A new methodology for a rapid and high-throughput comparison of molecular profiles and biological activity of phytoextracts.

To robustly discover and explore phytocompounds, it is necessary to evaluate the interrelationships between the plant species, plant tissue, and the extraction process on the extract composition and to predict its cytotoxicity. The present work evaluated how Fourier Transform InfraRed spectroscopy can acquire the molecular profile of aqueous and ethanol-based extracts obtained from leaves, seeds, and flowers of Cynara Cardunculus, and ethanol-based extracts from Matricaria chamomilla flowers, as well the impact of these extracts on the viability of mammalian cells. The extract molecular profile enabled to predict the extraction yield, and how the plant species, plant tissue, and extraction process affected the extract's relative composition. The molecular profile obtained from the culture media of cells exposed to extracts enabled to capture its impact on cells metabolism, at a higher sensitivity than the conventional assay used to determine the cell viability. Furthermore, it was possible to detect specific impacts on the cell's metabolism according to plant species, plant tissue, and extraction process. Since spectra were acquired on small volumes of samples (25 µL), after a simple dehydration step, and based on a plate with 96 wells, the method can be applied in a rapid, simple, high-throughput, and economic mode, consequently promoting the discovery of phytocompounds.

Predicting Cellular Rejection of Renal Allograft Based on the Serum Proteomic Fingerprint.

Kidney transplantation is an essential medical procedure that significantly enhances the survival rates and quality of life for patients with end-stage kidney disease. However, despite advancements in immunosuppressive therapies, allograft rejection remains a leading cause of organ loss. Notably, predictions of cellular rejection processes primarily rely on biopsy analysis, which is not routinely performed due to its invasive nature. The present work evaluates if the serum proteomic fingerprint, as acquired by Fourier Transform Infrared (FTIR) spectroscopy, can predict cellular rejection processes. We analyzed 28 serum samples, corresponding to 17 without cellular rejection processes and 11 associated with cellular rejection processes, as based on biopsy analyses. The leave-one-out-cross validation procedure of a Naïve Bayes model enabled the prediction of cellular rejection processes with high sensitivity and specificity (AUC > 0.984). The serum proteomic profile was obtained in a high-throughput mode and based on a simple, rapid, and economical procedure, making it suitable for routine analyses and large-scale studies. Consequently, the current method presents a high potential to predict cellular rejection processes translatable to clinical scenarios, and that should continue to be explored.

The Characteristics and Laboratory Findings of SARS-CoV-2 Infected Patients during the First Three COVID-19 Waves in Portugal-A Retrospective Single-Center Study.

Background and Objectives: Given the wide spectrum of clinical and laboratory manifestations of the coronavirus disease 2019 (COVID-19), it is imperative to identify potential contributing factors to patients' outcomes. However, a limited number of studies have assessed how the different waves affected the progression of the disease, more so in Portugal. Therefore, our main purpose was to study the clinical and laboratory patterns of COVID-19 in an unvaccinated population admitted to the intensive care unit, identifying characteristics associated with death, in each of the first three waves of the pandemic. Materials and Methods: This study included 337 COVID-19 patients admitted to the intensive care unit of a single-center hospital in Lisbon, Portugal, between March 2020 and March 2021. Comparisons were made between three COVID-19 waves, in the second (n = 325) and seventh (n = 216) days after admission, and between discharged and deceased patients. Results: Deceased patients were considerably older (p = 0.021) and needed greater ventilatory assistance (p = 0.023), especially in the first wave. Differences between discharged and deceased patients' biomarkers were minimal in the first wave, on both analyzed days. In the second wave significant differences emerged in troponins, lactate dehydrogenase, procalcitonin, C-reactive protein, and white blood cell subpopulations, as well as platelet-to-lymphocyte and neutrophil-to-lymphocyte ratios (all p < 0.05). Furthermore, in the third wave, platelets and D-dimers were also significantly different between patients' groups (all p < 0.05). From the second to the seventh days, troponins and lactate dehydrogenase showed significant decreases, mainly for discharged patients, while platelet counts increased (all p < 0.01). Lymphocytes significantly increased in discharged patients (all p < 0.05), while white blood cells rose in the second (all p < 0.001) and third (all p < 0.05) waves among deceased patients. Conclusions: This study yields insights into COVID-19 patients' characteristics and mortality-associated biomarkers during Portugal's first three COVID-19 waves, highlighting the importance of considering wave variations in future research due to potential significant outcome differences.

