Tracking the antibody immunome in sporadic colorectal cancer by using antigen self-assembled protein arrays

© 2021 by the authors.Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI14/01538, FIS PI17/01930 and CB16/12/00400. We also acknowledge Fondos FEDER (EU) “Una manera de hacer Europa” and Junta Castilla-León (COVID19 grant COV20EDU/00187). Fundación Solórzano FS/38-2017. The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023, of the PE I + D + I 2017-2020, funded by ISCIII and FEDER. CNPq-National Council for Scientific and Technological Development (Brazil) (306258/2019-6) and FAPERJ-Foundation for Research Support of Rio de Janeiro State for the financial support (E-26/201.670/2017 and 210.379/2018). M. González-González is supported by MINECOPTA2019-017870-I.A. Landeira-Viñuela is supported by VIII Centenario-USAL PhD Program. P.J.-V. is supported by JCYL PhD Program and scholarship JCYL-EDU/601/2020. P.D. and E.B. are supported by a JCYL-EDU/346/2013 Ph.D. scholarship

Systematic evaluation of plasma signaling cascades by functional proteomics approaches: SARS-CoV-2 infection as model

Purpose Acute phase reactants (APRs) play a critical role in inflammation. The difference in their physiological functions or the different dynamic ranges of these proteins in plasma makes it difficult to detect them simultaneously and to use several of these proteins as a tool in clinical practice. Experimental Design A novel multiplex assay has been designed and optimized to carry out a high-throughput and simultaneous screening of APRs, allowing the detection of each of them at the same time and in their corresponding dynamic range. Results Using Sars-CoV-2 infection as a model, it has been possible to profile different patterns of acute phase proteins that vary significantly between healthy and infected patients. In addition, severity profiles (acute respiratory distress syndrome and sepsis) have been established. Conclusions and Clinical Relevance Differential profiles in acute phase proteins can serve as a diagnostic and prognostic tool, among patient stratification. The design of this new platform for their simultaneous detection paves the way for them to be more extensive use in clinical practice.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI18/00682, FIS PI21/01545 and CB16/12/00400. We also acknowledge Fondos FEDER (EU) and Junta Castilla-León (COVID-19 grant COV20EDU/00187). The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023, of the PE I+D+I 2017–2020, funded by ISCIII and FEDER. This research work was funded by the European Commission -NextGenerationEU, through CSIC's Global Health Platform (PTI Salud Global). This research work was performed in the framework of the Nanomedicine CSIC HUB (ref. 202180E048). AL-V is supported by VIII Centenario-USAL PhD Program. PJ-V is supported by JCYL PhD Program “Nos Impulsa-JCYL” and scholarship JCYL-EDU/601/2020.Peer reviewe

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

BACKGROUND & AIMS: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. METHODS: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. RESULTS: Colonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. CONCLUSIONS: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization

Effectiveness of an mHealth intervention combining a smartphone app and smart band on body composition in an overweight and obese population: Randomized controlled trial (EVIDENT 3 study)

