Membrane proteins constitute the filter that controls the cellular traffic of nutrients, ions and other essential molecules, as well as the transmission of signals across the membrane. These proteins interact with other proteins in the cytosol, cytoskeleton or the extracellular side of the membrane, giving rise to complex interactomes that are distributed throughout the various lipid microdomains of the membrane plane. In this manner, complex networks of protein-protein and protein-lipid interactions are formed which regulate the most diverse biological functions, and disturbance of these networks can lead to disease. Therefore, characterization of these interactomes is a priority for current biomedical sciences. Traditionally, such studies have largely depended on solubilization/dissociation of the essential components of multiprotein complexes with detergents of various strength. However, this technique may result in the loss of certain components of such complexes, especially those whose binding is weak or transient. Moreover, protein solubilization can lead to the formation of non-native spurious interactions. As an alternative, proximity labelling (PL) techniques have been developed in recent years that can identify interactors of the protein of interest in a native cellular environment, prior to solubilization. In this article, we review the recent advances in PL and explore the new possibilities they offer for the characterization of membrane interactomes. SIGNIFICANCE: Membranes establish a series of complex protein-protein and protein-lipid interactions that are essential for cell physiology. For decades, they have been one of the central objects of study in Cell and Molecular Biology. However, knowledge of the structure of membrane proteins and their respective interactomes lags far behind that of soluble proteins, mainly due to technical difficulties in their handling and characterization caused by their insolubility. Recent research has developed various techniques to study these proteins in their native cellular environment. In this review article we address the application to membrane proteins of the so-called 'proximity labeling methods', which allows neighborhood relationships to be established between proteins in intact cells. The scarcity of alternatives for study of the components of membrane complexes make these methods especially attractive for analyzing this type of membrane associated supramolecular structures.

While macrophages are well-known to polarize across the inflammatory spectrum, neutrophils have only recently been found to activate in a similar fashion in response to pro- or anti-inflammatory stimuli. Matrix metalloproteinase (MMP)-12 mediates neutrophil physiology with direct signaling mechanisms yet to be investigated. We hypothesized MMP-12 may modify neutrophil signaling. Bone marrow neutrophils were stimulated with interleukin (IL-1β; pro-inflammatory), IL-4 (anti-inflammatory), or MMP-12. The secretome was mapped by multi-analyte profiling and intracellular signaling evaluated by array. IL-1β induced a cytokine-mediated inflammatory LPS-like signalome, with upregulation of pro-inflammatory cytokines such as interferon gamma (IFNγ,15.2-fold,p = 0.001), chemokine (C-X-C motif) ligand 1 (CXCL1,8.4-fold,p = 0.005), and tumor necrosis factor alpha (TNFα,11.2-fold,p = 0.004). IL-4 induced strong intracellular signaling with upregulation of mitogen-activated protein kinase kinase (MEK1;1.9-fold,p = 0.0005) and downregulation of signal transducer and activator of transcription 4 (STAT4;0.77-fold,0.001). MMP-12 increased IL-4 secretion 20-fold and induced a robust apoptotic neutrophil signalome with upregulation of forkhead box O1 (FOXO1;1.4-fold,p < 0.0001) and downregulation of WNT signaling with MMP-12 cleavage of the adherens junction components β-catenin, cahderin-3, and catenin-α2. In conclusion, neutrophils shifted phenotype by stimuli, with MMP-12 inducing a unique apoptotic signalome with higher resemblance to the anti-inflammatory signalome. SIGNIFICANCE: This study revealed that neutrophils demonstrate unique polarization signaling responses to specific stimuli, with the matrix metalloproteinase (MMP)-12 signalome showing similarity to the IL-4 signalome. MMP-12 polarized neutrophils towards a strong apoptotic signature by upregulating FOXO1 and downregulating WNT signaling. Our results highlight that neutrophils display more plasticity than previously appreciated.