Antimicrobial evaluation of the Cynara cardunculus extract in Helicobacter pylori cells using mid-infrared spectroscopy and chemometric methods.

The treatment effectiveness of gastric diseases caused by the bacteria Helicobacter pylori is failing due to high resistance to some antibiotics. Consequently, it is urgent to develop an accurate methodology to screen new antimicrobial agents. METHODS AND RESULTS: A preliminary assay, using both therapeutic-based antibiotics (clarithromycin and metronidazole), was conducted to optimize experimental conditions in terms of the sensibility of the Fourier-transform mid-infrared (MIR-FTIR) spectroscopy associated with chemometric methods. Principal component analysis was applied to understand how the Cynara extract concentration acts differentially against H. pylori bacteria. The partial least squares model, characterized by R2  = 0.98, and root mean square error cross-validation, 0.011, was developed for the spectral regions (3600-2500 cm-1 and 2000-698 cm-1 ). CONCLUSIONS: MIR-FTIR spectroscopy associated with chemometric methods can be considered a suitable approach to discover and analyse the promissory antimicrobial agents based on the biomolecular changes observed according to the Cynara extract. SIGNIFICANCE AND IMPACT OF THE STUDY: MIR-FTIR spectroscopy and chemometric methods allowed to register the biomolecular changes due to the potential antimicrobial drugs at reduced concentrations comparatively to the conventional assay based on an agar-dilution method, being considered a useful approach to develop a platform to discover new bioactive molecules, allowing to reduce time and costs related to the exploratory step.

Antigenic and conserved peptides from diverse Helicobacter pylori antigens.

Since the revolutionary finding of Helicobacter pylori as a common bacterial infection, that a high research effort for its eradication has been conducted. Epitope based-vaccine presents advantages over protein-based, as they can be designed to contain epitopes from diverse proteins, therefore, more easily representing the immune-variability of the bacterial population, while minimizing the toxicity associated to some whole proteins. In the present work, an iterative method, to design antigenic and conserved B-epitopes from diverse virulent factors of H. pylori, was established. The method considered the trade-off between epitopes antigenicity and conservation among the bacterial population. For the method validation, five virulent factors from H. pylori were selected. From each virulent factor, two epitopes were predicted, each with twelve residues of aminoacids. The corresponding ten peptides were synthesised and evaluated by enzyme-linked immunosorbent assay using polyclonal antibodies raised against a specific H. pylori strain. All ten peptides were recognised by the antibodies and were consequently antigenic and conserved. This result could strongly contribute to the design of a multivalent epitope-based vaccine, representing the immunogenetic variability within the bacterial population, leading to a sustained and effective immunogenic protection.

Fast identification of off-target liabilities in early antibiotic discovery with Fourier-transform infrared spectroscopy.

Structural modifications of known antibiotic scaffolds have kept the upper hand on resistance, but we are on the verge of not having antibiotics for many common infections. Mechanism-based discovery assays reveal novelty, exclude off-target liabilities, and guide lead optimization. For that, we developed a fast and automatable protocol using high-throughput Fourier-transform infrared spectroscopy (FTIRS). Metabolic fingerprints of Staphylococcus aureus and Escherichia coli exposed to 35 compounds, dissolved in dimethyl sulfoxide (DMSO) or water, were acquired. Our data analysis pipeline identified biomarkers of off-target effects, optimized spectral preprocessing, and identified the top-performing machine learning algorithms for off-target liabilities and mechanism of action (MOA) identification. Spectral bands with known biochemical associations more often yielded more significant biomarkers of off-target liabilities when bacteria were exposed to compounds dissolved in water than DMSO. Highly discriminative models distinguished compounds with predominant off-target effects from antibiotics with well-defined MOA (AUROC > 0.87, AUPR > 0.79, F1 > 0.81), and from the latter predicted their MOA (AUROC > 0.88, AUPR > 0.70, F1 > 0.70). The compound solvent did not affect predictive models. FTIRS is fast, simple, inexpensive, automatable, and highly effective at predicting MOA and off-target liabilities. As such, FTIRS mechanism-based screening assays can be applied for hit discovery and to guide lead optimization during the early stages of antibiotic discovery.

