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First ever lidar observation in Tajikistan were conducted during the Central Asian Dust Experiment 2015–2016 (CADEX) in Dushanbe. Analysis of layer-mean optical properties revealed frequently low lidar ratios at enhanced depolarization ratios. These cases were categorized as background aerosol since they occurred at low extinction conditions. Such optical properties are similar to dried and therefore cubic-like shaped sea salt particles. This led to the hypothesis that Central Asian background aerosol and its optical properties are influenced by dry lakes and saline playas which are frequent in Central Asia. The goal of this study is to extend the existing data analysis with a backward trajectory-based time– height resolved air mass source attribution tool which was not yet available at the time of the CADEX campaign and its data analysis. Despite on average similar air mass origins for all cases irrespective of their optical properties, results suggest slightly more southern and south-western influenced air masses for cases with larger lidar ratios than for cases with lower lidar ratios.

Focusing on Tajikistan, a region facing critical environmental challenges, this extended abstract provides insights into the relationship between aerosols and clouds in Central Asia by means of lidar observations. Since 2019, a novel Dual-Field-of-View (DFOV) Raman polarization lidar system in Dushanbe has provided highly-resolved data on aerosol and cloud microphysical properties. Given the limited observational experiments in Central Asia, these measurements might play a crucial role in addressing climaterelated concerns. In fact, this technological deployment might not only provide a better picture regarding the spatio-temporal distribution of mineral dust and urban emissions, but also improve our understanding regarding the complex interactions between aerosol and clouds. One of the most uncertain aspects when predicting future temperature and precipitation patterns. Furthermore, an extended trajectory-based source attribution tool has been implemented to track air masses in Tajikistan, providing enhanced support for this and future studies. This research focuses on data evaluation and analysis, building upon established lidar methodologies. The resulting insights can potentially contribute to a better understanding of the relationship between aerosols and clouds in the dry continental conditions that persists in this region.

Content The objective of this study was to evaluate different concentrations of growth hormone ( GH) on the development of bovine preantral follicles cultured included in the ovarian tissue ( in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium ( α- MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy ( MET). The parameters underwent anova (Tukey′s and Dunnett′s tests) and chi-square test ( χ2). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ. [ABSTRACT FROM AUTHOR]

Contents This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 ( IGF-1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α- MEM+, supplemented with different concentrations of human recombinant IGF-1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO2 in 24-well plates with total replacement of the medium every 2 days. Non-cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy ( ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF-1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF-1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF-1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF-1 tested. Ultrastructurally, the non-cultured control and IGF-1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF-1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro. [ABSTRACT FROM AUTHOR]

El transporte aéreo es un elemento indispensable en el desarrollo de un turismo globalizado. Las previsiones de crecimiento de ambos sectores son unánimes. En este trabajo se analiza la sostenibilidad del transporte aéreo desde una perspectiva energética y se compara el grado de criticidad de la disponibilidad de petróleo, frente a la de la amenaza del cambio climático por efecto de las emisiones de gases. Se analizan la evolución y proyecciones de los sectores de la energía, aviación y el turismo, elaborando una visión integral de las cuestiones de sostenibilidad energética y medioambiental. Se emplea una metodología mixta, triangulando el análisis de las proyecciones de entidades de la energía, el transporte aéreo y el turismo con la utilización de métodos cualitativos de observación participante y entrevistas a expertos.El estudio confirma la mayor urgencia por trabajar en la prevención de una posible crisis medioambiental frente a una de escasez energética. Dado que el paradigma energético de la propulsión de aeronaves no cambiará en el medio plazo, y que la aviación y el turismo continuarán creciendo, la relevancia de ambos como contribuidores al problema del cambio climático irá en aumento. Las mejoras en otros sectores de mayor consumo energético y generación de emisiones relativizarán la importancia del crecimiento de las emisiones globales del transporte aéreo. Los esquemas de compensación de emisiones serán necesarios para permitir al sector progresar hacia una estabilización de las mismas, advirtiendo de las dificultades y riesgos de su implementación si se convierten en un puro instrumento mercantil.