Objective of this study is to find the optimal conditions for preparing the samples, resulting in quality, reproducible and specific MS spectra of the ticks, with a shelf life in 70% ethanol of more than ten years. Amblyomma (Am.) variegatum species which had been stored in alcohol for more than twenty years and for which numerous specimens were available were used to compare the performance of four protocols tested. Spectra of insufficient quality were obtained from Am. variegatum legs preserved in alcohol for long periods with the reference protocol, named DO that we had set up years ago. The same observation was made on the spectra from Am. variegatum legs from dry (evaporated alcohol, DO-mod protocol). With new protocols named ReDO and PReDO the spectra were of good quality with high intensities (> 3000 a.u.). Blind testing showed that 94%, and 93% of the spectra were correctly identified with relevant log score values (LSVs ≥1.8), respectively for ReDO and PReDO protocols. All soft ticks treated in this study by PReDO protocol exhibited low intensity spectra with background noise. This study revealed that MALDI-TOF MS is able to identify hard ticks stored during decades in alcohol or dry (evaporated alcohol). SIGNIFICANCE OF THE STUDY: The correct identification of ticks, including vectors responsible for the transmission of infectious diseases in humans and animals is essential for their control. MALDI-TOF MS, a proteomic tool that has emerged in recent years, has become an innovative, accurate and alternative tool for the identification of arthropods, including ticks. However, previous studies reported that preservation of arthropods in alcohol modified the MS spectra obtained from specimens of the same species freshly collected or frozenly stored. In this study, a standard protocol was established for the identification of tick collections which had been stored for more than ten years in alcohol. Four different protocols were assessed. The analysis of the results showed that among the four protocols tested, two protocols named ReDO (Rehydration and incubation of the legs in 40 μl of HPLC water for 12 h in a dry bath at 37°) and PreDO (Drying of the legs for 12 h in a dry bath at 37 °C followed by rehydration and incubation in 40 μl of HPLC water for 12 h.) seem to be more appropriate for the MALDI-TOF MS identification of ticks from old collections preserved in alcohol or dry. This study is promising for the future, as it will make it possible to create a MALDI-TOF MS database from a wide range of ticks which have been stored for a long time in alcohol or which are dry stored in laboratories and museums around the world.

Aeromonas hydrophila is a widespread opportunistic pathogen of aquatic fishes in freshwater habitats. The current emergence of antimicrobial-resistant A. hydrophila has been reported in the world while the bacterial antibiotics adaptive mechanism remains poorly explored. In this study, using quantitative proteomics technology, the behavior of A. hydrophila was investigated by comparing the differentially expression proteins between with and without kanamycin (KAN) treatment. A total of 374 altered proteins including 184 increasing and 190 proteins decreasing abundances were quantified when responding to KAN stress. The bioinformatics analysis showed that stress related proteins were hub proteins that significantly increased to reduce the pressure from the misreading of mRNA caused by KAN. Moreover, several metallic pathways, such as oxidative phosphorylation and TCA cycle pathways may affect KAN resistance. Finally, eight selected genes were deleted and their antibiotics susceptibilities to kanamycin were valued, respectively. Results showed that OmpA II family protein A0KI26, and two-component system protein AtoC may involve in the KAN resistance in this study. In general, our results provide an insight into the behaviors of bacterial responding to KAN stress, and demonstrate the intrinsic antibiotics adaptive mechanism of A. hydrophila. BIOLOGICAL SIGNIFICANCE: In this study, the differentially expressed proteins (DEPs) of A. hydrophila strain between with and without kanamycin (KAN) were compared by using a data-independent acquisition (DIA) - based quantitative proteomics method. Bioinformatics analysis showed that stress - related proteins are hub proteins that significantly increased under KAN stress. Moreover, several metallic pathways, such as oxidative phosphorylation and citrate cycle (TCA cycle) pathways, can affect KAN resistance. Finally, our antibiotics susceptibility assay showed that the protein A0KI26 of the OmpA II family, and the AtoC of the two-component system may involve in KAN resistance in this study. These results provide insights into the antibiotics adaptation mechanism of A. hydrophila when responding to KAN stress.

