Co-expression Of Proteins Research Articles

Effect of Heat Shock Proteins on Life Span and Behavioral Functions of Animals with FUS Proteinopathy

Abstract—It is known that chaperones and, in particular, 70 kDa heat shock proteins (HSP70), are an integral part of cellular proteostasis. To assess the role of HSP70 in the neurodegenerative process, we used a new hybrid strain of mice with co-expression of the aberrant ΔFUS protein (1–359) and human HSP70. The mice of the new hybrid line are characterized by a longer lifespan, as well as an increase in the duration of the symptomatic stage as compared to control animals. In addition, according to the results of physiological tests, it can be concluded that HSP70 has a positive effect on muscle strength at the symptomatic stage of the model disease.

Identification of plasma proteins relating to brain neurodegeneration and vascular pathology in cognitively normal individuals.

IntroductionThis study aims to first discover plasma proteomic biomarkers relating to neurodegeneration (N) and vascular (V) damage in cognitively normal individuals and second to discover proteins mediating sex‐related difference in N and V pathology.MethodsFive thousand and thirty‐two plasma proteins were measured in 1061 cognitively normal individuals (628 females and 433 males), nearly 90% of whom had magnetic resonance imaging measures of hippocampal volume (as N) and white matter hyperintensities (as V).ResultsDifferential protein expression analysis and co‐expression network analysis revealed different proteins and modules associated with N and V, respectively. Furthermore, causal mediation analysis revealed four proteins mediated sex‐related difference in N and one protein mediated such difference in V damage.DiscussionOnce validated, the identified proteins could help to select cognitively normal individuals with N and V pathology for Alzheimer's disease clinical trials and provide targets for further mechanistic studies on brain sex differences, leading to sex‐specific therapeutic strategies.

Enrichment of Phosphorylated Tau (Thr181) and Functionally Interacting Molecules in Chronic Traumatic Encephalopathy Brain-derived Extracellular Vesicles.

Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of mild repetitive brain injury. The initial neuropathologic changes of CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau). Extracellular vesicles (EVs) are known to carry pathogenic molecules, such as tau in Alzheimer’s disease and CTE suggesting their contribution in pathogenesis. We therefore examined the protein composition of EVs separated from CTE and an age-matched control brain tissues by tandem mass tag -mass spectrometry. The reporter ion intensity was used to quantify the identified molecules. A total of 516 common proteins were identified among three sets of experiments. Weighted protein co-expression network analysis identified 18 unique modules of co-expressed proteins. Two modules were significantly correlated with total tau (t-tau) and p-tau protein in the isolated EVs and enriched in cellular components and biological processes for synaptic vesicle secretion and multivesicular body-plasma membrane fusion. The p-tau (Thr181) level is significantly higher in CTE EVs compared to control EVs and can distinguish the two groups with 73.6% accuracy. A combination of t-tau or p-tau (Thr181) with SNAP-25, PLXNA4 or UBA1, enhanced the accuracy to 96.3, 93.8 and 93.8%, respectively. Bioinformatic protein-protein interaction analysis revealed the functional interaction of SNAP-25 and PLXNA4 with tau, suggesting their interaction in CTE EVs. These data indicate the future application of identified EV proteins for monitoring the CTE risk assessments and understanding the EV-mediated disease progression mechanism.

Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

DOCK8 Serves as a Prognostic Biomarker and Is Related to Immune Infiltration in Patients With HPV Positive Head and Neck Squamous Cell Carcinoma.