In this work we evaluate the possibilities to assess aerosol-cloud interactions in short time scales (2 min.) on an observational base. Retrievals of the cloud effective radius and number concentration in a liquid-water cloud by using the multiple scattering technique Dual-FOV Polarization lidar, together with the aerosol extinction coefficient in the boundary layer has shown a correspondence between the aerosol and cloud properties in the 6 hours case presented, obtaining a value of ACIN = 0.76 ± 0.29, which corroborates the potential of lidar observations to study the relation between aerosols and clouds on small scales.

Canadian wildfire smoke was detected in the troposphere and lower stratosphere over Europe in August and September 2017. Lidar measurements from various stations of the European Aerosol Research Lidar Network (EARLINET) observed the stratospheric smoke layer. Triple-wavelength (355, 532, and 1064 nm) lidar measurements of the depolarization and the lidar ratio are reported from Leipzig, Germany. The particle linear depolarization ratio of the wildfire smoke in the stratosphere had an exceptional strong wavelength dependence reaching from 0.22 at 355 nm, to 0.18 at 532 nm, and 0.04 at 1064 nm. The lidar ratio increased with wavelength from 40±16 sr at 355 nm, to 66±12 sr at 532 nm, and 92±27 sr at 1064 nm. The development of the stratospheric smoke plume over several months was studied by long-term lidar measurements in Cyprus. The stratospheric smoke layers increased in altitude up to 24 km height.

Since 2010, the Raman dual-FOV lidar system permits the retrieval of microphysical properties of liquid-water clouds during nighttime. A new robust lidar depolarization approach was recently introduced, which permits the retrieval of these properties as well, with high temporal resolution and during daytime. To implement this approach, the lidar system was upgraded, by adding a three channel depolarization receiver. The first preliminary retrieval results and a comparison between both methods is presented.

The Latin American Lidar Network, ALINE a.k.a LALINET is a federation lidar network established in 2008 which became a member of GALION/GAW program in 2013. Currently the network consists of 9 operational stations with the perspective of two more stations to be included. The network today covers more than 18 million Km2 and spans in latitude from -52° to 21° and in longitude from -78° to -47°. It should cover a larger area in the future as planned with the inclusion of more active stations.

Background: Abdominal aortic aneurysm (AAA) is characterized by weakening and dilatation of the aortic wall in the abdomen. The aim of this study was to gain insight into cell-specific mechanisms involved in AAA pathophysiology by analyzing the (phospho)proteome of vascular smooth muscle cells derived from patients with AAA compared with those of healthy donors., Methods: A (phospho)proteomics analysis based on tandem mass spectrometry was performed on vascular smooth muscle cells derived from patients with AAA (n=24) and healthy, control individuals (C-SMC, n=8). Following protein identification and quantification using MaxQuant, integrative inferred kinase activity analysis was used to calculate kinase activity scores., Results: Expression differences between vascular smooth muscle cells derived from patients with AAA and healthy, control individuals were predominantly found in proteins involved in ECM (extracellular matrix) remodeling (THSD4 [thrombospondin type-1 domain-containing protein 4] and ADAMTS1 [A disintegrin and metalloproteinase with thrombospondin motifs 1]), energy metabolism (GYS1 [glycogen synthase 1] and PCK2 [phosphoenolpyruvate carboxykinase 2, mitochondrial]), and contractility (CACNA2D1 [calcium voltage-dependent channel subunit α-2/δ-1] and TPM1 [tropomyosin α-1 chain]). Phosphorylation patterns on proteins related to actin cytoskeleton organization dominated the phosphoproteome of vascular smooth muscle cells derived from patients with AAA . Besides, phosphorylation changes on proteins related to energy metabolism (GYS1), contractility (PARVA [α-parvin], PPP1R12A [protein phosphatase 1 regulatory subunit 12A], and CALD1 [caldesmon 1]), and intracellular communication (GJA1 [gap junction α-1 protein]) were seen. Kinase activity of NUAK1 (NUAK family SNF1-like kinase 1), FYN (tyrosine-protein kinase Fyn), MAPK7 (mitogen-activated protein kinase 7), and STK10 (serine/threonine kinase 10) was different in vascular smooth muscle cells derived from patients with AAA compared with those from healthy, control individuals., Conclusions: This study revealed changes in expression and phosphorylation levels of proteins involved in various processes responsible for AAA progression and development (eg, energy metabolism, ECM remodeling, actin cytoskeleton organization, contractility, intracellular communication, and cell adhesion). These newly identified proteins, phosphosites, and related kinases provide further insight into the underlying mechanism of vascular smooth muscle cell dysfunction within the aneurysmal wall. Our omics data thereby offer the opportunity to study the relevance, either as drug target or biomarker, of these proteins in AAA development., Competing Interests: None.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.

