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The selection of a suitable proteotypic peptide remains a challenge for designing a targeted quantitative proteomics assay. Although the criteria are well-established in the literature, the selection of these peptides is often performed in a subjective and time-consuming manner. Here, we have developed a practical and semiautomated workflow implemented in an open-source program named Typic. Typic is designed to run in a command line and a graphical interface to help selecting a list of proteotypic peptides for targeted quantitation. The tool combines the input data and downloads additional data from public repositories to produce a file per protein as output. Each output file includes relevant information to the selection of proteotypic peptides organized in a table, a colored ranking of peptides according to their potential value as targets for quantitation and auxiliary plots to assist users in the task of proteotypic peptides selection. Taken together, Typic leads to a practical and straightforward data extraction from multiple data sets, allowing the identification of most suitable proteotypic peptides based on established criteria, in an unbiased and standardized manner, ultimately leading to a more robust targeted proteomics assay.

SUMMARYThe poor prognosis of head and neck cancer (HNC) is associated with the presence of metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identified a nodal metastasis signature in the blood and saliva. In summary, we present the deepest proteome characterization wiring multiple sampling sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.

Purpose In the past decade, resting-state functional connectivity, acquired using functional magnetic resonance imaging (fMRI), has emerged as a popular measure of tinnitus, especially as related to self-reported handicap or psychological reaction. The goal of this study was to assess replicability of neural correlates of tinnitus, namely, resting-state functional connectivity, in the same individuals acquired over 2 sessions. Method Data were collected at 2 different sites (University of Illinois at Urbana–Champaign and Joint Base San Antonio Wilford Hall Ambulatory Surgical Center) using similar 3T magnets and similar data acquisition paradigms. Thirty-six patients (all civilians) were scanned using resting-state fMRI at the University of Illinois at Urbana–Champaign. Ten patients, active-duty Service members and Veterans, were scanned at the Wilford Hall Ambulatory Surgical Center and the Department of Defense Hearing Center of Excellence. Each participant was scanned twice, a week apart, using identical protocols of 10 min resting-state fMRI. Results Tinnitus handicap scores using the Tinnitus Functional Index and the Tinnitus Primary Function Questionnaire ranged between no or mild handicap to moderately severe handicap but did not significantly differ between visits. We examined the default mode, dorsal attention, and auditory resting-state networks and found that the strength of the within-network functional connections across visit was similar for the attention and default mode networks but not for the auditory network. In addition, the functional connection between the attention network and precuneus, a region of the default mode network, was also replicable across visits. Conclusions Our results show that resting-state fMRI measures are replicable and reliable in patients with a subjective condition, although some networks and functional connections may be more stable than others. This paves the way for using resting-state fMRI to measure the efficacy of tinnitus interventions and as a tool to help propose better management options.

Highlights • SILAC proteomics approach yielded important cisplatin resistance biomarkers in NSCLC. • Cisplatin resistance upregulated NER-based repair and downregulated mRNA splicing-related pathways. • STAT3 KO conferred epithelial morphology and sensitized to mTOR inhibition. • Cisplatin responsiveness is dependent on STAT3 and mTOR crosstalk in NSCLC cells. • Synergistically inhibition of STAT3 and mTOR is possibly a potential approach against NSCLC., Lung cancer is the second leading cause of cancer death worldwide and is strongly associated with cisplatin resistance. The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells and coordinates critical cellular processes as survival, self-renewal, and inflammation. In several types of cancer, STAT3 controls the development, immunogenicity, and malignant behavior of tumor cells while it dictates the responsiveness to radio- and chemotherapy. It is known that STAT3 phosphorylation at Ser727 by mechanistic target of rapamycin (mTOR) is necessary for its maximal activation, but the crosstalk between STAT3 and mTOR signaling in cisplatin resistance remains elusive. In this study, using a proteomic approach, we revealed important targets and signaling pathways altered in cisplatin-resistant A549 lung adenocarcinoma cells. STAT3 had increased expression in a resistance context, which can be associated with a poor prognosis. STAT3 knockout (SKO) resulted in a decreased mesenchymal phenotype in A549 cells, observed by clonogenic potential and by the expression of epithelial-mesenchymal transition markers. Importantly, SKO cells did not acquire the mTOR pathway overactivation induced by cisplatin resistance. Consistently, SKO cells were more responsive to mTOR inhibition by rapamycin and presented impairment of the feedback activation loop in Akt. Therefore, rapamycin was even more potent in inhibiting the clonogenic potential in SKO cells and sensitized to cisplatin treatment. Mechanistically, STAT3 partially coordinated the cisplatin resistance phenotype via the mTOR pathway in non-small cell lung cancer. Thus, our findings reveal important targets and highlight the significance of the crosstalk between STAT3 and mTOR signaling in cisplatin resistance. The synergic inhibition of STAT3 and mTOR potentially unveil a potential mechanism of synthetic lethality to be explored for human lung cancer treatment.