Simultaneous elucidation of antibiotic mechanism of action and potency with high-throughput Fourier-transform infrared (FTIR) spectroscopy and machine learning.

The low rate of discovery and rapid spread of resistant pathogens have made antibiotic discovery a worldwide priority. In cell-based screening, the mechanism of action (MOA) is identified after antimicrobial activity. This increases rediscovery, impairs low potency candidate detection, and does not guide lead optimization. In this study, high-throughput Fourier-transform infrared (FTIR) spectroscopy was used to discriminate the MOA of 14 antibiotics at pathway, class, and individual antibiotic level. For that, the optimal combinations and parametrizations of spectral preprocessing were selected with cross-validated partial least squares discriminant analysis, to which various machine learning algorithms were applied. This coherently resulted in very good accuracies, independently of the algorithms, and at all levels of MOA. Particularly, an ensemble of subspace discriminants predicted the known pathway (98.6%), antibiotic classes (100%), and individual antibiotics (97.8%) with exceptional accuracy, and similar results were obtained for simulated novel MOA. Even at very low concentrations (1 µg/mL) and growth inhibition (15%), over 70% pathway and class accuracy was achieved, suggesting FTIR spectroscopy can probe the grey chemical matter. Prediction of inhibitory effect was also examined, for which a squared exponential Gaussian process regression yielded a root mean square error of 0.33 and a R2 of 0.92, indicating that metabolic alterations leading to growth inhibition are intrinsically reflected on FTIR spectra beyond cell density. KEY POINTS: • Antibiotic MOA and potency estimated with high-throughput FTIR spectroscopy • Sub-inhibitory MOA identification suggests ability to explore grey chemical matter • Data analysis optimization improved MOA identification at antibiotic level by 38.

High-throughput FTIR-based bioprocess analysis of recombinant cyprosin production.

To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted. Principal component analysis (PCA) enabled capturing the general metabolic status of the yeast cells, as replicated samples appear grouped together in the score plot and groups of culture samples according to the main growth phase can be clearly distinguished. The PCA-loading vectors also revealed spectral regions, and the corresponding chemical functional groups and biomolecules that mostly contributed for the cell biomolecular fingerprint associated with the culture growth phase. These data were corroborated by the analysis of the samples' second derivative spectra. Partial least square (PLS) regression models built based on the MIR spectra showed high predictive ability for estimating the bioprocess critical variables: biomass (R 2 = 0.99, RMSEP 2.8%); cyprosin activity (R 2 = 0.98, RMSEP 3.9%); glucose (R 2 = 0.93, RMSECV 7.2%); galactose (R 2 = 0.97, RMSEP 4.6%); ethanol (R 2 = 0.97, RMSEP 5.3%); and acetate (R 2 = 0.95, RMSEP 7.0%). In conclusion, high-throughput MIR spectroscopy and multivariate data analysis were effective in identifying the main growth phases and specific cyprosin production phases along the yeast culture as well as in quantifying the critical variables of the process. This knowledge will promote future process optimization and control the recombinant cyprosin bioprocess according to Quality by Design framework.

A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses.

Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R (2) ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R (2) = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.

Bridging the gap between target-based and phenotypic-based drug discovery.

