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Monastrell variety, also known as Mourvèdre, is cultivated mainly in the southeast of Spain in more than 45,000 ha. It is an ancient variety, whose origins are not known with certainty, highly adapted to the dry and warm climate of the southeast Spanish. The existence of a great clonal diversity is well known but, although some clonal selection programs have been carried out (Murcia, Bandol, etc.), the genetic diversity has not been evaluated to date. The measure of this diversity is urgent, since very old vines have been plucked in recent years. We have initiated a project to estimate the genetic diversity of the conserved clones of this variety through GBS (Genotyping by Sequencing). To carry out this analysis, a survey was carried out covering the whole area of Monastrell cultivation in the Alicante Denomination of Origin (D.O.). We selected 46 vines from 46 different vineyards, always prioritizing the oldest vines at each sampling site. The GBS has allowed us to obtain between 0.54 and 1.17 million of reads per sample, of which between 73 and 78% have been mapped to the reference genome developed by the French-Italian consortium (vvinifera.IGGP 12x) with a mapping quality above threshold. More than 100,000 SNPs have been identified, ranging from 14,000 to 43,000 per sample, with a coverage greater than 10X. A high percentage of these variants are polymorphic among Monastrell clones, confirming the existence of significant levels of genetic diversity. In addition, this analysis provides a high number of high quality SNPs well distributed across the genome, suitable for clone genotyping, which will allow the design of strategies to optimize its conservation and use.

Mesenchymal stromal cells (MSCs) are multipotent, progenitor cells that reside in tissues across the human body, including the periodontal ligament (PDL) and gingiva. They are a promising therapeutic tool for various degenerative and inflammatory diseases. However, different heterogeneity levels caused by tissue-to-tissue and donor-to-donor variability, and even intercellular differences within a given MSCs population, restrict their therapeutic potential. There are considerable efforts to decipher these heterogeneity levels using different "omics" approaches, including single-cell transcriptomics. Previous studies applied this approach to compare MSCs isolated from various tissues of different individuals, but distinguishing between donor-to-donor and tissue-to-tissue variability is still challenging. In this study, MSCs were isolated from the PDL and gingiva of 5 periodontally healthy individuals and cultured in vitro. A total of 3,844 transcriptomes were generated using single-cell mRNA sequencing. Clustering across the 2 different tissues per donor identified PDL- and gingiva-specific and tissue-spanning MSCs subpopulations with unique upregulated gene sets. Gene/pathway enrichment and protein-protein interaction (PPI) network analysis revealed differences restricted to several cellular processes between tissue-specific subpopulations, indicating a limited tissue-of-origin variability in MSCs. Gene expression, pathway enrichment, and PPI network analysis across all donors' PDL- or gingiva-specific subpopulations showed significant but limited donor-to-donor differences. In conclusion, this study demonstrates tissue- and donor-specific variabilities in the transcriptome level of PDL- and gingiva-derived MSCs, which seem restricted to specific cellular processes. Identifying tissue-specific and tissue-spanning subpopulations highlights the intercellular differences in dental tissue–derived MSCs. It could be reasonable to control MSCs at a single-cell level to ensure their properties before transplantation. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Ventilator settings typically used for high-frequency oscillatory ventilation (HFO) in adults provide acceptable gas exchange but may not take best advantage of its lung-protective aspects. We provide guidelines for HFO in adults with acute respiratory distress syndrome that should optimize the lung-protective characteristics of this ventilation mode. DESIGN: Roundtable discussions, iterative revisions, and consensus. SETTING: Five academic medical centers. PATIENTS: Not applicable. INTERVENTIONS: Participants addressed how to best maintain ventilation through combinations of oscillation pressure amplitude, frequency, and the use of an endotracheal tube cuff leak, and to maintain oxygenation through combinations of recruitment maneuvers, mean airway pressure, and oxygen concentration. The guiding principles were to provide lung protective ventilation by minimizing the size of tidal volumes, and balance the risks and benefits of lung recruitment and distension. MAIN RESULTS: HFO may provide smaller tidal volumes and more complete lung recruitment than conventional modes. To optimize these features, we recommend use of the maximum pressure-oscillation amplitude coupled with the highest tolerated frequency, targeting a pH of only 7.25-7.35. This will yield a smaller tidal volume than typical HFO settings where frequency is limited to 6 Hz or less and pressure amplitude is submaximal. Lung recruitment can be achieved with the use of recruitment maneuvers, especially during the first several days of HFO. Recruitment may be augmented or sustained with generous mean airway pressures. These may either be chosen from a table of recommended mean airway pressure and oxygen concentration combinations, or individually titrated based on the oxygenation response of each patient. CONCLUSIONS: Modification of the goals and tactics of HFO use may better protect against ventilator-associated lung injury. Further clinical trials are needed to compare the effects on patient outcome of the best use of HFO compared to the most protective use of conventional modes in adult acute respiratory distress syndrome. [ABSTRACT FROM AUTHOR]