Accurate genome annotation, the foundation of life science research in the genome era, is hampered by limited known gene models, nonstandard start codons, and the limited homology of annotated genes in other organisms. LysargiNase mirrors trypsin at the cleavage sites, providing the opportunity to identify peptides other than tryptic peptides. In this study, we used an in-house developed acetylated LysargiNase (Ac-LysargiNase) with higher activity and stability in non-pathogenic Mycolicibacterium smegmatis MC2 155 to supplement the widely used trypsin in proteomic studies. We identified 27,582 peptides from 3844 annotated proteins and 332 novel genome search-specific peptides (GSSPs). Among these GSSPs, 88 peptides were annotated in another M.smegmatis genome database, and 41 were verified as novel peptides by predicted theoretical spectra and their corresponding 15N-labeling spectra. Further analysis revealed that 17 verified GSSPs corrected the N-terminus of the 13 annotated genes. The other 24 verified GSSPs helped identify 17 novel open reading frames (ORFs) missed in previously annotated M. smegmatis genomes. Among these novel ORFs, four relatively small proteins with amino acid residues less than 100 and three were precisely identified with C-terminal peptides. Ac-LysargiNase helps with genome reannotation by identifying new genes and events in proteogenomic studies. SIGNIFICANCE: Correct genomic annotation is vital in the field of life sciences. The nonstandard start codons seriously affect the confirmation of the translation initiation sites (TISs) of an open reading frame (ORF), and unknown structural genes are easily missed in automated gene prediction. Although proteogenomics presents new avenues for validating gene expression and gene structure refinement based on conventional tryptic peptides, determining the TISs and potential encoding genes is complicated. Thus, validation of TISs and encoding ORFs is crucial and urgent. Therefore, we recommend Ac-LysargiNase, a mirror enzyme of trypsin that can identify additional novel peptides for N-terminal correction and ORF identification.

Japanese encephalitis (JE) is just an acute encephalitis syndrome contributed to Japanese encephalitis virus (JEV) infection. It the chief causes of viral encephalitis in Asia. In recent years, association of JEV infection with neurological problems such as Guillain-Barré syndrome(GBS) had reported. Nevertheless, its potential pathogenic mechanism has not previously been reported. Therefore, it is urgent to study the relationship between peripheral nerve injury (PNI) and JEV infection. Here, we use the liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique to make out the protein expression levels of mice sciatic nerve between JEV infection group and the sham group. In general, 4303 proteins were designated by MS, and 187 differentially expressed proteins(DEPs) were found. There were 105 proteins up-regulated in the injured sciatic nerve, and 82 proteins were down-regulated. Functional enrichment analysis of DEPs showed that the up-regulated proteins were mainly related to immune regulatory response, and down-regulated proteins were related to ribosomal structural components and translation. SIGNIFICANCE: The Japanese encephalitis virus, a member of the flavivirus, is a Mosquito borne virus. It leads to central nervous system injury by the immune response and inflammation in the brain. In addition, the virus also gave rise to PNI. It is a major public health problem in Asia. The diversity of clinical symptoms has brought serious challenges to the diagnosis and treatment of the disease. Label-Free Proteomics was undertaken to explore the potential mechanisms between JEV and peripheral nervous system in this study. It provided strong evidence that tissue damage is caused by the immune-mediated mechanisms rather than the virus, which offers a basis for the prevention of the disease and further looking for treatment targets.

A major pathological mechanism involved in vascular remodeling diseases is the proliferation and migration of vascular smooth muscle cells. The lipid distribution of golden hamsters is similar to that of humans, which makes them an excellent study model for studying the pathogenesis and molecular characteristics of vascular remodeling diseases. We performed proteomic analysis on Sprague Dawley rat VSMCs (rVSMCs) and restenosis hamsters with low-density lipoprotein receptor (LDLR) deficiency as part of this study. We have also performed the enrichment analysis of differentially modified proteins in regards to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein domain. 1070 differentially abundant proteins were assessed in rVSMCs before and after platelet-derived growth factor-BB (PDGF-BB) stimulation. Specifically, 1246 proteins displayed significant differences in the restenosis model in LDLR-deficient hamsters. An analysis of crosstalk between LDLR+/- hamsters artery restenosis and proliferating rVSMCs revealed 130 differentially expressed proteins, including 67 up-regulated proteins and 63 downregulated proteins. Enrichment analysis with KEGG showed differential proteins to be mainly concentrated in metabolic pathways. There are numerous differentially abundant proteins but particularly two proteins (phosphofructokinase 1 of liver type and lactate dehydrogenase A) were found to be up-regulated by PDGF-BB stimulation of rVSMCs and in a restenosis model of hamsters with LDLR+/- expression. SIGNIFICANCE: Based on bioinformatics, we have found glycolysis pathway plays an important role in both the LDLR+/- hamsters restenosis model and the proliferation of rVSMCs. Some key glycolysis enzymes may likely be developed either as new biomarkers or drug targets for vascular remodeling diseases.