INTRODUCTION: The unparalleled progress in science of the last decades has brought a better understanding of the molecular mechanisms of diseases. This promoted drug discovery processes based on a target approach. However, despite the high promises associated, a critical decrease in the number of first-in-class drugs has been observed. AREAS COVERED: This review analyses the challenges, advances, and opportunities associated with the main strategies of the drug discovery process, i.e. based on a rational target approach and on an empirical phenotypic approach. This review also evaluates how the gap between these two crossroads can be bridged toward a more efficient drug discovery process. EXPERT OPINION: The critical lack of knowledge of the complex biological networks is leading to targets not relevant for the clinical context or to drugs that present undesired adverse effects. The phenotypic systems designed by considering available molecular mechanisms can mitigate these knowledge gaps. Associated with the expansion of the chemical space and other technologies, these designs can lead to more efficient drug discoveries. Technological and scientific knowledge should also be applied to identify, as early as possible, both drug targets and mechanisms of action, leading to a more efficient drug discovery pipeline.

Exosomes and microvesicles in kidney transplantation: the long road from trash to gold.

Kidney transplantation significantly enhances the survival rate and quality of life of patients with end-stage kidney disease. The ability to predict post-transplantation rejection events in their early phases can reduce subsequent allograft loss. Therefore, it is critical to identify biomarkers of rejection processes that can be acquired on routine analysis of samples collected by non-invasive or minimally invasive procedures. It is also important to develop new therapeutic strategies that facilitate optimisation of the dose of immunotherapeutic drugs and the induction of allograft immunotolerance. This review explores the challenges and opportunities offered by extracellular vesicles (EVs) present in biofluids in the discovery of biomarkers of rejection processes, as drug carriers and in the induction of immunotolerance. Since EVs are highly complex structures and their composition is affected by the parent cell's metabolic status, the importance of defining standardised methods for isolating and characterising EVs is also discussed. Understanding the major bottlenecks associated with all these areas will promote the further investigation of EVs and their translation into a clinical setting.

Simplifying Data Analysis in Biomedical Research: An Automated, User-Friendly Tool.

Robust data normalization and analysis are pivotal in biomedical research to ensure that observed differences in populations are directly attributable to the target variable, rather than disparities between control and study groups. ArsHive addresses this challenge using advanced algorithms to normalize populations (e.g., control and study groups) and perform statistical evaluations between demographic, clinical, and other variables within biomedical datasets, resulting in more balanced and unbiased analyses. The tool's functionality extends to comprehensive data reporting, which elucidates the effects of data processing, while maintaining dataset integrity. Additionally, ArsHive is complemented by A.D.A. (Autonomous Digital Assistant), which employs OpenAI's GPT-4 model to assist researchers with inquiries, enhancing the decision-making process. In this proof-of-concept study, we tested ArsHive on three different datasets derived from proprietary data, demonstrating its effectiveness in managing complex clinical and therapeutic information and highlighting its versatility for diverse research fields.

Discovery of Delirium Biomarkers through Minimally Invasive Serum Molecular Fingerprinting.

Delirium presents a significant clinical challenge, primarily due to its profound impact on patient outcomes and the limitations of the current diagnostic methods, which are largely subjective. During the COVID-19 pandemic, this challenge was intensified as the frequency of delirium assessments decreased in Intensive Care Units (ICUs), even as the prevalence of delirium among critically ill patients increased. The present study evaluated how the serum molecular fingerprint, as acquired by Fourier-Transform InfraRed (FTIR) spectroscopy, can enable the development of predictive models for delirium. A preliminary univariate analysis of serum FTIR spectra indicated significantly different bands between 26 ICU patients with delirium and 26 patients without, all of whom were admitted with COVID-19. However, these bands resulted in a poorly performing Naïve-Bayes predictive model. Considering the use of a Fast-Correlation-Based Filter for feature selection, it was possible to define a new set of spectral bands with a wider coverage of molecular functional groups. These bands ensured an excellent Naïve-Bayes predictive model, with an AUC, a sensitivity, and a specificity all exceeding 0.92. These spectral bands, acquired through a minimally invasive analysis and obtained rapidly, economically, and in a high-throughput mode, therefore offer significant potential for managing delirium in critically ill patients.

Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs.

Genotoxicity is an important information that should be included in human biomonitoring programmes. However, the usually applied cytogenetic assays are laborious and time-consuming, reason why it is critical to develop rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p < 0.01) and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p < 0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from the exposure with an accuracy, sensitivity, and specificity of 92 %, 93 % and 91 %, respectively. All these parameters were achieved based on 1 µL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be conclude that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.

The Impact of the Serum Extraction Protocol on Metabolomic Profiling Using UPLC-MS/MS and FTIR Spectroscopy.

Biofluid metabolomics is a very appealing tool to increase the knowledge associated with pathophysiological mechanisms leading to better and new therapies and biomarkers for disease diagnosis and prognosis. However, due to the complex process of metabolome analysis, including the metabolome isolation method and the platform used to analyze it, there are diverse factors that affect metabolomics output. In the present work, the impact of two protocols to extract the serum metabolome, one using methanol and another using a mixture of methanol, acetonitrile, and water, was evaluated. The metabolome was analyzed by ultraperformance liquid chromatography associated with tandem mass spectrometry (UPLC-MS/MS), based on reverse-phase and hydrophobic chromatographic separations, and Fourier transform infrared (FTIR) spectroscopy. The two extraction protocols of the metabolome were compared over the analytical platforms (UPLC-MS/MS and FTIR spectroscopy) concerning the number of features, the type of features, common features, and the reproducibility of extraction replicas and analytical replicas. The ability of the extraction protocols to predict the survivability of critically ill patients hospitalized at an intensive care unit was also evaluated. The FTIR spectroscopy platform was compared to the UPLC-MS/MS platform and, despite not identifying metabolites and consequently not contributing as much as UPLC-MS/MS in terms of information concerning metabolic information, it enabled the comparison of the two extraction protocols as well as the development of very good predictive models of patient's survivability, such as the UPLC-MS/MS platform. Furthermore, FTIR spectroscopy is based on much simpler procedures and is rapid, economic, and applicable in the high-throughput mode, i.e., enabling the simultaneous analysis of hundreds of samples in the microliter range in a couple of hours. Therefore, FTIR spectroscopy represents a very interesting complementary technique not only to optimize processes as the metabolome isolation but also for obtaining biomarkers such as those for disease prognosis.

Epigenetic and Drug Response Modulation of Epigalocaten-In-3-Gallate in Staphylococcus aureus with Divergent Resistance Phenotypes.

Healthcare-associated methicillin-resistant Staphylococcus aureus infections represent extremely high morbidity and mortality rates worldwide. We aimed to assess the antimicrobial potential and synergistic effect between Epigalocatenin-3-gallate (EGCG) and different antibiotics in S. aureus strains with divergent resistance phenotypes. EGCG exposure effects in epigenetic and drug resistance key modulators were also evaluated. S. aureus strains (n = 32) were isolated from infected patients in a Lisbon hospital. The identification of the S. aureus resistance phenotype was performed through automatized methods. The antibiotic synergistic assay was performed through disk diffusion according to EUCAST guidelines with co-exposure to EGCG (250, 100, 50 and 25 µg/mL). The bacteria's molecular profile was assessed through FTIR spectroscopy. The transcriptional expression of OrfX, SpdC and WalKR was performed by using qRT-PCR. FTIR-spectroscopy analysis enabled the clear discrimination of MRSA/MSSA strains and the EGCG exposure effect in the bacteria's molecular profiles. Divergent resistant phenotypes were associated with divergent transcriptional expression of the epigenetic modulator OrfX, particularly in MRSA strains, as well as the key drug response modulators SpdC and WalKR. These results clearly demonstrate that EGCG exposure alters the expression patterns of key epigenetic and drug response genes with associated divergent-resistant profiles, which supports its potential for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms.

High-throughput analysis of the plasmid bioproduction process in Escherichia coli by FTIR spectroscopy.