Purpose:Dedicator of cytokinesis 8 (DOCK8) was reported to have a vital link to immunoregulation. However, the mechanisms by which it drives immune infiltration in cancer remain uncertain. We tried to assess the role of DOCK8 in patients with cancer, especially human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC).Methods:Data on the expression and survival of DOCK8 in patients with various cancers were analyzed using the Oncomine and TIMER databases. The TIMER database assessed the relationship of DOCK8 with immune infiltration levels and various markers of multiple immune cells. Gene set enrichment analysis revealed tumor-associated biological processes related to DOCK8. ENCODE database was used to explore relevant transcription factors of DOCK8, and a PPI network was constructed using GENEMINIA. The expression and survival role of DOCK8 was confirmed in patients from independent GEO datasets.Results:We determined that DOCK8 expression was upregulated or downregulated in various cancers unlike in healthy tissues. A high expression of DOCK8 was significantly correlated with a favorable prognosis in HPV-positive HNSCC and lung adenocarcinoma (LUAD). Furthermore, multivariate Cox regression analysis revealed that DOCK8 was an independent prognostic factor of HPV-positive HNSCC. Additionally, elevated DOCK8 expression was positively correlated with multiple immune cell infiltration levels and immune marker expression associated with particular immune cell subsets. Also, 14 pathways involved in immune activities and carcinogenesis, 22 potential TFs, and co-expression proteins of DOCK8 indicated DOCK8 to be related to tumor-associated biological processes. Ultimately, we verified that DOCK8 is upregulated and confers a favorable overall survival and progression-free survival status in patients with HPV-positive HNSCC.Conclusion:These results elucidate that high expression of DOCK8 indicates a favorable prognosis in patients with HPV-positive HNSCC as well as increased microenvironmental immune infiltration levels. It would provide new insights into the prognosis predicting and clinical regimen decision making in patients with HPV-positive HNSCC.

Alimentary system is directly attacked by SARS-COV-2 and further prevents immune dysregulation caused by COVID-19.

SARS-COV-2 causes digestive system symptom, the effect of which remains equivocal. Patients with COVID-19 were classified into four groups according to symptom. The study traced the onset and duration of symptoms, compared laboratory examinations and conducted bioinformatic analysis. Immune indices were further analysed. By March 16, 25 patients with COVID-19 and 13 with suspect COVID-19 were admitted to West China Hospital, Sichuan University. Digestive system symptom group had the highest level of ESR (mm/h, P<.0001), serum ferritin (ng/ml, P<.0001), hepatic enzymes (P<.05) and retentive lymphocyte count/percentage (P<.05) and its subsets (P<.05). Combined group (respiratory combined with subsequent digestive system symptom) had the highest level of IL-6 (pg/ml, P=.0046), CRP (mg/L, P=.0004) and moderate lymphocyte depletion. Respiratory system symptom and asymptomatic groups suffered the most from lymphocyte depletion (P<.05). Bioinformatic analysis indicated co-expression of binding related proteins of SARS-COV-2 (ACE2, TMPRSS2 and Furin) in small intestine. CD147 was extensively expressed in alimentary tract. CTSL, PIKfyve, TPC2 and CTSB could be detected with ≥moderate expressions in a variety of organs including alimentary system. Alimentary system is possibly attacked by SARS-COV-2 other than hyperinflammation or immune dysregulation caused by it. Involvement of alimentary system might further protect mild and moderate cases from lymphocyte depletion caused by COVID-19.

Human eggs, zygotes, and embryos express the receptor angiotensin 1-converting enzyme 2 and transmembrane serine protease 2 protein necessary for severe acute respiratory syndrome coronavirus 2 infection