Introduction: Oral squamous cell carcinoma (OSCC) ranks among the top 10 leading causes of cancer worldwide, with 5-year survival rate of about 50%, high lymph node metastasis, and relapse rates. The OSCC diagnosis, prognosis, and treatment are mostly based on the clinical TNM classification. There is an urgent need for the discovery of biomarkers and therapeutic targets to assist in the clinical decision-making process. Areas covered: We summarize proteomic studies of the OSCC tumor, immune microenvironment, potential liquid biopsy sites, and post-translational modifications trying to retrieve information in the discovery and verification or (pre)validation phases. The search strategy was based on the combination of MeSH terms and expert refinement. Expert opinion: Untargeted combined with targeted proteomics are strategies that provide reliable and reproducible quantitation of proteins and are the methods of choice of many groups worldwide. Undoubtedly, proteomics has been contributing to the understanding of OSCC progression and uncovers potential candidates as biomarker or therapeutic targets. Nevertheless, none of these targets are available in the clinical practice yet. The scientific community needs to overcome the limitations by investing in robust experimental designs to strengthen the value of the findings, leveraging the translation of knowledge, and further supporting clinical decisions.

Lung cancer is the leading cause of cancer‑associated death worldwide and exhibits intrinsic and acquired therapeutic resistance to cisplatin (CIS). The present study investigated the role of mTOR signaling and other signaling pathways after metformin (MET) treatment in control and cisplatin‑resistant A549 cells, mapping pathways and possible targets involved in CIS sensitivity. MTT, flow cytometry, clonogenic assay, western blotting, proteomic analysis using the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) approach and reverse transcription‑quantitative PCR were performed. The results revealed that CIS treatment induced mTOR signaling pathway overactivation, and the mTOR status was restored by MET. MET and the mTOR inhibitor rapamycin (RAPA) decreased the viability in control and resistant cells, and decreased the cell size increase induced by CIS. In control cells, MET and RAPA decreased colony formation after 72 h and decreased IC50 values, potentiating the effects of CIS. Proteomics analysis revealed important pathways regulated by MET, including transcription, RNA processing and IL‑12‑mediated signaling. In CIS‑resistant cells, MET regulated the apoptotic process, oxidative stress and G2/M transition. Annexin 4 (ANXA4) and superoxide dismutase 2 (SOD2), involved in apoptosis and oxidative stress, respectively, were chosen to validate the SILAC analysis and may represent potential therapeutic targets for lung cancer treatment. In conclusion, the chemosensitizing and antiproliferative effects of MET were associated with mTOR signaling and with potential novel targets, such as ANXA4 and SOD2, in human lung cancer cells.

Made available in DSpace on 2021-06-25T10:24:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-23 In this study, a novel label-free electrochemical biosensor based on the zeolitic imidazole framework (ZIF-8) was developed for monitoring protein-protein interactions (PPIs). ZIF-8 was deposited on interdigitated electrodes and employed as a transducing material and simultaneously carried the thioredoxin-1 (Trx-1) protein, followed by the deposition of increased concentrations of the cytoplasmic domain of a disintegrin and metalloproteinase 17 (ADAM17cyto) known as the Trx-1 binding partner. Structural and morphological characterizations were used to validate and verify the formation of ZIF-8. The ZIF-8 crystals showed a rhombic dodecahedral structure with mainly exposed (011) facets, a mean particle size of 205 (±22) nm, and a ZIF-8 film thickness around 61 (±6) nm. The interaction between Trx-1 and ADAM17cyto proteins was analyzed through electrochemical impedance spectroscopy (EIS). The results indicate a linear and inverse relationship between the impedance responses at 0.1 Hz for ADAM17cyto concentrations from 50 nM to 8 μM, with a coefficient of variation from 1.0% to 11.4%. The proposed biosensor also displayed a significant selectivity and stability verified by using ADAM17cyto mutant and BSA as controls. As a proof-of-concept, we compared the results with a widely used type of PPI assay based on antibody recognition, the solid-phase binding assay, using the same proteins. The solid-phase binding assay was able to detect a significant binding only in ADAM17cyto concentrations above 0.5 μM, with a coefficient of variation varying from 5.4% to 27.5%. The results demonstrate that the developed biosensor was 10× more sensitive and reproducible than the conventional solid-phase binding assay. Furthermore, the developed electrochemical biosensor based on ZIF-8 provides a faster, label-free, and low-cost detection analysis, representing a novel strategy in detecting PPIs. Laboratório Nacional de Biociências (LNBio) Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) Laboratório Nacional de Nanotecnologia (LNNano) Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) Departamento de Biologia Molecular e Funcional Instituto de Biologia (IB) Universidade de Campinas (UNICAMP) Departamento de Físico-Química Instituto de Química (IQ) Universidade de Campinas (UNICAMP) Programa de Pós Graduação em Ciência e Tecnologia de Materiais (POSMAT) Universidade Estadual Paulista (UNESP) Programa de Pós Graduação em Ciência e Tecnologia de Materiais (POSMAT) Universidade Estadual Paulista (UNESP)

