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Aims Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. Methods and results Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-β and inflammation in the disease. Conclusion The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Fondazione Gigi & Pupa Ferrari ONLUS Italian Ministry of Health-Ricerca Corrente. Background Heart failure (HF) is defined as one of the main clinical and public health burdens with multifactorial symptoms and high worldwide prevalence. Dilated cardiomyopathy (DCM) is one of the leading causes of HF and is responsible for over half of all left ventricular assist device implantations and heart transplantations. DCM is regarded as a multiple disease entity linked to a number of genetic, nutritional, inflammatory, infective, metabolic, and environmental causes. Among the metabolic processes associated with HF, iron metabolism disorders, ranging from iron deficiency to iron overload, are gaining attention. However, the understanding of iron mechanisms, at cardiac level, involved in HF and particularly in DCM, remains elusive. Purpose We aim to evaluate the possible dysregulation of iron metabolism-linked genes in DCM, through a quantitative analysis of public myocardial gene expression data at tissue and cell levels. Methods Bulk RNA sequencing (RNA-seq) and single-nucleus RNA-seq (snRNA-seq) datasets, respectively of 436 and 38 left ventricle samples from adult non-failed (NF) and DCM subjects, were retrieved from public studies. We defined a list of 272 genes directly related to intramyocardial iron metabolism and performed the differential gene expression analysis both at whole cardiac tissue level and in cardiomyocytes, fibroblasts, myeloid, endocardial, and endothelial cells. Results In the overall myocardial tissue, we found 44 iron-linked genes differentially expressed between DCM and NF subjects. To better understand iron genes abnormalities in the myocardium of DCM patients we deepened the analysis from tissue to single-cell level. Among the five considered cell types (cardiomyocytes, fibroblasts, myeloid, endocardial, and endothelial cells) at least 9% of iron-linked expressed genes were significantly regulated in DCM when compared to NF. Specifically, the iron metabolism in DCM cardiomyocytes is altered at several levels (Figure 1), including: 1) imbalance of Fe3+ internalization (SCARA5 down-regulation) and reduction of internal conversion from Fe3+ to Fe2+ (STEAP3 down-regulation), 2) increase of iron consumption to produce hemoglobin (HBA1/2 up-regulation), 3) higher heme synthesis and externalization (ALAS2 and ABCG2 up-regulation), 4) lower cleavage of heme to Fe2+, biliverdin and carbon monoxide (HMOX2 down-regulation), and 5) positive regulation of hepcidin (BMP6 up-regulation). Conclusions In conclusion, the present study provides significant progress in knowledge of iron metabolism changes in DCM disease, which likely have a clinical meaning. Indeed, our data show that regulation of intracellular iron homeostasis is dysfunctional in DCM patients’ cardiac tissues, supporting, but not providing, the hypothesis of a direct effect of iron in the disease progression.

BackgroundPatients with acute myocardial infarction (AMI) are at increased risk of recurrent cardiovascular events. Aortic valve sclerosis (AVSc), which reflects a systemic damage, may serve as a marker of risk. The aim of the present study is to better stratify sub-groups of AMI patients with specific probabilities of recurrent AMI and to evaluate the importance of AVSc in this setting.MethodsWe analyzed 2120 AMI patients admitted at Centro Cardiologico Monzino (2010-2019) who underwent echocardiographic evaluation for AVSc assessment. Topological data analysis (TDA) was used to stratify sub-groups of patients experiencing recurrent AMI and a random forest procedure to evaluate the importance of baseline clinical characteristics. Kaplan-Meier and Cox regression analyses were used to evaluate recurrent AMI incidence, for up to 10 years.ResultsTDA highlighted the presence of 8 clusters of patients with specific risks of recurrent AMI. The evaluation of time-to-event curves allowed us to combine these clusters into three super-clusters (i.e., low-, moderate-, and high-risk) and a random forest procedure showed AVSc as the most relevant variable to discriminate the three classes of risk. AVSc was detected in 1000 (47%) patients. After full adjustment, we found a significant association of AVSc with recurrent AMI (hazard ratio (HR) 1.42, 95%CI:1.03-1.97). Stratifying by age, this association was significant only for patients younger than 75 years after 5 years of follow-up (HR 1.59, 95%CI:1.03-2.46).ConclusionsAVSc has been identified to be the most relevant variable to recognize patients at high risk of recurrent AMI. AVSc is frequently detected in AMI patients and is strongly associated with recurrent AMI, especially in patients younger than 75 years old. The presence of AVSc should be taken into consideration to improve the risk stratification and clinical management of AMI patients.

