Background The COVID-19 pandemic is challenging advanced health systems, which are dealing with an overwhelming number of patients in need of intensive care for respiratory failure, often requiring intubation. Prone positioning in intubated patients is known to reduce mortality in moderate-to-severe acute respiratory distress syndrome. We aimed to investigate feasibility and effect on gas exchange of prone positioning in awake, non-intubated patients with COVID-19-related pneumonia. MethodsIn this prospective, feasibility, cohort study, patients aged 18-75 years with a confirmed diagnosis of COVID-19related pneumonia receiving supplemental oxygen or non-invasive continuous positive airway pressure were recruited from San Gerardo Hospital, Monza, Italy. We collected baseline data on demographics, anthropometrics, arterial blood gas, and ventilation parameters. After baseline data collection, patients were helped into the prone position, which was maintained for a minimum duration of 3 h. Clinical data were re-collected 10 min after prone positioning and 1 h after returning to the supine position. The main study outcome was the variation in oxygenation (partial pressure of oxygen [PaO 2 ]/fractional concentration of oxygen in inspired air [FiO 2 ]) between baseline and resupination, as an index of pulmonary recruitment. This study is registered on ClinicalTrials.gov, NCT04365959, and is now complete. Findings Between March 20 and April 9, 2020, we enrolled 56 patients, of whom 44 (79%) were male; the mean age was 57•4 years (SD 7•4) and the mean BMI was 27•5 kg/m² (3•7). Prone positioning was feasible (ie, maintained for at least 3 h) in 47 patients (83•9% [95% CI 71•7 to 92•4]). Oxygenation substantially improved from supine to prone positioning (PaO 2 /FiO 2 ratio 180•5 mm Hg [SD 76•6] in supine position vs 285•5 mm Hg [112•9] in prone position; p<0•0001). After resupination, improved oxygenation was maintained in 23 patients (50•0% [95% CI 34•9-65•1]; ie, responders); however, this improvement was on average not significant compared with before prone positioning (PaO 2 /FiO 2 ratio 192•9 mm Hg [100•9] 1 h after resupination; p=0•29). Patients who maintained increased oxygenation had increased levels of inflammatory markers (C-reactive protein: 12•7 mg/L [SD 6•9] in responders vs 8•4 mg/L [6•2] in non-responders; and platelets: 241•1 × 10³/µL [101•9] vs 319•8 × 10³/µL [120•6]) and shorter time between admission to hospital and prone positioning (2•7 days [SD 2•1] in responders vs 4•6 days [3•7] in non-responders) than did those for whom improved oxygenation was not maintained. 13 (28%) of 46 patients were eventually intubated, seven (30%) of 23 responders and six (26%) of 23 non-responders (p=0•74). Five patients died during follow-up due to underlying disease, unrelated to study procedure. Interpretation Prone positioning was feasible and effective in rapidly ameliorating blood oxygenation in awake patients with COVID-19-related pneumonia requiring oxygen supplementation. The effect was maintained after resupina...

Imatinib is effective for the treatment of chronic myeloid leukemia (CML). However even undetectable BCR-ABL1 by Q-RT-PCR does not equate to eradication of the disease. Digital-PCR (dPCR), able to detect 1 BCR-ABL1 positive cell out of 10 7 , has been recently developed. The ISAV study is a multicentre trial aimed at validating dPCR to predict relapses after imatinib discontinuation in CML patients with undetectable Q-RT-PCR. CML patients under imatinib therapy since more than 2 years and with undetectable PCR for at least 18 months were eligible. Patients were monitored by standard Q-RT-PCR for 36 months. Patients losing molecular remission (two consecutive positive Q-RT-PCR with at least 1 BCR-ABL1/ABL1 value above 0.1%) resumed imatinib. The study enrolled 112 patients, with a median follow-up of 21.6 months. Fifty-two of the 108 evaluable patients (48.1%), relapsed; 73.1% relapsed in the first 9 months but 14 late relapses were observed between 10 and 22 months. Among the 56 not-relapsed patients, 40 (37.0% of total) regained Q-RT-PCR positivity but never lost MMR. dPCR results showed a significant negative predictive value ratio of 1.115 [95% CI: 1.013-1.227]. An inverse relationship between patients age and risk of relapse was evident: 95% of patients <45 years relapsed versus 42% in the class 45 to <65 years and 33% of patients 65 years [P(v 2 ) < 0.0001]. Relapse rates ranged between 100% (<45 years, dPCR1) and 36% (>45 years, dPCR-). Imatinib can be safely discontinued in the setting of continued PCR negativity; age and dPCR results can predict relapse.

