Search

BackgroundLung transplant (LUTX) candidates have subclinical right ventricular (RV) dysfunction, which has not yet been assessed by speckle-tracking echocardiography (STE)-derived RV free-wall longitudinal strain (RVFWLS). To evaluate the prevalence of RV dysfunction by RVFWLS and its relationship with conventional RV echocardiographic indexes in LUTX candidates.MethodsIn a single-center prospective observational cohort study, from January 2021 to March 2023 consecutive LUTX candidates underwent cardiac catheterization, radionuclide ventriculography, standard and STE. The diagnostic accuracy of RV ejection fraction by ventriculography (RVEF), tricuspid annular plane excursion (TAPSE), fractional area change (FAC), tricuspid peak annulus systolic velocity (S') versus RVFWS were computed.ResultsThirty-four patients (female, 41%) with a mean age of 48 [36-59] years old enlisted for pulmonary fibrosis (35%) and cystic fibrosis (30%) were included. At cardiac catheterization, only 7 (23%) had pulmonary hypertension. Around 15-25% presented right heart enlargement. Tricuspid regurgitation was present in 20 (60%) of the patients. Median RVFWLS was -20.1% [-22.5%--17%], being impaired (> -20%) in 16 (47%) of the patients. RVFWLS identified the highest percentage (47%) of RV dysfunction, compared to TAPSE (32%), S' (27%), FAC (26%), and ventriculography (15%), which had very low sensitivity for detecting RV dysfunction compared to RVFWLS.ConclusionsIn patients enlisted for LUTX, RV dysfunction assessed by STE-derived RVFWLS is highly prevalent. STE can detect RV dysfunction better than standard two-dimensional echocardiography and ventriculography. Further studies are urgently needed to define the clinical implications and the prognostic value of RV dysfunction measured with RVFWLS.

BackgroundThe coronavirus disease 2019 (COVID-19) presents an urgent threat to global health. Identification of predictors of poor outcomes will assist medical staff in treatment and allocating limited healthcare resources.AimsThe primary aim was to study the value of D-dimer as a predictive marker for in-hospital mortality.MethodsThis was a cohort study. The study population consisted of hospitalized patients (age >18 years), who were diagnosed with COVID-19 based on real-time PCR at 9 hospitals during the first COVID-19 wave in Lombardy, Italy (Feb-May 2020). The primary endpoint was in-hospital mortality. Information was obtained from patient records. Statistical analyses were performed using a Fine-Gray competing risk survival model. Model discrimination was assessed using Harrell's C-index and model calibration was assessed using a calibration plot.ResultsOut of 1049 patients, 507 patients (46%) had evaluable data. Of these 507 patients, 96 died within 30 days. The cumulative incidence of in-hospital mortality within 30 days was 19% (95CI: 16%-23%), and the majority of deaths occurred within the first 10 days. A prediction model containing D-dimer as the only predictor had a C-index of 0.66 (95%CI: 0.61-0.71). Overall calibration of the model was very poor. The addition of D-dimer to a model containing age, sex and co-morbidities as predictors did not lead to any meaningful improvement in either the C-index or the calibration plot.ConclusionThe predictive value of D-dimer alone was moderate, and the addition of D-dimer to a simple model containing basic clinical characteristics did not lead to any improvement in model performance.

BackgroundThis paper is aimed to (i) develop an innovative classification of COPD, multi-dimensional phenotype, based on a multidimensional assessment; (ii) describe the identified multi-dimensional phenotypes.MethodsAn exploratory factor analysis to identify the main classificatory variables and, then, a cluster analysis based on these variables were run to classify the COPD-diagnosed 514 patients enrolled in the STORICO (trial registration number: NCT03105999) study into multi-dimensional phenotypes.ResultsThe circadian rhythm of symptoms and health-related quality of life, but neither comorbidity nor respiratory function, qualified as primary classificatory variables. Five multidimensional phenotypes were identified: the MILD COPD characterized by no night-time symptoms and the best health status in terms of quality of life, quality of sleep, level of depression and anxiety, the MILD EMPHYSEMATOUS with prevalent dyspnea in the early-morning and day-time, the SEVERE BRONCHITIC with nocturnal and diurnal cough and phlegm, the SEVERE EMPHYSEMATOUS with nocturnal and diurnal dyspnea and the SEVERE MIXED COPD distinguished by higher frequency of symptoms during 24h and worst quality of life, of sleep and highest levels of depression and anxiety.ConclusionsOur results showed that properly collected respiratory symptoms play a primary classificatory role of COPD patients. The longitudinal observation will disclose the discriminative and prognostic potential of the proposed multidimensional phenotype.Trial registrationTrial registration number: NCT03105999, date of registration: 10th April 2017.