Background: Mobile health (mHealth) is currently among the supporting elements that may contribute to an improvement in health markers by helping people adopt healthier lifestyles. mHealth interventions have been widely reported to achieve greater weight loss than other approaches, but their effect on body composition remains unclear. Objective: This study aimed to assess the short-term (3 months) effectiveness of a mobile app and a smart band for losing weight and changing body composition in sedentary Spanish adults who are overweight or obese. Methods: A randomized controlled, multicenter clinical trial was conducted involving the participation of 440 subjects from primary care centers, with 231 subjects in the intervention group (IG; counselling with smartphone app and smart band) and 209 in the control group (CG; counselling only). Both groups were counselled about healthy diet and physical activity. For the 3-month intervention period, the IG was trained to use a smartphone app that involved self-monitoring and tailored feedback, as well as a smart band that recorded daily physical activity (Mi Band 2, Xiaomi). Body composition was measured using the InBody 230 bioimpedance device (InBody Co., Ltd), and physical activity was measured using the International Physical Activity Questionnaire. Results: The mHealth intervention produced a greater loss of body weight (–1.97 kg, 95% CI –2.39 to –1.54) relative to standard counselling at 3 months (–1.13 kg, 95% CI –1.56 to –0.69). Comparing groups, the IG achieved a weight loss of 0.84 kg more than the CG at 3 months. The IG showed a decrease in body fat mass (BFM; –1.84 kg, 95% CI –2.48 to –1.20), percentage of body fat (PBF; –1.22%, 95% CI –1.82% to 0.62%), and BMI (–0.77 kg/m2, 95% CI –0.96 to 0.57). No significant changes were observed in any of these parameters in men; among women, there was a significant decrease in BMI in the IG compared with the CG. When subjects were grouped according to baseline BMI, the overweight group experienced a change in BFM of –1.18 kg (95% CI –2.30 to –0.06) and BMI of –0.47 kg/m2 (95% CI –0.80 to –0.13), whereas the obese group only experienced a change in BMI of –0.53 kg/m2 (95% CI –0.86 to –0.19). When the data were analyzed according to physical activity, the moderate-vigorous physical activity group showed significant changes in BFM of –1.03 kg (95% CI –1.74 to –0.33), PBF of –0.76% (95% CI –1.32% to –0.20%), and BMI of –0.5 kg/m2 (95% CI –0.83 to –0.19). Conclusions: The results from this multicenter, randomized controlled clinical trial study show that compared with standard counselling alone, adding a self-reported app and a smart band obtained beneficial results in terms of weight loss and a reduction in BFM and PBF in female subjects with a BMI less than 30 kg/m2 and a moderate-vigorous physical activity level. Nevertheless, further studies are needed to ensure that this profile benefits more than others from this intervention and to investigate modifications of this intervention to achieve a global effect

Human colon-derived soluble factors modulate gut microbiota composition.

The commensal microbiota modulates immunological and metabolic aspects of the intestinal mucosa contributing to development of human gut diseases including inflammatory bowel disease. The host/microbiota interaction often referred to as a crosstalk, mainly focuses on the effect of the microbiota on the host neglecting effects that the host could elicit on the commensals. Colonic microenvironments from three human healthy controls (obtained from the proximal and distal colon, both in resting conditions and after immune - IL-15- and microbiota - LPS-in vitro challenges) were used to condition a stable fecal population. Subsequent 16S rRNA gene-based analyses were performed to study the effect induced by the host on the microbiota composition and function. Non-supervised principal component analysis (PCA) showed that all microbiotas, which had been conditioned with colonic microenvironments clustered together in terms of relative microbial composition, suggesting that soluble factors were modulating a stable fecal population independently from the treatment or the origin. Our findings confirmed that the host intestinal microenvironment has the capacity to modulate the gut microbiota composition via yet unidentified soluble factors. These findings indicate that an appropriate understanding of the factors of the host mucosal microenvironment affecting microbiota composition and function could improve therapeutic manipulation of the microbiota composition

Vat 3D printing of full-alginate hydrogels via thiol-ene reactions towards tissue engineering applications

The rise of 3D printing has given an important impulse to the medical field, envisaging the possibility of creating artificially engineered tissues/organs perfectly suiting the tissue defects of patients using their own cells; this approach could in the future overcome the lack of tissue donors and decrease the possible dangerous tissue rejection. Different 3D printing technologies can be considered for the building of scaffolds; despite these promises, very few inks for light-induced 3D printing are nowadays available on the market. Herein, for the first time, the alginate backbone is completely functionalized with thiol and alkene groups (separately) to create an innovative full-alginate ink for digital light processing (DLP) printers. The alginate hydrogel is produced with the more biocompatible thiol-ene reaction instead of the most commonly used radical photopolymerization based on (meth)acrylates and without any addition of small crosslinkers to the printable formulation. Simple synthetic "two-reactions" or "one-pot" strategies are explored to functionalize alginate with thiol/alkene groups that are able to undergo click reactions. High levels of reproducibility of the modification strategy are obtained. The hydrogels are characterized by studying their formulation reactivity, mechanical properties, swelling kinetics and morphological appearance, placing the resulting hydrogel into the stiffer scaffold category. The selected hydrogel formulation, tested as the ink for DLP 3D printing, demonstrates good processability and geometry fidelity with the possibility of forming 3D suspended structures. In the end, cell attachment and proliferation are evaluated on the hydrogel, certifying the possible use of the ink for the creation of tissue/organ substitutes (e.g., intestines or tendons) in tissue engineering applications