The efficacy of costimulation blockade with CTLA4-Ig (belatacept) in transplantation is limited due to T cell-mediated rejection, which also persists after induction with anti-thymocyte globulin (ATG). Here, we investigate why ATG fails to prevent costimulation blockade-resistant rejection and how this barrier can be overcome. ATG did not prevent graft rejection in a murine heart transplant model of CTLA4-Ig therapy and induced a pro-inflammatory cytokine environment. While ATG improved the balance between regulatory T cells (Treg) and effector T cells in the spleen, it had no such effect within cardiac allografts. Neutralizing IL-6 alleviated graft inflammation, increased intragraft Treg frequencies, and enhanced intragraft IL-10 and Th2-cytokine expression. IL-6 blockade together with ATG allowed CTLA4-Ig therapy to achieve long-term, rejection-free heart allograft survival. This beneficial effect was abolished upon Treg depletion. Combining ATG with IL-6 blockade prevents costimulation blockade-resistant rejection, thereby eliminating a major impediment to clinical use of costimulation blockers in transplantation., (© 2024. The Author(s).)

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene ( MECP2 ), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6 . We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.

Alternative transcription increases transcriptome complexity by expression of multiple transcripts per gene. Annotation and quantification of transcripts using short-read sequencing is non-trivial. Long-read sequencing aims at overcoming these problems by sequencing full-length transcripts. Activation of brown adipose tissue (BAT) thermogenesis involves major transcriptomic remodeling and positively affects metabolism via increased energy expenditure. We benchmark Oxford Nanopore Technology (ONT) long-read sequencing protocols to Illumina short-read sequencing assessing alignment characteristics, gene and transcript detection and quantification, differential gene and transcript expression, transcriptome reannotation, and differential transcript usage (DTU). We find ONT sequencing is superior to Illumina for transcriptome reassembly, reducing the risk of false-positive events by unambiguously mapping reads to transcripts. We identified novel isoforms of genes undergoing DTU in cold-activated BAT including Cars2 , Adtrp , Acsl5 , Scp2 , Aldoa , and Pde4d , validated by real-time PCR. The reannotated murine BAT transcriptome established here provides a framework for future investigations into the regulation of BAT., Competing Interests: The authors declare no competing interest., (© 2023 The Author(s).)