Patients with severe community-acquired pneumonia (SCAP) and life-threatening acute respiratory failure may require invasive mechanical ventilation (IMV). Since use of IMV is often associated with significant morbidity and mortality, we assessed whether patients invasively ventilated would represent a target population for interventions aimed at reducing mortality of SCAP.We prospectively recruited consecutive patients with SCAP for 12 years. We assessed the characteristics and outcomes of patients invasively ventilated at presentation of pneumonia, compared with those without IMV, and determined the influence of risks factors on mortality with a multivariate weighted logistic regression using a propensity score.Among 3,719 patients hospitalized with CAP, 664 (18%) had criteria for SCAP, and 154 (23%) received IMV at presentation of pneumonia; 198 (30%) presented with septic shock. In 370 (56%) cases SCAP was diagnosed based solely on the presence of 3 or more IDSA/ATS minor criteria. Streptococcus pneumoniae was the main pathogen in both groups. The 30-day mortality was higher in the IMV, compared to non-intubated patients (51, 33%, vs. 94, 18% respectively, p

Stenotrophomonas maltophilia and Burkholderia cepacia complex (Bcc) have been increasingly recognized as relevant pathogens in hospitalized, immunocompromised and cystic fibrosis (CF) patients. As a result of complex mechanisms, including biofilm formation and multidrug resistance phenotype, S. maltophilia and Bcc respiratory infections are often refractory to therapy, and have been associated with a worse outcome in CF patients. Here we demonstrate for the first time that N-acetylcysteine (NAC), a mucolytic agent with antioxidant and anti-inflammatory properties, may exhibit antimicrobial and antibiofilm activity against these pathogens. The antimicrobial and antibiofilm activity of high NAC concentrations, potentially achievable by topical administration, was tested against a collection of S. maltophilia (n = 19) and Bcc (n = 19) strains, including strains from CF patients with acquired resistance traits. Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) ranged from 16 to 32 mg/ml and from 32 to >32 mg/ml, respectively. Sub-MIC concentrations (i.e., 0.25 × MIC) slowed down the growth kinetics of most strains. In time-kill assays, 2-day-old biofilms were more affected than planktonic cultures, suggesting a specific antibiofilm activity of NAC against these pathogens. Indeed, a dose- and time-dependent antibiofilm activity of NAC against most of the S. maltophilia and Bcc strains tested was observed, with a sizable antibiofilm activity observed also at 0.5 and 1 × MIC NAC concentrations. Furthermore, at those concentrations, NAC was also shown to significantly inhibit biofilm formation with the great majority of tested strains.

Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA.

In translational cancer medicine, implicated pathways and the relevant master genes are of focus. Exome's specificity, processing-time, and cost advantage makes it a compelling tool for this purpose. However, analysis of exome lacks reliable combinatory analysis tools and techniques. In this paper we present XomAnnotate--a meta- and functional-analysis software for exome. We compared UnifiedGenotyper, Freebayes, Delly, and Lumpy algorithms that were designed for whole-genome and combined their strengths in XomAnnotate for exome data through meta-analysis to identify comprehensive mutation profile (SNPs/SNVs, short inserts/deletes, and SVs) of patients. The mutation profile is annotated followed by functional analysis through pathway enrichment and network analysis to identify most critical genes and pathways implicated in the disease genesis. The efficacy of the software is verified through MDS and clustering and tested with available 11 familial non-BRCA1/BRCA2 breast cancer exome data. The results showed that the most significantly affected pathways across all samples are cell communication and antigen processing and presentation. ESCO1, HYAL1, RAF1 and PRKCA emerged as the key genes. Network analysis further showed the purine and propanotate metabolism pathways along with RAF1 and PRKCA genes to be master regulators in these patients. Therefore, XomAnnotate is able to use exome data to identify entire mutation landscape, pathways, and the master genes accurately with wide concordance from earlier microarray and whole-genome studies--making it a suitable biomedical software for using exome in next-generation translational medicine.

