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All sources NASA NTRS DOE OSTI

DOE OSTI2021

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 ( pfhrp2/3 ) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp 2/3 deletions. Samples were assayed for HRP2, pan- Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2011

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗

DOE OSTI2021

Discovery and characterization of bromodomain 2–specific inhibitors of BRDT

Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure–activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1991

Methods development for total organic carbon accountability

This report describes the efforts completed during the contract period beginning November 1, 1990 and ending April 30, 1991. Samples of product hygiene and potable water from WRT 3A were supplied by NASA/MSFC prior to contract award on July 24, 1990. Humidity condensate samples were supplied on August 3, 1990. During the course of this contract chemical analyses were performed on these samples to qualitatively determine specific components comprising, the measured organic carbon concentration. In addition, these samples and known standard solutions were used to identify and develop methodology useful to future comprehensive characterization of similar samples. Standard analyses including pH, conductivity, and total organic carbon (TOC) were conducted. Colorimetric and enzyme linked assays for total protein, bile acid, B-hydroxybutyric acid, methylene blue active substances (MBAS), urea nitrogen, ammonia, and glucose were also performed. Gas chromatographic procedures for non-volatile fatty acids and EPA priority pollutants were also performed. Liquid chromatography was used to screen for non-volatile, water soluble compounds not amenable to GC techniques. Methods development efforts were initiated to separate and quantitate certain chemical classes not classically analyzed in water and wastewater samples. These included carbohydrates, organic acids, and amino acids. Finally, efforts were initiated to identify useful concentration techniques to enhance detection limits and recovery of non-volatile, water soluble compounds.

Benson, Brian L.↗

NASA NTRS2000

Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Karr, Laurel↗

DOE OSTI2023

Identification of and Structural Insights into Hit Compounds Targeting N -Myristoyltransferase for Cryptosporidium Drug Development

Each year, approximately 50,000 children under 5 die as a result of diarrhea caused by Cryptosporidium parvum, a protozoan parasite. There are currently no effective drugs or vaccines available to cure or prevent Cryptosporidium infection, and there are limited tools for identifying and validating targets for drug or vaccine development. We previously reported a high throughput screening (HTS) of a large compound library against Plasmodium N-myristoyltransferase (NMT), a validated drug target in multiple protozoan parasite species. To identify molecules that could be effective against Cryptosporidium, we counter-screened hits from the Plasmodium NMT HTS against Cryptosporidium NMT. We identified two potential hit compounds and validated them against CpNMT to determine if NMT might be an attractive drug target also for Cryptosporidium. We tested the compounds against Cryptosporidium using both cell-based and NMT enzymatic assays. We then determined the crystal structure of CpNMT bound to Myristoyl-Coenzyme A (MyrCoA) and structures of ternary complexes with MyrCoA and the hit compounds to identify the ligand binding modes. The binding site architectures display different conformational states in the presence of the two inhibitors and provide a basis for rational design of selective inhibitors.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

High-throughput virtual laboratory for drug discovery using massive datasets

Time-to-solution for structure-based screening of massive chemical databases for COVID-19 drug discovery has been decreased by an order of magnitude, and a virtual laboratory has been deployed at scale on up to 27,612 GPUs on the Summit supercomputer, allowing an average molecular docking of 19,028 compounds per second. Over one billion compounds were docked to two SARS-CoV-2 protein structures with full optimization of ligand position and 20 poses per docking, each in under 24 hours. GPU acceleration and high-throughput optimizations of the docking program produced 350× mean speedup over the CPU version (50× speedup per node). GPU acceleration of both feature calculation for machine-learning based scoring and distributed database queries reduced processing of the 2.4 TB output by orders of magnitude. The resulting 50× speedup for the full pipeline reduces an initial 43 day runtime to 21 hours per protein for providing high-scoring compounds to experimental collaborators for validation assays.

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2024

RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer

Chronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrP Sc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer. A total of 23 platelet samples derived from CWD-positive deer (ELISA +/IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10 -2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10 -3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels. Incorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component signifcantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Building a Computational and Experimental Rapid Response Pipeline to Counter the Coronavirus Disease 2019 Outbreak and Emerging Biothreats

