Effect of Arx(GCG)7 mutation in the olfactory bulb

Tutor: Manuel Álvarez Dolado y Co-Tutor: Ana Moral Rama.La formación de la estructura del bulbo olfativo, organizada en diferentes capas interconectadas, está finamente controlada por multitud de factores. El trabajo previo de Yoshihara et al. demostró que el factor de transcripción Arx se requiere para el correcto desarrollo del bulbo olfativo. No obstante, este grupo realizó su estudio en ratones con una mutación Knock-out para Arx, la cual no se encuentra de forma natural en roedores o humanos. La mutación Arx(GCG) 7 es la mas frecuente en humanos y está relacionada con una encefalopatía epiléptica infantil denominada Síndrome de West. En el presente trabajo hemos estudiado el posible efecto de esta mutación en la morfología del bulbo olfativo para entender mejor esta patología. Los ratones macho Arx(GCG)7 con 18-21 dias de edad presentaron evidentes anomalías en la citoarquitectura del bulbo olfativo: (1) reducción del tamaño de la capa plexiforme interna, (2) pérdida de células periglomerulares tiroxina hidroxilasapositivas, (3) pérdida de células periglomerulares calbindina-positivas, y (4) reducción en el tamaño de los glomérulos de la capa glomerular. Por ello, concluimos que la correcta actividad de Arx es esencial para la generación y diposición de las interneuronas periglomerulares del bulbo olfativo y probablemente para una adecuada inervación axonal en la capa plexiforme interna.Peer Reviewe

Effects of intubation timing in patients with COVID-19 throughout the four waves of the pandemic : a matched analysis

The primary aim of our study was to investigate the association between intubation timing and hospital mortality in critically ill patients with COVID-19-associated respiratory failure. We also analysed both the impact of such timing throughout the first four pandemic waves and the influence of prior non-invasive respiratory support on outcomes. This is a secondary analysis of a multicentre, observational and prospective cohort study that included all consecutive patients undergoing invasive mechanical ventilation due to COVID-19 from across 58 Spanish intensive care units (ICU) participating in the CIBERESUCICOVID project. The study period was between 29 February 2020 and 31 August 2021. Early intubation was defined as that occurring within the first 24 h of intensive care unit (ICU) admission. Propensity score (PS) matching was used to achieve balance across baseline variables between the early intubation cohort and those patients who were intubated after the first 24 h of ICU admission. Differences in outcomes between early and delayed intubation were also assessed. We performed sensitivity analyses to consider a different timepoint (48 h from ICU admission) for early and delayed intubation. Of the 2725 patients who received invasive mechanical ventilation, a total of 614 matched patients were included in the analysis (307 for each group). In the unmatched population, there were no differences in mortality between the early and delayed groups. After PS matching, patients with delayed intubation presented higher hospital mortality (27.3% versus 37.1%, p =0.01), ICU mortality (25.7% versus 36.1%, p=0.007) and 90-day mortality (30.9% versus 40.2%, p=0.02) when compared to the early intubation group. Very similar findings were observed when we used a 48-hour timepoint for early or delayed intubation. The use of early intubation decreased after the first wave of the pandemic (72%, 49%, 46% and 45% in the first, second, third and fourth wave, respectively; first versus second, third and fourth waves p<0.001). In both the main and sensitivity analyses, hospital mortality was lower in patients receiving high-flow nasal cannula (n=294) who were intubated earlier. The subgroup of patients undergoing NIV (n=214) before intubation showed higher mortality when delayed intubation was set as that occurring after 48 h from ICU admission, but not when after 24 h. In patients with COVID-19 requiring invasive mechanical ventilation, delayed intubation was associated with a higher risk of hospital mortality. The use of early intubation significantly decreased throughout the course of the pandemic. Benefits of such an approach occurred more notably in patients who had received high-flow nasal cannul

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Occurrence of SARS-CoV-2 viremia is associated with genetic variants of genes related to COVID-19 pathogenesis