Adenosine deaminase acting on RNA ADAR1 promotes A-to-I conversion in double-stranded and structured RNAs. ADAR1 has two isoforms transcribed from different promoters: cytoplasmic ADAR1p150 is interferon-inducible while ADAR1p110 is constitutively expressed and primarily localized in the nucleus. Mutations in ADAR1 cause Aicardi - Goutières syndrome (AGS), a severe autoinflammatory disease associated with aberrant IFN production. In mice, deletion of ADAR1 or the p150 isoform leads to embryonic lethality driven by overexpression of interferon-stimulated genes. This phenotype is rescued by deletion of the cytoplasmic dsRNA-sensor MDA5 indicating that the p150 isoform is indispensable and cannot be rescued by ADAR1p110. Nevertheless, editing sites uniquely targeted by ADAR1p150 remain elusive. Here, by transfection of ADAR1 isoforms into ADAR-less mouse cells we detect isoform-specific editing patterns. Using mutated ADAR variants, we test how intracellular localization and the presence of a Z-DNA binding domain-α affect editing preferences. These data show that ZBDα only minimally contributes to p150 editing-specificity while isoform-specific editing is primarily directed by the intracellular localization of ADAR1 isoforms. Our study is complemented by RIP-seq on human cells ectopically expressing tagged-ADAR1 isoforms. Both datasets reveal enrichment of intronic editing and binding by ADAR1p110 while ADAR1p150 preferentially binds and edits 3'UTRs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Treg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development., Objective: To evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds., Materials and Methods: We used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds., Results: We show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils., Conclusions: BX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases., Competing Interests: With regards to the authors disclosure of potential conflicts of interest we would like to indicate that WP received honoraria from Novartis, Astra Zeneca and Roche, GSK and Bristol-Myers Squibb outside the submitted work and GG reports receiving personal fees from Bencard, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tauber, Kratzer, Schatzlmaier, Smole, Köhler, Rausch, Kranich, Trapin, Neunkirchner, Zabel, Jutz, Steinberger, Gadermaier, Brocker, Stockinger, Derdak and Pickl.)

Previous studies have demonstrated an involvement of chromatin-remodelling SWI/SNF complexes in the development of prostate cancer, suggesting both tumor suppressor and oncogenic activities. SMARCD1/BAF60A, SMARCD2/BAF60B, and SMARCD3/BAF60C are mutually exclusive accessory subunits that confer functional specificity and are components of all known SWI/SNF subtypes. To assess the role of SWI/SNF in prostate tumorigenesis, we studied the functions and functional relations of the SMARCD family members. Performing RNA-seq in LnCAP cells grown in the presence or absence of dihydrotestosterone, we found that the SMARCD proteins are involved in the regulation of numerous hormone-dependent AR-driven genes. Moreover, we demonstrated that all SMARCD proteins can regulate AR-downstream targets in androgen-depleted cells, suggesting an involvement in the progression to castration-resistance. However, our approach also revealed a regulatory role for SMARCD proteins through antagonization of AR-signalling. We further demonstrated that the SMARCD proteins are involved in several important cellular processes such as the maintenance of cellular morphology and cytokinesis. Taken together, our findings suggest that the SMARCD proteins play an important, yet paradoxical, role in prostate carcinogenesis. Our approach also unmasked the complex interplay of paralogue SWI/SNF proteins that must be considered for the development of safe and efficient therapies targeting SWI/SNF.