Monitoring plasmid production systems is a lab intensive task. This article proposes a methodology based on FTIR spectroscopy and the use of chemometrics for the high-throughput analysis of the plasmid bioproduction process in E. coli. For this study, five batch cultures with different initial medium compositions are designed to represent different biomass and plasmid production behavior, with the maximum plasmid and biomass concentrations varying from 11 to 95 mg L(-1) and 6.8 to 12.8 g L(-1), respectively, and the plasmid production per biomass varying from 0.4 to 5.1 mg g(-1). After a short sample processing consisting of centrifugation and dehydration, the FTIR spectra are recorded from the collected cellular biomass using microtiter plates with 96 wells. After spectral pre-processing, the predictive FTIR spectra models are derived by using partial least squares (PLS) regression with the wavenumber selection performed by a Monte-Carlo strategy. Results show that it is possible to improve the PLS models by selecting specific spectral ranges. For the plasmid model, the spectral regions between 590-1,130, 1,670-2,025, and 2,565-3,280 cm(-1) are found to be highly relevant. Whereas for the biomass, the best wavenumber selections are between 900-1,200, 1,500-1,800, and 2,850-3,200 cm(-1). The optimized PLS models show a high coefficient of determination of 0.91 and 0.89 for the plasmid and biomass concentration, respectively. Additional PLS models for the prediction of the carbon sources glucose and glycerol and the by-product acetic acid, based on metabolism-induced correlations between the nutrients and the cellular biomass are also established.

Proteomics for Biomarker Discovery for Diagnosis and Prognosis of Kidney Transplantation Rejection.

Renal transplantation is currently the treatment of choice for end-stage kidney disease, enabling a quality of life superior to dialysis. Despite this, all transplanted patients are at risk of allograft rejection processes. The gold-standard diagnosis of graft rejection, based on histological analysis of kidney biopsy, is prone to sampling errors and carries high costs and risks associated with such invasive procedures. Furthermore, the routine clinical monitoring, based on urine volume, proteinuria, and serum creatinine, usually only detects alterations after graft histologic damage and does not differentiate between the diverse etiologies. Therefore, there is an urgent need for new biomarkers enabling to predict, with high sensitivity and specificity, the rejection processes and the underlying mechanisms obtained from minimally invasive procedures to be implemented in routine clinical surveillance. These new biomarkers should also detect the rejection processes as early as possible, ideally before the 78 clinical outputs, while enabling balanced immunotherapy in order to minimize rejections and reducing the high toxicities associated with these drugs. Proteomics of biofluids, collected through non-invasive or minimally invasive analysis, e.g., blood or urine, present inherent characteristics that may provide biomarker candidates. The current manuscript reviews biofluids proteomics toward biomarkers discovery that specifically identify subclinical, acute, and chronic immune rejection processes while allowing for the discrimination between cell-mediated or antibody-mediated processes. In time, these biomarkers will lead to patient risk stratification, monitoring, and personalized and more efficient immunotherapies toward higher graft survival and patient quality of life.

Infection Biomarkers Based on Metabolomics.

Current infection biomarkers are highly limited since they have low capability to predict infection in the presence of confounding processes such as in non-infectious inflammatory processes, low capability to predict disease outcomes and have limited applications to guide and evaluate therapeutic regimes. Therefore, it is critical to discover and develop new and effective clinical infection biomarkers, especially applicable in patients at risk of developing severe illness and critically ill patients. Ideal biomarkers would effectively help physicians with better patient management, leading to a decrease of severe outcomes, personalize therapies, minimize antibiotics overuse and hospitalization time, and significantly improve patient survival. Metabolomics, by providing a direct insight into the functional metabolic outcome of an organism, presents a highly appealing strategy to discover these biomarkers. The present work reviews the desired main characteristics of infection biomarkers, the main metabolomics strategies to discover these biomarkers and the next steps for developing the area towards effective clinical biomarkers.

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori.

BACKGROUND: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori-associated gastric diseases. MATERIAL AND METHODS: Immunoassays were performed on two-dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. RESULTS: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl-(acyl-carrier-protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl-CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. CONCLUSIONS: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti-H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.