Xiangshui' lemon exhibits gametophyte self-incompatibility: after self-pollination, the growth of a pollen tube in the style is inhibited. The aim of this work was to study the effect of pollination on the ubiquitylome and proteome in 'Xiangshui' lemon pistil. Here, by using label-free quantification, enrichment by anti-diglycine lysine antibody conjugated agarose beads, high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) and the method of shotgun proteomics, we investigated the quantitative proteome, ubiquitylome, and crosstalk between the two datasets in the 'Xiangshui' lemon pistil after pollination. In total, 4682 protein species, 1956 ubiquitinated protein species and 6922 ubiquitination sites were quantified. A principal component analysis revealed that there were some differences in the ubiquitylome and proteome of CK, self-pollination and cross-pollination. The results of the crosstalk showed that the proteome and ubiquitylome were negatively correlated. After self-pollination, the abundance of the ubiquitinated protein species related to programmed cell death, cell aging and sphingolipid metabolic pathways was changed. Furthermore, we analyzed the proteasome pathway in the pistil after self-pollination and further discussed the mechanism by which ubiquitination events affect S-RNase in the style. Overall, pollination greatly changed the ubiquitinated protein species abundance in the pistils of 'Xiangshui' lemon. Accordingly, our work provides a systematic analysis of the proteome and ubiquitylome in 'Xiangshui' lemon pistils under self-incompatibility and sheds light for future studies on the function and mechanism of ubiquitination during self-incompatibility in 'Xiangshui' lemon. SIGNIFICANCE: As a mechanism to prevent self-pollination, self-incompatibility (SI) exists widely in plant species. Although a large number of SI-related genes have been found in fruit trees, there are few studies on the PTMs that affect and are involved in fruit tree responses to SI. This study highlights the fact that the effects of pollination on proteome and ubiquitylome in the 'Xiangshui' lemon pistil, we discussed the correlation between transcriptome and proteome, ubiquitylome and proteome, and we analyzed the expression and the changes of ubiquitination levels of SI related proteins and pathway after self- and cross pollination, and the changes of ubiquitination level of 26S proteasome pathway after cross-pollination. This study provides new insights into the ubiquitination pathway of SI in 'Xiangshui' lemon.

Constitutively active K-Ras oncogene mutation at G12V changes the proteome of cells and activates macroautophagy for cell advantage. Inhibition of macroautophagy impairs K-Ras mediated tumor progression to a limited extent with increase of spontaneous tumors due to poorly understood mechanisms. Here, we show that inhibition of macroautophagy in K-Ras G12V mouse embryonic fibroblasts (MEFs) hyper activates chaperon mediated autophagy (CMA). Quantitative identification of CMA substrates through co-immunoprecipitation of CMA component heat shock cognate 70 (Hsc70) demonstrates a shift of proteins from macroautophagy to CMA mediated degradation. However, macroautophagy impairment show significant inhibition on proliferation and CMA hyper activation provides a basal support to macroautophagy-inhibited MEFs for survival. On the other hand, K-Ras G12V MEFs impaired of CMA reduces number of Hsc70 clients but activated macroautophagy significantly compensated CMA loss. Nonetheless, co-inhibition of CMA and macroautophagy had a synergistic detrimental effect on both proliferation and survival of MEFs expressing K-Ras G12V mutant. Our results point to K-Ras G12V MEFs dependency on macroautophagy and CMA partly compensates its loss for survival but not hyper-proliferation; implicating that targeting both macroautophagy and CMA as a promising therapeutic target in G12V mutation associated K-Ras cancers. SIGNIFICANCE: The present study provides a framework of Hsc70 interacting proteins, which differentially interact with Hsc70 in response to autophagy alterations. The role of proteins accumulation and induced proteo-toxicity could be underlying factor in macroautophagy and CMA co-inhibited K-Ras G12V MEFs phenotype. Our study provides rational for adaptive mechanisms in K-Ras tumors inhibited with different autophagy pathways and also supports targeting both macroautophagy and CMA simultaneously as therapeutic target. At the same time current study will help in characterizing the underlying cellular processes that may play a role in escaping tutor suppressor role CMA and macroautophagy in cancers harboring K-Ras G12V mutation that may be further utilized to identify molecular targets for K-Ras-driven cancers.

