Quality attributes of CTVad1, a nanoemulsified adjuvant for phase I clinical trial of SpiN COVID-19 vaccine.

Aim: To develop, characterize and evaluate an oil/water nanoemulsion with squalene (CTVad1) to be approved as an adjuvant for the SpiN COVID-19 vaccine clinical trials. Materials & methods: Critical process parameters (CPPs) of CTVad1 were standardized to meet the critical quality attributes (CQAs) of an adjuvant for human use. CTVad1 and the SpiN-CTVad1 vaccine were submitted to physicochemical, stability, in vitro and in vivo studies. Results & conclusion: All CQAs were met in the CTVad1 production process. SpiN- CTVad1 met CQAs and induced high levels of antibodies and specific cellular responses in in vivo studies. These results represented a critical step in the process developed to meet regulatory requirements for the SpiN COVID-19 vaccine clinical trial.

Proteomic analysis reveals stage-specific reprogramed metabolism for the primary breast cancer cell lines MGSO-3 and MACL-1.

Breast cancer is the most prevalent cancer in women worldwide. Its molecular subtypes are based on the presence/absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). MACL-1 and MGSO-3 are cell lines derived from primary tumor sites of patients diagnosed with luminal A subtype carcinoma (ER+/PR+/HER2-) and ductal carcinoma in situ (ER-/PR-/HER2+), respectively. However, these cell lines lost the expression of these markers over cell culturing, and both have triple-negative phenotypes (ER-/PR-/HER2-), which has the poorest prognosis. Here, we sought to study the proteome signature of MGSO-3 and MACL-1, comparing them with the epithelial cell line MCF-10A and the well-established metastatic-derived breast cancer cell line MDA-MB-231. Our results showed that proteins associated with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) were upregulated in MGSO-3 and MACL-1 cells. These cell lines also showed upregulation of pro-apoptotic proteins when compared with MDA-MB-231. The molecular differences highlighted in this study may clarify the molecular basis behind cancer cells functioning and may reveal novel signatures across the breast cancer cell models.

Pathological cardiac remodeling seen by the eyes of proteomics.

Cardiac remodeling involves cellular and structural changes that occur as consequence of multifactorial events to maintain the homeostasis. The progression of pathological cardiac remodeling involves a transition from adaptive to maladaptive changes that eventually leads to impairment of ventricular function and heart failure. In this scenario, proteins are key elements that orchestrate molecular events as increased expression of fetal genes, neurohormonal and second messengers' activation, contractile dysfunction, rearrangement of the extracellular matrix and alterations in heart geometry. Mass spectrometry based-proteomics has emerged as a sound method to study protein dysregulation and identification of cardiac diseases biomarkers in plasma. In this review, we summarize the main findings related to large-scale proteome modulation of cardiac cells and extracellular matrix occurred during pathological cardiac remodeling. We describe the recent proteomic progresses in the selection of protein targets and introduce the renin-angiotensin system as an interesting target for the treatment of pathological cardiac remodeling.

Cardiomyocyte Proteome Remodeling due to Isoproterenol-Induced Cardiac Hypertrophy during the Compensated Phase.

PURPOSE: Although the pathophysiological response of cardiac tissue to pro-hypertrophic stimulus is well characterized, a comprehensive characterization of the molecular events underlying the pathological hypertrophy in cardiomyocytes during the early compensated cardiac hypertrophy is currently lacking. EXPERIMENTAL DESIGN: A quantitative label-free proteomic analysis of cardiomyocytes isolated was conducted from mice treated subcutaneously with isoproterenol (ISO) during 7 days in comparison with cardiomyocytes from control animals (CT). RESULTS: Canonical pathway analysis of dysregulated proteins indicated that ISO-hypertrophy drives the activation of actin cytoskeleton and integrin-linked kinase (ILK) signaling, and inhibition of the sirtuin signaling. Alteration in cardiac contractile function and calcium signaling are predicted as downstream effects of ISO-hypertrophy probably due to the upregulation of key elements such as myosin-7 (MYH7). Confocal microscopy corroborated that indeed ISO-treatment led to increased abundance of MYH7. Potential early markers for cardiac hypertrophy as APBB1, GOLGA4, HOOK1, KATNA1, KIFBP, MAN2B2, and SLC16A1 are also reported. CONCLUSIONS AND CLINICAL RELEVANCE: The data consist in a complete molecular mapping of ISO-induced compensated cardiac hypertrophy model at cardiomyocyte level. Marker candidates reported may assist early diagnosis of cardiac hypertrophy and ultimately heart failure.

