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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

The POINTER Imaging baseline cohort: Associations between multimodal neuroimaging biomarkers, cardiovascular health, and cognition

Abstract INTRODUCTION The U.S. Study to Protect Brain Health Through Lifestyle Intervention to Reduce Risk (U.S. POINTER) is evaluating lifestyle interventions in older adults at risk for cognitive decline and dementia. Here we characterize the baseline data set of the POINTER Imaging ancillary study. METHODS Participants underwent health and cognitive assessments and neuroimaging with multimodal positron emission tomography (PET) (beta‐amyloid [Aβ] and tau) and magnetic resonance imaging (MRI). Framingham risk score (FRS) was used to quantify cardiovascular disease (CVD) risk. RESULTS A total of 1052 participants (31% from underrepresented ethnoracial groups) were enrolled. Compared to Aβ−, Aβ+ (29%) participants were older, had higher apolipoprotein E (APOE) ε4 carriage rate and white matter hyperintensity volume, and greater temporal tau. FRS was related to MRI measures, but not AD biomarkers. FRS and tau had independent effects on cognition. DISCUSSION In this heterogenous, at‐risk cohort, CVD risk was related to more abnormal brain structure and poorer cognition, representing a putative non‐AD (Alzheimer's disease) pathway to brain injury and cognitive decline. Highlights The U.S. Study to Protect Brain Health Through Lifestyle Intervention to Reduce Risk (U.S. POINTER) cohort is enriched for cardiovascular disease (CVD) and poor lifestyle POINTER Imaging collected multimodal neuroimaging data in this unique, at‐risk cohort Amyloid burden was related to age, apolipoprotein E (APOE) ε4 carriage, and measures of disease progression Associations between amyloid and tau, and tau and cognition, were relatively weak CVD risk and tau pathology were independently related to memory

Neurosciences & Neurology↗
DOE OSTI2020

Benchmark Dose Analysis of DNA Damage Biomarker Responses Provides Compound Potency and Adverse Outcome Pathway Information for the Topoisomerase II Inhibitor Class of Compounds

Genetic toxicology data have traditionally been utilized for hazard identification to provide a binary call for a compound's risk. Recent advances in the scientific field, especially with the development of high‐throughput methods to quantify DNA damage, have influenced a change of approach in genotoxicity assessment. The in vitro MultiFlow® DNA Damage Assay is one such method which multiplexes γH2AX, p53, phospho‐histone H3 biomarkers into a single‐flow cytometric analysis (Bryce et al., [2016]: Environ Mol Mutagen 57:546–558). This assay was used to study human TK6 cells exposed to each of eight topoisomerase II poisons for 4 and 24 hr. Using PROAST v65.5, the Benchmark Dose approach was applied to the resulting flow cytometric datasets. With “compound” serving as covariate, all eight compounds were combined into a single analysis, per time point and endpoint. The resulting 90% confidence intervals, plotted in Log scale, were considered as the potency rank for the eight compounds. The in vitro MultiFlow data showed a maximum confidence interval span of 1Log, which indicates data of good quality. Patterns observed in the compound potency rank were scrutinized by using the expert rule‐based software program Derek Nexus, developed by Lhasa Limited. Compound sub‐classification and structural alerts were considered contributory to the potencies observed for the topoisomerase II poisons studied herein. The Topo II poison Adverse Outcome Pathway was evaluated with MultiFlow endpoints serving as Key Events. The step‐wise approach described herein can be considered as a foundation for risk assessment of compounds within a specific mode of action of interest. Environ. Mol. Mutagen. 2020. © 2020 Wiley Periodicals, Inc.

Wheeldon, Ryan P.↗
DOE OSTI2020

Detection of a multi–disease biomarker in saliva with graphene field effect transistors

Human carbonic anhydrase 1 (CA1) has been suggested as a biomarker for identification of several diseases including cancers, pancreatitis, diabetes, and Sjogren’s syndrome. However, the lack of a rapid, cheap, accurate, and easy-to-use quantification technique has prevented widespread utilization of CA1 for practical clinical applications. To this end, we present a label-free electronic biosensor for detection of CA1 utilizing highly sensitive graphene field effect transistors (G-FETs) as a transducer and specific RNA aptamers as a probe. The binding of CA1 with aptamers resulted in a positive shift in Dirac voltage V D of the G-FETs, the magnitude of which depended on target concentration. These aptameric G-FET biosensors showed the binding affinity (K D ) of ~2.3 ng/ml (70 pM), which is four orders lower than that reported using a gel shift assay. This lower value of K D enabled us to achieve a detection range (10 pg/ml - 100 ng/ml) which is well in line with the clinically relevant range. These highly sensitive devices allowed us to further prove their clinical relevance by successfully detecting the presence of CA1 in human saliva samples. In conclusion, the utilization of this label-free biosensor could facilitate the early stage identification of various diseases associated with changes in concentration of CAs.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2020

