Proteome Changes Induced by Iprodione Exposure in the Pesticide-Tolerant Pseudomonas sp. C9 Strain Isolated from a Biopurification System.

Iprodione is a pesticide that belongs to the dicarboximide fungicide family. This pesticide was designed to combat various agronomical pests; however, its use has been restricted due to its environmental toxicity and risks to human health. In this study, we explored the proteomic changes in the Pseudomonas sp. C9 strain when exposed to iprodione, to gain insights into the affected metabolic pathways and enzymes involved in iprodione tolerance and biodegradation processes. As a result, we identified 1472 differentially expressed proteins in response to iprodione exposure, with 978 proteins showing significant variations. We observed that the C9 strain upregulated the expression of efflux pumps, enhancing its tolerance to iprodione and other harmful compounds. Peptidoglycan-binding proteins LysM, glutamine amidotransferase, and protein Ddl were similarly upregulated, indicating their potential role in altering and preserving bacterial cell wall structure, thereby enhancing tolerance. We also observed the presence of hydrolases and amidohydrolases, essential enzymes for iprodione biodegradation. Furthermore, the exclusive identification of ABC transporters and multidrug efflux complexes among proteins present only during iprodione exposure suggests potential counteraction against the inhibitory effects of iprodione on downregulated proteins. These findings provide new insights into iprodione tolerance and biodegradation by the Pseudomonas sp. C9 strain.

An Aphid Pest Superclone Benefits From a Facultative Bacterial Endosymbiont in a Host-Dependent Manner, Leading to Reproductive and Proteomic Changes.

The English grain aphid, Sitobion avenae, is a significant agricultural pest affecting wheat, barley, and oats. In Chile, the most prevalent and persistent clone (superclone) of S. avenae harbors the facultative endosymbiont bacterium Regiella insecticola. To determine the role of this bacterium in the reproductive success of this superclone, the presence of R. insecticola was manipulated to assess its impact on (1) the reproductive performance of this clone on two host plant species (wheat and barley), (2) the production of winged morphs, (3) changes in the insects' proteomic profiles, and (4) the root/shoot ratio of plant. It was found that the reproductive performance of this S. avenae superclone varied across host plants, depending on the presence of the facultative bacterial endosymbiont. Aphids infected with R. insecticola showed higher reproductive success on wheat, while the opposite effect was observed on barley. Aphid biomass was greater when infected with R. insecticola, particularly on barley. Additionally, aphids harboring R. insecticola exhibited a higher proportion of winged individuals on both host plants. Protein regulation in aphids on wheat was lower compared to those on barley. A higher root/shoot biomass ratio was observed in wheat plants compared to barley when infested by R. insecticola-infected aphid. Thus, R. insecticola significantly influences the reproductive performance and proteomic profile of a S. avenae superclone, with these effects shaped by the host plant. This suggests that the interaction between the host plant and the facultative endosymbiont contributes to the ecological success of this superclone.

The Modulation of Septic Shock: A Proteomic Approach.

Sepsis poses a significant challenge due its lethality, involving multiple organ dysfunction and impaired immune responses. Among several factors affecting sepsis, monocytes play a crucial role; however, their phenotype, proteomic profile, and function in septic shock remain unclear. Our aim was to fully characterize the subpopulations and proteomic profiles of monocytes seen in septic shock cases and discuss their possible impact on the disease. Peripheral blood monocyte subpopulations were phenotype based on CD14/CD16 expression by flow cytometry, and proteins were extracted from the monocytes of individuals with septic shock and healthy controls to identify changes in the global protein expression in these cells. Analysis using 2D-nanoUPLC-UDMSE identified 67 differentially expressed proteins in shock patients compared to controls, in which 44 were upregulated and 23 downregulated. These proteins are involved in monocyte reprogramming, immune dysfunction, severe hypotension, hypo-responsiveness to vasoconstrictors, vasodilation, endothelial dysfunction, vascular injury, and blood clotting, elucidating the disease severity and therapeutic challenges of septic shock. This study identified critical biological targets in monocytes that could serve as potential biomarkers for the diagnosis, prognosis, and treatment of septic shock, providing new insights into the pathophysiology of the disease.

