Resultados de investigación

Supplemental material, sj-docx-1-tam-10.1177_17588359211072621 for Prognostic value of the immune target CEACAM6 in cancer: a meta-analysis by Miguel Burgos, Iván Cavero-Redondo, Celia Álvarez-Bueno, Eva María Galán-Moya, Atanasio Pandiella, Eitan Amir and Alberto Ocaña in Therapeutic Advances in Medical Oncology, Instituto de Salud Carlos III, Peer reviewed

Supplemental material, sj-docx-2-tam-10.1177_17588359211072621 for Prognostic value of the immune target CEACAM6 in cancer: a meta-analysis by Miguel Burgos, Iván Cavero-Redondo, Celia Álvarez-Bueno, Eva María Galán-Moya, Atanasio Pandiella, Eitan Amir and Alberto Ocaña in Therapeutic Advances in Medical Oncology, Instituto de Salud Carlos III, Peer reviewed

Retinal neurodegenerative diseases are the leading causes of visual impairment and irreversible blindness worldwide. Although the retinal response to injury remains closely similar between different retinal neurodegenerative diseases, available therapeutic alternatives are only palliative, too expensive, or very specific, such as gene therapy. In that sense, the development of broad-spectrum neuroprotective therapies seems to be an excellent option. In this regard, it is essential to identify molecular targets involved in retinal degeneration, such as cell death mechanisms. Apoptosis has been considered as the primary cell death mechanism during retinal degeneration; however, recent studies have demonstrated that the only use of anti-apoptotic drugs is not enough to confer good neuroprotection in terms of cell viability and preservation. For that reason, the interrelationship that exists between apoptosis and other cell death mechanisms needs to be characterized deeply to design future therapeutic options that simultaneously block the main cell death pathways. In that sense, the study aimed to characterize the programmed cell death (in terms of apoptosis and necroptosis) and autophagy response and modulation in retinal neurodegenerative diseases, using an in vitro model of spontaneous retinal neurodegeneration. For that purpose, we measured the mRNA relative expression through qPCR of a selected pool of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, and CASP9), necroptosis (MLKL, RIPK1, and RIPK3), and autophagy (ATG7, BCLIN1, LC3B, mTOR, and SQSTM1); besides, the immunoexpression of their encoding proteins (Casp3, MLKL, RIPK1, LC3B, and p62) were analyzed using immunohistochemistry. Our results showed an increase of pro-apoptotic and pro-necroptotic related genes and proteins during in vitro retinal neurodegeneration. Besides, we describe for the first time the modulation between programmed cell death mechanisms and autophagy in an in vitro retinal neurodegeneration model. This study reinforces the idea that cell death mechanisms are closely interconnected and provides new information about molecular signaling and autophagy along the retinal degeneration process., Peer reviewed

Supplementary Figures.docx Supplementary Materials and Methods.docx Supplementary Table Legends.docx Supplementary Table S1.docx Supplementary Table S2.xlsx Supplementary Table S3.xlsx Supplementary Table S4.docx Supplementary Table S5.xlsx Supplementary Table S6.xlsx, Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations., Peer reviewed

Supplementary Table S10. Comparative analysis of -omics platforms Supplementary Table S10. Comparative analysis of -omics platforms Supplementary Table S7.xlsx Supplementary Table S8.xlsx Supplementary Table S9.xlsx, Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations., Peer reviewed

Additional file 1: Table S1. List of primer sequences used for RT-PCR analysis. F: Forward primer. R: Reverse primer., Instituto de Salud Carlos III Gerencia Regional de Salud, Junta de Castilla y León Asociación Española Contra el Cancer (AECC) Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 2: Fig. S1. Efficiency of cleavage reaction. (A) XBP1 and (B) BLOC1S1 mRNA levels measured by qRT-PCR using the cDNAs synthesized with oligo (dT), and primers mapping the cleavage site of IRE1 from mock and IRE1-treated samples., Instituto de Salud Carlos III Gerencia Regional de Salud, Junta de Castilla y León Asociación Española Contra el Cancer (AECC) Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 3: Table S2. List of differential usage of exons from RNA-seq., Instituto de Salud Carlos III Gerencia Regional de Salud, Junta de Castilla y León Asociación Española Contra el Cancer (AECC) Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 4: Fig. S2. Reactome pathway analysis using RNA-seq data. Bar chart representing the most significantly enriched pathways. FDR ≤ 0.05., Instituto de Salud Carlos III Gerencia Regional de Salud, Junta de Castilla y León Asociación Española Contra el Cancer (AECC) Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 5: Fig. S3. Validation of putative IRE1 substrates. Exon-usage plots of the 28 remaining putative mRNAs, showing the number of reads in mock (red) and IRE1-treated (blue) samples. The black arrows represent the site of primers used in the 5´ region of the putative IRE1-substrates. Red arrows represent the site of primers mapping the predicted cleavage site. Right panel of each exon-usage plot shows the abundance of mRNA in the corresponding target. All results are presented as the means ± SD of three experiments. (*p < 0.05, **p < 0.01, ***p < 0.001)., Instituto de Salud Carlos III Gerencia Regional de Salud, Junta de Castilla y León Asociación Española Contra el Cancer (AECC) Consejería de Educación, Junta de Castilla y León, Peer reviewed