Mechanistic Models as Framework for Understanding Biomarker Disposition: Prediction of Creatinine‐Drug Interactions

Creatinine is widely used as a biomarker of glomerular filtration, and, hence, renal function. However, transporter‐mediated secretion also contributes to its renal clearance, albeit to a lesser degree. Inhibition of these transporters causes transient serum creatinine elevation, which can be mistaken as impaired renal function. The current study developed mechanistic models of creatinine kinetics within physiologically based framework accounting for multiple transporters involved in creatinine renal elimination, assuming either unidirectional or bidirectional‐OCT2 transport (driven by electrochemical gradient). Robustness of creatinine models was assessed by predicting creatinine‐drug interactions with 10 perpetrators; performance evaluation accounted for 5% intra‐individual variability in serum creatinine. Models showed comparable predictive performances of the maximum steady‐state effect regardless of OCT2 directionality assumptions. However, only the bidirectional‐OCT2 model successfully predicted the minimal effect of ranitidine. The dynamic nature of models provides clear advantage to static approaches and most advanced framework for evaluating interplay between multiple processes in creatinine renal disposition.

Scotcher, Daniel↗
DOE OSTI2023

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis

Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producingEscherichia coli(STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent results have shown that HNF4a defective function and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein an integrative analysis of hepatic RNAseq and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to identify plasma protein signatures for mild and severe AH patients. Alcohol-related liver disease cirrhosis (ALD)(AC), non-alcoholic fatty liver disease (NALFD), and healthy subjects (HC) were used as comparator groups. Identified proteins primarily involved in hepatocellular function were decreased in AH patients which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified including thrombin (THRB), hepatocyte growth factor alpha (HGFA), clusterin (CLUS), human serum factor H-related protein (FHR1) and kallistatin (KAIN), which exhibited large abundance shifts between severe and non-severe AH. The combination of THRB and HGFA discriminated between severe and non-severe AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Long-Term Outcomes of Radical Radiation Therapy with Hypoxia Modification with Biomarker Discovery for Stratification: 10-Year Update of the BCON (Bladder Carbogen Nicotinamide) Phase 3 Randomized Trial (ISRCTN45938399)

Many muscle-invasive bladder cancers are hypoxic, which limits the efficacy of radiation therapy. Hypoxia modification using carbogen and nicotinamide has been tested in a phase 3 trial, Bladder Carbogen Nicotinamide. We present mature follow-up data with biomarker predictions of outcomes.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2023

Identification of blood protein biomarkers associated with prostate cancer risk using genetic prediction models: analysis of over 140,000 subjects

Prostate cancer (PCa) brings huge public health burden in men. A growing number of conventional observational studies report associations of multiple circulating proteins with PCa risk. However, the existing findings may be subject to incoherent biases of conventional epidemiologic studies. To better characterize their associations, herein, we evaluated associations of genetically predicted concentrations of plasma proteins with PCa risk. In this study, we developed comprehensive genetic prediction models for protein levels in plasma. After testing 1308 proteins in 79 194 cases and 61 112 controls of European ancestry included in the consortia of BPC3, CAPS, CRUK, PEGASUS, and PRACTICAL, 24 proteins showed significant associations with PCa risk, including 16 previously reported proteins and eight novel proteins. Of them, 14 proteins showed negative associations and 10 showed positive associations with PCa risk. For 18 of the identified proteins, potential functional somatic changes of encoding genes were detected in PCa patients in The Cancer Genome Atlas (TCGA). Genes encoding these proteins were significantly involved in cancer-related pathways. We further identified drugs targeting the identified proteins, which may serve as candidates for drug repurposing for treating PCa. In conclusion, this study identifies novel protein biomarker candidates for PCa risk, which may provide new perspectives on the etiology of PCa and improve its therapeutic strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Normalization of CSF pTau measurement by Aβ 40 improves its performance as a biomarker of Alzheimer’s disease

Alzheimer’s disease (AD)-related tauopathy can be measured with CSF phosphorylated tau (pTau) and tau PET. We aim to investigate the associations between these measurements and their relative ability to predict subsequent disease progression. In 219 cognitively unimpaired and 122 impaired Alzheimer’s Disease Neuroimaging Initiative participants with concurrent amyloid-β (Aβ) PET ( 18 F-florbetapir or 18 F-florbetaben), 18 F-flortaucipir (FTP) PET, CSF measurements, structural MRI, and cognition, we examined inter-relationships between these biomarkers and their predictions of subsequent FTP and cognition changes. The use of a CSF pTau/Aβ 40 ratio eliminated positive associations we observed between CSF pTau alone and CSF Aβ 42 in the normal Aβ range likely reflecting individual differences in CSF production rather than pathology. Use of the CSF pTau/Aβ 40 ratio also increased expected associations with Aβ PET, FTP PET, hippocampal volume, and cognitive decline compared to pTau alone. In Aβ+ individuals, abnormal CSF pTau/Aβ 40 only individuals (26.7%) were 4 times more prevalent (p < 0.001) than abnormal FTP only individuals (6.8%). Furthermore, among individuals on the AD pathway, CSF pTau/Aβ 40 mediates the association between Aβ PET and FTP PET accumulation, but FTP PET is more closely linked to subsequent cognitive decline than CSF pTau/Aβ 40 . Together, these findings suggest that CSF pTau/Aβ 40 may be a superior measure of tauopathy compared to CSF pTau alone, and CSF pTau/Aβ 40 enables detection of tau accumulation at an earlier stage than FTP among Aβ+ individuals.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

The influence of spaceflight on the astronaut salivary microbiome and the search for a microbiome biomarker for viral reactivation

Background: Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. Results: Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella , Megasphera , and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria , Absconditabacteria , and Abiotrophia and a negative correlation between Oribacterium , Veillonella , and Haemophilus . There was also a significant positive correlation between microbiome richness and EBV viral titers. Conclusions: This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

In search of autophagy biomarkers in breast cancer: Receptor status and drug agnostic transcriptional changes during autophagy flux in cell lines

Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER + Patients (RSF ER + ). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa . Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Peripheral Blood Transcript Signatures after Internal 131 I-mIBG Therapy in Relapsed and Refractory Neuroblastoma Patients Identifies Early and Late Biomarkers of Internal 131 I Exposures

131 I-metaiodobenzylguanidine ( 131 I-mIBG) is a targeted radiation therapy developed for the treatment of advanced neuroblastoma. We have previously shown that this patient cohort can be used to predict absorbed dose associated with early 131 I exposure, 72 h after treatment. We now expand these studies to identify gene expression differences associated with 131 I-mIBG exposure 15 days after treatment. Total RNA from peripheral blood lymphocytes was isolated from 288 whole blood samples representing 59 relapsed or refractory neuroblastoma patients before and after 131 I-mIBG treatment. We found that several transcripts predictive of early exposure returned to baseline levels by day 15, however, selected transcripts did not return to baseline. At 72 h, all 17 selected pathway-specific transcripts were differentially expressed. Transcripts CDKN1A (P < 0.000001), FDXR (P < 0.000001), DDB2 (P < 0.000001), and BBC3 (P < 0.000001) showed the highest up-regulation at 72 h after 131 I-mIBG exposure, with mean log2 fold changes of 2.55, 2.93, 1.86 and 1.85, respectively. At day 15 after 131I-mIBG, 11 of the 17 selected transcripts were differentially expressed, with XPC, STAT5B, PRKDC, MDM2, POLH, IGF1R, and SGK1 displaying significant up-regulation at 72 h and significant down-regulation at day 15. Interestingly, transcripts FDXR (P = 0.01), DDB2 (P = 0.03), BCL2 (P = 0.003), and SESN1 (P < 0.0003) maintained differential expression 15 days after 131 I-mIBG treatment. These results suggest that transcript levels for DNA repair, apoptosis, and ionizing radiation-induced cellular stress are still changing by 15 days after 131 I-mIBG treatment. Our studies showcase the use of biodosimetry gene expression panels as predictive biomarkers following early (72 h) and late (15 days) internal 131 I exposure. Our findings also demonstrate the utility of our transcript panel to differentiate exposed from non-exposed individuals up to 15 days after exposure from internal 131 I.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2022

Quantile-Specific Heritability of Inflammatory and Oxidative Stress Biomarkers Linked to Cardiovascular Disease

