Analysis Of Protein Profiles Research Articles

Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Polymerase chain reaction-based typing methods and protein profiling analysis of Acinetobacter baumannii isolated from environmental and clinical sources from South India.

Acinetobacter baumannii is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in infected individuals' blood, pus, and respiratory pathways. Due to its resilient nature, it is known to be a causative agent for outbreaks. Therefore, it is crucial to understand the genetic similarity between clinical and environmental isolates. The study aimed to find the genetic relationships between clinical and environmental isolates using PCR-based typing methods such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), random amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic sequence-based PCR (Rep-PCR). Additionally, outer membrane protein (OMP) and whole cell protein (WCP) profiles were also used. The PCR-based methods, ERIC-PCR and Rep-PCR, showed decreased genetic similarity between clinical and environmental isolates (66% and 58%, respectively). However, RAPD showed relatively higher genetic similarity (91%). The OMP and WCP profiles showed varied banding patterns between the clinical and environmental isolates in the 29-43 kDa region. The PCR-based methods proved to be a reliable and reproducible technique. The OMP and WCP profiles, though not as discriminatory as the molecular typing methods, could help identify the most and least commonly occurring protein bands and thus help in typing clinical and environmental A. baumannii isolates.

Analysis of inflammatory protein profiles in the circulation of COVID-19 patients identifies patients with severe disease phenotypes

BackgroundThe coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can present with a broad range of clinical manifestations, ranging from asymptomatic to severe multiple organ failure. The severity of the disease can vary depending on factors such as age, sex, ethnicity, and pre-existing medical conditions. Despite multiple efforts to identify reliable prognostic factors and biomarkers, the predictive capacity of these markers for clinical outcomes remains poor. Circulating proteins, which reflect the active mechanisms in an individual, can be easily measured in clinical practice and therefore may be useful as biomarkers for COVID-19 disease severity. In this study, we sought to identify protein biomarkers and endotypes for COVID-19 severity and evaluate their reproducibility in an independent cohort. MethodsWe investigated a cohort of 153 Greek patients with confirmed SARS-CoV-2 infection in which plasma protein levels were measured using the Olink Explore 1536 panel, which consists of 1472 proteins. We compared the protein profiles from severe and moderate COVID-19 patients to identify proteins associated with disease severity. To evaluate the reproducibility of our findings, we compared the protein profiles of 174 patients with comparable COVID-19 severities in a US COVID-19 cohort to identify proteins consistently correlated with COVID-19 severity in both groups. ResultsWe identified 218 differentially regulated proteins associated with severity, 20 proteins were also replicated in an external cohort which we used for validation. Moreover, we performed unsupervised clustering of patients based on 97 proteins with the highest log2 fold changes in order to identify COVID-19 endotypes. Clustering of patients based on differentially regulated proteins revealed the presence of three clinical endotypes. While endotypes 2 and 3 were enriched for severe COVID-19 patients, endotypes 3 represented the most severe form of the disease. ConclusionsThese results suggest that identified circulating proteins may be useful for identifying COVID-19 patients with worse outcomes, and this potential utility may extend to other populations. Trial registrationNCT04357366.

Proteins journey-from marine to freshwater ecosystem: blood plasma proteomic profiles of pink salmon Oncorhynchus gorbuscha Walbaum, 1792 during spawning migration.