The Prep1 (Pknox1) homeodomain transcription factor is essential at multiple stages of embryo development. In the E11.5 embryo trunk, we previously estimated that Prep1 binds about 3,300 genomic sites at a highly specific decameric consensus sequence, mainly governing basal cellular functions. We now show that in embryonic stem (ES) cells Prep1 binding pattern only partly overlaps that of the embryo trunk, with about 2,000 novel sites. Moreover, in ES cells Prep1 still binds mostly to promoters, as in total embryo trunk but, among the peaks bound exclusively in ES cells, the percentage of enhancers was three-fold higher. RNA-seq identifies about 1800 genes down-regulated in Prep1-/- ES cells which belong to gene ontology categories not enriched in the E11.5 Prep1i/i differentiated embryo, including in particular essential components of the Wnt and Fgf pathways. These data agree with aberrant Wnt and Fgf expression levels in the Prep1-/- ES cells with a deficient embryoid bodies (EBs) formation and differentiation. Re-establishment of the Prep1 level rescues the phenotype.

A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.

Chronic Obstructive Pulmonary Disease (COPD) is a common disease with significant health and economic consequences. This study assesses the burden of COPD in the general population, and the influence of exacerbations (E-COPD) on disease progression and costs.This is a secondary data analysis of healthcare administrative databases of the region of Lombardy, in northern Italy. The study included ≥ 40 year-old patients hospitalized for a severe E-COPD (index event) during 2006. Patients were classified in relation to the number and type of E-COPD experienced in a three-year pre-index period. Subjects were followed up until December 31st, 2009, collecting data on healthcare resource use and vital status.15857 patients were enrolled -9911 males, mean age: 76 years (SD 10). Over a mean follow-up time of 2.4 years (1.36), 81% of patients had at least one E-COPD with an annual rate of 3.2 exacerbations per person-year and an all-cause mortality of 47%. A history of exacerbation influenced the occurrence of new E-COPD and mortality after discharge for an E-COPD. On average, the healthcare system spent 6725€ per year per person (95%CI 6590-6863). Occurrence and type of exacerbations drove the direct healthcare cost. Less than one quarter of patients presented claims for pulmonary function tests.COPD imposes a substantial burden on healthcare systems, mainly attributable to the type and occurrence of E-COPD, or in other words, to the exacerbator phenotypes. A more tailored approach to the management of COPD patients is required.

Prep1 and Meis1 ortholog TALE transcription factors have opposing roles in tumorigenesis: Meis1 serves as an oncogene, Prep1 as a tumor suppressor. We now report that, Meis1 overexpression in primary Prep1-deficient (Prep1i/i) embryonic hematopoietic cells increases self-renewal potential of cells in vitro but not in vivo, whereas leukemia is instead obtained when Meis1 is combined with another oncogene, HoxA9. Prep1i/i Meis1-HoxA9-generated leukemic cells are less differentiated and grow more aggressively after the second passage in the mouse. These data indicate that Prep1 represents a barrier to the transforming activity of Meis1 in vitro, but its absence is not sufficient to induce early leukemogenesis. On the other hand, the Prep1i/i background appears to favor the insurgence of mutations that cause a more aggressive Meis1-HoxA9-generated leukemia. Indeed, the Prep1i/i leukemic cells upregulate the Polycomb protein Bmi-1 and expectedly down-regulate the Ink4a/Arf locus products. Finally, an important feature contributed by the Prep1i/i background is the post-transcriptional increase in Meis1 protein level.

Progress in experimental stroke and translational medicine could be accelerated by high-resolution in vivo imaging of disease progression in the mouse cortex. Here, we introduce optical microscopic methods that monitor brain injury progression using intrinsic optical scattering properties of cortical tissue. A multi-parametric Optical Coherence Tomography (OCT) platform for longitudinal imaging of ischemic stroke in mice, through thinned-skull, reinforced cranial window surgical preparations, is described. In the acute stages, the spatiotemporal interplay between hemodynamics and cell viability, a key determinant of pathogenesis, was imaged. In acute stroke, microscopic biomarkers for eventual infarction, including capillary non-perfusion, cerebral blood flow deficiency, altered cellular scattering, and impaired autoregulation of cerebral blood flow, were quantified and correlated with histology. Additionally, longitudinal microscopy revealed remodeling and flow recovery after one week of chronic stroke. Intrinsic scattering properties serve as reporters of acute cellular and vascular injury and recovery in experimental stroke. Multi-parametric OCT represents a robust in vivo imaging platform to comprehensively investigate these properties.