Angiotensin-(1-7) oral treatment after experimental myocardial infarction leads to downregulation of CXCR4.

Myocardial infarction triggers cellular events that starts with the activation of inflammatory response and fibrogenic pathways involved in cardiac tissue remodeling. Angiotensin-(1-7) (Ang-(1-7)) is an endogenous heptapeptide from the renin-angiotensin system with a cardioprotective role due to its anti-inflammatory and anti-fibrotic activities in cardiac cells. Although the beneficial aspects of Ang-(1-7) in animal models of cardiac ischemia have been reported, the molecular events underlying Ang-(1-7) cardioprotective effect remains elusive. This study investigated the impact of oral treatment with Ang-(1-7) included in hydroxypropyl ß-cyclodextrin (HPßCD/Ang-(1-7)) on the cardiac proteome dysregulation due to experimental myocardial infarction. Wistar male rats were submitted to experimental myocardial infarction and treated daily with HPßCD/Ang-(1-7) during 7 days or 60 days by gavage. Our results showed that HPßCD/Ang-(1-7) treatment ameliorates the post-infarction condition due to the modulation of proteins that initially favor the resolution of inflammation and mitochondrial dysfunction. Moreover, this study reported for the first time that Ang-(1-7) treatment after experimental myocardial infarction leads to the downregulation of the C-X-C chemokine receptor type 4 (CXCR4). SIGNIFICANCE: Myocardial infarction triggers a sequence of cellular and molecular events that starts with an intense inflammatory response that is resolved in the proliferative phase. Prolonged inflammatory phase can lead to adverse cardiac repair and heart failure. In this context, we proposed a post-MI treatment using Ang-(1-7) included in HPßCD and administrated orally. We observed that HPßCD/Ang-(1-7) treatment led to CXCR4 downregulation, highlighting this C-X-C chemokine receptor as a potential therapeutic target for ischemic heart diseases.

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics.

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

Thalamic Proteome Changes and Behavioral Impairments in Thiamine-deficient Rats.

Thiamine deficiency (TD) has been used as an experimental model in rodents to study the molecular mechanisms of neurodegeneration and its association with behavioral changes. The aims of the present study were to investigate the spatial cognitive performance of pyrithiamine-induced thiamine deficiency (PTD) in adult male rats and disclose the thalamic proteome alterations caused by a severe TD episode. After the onset of the neurological signs, such as seizure and/or loss of righting reflex, the TD treatment was interrupted. Following 15 days of recovery, all rats were submitted to the spatial cognitive tasks in the Morris Water Maze (MWM). The results show that the PTD rats exhibited deficits during the learning process, which was reverted by repeated training. However, despite the spatial cognitive recovery, some protein changes were not reversible. The proteomic analysis, using label-free quantification, revealed deregulation of 183 thalamic proteins. Using bioinformatic tools, these proteins were categorized according to Gene Ontology functional annotation and metabolic pathways. We show that a severe TD affects proteins involved in different biological processes, such as, oxidative stress, neurotransmitter synthesis and synaptic vesicle cycle. These could explain the outcome in neurotransmitter release changes caused by TD, previously observed by our group and by other authors. These findings disclose the role of key proteins and metabolic pathways probably involved in the neurodegeneration process induced by TD. These proteins represent relevant molecular targets for future studies focusing also on the molecular basis of selective vulnerability of some brain areas to TD insult.

Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest.

Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and quantify enzymatic and non-enzymatic polypeptides present in secretomes of several fungi species. The resulting data have widened the scientific understanding of the way filamentous fungi perform biomass degradation and offered novel perspectives for biotechnological applications. The present review discusses proteomics approaches that have been applied to the study of fungal secretomes, focusing on two of the most studied filamentous fungi genera: Trichoderma and Aspergillus.