ObjectiveTo study messenger ribonucleic acid (mRNA) and protein expressions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptors (angiotensin 1-converting enzyme 2 [ACE2] and CD147) and proteases (transmembrane serine protease 2 [TMPRSS2] and cathepsin L [CTSL]) in human oocytes, embryos, and cumulus (CCs) and granulosa cells (GCs).DesignResearch study.SettingClinical in vitro fertilization (IVF) treatment center.PatientsPatients undergoing IVF were treated at the Colorado Center for Reproductive Medicine.InterventionsOocytes (germinal vesicle and metaphase II [MII]) and embryos (1-cell [1C] and blastocyst [BL]) were donated for research at the disposition by the patients undergoing IVF. Follicular cells (CC and GC) were collected from women undergoing egg retrieval after ovarian stimulation without an ovulatory trigger for in vitro maturation/IVF treatment cycles.Main Outcome MeasuresPresence or absence of ACE2, CD147, TMPRSS2, and CTSL mRNAs detected using quantitative reverse transcription polymerase chain reaction and proteins detected using capillary Western blotting in human oocytes, embryos, and ovarian follicular cells.ResultsThe quantitative reverse transcription polymerase chain reaction analysis revealed high abundance of ACE2 gene transcripts in germinal vesicle and MII oocytes than in CC, GC, and BL. ACE2 protein was present only in the MII oocytes, and 1C and BL embryos, but other ACE2 protein variants were observed in all the samples. TMPRSS2 protein was present in all the samples, whereas mRNA was observed only in the BL stage. All the samples were positive for CD147 and CTSL mRNA expressions. However, CCs and GCs were the only samples that showed coexpression of both CD147 and CTSL proteins in low abundance.ConclusionsCCs and GCs are the least susceptible to SARS-CoV-2 infection because of lack of the required combination of receptors and proteases (ACE2/TMPRSS2 or CD147/CTSL) in high abundance. The coexpression of ACE2 and TMPRSS2 proteins in the MII oocytes, zygotes, and BLs demonstrated that these gametes and embryos have the cellular machinery required and, thus, are potentially susceptible to SARS-CoV-2 infection if exposed to the virus. However, we do not know whether the infection occurs in vivo or in vitro in an assisted reproductive technology setting yet.

Combined strategies to improve the expression of acidic mammalian chitinase in Pichia pastoris for the production of N, N'-diacetylchitobiose

Acidic mammalian chitinase (AMCase) could hydrolyze chitin into (GlcNAc)2 at pH 2.0. Therefore, it was essential to develop an engineered strain with a high-yield AMCase. Herein, multiple strategies including codon optimization, multiple expression cassettes insertion, co-expression of helper proteins, were developed to construct a recombinant Pichia pastoris with the high-level secretion of AMCase. The recombinant strain harboring four copies of codon-optimized AMCase-encoding gene ChiD and co-expression of HAC1 presented the highest chitinase activity of 570 U/L in high density fermentation, which was 934 folds higher than that in E. coli reported before. Then, the recombinant AMCase was applied to hydrolyze colloidal chitin with a conversion rate of 90 % at pH 2.0, which could save half of the water consumption during the preparation of colloidal chitin. Finally, we also demonstrated that the hydrolysate of AMCase could promote seed germination of wheat and rice, improving the growth of roots and seedling.

β-Catenin/Lin28/let-7 regulatory network determines type II alveolar epithelial stem cell differentiation phenotypes following thoracic irradiation.

The contribution of type II alveolar epithelial stem cells (AEC II) to radiation-induced lung fibrosis (RILF) is largely unknown. Cell differentiation phenotypes are determined by the balance between Lin28 and lethal-7 microRNA (let-7 miRNA). Lin28 is activated by β-catenin. The aim of this study was to track AEC II phenotypes at different phases of injury following thoracic irradiation and examine the expression of β-catenin, Lin28 and let-7 to identify their role in AEC II differentiation. Results showed that coexpression of prosurfactant protein C (proSP-C, an AEC II biomarker) and HOPX (homeobox only protein X, an AEC I biomarker) or vimentin (a differentiation marker) was detected in AEC II post-irradiation. The protein expression levels of HOPX and proSP-C were significantly downregulated, but vimentin was significantly upregulated following irradiation. The expression of E-cadherin, which prevents β-catenin from translocating to the nucleus, was downregulated, and the expression of β-catenin and Lin28 was upregulated after irradiation (P < 0.05 to P < 0.001). Four let-7 miRNA members (a, b, c and d) were upregulated in irradiated lungs (P < 0.05 to P < 0.001), but let-7d was significantly downregulated at 5 and 6 months (P < 0.001). The ratios of Lin28 to four let-7 members were low during the early phase of injury and were slightly higher after 2 months. Intriguingly, the Lin28/let-7d ratio was strikingly increased after 4 months. We concluded that β-catenin contributed to RILF by promoting Lin28 expression, which increased the number of AEC II and the transcription of profibrotic molecules. In this study, the downregulation of let-7d miRNA by Lin28 resulted in the inability of AEC II to differentiate into type I alveolar epithelial cells (AEC I).

Identification of 6 Hub Proteins and Protein Risk Signature of Colorectal Cancer.