Background: Military service personnel are at increased risk for developing tinnitus due to heightened exposure to acoustic trauma. The auditory disorder is a leading service-connected disability among veterans and is highly comorbidly diagnosed with posttraumatic stress disorder (PTSD). The biopsychosocial model illustrates that chronic health conditions are exacerbated or maintained by psychiatric distress. Therefore, alleviation of such psychiatric distress can have beneficial impacts on health conditions, such as tinnitus. The aim of this study was to determine whether individuals with both disorders who receive evidence-based therapy for PTSD, will experience decreases in both PTSD and tinnitus-related distress.Methods: Veterans with comorbid bothersome tinnitus and PTSD received Cognitive Processing Therapy (CPT) and were assessed for PTSD, tinnitus-related distress, and depression at baseline and one month post-treatment follow-up.Results: At post-treatment follow-up, participants demonstrated significant decreases in PTSD symptoms compared to their baseline scores. Participants also demonstrated decreased tinnitus-related distress and depression, with high effect sizes.Discussion: This pilot study demonstrated the benefit of addressing psychiatric distress, such as PTSD, to simultaneously target bothersome tinnitus. Although not statistically significant due to the small sample size, large effect sizes indicate that tinnitus-related distress decreased as a function of receiving evidence-based therapy for PTSD. Future clinical trials should increase sample sizes and compare effects to control conditions.Trial RegistrationClinicalTrials.gov. NCT03702166. Registered October 10, 2018, https://clinicaltrials.gov/ct2/show/NCT03702166?cond=tinnitus&cntry=US&state=US%3ATX&city=San+Antonio&draw=1&rank=1

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging because changes can occur simultaneously at protease, their inhibitor, and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics, and peptidase predictions for studying proteolytic events in the saliva of 79 patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph-node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (area under the receiver operating characteristic curve > 0.85). In addition, endopeptidases and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from public repositories, reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis were further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by selected reaction monitoring–mass spectrometry, while minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of patients with OSCC and lymph-node metastasis and, at least in part, is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis., Graphical Abstract, Highlights • Proteolysis is accentuated in the saliva of patients with OSCC and lymph-node metastasis. • Differential saliva peptidome outperformed proteomics in patient classification. • Proteases implicated in differential proteolysis correlated with prognostic factors. • Reduced levels of protease inhibitors disturb the proteolytic balance in the saliva., In Brief Mass spectrometry–based peptidomics has been applied to the saliva of patients with oral squamous cell carcinoma, enabling the discovery of signatures correlated with poor prognostic factors, including lymph-node metastasis. Sequence analysis of differential peptides allowed the prediction of proteases implicated in endogenous proteolysis and associated with prognosis in head and neck cancers. The results of combined saliva peptidomics and proteomics revealed that the accentuated proteolysis in patients with oral squamous cell carcinoma and lymph-node metastasis concurs with reduced levels of protease inhibitors in saliva.

Tailoring genotypes for the variety of environmental scenarios associated with climate change requires modelling of the genetic variability of adaptation mechanisms to environmental cues. A large number of physiological mechanisms have been described and modelled, e.g. at transcript, metabolic or hormonal levels, but they remain to be assembled into whole-plant and canopy models. A ‘bottom-up’ approach combining physiological mechanisms leads to a near-infinite number of combinations and to an unmanageable number of parameters, so more parsimonious approaches are required. We propose that natural selection has constrained the large diversity of mechanisms into consistent strategies, in such a way that not all combinations of mechanisms are possible. These constraints, and resulting feedbacks, result in integrative ‘meta-mechanisms’, e.g. response curves of traits to environmental conditions, measurable via high-throughput phenotyping, and resulting in robust and stable equations with heritable genotype-dependent parameters. Examples are provided for the responses of developmental traits to temperature, for the response of growth and yield to water deficit and evaporative demand, and for the response of tillering to light and temperature. In these examples, it was inoperative to combine upstream mechanisms into whole-plant mechanisms, whereas the evolutionary constraints on the combinations of physiological mechanisms render possible the use of genotype-specific response curves at plant or canopy levels. These can be used for a new generation of crop models capable of simulating the behaviour of thousands of genotypes. This has significant consequences for plant modelling and its use in genetics and breeding.