Milk is a nutrient-rich biofluid that contains several biocomponents with distinctive functions, including extracellular vesicles (EV). Milk EV have been associated with the regulation of the newborn's immune system and to influence essential cellular development. The EV proteome comprises the protein constituents and cargo; changes in these compartments could impact their role mediating communication. The ratio of dietary ω-6 to ω-3 polyunsaturated fatty acids (PUFA) is known to affect health and inflammation, and to induce changes in milk fatty acid composition, but no reports have included the milk EV fraction so far. We isolated EV from milk samples obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet or a test diet enriched in ω-3 (ω6:ω3 = 4:1). Small milk-derived EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Using a TMT-based high-resolution quantitative approach, the proteomics analysis revealed variations in the milk EV proteome within the diet groups with differences in the abundance of spondin-2 and 78 kDa glucose-regulated protein. Future studies are encouraged to explore further dietary effects on milk EV composition and their relation to the offspring's development. SIGNIFICANCE: Milk EV are known as key players mediating the regulation of the infant's immune system and growth. The EV proteome comprises the protein constituents and protein cargo, and any changes in this system could impact their role in intercellular communication. This study aimed at evaluating how different ω-6:ω-3 ratios in the maternal diet could translate to the milk EV proteome. This is relevant for basic research, but also has applied aspects in animal nutrition and health and may provide new perspectives for feeding additives.

The Imbabura treefrog (Boana picturata) is an underexplored source of bioactive peptides. The combination of molecular cloning and mass spectrometry allowed us to identify three new peptide families, named "Picturins" (PTR), "Pictuseptins" (PTS), and "Boanins" (BNS). PTR is composed of three 25-mer peptides, characterized by the N-terminal sequence: GVFKDALKQ and the C-terminal sequence: AANALKPK. The sequences of PTR-1, -2 and - 3 are highly conserved only showing two divergent sites: (L/F) in position 10 and (K/Q) in position 17. PTS gathers six peptides. PTS -1, -2 and - 4 have 22 amino acid residues in length, while PTS -3, -5 and - 6 are composed of 26 residues. Whereas BNS are four 28-37 mer peptides, showing two conserved regions: the N-terminal sequence FLGAL and the C-terminal sequence KALNP. PTR-1 to 3 and PTS -1 to -3 were chemically synthetized and their antimicrobial and haemolytic activity was assessed. PTR displayed moderate activity against Escherichia coli (MIC 24.80 to 48.95 μM), while PTS showed a broad antimicrobial and antifungal effect. PTS-1 was the most active peptide against E. coli (6.8 μM) followed by PTS-3 (11.7 μM) and PTS-2 (14.24 μM). These peptides also showed low haemolytic activity, pointing to a favorable selectivity. Overall, new unique non-hemolytic and cationic peptide sequences were characterized that could be valuable for the next-generation of anti-infective drugs. Future functional studies should explore the pharmacological potential of Boanins to include them as antimicrobial scaffolds. BIOLOGICAL SIGNIFICANCE: Nature-inspired solutions have shown their importance mainly for the development of the pharmaceutical industry. Frog skin peptides are excellent examples of the biomedical potential of naturally evolved molecules for specific targets, including multi-resistant bacteria. The characterization of new chemical entities from poorly studied skin secretions of Ecuadorian biodiversity, such as B. picturata, represents an unprecedented opportunity to identify candidates to tackle global concerns, for instance, antibiotic resistance.

The study of chemical speciation and the refinement and expansion of omics-based methods are both consolidated and highly active research fields. Although well established, such fields are extremely dynamic and are driven by the emergence of new strategies and improvements in instrumentation. In the case of omics-based studies, subareas including lipidomics, proteomics, metallomics, metabolomics and foodomics have emerged. Here, speciomics is being proposed as an "umbrella" term, that incorporates all of these subareas, to capture studies where the evaluation of chemical species is carried out using omics approaches. This paper contextualizes both speciomics and the speciome, and reviews omics applications used for species identification through examination of proteins, metalloproteins, metabolites, and nucleic acids. In addition, some implications from such studies and a perspective for future development of this area are provided. SIGNIFICANCE: The synergic effect between chemical speciation and omics is highlighted in this work, demonstrating an emerging area of research with a multitude of possibilities in terms of applications and further developments. This work not only defines and contextualizes speciomics and individual speciomes, but also demonstrates with some examples the great potential of this new interdisciplinary area of research.