IntroductionSARS-CoV-2 viral load has been related to COVID-19 severity. The main aim of this study was to evaluate the relationship between SARS-CoV-2 viremia and SNPs in genes previously studied by our group as predictors of COVID-19 severity.Materials and methodsRetrospective observational study including 340 patients hospitalized for COVID-19 in the University Hospital La Princesa between March 2020 and December 2021, with at least one viremia determination. Positive viremia was considered when viral load was above the quantifiable threshold (20 copies/ml). A total of 38 SNPs were genotyped. To study their association with viremia a multivariate logistic regression was performed.ResultsThe mean age of the studied population was 64.5 years (SD 16.6), 60.9% patients were male and 79.4% white non-Hispanic. Only 126 patients (37.1%) had at least one positive viremia. After adjustment by confounders, the presence of the minor alleles of rs2071746 (HMOX1; T/T genotype OR 9.9 p &lt; 0.0001), rs78958998 (probably associated with SERPING1 expression; A/T genotype OR 2.3, p = 0.04 and T/T genotype OR 12.9, p &lt; 0.0001), and rs713400 (eQTL for TMPRSS2; C/T + T/T genotype OR 1.86, p = 0.10) were associated with higher risk of viremia, whereas the minor alleles of rs11052877 (CD69; A/G genotype OR 0.5, p = 0.04 and G/G genotype OR 0.3, p = 0.01), rs2660 (OAS1; A/G genotype OR 0.6, p = 0.08), rs896 (VIPR1; T/T genotype OR 0.4, p = 0.02) and rs33980500 (TRAF3IP2; C/T + T/T genotype OR 0.3, p = 0.01) were associated with lower risk of viremia.ConclusionGenetic variants in HMOX1 (rs2071746), SERPING1 (rs78958998), TMPRSS2 (rs713400), CD69 (rs11052877), TRAF3IP2 (rs33980500), OAS1 (rs2660) and VIPR1 (rs896) could explain heterogeneity in SARS-CoV-2 viremia in our population

Characterization of the Physiological Role of the Deubiquitinase OTUB1 in Mice

Protein ubiquitination is a key regulatory process in a plethora of physiological and pathological pathways. During ubiquitination, the protein ubiquitin is covalently attached to a substrate protein forming mono- or polyubiquitin chains. Ubiquitination can be reversed via deubiquitinases (DUBs). OTUB1 (ovarian tumor domain-containing ubiquitin aldehyde binding protein 1) is one of the most highly expressed deubiquitinases in cells with a unique enzymatic activity cleaving Lys48 ubiquitin chains and preventing Lys48 and Lys63 ubiquitin chain formation non-enzymatically. OTUB1 regulates various pathways and processes in vitro, like proinflammatory and profibrotic signaling, DNA damage response, proliferation and apoptosis. The cellular oxygen sensor factor inhibiting HIF (FIH) is an asparagine hydroxylase controlling the transactivation activity of the hypoxia inducible factor (HIF), key regulator of the transcriptional response to hypoxia. Our group recently demonstrated that FIH hydroxylates OTUB1 on Asn22, regulating cellular energy metabolism. This thesis aims to investigate the interaction between FIH and OTUB1 in more detail and to assess if OTUB1 is a possible physiological target of FIH. The molecular interaction between FIH and OTUB1 showed a denaturing-resistant, likely covalently linked protein heterodimer, highly sensitive to hypoxia and depending on FIH enzymatic activity. Several studies have investigated the role of OTUB1 in diseases in vivo. However, the physiological function of OTUB1 is unknown. To understand the in vivo role of OTUB1 and to assess if it is a likely physiologically relevant FIH target, we characterized mice with constitutive (Otub1-/-) and conditional whole-body Otub1 (wbOtub1-/-) deletion. Otub1-/-mice died perinatally due to asphyxiation and showed decreased alveolar space, increased lung cell proliferation and cardiac hypertrophy. wbOtub1-/- mice showed pulmonary hyperventilation and cardiac hypertrophy with left ventricular dysfunction. Additionally, adult animals presented with decreased body weight, increased energy expenditure and glucose metabolism. Furthermore, the AKT/mTOR signaling was increased in cells and tissues without Otub1. Overall, these results demonstrate that OTUB1 is essential for development, respiration, cardiac function and energy metabolic homeostasis in vivo. Interestingly, Hif1an (the gene encoding FIH) homozygous knockout (KO) mice display a metabolic phenotype that is virtually identical to the metabolic alterations observed in Otub1 KO mice. In summary, we found that FIH forms a stable complex with OTUB1 at a cellular level. Interestingly, the metabolic and respiratory phenotypes of Otub1 KO mice support that the FIH-dependent regulation of OTUB1 is physiologically relevant. Our studies of Otub1 KO mice will further help evaluating the potential of OTUB1 as a therapeutic target and possible role in diseases

The functional interplay between the HIF pathway and the ubiquitin system – more than a one-way road

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the beststudied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis

OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control

OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation. Keywords: FIH; HIF1AN; deubiquitinating enzyme; hypoxia; respiratory distress syndrome; respiratory failure

Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis