ABSTRACT
Bacteriophage ÏX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ÏX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ÏX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ÏX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ÏX174 replication; moreover, their absence has no discernible effect on ÏX174 fecundity. These results suggest IbpA/B upregulation is a response to ÏX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Virus Replication , Escherichia coli/virology , Escherichia coli/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Bacteriophage phi X 174/genetics , Bacteriophage phi X 174/physiology , Bacteriophage phi X 174/metabolism , Heat-Shock Proteins, Small/metabolism , Heat-Shock Proteins, Small/genetics , Heat-Shock ProteinsABSTRACT
Biofluids such as blood plasma are rich reservoirs of potential biomarkers for disease diagnosis, prognosis, and prediction of treatment response. However, mass spectrometry analysis of circulating plasma proteins remains challenging. The introduction of data-independent acquisition mass spectrometry (DIA-MS) is an important step toward addressing detection of less abundant plasma proteins. Numerous plasma peptide MS/MS spectral library datasets produced from extensive plasma fractionation are accessible from public archives, and these can be repurposed as spectral reference libraries to increase the depth of proteomic analysis when DIA-MS is used. Here we describe the workflow that relies on reusing the existing spectral reference libraries by populating them with locally obtained peptide MS/MS data acquired by DIA-MS. This approach was demonstrated effectively to identify putative plasma biomarkers of response to neoadjuvant chemotherapy in the setting of pancreatic ductal adenocarcinoma (PDAC) (O'Rourke et al., J Proteomics 231:103998, 2021).
Subject(s)
Biomarkers, Tumor , Pancreatic Neoplasms , Proteomics , Tandem Mass Spectrometry , Humans , Biomarkers, Tumor/blood , Proteomics/methods , Tandem Mass Spectrometry/methods , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/blood , Peptide Library , Carcinoma, Pancreatic Ductal/blood , Carcinoma, Pancreatic Ductal/drug therapy , Blood Proteins/analysis , Blood Proteins/metabolism , Neoadjuvant Therapy/methodsABSTRACT
AIMS: Therapeutic drug monitoring (TDM) has led to significant improvements in individualized medical care, although its implementation in oncology has been limited to date. Tyrosine kinase inhibitors (TKIs) are a group of therapies for which TDM has been suggested. Osimertinib is one such therapy used in the treatment of epidermal growth factor receptor (EGFR) mutation-driven lung cancer. Herein, we describe a prospective pilot study involving 21 patients on osimertinib primarily as a preliminary evaluation of drug levels in a real-world setting. METHODS: Concentrations of the drug and its primary metabolites were measured with a validated liquid chromatography-mass spectrometry (LC-MS) assay across serial timepoints. As part of this study, inter-individual variability by dose and ethnicity as well as intra-individual variability across timepoints are explored. Furthermore, we attempted to validate dried blood spot (DBS)-based quantitation as an accurate alternative to plasma quantitation. RESULTS: Successful quantitation of osimertinib and primary metabolites was achieved for our subjects. Compound plasma levels were highly correlated to DBS levels. There was no significant difference in concentrations with ethnicity or dosing or intra-individual variability across timepoints. CONCLUSIONS: As such, we demonstrate that TDM for osimertinib is practical for future trials. We also validated the use of DBS as an alternative to conventional quantitation for exploration of TDM for osimertinib in larger trials and for other targeted therapies.
Subject(s)
Acrylamides , Aniline Compounds , Carcinoma, Non-Small-Cell Lung , Dried Blood Spot Testing , Drug Monitoring , ErbB Receptors , Lung Neoplasms , Mutation , Protein Kinase Inhibitors , Humans , Aniline Compounds/blood , Aniline Compounds/therapeutic use , Aniline Compounds/pharmacokinetics , Acrylamides/blood , Acrylamides/therapeutic use , Pilot Projects , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/blood , Drug Monitoring/methods , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/blood , Dried Blood Spot Testing/methods , ErbB Receptors/genetics , ErbB Receptors/antagonists & inhibitors , Male , Female , Middle Aged , Prospective Studies , Aged , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacokinetics , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Chromatography, Liquid/methods , Aged, 80 and over , Adult , Indoles , PyrimidinesABSTRACT
BACKGROUND: Triple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. METHODS: Immunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. RESULTS: Metabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. CONCLUSION: In this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.
Subject(s)
Autophagy , Cell Proliferation , Triple Negative Breast Neoplasms , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/metabolism , Autophagy/drug effects , Humans , Animals , Cell Line, Tumor , Female , Cell Proliferation/drug effects , Mice , Xenograft Model Antitumor Assays , Drug Resistance, Neoplasm/drug effects , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Cell Movement/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic useABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Late presentation of disease at the time of diagnosis is one of the major reasons for dismal prognostic outcomes for PDAC patients. Currently, there is a lack of clinical biomarkers, which can be used to diagnose PDAC patients at an early resectable stage. This study performed proteomic mass spectrometry to identify novel blood-based biomarkers for early diagnosis of PDAC. Serum specimens from 88 PDAC patients and 88 healthy controls (60 discovery cohort and 28 validation cohort) were analyzed using data independent acquisition high resolution mass spectrometry to identify candidate biomarker proteins. A total of 249 proteins were identified and quantified by the mass spectrometric analysis. Six proteins were markedly (>1.5 fold) and significantly (p < .05; q < 0.1) increased in PDAC patients compared to healthy controls in discovery cohort. Notably, four of these six proteins were significantly upregulated in an independent validation cohort. The top three upregulated proteins (i.e., Polymeric Immunoglobulin Receptor [PIGR], von Willebrand Factor [vWF], and Fibrinogen) were validated using enzyme linked immunosorbent assay, which led to selection of PIGR and vWF as a diagnostic biomarker panel for PDAC. The panel showed high ability to diagnose early stage (stage I and II) PDAC patients (area under the curve [AUC]: 0.8926), which was further improved after the addition of clinically used prognostic biomarker (Ca 19-9) to the panel (AUC: 0.9798). In conclusion, a novel serum protein biomarker panel for early diagnosis of PDAC was identified.
Subject(s)
Biomarkers, Tumor , Carcinoma, Pancreatic Ductal , Early Detection of Cancer , Pancreatic Neoplasms , Proteomics , Humans , Biomarkers, Tumor/blood , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/blood , Female , Male , Early Detection of Cancer/methods , Carcinoma, Pancreatic Ductal/blood , Carcinoma, Pancreatic Ductal/diagnosis , Middle Aged , Aged , Proteomics/methods , Receptors, Polymeric Immunoglobulin/blood , von Willebrand Factor/analysis , von Willebrand Factor/metabolism , Fibrinogen/analysis , Fibrinogen/metabolism , Case-Control Studies , Adult , Blood Proteins/analysisABSTRACT
BACKGROUND: Osimertinib is an oral small-molecule tyrosine kinase receptor inhibitor used to treat non-small cell lung cancer (NSCLC) with a sensitizing epidermal growth factor receptor mutation. Patients may experience drug toxicity and require dose deescalation. The study aimed to quantitate osimertinib and its 2 active metabolites, AZ5104 and AZ7550, in microsampled dried blood spots (DBS) collected from patients with NSCLC using a hemaPEN device and compare them with plasma drug levels. METHODS: A 6-min ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated using plasma and DBS. The accuracy, selectivity, matrix effect, recovery, and stability were assessed using bioanalytical validation criteria. The hematocrit effect was investigated in DBS. Drug levels were measured in 15 patients with NSCLC, and the Bland-Altman method was used to compare measurements between plasma and DBS. RESULTS: The validated assay determined accurate and precise quantities, respectively, for osimertinib in both plasma (93.2%-99.3%; 0.2%-2.3%) and DBS (96.7%-99.6%; 0.5%-10.3%) over a concentration of 1-729 ng/mL. The osimertinib metabolites, AZ5104 and AZ7550, were similarly validated in accordance with bioanalytical guidelines. For 30%-60% patient hematocrit, no hematocrit bias was observed with DBS for all analytes. The Bland-Altman method showed high concordance between plasma and DBS analyte levels. Stability experiments revealed that osimertinib and its metabolites were poorly stable in plasma at room temperature, whereas all analytes were stable in DBS for 10 days at room temperature. CONCLUSIONS: The measurement of osimertinib, AZ5104, and AZ7550 from hemaPEN microsampled DBS is a convenient and reliable approach for therapeutic drug monitoring that produces measurements consistent with plasma drug levels.
Subject(s)
Acrylamides , Aniline Compounds , Carcinoma, Non-Small-Cell Lung , Dried Blood Spot Testing , Lung Neoplasms , Tandem Mass Spectrometry , Humans , Aniline Compounds/blood , Dried Blood Spot Testing/methods , Acrylamides/blood , Tandem Mass Spectrometry/methods , Lung Neoplasms/drug therapy , Lung Neoplasms/blood , Chromatography, High Pressure Liquid/methods , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/blood , Drug Monitoring/methods , Reproducibility of Results , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/pharmacokinetics , Indoles , PyrimidinesABSTRACT
BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.