Single-cell proteomics: the critical role of nanotechnology

In single-cell analysis, biological variability can be attributed to individual cells, their specific state, and the ability to respond to external stimuli, which are determined by protein abundance and their relative alterations. Mass spectrometry (MS)-based proteomics (e.g., SCoPE-MS and SCoPE2) can be used as a non-targeted method to detect molecules across hundreds of individual cells. To achieve high-throughput investigation, novel approaches in Single-Cell Proteomics (SCP) are needed to identify and quantify proteins as accurately as possible. Controlling sample preparation prior to LC-MS analysis is critical, as it influences sensitivity, robustness, and reproducibility. Several nanotechnological approaches have been developed for the removal of cellular debris, salts, and detergents, and to facilitate systematic sample processing at the nano- and microfluidic scale. In addition, nanotechnology has enabled high-throughput proteomics analysis, which have required the improvement of software tools, such as DART-ID or DO-MS, which are also fundamental for addressing key biological questions. Single-cell proteomics has many applications in nanomedicine and biomedical research, including advanced cancer immunotherapies or biomarker characterization, among others; and novel methods allow the quantification of more than a thousand proteins while analyzing hundreds of single cells.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI21/01545 and CB16/12/00400. We also acknowledge Fondos FEDER (EU) and Junta Castilla-León (COVID-19 grant COV20EDU/00187). The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023, of the PE I + D + I 2017–2020, funded by ISCIII and FEDER. This research work was funded by the European Commission-NextGenerationEU, through CSIC’s Global Health Platform (PTI + Salud Global). This work was performed in the framework of NanoMedicine CSIC HUB (ref. 202180E048). CA-H is supported by Instituto de Investigación Biomédica de Salamanca, IBSAL (Programa Puente Contratos Predoctorales-2021). PJ-V is supported by JCYL PhD Program “Nos Impulsa-JCYL” and scholarship JCYL-EDU/601/2020. AL-V is supported by VIII Centenario-USAL PhD Program

Single-Step Glycosylations with 13C-Labelled Sulfoxide Donors: A Low-Temperature NMR Cartography of the Distinguishing Mechanistic Intermediates

Glycosyl sulfoxides have gained recognition in the total synthesis of complex oligosaccharides and as model substrates for dissecting the mechanisms involved. Reactions of these donors are usually performed under pre-activation conditions, but an experimentally more convenient single-step protocol has also been reported, whereby activation is performed in the presence of the acceptor alcohol; yet, the nature and prevalence of the reaction intermediates formed in this more complex scenario have comparatively received minimal attention. Herein, a systematic NMR-based study employing both C-labelled and unlabelled glycosyl sulfoxide donors for the detection and monitoring of marginally populated intermediates is reported. The results conclusively show that glycosyl triflates play a key role in these glycosylations despite the presence of the acceptor alcohol. Importantly, the formation of covalent donor/acceptor sulfonium adducts was identified as the main competing reaction, and thus a non-productive consumption of the acceptor that could limit the reaction yield was revealed.This investigation was supported by research grants of the Spanish “Plan Nacional” (MINECO)CTQ2016-79255-P,PID2019- 107476GB-I00 and RTI2018-094862-B-100. A.G.S. thanks the EU commission for aMSCA-IF postdoctoral fellowsh ip (H2020- MSCA-IF-2018, 841824). L.M.-J.and L.D.-C. thank the Spanish Ministerio de Economía, Industria y Competitividad and Minis- terio de Ciencia,Innovación y Universidades foranFPI fellow- ship each (BES-2014-070232 and BES-2017-080618)