Background Colorectal cancer (CRC) is the second most common cause of cancer death in the United States and the third most common cancer globally. The incidence of CRC tends to be younger, and we urgently need a reliable prognostic assessment strategy. Methods Protein expression profile and clinical information of 390 CRC patients/samples were downloaded from the TCPA and TCGA database, respectively. The Kaplan-Meier, Cox regression, and Pearson correlation analysis were applied in this study. Results Based on the TCPA and TCGA database, we screened 6 hub proteins and first constructed protein risk signature, all of which were significantly associated with CRC patients' overall survival (OS). The risk score was an independent prognostic factor and significantly related with the size of the tumor in situ (T). 6 hub proteins were differentially expressed in cancer and normal tissues and in different CRC stages, which were validated at the ONCOMINE database. Next, 40 coexpressed proteins of 6 hub proteins were extracted from the TCPA database. In the protein-protein interaction (PPI) network, HER1, HER2, and CTNNB1 were at the center. Function enrichment analysis illustrated that 46 proteins were mainly involved in the EGFR (HER1) tyrosine kinase inhibitor resistance pathway. Conclusion Studies indicated that 6 hub proteins might be considered as new targets for CRC therapies, and the protein risk signature can be used to predict the OS of CRC patients.

Characterization and oncolytic virus targeting of FAP-expressing tumor-associated pericytes in glioblastoma

Cancer-associated fibroblasts (CAFs) are activated fibroblasts constituting the major stromal components in many types of cancer. CAFs contribute to hallmarks of cancer such as proliferation, invasion and immunosuppressive tumor microenvironment, and are associated with poor prognosis of patients with cancer. However, in glioblastoma (GBM), the most common and aggressive primary malignant brain tumor, our knowledge about CAFs or CAF-like stromal cells is limited. Here, using commonly accepted CAF markers, we characterized CAF-like cell populations in clinical glioma specimens and datasets along with mouse models of GBM. We found that tumor-associated pericytes marked by co-expression of fibroblast activation protein α (FAP) and PDGFRβ represent major stromal cell subsets in both human GBM and mouse GBM models, while a fraction of mesenchymal neoplastic cells also express FAP in patient tumors. Since oncolytic viruses can kill cancer cells and simultaneously modulate the tumor microenvironment by impacting non-neoplastic populations such as immune cells and tumor vasculature, we further investigated the ability of oncolytic viruses to target GBM-associated stromal cells. An oncolytic adenovirus, ICOVIR15, carrying ∆24-E1A and an RGD-fiber, infects and depletes FAP+ pericytes as well as GBM cells in murine GBM. Our study thus identifies FAP+/PDGFRβ+ pericytes as a major CAF-like stromal cell population in GBM, and highlights the unique property of this oncolytic adenovirus to target both GBM cells and GBM-associated stromal FAP+ cells.

Thylakoid Transit Peptide Is Related to the Expression and Localization of NdhB Subunits in Soybean

The chloroplast NAD(P)H dehydrogenase (NDH) complex, as one of the most important photosynthesis protein complexes in thylakoid membrane, is involved in photosystem I (PSI) cyclic electron transport (CEF). Under abiotic environmental stress, the photosynthetic apparatus is susceptible to the damage caused by the strong light illumination. However, the enhancement of NDHdependent CEF could facilitate the alleviation of the damage to the photosynthetic apparatus. The NdhB subunit encoded by chloroplast genome is one of most important subunits of NDH complex and consists of 510 amino acids. Here, according to cloning ndhB from Melrose (cultivated soybean), ACC547 (wild salt-tolerant soybean), S113-6 and S111-9 (hybrid descendant), based on the comparison and analysis of the sequences of NdhB subunits, we found that there is a novel thylakoid transit peptide of NdhB subunit in S111-9. In addition, crosslink immunoprecipitation, immunogold labeling and co-expression of GFP fusion protein indicated that the novel thylakoid transit peptide is favorable to the expression and localization of NdhB subunit in chloroplast. Therefore, we suggest that this novel thylakoid transit peptide plays the same role as chaperonin and contributes to facilitating the expression and localization of NdhB subunit.