The Prep1 homeodomain transcription factor has recently been recognized as a tumor suppressor. Among other features, haploinsufficiency of Prep1 is able to strongly accelerate the B-lymphomagenesis in EμMyc mice. Now we report that this occurs concomitantly with a change in the type of B-cell lymphomas generated by the Myc oncogene. Indeed, the tumors generated in the EμMyc-Prep1(+/-) mice are much more immature, being mostly made up of Pro-B or Pre-B cells, while those in the EμMyc-Prep1(+/+) mice are more differentiated being invariably IgM(+). Moreover, we show that Prep1 is in fact required for the differentiation of Pro-B and Pre-B cells into IgM(+) lymphocytes and/or their proliferation, thus showing also how a normal function of Prep1 affects EμMyc lymphomagenesis. Finally, we show that the haploinsufficiency of Prep1 is accompanied with a major decrease of Myc-induced apoptosis and that the haploinsufficieny is sufficient for all these effects because the second allele of Prep1 is not lost even at late stages. Therefore, the tumor-suppressive activity of Prep1 is intertwined with both the interference with Myc-induced apoptosis as well as with natural developmental functions of the protein.

The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (-IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(-)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (-)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(-)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (-)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(-)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-κB/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage.

MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs) and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5' upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to -1.8, possibly -5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the -5.0 Kb promoter. A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the -3 to -5 Kb of the human upstream region.

Homeobox genes are essential for embryonic patterning and cell fate determination. They are regulated mostly at the transcriptional level. In particular, Prep1 regulates Hox transcription in association with Pbx proteins. Despite its nuclear role as a transcription factor, Prep1 is located in the cytosol of mouse oocytes from primary to antral follicles. The homeodomain factor Bicoid (Bcd) has been shown to interact with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Caudal mRNA and to drive Drosophila embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function.In this paper we show by confocal microscopy and deconvolution analysis that Prep1 and 4EHP co-localize in the cytosol of growing mouse oocytes, demonstrating their interaction by co-immunoprecipitation and pull-down experiments. A functional 4EHP-binding motif present in Prep1 has been also identified by mutagenesis analysis. Moreover, Prep1 inhibits (>95%) the in vitro translation of a luciferase reporter mRNA fused to the Hoxb4 3'UTR, in the presence of 4EHP. RNA electrophoretic mobility shift assay was used to demonstrate that Prep1 binds the Hoxb4 3'UTR. Furthermore, conventional histology and immunohistochemistry has shown a dramatic oocyte growth failure in hypomorphic mouse Prep1(i/i) females, accompanied by an increased production of Hoxb4. Finally, Hoxb4 overexpression in mouse zygotes showed a slow in vitro development effect.Prep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation. Mechanistically, the Prep1-4EHP interaction might bridge the 3'UTR of Hoxb4 mRNA to the 5' cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

Prep1 and Pbx2 are the main homeodomain DNA-binding proteins of the TALE (three amino acid loop extension) family expressed in the thymus. We previously reported reduced Pbx2 expression and defective thymocyte maturation in Prep1 hypomorphic mice. To further investigate the role of this homeodomain DNA-binding protein in T cell development, we generated transgenic mice expressing the N-terminal fragment of Pbx1 (Pbx1NT) under the control of the Lck proximal promoter.Pbx1NT causes Prep1 cytosolic sequestration, abolishes Prep1-dependent DNA-binding activity and results in reduced Pbx2 expression in developing thymocytes. Transgenic thymi reveal increased numbers of CD4(-) CD8(-) CD44(-) (DN3 and DN4) thymocytes, due to a higher frequency of DN2 and DN4 Pbx1NT thymocytes in the S phase. Transgenic thymocytes however do not accumulate at later stages, as revealed by a normal representation of CD4/CD8 double positive and single positive thymocytes, due to a higher rate of apoptotic cell death of DN4 Pbx1NT thymocytes.The results obtained by genetic (Prep1 hypomorphic) and functional (Pbx1NT transgenic) inactivation of Prep1 support nonredundant roles for this homeodomain protein during different stages of T cell development.

BackgroundThe urokinase receptor (uPAR/CD87) is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA) and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1.Methodology/principal findingsIn this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments.Conclusions/significanceThese results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.