The shedding of ectodomains is a crucial mechanism in many physiological and pathological events. A disintegrin and metalloprotease-17 (ADAM17) is a key sheddase involved in essential processes, such as development, regeneration, and immune defense. ADAM17 exists in two conformations which differ in their disulfide connection in the membrane-proximal domain (MPD). Protein-disulfide isomerases (PDIs) on the cell surface convert the open MPD into a rigid closed form, which corresponds to inactive ADAM17. ADAM17 is expressed in its open activatable form in the endoplasmic reticulum (ER) and consequently must be protected against ER-resident PDI activity. Here, we show that the chaperone 78-kDa glucose-regulated protein (GRP78) protects the MPD against PDI-dependent disulfide-bond isomerization by binding to this domain and, thereby, preventing ADAM17 inhibition.

Background Peripheral aneurysms are not included in the diagnostic criteria for Marfan syndrome (MFS); however, their real prevalence in MFS is unknown. Furthermore, they are commonly seen in other genetic entities such as Loeys-Dietz syndrome. We aimed to investigate the prevalence of peripheral aneurysm in Marfan syndrome. Methods Patients with clinical criteria of Marfan syndrome and identified FBN1 mutation were evaluated. Only patients with either MRI or CT angiography assessing peripheral vessels were included in this study. MRI and CT angiography studies were retrospectively evaluated to detect the presence of peripheral aneurysms. Aortic dissection-related arterial dilations were excluded. Aortic events and those related to aneurysm complications were collected during follow-up. Results Two hundred and nine patients with Marfan and FBN1 mutation were evaluated. Of these 136 (65.1%) had undergone either MRA or CTA with peripheral artery study during follow-up. Mean age at the last follow-up visit was 42.4±14.1yrs; 54.4% were men, and mean follow-up 7.3±3.1 years. Sixty-six aneurysms were identified in 42 (30.9%) patients. The most common locations were the iliac arteries in 23. The rest were: renal (7), vertebral (5), splenic (5), coeliac (3), brachiocephalic (1), subclavian (3), carotid (3), axillary (2), internal mammary (3), femoral (2), hypogastric (3), bronchial (2), coronary (1), hepatic (1), lumbar (1), gastroduodenal (1) and popliteal (1). Twenty-six patients (61.9%) had more than one peripheral aneurysm, and only 4 required surgery. Patients with peripheral aneurysms were older (47.2±14.3yrs vs 40.2±13.6yrs, p=0.06) and more frequently men (69.0% vs 47.9% p=0.026). Although patients with peripheral aneurysms did not more frequently have aortic dissection (16.7% vs 17.0%, p=0.586), they did more frequently have aortic surgery (73.8% vs 47.9% p=0.05). Conclusions Peripheral aneurysms are present in one third of Marfan syndrome patients and are related to age and more advanced aortic disease. Systematic use of whole-body vascular assessment in Marfan patients can provide a comprehensive evaluation of the entire arterial system, identifying other sites of vascular involvement at risk of potential complications, and the subgroup of patients with more aggressive vascular disease expression.