Plant-made dengue virus-like particles produced by co-expression of structural and non-structural proteins induce a humoral immune response in mice.

SummaryDengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus‐like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co‐expression of DENV structural proteins (SP) and a truncated version of the non‐structural proteins (NSPs), lacking NS5 that contains the RNA‐dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM‐E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant‐made DENV1‐SP + NSP VLPs led to a higher antibody response in mice compared with DENV‐E domain III displayed inside bluetongue virus core‐like particles and a DENV‐E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses.

Immunohistochemical expression of BRCA1 and BRCA2 in a cohort of Ugandan men with prostate cancer: an analytical cross-sectional study

BackgroundMutation of the tumour suppressor genes BRCA1 and BRCA2 is thought to cause early development of prostate cancer which has poor prognosis. The purpose of this study was to determine the expression of BRCA1/2 and correlate it with clinicopathological factors for patients with prostate cancer in uganda.MethodsRetrospectively, we used immunohistochemistry to evaluate the expression of BRCA1/2 antibodies in tissue blocks of 188 patients with prostate cancer who were diagnosed between January 2005 and December 2014 in the Department of Pathology, Makerere College of Health Sciences. The Chi-Square test was used to determine the association of the categorical variables, whereas t-test was used to compare groups of mean of the variables in the study.ResultsExpression of BRCA1 and BRCA2 was found in 26.1% and 22.9% cases, respectively. Co-expression of BRCA1 and BRCA2 was found in only 7.4%. Gleason score was associated with expression of BRCA1 and BRCA2 (P = 0.013, P = 0.041, respectively). Age was not associated with BRCA1 and BRCA2 expression; P = 0.543, P = 0.091, respectively. Likewise, PSA was not associated with BRCA1 and BRCA2 expression; P = 0.446, P = 0.399, respectively.ConclusionBRCA1 and BRCA2 proteins in this study were expressed more in cases with poorly differentiated prostate cancer than in cases with either well or moderately differentiated prostate cancer. Co-expression of BRCA1 and BRCA2 proteins in the same patient in our study was 3 times less than either BRCA1 or BRCA2 alone.

Identification and Reporting of Cell of Origin, Double-/Triple-Hit and Double Expressor Lymphoma in a Real-World Cohort of Diffuse Large B-Cell Lymphoma Patients