The pink salmon (Oncorhynchus gorbuscha) is a commercial anadromous fish species of the family Salmonidae. This species has a 2-year life cycle that distinguishes it from other salmonids. It includes the spawning migration from marine to freshwater environments, accompanied by significant physiological and biochemical adaptive changes in the body. This study reveals and describes variability in the blood plasma proteomes of female and male pink salmon collected from three biotopes-marine, estuarine and riverine-that the fish pass through in spawning migration. Identification and comparative analysis of blood plasma protein profiles were performed using proteomics and bioinformatic approaches. The blood proteomes of female and male spawners collected from different biotopes were qualitatively and quantitatively distinguished. Females differed primarily in proteins associated with reproductive system development (certain vitellogenin and choriogenin), lipid transport (fatty acid binding protein) and energy production (fructose 1,6-bisphosphatase), and males in proteins involved in blood coagulation (fibrinogen), immune response (lectins) and reproductive processes (vitellogenin). Differentially expressed sex-specific proteins were implicated in proteolysis (aminopeptidases), platelet activation (β- and γ-chain fibrinogen), cell growth and differentiation (a protein containing the TGF_BETA_2 domain) and lipid transport processes (vitellogenin and apolipoprotein). The results are of both fundamental and practical importance, adding to existing knowledge of the biochemical adaptations to spawning of pink salmon, a representative of economically important migratory fish species.

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5 CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.

CAR +and CAR −T cells differentiate into an NK-like subset that is associated with increased inflammatory cytokines following infusion

Abstract Chimeric antigen receptor (CAR) T cells have demonstrable efficacy in treating B-cell malignancies. Factors such as product composition, lymphodepletion and immune reconstitution are known to influence CAR +T cell survival and persistence, however the determinants of CAR +T cells’ fate and differentiation and how the patients’ reconstituting immune system influence therapy outcomes remain poorly understood. We applied single cell multi-omics analysis of RNA, protein and clonal profiles of CAR +T and PBMC in patients receiving donor-derived products from a piggybac CAR19 clinical trial (N=8 patients). We used these data to reconstruct a differentiation trajectory, which explained the observed phenotype and fate of CAR +and CAR −T cells within the first 30 days of post-infusion. Following initial lympho-depletion, endogenous CAR −CD8 +, γδ T cells and CAR+ T cells clonally expanded, and differentiated across heterogenous phenotypes. They transitioned from a dominant resting or proliferating state into precursor of exhausted T cells, and finally into a terminal NK-like phenotype. The subset of terminal NK-like CAR +T cells was associated with increased serum concentrations of proinflammatory cytokines including IL-12 and IL-18. Validation on a published cohort (61 patients) showed that this subset was associated with clinical outcome at 6 months. Finally, we analysed in detail one patient that developed CAR T cell induced lymphoma and discussed the impact of early phase therapy in determining this unexpected outcome. These results demonstrate that CAR- and CAR+ T cells share a differentiation profile which may be determined by non-CAR derived signals and which may influence patients’ immune-recovery and outcome. National Health And Medical Research Funding (Australia) (APP1121643, APP1128416) BD Bioscience

Fixed Time-Point Analysis Reveals Repetitive Mild Traumatic Brain Injury Effects on Resting State Functional Magnetic Resonance Imaging Connectivity and Neuro-Spatial Protein Profiles.

Repetitive mild traumatic brain injuries (rmTBIs) are serious trauma events responsible for the development of numerous neurodegenerative disorders. A major challenge in developing diagnostics and treatments for the consequences of rmTBI is the fundamental knowledge gaps of the molecular mechanisms responsible for neurodegeneration. It is both critical and urgent to understand the neuropathological and functional consequences of rmTBI to develop effective therapeutic strategies. Using the Closed-Head Impact Model of Engineered Rotational Acceleration, or CHIMERA, we measured neural changes following injury, including brain volume, diffusion tensor imaging, and resting-state functional magnetic resonance imaging coupled with graph theory and functional connectivity analyses. We determined the effect of rmTBI on markers of gliosis and used NanoString-GeoMx to add a digital-spatial protein profiling analysis of neurodegenerative disease-associated proteins in gray and white matter regions. Our analyses revealed aberrant connectivity changes in the thalamus, independent of microstructural damage or neuroinflammation. We also identified distinct changes in the levels of proteins linked to various neurodegenerative processes including total and phospho-tau species and cell proliferation markers. Together, our data show that rmTBI significantly alters brain functional connectivity and causes distinct protein changes in morphologically intact brain areas.

Discovery and Mechanism of Azatryptanthrin Derivatives as Novel Anti-Phytopathogenic Bacterial Agents for Potent Bactericide Candidates.