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs, especially where protective candidates can be rapidly identified or developed for emerging biothreats and escape variants. Current cutting-edge technologies for this purpose still rely on pathogen-exposed convalescent volunteers and a large screening effort to find a proverbial needle in a haystack. Computational design of protective antibodies based on pre-existing templates skips those requirements and allows for greater control over the breadth and target epitope, while also co-optimizing for potency and developability or other biophysical characteristics. We approached this problem by building and expanding an in vitro experimental rapid antibody production and characterization pipeline to support development of an autonomous, closed loop, active learning software system based on structural simulation and ground truth experimental data to design and evaluate antibody antigen interactions. Starting from early in the pandemic, we targeted SARS-CoV-2, the causative agent of COVID-19, by re-purposing neutralizing antibodies against SARS-CoV-1 that had been identified in the wake of that outbreak in the early 2000’s. We successfully re-targeted three different anti-SARS-CoV-1 antibodies to neutralize SARS-CoV-2 in vitro, where the antibodies were generated externally and tested through conventional binding and neutralization assays internally or with collaborators. As antibodies were identified from the blood of humans infected with SARS-CoV-2, we shifted to collaborate with academic partners to develop improved versions of their human-derived antibodies. This work reached its most important stage in rapid response to the emergence of the Omicron variant of concern (VOC) in late 2021. In a matter of weeks, enabled by on demand innovation to our screening pipeline, we computationally designed and experimentally characterized derivative antibodies of COV2-2130, one of two antibodies from Vanderbilt that form the basis of the AstraZeneca Evusheld prophylactic drug product. This drug product suffers a serious loss of efficacy against Omicron BA.1 and BA.1.1, the first Omicron strains. Due to tight integration of computational design and experimental evaluation, we were able to identify a pair of designs with potent neutralization of the main targets Omicron BA.1 and BA.1.1; but also the earlier Delta variant, and subsequent Omicron strains including BA.2, BA.4, BA.5, and BA.2.75, demonstrating that our multi-target design process can, by its nature, produce robust antibody designs that strictly improve over the parental antibody. These results, recognized by a 2022 Director’s Science and Technology award, have enabled the follow-on GUIDE program, to commence in FY23. While earlier design campaigns were substantially outsourced, we have engineered better and faster processes internally to better compliment, calibrate, and speed computational designs. As part of the follow-on GUIDE program, we will stand up a rapid and high-throughput antibody production and characterization facility staffed with the expertise and capabilities to foster our current collaboration across PLS and ENG as well as other partnerships toward computational design of biologics.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Imaging of multiple fluorescent proteins in canopies enables synthetic biology in plants

Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields. Genetically encoded fluorescent proteins (FPs) have revolutionized plant biology paradigms in gene expression, protein trafficking and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 25 years ago, modern phenomics has largely ignored fluorescence as a transgene expression device despite the burgeoning FP colour palette available to plant biologists. Here, we show a new platform for stand-off imaging of plant canopies expressing a wide variety of FP genes. The platform-the fluorescence-inducing laser projector (FILP)-uses an ultra-low-noise camera to image a scene illuminated by compact diode lasers of various colours, coupled with emission filters to resolve individual FPs, to phenotype transgenic plants expressing FP genes. Each of the 20 FPs screened in plants were imaged at >3 m using FILP in a laboratory-based laser range. We also show that pairs of co-expressed fluorescence proteins can be imaged in canopies. The FILP system enabled a rapid synthetic promoter screen: starting from 2000 synthetic promoters transfected into protoplasts to FILP-imaged agroinfiltrated Nicotiana benthamiana plants in a matter of weeks, which was useful to characterize a water stress-inducible synthetic promoter. FILP canopy imaging was also accomplished for stably transformed GFP potato and in a split-GFP assay, which illustrates the flexibility of the instrument for analysing fluorescence signals in plant canopies.

abiotic stress↗

DOE OSTI2025

Ultra-sensitive radon assay using an electrostatic chamber in a recirculating system

Rare event searches such as neutrinoless double beta decay and Weakly Interacting Massive Particle detection require ultra-low background detectors. Radon contamination is a significant challenge for these experiments, which employ highly sensitive radon assay techniques to identify and select low-emission materials. This work presents the development of ultra-sensitive electrostatic chamber (ESC) instruments designed to measure radon emanation in a recirculating gas loop, for future lower background experiments. Unlike traditional methods that separate emanation and detection steps, this system allows continuous radon transport and detection. This is made possible with a custom-built recirculation pump. A Python-based analysis framework, PyDAn, was developed to process and fit time-dependent radon decay data. Radon emanation rates are given for various materials measured with this instrument. A radon source of known activity provides an absolute calibration, enabling statistically-limited minimal detectable activities of 20 uBq. These devices are powerful tools for screening materials in the development of low-background particle physics experiments.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2023

Integrated data-driven and experimental approaches to accelerate lead optimization targeting SARS-CoV- 2 main protease

Identification of potential therapeutic candidates can be expedited by integrating computational modeling with domain aware machine learning (ML) approaches followed by experimental validation. Generative deep learning models have been recently developed that can generate thousands of new candidates, but their physiochemical properties are typically not optimized. Using our deep learning models and a scaffold as a starting point, we generated tens of thousands of compounds for SARS-CoV-2 M pro that preserve the core scaffold. Here we utilized and implemented several computational tools such as structural alert and toxicity analysis, high throughput virtual screening, ML-based 3D quantitative structure–activity relationships, multi-parameter optimization, and graph neural networks on libraries of generated candidates to predict biological activity and binding affinity a priori. From these collective computational results, eight promising candidates were identified and tested experimentally using Native Mass Spectrometry (MS) and FRET-based functional assays. Two compounds, with quinazoline-2-thiol and acetylpiperidine core moiety showed IC 50 values in the low micromolar range: 2.95±0.0017 µM and 3.41±0.0015 µM, respectively. The molecular dynamics simulations further highlight that binding of these compounds results in allosteric modulations in the chain B and the interface domains of the M pro . The key fragments from these top hits can be used as input for closed loop lead optimization in the integrated pipeline.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Systematic review of type 1 diabetes biomarkers reveals regulation in circulating proteins related to complement, lipid metabolism, and immune response