Background The distinction of diffuse large B-cell lymphoma (DLBCL) into cell-of-origin (COO) subgroups (germinal center B-cell-like [GCB] or activated B-cell-like [ABC]) based on gene expression profiling is associated with prognosis and has potential therapeutic implications to mitigate the worse outcome for patients with DLBCL. Other phenotypic and molecular/cytogenetic features such as concurrent translocations of oncogene MYC and BCL2 and/or BCL6 (so-called double-/triple-hit lymphoma, DHL/THL) and coexpression of MYC and BCL2 proteins (so-called double-expressor lymphoma, DEL) are also recognized to have great prognostic impact (Swerdlow SH et al. Blood 2016;127(20):2375-2390). This study investigated the prevalence of COO, DHL/THL and DEL in a real-world cohort of patients with DLBCL who had documented results of diagnostic testing. Methods This study used the Flatiron Health electronic health record-derived de-identified database to abstract information on patients diagnosed with DLBCL between 2011-2019. Information on diagnostic testing from immunohistochemistry (IHC) for expression of MYC, BCL2, BCL6, CD10 or MUM1, and from fluorescence in situ hybridization (FISH) or karyotype analysis for rearrangement of MYC, BCL2 or BCL6 was abstracted from pathology reports or clinical visit notes, where available. We calculated the proportions of COO subgroups (GCB vs. ABC) that were derived from IHC testing results according to Hans algorithm (Hans C et al., Blood 2004;103(1):275-282), DHL/THL, and DEL. We also examined concordance of COO classification derived from IHC testing results with that directly reported by the healthcare providers. Differences in patient characteristics between IHC testing results-derived COO subgroups (GCB vs. ABC) were assessed using chi-square tests. Results 4400 patients had documented results of IHC and 73% (n=3194) can be classified into either GCB or ABC DLBCL (GCB/ABC ratio of 1.38). 3205 patients had documented results of FISH or karyotype analysis and 8% (n=245) were DHL/THL; only 33 patients were DEL. Within the GCB DLBCL patients derived from IHC testing results (n=1854), 163 patients were DHL/THL and 11 were DEL, whereas 24 DHL/THL and 18 DEL were identified within the ABC type (n=1340). When comparing COO classification derived from IHC testing results (n=3194) with that directly reported by the healthcare providers (n=2765), additional 695 and 439 patients can be classified as GCB and ABC DLBCL by IHC, respectively (Table). Univariate analysis showed that patients who were non-White ethnic group, diagnosed in academic centers, with lower body mass index but elevated serum lactate dehydrogenase levels and worse ECOG performance status, and without transformation from a prior indolent lymphoid malignancy, were more likely to be associated with ABC DLBCL (for all variables, p&amp;lt;0.05). There were no clinically meaningful and/or statistically significant differences in IHC testing results-derived COO classification (GCB vs. ABC) by age, gender, year of DLBCL diagnosis, geographic location of residency, type of insurance plan, tumor group stage, documentation of extranodal site or any other primary cancer history at the time of diagnosis. Discussion In this large real-world DLBCL cohort, a lower-than-expected proportion of DEL patients were identified vs. the 20-35% reported in the literature (Karube K and Campo E. Hematology 2015;52(2):97-106). This is likely due to our cohort of patients requiring clear evidence of coexpression for MYC and BCL2 (≥40% and &amp;gt;50%, respectively) that are not related to underlying chromosomal rearrangements, and few pathologists reported levels of percent staining for IHC testing among those with documented positive results of MYC/BCL2 protein coexpression. In addition, results from this study showed that only half of cases had COO classification documented by healthcare providers, despite available IHC results. Although this study indicated lack of details in the reporting of diagnostic testing (e.g. COO identification, levels of percent staining, methods for DLBCL subgroup identification), findings should be interpreted with caution, as patients with DLBCL might have been tested but not documented in the electronic health record system or might have biomarker testing performed at sites outside of the Flatiron Health network. Disclosures Yang: F. Hoffmann-La Roche: Current Employment. Zhang:F. Hoffmann-La Roche: Current Employment. Xiao:F. Hoffmann-La Roche: Current Employment. Hammer:Roche: Consultancy, Honoraria, Research Funding; Caris Lifesciences: Honoraria; PER Med education: Honoraria; PathEdEx: Current equity holder in private company. Prime:F. Hoffmann-La Roche: Current Employment.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI &amp;lt; 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI &amp;gt; 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Identifying a Potential Key Gene, TIMP1, Associated with Liver Metastases of Uveal Melanoma by Weight Gene Co-Expression Network Analysis.

PurposeUveal melanoma (UM) is a primary intraocular tumor in adults, with a high percentage of metastases to the liver. Identifying potential key genes may provide information for early detection and prognosis of UM metastasis.Patients and MethodsDifferentially expressed genes (DEGs) were identified using the GSE22138 dataset. Weighted gene co-expression network analysis was used to construct co-expression modules. Functional enrichment analysis was performed for DEGs and genes of key modules. Hub genes were screened by co-expression network and protein–protein interaction network (PPI), and validated by survival analysis in The Cancer Genome Atlas database. Gene set enrichment analysis (GSEA) was used to explore the potential metastasis mechanism of UM. Transient transfection was used to investigate the effect of TIMP1 on the proliferation, migration, and invasion of UM cells.ResultsIn total, 552 DEGs were identified between primary and metastatic UM and mainly enriched in extracellular matrix, cellular senescence and focal adhesion pathway. A weighted gene co‑expression network was built to identify key gene modules associated with UM metastasis (n=36). The turquoise module is positively correlated with metastasis and genes in this module were mainly enriched in peptidyl-tyrosine autophosphorylation and regulation of organ growth. The hub gene TIMP1 was screened out by co-expression network and PPI analysis. High expression of TIMP1 was related to p53 pathway by GSEA and short overall survival time. Experimental results indicated that overexpression of TIMP1 inhibited the proliferation and migration, while it had no significant effect on invasion of UM cells.ConclusionOur study indicates that TIMP1 might be associated with metastasis in UM, which might have important significance for identifying patients with high risk of metastasis and predicting the prognosis of UM.

