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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

To investigate the effects of stabilizing G-quadruplexes on Escherichia coli, cells were grown in the presence of the G-quadruplex stabilizing compound, NMM, and RNA-seq was performed. This was done for the control cell strain (MG1655 dtolC) as well as a strain with a decrease in a translation elongation factor (MG1655 dtolC tufA::kan). TPM values were used for differential gene expression analysis between each growth condition. All samples were grown and sequenced in triplicate.

Keck, James L.↗
DOE OSTI2024

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora

Abstract The oomycete Phytophthora palmivora infects the fruit of cacao trees ( Theobroma cacao ) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora , yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue- P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis -regulatory modules likely to be involved in cacao defense against P. palmivora . This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora -resistant cacao.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Adipose-specific knockout of Protein Kinase D1 suppresses de novo lipogenesis in mice via SREBP1c-dependent signaling

Highlights: • Protein Kinase D1 is a functional regulator in adipocyte and white adipose tissue. • PKD1 drives de novo lipogenesis in adipocyte. • SREBP1c signaling regulates lipogenesis in adipocyte. • PKD1-mediated de novo lipogenesis relies on SREBP1c. Having healthy adipose tissue is essential for metabolic health, as excessive adipose tissue in the body can cause its dysregulation and driving chronic metabolic diseases. Protein kinase D1 (PKD1) is considered to be a key kinase in signal transduction, which regulates multiple cellular functions, but its physiological functions in adipose are still not fully understood. This study aimed at elucidating the function of adipocyte PKD1 on lipogenesis. From RNA-Sequencing data, we found that the fatty acid biosynthesis pathway in white adipose tissue lacking PKD1 was significantly affected. Critical rate-limiting enzymes for de novo lipogenesis in adipocytes, such as FASN, ACCα, and SCD1, were significantly repressed after deleting PKD1 in vivo and in vitro. Further studies revealed that blockade of PKD1 significantly increased phosphorylation of SREBP1c at serine 372 site. Co-immunoprecipitation analysis showed that PKD1 interacts with SREBP1c in vitro and in vivo. Importantly, overexpression of SREBP1c reversed the inhibition of FASN and ACCα expression caused by PKD1 silencing. Together, adipocyte PKD1 promotes de novo lipogenesis via SREBP1c-dependent manner in visceral white adipose tissue and might provide a new target for the development of anti-obesity therapies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Mef2c regulates bone mass through Sost -dependent and -independent mechanisms

We report Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2 cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost -/- ; Mef2 cfl/fl ; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost-/- alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2 cfl/fl ; Dmp1-Cre and Mef2 cfl/fl ; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2 cfl/fl ; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2 cfl/fl ; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp -/- mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost -/- ; Mef2 cfl/fl ; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

TransPlatformer

We propose TransPlatformer for translating toxicogenomics from one platform to another. Transcriptomic profiling has evolved through multiple generations of technology, from microarrays (e.g., Affymetrix, CodeLink) to more recent high-throughput sequencing and targeted panels such as S1500+. Microarrays, which dominated gene expression studies in the early 2000s, provided affordable and high-throughput transcript quantification but suffered from cross-hybridization issues and limited dynamic range . RNA-Seq, introduced in the late 2000s, revolutionized transcriptomics by enabling unbiased and comprehensive gene expression analysis, albeit at higher costs and computational demands . Despite advances, many studies rely on historical microarray data, necessitating the translation of legacy data into modern platforms to ensure continuity and comparability. This translation is complicated by factors such as platform-specific probe design, differences in transcript coverage, and batch effects . Existing methods for cross-platform mapping include statistical normalization, machine learning models, and biological anchoring approaches. The ability to translate transcriptomic data between platforms has broad implications, including enhanced meta-analyses, improved toxicological modeling, and better integration of historical datasets with contemporary research. TransPlatformer seeks to contribute to this effort by evaluating translation methodologies and proposing novel strategies to improve cross-platform gene expression harmonization. In this repository there are code examples for TransPlatformer implementation

Cong, Guojing↗
DOE OSTI2026

Single-cell proteomics of Arabidopsis leaf mesophyll reveals dynamic protein responses to water-deficit stress

