s. DNGR-1 has been involved in both cross-presentation of dead cell-associated antigens and processes of disease tolerance, but its role in antitumor immunity has not been clarified yet. METHODS: B16 and MC38 tumor cell lines were inoculated subcutaneously into wild-type (WT) and DNGR-1-deficient mice. To overexpress Flt3L systemically, we performed gene therapy through the hydrodynamic injection of an Flt3L-encoding plasmid. To characterize the immune response, we performed flow cytometry and RNA-Seq of tumor-infiltrating cDC1s. RESULTS: Here, we found that cross-presentation of tumor antigens in the steady state was DNGR-1-independent. However, on Flt3L systemic overexpression, tumor growth was delayed in DNGR-1-deficient mice compared with WT mice. Of note, this protection was recapitulated by anti-DNGR-1-blocking antibodies in mice following Flt3L gene therapy. This improved antitumor immunity was associated with Batf3-dependent enhanced accumulation of CD8+ T cells and cDC1s within tumors. Mechanistically, the deficiency in DNGR-1 boosted an Flt3L-induced specific inflammatory gene signature in cDC1s, including Ccl5 expression. Indeed, the increased infiltration of cDC1s within tumors and their protective effect rely on CCL5/CCR5 chemoattraction. Moreover, FLT3LG and CCL5 or CCR5 gene expression signatures correlate with an enhanced cDC1 signature and a favorable overall survival in patients with cancer. Notably, cyclophosphamide elevated serum Flt3L levels and, in combination with the absence of DNGR-1, synergized against tumor growth. CONCLUSION: DNGR-1 limits the accumulation of tumor-infiltrating cDC1s promoted by Flt3L. Thus, DNGR-1 blockade may improve antitumor immunity in tumor therapy settings associated to high Flt3L expression.

Gene therapy with secreted acid alpha-glucosidase rescues Pompe disease in a novel mouse model with early-onset spinal cord and respiratory defects.

BACKGROUND: Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. METHODS: Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). FINDINGS: Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. INTERPRETATION: These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. FUNDING: This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics.

Sox17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels.

RATIONALE: The molecular mechanisms underlying the formation of coronary arteries during development and during cardiac neovascularization after injury are poorly understood. However, a detailed description of the relevant signaling pathways and functional TFs (transcription factors) regulating these processes is still incomplete. OBJECTIVE: The goal of this study is to identify novel cardiac transcriptional mechanisms of coronary angiogenesis and vessel remodeling by defining the molecular signatures of coronary vascular endothelial cells during these complex processes. METHODS AND RESULTS: We demonstrate that Nes-gfp and Nes-CreERT2 transgenic mouse lines are novel tools for studying the emergence of coronary endothelium and targeting sprouting coronary vessels (but not ventricular endocardium) during development. Furthermore, we identify Sox17 as a critical TF upregulated during the sprouting and remodeling of coronary vessels, visualized by a specific neural enhancer from the Nestin gene that is strongly induced in developing arterioles. Functionally, genetic-inducible endothelial deletion of Sox17 causes deficient cardiac remodeling of coronary vessels, resulting in improper coronary artery formation. CONCLUSIONS: We demonstrated that Sox17 TF regulates the transcriptional activation of Nestin's enhancer in developing coronary vessels while its genetic deletion leads to inadequate coronary artery formation. These findings identify Sox17 as a critical regulator for the remodeling of coronary vessels in the developing heart.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade.

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

PGC-1α deficiency causes spontaneous kidney inflammation and increases the severity of nephrotoxic AKI.

PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α, PPARGC1A) regulates the expression of genes involved in energy homeostasis and mitochondrial biogenesis. Here we identify inactivation of the transcriptional regulator PGC-1α as a landmark for experimental nephrotoxic acute kidney injury (AKI) and describe the in vivo consequences of PGC-1α deficiency over inflammation and cell death in kidney injury. Kidney transcriptomic analyses of WT mice with folic acid-induced AKI revealed 1398 up- and 1627 downregulated genes. Upstream transcriptional regulator analyses pointed to PGC-1α as the transcription factor potentially driving the observed expression changes with the highest reduction in activity. Reduced PGC-1α expression was shared by human kidney injury. Ppargc1a-/- mice had spontaneous subclinical kidney injury characterized by tubulointerstitial inflammation and increased Ngal expression. Upon AKI, Ppargc1a-/- mice had lower survival and more severe loss of renal function, tubular injury, and reduction in expression of mitochondrial PGC-1α-dependent genes in the kidney, and an earlier decrease in mitochondrial mass than WT mice. Additionally, surviving Ppargc1a-/- mice showed higher rates of tubular cell death, compensatory proliferation, expression of proinflammatory cytokines, NF-κB activation, and interstitial inflammatory cell infiltration. Specifically, Ppargc1a-/- mice displayed increased M1 and decreased M2 responses and expression of the anti-inflammatory cytokine IL-10. In cultured renal tubular cells, PGC-1α targeting promoted spontaneous cell death and proinflammatory responses. In conclusion, PGC-1α inactivation is a key driver of the gene expression response in nephrotoxic AKI and PGC-1α deficiency promotes a spontaneous inflammatory kidney response that is magnified during AKI. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978.

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.

Nickel-resistance determinants in Acidiphilium sp. PM identified by genome-wide functional screening.

Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.

Role of vermicompost chemical composition, microbial functional diversity, and fungal community structure in their microbial respiratory response to three pesticides.

The relationships between vermicompost chemical features, enzyme activities, community-level physiological profiles (CLPPs), fungal community structures, and its microbial respiratory response to pesticides were investigated. Fungal community structure of vermicomposts produced from damaged tomato fruits (DT), winery wastes (WW), olive-mill waste and biosolids (OB), and cattle manure (CM) were determined by denaturing gradient gel electrophoresis of 18S rDNA. MicroResp™ was used for assessing vermicompost CLPPs and testing the microbial response to metalaxyl, imidacloprid, and diuron. Vermicompost enzyme activities and CLPPs indicated that WW, OB, and DT had higher microbial functional diversity than CM. The microbiota of the former tolerated all three pesticides whereas microbial respiration in CM was negatively affected by metalaxyl and imidacloprid. The response of vermicompost microbiota to the fungicide metalaxyl was correlated to its fungal community structure. The results suggest that vermicomposts with higher microbial functional diversity can be useful for the management of pesticide pollution in agriculture.

Environmental transcriptome analysis reveals physiological differences between biofilm and planktonic modes of life of the iron oxidizing bacteria Leptospirillum spp. in their natural microbial community.

BACKGROUND: Extreme acidic environments are characterized by their high metal content and lack of nutrients (oligotrophy). Macroscopic biofilms and filaments usually grow on the water-air interface or under the stream attached to solid substrates (streamers). In the Río Tinto (Spain), brown filaments develop under the water stream where the Gram-negative iron-oxidizing bacteria Leptospirillum spp. (L. ferrooxidans and L. ferriphilum) and Acidithiobacillus ferrooxidans are abundant. These microorganisms play a critical role in bioleaching processes for industrial (biominery) and environmental applications (acid mine drainage, bioremediation). The aim of this study was to investigate the physiological differences between the free living (planktonic) and the sessile (biofilm associated) lifestyles of Leptospirillum spp. as part of its natural extremely acidophilic community. RESULTS: Total RNA extracted from environmental samples was used to determine the composition of the metabolically active members of the microbial community and then to compare the biofilm and planktonic environmental transcriptomes by hybridizing to a genomic microarray of L. ferrooxidans. Genes up-regulated in the filamentous biofilm are involved in cellular functions related to biofilm formation and maintenance, such as: motility and quorum sensing (mqsR, cheAY, fliA, motAB), synthesis of cell wall structures (lnt, murA, murB), specific proteases (clpX/clpP), stress response chaperons (clpB, clpC, grpE-dnaKJ, groESL), etc. Additionally, genes involved in mixed acid fermentation (poxB, ackA) were up-regulated in the biofilm. This result, together with the presence of small organic acids like acetate and formate (1.36 mM and 0.06 mM respectively) in the acidic (pH 1.8) water stream, suggests that either L. ferrooxidans or other member of the microbial community are producing acetate in the acidophilic biofilm under microaerophilic conditions. CONCLUSIONS: Our results indicate that the acidophilic filaments are dynamic structures in which different mechanisms for biofilm formation/dispersion are operating. Specific transcriptomic fingerprints can be inferred for both planktonic and sessile cells, having the former a more active TCA cycle, while the mixed acid fermentation process dominate in the latter. The excretion of acetate may play a relevant ecological role as a source of electron donor for heterotrophic Fe3+ reducers like some Alphaproteobacteria, Acidobacterium spp. and Sulfobacillus spp., also present in the biofilm. Additionally, acetate may have a negative effect on bioleaching by inhibiting the growth of chemolithotrophic bacteria.