Impact of maternal protein restriction on the proteomic landscape of male rat lungs across the lifespan.

The developmental origins of healthy and disease (DOHaD) concept has demonstrated a higher rate of chronic diseases in the adult population of individuals whose mothers experienced severe maternal protein restriction (MPR). Using proteomic and in silico analyses, we investigated the lung proteomic profile of young and aged rats exposed to MPR during pregnancy and lactation. Our results demonstrated that MPR lead to structural and immune system pathways changes, and this outcome is coupled with a rise in the PI3k-AKT-mTOR signaling pathway, with increased MMP-2 activity, and CD8 expression in the early life, with long-term effects with aging. This led to the identification of commonly or inversely differentially expressed targets in early life and aging, revealing dysregulated pathways related to the immune system, stress, muscle contraction, tight junctions, and hemostasis. We identified three miRNAs (miR-378a-3p, miR-378a-5p, let-7a-5p) that regulate four proteins (ACTN4, PPIA, HSPA5, CALM1) as probable epigenetic lung marks generated by MPR. In conclusion, MPR impacts the lungs early in life, increasing the possibility of long-lasting negative outcomes for respiratory disorders in the offspring.

Transcriptome and proteome changes triggered by overexpression of the transcriptional regulator Maf1 in the human pathogen Leishmania major.

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.

Proteomic Profiling of HDL in Newly Diagnosed Breast Cancer Based on Tumor Molecular Classification and Clinical Stage of Disease.

The association between high-density lipoprotein (HDL) cholesterol and breast cancer (BC) remains controversial due to the high complexity of the HDL particle and its functionality. The HDL proteome was determined in newly diagnosed BC classified according to the molecular type [luminal A or B (LA or LB), HER2, and triple-negative (TN)] and clinical stage of the disease. Women (n = 141) aged between 18 and 80 years with BC, treatment-naïve, and healthy women [n = 103; control group (CT)], matched by age and body mass index, were included. Data-independent acquisition (DIA) proteomics was performed in isolated HDL (D = 1.063-1.21 g/mL). Results: Paraoxonase1, carnosine dipeptidase1, immunoglobulin mMu heavy chain constant region (IGHM), apoA-4, and transthyretin were reduced, and serum amyloid A2 and tetranectin were higher in BC compared to CT. In TNBC, apoA-1, apoA-2, apoC-2, and apoC-4 were reduced compared to LA, LB, and HER2, and apoA-4 compared to LA and HER2. ComplementC3, lambda immunoglobulin2/3, serpin3, IGHM, complement9, alpha2 lysine rich-glycoprotein1, and complement4B were higher in TNBC in comparison to all other types; complement factor B and vitamin D-binding protein were in contrast to LA and HER2, and plasminogen compared to LA and LB. In grouped stages III + IV, tetranectin and alpha2-macroglobulin were reduced, and haptoglobin-related protein; lecithin cholesterol acyltransferase, serum amyloid A1, and IGHM were increased compared to stages I + II. Conclusions: A differential proteomic profile of HDL in BC based on tumor molecular classification and the clinical stage of the disease may contribute to a better understanding of the association of HDL with BC pathophysiology, treatment, and outcomes.

Bulk serum extracellular vesicles from stressed mice show a distinct proteome and induce behavioral and molecular changes in naive mice.

Chronic stress can trigger several pathologies including mood disorders for which no clear diagnostic molecular markers have been established yet. Attractive biomarker sources are extracellular vesicles (EVs). Evs are released by cells in health and disease and contain genetic material, proteins and lipids characteristic of the cell state. Here we show that Evs recovered from the blood of animals exposed to a repeated interrupted stress protocol (RIS) have a different protein profile compared to those obtained from control animals. Proteomic analysis indicated that proteins differentially present in bulk serum Evs from stressed animals were implicated in metabolic and inflammatory pathways and several of them were previously related to psychiatric disorders. Interestingly, these serum Evs carry brain-enriched proteins including the stress-responsive neuronal protein M6a. Then, we used an in-utero electroporation strategy to selectively overexpress M6a-GFP in brain neurons and found that M6a-GFP could also be detected in bulk serum Evs suggesting a neuronal origin. Finally, to determine if these Evs could have functional consequences, we administered Evs from control and RIS animals intranasally to naïve mice. Animals receiving stress EVs showed changes in behavior and brain M6a levels similar to those observed in physically stressed animals. Such changes could therefore be attributed, or at least in part, to EV protein transfer. Altogether these findings show that EVs may participate in stress signaling and propose proteins carried by EVs as a valuable source of biomarkers for stress-induced diseases.

Proteomic Profiling of Cocos nucifera L. Zygotic Embryos during Maturation of Dwarf and Tall Cultivars: The Dynamics of Carbohydrate and Fatty Acid Metabolism.

There is a limited number of studies analyzing the molecular and biochemical processes regulating the metabolism of the maturation of Cocos nucifera L. zygotic embryos. Our research focused on the regulation of carbohydrate and lipid metabolic pathways occurring at three developmental stages of embryos from the Mexican Pacific tall (MPT) and the Yucatan green dwarf (YGD) cultivars. We used the TMT-synchronous precursor selection (SPS)-MS3 strategy to analyze the dynamics of proteomes from both embryos; 1044 and 540 proteins were determined for the MPT and YGD, respectively. A comparison of the differentially accumulated proteins (DAPs) revealed that the biological processes (BP) enriched in the MPT embryo included the glyoxylate and dicarboxylate metabolism along with fatty acid degradation, while in YGD, the nitrogen metabolism and pentose phosphate pathway were the most enriched BPs. Findings suggest that the MPT embryos use fatty acids to sustain a higher glycolytic/gluconeogenic metabolism than the YGD embryos. Moreover, the YGD proteome was enriched with proteins associated with biotic or abiotic stresses, e.g., peroxidase and catalase. The goal of this study was to highlight the differences in the regulation of carbohydrate and lipid metabolic pathways during the maturation of coconut YGD and MPT zygotic embryos.

From Proteome to Potential Drugs: Integration of Subtractive Proteomics and Ensemble Docking for Drug Repurposing against Pseudomonas aeruginosa RND Superfamily Proteins.

Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat as a nosocomial pathogen due to its robust resistance mechanisms and virulence factors. This study integrates subtractive proteomics and ensemble docking to identify and characterize essential proteins in P. aeruginosa, aiming to discover therapeutic targets and repurpose commercial existing drugs. Using subtractive proteomics, we refined the dataset to discard redundant proteins and minimize potential cross-interactions with human proteins and the microbiome proteins. We identified 12 key proteins, including a histidine kinase and members of the RND efflux pump family, known for their roles in antibiotic resistance, virulence, and antigenicity. Predictive modeling of the three-dimensional structures of these RND proteins and subsequent molecular ensemble-docking simulations led to the identification of MK-3207, R-428, and Suramin as promising inhibitor candidates. These compounds demonstrated high binding affinities and effective inhibition across multiple metrics. Further refinement using non-covalent interaction index methods provided deeper insights into the electronic effects in protein-ligand interactions, with Suramin exhibiting superior binding energies, suggesting its broad-spectrum inhibitory potential. Our findings confirm the critical role of RND efflux pumps in antibiotic resistance and suggest that MK-3207, R-428, and Suramin could be effectively repurposed to target these proteins. This approach highlights the potential of drug repurposing as a viable strategy to combat P. aeruginosa infections.

Different Proteomic Profiles Regarding Antihypertensive Therapy in Preeclampsia Pregnant.

Preeclampsia (PE) is a hypertensive pregnancy syndrome associated with target organ damage and increased cardiovascular risks, necessitating antihypertensive therapy. However, approximately 40% of patients are nonresponsive to treatment, which results in worse clinical outcomes. This study aimed to compare circulating proteomic profiles and identify differentially expressed proteins among 10 responsive (R-PE), 10 nonresponsive (NR-PE) patients, and 10 healthy pregnant controls (HP). We also explored correlations between these proteins and clinical data. Plasma protein relative quantification was performed using mass spectrometry, followed by bioinformatics analyses with the UniProt database, PatternLab for Proteomics 4.0, and MetaboAnalyst software (version 6.0). Considering a fold change of 1.5, four proteins were differentially expressed between NR-PE and R-PE: one upregulated (fibronectin) and three downregulated (pregnancy-specific beta-1-glycoprotein 1, complement C4B, and complement C4A). Between NR-PE and HP, six proteins were differentially expressed: two upregulated (clusterin and plasmin heavy chain A) and four downregulated (apolipoprotein L1, heparin cofactor II, complement C4B, and haptoglobin-related protein). Three proteins were differentially expressed between R-PE and HP: one downregulated (transthyretin) and two upregulated (apolipoprotein C1 and hemoglobin subunit beta). These findings suggest a complex interplay of these proteins involved in inflammatory, immune, and metabolic processes with antihypertensive therapy responsiveness and PE pathophysiology.

Proteomic Biomarkers Associated with Low Bone Mineral Density: A Systematic Review.

Osteoporosis is a globally relevant public health issue. Our study aimed to summarize the knowledge on the proteomic biomarkers for low bone mineral density over the last years. We conducted a systematic review following the PRISMA guidelines; the scoured databases were PubMed, Web of Sciences, Scopus, and EBSCO, from inception to 2 June 2023. A total of 610 relevant studies were identified and 33 were assessed for eligibility. Finally, 29 studies met the criteria for this systematic review. The risk of bias was evaluated using the Joanna Briggs Institute Critical Appraisal Checklist tool. From the studies selected, 154 proteins were associated with changes of bone mineral density, from which only 10 were reported in at least two articles. The protein-protein network analysis indicated potential biomarkers involved in the skeletal system, immune system process, regulation of protein metabolic process, regulation of signaling, transport, cellular component assembly, cell differentiation, hemostasis, and extracellular matrix organization. Mass spectrometry-based proteomic profiling has allowed the discovery of new biomarkers with diagnostic potential. However, it is necessary to compare and validate the potential biomarkers in different populations to determine their association with bone metabolism and evaluate their translation to the clinical management of osteoporosis.

Proteome and metabolome of Caryocar brasiliense camb. fruit and their interaction during development.

Considered the symbol fruit of the Brazilian Cerrado, pequi (Caryocar brasiliense Camb.) is an exotic and much-appreciated fruit with an internal mesocarp (edible part) with an eye-catching golden yellow color. In an unprecedented way, this study characterized the proteome throughout pequi development. The most influential and essential transcription factors operating in the regulation of pequi ripening identified were members of the MAD-box family. A group of proteins related to the methionine cycle indicates the high consumption and recycling of methionine. However this consumption does not occur mainly for the biosynthesis of ethylene, a process dependent on methionine consumption. In the bioactive compounds presented, different proteins could be correlated with the presence of these phytochemicals, such as monodehydroascorbate reductase and ascorbate peroxidase in ascorbic acid recycling; pyruvate kinase, fructose bisphosphate aldolase and phytoene synthase with carotenoid biosynthesis; S-adenosylmethionine synthase 1 as a donor of methyl groups in the formation of trigonelline and aspartate aminotransferase as a biomarker of initial regulation of the trigonelline biosynthetic pathway; phenylalanine ammonia lyase, chorismate synthesis and chalcone-flavononone isomerase in the biosynthesis of phenolic compounds. Among the volatile organic compounds identified, the majority compound in pequi was ethyl hexanoate ester, with an area of 50.68 % in the ripe fruit, and in this group of esters that was the most representative, alcohol dehydrogenase, a fundamental enzyme in the synthesis of esters, was identified with an increase of approximately 7.2 times between the first and last stages. Therefore, an extensive group of proteins and some metabolites can serve as biomarkers of ripening in pequi, as most were more expressed in the last stage, which is the ripe fruit suitable for consumption.

Extracellular Matrix Protein Signatures of the Outer and Inner Zones of the Rat Adrenal Cortex.

This study analyzes the extracellular matrix (ECM) signatures of the outer (OF = capsule + subcapsular + zona glomerulosa cells) and inner fractions (IF = zona fasciculata cells) of the rat adrenal cortex, which comprise two distinct microenvironment niches. Proteomic profiles of decellularized OF and IF samples, male and female rats, identified 252 proteins, with 32 classified as ECM-component and ECM-related. Among these, 25 proteins were differentially regulated: 17 more abundant in OF, including Col1a1, Col1a2, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, Lama5, Lamb2, Lamc1, Eln, Emilin, Fbln5, Fbn1, Fbn2, Nid1, and Ltbp4, and eight more abundant in IF, including Col4a1, Col4a2, Lama2, Lama4, Lamb1, Fn1, Hspg2, and Ecm1. Eln, Tnc, and Nid2 were abundant in the female OF, while Lama2, Lama5, Lamb2, and Lamc1 were more abundant in the male IF. The complex protein signature of the OF suggests areas of tissue stress, stiffness, and regulatory proteins for growth factor signaling. The higher concentrations of Col4a1 and Col4a2 and their role in steroidogenesis should be further investigated in IF. These findings could significantly enhance our understanding of adrenal cortex functionality and its implications for human health and disease. Key findings were validated, and data are available in ProteomeXchange (PXD046828).

Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

Discovery of candidate biomarkers from plasma-derived extracellular vesicles of patients with cirrhosis and hepatocellular carcinoma: an exploratory proteomic study.

Extracellular vesicles (EVs) represent an attractive source of biomarkers due to their biomolecular cargo. The aim of this study was to identify candidate protein biomarkers from plasma-derived EVs of patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Plasma-derived EVs from healthy participants (HP), LC, and HCC patients (eight samples each) were subjected to label-free quantitative proteomic analysis using LC-MS/MS. A total of 248 proteins were identified, and differentially expressed proteins (DEPs) were obtained after pairwise comparison. We found that DEPs mainly involve complement cascade activation, coagulation pathways, cholesterol metabolism, and extracellular matrix components. By choosing a panel of up- and down-regulated proteins involved in cirrhotic and carcinogenesis processes, TGFBI, LGALS3BP, C7, SERPIND1, and APOC3 were found to be relevant for LC patients, while LRG1, TUBA1C, TUBB2B, ACTG1, C9, HP, FGA, FGG, FN1, PLG, APOB and ITIH2 were associated with HCC patients, which could discriminate both diseases. In addition, we identified the top shared proteins in both diseases, which included LCAT, SERPINF2, A2M, CRP, and VWF. Thus, our exploratory proteomic study revealed that these proteins might play an important role in the disease progression and represent a panel of candidate biomarkers for the prognosis and diagnosis of LC and HCC.

Maternal protein restriction combined with postnatal sugar consumption alters liver proteomic profile and metabolic pathways in adult male offspring rats.

This study investigated the impact of maternal protein restriction (MPR) and early postnatal sugar consumption (SUG) on the liver health of adult male descendant rats. Male offspring of mothers fed a normal protein diet (NPD) or a low protein diet (LPD) were divided into four groups: Control (CTR), Sugar Control (CTR + SUG), LPD during gestation and lactation (GLLP), and LPD with sugar (GLLP + SUG). Sugar consumption (10% glucose diluted in water) began after weaning on day 21 (PND 21), and at 90 days (PND 90), rats were sacrificed for analysis. Sugar intake reduced food intake and increased water consumption in CTR + SUG and GLLP + SUG compared to CTR and GLLP. GLLP and GLLP + SUG groups showed lower body weight and total and retroperitoneal fat compared to CTR and CTR + SUG. CTR + SUG and GLLP + SUG groups exhibited hepatocyte vacuolization associated with increased hepatic glycogen content compared to CTR and GLLP. Hepatic catalase activity increased in GLLP compared to CTR. Proteomic analysis identified 223 differentially expressed proteins (DEPs) among experimental groups. While in the GLLP group, the DEPs enriched molecular pathways related to cellular stress, glycogen metabolic pathways were enriched in the GLLP + SUG and CTR + SUG groups. The association of sugar consumption amplifies the effects of MPR, deregulating molecular mechanisms related to metabolism and the antioxidant system.

Ketogenic diet administration later in life improves memory by modifying the synaptic cortical proteome via the PKA signaling pathway in aging mice.

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated in vivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.

Leishmaniinae: Evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas.

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

Robust assessment of sample preparation protocols for proteomics of cells and tissues.

In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 µg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out.

Effect of Hydroxyurea on Morphology, Proliferation, and Protein Expression on Taenia crassiceps WFU Strain.

Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.