Background: Early cutaneous squamous cell carcinomas (cSCCs) generally show epithelial differentiation features and good prognosis, whereas advanced cSCCs present mesenchymal traits associated with tumor relapse, metastasis, and poor survival. Currently, the mechanisms involved in cSCC progression are unclear, and the established markers are suboptimal for accurately predicting the clinical course of the disease., Methods: Using a mouse model of cSCC progression, expression microarray analysis, immunofluorescence and flow cytometry assays, we have identified a prognostic biomarker of tumor relapse, which has been evaluated in a cohort of cSCC patient samples. Phosphoproteomic analysis have revealed signaling pathways induced in epithelial plastic cancer cells that promote epithelial-mesenchymal plasticity (EMP) and tumor progression. These pathways have been validated by genetic and pharmacological inhibition assays., Results: We show that the emergence of epithelial cancer cells expressing integrin αV (ITGAV) promotes cSCC progression to a mesenchymal state. Consistently, ITGAV expression allows the identification of patients at risk of cSCC relapse above the currently employed clinical histopathological parameters. We also demonstrate that activation of insulin-like growth factor-1 receptor (IGF1R) pathway in epithelial cancer cells is necessary to induce EMP and mesenchymal state acquisition in response to tumor microenvironment-derived factors, while promoting ITGAV expression. Likewise, ITGAV knockdown in epithelial plastic cancer cells also blocks EMP acquisition, generating epithelial tumors., Conclusions: Our results demonstrate that ITGAV is a prognostic biomarker of relapse in cSCCs that would allow improved patient stratification. ITGAV also collaborates with IGF1R to induce EMP in epithelial cancer cells and promotes cSCC progression, revealing a potential therapeutic strategy to block the generation of advanced mesenchymal cSCCs., (© 2024. The Author(s).)

Summary: Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here, we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools., Availability and Implementation: sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Hypertrophic cardiomyopathy (HCM) is caused by sarcomere gene mutations (genotype-positive HCM) in ≈50% of patients and occurs in the absence of mutations (genotype-negative HCM) in the other half of patients. We explored how alterations in the metabolomic and lipidomic landscape are involved in cardiac remodeling in both patient groups., Methods: We performed proteomics, metabolomics, and lipidomics on myectomy samples (genotype-positive N=19; genotype-negative N=22; and genotype unknown N=6) from clinically well-phenotyped patients with HCM and on cardiac tissue samples from sex- and age-matched and body mass index-matched nonfailing donors (N=20). These data sets were integrated to comprehensively map changes in lipid-handling and energy metabolism pathways. By linking metabolomic and lipidomic data to variability in clinical data, we explored patient group-specific associations between cardiac and metabolic remodeling., Results: HCM myectomy samples exhibited (1) increased glucose and glycogen metabolism, (2) downregulation of fatty acid oxidation, and (3) reduced ceramide formation and lipid storage. In genotype-negative patients, septal hypertrophy and diastolic dysfunction correlated with lowering of acylcarnitines, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines. In contrast, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines were positively associated with septal hypertrophy and diastolic impairment in genotype-positive patients., Conclusions: We provide novel insights into both general and genotype-specific metabolic changes in HCM. Distinct metabolic alterations underlie cardiac disease progression in genotype-negative and genotype-positive patients with HCM., Competing Interests: Disclosures Dr Margulies is a consultant for Bristol Myers Squibb and receives research support from Amgen.