Background & Aims: Previous single-cell RNA-sequencing analyses have shown that Trem2-expressing macrophages are present in the liver during obesity, non-alcoholic steatohepatitis (NASH) and cirrhosis. Herein, we aimed to functionally characterize the role of bone marrow-derived TREM2-expressing macrophage populations in NASH., Methods: We used bulk RNA sequencing to assess the hepatic molecular response to lipid-dependent dietary intervention in mice. Spatial mapping, bone marrow transplantation in two complementary murine models and single-cell sequencing were applied to functionally characterize the role of TREM2 + macrophage populations in NASH., Results: We found that the hepatic transcriptomic profile during steatohepatitis mirrors the dynamics of recruited bone marrow-derived monocytes that already acquire increased expression of Trem2 in the circulation. Increased Trem2 expression was reflected by elevated levels of systemic soluble TREM2 in mice and humans with NASH. In addition, soluble TREM2 levels were superior to traditionally used laboratory parameters for distinguishing between different fatty liver disease stages in two separate clinical cohorts. Spatial transcriptomics revealed that TREM2 + macrophages localize to sites of hepatocellular damage, inflammation and fibrosis in the steatotic liver. Finally, using multiple murine models and in vitro experiments, we demonstrate that hematopoietic Trem2 deficiency causes defective lipid handling and extracellular matrix remodeling, resulting in exacerbated steatohepatitis, cell death and fibrosis., Conclusions: Our study highlights the functional properties of bone marrow-derived TREM2 + macrophages and implies the clinical relevance of systemic soluble TREM2 levels in the context of NASH., Lay Summary: Our study defines the origin and function of macrophages (a type of immune cell) that are present in the liver and express a specific protein called TREM2. We find that these cells have an important role in protecting against non-alcoholic steatohepatitis (a progressive form of fatty liver disease). We also show that the levels of soluble TREM2 in the blood could serve as a circulating marker of non-alcoholic fatty liver disease., Competing Interests: Conflict of interest The authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Autophagy is a controlled mechanism of intracellular self-digestion with functions in metabolic adaptation to stress, in development, in proteostasis and in maintaining cellular homeostasis in ageing. Deletion of autophagy in epidermal keratinocytes does not prevent the formation of a functional epidermis and the permeability barrier but causes increased susceptibility to damage stress and metabolic alterations and accelerated ageing phenotypes. We here investigated how epidermal autophagy deficiency using Keratin 14 driven Atg7 deletion would affect the lipid composition of the epidermis of young and old mice. Using mass spectrometric lipidomics we found a reduction of age-related accumulation of storage lipids in the epidermis of autophagy-deficient mice, and specific changes in chain length and saturation of fatty acids in several lipid classes. Transcriptomics and immunostaining suggest that these changes are accompanied by changes in expression and localisation of lipid and fatty acid transporter proteins, most notably fatty acid binding protein 5 (FABP5) in autophagy knockouts. Thus, maintaining autophagic activity at an advanced age may be necessary to maintain epidermal lipid homeostasis in mammals., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Mature microRNAs are bound by a member of the Argonaute (Ago1-4) protein family, forming the core of the RNA-induced silencing complex (RISC). Association of RISC with target mRNAs results in ribonucleoprotein (RNP) assembly involved in translational silencing or RNA degradation. Yet, the dynamics of RNP assembly and its underlying functional implications are unknown. Here, we have characterized the role of the RNA-binding protein Staufen2, a candidate Ago interactor, in RNP assembly. Staufen2 depletion resulted in the upregulation of Ago1/2 and the RISC effector proteins Ddx6 and Dcp1a. This upregulation was accompanied by the displacement of Ago1/2 from processing bodies, large RNPs implicated in RNA storage, and subsequent association of Ago2 with polysomes. In parallel, Staufen2 deficiency decreased global translation and increased dendritic branching. As the observed phenotypes can be rescued by Ago1/2 knockdown, we propose a working model in which both Staufen2 and Ago proteins depend on each other and contribute to neuronal homeostasis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

The function of the skin as a barrier against a dry environment evolved in a common ancestor of terrestrial vertebrates such as mammals and birds. However, it is unknown which elements of the genetic program of skin barrier formation are evolutionarily ancient and conserved. In this study, we determined the transcriptomes of chicken keratinocytes (KCs) grown in monolayer culture and in an organotypic model of avian skin. The differentiation-associated changes in global gene expression were compared with previously published transcriptome changes of human KCs cultured under equivalent conditions. We found that specific keratins and genes of the epidermal differentiation complex were upregulated during the differentiation of both chicken and human KCs. Likewise, the transcriptional upregulation of genes that control the synthesis and transport of lipids, anti-inflammatory cytokines of the IL-1 family, protease inhibitors, and other regulators of tissue homeostasis was conserved in the KCs of both species. However, some avian KC differentiation-associated transcripts lack homologs in mammals and vice versa, indicating a genetic basis for taxon-specific skin features. The results of this study reveal an evolutionarily ancient program in which dynamic gene transcription controls the metabolism and transport of lipids as well as other core processes during terrestrial skin barrier formation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The COVID-19 pandemic has caused a global mechanical ventilator shortage for treatment of severe acute respiratory failure. Development of novel breathing devices has been proposed as a low cost, rapid solution when full-featured ventilators are unavailable. Here we report the design, bench testing and preclinical results for an 'Automated Bag Breathing Unit' (ABBU). Output parameters were validated with mechanical test lungs followed by animal model testing., Results: The ABBU design uses a programmable motor-driven wheel assembled for adult resuscitation bag-valve compression. ABBU can control tidal volume (200-800 ml), respiratory rate (10-40 bpm), inspiratory time (0.5-1.5 s), assist pressure sensing (- 1 to - 20 cm H 2 O), manual PEEP valve (0-20 cm H 2 O). All set values are displayed on an LCD screen. Bench testing with lung simulators (Michigan 1600, SmartLung 2000) yielded consistent tidal volume delivery at compliances of 20, 40 and 70 (mL/cm H 2 O). The delivered fraction of inspired oxygen (FiO 2 ) decreased with increasing minute ventilation (V E ), from 98 to 47% when V E was increased from 4 to 16 L/min using a fixed oxygen flow source of 5 L/min. ABBU was tested in Berkshire pigs (n = 6, weight of 50.8 ± 2.6 kg) utilizing normal lung model and saline lavage induced lung injury. Arterial blood gases were measured following changes in tidal volume (200-800 ml), respiratory rate (10-40 bpm), and PEEP (5-20 cm H 2 O) at baseline and after lung lavage. Physiological levels of PaCO 2 (≤ 40 mm Hg [5.3 kPa]) were achieved in all animals at baseline and following lavage injury. PaO 2 increased in lavage injured lungs in response to incremental PEEP (5-20 cm H 2 O) (p < 0.01). At fixed low oxygen flow rates (5 L/min), delivered FiO 2 decreased with increased V E ., Conclusions: ABBU provides oxygenation and ventilation across a range of parameter settings that may potentially provide a low-cost solution to ventilator shortages. A clinical trial is necessary to establish safety and efficacy in adult patients with diverse etiologies of respiratory failure., (© 2021. The Author(s).)