Abstract PO-084: Protein marker heterogeneity in breast cancer subtypes measured using immunofluorescence protein multiplexing and quantitative, single cell image analysis

Abstract The extent of intra-tumoral heterogeneity - the variation in the composition of cells in a given tumor and those in the local tumor microenvironment - could have potential impact on diagnosis, treatment planning and subsequent response to treatment. To evaluate the extent of cancer cellular heterogeneity, we conducted quantitative protein marker multiplex imaging to study the variations in protein marker expression patterns on individual cells and spatial localizations. A multiplexed immunofluorescence imaging platform (MxIF, Cell DIVE) was used to measure the cellular expression of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor 2 (HER2), Ki67, p53, p21WAF1 and p16INK4A in cancer epithelium. Analysis was conducted on a tissue microarray (TMA) representing subtypes classified as Luminal A-like, Luminal B-like (HER2-negative), Luminal B-like (HER2-positive), HER2-positive (non-luminal) or Triple-negative based on tumor grade and immune activity according to the St. Gallen surrogate classification. Of the 101 cores from 59 cases studied, high levels of heterogeneity were observed in ER and PR expression among the hormonal receptor-positive tumors. As expected Luminal A-like cancers exhibited higher proportions of individual cells co-expressing ER and PR, while cells in Luminal B-like, HER2-negative cancers showed ER expression only. Luminal B-like, HER2-positive cores were composed of cells with strong HER2 staining, and some cells co-expressing PR and HER2. Single cells with strong ER and HER2 labelling were rarely observed. Spatial visualizations illustrated that cells with similar expression signatures tend to be clustered together. Among cases which showed p53 overexpression with immunohistochemistry, the overall MxIF-measured p53 level was highest in TNBC compared to HER2+ and Luminal B-like cases. TNBC exhibited the highest proliferative fraction and most incidence of abnormal p53 and p16. We did not observe an association of p21 expression to P53 or P16 patterns, yet a slightly higher proportion of Luminal B-like cancers showed increased P21 levels compared to the other subtypes. Our study demonstrated the application of protein marker multiplexing and quantitative image analysis in measuring heterogeneity of protein co-expression signatures within breast cancer subtypes. Our next step is to apply the methods developed here to study a cohort where molecular profiling and radiomics were conducted (Bayani et al.) to reveal the extent of heterogeneity of breast cancer with a multi-omics approach. Citation Format: Alison M. Cheung, Dan Wang, Kela Liu, Fiona Ginty, Sharon Nofech-Mozes, Jane Bayani, John M.S. Bartlett, Anne Martel, Martin J. Yaffe. Protein marker heterogeneity in breast cancer subtypes measured using immunofluorescence protein multiplexing and quantitative, single cell image analysis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-084.

Swath-MS Co-Expression Profiles Reveal Paralogue Interference in Protein Complex Evolution

Understanding the conservation and evolution of protein complexes is of critical value to decode their function in physiological and pathological processes. One prominent proposal posits gene duplication as a potential mechanism for protein complex evolution. In this study we take advantage of large-scale proteome expression datasets to systematically investigate the role of paralogues, and specifically self-interacting paralogues, in shaping the evolutionary trajectories of protein complexes. First, we show that protein co-expression derived from quantitative proteomic matrices is a good indicator for complex membership and is conserved across species. Second, we suggest that paralogues are commonly strongly co-expressed and that for the subset of paralogues that show diverging co-expression patterns, the divergent co-expression patterns reflect both sequence and functional divergence. Finally, on this basis, we show that homomeric paralogues known to be part of protein complexes display a unique co-expression pattern distribution, with a subset of them being highly diverging. These findings support the idea that homomeric paralogues can avoid cross-interference by diversifying their expression patterns, and corroborates the role of this mechanism as a force shaping protein complex evolution and specialization.

Co-expression, purification and bioassay of three avian viral antigens

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.