The natural alkaloids of tryptanthrin and their derivatives have a wide range of biological activities. In this research, four series of azatryptanthrin derivatives containing 4-aza/3-aza/2-aza/1-aza tryptanthrin were prepared by condensation cyclization reaction against plant pathogens to develop a new natural product-based bacterial pesticide. Compound 4Aza-8 displayed a remarkable growth inhibitory effect on pathogenic bacteria of Xanthomonas axonopodis pv. citri (Xac), Xanthomonas oryzae pv. Oryzae (Xoo), and Pseudomonas syringae pv. actinidiae (Psa) with the final corrected EC50 values of 0.312, 1.91, and 18.0 μg/mL, respectively, which were greatly superior than that of tryptanthrin (Tryp). Moreover, 4Aza-8 also showed effective therapeutic and protective activities in vivo on citrus canker. Further mechanism studies on Xac elucidated that compound 4Aza-8 was able to affect the growth curve of Xac and the formation of biofilm, cause severe shrinkage in bacterial morphology, increase reactive oxygen species levels, and induce apoptosis in bacterial cells. Quantitative analysis of differential protein profiles found that the major differences were mainly concentrated on the endometrial protein in the bacterial secretion system pathway, which blocked the membrane transport and affected the transfer of DNA to the host cell. In summary, these research results suggest that 4Aza-8 represents a promising anti-phytopathogenic-bacteria agent, which is worth being further investigated as a bactericide candidate.

Long- and short-term acclimation of the photosynthetic apparatus to salinity in Chlamydomonas reinhardtii. The role of Stt7 protein kinase.

Salt stress triggers an Stt7-mediated LHCII-phosphorylation signaling mechanism similar to light-induced state transitions. However, phosphorylated LHCII, after detaching from PSII, does not attach to PSI but self-aggregates instead. Salt is a major stress factor in the growth of algae and plants. Here, our study mainly focuses on the organization of the photosynthetic apparatus to the long-term responses of Chlamydomonas reinhardtii to elevated NaCl concentrations. We analyzed the physiological effects of salt treatment at a cellular, membrane, and protein level by microscopy, protein profile analyses, transcripts, circular dichroism spectroscopy, chlorophyll fluorescence transients, and steady-state and time-resolved fluorescence spectroscopy. We have ascertained that cells that were grown in high-salinity medium form palmelloids sphere-shaped colonies, where daughter cells with curtailed flagella are enclosed within the mother cell walls. Palmelloid formation depends on the presence of a cell wall, as it was not observed in a cell-wall-less mutant CC-503. Using the stt7 mutant cells, we show Stt7 kinase-dependent phosphorylation of light-harvesting complex II (LHCII) in both short- and long-term treatments of various NaCl concentrations-demonstrating NaCl-induced state transitions that are similar to light-induced state transitions. The grana thylakoids were less appressed (with higher repeat distances), and cells grown in 150 mM NaCl showed disordered structures that formed diffuse boundaries with the flanking stroma lamellae. PSII core proteins were more prone to damage than PSI. At high salt concentrations (100-150 mM), LHCII aggregates accumulated in the thylakoid membranes. Low-temperature and time-resolved fluorescence spectroscopy indicated that the stt7 mutant was more sensitive to salt stress, suggesting that LHCII phosphorylation has a role in the acclimation and protection of the photosynthetic apparatus.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Haemolymph Proteins on Stressed Cockroach Periplaneta Americana

Immune responses in insects are humoral and cell mediated and the haemolymph proteins are found to be involved in humoral immune responses. In this experiment cockroaches were divided into two groups as control and wounded and the haemolymph samples were collected at different time intervals and subjected for quantitative analysis of proteins, proteolytic activity and protein profile. Haemolymph protein concentration reduced initially after injury then they recovered after 24 hrs. Proteolytic activity was found to be the highest during the first 24hrs of the wounded insects. Seven protein bands were observed in PAGE gel electrophoresis of the wounded and control cockroaches. A 115 kDa protein band was found to appear in the wounded P.americana after 8 hours is an important finding of this experiment. Some of the protein bands were reduced in the wounded cockroach and another important finding was protein retaining to normal level in the haemolymph after 24hrs and 80 and 76kDa protein bands were thicker in this group. Cockroach recovering after wounding is revealed after 8hrs after wounding.