Angelman syndrome (AS) is a neurogenetic disorder characterized by severe developmental delay with absence of speech, happy disposition, frequent laughter, hyperactivity, stereotypies, ataxia and seizures with specific EEG abnormalities. There is a 10-15% of patients with an AS phenotype whose genetic cause remains unknown (Angelman-like syndrome, AS-like). Whole-exome sequencing (WES) was performed on a cohort of 14 patients with clinical features of AS and no molecular diagnosis. As a result, we identified 10 de novo and 1 X-linked pathogenic/likely pathogenic variants in 10 neurodevelopmental genes (SYNGAP1, VAMP2, TBL1XR1, ASXL3, SATB2, SMARCE1, SPTAN1, KCNQ3, SLC6A1 and LAS1L) and one deleterious de novo variant in a candidate gene (HSF2). Our results highlight the wide genetic heterogeneity in AS-like patients and expands the differential diagnosis., Competing Interests: The authors have declared that no competing interests exist.

Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity. KEY MESSAGES: LMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo. LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes. LMO3 increases nuclear coactivator 1 (Ncoa1). LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1., (© 2021. The Author(s).)

Dendritic cell (DC) development is orchestrated by lineage-determining transcription factors (TFs). Although, members of the activator-protein-1 (AP-1) family, including Batf3, have been implicated in conventional (c)DC specification, the role of Jun proteins is poorly understood. Here, we identified c-Jun and JunB as essential for cDC1 fate specification and function. In mice, Jun proteins regulate extrinsic and intrinsic pathways, which control CD8α cDC1 diversification, whereas CD103 cDC1 development is unaffected. The loss of c-Jun and JunB in DC progenitors diminishes the CD8α cDC1 pool and thus confers resistance to Listeria monocytogenes infection. Their absence in CD8α cDC1 results in impaired TLR triggering and antigen cross-presentation. Both TFs are required for the maintenance of the CD8α cDC1 subset and suppression of cDC2 identity on a transcriptional and phenotypic level. Taken together, these results demonstrate the essential role of c-Jun and JunB in CD8α cDC1 diversification, function, and maintenance of their identity., (© 2021. The Author(s).)