The majority of patients with resected stage II-IIIA non-small cell lung cancer (NSCLC) are treated with platinum-based adjuvant chemotherapy (ACT) in a one-size-fits-all approach. However, a significant number of patients do not derive clinical benefit, and no predictive patient selection biomarker is currently available. Using mass spectrometry-based proteomics, we have profiled tumour resection material of 2 independent, multi-centre cohorts of in total 67 patients with NSCLC who underwent ACT. Unsupervised cluster analysis of both cohorts revealed a poor response/survival sub-cluster composed of ~ 25% of the patients, that displayed a strong epithelial-mesenchymal transition signature and stromal phenotype. Beyond this stromal sub-population, we identified and validated platinum response prediction biomarker candidates involved in pathways relevant to the mechanism of action of platinum drugs, such as DNA damage repair, as well as less anticipated processes such as those related to the regulation of actin cytoskeleton. Integration with pre-clinical proteomics data supported a role for several of these candidate proteins in platinum response prediction. Validation of one of the candidates (HMGB1) in a third independent patient cohort using immunohistochemistry highlights the potential of translating these proteomics results to clinical practice., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Ovarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood., Methods: The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer., Results: In this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response., Conclusion: HOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors., (© 2024. The Author(s).)

Complex cross-talk occurs between gastrointestinal nematodes and gut symbiotic microbiota, with consequences for animal metabolism. To investigate the connection between methane production and endoparasites, this study evaluated the effect of mixed infection with Haemonchus contortus and Trichostrongylus colubriformis on methanogenic and methanotrophic community in rumen microbiota of lambs using shotgun metagenomic and real-time quantitative PCR (qPCR). The rumen content was collected from six Santa Inês lambs, (7 months old) before and after 42 days infection by esophageal tube. The metagenomic analysis showed that the infection affected the microbial community structure leading to decreased abundance of methanotrophs bacteria, i.e. α-proteobacteria and β-proteobacteria, anaerobic methanotrophic archaea (ANME), protozoa, sulfate-reducing bacteria, syntrophic bacteria with methanogens, geobacter, and genes related to pyruvate, fatty acid, nitrogen, and sulfur metabolisms, ribulose monophosphate cycle, and Entner-Doudoroff Pathway. Additionally, the abundance of methanogenic archaea and the mcrA gene did not change. The co-occurrence networks enabled us to identify the interactions between each taxon in microbial communities and to determine the reshaping of rumen microbiome associations by gastrointestinal nematode infection. Besides, the correlation between ANMEs was lower in the animal's postinfection. Our findings suggest that gastrointestinal parasites potentially lead to decreased methanotrophic metabolism-related microorganisms and genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors. Our findings revealed that acetone protein precipitation coupled to ISD outperformed the other methods in terms of the number of identified proteins (2246) and method reproducibility (correlation coefficient between replicates (r = 0.94, CV = 19%). Overall, especially small proteins such as those from the classes mentioned above were better identified using ISD workflows. Concluding, herein we report that secretome protein precipitation coupled to ISD is the method of choice for high-throughput secretome proteomics via single shot nanoLC-MS/MS., (© 2024 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

Micronutrient deficiencies can develop in critically ill patients, arising from factors such as decreased intake, increased losses, drug interactions, and hypermetabolism. These deficiencies may compromise important immune functions, with potential implications for patient outcomes. Alternatively, micronutrient blood levels may become low due to inflammation-driven redistribution rather than consumption. This explorative pilot study investigates blood micronutrient concentrations during the first three weeks of ICU stay in critically ill COVID-19 patients and evaluates the impact of additional micronutrient administration. Moreover, associations between inflammation, disease severity, and micronutrient status were explored. We measured weekly concentrations of vitamins A, B6, D, and E; iron; zinc; copper; selenium; and CRP as a marker of inflammation state and the SOFA score indicating disease severity in 20 critically ill COVID-19 patients during three weeks of ICU stay. Half of the patients received additional (intravenous) micronutrient administration. Data were analyzed with linear mixed models and Pearson's correlation coefficient. High deficiency rates of vitamins A, B6, and D; zinc; and selenium (50-100%) were found at ICU admission, along with low iron status. After three weeks, vitamins B6 and D deficiencies persisted, and iron status remained low. Plasma levels of vitamins A and E, zinc, and selenium improved. No significant differences in micronutrient levels were found between patient groups. Negative correlations were identified between the CRP level and levels of vitamins A and E, iron, transferrin, zinc, and selenium. SOFA scores negatively correlated with vitamin D and selenium levels. Our findings reveal high micronutrient deficiency rates at ICU admission. Additional micronutrient administration did not enhance levels or expedite their increase. Spontaneous increases in vitamins A and E, zinc, and selenium levels were associated with inflammation resolution, suggesting that observed low levels may be attributed, at least in part, to redistribution rather than true deficiencies.