Plasma protein profiling analysis in patients with atrial fibrillation before and after three different ablation techniques.

There are controversies on the pathophysiological alteration in patients with atrial fibrillation (AF) undergoing pulmonary vein isolation using different energy sources. We evaluated the changes in plasma proteins in acute phase post-ablation in patients receiving cryoballoon ablation, radiofrequency balloon ablation, or radiofrequency ablation. Blood samples from eight healthy controls and 24 patients with AF were taken on the day of admission, day 1, and day 2 post-ablation and analyzed by the Olink proximity extension assay. Proteins were identified and performed with enrichment analysis. Protein-protein interaction network and module analysis were conducted using Cytoscape software. Of 181 proteins, 42 proteins in the cryoballoon group, 46 proteins in the radiofrequency balloon group, and 43 proteins in the radiofrequency group significantly changed after ablation. Most of the proteins altered significantly on the first day after ablation. Altered proteins were mainly involved in cytokine-cytokine receptor interaction. Both balloon-based ablations showed a similar shift toward enhancing cell communication and regulation of signaling while inhibiting neutrophil chemotaxis. However, radiofrequency ablation presented a different trend. Seed proteins, including osteopontin, interleukin-6, interleukin-10, C-C motif ligand 8, and matrix metalloproteinase-1, were identified. More significant proteins associated with hemorrhage and coagulation were selected in balloon-based ablations by machine learning. Plasma protein response after three different ablations in patients with AF mainly occurred on the first day. Radiofrequency balloon ablation shared similar alteration in protein profile as cryoballoon ablation compared with radiofrequency ablation, suggesting that lesion size rather than energy source is the determinant in pathophysiological responses to the ablation.

Protein profile analysis of tear fluid with hyphenated HPLC-UV LED-induced fluorescence detection for the diagnosis of dry eye syndrome.

Tear fluid contains organic and inorganic constituents, variations in their relative concentrations could provide valuable information and can be useful for the detection of several ophthalmological diseases. This report describes the application of the lab-assembled light-emitting diode (LED)-based high-performance liquid chromatography system for protein profiling of tear fluids to diagnose dry eye disease. Principal Component Analysis (PCA), match/no-match, and Artificial Neural Network (ANN) based binary classification of protein profile data were performed for disease diagnosis. Results from the match/no-match test of the protein profile data showed 94.4% sensitivity and 87.8% specificity. ANN with the leaving one out procedure has given 91.6% sensitivity and 93.9% specificity.

Analysis and comparison of tear protein profiles in dogs using different tear collection methods

BackgroundTear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS).MethodsThe tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots’ expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsTear protein concentrations ranged from 2.80 to 4.03 μg/μL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins.ConclusionsThe recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

Development of yogurts with mixture of sheep and bovine milk: effect on chemical physical characteristics, protein profile and antioxidant activity

Yogurt is one of the most popular fermented dairy products and an excellent medium for new ingredients/food source. This study sought to evaluate and characterize bovine milk (BM) and sheep milk (SM) and yogurt made from bovine (BM) and sheep (SM) milk, and BM:SM mixture with the respective 100:0 fractions: 75:25, 50:50, 25:75 and 0:100, and evaluate their influence on physicochemical parameters, antioxidant, protein profile, microstructure and color analysis for yogurts. Yogurts with higher levels of acidity, protein, fat, total solids, ash and calcium were the ones made from SM.. Regarding the color attributes, all yogurts showed yellow-green characteristics after 21 days of storage by evaluating parameters a* and b*, with a predominance of bovine milk. The antioxidant profile showed a higher concentration at 100:0 and 0:100, but bovine milk has a higher DPPH (2,2-diphenyl-1-picrylhydrazyl) value, while yogurts maintained constant DPPH values ​​during storage, being higher at 0:100. Scanning electron microscopy (SEM) showed that BM (100:0) has a more compact structure, possibly due to the larger size of the fat particles and the lower protein concentration compared to SM (0:100). Sheep milk presented better nutritional value, showing its potential in the manufacture of products.