We have examined glucose homeostasis in mice hypomorphic for the homeotic transcription factor gene Prep1. Prep1-hypomorphic (Prep1 i/i ) mice exhibit an absolute reduction in circulating insulin levels but normal glucose tolerance. In addition, these mice exhibit protection from streptozotocin-induced diabetes and enhanced insulin sensitivity with improved glucose uptake and insulin-dependent glucose disposal by skeletal muscle. This muscle phenotype does not depend on reduced expression of the known Prep1 transcription partner, Pbx1. Instead, in Prep1 i/i muscle, we find normal Pbx1 but reduced levels of the recently identified novel Prep1 interactor p160. Consistent with this reduction, we find a muscle-selective increase in mRNA and protein levels of PGC-1␣, accompanied by enhanced expression of the GLUT4 transporter, responsible for insulin-stimulated glucose uptake in muscle. Indeed, using L6 skeletal muscle cells, we induced the opposite effects by overexpressing Prep1 or p160, but not Pbx1. In vivo skeletal muscle delivery of p160 cDNA in Prep1 i/i mice also reverses the molecular phenotype. Finally, we show that Prep1 controls the stability of the p160 protein. We conclude that Prep1 controls insulin sensitivity through the p160-GLUT4 pathway.

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. Targeting of proteins by conjugation of Small Ubiquitin-like MOdifier (SUMO)1 is a key mechanism for regulating many cellular processes (1, 2), for example the activity of transcription factors (3). Other regulated processes are DNA repair, protein transport, protein-protein interaction, cell cycle progression, and RNA metabolism (4 -6). SUMO proteins are ubiquitously expressed throughout the eukaryotic kingdom. Yeast, Caenorhabditis elegans, and Drosophila melanogaster carry a single SUMO gene, whereas plants and vertebrates have several SUMO genes (5). In particular, humans express four distinct SUMO family members: SUMO-1, SUMO-2, SUMO-3, and SUMO-4 (7, 8). SUMO-1 is an 11.6 kDa protein. It shares about 47% homology with SUMO-2 and SUMO-3 that, on the contrary, differ from each other only by three amino-terminal residues and form a distinct subfamily known as SUMO-2/-3 (9). Despite the low sequence homology, SUMO-1 and SUMO-2/-3 share a similar protein size, tertiary structure, and a carboxyl-terminal diglycine motif (10, 11). At the cellular level, different amounts of free SUMO-1 and SUMO-2/-3 are present. The majority of SUMO-1 in fact is conjugated to substrates, whereas the conjugation of SUMO-2/-3 is strongly induced in response to various stresses (10). Finally SUMO-1 and SUMO-2/-3 serve distinct functions as they modify different target proteins (5). Unlike SUMO-1, SUMO-2, and SUMO-3, which are ubiquitously expressed (7), SUMO-4 isoform has yet to be characterized. It seems to be expressed mainly in the kidney, lymph nodes, and spleen, but its role still remains unclear because its mature form has never been reported in vivo (7,12).Several SUMO targets are known; they are mostly nuclear proteins presenting a consensus acceptor site: ⌿KXE (in which ⌿ is an aliphatic branched amino acid and X is any amino acid) (5). The mutation of this site abolishes sumoylation of substrates and is commonly used to understand the biological implication of the substrate modification. Also SUMO-2/-3 present a conserved lysine in this motif, and they form polymeric SUMO chains (13,14). SUMO-1, however, lacks this consensus site and is not thought to form chains even if rec...

Prep1 is known to interact in vivo with Pbx1 to regulate development and organogenesis. We have identified a novel Prep1-interacting protein, p160 c-Myb binding protein (p160). p160 and Pbx1 compete for Prep1 in vitro, and p160 inhibits Prep1-dependent HoxB2 expression in retinoic acid-treated NT2-D1 cells. The Nterminal physiologically truncated form of p160, p67, binds the sequence 63LFPLL67 in the HR1 domain of Prep1. Mutation of both L63 and L66 impairs the binding of Prep1 to both p160/p67 and Pbx1. The sequences required to bind Prep1 are mainly located in residues 51 to 151. Immunofluorescence colocalization and coimmunoprecipitation of endogenous p160 and Prep1 are induced by ActD, which translocates p160 from the nucleolus to the nucleoplasm. These data therefore show that p160 is a novel regulator of Prep1-Pbx1 transcriptional activity.