Genome-wide analysis reveals increased levels of transcripts related with infectivity in peanut lectin non-agglutinated promastigotes of Leishmania infantum.

Metacyclic promastigotes are transmitted during bloodmeals after development inside the gut of the sandfly vector. The isolation from axenic cultures of procyclic and metacyclic promastigotes by peanut lectin agglutination followed by differential centrifugation is controversial in Leishmania infantum. The purpose of this study has been to isolate both fractions simultaneously from the same population in stationary phase of axenic culture and compare their expression profiles by whole-genome shotgun DNA microarrays. The 317 genes found with meaningful values of stage-specific regulation demonstrate that negative selection of metacyclic promastigotes by PNA agglutination is feasible in L. infantum and both fractions can be isolated. This subpopulation up-regulates a cysteine peptidase A and several genes involved in lipophosphoglycan, proteophosphoglycan and glycoprotein biosynthesis, all related with infectivity. In fact, we have confirmed the increased infection rate of PNA(-) promastigotes by U937 human cell line infection experiments. These data support that metacyclic promastigotes are related with infectivity and the lack of agglutination with PNA is a phenotypic marker for this subpopulation.

[Hemithyroidectomy in a unilateral goiter: a valid therapeutic option. Review of the contralateral hemithyroid ten years after treatment]. / Hemitiroidectomía en el bocio unilateral: una opción terapéutica válida. Revisión del hemitiroides contralateral a los diez años del tratamiento.

INTRODUCTION: Nodular goiter (NG) is frequent among the general population and is considered a diffuse disease. Although NGs are rarely unilateral, they pose a dilemma in terms of the extent of the thyroidectomy. The aim of the present study was to evaluate the remaining thyroid in patients with NG compared with those with follicular adenoma who underwent hemithyroidectomy. PATIENTS AND METHODS: Patients who underwent surgery for unilateral NG with over 10 years of postoperative follow-up and normal findings on ultrasonography of the contralateral thyroid lobe were selected to form the study group (SG). Patients with follicular adenoma (with normal contralateral ultrasonography) who underwent hemithyroidectomy during the same period were selected to form the control group (CG). The selected patients underwent clinical, laboratory and ultrasound examinations. Both groups were compared statistically. No significant differences were found in age, gender, anesthetic risk, side, postoperative complications, length of hospital stay, or postoperative outcome. RESULTS: Less than 10% of the patients reported symptoms, and all symptoms were of little significance. Ultrasonographic nodules were found in the remaining thyroid lobe in 70% of patients in the SG and in 60% of those in the CG, with no statistically significant differences. The mean size of the largest nodule was 13.58 +/- 8.01 in the SG and 9.15 +/- 5.93 in the GC (p = 0.048). No differences were found in the anterior-posterior, transverse or longitudinal diameters of the remaining lobe. None of the patients underwent reintervention for nodular disease. CONCLUSIONS: After hemithyroidectomy, both groups of patients developed nodules in the remaining thyroid lobe, with no statistically significant differences. Hemithyroidectomy due to unilateral NG involves less risk to the patient and therefore we consider it to be a valid option. Long-term ultrasonographic follow-up seems advisable.