Model-based assessment of drug pharmacokinetics in liver disease requires quantification of abundance and disease-related changes in hepatic enzymes and transporters. This study aimed to assess performance of three label-free methods [high N (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA)] against QconCAT-based targeted data in healthy and diseased (cancer and cirrhosis) liver tissue. Measurements were compared across methods and disease-to-control ratios provided a 'disease perturbation factor' (DPF) for each protein. Mean label-free measurements of targets correlated well (Pearson's coefficient, r = 0.91-0.98 p < 0.001) and with targeted data (r = 0.65-0.95, p < 0.001). Concordance with targeted data was generally moderate (Lin's concordance coefficient, ρc = 0.46-0.92), depending on methodology. Moderate precision and accuracy were observed for label-free methods (average fold error, AFE = 1.44-1.68; absolute average fold error, AAFE = 2.44-3.23). The DPF reconciled the data and indicated downregulated expression in cancer and cirrhosis, consistent with an inflammatory effect. HiN estimated perturbation consistently with targeted data (AFEHiN = 1.07, AAFEHiN = 1.57), whereas iBAQ overestimated (AFEiBAQ = 0.81, AAFEiBAQ = 1.67) and TPA underestimated (AFETPA = 1.37, AAFETPA = 1.65) disease effect. Progression from mild to severe cirrhosis was consistent with progressive decline in expression, reproduced by HiN but overestimated by iBAQ and underestimated by TPA (AFEHiN = 0.98, AFEiBAQ = 0.60, AFETPA = 1.24). DPF data confirmed non-uniform disease effect on drug-elimination pathways and progressive impact of disease severity. SIGNIFICANCE: This study demonstrated good correlation and moderate concordance between intensity-based label-free proteomic methods (HiN, iBAQ and TPA) and targeted data. Label-free measurements tended to overestimate abundance, but differences were reconciled using a disease perturbation factor (DPF) for each protein. With targeted data as a reference, HiN defined disease perturbation and the impact of disease progression consistently, indicating that the use of 'razor' peptides for quantification against an exogenous standard provides biologically sensible quantitative fingerprints of disease. Disease-driven perturbations in expression relative to healthy baseline are incorporated into drug kinetic models used to predict drug exposure in disease populations where clinical studies may not be feasible.

The prevalence of obesity has increased significantly worldwide. Therefore, this study aimed to evaluate the influence of obesity on the proteomic profile of periodontal ligament (PDL) tissues of rat first maxillary molars (1 M) submitted to orthodontic tooth movement (OTM). Ten Holtzman rats were distributed into two groups (n = 5): the M group (OTM), and the OM group (obesity induction plus OTM). Obesity was induced by a high-fat diet for the entire experimental periods After that period, the animals were euthanized and the hemimaxillae removed and processed for laser capture microdissection of the PDL tissues of the 1 M. Peptide extracts were obtained and analyzed by LC-MS/MS. Data are available via ProteomeXchange with identifier PXD033647. Out of the 109 proteins with differential abundance, 49 were identified in the OM group, including Vinculin, Cathepsin D, and Osteopontin, which were selected for in situ localization by immunohistochemistry analysis (IHC). Overall, Gene Ontology (GO) analysis indicated that enriched proteins were related to the GO component cellular category. IHC validated the trends for selected proteins. Our study highlights the differences in the PDL proteome profiling of healthy and obese subjects undergoing OTM. These findings may provide valuable information needed to better understand the mechanisms involved in tissue remodeling in obese patients submitted to orthodontic treatment. SIGNIFICANCE: The prevalence of obesity is increasing worldwide. Emerging findings in the field of dentistry suggest that obesity influences the tissues around the teeth, especially those in the periodontal ligament. Therefore, evaluation of the effect of obesity on periodontal tissues remodeling during orthodontic tooth movement is a relevant research topic. To our knowledge, this is the first study to evaluate proteomic changes in periodontal ligament tissue in response to the association between orthodontic tooth movement and obesity. Our study identified a novel protein profile associated with obesity by using laser microdissection and proteomic analysis, providing new information to increase understanding of the mechanisms involved in obese patients undergoing orthodontic treatment which can lead to a more personalized orthodontic treatment approach.