Purpose: Heritability (h 2 , the proportion of the phenotypic variance attributable to additive genetic effects) is traditionally assumed to be constant throughout the distribution of the phenotype. However, the heritabilities of circulating C-reactive protein, interleukin-6, plasminogen activator inhibitor type-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) concentrations depend upon whether the phenotype is high or low relative to their distributions (quantile-dependent expressivity), which may account for apparent gene–environment interactions. Whether the heritabilities of other inflammatory biomarkers linked to cardiovascular disease are quantile-dependent remain to be determined. Patients and Methods: Quantile-specific offspring-parent (β OP ) and full-sib regression slopes (β FS ) were estimated by applying quantile regression to the age- and sex-adjusted phenotypes of families surveyed as part of the Framingham Heart Study. Quantile-specific heritabilities were calculated as: h 2 =2β OP /(1+r spouse ) and h 2 ={(1+8r spouse β FS ) 0.5 – 1}/(2r spouse ). Results: Heritability (h 2 ± SE) of lipoprotein-associated phospholipase A2 (Lp-PLA 2 ) mass concentrations increased from 0.11 ± 0.03 at the 10 th percentile, 0.08 ± 0.03 at the 25 th , 0.12 ± 0.03 at the 50 th , 0.20 ± 0.04 at the 75 th , and 0.26 ± 0.06 at the 90 th percentile, or 0.0023 ± 0.0006 per each one-percent increase in the phenotype distribution (P linear trend = 0.0004). Similarly, h 2 increased 0.0029 ± 0.0011 (P linear trend = 0.01) for sP-selectin, 0.0032 ± 0.0009 (P linear trend = 0.0001) for soluble intercellular adhesion molecule 1 (sICAM-1), and 0.0026 ± 0.0006 for tumor necrosis factor receptor 2 (TNFR2) (P linear trend = 5.0 × 10 -6 ) per each one-percent increase in their distributions when estimated from β OP . Osteoprotegerin and soluble ST2 heritability also increased significantly with increasing percentiles of their distributions when estimated from β FS . Lp-PLA 2 activity, CD40 ligand, TNFα, interleukin-18, and myeloperoxidase heritability showed no significant quantile-dependence. Conclusion: The heritabilities of circulating Lp-PLA 2 -mass, sP-selectin, sICAM-1, TNFR2, osteoprotegerin and soluble ST2 concentrations are quantile-dependent, which may contribute to purported genetic modulations of: 1) sP-selectin’s relationships to venous thrombosis, pulmonary hypertension, type 2 diabetes and atorvastatin treatment; 2) sICAM-I’s relationships to brain abscess and atorvastatin treatment; and 3) Lp-PLA 2 ’s relationships to myocardial infarction and preeclampsia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Elucidating algal-bacterial community interactions by tracking volatile biomarkers (Final Report)

This feasibility study project aimed to lay further groundwork for studying and understanding how microorganisms interact with each other at the molecular level using model algal-bacterial co-cultures. A better understanding of such interactions is critical to optimizing and operating biotic platforms for a number of applications, including bioenergy, algal bioproducts and agriculture, and carbon capture. Here we explored the feasibility of characterizing and following these interactions by identifying and tracking volatile metabolites and biomarkers in the complex gaseous headspace of such cultures. We set up an experimental platform using a co-culture of Phaeodactylum tricornutum and Marinobacter subspecies 3-2 as a model system, used solid-phase microextraction fibers for volatile collection at various time points throughout culture growth, and employed gas chromatography mass spectrometry-based instrumentation available at LLNL for compound identification and quantification. We found that it is feasible to determine volatile profiles from the growth of Phaeodactylum tricornutum and Marinobacter subspecies 3-2 using our methodology and monitor profile changes over time. We also observed that volatiles distinguishing between cultures of the individual species and the co-cultures could be detected. We expect that results of this project will feed into the ongoing LLNL’s DOE BER funded Biofuels Scientific Focus Area (SFA) on algal-bacterial interactions and pave the way for future expanded volatile studies supported by DOE. The approach and technologies developed here would also be applicable and transferable to other microbial communities under study, such as cyanobacteria, rhizosphere communities and biofilms.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Nanopore Activity Assays for Detection of Biomarker Protease Activity: Design and Testing of Substrates for Both Nanopore Sequencing and PCR-Based Detection Methods

The work performed in this project has demonstrated the ability to construct proteolytic enzyme substrates that are PCR and sequencing-readable reporter molecules. Specifically, the goal was to detect those reporter molecules via PCR and Oxford Nanopore Technologies MinION sequencing methods following exposure to the biomarker protease thrombin. The assay development focused on binding the constructed peptide-oligonucleotide chimera to immobilized streptavidin. The action of thrombin on the peptide portion of the molecule released the oligonucleotide for detection. Detection of protease activity was demonstrated in a concentration-dependent manner using MALDI-MS, RT-PCR and DNA sequencing. Additional steps to remove background release of reporter molecules during the assay was used to improve the difference in detected oligonucleotide reporter following protease activity. Additional steps in assay development will be to (1) test the assay in an appropriate matrix, (2) investigate detection using additional DNA sequencing platforms and (3) demonstrate multiplexed detection of multiple protease markers in a single reaction.

59 BASIC BIOLOGICAL SCIENCES↗