Background The impact of recent advances on the management of acute aortic syndrome (AAS) is usually reported by centres with great experience in aortic diseases. Current data on the management of this specific disease in Spain remains unknown. Purpose The Spanish Registry of Acute Aortic Syndrome (RESA-III) was established to assess current results in the management of AAS in a large cohort of hospitals from the same geographical area. Methods All patients admitted for AAS to 29 Spanish tertiary hospitals were enroled over 18 months (2017/2018). Results 574 patients, (68% men; mean age 64±14y; range 18–99) were prospectively and consecutively included. Aortic dissection was the underlying disease in 474 (82.6%) (375 type A, 99 type B), aortic haematoma in 76 (13.2%) (43 type A and 33 type B) and penetrating ulcer in 24 (4.2%) (7 type A and 17 type B). From the subgroup of type A AAS (74% n=425), 81% underwent surgical treatment and medical management was chosen in the remaining 19%. 78 patients did not undergo surgery principally because of severe comorbidities (n=34) or advanced age (n=24), patient refusal (n=7), or presence of an intramural haematoma (n=2). Regarding the cohort of patients with type B AAS (26% n=149), 52% were managed only medically, 37% with endovascular treatment, and 11% underwent open surgery. Endovascular treatment was indicated owing to recurrent pain (n=19), progressive vessel dilation (n=9), dissection expansion (n=5), peripheral (n=5) or visceral ischaemia (n=4), high blood pressure (n=18), peripheral bleeding (n=17) or haemodynamical instability (n=8). Overall type A mortality during hospitalisation was 36.4%; 26.4% in surgically treated and 79.4% in medically-treated patients (p=0.001). In type B AAS, overall mortality was 19.1%; 21.9% in the treated medically subgroup, 43.8% in those treated with open surgery and 7.8% in the endovascular treatment cohort (p=0.004). Conclusion Despite significant advances in acute aortic syndrome diagnosis and management, in-hospital mortality remains high. In type A AAS, medical management rate was too high (19%); however, in type B AAS, endovascular treatment yielded excellent results with less mortality than medical management (7.8% vs 19.1%, respectively). Our data support the need for continued improvement in the management of acute aortic syndrome.

Background The most common cardiovascular complications in Marfan syndrome (MFS) are aortic root dilation and type A aortic dissections. Elective aortic root surgery is indicated when maximum aortic diameter is larger than a defined threshold or in the case of fast-progressing dilation. However, maximum aortic diameter is limited for the prediction of aortic events. Indeed, a large international registry of acute aortic syndromes reported that as much as 40% of aortic dissections happen with maximum aortic diameter lower than 50 mm. Consequently, there is a need for new, non-invasive biomarkers to improve the prediction of aortic complications. Purpose The aim of the present study was to assess if proximal aorta circumferential and longitudinal strain and ascending aorta distensibility were associated with progressive aortic dilation and incidence of aortic events in Marfan syndrome patients. Methods Eighty seven Marfan syndrome patients free from previous cardiac/aortic surgery or dissection, were prospectively included in a multicenter follow-up. Patients were diagnosed by original Ghent criteria. Proximal aorta longitudinal and circumferential strain and distensibility were computed from baseline cine CMR images by means of feature-tracking. The predictive capacity of each stiffness biomarkers was separately tested with multivariable linear regression analysis (aortic growth) and with Cox logistic regression analysis (aortic events), both corrected for clinical and demographic variables, including baseline maximum aortic diameter. Results During a follow-up of 81.6±17 months, mean diameter growth-rate was 0.65±0.67 mm/year and z-score growth rate was 0.07±0.13 / year. Elective aortic root replacement was performed in 11 patients while two patients presented type A aortic dissection.Baseline proximal aorta longitudinal strain was independently related to diameter growth-rate (p=0.001), z-score growth-rate (p=0.018) and aortic events (p=0.018). Conversely, neither circumferential strain nor distensibility were independent predictors of diameter growth-rate (p=0.385 and p=0.381, respectively), z-score growth-rate (p=0.515 and p=0.484, respectively) and aortic events (p=0.064 and p=0.205, respectively). Conclusions Proximal aorta longitudinal strain predicts aortic root dilation and major aortic events in Marfan syndrome patients beyond aortic root diameter and clinical and demographic characteristics. Acknowledgement/Funding ISCIII PI14/0106, La Maratό de TV3 (20151330) and CIBERCV. Guala A. FP7/People n° 267128