Lataste's viper (Vipera latastei) is a venomous European viper endemic to the Iberian Peninsula, recognised as medically important by the World Health Organization. To date, no comprehensive characterisation of this species' venom has been reported. Here, we analysed the venoms of juvenile and adult specimens of V. latastei from two environmentally different populations from northern Portugal. Using bottom-up venomics, we produced six venom proteomes (three per population) from vipers belonging to both age classes (i.e., two juveniles and four adults), and RP-HPLC profiles of 54 venoms collected from wild specimens. Venoms from juveniles and adults differed in their chromatographic profiles and relative abundances of their toxins, suggesting the occurrence of ontogenetic changes in venom composition. Specifically, snake venom metalloproteinase (SVMP) was the most abundant toxin family in juvenile venoms, while snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins were the main toxins comprising adult venoms. The RP-HPLC venom profiles were found to vary significantly between the two sampled localities, indicating geographic variability. Furthermore, the presence/absence of certain peaks in the venom chromatographic profiles appeared to be significantly correlated also to factors like body size and sex of the vipers. Our findings show that V. latastei venom is a variable phenotype. The intraspecific differences we detected in its composition likely mirror changes in the feeding ecology of this species, taking place during different life stages and under different environmental pressures. SIGNIFICANCE: Lataste's viper (Vipera latastei) is a medically important viper endemic to the Iberian Peninsula, inhabiting different habitats and undergoing a marked ontogenetic dietary shift. In the current study, we report the first proteomic analysis of V. latastei venom from two environmentally different localities in northern Portugal. Our bottom-up venomic analyses show that snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins are the major components of adult V. latastei venom. The comparative analysis of young and adult venoms suggests the occurrence of ontogenetic shift in toxin abundances, with snake venom metalloproteinases (SVMPs) being the predominant toxins in juvenile venoms. Moreover, geographic venom variation between the two studied populations is also detected, with our statistical analyses suggesting that factors like body size and sex of the vipers are possibly at play in its determination. Our work represents the first assessment of the composition of V. latastei venom, and the first step towards a better understanding of the drivers behind its variability.

Rhipicephalus microplus is the most serious tick parasite for the livestock industry in tropical and subtropical regions. A cost-effective control method to manage the infestation of this parasite involves the use of chemicals such as ivermectin. However, massive overuse of ivermectin over recent decades has selected for ivermectin-resistant populations of R. microplus. Here, we carried out a comparative proteomic analysis of the midgut of ivermectin-susceptible versus ivermectin-resistant ticks using tandem mass tags coupled to synchronous precursor selection. In susceptible ticks, there was an over-representation of proteins associated with blood digestion and anticoagulation. In contrast, resistant ticks exhibited an over-accumulation of proteins involved in phase I and phase II of the detoxification metabolism, including cytochrome P450, glutathione-S-transferase, and ABC transporters, as well as many ribosomal and other translation-related proteins. This information provides new clues about the mechanisms of ivermectin resistance in R. microplus as well as suggesting potential novel molecular targets to cope with ivermectin-resistant populations of R. microplus. SIGNIFICANCE: Cattle farming is an important primary economic activity for food production all over the globe. However, this activity also has detrimental environmental impacts, including the overuse of ivermectin and other chemicals used to control parasite infestations. The overuse of ivermectin selected for parasites with resistance to this chemical, including tick species like R. microplus. There has been extensive to understand the mechanisms that mediate ivermectin resistance in arthropods, but many gaps remain for the full comprehension of this phenomenon. Understanding the biochemistry behind ivermectin resistance could provide new alternatives to fight these parasites. We therefore consider that determining the metabolic mechanisms involved in ivermectin resistance is of great relevance. The comparative proteomic analysis here reported shows the relevance of the active detoxifying metabolism in the midgut of resistant ticks, which may be key for the development of novel control methods.

Increased fructose consumption has been associated with the development of metabolic diseases due to the modification in protein expression, altering metabolic and signaling pathways. Curcumin is a natural compound with a regulatory effect on genes and metabolic pathways. To identify the fructose-induced protein expression changes and the effect of curcumin on the change of protein expression in the liver of mice fed a standard diet and a high fructose diet, to elucidate the global role of curcumin. Four groups (n = 4/group) of male mice (C57BL6J) of six-weeks-old were formed. One group received a standard diet (C); another received curcumin at 0.75% w/w in the feed (C + C); one more received 30% w/v fructose in drinking water (F); and one group received 30% w/v fructose in drinking water and 0.75% w/w curcumin in food (F + C); for 15 weeks. Proteomic analysis was performed by LC-MS/MS, using the label-free technique with the MaxQuant programs for identification and Perseus for expression change analysis. Differentially expressed proteins (fold change ≥1.5 and p < 0.5) were analyzed by gene ontology and KEGG. A total of 1047 proteins were identified, of which 113 changed their expression in mice fed fructose, compared to the control group, and curcumin modified the expression of 64 proteins in mice fed fructose and curcumin compared to mice that only received fructose. Curcumin prevented the change of expression of 13 proteins involved in oxidative phosphorylation (NDUFB8, NDUFB3, and ATP5L) in the cellular response to stress (PSMA5, HIST1H1D) and lipid metabolism (THRSP, DGAT1, ECI1, and ACOT13). Curcumin in mice fed the standard diet increased the expression of proteins related to oxidative phosphorylation, ribosomes, and PPAR pathways. In addition to fructose, increased expression of proteins involved in oxidative phosphorylation, ribosomes, lipid metabolism, and carbon metabolism. However, curcumin prevented expression change in 13 hepatic proteins of fructose-fed mice involved in oxidative phosphorylation, cellular stress response, and lipid metabolism. SIGNIFICANCE: Curcumin is a natural compound with a regulatory effect on proteins and metabolic pathways. So, curcumin prevents the change of expression in 13 hepatic proteins of fructose-fed mice involved in oxidative phosphorylation, cellular stress response and lipid metabolism, as a supplement with protector activity on fructose-induced toxic effects.