Targeting HER3 or MEK overcomes acquired Trastuzumab resistance in HER2-positive gastric cancer-derived xenograft

Acquired Trastuzumab resistance is a complicated and disastrous event for HER2-positive gastric cancer (GC). In this study, we successfully established a GC PDX model with Trastuzumab sensitivity (176P) and induced a homologous model with acquired Trastuzumab resistance (176R), then comprehensively delineated the landscape of Trastuzumab resistance mechanisms using single-cell transcriptome sequencing, as well as protein profiling and genomic variation analysis. According to multi-omics study, different gene expression profiles, rather than genetic changes, contributed to acquired Trastuzumab resistance. The mechanisms underlying acquired Trastuzumab resistance present great complexity as multiple molecules and pathways were involved, including ERBB family, MAPK, PI3K/AKT, JAK/STAT, and cell cycle pathways. Through phenotypical and molecular validation, we found that Trastuzumab combined with HER3-targeted antibody or MEK inhibitor demonstrated excellent antitumor activity and good tolerance, which may serve as promising strategies for overcoming acquired Trastuzumab resistance.

Kekerabatan merpati hias (Columba livia) dilihat dari profil protein darah

Objective: The purpose of this study was to predict the kinship of ornamental pigeons based on blood proteins. Methods: The research method used is exploratory descriptive with a laboratory observation-based approach. Research procedures include: Blood Sampling, Blood Protein Isolation, Purification, Measurement of concentration with a nanodrop spectrophotometer and Electrophoresis. Data analysis includes: Protein Band Profile and Determination of Protein Molecular Weight with RF formula and Relationship with MVSP version 3.22 program. Results : The protein profile in the plasma of ornamental pigeons is different from one type of pigeon to another. some of the protein appears in the ornamental pigeon species but does not appear in other breeds. Likewise with the target protein to be analyzed, some are expressed and some are not expressed in ornamental pigeons. Results based on the analysis of protein profiles and dendograms of the four specific proteins (hemoglobin, transferrin, posttransferrin, and immunoglobulin) contained in erythrocytes and blood plasma, it can be seen that there are two clusters. Cluster 1 is occupied by varieties of ornamental pigeons Moven and cluster 2 is occupied by varieties of ornamental pigeons Lahore, American fantail, Vienna, Indian Fantail, King, Capucine, Gondok and Modena. Conclusions: The results of the ornamental pigeon kinship prediction based on blood protein showed that each variety had different kinship relationships. King, Capucine, Gondok and Modena pigeons have the closest relationship, and Moven pigeons have the most distant relationship from the researched ornamental pigeons.

EXTH-74. ATR INHIBITION IN EXPERIMENTAL GLIOMA

Abstract Glioblastoma are incurable primary tumors of the central nervous system. Novel treatment options are urgently needed. Recently, we investigated molecular effects of disrupting the (b)HLH network, a frequently altered and tumor-promoting transcriptional network in glioma, by introducing a dominant-negative variant of the E47 protein. The identified downstream anti-tumor effects included the DNA damage response pathway. In the present study, we aimed at investigating the anti-glioma effects of ATR inhibition in vitro and in vivo with a particular focus on treatment-induced vulnerabilities and potential combination therapies. We performed acute cytotoxicity, clonogenic survival assays, flow cytometry-based determination of cell cycle status and apoptosis induction in human and murine glioma cell lines in vitro, and investigated tumor growth in the SMA560/VMDk syngeneic mouse model in vivo. Furthermore, we applied RNA sequencing, DigiWest protein profiling analyses and genome-wide CRISPR/Cas9 loss-of function and activation screens to determine potential combination therapies. Combination treatments were further analyzed for synergism by the Bliss and ZIP synergy models. ATR inhibition in glioma cells resulted in reduced viability and clonogenic survival. However, differential patterns of gene expression deregulation (e.g. p53 signaling) and differing modes of cell cycle arrest in distinct glioma model systems argue for a substantial heterogeneity with regard to response to ATR inhibition. Based on our data we designed combinatorial treatments. We will present novel insights into promising combinatorial treatments using ATR inhibition and their underlying molecular mechanisms.