Introduction Marfan syndrome (MFS) is a hereditary connective tissue disorder caused by mutation in the FBN1 gene. Main pulmonary artery (MPA) dilation is very prevalent in MFS patients. Indeed, the old Ghent nosology considered main pulmonary artery (MPA) dilation as diagnostic criterion of MFS patients. Although clinical complications related to pulmonary dilation in MFS are rare, this may potentially lead to MPA dissection or be a marker of vascular disease in MFS. Studies regarding potential causes of MPA dilation in MFS patients are very scarce. Purpose Through 4D flow CMR, we aimed to assess whether flow abnormalities exist in the MPA of MFS patients and their relation to local diameter. Methods Fifty-five consecutive Marfan syndrome adults (MFS) and 22 healthy volunteers (HV) were prospectively enrolled. All subjects underwent non-contrast-enhanced 4D flow-MRI, obtaining 4D flow field and a 3D angiography. The MPA was segmented from the 3D angiography, and the segmentation was used to mask 4D velocity data. Four, equidistant analysis planes were placed in the MPA between the pulmonary valve and the pulmonary artery bifurcation. Common descriptors of large arteries hemodynamics were computed at each plane: maximum velocity, systolic flow reversal ratio (a descriptor of the amount of systolic backward flow) and circumferentially-averaged axial and circumferential wall shear stress (WSS). Pulmonary artery diameters were measured on axial images. MPA dilation was defined as a diameter larger than 27 mm in women and 29 mm in men. Systolic (SBP) and diastolic (DBP) systemic blood pressure were measured at the brachial artery with a calibrated cuff immediately after the scan. Results Compared with HV, MFS patients presented similar age, BSA, SBP and maximum blood velocity, but had larger MPA diameter (27.8 vs 25.1 mm, p Figure 1 Conclusions Dilation of the main pulmonary artery is prevalent in Marfan syndrome patients. Abnormal increase in systolic vortexes and reduction in axial WSS were present in dilated and non-dilated MPA in MFS patients. Axial and circumferential WSS were independently related to MPA diameter. The eventual predictive role of abnormal pulmonary flow pattern in pulmonary artery dilation in MFS patients remain to be established Acknowledgement/Funding Instituto de Salud Carlos III (PI14/0106), La Maratό de TV3 (20151330), CIBERCV and FP7/People (267128)

International audience; Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

Numerical results included in Fig. 2.(DOCX 27 kb)

Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely associated with lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are membrane-bound particles that play a role in intercellular communication and impact cancer development and progression. Thus, profiling EVs would be of great significance to decipher their role in OSCC metastasis. For that purpose, we used a reductionist approach to map the proteomic, miRNA, metabolomic, and lipidomic profiles of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites, and 63 lipids differentially abundant between LN1 cell– and SCC-9 cell–derived EVs. A multi-omics integration identified 11 ‘hub proteins’ significantly decreased at the metastatic site compared with primary tumor–derived EVs. We confirmed the validity of these findings with analysis of data from multiple public databases and found that low abundance of seven ‘hub proteins’ in EVs from metastatic lymph nodes (ALDH7A1, CAD, CANT1, GOT1, MTHFD1, PYGB, and SARS) is correlated with reduced survival and tumor aggressiveness in patients with cancer. In summary, this multi-omics approach identified proteins transported by EVs that are associated with metastasis and which may potentially serve as prognostic markers in OSCC., Graphical Abstract, Highlights • Proteomic, miRNA, metabolomic, and lipidomic profiles were mapped in oral cancer EVs. • The molecular profile of EVs was associated with the lymph node metastatic phenotype. • A multi-omics integrative analysis revealed 11 highly connected ‘hub proteins.’ • ‘Hub proteins’ from EVs are candidates as prognostic markers in oral cancer., In Brief A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Differentially abundant molecules associated with the metastatic phenotype were enriched for key processes and pathways. An integrative analysis revealed 11 ‘hub proteins’ that are correlated with reduced survival and tumor aggressiveness in patients with cancer according to public databases. These EV molecules are candidates as prognostic markers in oral cancer.

A field experiment was conducted from 2005 to 2008 in Fulton County, Western Illinois with biosolids from conventional wastewater treatment applied as corn fertilizer in a series of P rates (0, 163, 325, 488, 650 kg P ha(-1)) along with commercial P fertilizer - triple superphosphate P (TSP) as reference to assess biosolids-P plant availability and potential loss to waterbodies through runoff. Air-dried biosolids and TSP were incorporated into surface soil at end of 2005, and corn (Zea mays) was planted for three consecutive years (2006-2008). Concentrations of soil extractable P except for Mehlich-3 P were always lower in the biosolids than TSP treatments at the same P rates. The soil potentially available P in water extractable P (WEP) and Olsen P derived from biosolids-P estimated by the exponential depletion model was 2-4% and 15-24% of total P in the applied biosolids, respectively. The residence time of biosolids-induced WEP and Olsen P in Midwest soil under annual corn cropping was 5 and 2 years, respectively. Corn tissue analysis showed lower increase in P concentration by biosolids-P than TSP. The elevation rate of soluble reactive P (SRP) concentration in simulated runoff was less by biosolids than TSP. Based on the data in this study, the plant availability and environmental risk of biosolids-P are lower than those of TSP in the Midwest soil, thus use of biosolids as P nutrient for corn would not cause a major impairment to water sources even P applied through biosolids was not completely used by annual crop.