Type 1 diabetes (T1D) results from an autoimmune attack of the pancreatic β cells that progresses to dysglycemia and symptomatic hyperglycemia. Current biomarkers to track this evolution are limited, with development of islet autoantibodies marking the onset of autoimmunity and metabolic tests used to detect dysglycemia. Therefore, additional biomarkers are needed to better track disease initiation and progression. Multiple clinical studies have used proteomics to identify biomarker candidates. However, most of the studies were limited to the initial candidate identification, which needs to be further validated and have assays developed for clinical use. Here we curate these studies to help prioritize biomarker candidates for validation studies and to obtain a broader view of processes regulated during disease development. This systematic review was registered with Open Science Framework (https://doi.org/10.17605/OSF.IO/N8TSA). Using PRISMA guidelines, we conducted a systematic search of proteomics studies of T1D in the PubMed to identify putative protein biomarkers of the disease. Studies that performed mass spectrometry-based untargeted/targeted proteomic analysis of human serum/plasma of control, pre-seroconversion, post-seroconversion, and/or T1D-diagnosed subjects were included. For unbiased screening, 3 reviewers screened all the articles independently using the pre-determined criteria. A total of 13 studies met our inclusion criteria, resulting in the identification of 266 unique proteins, with 31 (11.6%) being identified across 3 or more studies. The circulating protein biomarkers were found to be enriched in complement, lipid metabolism, and immune response pathways, all of which are found to be dysregulated in different phases of T1D development. We found 2 subsets: 17 proteins (C3, C1R, C8G, C4B, IBP2, IBP3, ITIH1, ITIH2, BTD, APOE, TETN, C1S, C6A3, SAA4, ALS, SEPP1 and PI16) and 3 proteins (C3, CLUS and C4A) have consistent regulation in at least 2 independent studies at post-seroconversion and post-diagnosis compared to controls, respectively, making them strong candidates for clinical assay development. Biomarkers analyzed in this systematic review highlight alterations in specific biological processes in T1D, including complement, lipid metabolism, and immune response pathways, and may have potential for further use in the clinic as prognostic or diagnostic assays.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2000

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗

DOE OSTI2020

Antibody dynamics to SARS‐CoV‐2 in asymptomatic COVID‐19 infections

Abstract Background The missing asymptomatic COVID‐19 infections have been overlooked because of the imperfect sensitivity of the nucleic acid testing (NAT). Globally understanding the humoral immunity in asymptomatic carriers will provide scientific knowledge for developing serological tests, improving early identification, and implementing more rational control strategies against the pandemic. Measure Utilizing both NAT and commercial kits for serum IgM and IgG antibodies, we extensively screened 11 766 epidemiologically suspected individuals on enrollment and 63 asymptomatic individuals were detected and recruited. Sixty‐three healthy individuals and 51 mild patients without any preexisting conditions were set as controls. Serum IgM and IgG profiles were further probed using a SARS‐CoV‐2 proteome microarray, and neutralizing antibody was detected by a pseudotyped virus neutralization assay system. The dynamics of antibodies were analyzed with exposure time or symptoms onset. Results A combination test of NAT and serological testing for IgM antibody discovered 55.5% of the total of 63 asymptomatic infections, which significantly raises the detection sensitivity when compared with the NAT alone (19%). Serum proteome microarray analysis demonstrated that asymptomatics mainly produced IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS‐CoV‐2. Different from strong and persistent N‐specific antibodies, S1‐specific IgM responses, which evolved in asymptomatic individuals as early as the seventh day after exposure, peaked on days from 17 days to 25 days, and then disappeared in two months, might be used as an early diagnostic biomarker. 11.8% (6/51) mild patients and 38.1% (24/63) asymptomatic individuals did not produce neutralizing antibody. In particular, neutralizing antibody in asymptomatics gradually vanished in two months. Conclusion Our findings might have important implications for the definition of asymptomatic COVID‐19 infections, diagnosis, serological survey, public health, and immunization strategies.

Lei, Qing↗

NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗

NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel↗

DOE OSTI2020

Discovery of a Natural Product That Binds to the Mycobacterium tuberculosis Protein Rv1466 Using Native Mass Spectrometry

Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound’s cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 μge/μL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 μM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis .

59 BASIC BIOLOGICAL SCIENCES↗