Identification of multienzymatic complexes in the Clonostachys byssicola secretomes produced in response to different lignocellulosic carbon sources.

Multienzymatic complexes with plant lignocellulose-degrading activities have recently been identified in filamentous fungi secretomes. Such complexes have potential biotechnological applications in the degradation of agro-industrial residues. Fungal species from the Clonostachys genus have been intensively investigated as biocontrol agents; however so far their use as producers of lignocellulose-degrading enzymes has not been extensively explored. Secretomes of Clonostachys byssicola following growth on different carbon sources (passion fruit peel, soybean hulls, cotton gin trash, banana stalk, sugarcane bagasse, orange peel, and a composition of soybean hulls: cotton gin trash:orange peel) were subjected to enzymatic assays. Remarkable differences were observed among the samples, especially regarding levels of mannanase and pectinase activities. Secretomes were then subjected to Blue Native PAGE in order to resolve putative protein complexes which subsequently had their composition revealed by trypsin digestion followed by LC-MS/MS analysis. The protein bands (named I, II, III and IV) were shown to be composed by holocellulolytic enzymes, mainly cellulases and xylanases as well as proteins involved in biocontrol processes, such as chitinases and proteases. The high diversity of proteins found in these multicatalytic assemblies confirms C. byssicola as a novel source of plant biomass-degrading enzymes.

Unveiling the Trypanosoma cruzi Nuclear Proteome.

Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions.

Secretomic survey of Trichoderma harzianum grown on plant biomass substrates.

The present work aims at characterizing T. harzianum secretome when the fungus is grown in synthetic medium supplemented with one of the four substrates: glucose, cellulose, xylan, and sugarcane bagasse (SB). The characterization was done by enzymatic assays and proteomic analysis using 2-DE/MALDI-TOF and gel-free shotgun LC-MS/MS. The results showed that SB induced the highest cellulolytic and xylanolytic activities when compared with the other substrates, while remarkable differences in terms of number and distribution of protein spots in 2-DE gels were also observed among the samples. Additionally, treatment of the secretomes with PNGase F revealed that most spot trails in 2-DE gels corresponded to N-glycosylated proteoforms. The LC-MS/MS analysis of the samples identified 626 different protein groups, including carbohydrate-active enzymes and accessory, noncatalytic, and cell-wall-associated proteins. Although the SB-induced secretome displayed the highest cellulolytic and xylanolytic activities, it did not correspond to a higher proteome complexity because CM-cellulose-induced secretome was significantly more diverse. Among the identified proteins, 73% were exclusive to one condition, while only 5% were present in all samples. Therefore, this study disclosed the variation of T. harzianum secretome in response to different substrates and revealed the diversity of the fungus enzymatic toolbox.

Blue native-PAGE analysis of Trichoderma harzianum secretome reveals cellulases and hemicellulases working as multienzymatic complexes.

Plant cell wall-degrading enzymes produced by microorganisms possess important biotechnological applications, including biofuel production. Some anaerobic bacteria are able to produce multienzymatic complexes called cellulosomes while filamentous fungi normally secrete individual hydrolytic enzymes that act synergistically for polysaccharide degradation. Here, we present evidence that the fungus Trichoderma harzianum, cultivated in medium containing the agricultural residue sugarcane bagasse, is able to secrete multienzymatic complexes. The T. harzianum secretome was firstly analyzed by 1D-BN (blue native)-PAGE that revealed several putative complexes. The three most intense 1D-BN-PAGE bands, named complexes [I], [II], and [III], were subsequently subjected to tricine SDS-PAGE that demonstrated that they were composed of smaller subunits. Zymographic assays were performed using 1D-BN-PAGE and 2D-BN/BN-PAGE demonstrating that the complexes bore cellulolytic and xylanolytic activities. The complexes [I], [II], and [III] were then trypsin digested and analyzed separately by LC-MS/MS that revealed their protein composition. Since T. harzianum has an unsequenced genome, a homology-driven proteomics approach provided a higher number of identified proteins than a conventional peptide-spectrum matching strategy. The results indicate that the complexes are formed by cellulolytic and hemicellulolytic enzymes and other proteins such as chitinase, cutinase, and swollenin, which may act synergistically to degrade plant cell wall components.