The former US Steel Corporation's South Works site in Chicago, IL, is a 230-ha bare brownfield consisting of steel mill slag fill materials that will need to be reclaimed to support and sustain vegetation. We conducted a case study to evaluate the suitability of biosolids and dredged sediments for capping the steel mill slag to establish good quality turfgrass vegetation. Eight study plots were established on a 0.4-ha parcel that received biosolids and dredged sediment blends of 0, 25, 50, or 100% biosolids (v/v). Turfgrass was successfully established and was thicker and greener in biosolids-amended sediments than in unamended sediments. Concentrations of N, P, K, and micronutrients in turfgrass tissues increased with increasing biosolids. Soil organic carbon, N, P, and micronutrients increased with increasing biosolids. Cadmium, Cu, Ni, and Zn concentrations in biosolids-amended sediments also increased with increasing biosolids but were far below phytotoxicity limits for turfgrass. Lead and Cr concentrations in biosolids-amended plots were comparable to concentrations in unamended sediments. Groundwater monitoring lysimeters and wells below the study site and near Lake Michigan were not affected by nutrients leaching from the amendments. Overall, the results from this case study demonstrated that blends of biosolids and dredged sediments could be successfully used for capping steel mill slag brownfield sites to establish good quality turfgrass vegetation.

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor−node−metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis., Oral cancer has region-specific histopathological and molecular characteristics, complicating its classification by the standard tumor-node-metastasis system. Here, the authors combine discovery and targeted proteomics with IHC to identify region-specific and saliva biomarkers for oral cancer prognosis.

In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap-specific messenger RNA (mRNA) (nucleoside-2'-O-)-methyltransferase 1 (hMTr1), which catalyzes methylation of the 2'-O -ribose of the first nucleotide of capped mRNAs. Pre-mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5'-end of the whole cellular pre-mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre-mRNA maturation was identified, including splicing factors, spliceosome-associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA-binding proteins and proteins involved in mRNA 5'- and 3'-end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre-mRNA synthesis.

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.

A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17.Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases.This unexpected Trx-1

Prostaglandin E2 (PGE2) is well known to modulate urinary bladder functions, but it is also thought to be involved in the pathophysiology of lower urinary tract dysfunctions, since high levels of PGE2 have been found in overactive bladder (OAB) patients. β-Adrenoceptors are major players in detrusor muscle relaxation, and the selective β3-adrenoceptor (AR) agonist mirabegron was recently approved for the treatment of overactive bladder (OAB). β-Adrenoceptor modulation of PGE2 excitatory effects on bladder detrusor muscle was investigated by i.v. mirabegron after intravesical PGE2 infusion in conscious rats. Non-voiding activity (NVA) was assessed under isovolumetric conditions. In addition, mirabegron and isoprenaline (0.01-10 μM) were studied on PGE2-increased micro-contractile activity during isometric tension recordings of intact isolated bladder muscle strips. Our investigations showed that PGE2 dramatically increased NVA in vivo and spontaneous micro-contractions in vitro. In vivo administration of mirabegron (0.1, 0.3 and 3 mg/kg) reduced PGE2-augmented NVA in dose-dependent manner, while the PGE2-increased micro-contractions in isolated bladder strips were poorly inhibited. Isoprenaline inhibited PGE2-augmented micro-contractions in a concentration-dependent manner and had a higher potency compared to mirabegron. The apparent pKB of 7.25 for metoprolol at the isoprenaline concentration-response curve for PGE2-augmented micro-contractions suggests a β1-AR-mediated.

In the resting and un-stimulated state, the bladder wall is not quiescent and discrete contractile events, microcontractions, can be recorded in almost all species. This activity contributes to the active element of compliance and to the basal resting tension. This intrinsic activity underpins the more complex phasic activity, non-voiding activity (NVA) that can be seen to increase progressively as the bladder is filled. The NVA represents the motor component of a motor sensory system that relays information to the CNS on bladder volume. Despite the importance of this intrinsic motor activity, little is known about the mechanisms involved in its generation and modulation. The present experiments were done on isolated hemi-bladders from normal rats and measurements made of the intrinsic motor activity. Detailed analysis of the resting state reveals the presence of discrete phasic contractile events, micro-contractions that range in amplitude from 0.1-0.6 mN. These events seem to occur randomly and the basal activity has the appearance of ‘noise’. An analysis of the frequency amplitude distribution of the contractile events, reveals that the total activity appears to be the sum of a number of discrete contractile units, each generating a phasic contraction about a specific mean value and with characteristic frequency. In a hemi-bladder, there are between 20-30 units generating the activity at rest. Using the timed integral of the activity (product of amplitude and frequency), it was noted that the activity was increased by the muscarinic agonist carbachol, but it was decreased by the β-adrenergic agonist isoprenaline. Stretching the preparations also increased the activity. Using these observations, a simple model of the structural and functional organisation of the isolated rat bladder is proposed: the wall appears to be arranged into a number of discrete motor units acting independently. However, the activity can be stimulated or inhibited by pharmacological agents and mechanically (stretch). The possible relevance of this activity, its relationship to NVA and in relation to the mode of action of drugs are discussed. [Corrected]