The signal transducer and activator of transcription 3 (STAT3) signalling pathway is activated through phosphorylation by Janus kinases in response to a diverse set of immunogenic and non-immunogenic triggers. Several distinct lines of evidence propose an intricate involvement of STAT3 in neural function relevant to behaviour in health and disease. However, in part due to the pleiotropic effects resulting from its DNA binding activity and the consequent regulation of expression of a variety of genes with context-dependent cellular consequences, the precise nature of STAT3 involvement in the neural mechanisms underlying psychopathology remains incompletely understood. Here, we focused on the midbrain serotonergic system, a central hub for the regulation of emotions, to examine the relevance of STAT3 signalling for emotional behaviour in mice by selectively knocking down raphe STAT3 expression using germline genetic (STAT3 KO) and viral-mediated approaches. Mice lacking serotonergic STAT3 presented with reduced negative behavioural reactivity and a blunted response to the sensitising effects of amphetamine, alongside alterations in midbrain neuronal firing activity of serotonergic neurons and transcriptional control of gene networks relevant for neuropsychiatric disorders. Viral knockdown of dorsal raphe (DR) STAT3 phenocopied the behavioural alterations of STAT3 KO mice, excluding a developmentally determined effect and suggesting that disruption of STAT3 signalling in the DR of adult mice is sufficient for the manifestation of behavioural traits relevant to psychopathology. Collectively, these results suggest DR STAT3 as a molecular gate for the control of behavioural reactivity, constituting a mechanistic link between the upstream activators of STAT3, serotonergic neurotransmission and psychopathology., (© 2020. The Author(s).)

Neurons have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their precise impact, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2), which both regulate synaptic transmission. Surprisingly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. Although Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, we show that Pum2 activates expression of key GABAergic synaptic components, e.g., the GABA A receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data argue for an important role of RBPs to maintain proteostasis in order to control distinct aspects of synaptic transmission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo., (© 2020 Federation of American Societies for Experimental Biology.)

The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo. We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 μM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts. In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The ectonucleotidase CD39 on human regulatory T-cells (Treg) is an important immune regulator which is dysregulated in autoimmune diseases and cancer immunosuppression. We here define that CD39 expression on Treg is independent of the Treg-specific transcription factors FOXP3 and HELIOS and promoted by canonical TGF-b- and mTOR-signaling. Furthermore, the TGF-b mediated upregulation of CD39 is counteracted by reactive oxygen species (ROS)-driven autophagy. In line, CD39 + peripheral blood Treg constitute a distinct lineage with low autophagic flux and absent ROS production. Patients with rare genetic defects in autophagy show supraphysiological levels of CD39 + Treg, validating our observations in vivo. These biological processes rely on a distinct transcriptional program with CD39 + Treg expressing low levels of two genes with putative involvement in autophagy, NEFL and PLAC8. Furthermore, the TGF-b downstream transcription factor SOX4 is selectively upregulated in CD39 + Treg. Overexpression of SOX4 in Treg strongly increases CD39 expression, while Crispr/Cas9-mediated knockout of SOX4 in Treg has the opposing effect. Thus, we identify a crucial role of SOX4 in immune regulation and provide new insights involving the interplay of tolerogenic cues and autophagy in Treg., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: The aim of the present work is the molecular diagnosis of three patients with deafness and retinal degeneration., Methods: Three patients from two unrelated families were initially analyzed with custom gene panels for Usher genes, non-syndromic hearing loss, or inherited syndromic retinopathies and further investigated by means of clinical or whole exome sequencing., Results: The study allowed us to detect likely pathogenic variants in PEX6 , a gene typically involved in peroxisomal biogenesis disorders (PBDs). Beside deaf-blindness, both families showed additional features: Siblings from Family 1 showed enamel alteration and abnormal peroxisome. In addition, the brother had mild neurodevelopmental delay and nephrolithiasis. The case II:1 from Family 2 showed intellectual disability, enamel alteration, and dysmorphism., Conclusions: We have reported three new cases with pathogenic variants in PEX6 presenting with milder forms of the Zellweger spectrum disorders (ZSD). The three cases showed distinct clinical features. Thus, expanding the phenotypic spectrum of PBDs and ascertaining exome sequencing is an effective strategy for an accurate diagnosis of clinically overlapping and genetically heterogeneous disorders such as deafness-blindness association., (Copyright © 2020 Molecular Vision.)