Lactucin, a Bitter Sesquiterpene from Cichorium intybus, Inhibits Cancer Cell Proliferation by Downregulating the MAPK and Central Carbon Metabolism Pathway.

Lung cancer, especially adenocarcinoma, is the second most occurring and highest fatality-causing cancer worldwide. Many natural anticancer compounds, such as sesquiterpene lactones (SLs), show promising anticancer properties. Herein, we examined Lactucin, an SL from the plant Cichorium intybus, for its cytotoxicity, apoptotic-inducing, cell cycle inhibiting capacity, and associated protein expression. We also constructed a biotinylated Lactucin probe to isolate interacting proteins and identified them. We found that Lactucin stops the proliferation of A549 and H2347 lung adenocarcinoma cell lines while not affecting normal lung cell MRC5. It also significantly inhibits the cell cycle at G0/G1 stage and induces apoptosis. The western blot analysis shows that Lactucin downregulates the MAPK pathway, cyclin, and cyclin-dependent kinases, inhibiting DNA repair while upregulating p53, p21, Bax, PTEN, and downregulation of Bcl-2. An increased p53 in response to DNA damage upregulates p21, Bax, and PTEN. In an activity-based protein profiling (ABPP) analysis of A549 cell's protein lysate using a biotinylated Lactucin probe, we found that Lactucin binds PGM, PKM, and LDHA PDH, four critical enzymes in central carbon metabolism in cancer cells, limiting cancer cells in its growth; thus, Lactucin inhibits cancer cell proliferation by downregulating the MAPK and the Central Carbon Metabolism pathway.

Comparative Analysis of Saliva and Plasma Proteins Patterns in Pregnant Cows-Preliminary Studies.

Simple SummaryOne of the most crucial topics about cattle breeding is pregnancy. During this state, there are many changes in protein expression and abundance. These changes find reflection not only in plasma protein patterns but also in saliva, which is easier to obtain than blood. The aim of this study was the analysis of plasma and salivary protein profiles in pregnant cows in order to search for valuable markers of pregnancy status. In this study, the presence of apolipoproteins possibly related to bovine pregnancy was confirmed both in plasma and saliva. This means that saliva can be considered a good source of information about the condition of the organism, including during pregnancy. It is possible that the comparison of salivary and plasma proteomes can be a helpful tool to assess the pregnancy status of cattle, and can be useful for developing rapid tests from saliva.Pregnancy is a physiological state that can be described, from a biochemical point of view, using protein patterns. The present study focused on the comparison of protein patterns between the saliva and plasma of pregnant cows to search for possible markers which are present both in plasma and saliva. Saliva and plasma were collected from healthy, pregnant (3–4 months) and non-pregnant (C; n = 4) cows aged between 4 and 8 years (P; n = 8) from the same farm. Biological material was analyzed using 2D electrophoresis and MS identification. Among identified spots, there were those which could be related to pregnancy (e.g., apolipoproteins I and II in all examined matrices or transforming growth factor-beta-induced protein ig-h3 in albumin-free plasma) as well as those which are responsible for regulating of cellular processes (e.g., pyruvate kinase and aspartate aminotransferase in all examined matrices, or lactate dehydrogenase, phosphoglycerate kinase, and NADH dehydrogenase in plasma). Further identification of common spots and those only specific to saliva as well as the comparison between other periods of pregnancy are necessary; it is already clear that saliva can be considered a valuable diagnostic matrix containing potential markers of physiological and pathological status.