Whey permeate (WP) is a lactose-rich waste effluent, generated during cheese manufacturing and further valorization steps, such as protein extraction. The production of ethanol by WP fermentation has been proposed to increase cost-competitiveness of dairy waste processing. In previous work, the Escherichia coli W strain was selected for its efficient growth in dairy waste and it was engineered to convert lactose into ethanol as the main fermentation product from WP and concentrated WP (CWP). To improve its performance, here the lactate dehydrogenase, fumarate reductase and pyruvate formate lyase fermentative routes were disrupted, obtaining new deletion strains. In test tubes, growth and fermentation profiles obtained in standard laboratory media and CWP showed large differences, and were affected by oxygen, medium and ethanologenic gene expression level. Among the tested strains, the one with triple deletion was superior in both high-oxygen and low-oxygen test tube fermentations, in terms of ethanol titer, rate and yield. The improved performance was due to a lower inhibition by medium acidification rather than an improved ethanol flux. The parent and triple deletion strains showed similar performance indexes in pH-controlled bioreactor experiments. However, the deletion strain showed lower base consumption and residual waste, in terms of both dry matter and chemical oxygen demand after distillation. It thus represents a step towards sustainable dairy wastewater valorization for bioenergy production by decreasing process operation costs.

Rationale—Calcific aortic valve stenosis (CAVS) is a pathological condition of the aortic valve with a prevalence of 3% in the general population. It is characterized by massive rearrangement of the extracellular matrix, mostly due to the accumulation of fibro-calcific deposits driven by valve interstitial cells (VIC), and no pharmacological treatment is currently available. The aim of this study was to evaluate the effects of P2Y2 receptor (P2RY2) activation on fibro-calcific remodeling of CAVS. Methods—We employed human primary VICs isolated from CAVS leaflets treated with 2-thiouridine-5′-triphosphate (2ThioUTP, 10 µM), an agonist of P2RY2. The calcification was induced by inorganic phosphate (2 mM) and ascorbic acid (50 µg/mL) for 7 or 14 days, while the 2ThioUTP was administered starting from the seventh day. 2ThioUTP was chronically administered for 5 days to evaluate myofibroblastic activation. Results—P2RY2 activation, under continuous or interrupted pro-calcific stimuli, led to a significant inhibition of VIC calcification potential (p < 0.01). Moreover, 2ThioUTP treatment was able to significantly reduce pro-fibrotic gene expression (p < 0.05), as well as that of protein α-smooth muscle actin (p = 0.004). Conclusions—Our data suggest that P2RY2 activation should be further investigated as a pharmacological target for the prevention of CAVS progression, acting on both calcification and myofibroblastic activation.

Mitral valve prolapse (MVP) associated with severe mitral regurgitation is a debilitating disease with no pharmacological therapies available. MicroRNAs (miRNA) represent an emerging class of circulating biomarkers that have never been evaluated in MVP human plasma. Our aim was to identify a possible miRNA signature that is able to discriminate MVP patients from healthy subjects (CTRL) and to shed light on the putative altered molecular pathways in MVP. We evaluated a plasma miRNA profile using Human MicroRNA Card A followed by real-time PCR validations. In addition, to assess the discriminative power of selected miRNAs, we implemented a machine learning analysis. MiRNA profiling and validations revealed that miR-140-3p, 150-5p, 210-3p, 451a, and 487a-3p were significantly upregulated in MVP, while miR-223-3p, 323a-3p, 340-5p, and 361-5p were significantly downregulated in MVP compared to CTRL (p ≤ 0.01). Functional analysis identified several biological processes possible linked to MVP. In addition, machine learning analysis correctly classified MVP patients from CTRL with high accuracy (0.93) and an area under the receiving operator characteristic curve (AUC) of 0.97. To the best of our knowledge, this is the first study performed on human plasma, showing a strong association between miRNAs and MVP. Thus, a circulating molecular signature could be used as a first-line, fast, and cheap screening tool for MVP identification.

Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.

Diabetes mellitus (DM) is one of the most common and costly disorders that affect humans around the world. Recently, clinicians and scientists have focused their studies on the effects of glycemic variability (GV), which is especially associated with cardiovascular diseases. In healthy subjects, glycemia is a very stable parameter, while in poorly controlled DM patients, it oscillates greatly throughout the day and between days. Clinically, GV could be measured by different parameters, but there are no guidelines on standardized assessment. Nonetheless, DM patients with high GV experience worse cardiovascular disease outcomes. In vitro and in vivo studies showed that high GV causes several detrimental effects, such as increased oxidative stress, inflammation, and apoptosis linked to endothelial dysfunction. However, the evidence that treating GV is beneficial is still scanty. Clinical trials aiming to improve the diagnostic and prognostic accuracy of GV measurements correlated with cardiovascular outcomes are needed. The present review aims to evaluate the clinical link between high GV and cardiovascular diseases, taking into account the underlined biological mechanisms. A clear view of this challenge may be useful to standardize the clinical evaluation and to better identify treatments and strategies to counteract this DM aspect.