In agricultural soils, a large portion of C in crop residues (i.e., non-harvested plant parts left in the field) is annually lost to atmosphere due to the low C use metabolism of soil microorganisms adapting to the environmental stress (moisture stress and substrate C and N imbalance). In this study, we tested the hypothesis that amending soil with biosolids (treated sewage sludge with high stable organic matter and low C:N ratio) can improve the C metabolism of microorganisms in agricultural soils through alleviation of microbial stress, leading to increased sequestration of crop residue-C in agricultural soils. Biosolids were applied at a mean annual rate of 4.2 kg m−2 (dry weight) to eight agricultural fields (biosolids-amended) for 13 years (1972–1984) in western Illinois. Four agricultural fields (unamended) received chemical fertilizer as control. We measured the sequestration rate of crop residue-C in the soils over the span of 34 years (1972–2006) using a 13C technique. We found dramatically greater sequestration rate of crop residue-C in biosolids-amended soil (32.5 ± 1.7% of total crop residue-C) versus unamended soil (11.8 ± 1.6%). Soil microbial metabolic quotient was significantly lower in biosolids-amended than in unamended fields, indicating that biosolid-amendment reduced soil microbial stress and improved microbial C metabolism. The study concludes use of a soil amendment with high stable C and low C:N is a valid approach to transform agricultural soils from current C-neutral status to a C sink. Biosolids represent a good choice of such soil amendments.

The objective of this study was to examine the effects of the type and intensity of nutritional stress, and of the statistical treatment of the data, on the genotype x environment (G x E) interaction for tropical maize (Zea mays). For this purpose, 39 hybrid combinations were evaluated under low- and high-nitrogen and -phosphorus availability. The plants were harvested at the V6 stage, and the shoot dry mass was estimated. The variance components and genetic values were assessed using the restricted maximum likelihood/best linear unbiased prediction method, and subsequently analyzed using the GGE biplot method. We observed differences in the performances of the hybrids depending on both the type and intensity of nutritional stress. The results of relationship between environments depended on whether genotypic values or phenotypic means were used. The selection of tropical maize genotypes against nutritional stress should be performed for each nutrient availability level within each type of nutritional stress. The use of phenotypic means for this purpose provides greater reliability than do genotypic values for the analysis of the G x E interaction using GGE biplot. Made available in DSpace on 2018-01-03T23:19:01Z (GMT). No. of bitstreams: 1 2016M.DeonGMREffects.pdf: 519873 bytes, checksum: e3d9abc79cc9b3333d0536409b6160c1 (MD5) Previous issue date: 2017-07-14

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrus, This work was supported by Sa˜ o Paulo Research Foundation (FAPESP grant 2011/20468-1). C.E.B. and A.F.Z.N. received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5–9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes., Author summary Previously thought to produce a harmless bout of fever associated with skin rash and muscle, or joint pain, the Zika virus (ZIKV) is an important cause of morbidity/mortality to human embryos/fetuses. Different from other models, here we report data from wild-type immunocompetent mice, rather than transgenic animals with suppressed immune responses. We found that intravascular ZIKV produced infection of maternal tissues, placentas and conceptuses, but that embryos/fetuses were comparatively much more affected than pregnant females, which seemed to tolerate well the viral challenge with no signs of encephalopathy. Importantly, 10.5 days post-coitum (dpc) embryos exposed to ZIKV at embryonic day 5 (second week in humans) displayed dysraphia, which is a regional failure of neural tube closure, and hydrocephalus, which is a symptom recently shown to precede microcephaly in humans. Characteristic phenotypes in more developed embryos/fetuses included abnormal articular postures analogous to arthrogryposis, which are typical human congenital contractures, gross and generalized malformations and intra uterine growth restriction (IUGR), rather than isolated microcephaly. Some developmental abnormalities and IUGR correlated with placental damage, suggesting that loss of placental function may play an important role in the disease. We believe our model is an asset in the search for useful concepts and prospective therapies for ZIKV because it better reproduces the human condition.