The protein kinase D (PKD) family members regulate the fission of cargo vesicles at the Golgi complex and play a pro-oncogenic role in triple-negative breast cancer (TNBC). Whether PKD facilitates the secretion of tumor-promoting factors in TNBC, however, is still unknown. Using the pharmacological inhibition of PKD activity and siRNA-mediated depletion of PKD2 and PKD3, we identified the PKD-dependent secretome of the TNBC cell lines MDA-MB-231 and MDA-MB-468. Mass spectrometry-based proteomics and antibody-based assays revealed a significant downregulation of extracellular matrix related proteins and pro-invasive factors such as LIF, MMP-1, MMP-13, IL-11, M-CSF and GM-CSF in PKD-perturbed cells. Notably, secretion of these proteins in MDA-MB-231 cells was predominantly controlled by PKD2 and enhanced spheroid invasion. Consistently, PKD-dependent secretion of pro-invasive factors was more pronounced in metastatic TNBC cell lines. Our study thus uncovers a novel role of PKD2 in releasing a pro-invasive secretome., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

The tyrosine kinase inhibitor sunitinib is an effective first-line treatment for patients with advanced renal cell carcinoma (RCC). Hypothesizing that a functional read-out by mass spectrometry-based (phospho, p-)proteomics will identify predictive biomarkers for treatment outcome of sunitinib, tumor tissues of 26 RCC patients were analyzed. Eight patients had primary resistant (RES) and 18 sensitive (SENS) RCC. A 78 phosphosite signature (p < 0.05, fold-change > 2) was identified; 22 p-sites were upregulated in RES (unique in RES: BCAR3, NOP58, EIF4A2, GDI1) and 56 in SENS (35 unique). EIF4A1/EIF4A2 were differentially expressed in RES at the (p-)proteome and, in an independent cohort, transcriptome level. Inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) as determined by INKA was higher in SENS. Posttranslational modifications signature enrichment analysis showed that different p-site-centric signatures were enriched (p < 0.05), of which FGF1 and prolactin pathways in RES and, in SENS, vanadate and thrombin treatment pathways, were most significant. In conclusion, the RCC (phospho)proteome revealed differential p-sites and kinase activities associated with sunitinib resistance and sensitivity. Independent validation is warranted to develop an assay for upfront identification of patients who are intrinsically resistant to sunitinib., (© 2023. The Author(s).)

Introduction: Extracellular vesicles (EVs) may propagate and modulate Alzheimer's disease (AD) pathology. We aimed to comprehensively characterize the proteome of cerebrospinal fluid (CSF) EVs to identify proteins and pathways altered in AD., Methods: CSF EVs were isolated by ultracentrifugation (Cohort 1) or Vn96 peptide (Cohort 2) from non-neurodegenerative controls (n = 15, 16) and AD patients (n = 22, 20, respectively). EVs were subjected to untargeted quantitative mass spectrometry-based proteomics. Results were validated by enzyme-linked immunosorbent assay (ELISA) in Cohorts 3 and 4, consisting of controls (n = 16, n = 43, (Cohort3, Cohort4)), and patients with AD (n = 24, n = 100)., Results: We found > 30 differentially expressed proteins in AD CSF EVs involved in immune-regulation. Increase of C1q levels in AD compared to non-demented controls was validated by ELISA (∼ 1.5 fold, p (Cohort 3) = 0.03, p (Cohort 4) = 0.005)., Discussion: EVs may be utilized as a potential biomarker and may play a so far unprecedented role in immune-regulation in AD., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Background: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM., Methods: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3 c.2373insG HCM mouse model., Results: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes., Conclusions: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM., Competing Interests: Disclosures E. Manders is an employee of CytoCypher BV. M. Helmes is the Director of CytoCypher BV. The other authors report no conflicts.

Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors.

Background: Alzheimer's disease (AD) cerebrospinal fluid (CSF) core biomarkers (Aβ42/40 ratio, p-tau, and t-tau) provide high diagnostic accuracy, even at the earliest stage of disease. However, these markers do not fully reflect the complex AD pathophysiology. Recent large scale CSF proteomic studies revealed several new AD candidate biomarkers related to metabolic pathways. In this study we measured the CSF levels of four metabolism-related proteins not directly linked to amyloid- and tau-pathways (i.e., pyruvate kinase, PKM; aldolase, ALDO; ubiquitin C-terminal hydrolase L1, UCHL1, and fatty acid-binding protein 3, FABP3) across the AD continuum. We aimed at validating the potential value of these proteins as new CSF biomarkers for AD and their possible involvement in AD pathogenesis, with specific interest on the preclinical phase of the disease., Methods: CSF PKM and ALDO activities were measured with specific enzyme assays while UCHL1 and FABP3 levels were measured with immunoassays in a cohort of patients composed as follows: preclinical AD (pre-AD, n = 19, cognitively unimpaired), mild cognitive impairment due to AD (MCI-AD, n = 50), dementia due to AD (ADdem, n = 45), and patients with frontotemporal dementia (FTD, n = 37). Individuals with MCI not due to AD (MCI, n = 30) and subjective cognitive decline (SCD, n = 52) with negative CSF AD-profile, were enrolled as control groups., Results: CSF UCHL1 and FABP3 levels, and PKM activity were significantly increased in AD patients, already at the pre-clinical stage. CSF PKM activity was also increased in FTD patients compared with control groups, being similar between AD and FTD patients. No difference was found in ALDO activity among the groups. UCHL1 showed good performance in discriminating early AD patients (pre-AD and MCI-AD) from controls (AUC ~ 0.83), as assessed by ROC analysis. Similar results were obtained for FABP3. Conversely, PKM provided the best performance when comparing FTD vs. MCI (AUC = 0.80). Combination of PKM, FABP3, and UCHL1 improved the diagnostic accuracy for the detection of patients within the AD continuum when compared with single biomarkers., Conclusions: Our study confirmed the potential role of UCHL1 and FABP3 as neurodegenerative biomarkers for AD. Furthermore, our results validated the increase of PKM activity in CSF of AD patients, already at the preclinical phase of the disease. Increased PKM activity was observed also in FTD patients, possibly underlining similar alterations in energy metabolism in AD and FTD., (© 2023. The Author(s).)

A comprehensive pan-human spectral library is critical for biomarker discovery using mass spectrometry (MS)-based proteomics. DPHL v.1, a previous pan-human library built from 1,096 data-dependent acquisition (DDA) MS data of 16 human tissue types, allows quantifying of 10,943 proteins. Here, we generated DPHL v.2 from 1,608 DDA-MS data. The data included 586 DDA-MS data acquired from 18 tissue types, while 1,022 files were derived from DPHL v.1. DPHL v.2 thus comprises data from 24 sample types, including several cancer types (lung, breast, kidney, and prostate cancer, among others). We generated four variants of DPHL v.2 to include semi-tryptic peptides and protein isoforms. DPHL v.2 was then applied to two colorectal cancer cohorts. The numbers of identified and significantly dysregulated proteins increased by at least 21.7% and 14.2%, respectively, compared with DPHL v.1. Our findings show that the increased human proteome coverage of DPHL v.2 provides larger pools of potential protein biomarkers., Competing Interests: T.G. is shareholder of Westlake Omics, Inc. N.X., X.Y., and W.L. were employees of Westlake Omics, Inc., when they participated in this project., (© 2023 The Author(s).)