Dysfunction of blood-brain barrier formed by endothelial cells of cerebral blood vessels, plays a key role in development of neurodegenerative disorders. Epicatechin exerts vasculo-protective effects through genomic modifications, however molecular mechanisms of action, particularly on brain endothelial cells, are largely unknow. This study aimed to use a multi-omic approach (transcriptomics of mRNA, miRNAs and lncRNAs, and proteomics), to provide novel in-depth insights into molecular mechanisms of how metabolites affect brain endothelial cells under lipid-stressed (as a model of BBB dysfunction) at physiological concentrations. We showed that metabolites can simultaneously modulate expression of protein-coding, non-coding genes and proteins. Integrative analysis revealed interactions between different types of RNAs and form functional groups of genes involved in regulation of processing like VEGF-related functions, cell signaling, cell adhesion and permeability. Molecular modeling of genomics data predicted that metabolites decrease endothelial cell permeability, increased by lipotoxic stress. Correlation analysis between genomic modifications observed and genomic signature of patients with vascular dementia and Alzheimer's diseases showed opposite gene expression changes. Taken together, this study describes for the first time a multi-omic mechanism of action by which (-)-epicatechin metabolites could preserve brain vascular endothelial cell integrity and reduce the risk of neurodegenerative diseases. SIGNIFICANCE: Dysfunction of the blood-brain barrier (BBB), characterized by dysfunction of endothelial cells of cerebral blood vessels, result in an increase in permeability and neuroinflammation which constitute a key factor in the development neurodegenerative disorders. Even though it is suggested that polyphenols can prevent or delay the development of these disorders, their impact on brain endothelial cells and underlying mechanisms of actions are unknow. This study aimed to use a multi-omic approach including analysis of expression of mRNA, microRNA, long non-coding RNAs, and proteins to provide novel global in-depth insights into molecular mechanisms of how (-)-epicatechin metabolites affect brain microvascular endothelial cells under lipid-stressed (as a model of BBB dysfunction) at physiological relevant conditions. The results provide basis of knowledge on the capacity of polyphenols to prevent brain endothelial dysfunction and consequently neurodegenerative disorders.

Microsporidium is a kind of intracellular fungal pathogen that greatly threatens the human health, breeding industry, and food security. All members of microsporidia possess a unique, highly specialized invasion organelle, described as the polar filament. Like "reversing a finger of gloves", the polar filament discharges out of mature spores to transform as the polar tube, and pathogenic sporoplasm is transported to host cell through polar tube to complete infection. During the invasion process, the structure of polar filament and polar tube has changed, so does the protein composition on them? In this study, we firstly proposed a purification method for polar filament and polar tube from microsporidium Nosema bombycis which was infected silkworm Bombyx mori, and it was also found that the structure of polar filament and polar tube was obviously different. Therefore, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia. SIGNIFICANCE: Microsporidia are obligate intracellular parasites that infect a wide variety of hosts, including humans. The polar filament is a unique invasion organelle for microsporidia, and it is also one of the important indexes of microsporidian taxonomy. The polar tube is deformed from the primitive polar filament in mature spores. During the germination, the polar filament turns into a polar tube, like "reversing a finger of gloves", through which pathogenic sporoplasm is transported to host cells to complete infection. Since the structure of the polar filament and polar tube has changed, what about their protein composition? In this study, it was the first time to purify the polar filament and the polar tube from microsporidium Nosema bombycis that was infected silkworm Bombyx mori, which provided new insights for studying the invasion organelle of microsporidia. Comparing the fine structure of polar filament and polar tube, we found that their structure was obviously different. Therefore, the protein composition of these two structures is supposed to be varied. In this case, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia.