ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Recent advances of small extracellular vesicle biomarkers in breast cancer diagnosis and prognosis.

Current clinical tools for breast cancer (BC) diagnosis are insufficient but liquid biopsy of different bodily fluids has recently emerged as a minimally invasive strategy that provides a real-time snapshot of tumour biomarkers for early diagnosis, active surveillance of progression, and post-treatment recurrence. Extracellular vesicles (EVs) are nano-sized membranous structures 50-1000 nm in diameter that are released by cells into biological fluids. EVs contain proteins, nucleic acids, and lipids which play pivotal roles in tumourigenesis and metastasis through cell-to-cell communication. Proteins and miRNAs from small EVs (sEV), which range in size from 50-150 nm, are being investigated as a potential source for novel BC biomarkers using mass spectrometry-based proteomics and next-generation sequencing. This review covers recent developments in sEV isolation and single sEV analysis technologies and summarises the sEV protein and miRNA biomarkers identified for BC diagnosis, prognosis, and chemoresistance. The limitations of current sEV biomarker research are discussed along with future perspective applications.

Comparison Study of Small Extracellular Vesicle Isolation Methods for Profiling Protein Biomarkers in Breast Cancer Liquid Biopsies.

Small extracellular vesicles (sEVs) are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Therefore, protein cargoes within sEVs are considered as a superior source for breast cancer (BC) biomarker discovery. Our study aimed to optimise the approach for sEV isolation and sEV proteomic analysis to identify potential sEV protein biomarkers for BC diagnosis. sEVs derived from BC cell lines, BC patients' plasma, and non-cancer controls were isolated using ultracentrifugation (UC), a Total Exosome Isolation kit (TEI), and a combined approach named UCT. In BC cell lines, the UC isolates showed a higher sEV purity and marker expression, as well as a higher number of sEV proteins. In BC plasma samples, the UCT isolates showed the highest proportion of sEV-related proteins and the lowest percentage of lipoprotein-related proteins. Our data suggest that the assessment of both the quantity and quality of sEV isolation methods is important in selecting the optimal approach for the specific sEV research purpose, depending on the sample types and downstream analysis.

Tryptophan Metabolism 'Hub' Gene Expression Associates with Increased Inflammation and Severe Disease Outcomes in COVID-19 Infection and Inflammatory Bowel Disease.

The epithelial barrier's primary role is to protect against entry of foreign and pathogenic elements. Both COVID-19 and Inflammatory Bowel Disease (IBD) show commonalities in symptoms and treatment with sensitization of the epithelial barrier inviting an immune response. In this study we use a multi-omics strategy to identify a common signature of immune disease that may be able to predict for more severe patient outcomes. Global proteomic approaches were applied to transcriptome and proteome. Further semi- and relative- quantitative targeted mass spectrometry methods were developed to substantiate the proteomic and metabolomics changes in nasal swabs from healthy, COVID-19 (24 h and 3 weeks post infection); serums from Crohn's disease patients (scored for epithelial leak), terminal ileum tissue biopsies (patient matched inflamed and non-inflamed regions, and controls). We found that the tryptophan/kynurenine metabolism pathway is a 'hub' regulator of canonical and non-canonical transcription, macrophage release of cytokines and significant changes in the immune and metabolic status with increasing severity and disease course. Significantly modified pathways include stress response regulator EIF2 signaling (p = 1 × 10-3); energy metabolism, KYNU (p = 4 × 10-4), WARS (p = 1 × 10-7); inflammation, and IDO activity (p = 1 × 10-6). Heightened levels of PARP1, WARS and KYNU are predictive at the acute stage of infection for resilience, while in contrast, levels remained high and are predictive of persistent and more severe outcomes in COVID disease. Generation of a targeted marker profile showed these changes in immune disease underlay resolution of epithelial barrier function and have the potential to define disease trajectory and more severe patient outcomes.

The Molecular Floodgates of Stress-Induced Senescence Reveal Translation, Signalling and Protein Activity Central to the Post-Mortem Proteome.

The transitioning of cells during the systemic demise of an organism is poorly understood. Here, we present evidence that organismal death is accompanied by a common and sequential molecular flood of stress-induced events that propagate the senescence phenotype, and this phenotype is preserved in the proteome after death. We demonstrate activation of "death" pathways involvement in diseases of ageing, with biochemical mechanisms mapping onto neurological damage, embryonic development, the inflammatory response, cardiac disease and ultimately cancer with increased significance. There is sufficient bioavailability of the building blocks required to support the continued translation, energy, and functional catalytic activity of proteins. Significant abundance changes occur in 1258 proteins across 1 to 720 h post-mortem of the 12-week-old mouse mandible. Protein abundance increases concord with enzyme activity, while mitochondrial dysfunction is evident with metabolic reprogramming. This study reveals differences in protein abundances which are akin to states of stress-induced premature senescence (SIPS). The control of these pathways is significant for a large number of biological scenarios. Understanding how these pathways function during the process of cellular death holds promise in generating novel solutions capable of overcoming disease complications, maintaining organ transplant viability and could influence the findings of proteomics through "deep-time" of individuals with no historically recorded cause of death.

Analysis of the Preserved Amino Acid Bias in Peptide Profiles of Iron Age Teeth from a Tropical Environment Enable Sexing of Individuals Using Amelogenin MRM.

The first dental proteomic profile of Iron Age individuals (ca. 2000-1000 years B.P.), collected from the site of Long Long Rak rock shelter in northwest Thailand is described. A bias toward the preservation of the positively charged aromatic, and polar amino acids is observed. It is evident that the 212 proteins identified (2 peptide, FDR <1%) comprise a palimpsest of alterations that occurred both ante-mortem and post-mortem. Conservation of amino acids within the taphonomically resistant crystalline matrix enabled the identification of both X and Y chromosome linked amelogenin peptides. A novel multiple reaction monitoring method using the sex specific amelogenin protein isoforms is described and indicate the teeth are of male origin. Functional analysis shows an enrichment of pathways associated with metabolic disorders and shows a capacity for harboring these conditions prior to death. Stable isotope analysis using carbon isotopes highlights the strongly C3 based (≈80%) diet of the Long Long Rak cemetery people, which probably comprised rice combined with protein from freshwater fish among other food items. The combination of proteomics and stable isotope analysis provides a complementary strategy for assessing the demography, diet, lifestyle, and possible diseases experienced by ancient populations.

Serological Epithelial Component Proteins Identify Intestinal Complications in Crohn's Disease.

Crohn's Disease (CD) is a relapsing inflammation of the gastrointestinal tract that affects a young working age population and is increasing in developing countries. Half of all sufferers will experience stricturing or fistulizing intestinal complications that require extensive surgical interventions and neither genes nor clinical risk factors can predict this debilitating natural history. We applied discovery and verification phase studies as part of an NCI-FDA modeled biomarker pipeline to identify differences in the low-mass (<25kDa) blood-serum proteome between CD behavioral phenotypes. A significant enrichment of epithelial component proteins was identified in CD patients with intestinal complications using quantitative proteomic profiling with label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). DAVID 6.7 (NIH) was used for functional annotation analysis of detected proteins and immunoblotting and multiple reaction monitoring (MRM) to verify a priori findings in a secondary independent cohort of complicated CD (CCD), uncomplicated inflammatory CD (ICD), Th1/17 pathway inflammation controls (rheumatoid arthritis), inflammatory bowel disease controls (ulcerative colitis), and healthy controls. Seventy-six high-confidence serum proteins were modulated in CCD versus ICD by LC-MS/MS (p < 0.05, FDR q<0.01), annotating to pathways of epithelial barrier homeostasis (p < 0.01). In verification phase, a putative serology panel developed from discovery proteomics data consisting of desmoglein-1, desmoplakin, and fatty acid-binding protein 5 (FABP5) distinguished CCD from all other groups (p = 0.041) and discriminated complication in CD (70% sensitivity and 72.5% specificity at score ≥1.907, AUC = 0.777, p = 0.007). An MRM assay secondarily confirmed increased FABP5 levels in CCD (p < 0.001). In a longitudinal subanalysis-cohort, FABP5 levels were stable over a two-month period with no behavioral changes (p = 0.099). These studies along the biomarker development pipeline provide substantial proof-of-principle that a blood test can be developed specific to transmural intestinal injury. Data are available via the PRIDE proteomics data repository under identifier PXD001821 and PeptideAtlas with identifier PASS00661.

Impaired Intestinal Permeability Contributes to Ongoing Bowel Symptoms in Patients With Inflammatory Bowel Disease and Mucosal Healing.

BACKGROUND & AIMS: Many patients with inflammatory bowel diseases (IBD) have ongoing bowel symptoms of diarrhea or abdominal pain despite mucosal healing. We investigated whether impaired intestinal permeability contributes to these symptoms. METHODS: We performed a prospective study of intestinal permeability, measured by endoscopic confocal laser endomicroscopy in 110 consecutive subjects (31 with ulcerative colitis [UC], 57 with Crohn's disease [CD], and 22 healthy individuals [controls]) in Sydney, Australia from May 2009 and September 2015. Symptomatic CD was defined by a CD Activity Index score of 150 or more and symptomatic UC by a partial Mayo score of 2 or more. Mucosal healing was defined as CD Endoscopic Index of Severity of 0 in CD or Mayo endoscopic sub-score of 0-1 for patients with UC. Intestinal permeability was quantified by the Confocal Leak Score (CLS; range: 0=no impaired permeability to 100=complete loss of barrier function). The primary endpoint was intestinal permeability in patients with symptomatic IBD in mucosal healing vs patients with asymptomatic IBD in mucosal healing. We determined the sensitivity and specificity of CLS in determining symptoms based on receiver operating characteristic analysis. RESULTS: Ongoing bowel symptoms were present in 16.3% of patients with IBD and mucosal healing (15.4% of patients with CD, 17.4% with UC). Patients with symptomatic IBD had a significantly higher median CLS (19.0) than patients with asymptomatic IBD (7.3; P < .001) or controls (5.9, P < .001). There were no significant differences between patients with IBD in remission vs controls (P = .261). Median CLS was significantly higher in patients with symptomatic than asymptomatic CD (17.7 vs 8.1; P = .009) and patients with symptomatic than asymptomatic UC (22.2 vs 6.9; P = .021). A CLS of 13.1 or more identified ongoing bowel symptoms in patients with IBD and mucosal healing with 95.2% sensitivity and 97.6% specificity; the receiver operating characteristic area under curve value was 0.88. Based on this cutoff, 36.2% of patients with IBD in mucosal healing have increased intestinal permeability. On regression analysis, every increase in CLS of 1.9 correlated with an additional diarrheal motion per day (P = .008). CONCLUSIONS: In a prospective study of intestinal permeability in patients with IBD and mucosal healing, we associated impaired intestinal permeability with ongoing bowel symptoms; increases in permeability correlated with increased severity of diarrhea. Resolution of mucosal permeability beyond mucosal healing might improve outcomes of patients with IDB (ANZCTR.org.au: ACTRN12613001248752).

Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective.

Breakdown of the protective gut barrier releases effector molecules and degradation products into the blood stream making serum and plasma ideal as a diagnostic medium. The enriched low mass proteome is unexplored as a source of differentiators for diagnosing and monitoring inflammatory bowel disease (IBD) activity, that is less invasive than colonoscopy. Differences in the enriched low mass plasma proteome (<25 kDa) were assessed by label-free quantitative mass-spectrometry. A panel of marker candidates were progressed to validation phase and "Tier-2" FDA-level validated quantitative assay. Proteins important in maintaining gut barrier function and homeostasis at the epithelial interface have been quantitated by multiple reaction monitoring in plasma and serum including both inflammatory; rheumatoid arthritis controls, and non-inflammatory healthy controls; ulcerative colitis (UC), and Crohn's disease (CD) patients. Detection by immunoblot confirmed presence at the protein level in plasma. Correlation analysis and receiver operator characteristics were used to report the sensitivity and specificity. Peptides differentiating controls from IBD originate from secreted phosphoprotein 24 (SPP24, p = 0.000086, 0.009); whereas those in remission and healthy can be differentiated in UC by SPP24 (p = 0.00023, 0.001), α-1-microglobulin (AMBP, p = 0.006) and CD by SPP24 (p = 0.019, 0.05). UC and CD can be differentiated by Guanylin (GUC2A, p = 0.001), and Secretogranin-1 (CHGB p = 0.035). Active and quiescent disease can also be differentiated in UC and CD by CHGB (p ≤ 0.023) SPP24 (p ≤ 0.023) and AMBP (UC p = 0.046). Five peptides discriminating IBD activity and severity had very little-to-no correlation to erythrocyte sedimentation rate, C-reactive protein, white cell or platelet counts. Three of these peptides were found to be binding partners to SPP24 protein alongside other known matrix proteins. These proteins have the potential to improve diagnosis and evaluate IBD activity, reducing the need for more invasive techniques. Data are available via ProteomeXchange with identifier PXD002821.

Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain.

Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson's disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Parkinson's disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson's disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson's disease.

Proteomics in Inflammatory Bowel Disease: Approach Using Animal Models.

Recently, proteomics studies have provided important information on the role of proteins in health and disease. In the domain of inflammatory bowel disease, proteomics has shed important light on the pathogenesis and pathophysiology of inflammation and has contributed to the discovery of some putative clinical biomarkers of disease activity. By being able to obtain a large number of specimens from multiple sites and control for confounding environmental, genetic, and metabolic factors, proteomics studies using animal models of colitis offered an alternative approach to human studies. Our aim is to review the information and lessons acquired so far from the use of proteomics in animal models of colitis. These studies helped understand the importance of different proteins at different stages of the disease and unraveled the different pathways that are activated or inhibited during the inflammatory process. Expressed proteins related to inflammation, cellular structure, endoplasmic reticulum stress, and energy depletion advanced the knowledge about the reaction of intestinal cells to inflammation and repair. The role of mesenteric lymphocytes, exosomes, and the intestinal mucosal barrier was emphasized in the inflammatory process. In addition, studies in animal models revealed mechanisms of the beneficial effects of some therapeutic interventions and foods or food components on intestinal inflammation by monitoring changes in protein expression and paved the way for some new possible inflammatory pathways to target in the future. Advances in proteomics technology will further clarify the interaction between intestinal microbiota and IBD pathogenesis and investigate the gene-environmental axis of IBD etiology.

Reverse-polynomial dilution calibration methodology extends lower limit of quantification and reduces relative residual error in targeted peptide measurements in blood plasma.

Matrix effect is the alteration of an analyte's concentration-signal response caused by co-existing ion components. With electrospray ionization (ESI), matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the electrospray ionization droplet. For biological matrices such as plasma, the interactions between droplet components is immensely complex and the effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: we investigated whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity); the amount of matrix components (concentration); and matrix effect, by comparing an E. coli digest matrix (∼2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, nondepleted human plasma matrices (∼10(7) proteome) for six human plasma peptide multiple reaction monitoring assays. Our data set demonstrated analyte-specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p < 0.01), with nonuniform effects on the ion signals of the analytes and their stable-isotope analogs. These matrix effects were then assessed for translation into relative residual error and precision effects in a low concentration (∼0-250 ng/ml) range across no-matrix, complex matrix, and highly complex matrix, when a standard addition stable isotope dilution calibration method was used. Relative residual error (%) and precision (CV%) by stable isotope dilution were within <20%; however, error in phospholipid-depleted and nondepleted plasma matrices were significantly higher compared with no-matrix (p = 0.006). Finally a novel reverse-polynomial dilution calibration method with and without phospholipid-depletion was compared with stable isotope dilution for relative residual error and precision. Reverse-polynomial dilution techniques extend the Lower Limit of Quantification and reduce error (p = 0.005) in low-concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed multiple reaction monitoring assay development within the FDA-National Cancer Institute (NCI) biomarker development pipeline.

Proteomics discovery of chemoresistant biomarkers for ovarian cancer therapy.

INTRODUCTION: Chemoresistance is a major challenge to current ovarian cancer chemotherapy. It is important to identify biomarkers to distinguish chemosensitive and chemoresistant patients. Areas covered: We review the medical literature, discuss MS-based technologies with respect to chemoresistant ovarian cancer and summarize the promising chemoresistant biomarkers identified. In addition, the challenges and future perspectives of biomarker discovery research are explored. With the employment of mass spectrometry-based (MS-based) proteomics, biomarker discovery of ovarian cancer has made great progress in the last decade. Many potential biomarkers were identified by MS-based proteomics technologies, some of which have been validated for further extensive studies in clinical settings. Expert commentary: The discovery of chemoresistant biomarkers is a newly developing area and may provide a clue for predicting chemotherapeutic response and discover therapeutic targets for paving the way of personalized medicine. Multiple complementary MS-based proteomics approaches hold promise for finding novel therapeutic targets in ovarian cancer treatment.

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling.

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 µl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 µg/µl in control samples, 0.96 ± 0.07 µg/µl in the BPH group, and 0.98 ± 0.07 µg/µl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.

Glycosylation in a mammalian expression system is critical for the production of functionally active leukocyte immunoglobulin-like receptor A3 protein.

The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2-4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by ß-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions.

In vivo localization of antibodies raised against Eimeria maxima wall forming bodies during sexual intracellular development.

SUMMARY Apicomplexan parasites cause devastating diseases in humans and livestock. Previously we demonstrated that antibodies targeting transmissible forms of the apicomplexan parasite, Eimeria, are effective at reducing parasite shedding thus preventing the transmission of the disease. However, the mechanisms responsible have not been fully defined. Moreover, there is no direct evidence that the parasite-specific IgG antibodies can reach the parasite developing in the enterocytes of the infected chicken host. This study summarizes our efforts using host immunity, parasite proteomics and 3D microscopy to provide a step forward in our understanding of how this immune response works. Eimeria maxima is an important pathogen of poultry and used as a surrogate for a number of human pathogens including Toxoplasma and Plasmodium. Our studies demonstrate that immunization with the purified wall forming bodies (WFBs) results in a production of parasite-specific IgG antibodies, which have the ability to reach in situ gametocytes in the intestinal lumen and permeate the enterocyte/parasite membranes in order to bind to the cytoplasmic Type 1 and Type 2 WFBs. This raises the intriguing possibility that via this process antibodies block the development of Eimeria maxima in vivo.

Prostate cancer-derived extracellular vesicles metabolic biomarkers: Emerging roles for diagnosis and prognosis.

Prostate cancer (PCa) is a global health concern, ranking as the most common cancer among men in Western countries. Traditional diagnostic methods are invasive with adverse effects on patients. Due to the heterogeneous nature of PCa and their multifocality, tissue biopsies often yield false-negative results. To address these challenges, researchers are exploring innovative approaches, particularly in the realms of proteomics and metabolomics, to identify more reliable biomarkers and improve PCa diagnosis. Liquid biopsy (LB) has emerged as a promising non-invasive strategy for PCa early detection, biopsy selection, active surveillance for low-risk cases, and post-treatment and progression monitoring. Extracellular vesicles (EVs) are lipid-bilayer nanovesicles released by all cell types and play an important role in intercellular communication. EVs have garnered attention as a valuable biomarker resource in LB for PCa-specific biomarkers, enhancing diagnosis, prognostication, and treatment guidance. Metabolomics provides insight into the body's metabolic response to both internal and external stimuli, offering quantitative measurements of biochemical alterations. It excels at detecting non-genetic influences, aiding in the discovery of more accurate cancer biomarkers for early detection and disease progression monitoring. This review delves into the potential of EVs as a resource for LB in PCa across various clinical applications. It also explores cancer-related metabolic biomarkers, both within and outside EVs in PCa, and summarises previous metabolomic findings in PCa diagnosis and risk assessment. Finally, the article addresses the challenges and future directions in the evolving field of EV-based metabolomic analysis, offering a comprehensive overview of its potential in advancing PCa management.

Proteome profiles of vaginal fluids from women affected by bacterial vaginosis and healthy controls: outcomes of rifaximin treatment.

OBJECTIVES: This study was designed to characterize the proteome of vaginal fluid (VF) from women with bacterial vaginosis (BV) in comparison with that from healthy women, and to evaluate the effect exerted by rifaximin vaginal tablets. METHODS: Women with BV (n = 39) and matched healthy controls (n = 41) were included in the study. BV patients were distributed among four groups receiving different doses of rifaximin. Vaginal rinsings were collected at the screening visit from all the participants and at a follow-up visit from BV-affected women. The VF proteome was analysed by tandem mass spectrometry using an Orbitrap mass analyser. RESULTS: A large number of human proteins were differentially expressed in women with BV in comparison with healthy women (n = 118) and in BV-affected women treated with rifaximin (n = 284). In both comparisons, a high proportion of the dysregulated proteins (∼20%) were involved in the innate immune response. Twenty-one of 24 proteins increased in abundance in women with BV versus healthy women and 31/59 proteins decreased after rifaximin treatment, suggesting a general reduction of the immune response resulting from the therapy. Major changes in protein abundance were found following treatment with 25 mg of rifaximin once daily for 5 days. CONCLUSIONS: BV is associated with a massive change in the VF proteome, mainly regarding the abundance of proteins involved in the innate immune response. Rifaximin at a dosage of 25 mg for 5 days modulated the vaginal proteome, counteracting the alterations associated with the BV condition.

Preliminary identification of differentially expressed tear proteins in keratoconus.

PURPOSE: To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. METHODS: Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. RESULTS: Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. CONCLUSIONS: The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options.

Proteomics and metabolomics in inflammatory bowel disease.

Genome-wide studies in inflammatory bowel disease (IBD) have allowed us to understand Crohn's disease and ulcerative colitis as forms of related autoinflammatory disorders that arise from a multitude of pathogenic origins. Proteomics and metabolomics are the offspring of genomics that possess unprecedented possibilities to characterize unknown pathogenic pathways. It has been about a decade since proteomics was first applied to IBD, and 5 years for metabolomics. These techniques have yielded novel and potentially important findings, but turning these results into beneficial patient outcomes remains challenging. This review recounts the history and context of clinical IBD developments before and after proteomics and metabolomics IBD in this field, discusses the challenges in consolidating high complexity data with physiological understanding, and provides an outlook on the emerging principles that will help interface the bioanalytical laboratory with IBD prognosis.