Introduction Osteopontin (OPN) is involved in ectopic calcification. Its circulating form is upregulated in coronary artery disease (CAD) patients. Circulating OPN levels positively correlate with oxidative stress, one of the major triggers of endothelial dysfunction. Endothelial dysfunction is, in turn, associated with reduced nitric oxide (NO) bioavailability due to the impaired arginine pathway. The aim of this study was to better understand the correlations between OPN, oxidative stress markers, and the arginine pathway metabolites. Methods and results ELISA and mass spectrometry techniques have been used to evaluate circulating OPN and arginine pathway/oxidative stress metabolites, respectively, in twenty-five control subjects and thirty-three patients with overt atherosclerosis. OPN positively correlates with 2,3-dinor-8isoPGF2a levels (p = 0.02), ornithine (p = 0.01), ADMA (p = 0.001), SDMA (p = 0.03), and citrulline (p = 0.008) levels only in CAD patients. In addition, citrulline positively correlated with ADMA (p = 0.02) levels, possibly as result of other sources of citrulline biosynthetic pathways. Conclusion The association between OPN and impaired arginine/NO pathway could play a role in the inhibition of endothelial NO synthase (eNOS) and/or in the arginase activation in the context of CAD patients. However, further studies are needed to verify the cause-effect relationship between OPN, oxidative stress, and arginine/NO pathway dysregulation.

Aging of the aortic valve, characterized by leaflet thickening and loss of extensibility, leads to progressive changes in valve function. These age-related mechanisms have not been evaluated yet in sex-specific calcific aortic valve stenosis (CAVS) onset and progression. Recent studies reported the association between high aortic valve calcification (AVC) load and male gender in patients with CAVS while women present faster progression than men. To evaluate these age- and sex-specific differences, we performed a systematic review and meta-analysis with meta-regression. A systematic search related to AVC measured by computed tomography and gender-specific differences was conducted according to PRISMA guidelines. Seven studies, enrolling 1859 men and 1055 women, were included in the quantitative synthesis. We found a significant difference between men and women both in AVC load and density. AVC load mean difference (MD), between men and women, was 1131 ± 243 AU (p < 0.0001; I2: 96.5 %, p < 0.001), while AVC density MD was 159 ± 20 AU/cm2 (p < 0.0001) without heterogeneity among the studies (I2: 23.5, p = 0.3). Meta-regression analyses showed that AVC load MD positively correlated with age and other cardiovascular risk factors such as diabetes, hypertension, and coronary artery disease presence. Our meta-analysis shows a significant association of incremental AVC load with male gender, regardless of the individual anatomical characteristics and the cardiovascular risk factors. Further studies are needed: i) to clarify if there are different sex-related pathophysiological processes driving the development and the progression of age-related CAVS, and ii) to determine if a sex-specific therapeutic strategy should be applied for CAVS treatment and/or prevention.

Background Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. Results Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. Conclusions This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis.

Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.

Background Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids. Findings We illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering. Conclusions Compared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures. Electronic supplementary material The online version of this article (doi:10.1186/s12575-016-0036-z) contains supplementary material, which is available to authorized users.

Small RNAs (sRNAs) are genetic tools for the efficient and specific tuning of target genes expression in bacteria. Inspired by naturally occurring sRNAs, recent works proposed the use of artificial sRNAs in synthetic biology for predictable repression of the desired genes. Their potential was demonstrated in several application fields, such as metabolic engineering and bacterial physiology studies. Guidelines for the rational design of novel sRNAs have been recently proposed. According to these guidelines, in this work synthetic sRNAs were designed, constructed and quantitatively characterized in Escherichia coli. An sRNA targeting the reporter gene RFP was tested by measuring the specific gene silencing when RFP was expressed at different transcription levels, under the control of different promoters, in different strains, and in single-gene or operon architecture. The sRNA level was tuned by using plasmids maintained at different copy numbers. Results demonstrated that RFP silencing worked as expected in an sRNA and mRNA expression-dependent fashion. A mathematical model was used to support sRNA characterization and to estimate an efficiency-related parameter that can be used to compare the performance of the designed sRNA. Gene silencing was also successful when RFP was placed in a two-gene synthetic operon, while the non-target gene (GFP) in the operon was not considerably affected. Finally, silencing was evaluated for another designed sRNA targeting the endogenous lactate dehydrogenase gene. The quantitative study performed in this work elucidated interesting performance-related and context-dependent features of synthetic sRNAs that will strongly support predictable gene silencing in disparate basic or applied research studies.

Whey is an abundant by-product of cheese production process and it is considered a special waste due to its high nutritional load and hypertrophic potential. Technologies for whey valorization are available. They can convert such waste into high-value products, like whey proteins. However, the remaining liquid (called permeate) is still considered as a polluting waste due to its high lactose concentration. The alcoholic fermentation of lactose into ethanol will simultaneously achieve two important goals: safe disposal of a pollutant waste and green energy production. This methodology paper illustrates the workflow carried out to design and realize an optimized microorganism that can efficiently perform the lactose-to-ethanol conversion, engineered via synthetic biology experimental and computational approaches.