Background The application of single-cell omics tools to biological systems can provide unique insights into diverse cellular populations and their heterogeneous responses to internal and external perturbations. Thus far, most single-cell studies in plant systems have been limited to RNA-sequencing approaches, which only provide indirect readouts of cellular functions. Results Here, we present a single-cell proteomics workflow for plant cells that integrates tape-sandwich protoplasting, piezoelectric cell sorting, nanoPOTS sample preparation, and ion mobility-based MS data acquisition method for label-free single-cell proteomics analysis of Arabidopsis leaf mesophyll cells. From a single leaf protoplast, over 3,000 proteins were quantified with high precision. The workflow is demonstrated to identify stress associated changes in protein abundance by analyzing 117 protoplasts from well-watered and water-deficit stressed plants. Additionally, we describe a new approach for constructing covarying protein networks at the single-cell level and demonstrate how single-cell protein covariation analysis can reveal previously unrecognized protein functions while also capturing stress-induced changes in protein–protein dynamics. Conclusions The label-free scProteomic approach presented here represents a significant advance through the demonstration of a facile protoplast isolation method combined with deep and precise proteomic coverage of Arabidopsis leaf mesophyll cell types. We believe this study will serve as an informative reference to future plant scProteomic investigations.

Arabidopsis↗
DOE OSTI2022

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification of candidate host-specificity genes in Exserohilum turcicum using comparative genomics and transcriptomics

Abstract Exserohilum turcicum causes northern corn leaf blight and sorghum leaf blight. While the same species cause disease in both crops, the strains are host-specific. Here, we report the sequence and de novo annotated assemblies of one sorghum- and one maize-specific E. turcicum strain. The strains were sequenced using the PacBio Sequel II system. The total genome length for both assemblies was between 44 and 45 Mb with N50 of ∼2.5 Mb. Ninety-eight percent of the Benchmarking Universal Single-Copy Orthologs (BUSCO) for both assemblies had complete status. The estimated number of genes was 11,762 and 12,029 in the sorghum- and maize-specific isolates, respectively. Funannotate, EffectorP, SignalP, and transcriptome data were used to create functional annotation of each genome. The whole-genome comparison identified ten large-scale inversions and three translocations between the maize- and sorghum-specific strains, along with homologous genes and gene duplications. RNA was sequenced from the maize- and sorghum-specific isolate 10 days post-inoculation in maize and sorghum and from axenic cultures. Gene expression data from planta and axenic growth experiments were compared for each strain. Candidate host-specificity genes were identified by combining results from whole-genome comparison, synteny analysis, gene annotations, and transcriptome data. Overall, this study identified several candidate host-specificity genes that provide insights into E. turcicum interaction with its hosts.

Krone, Mara J. (ORCID:0000000159006624)↗
DOE OSTI2024

Single-cell and spatiotemporal transcriptomic profiling of brain immune infiltration following Venezuelan equine encephalitis virus infection

Neurotropic alphaviruses such as Venezuelan equine encephalitis virus (VEEV) are critical human pathogens that continually expand to naïve populations and for which there are no licensed vaccines or therapeutics. VEEV is highly infectious via the aerosol route and is a recognized weaponizable biothreat that causes neurological disease in humans. The neuropathology of VEEV has been attributed to an inflammatory immune response in the brain yet the underlying mechanisms and specific immune cell populations involved are not fully elucidated. This study uses single-cell RNA sequencing to produce a comprehensive transcriptional profile of immune cells isolated from the brain over a time course of infection in a mouse model of VEEV. Analyses reveal differentially activated subpopulations of microglia, including a distinct type I interferon-expressing subpopulation. This is followed by the sequential infiltration of myeloid cells and cytotoxic lymphocytes, also comprising subpopulations with unique transcriptional signatures. We identify a subpopulation of myeloid cells that form a distinct localization pattern in the hippocampal region whereas lymphocytes are widely distributed, indicating differential modes of recruitment, including that to specific regions of the brain. Altogether, this study provides a high-resolution analysis of the immune response to VEEV in the brain and highlights potential avenues of investigation for therapeutics that target neuroinflammation in the brain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of over ten thousand candidate structured RNAs in viruses and phages

Structured RNAs play crucial roles in viruses, exerting influence over both viral and host gene expression. However, the extensive diversity of structured RNAs and their ability to act in cis or trans positions pose challenges for predicting and assigning their functions. While comparative genomics approaches have successfully predicted candidate structured RNAs in microbes on a large scale, similar efforts for viruses have been lacking. In this study, we screened over 5 million DNA and RNA viral sequences, resulting in the prediction of 10,006 novel candidate structured RNAs. These predictions are widely distributed across taxonomy and ecosystem. We found transcriptional evidence for 206 of these candidate structured RNAs in the human fecal microbiome. These candidate RNAs exhibited evidence of nucleotide covariation, indicative of selective pressure maintaining the predicted secondary structures. Our analysis revealed a diverse repertoire of candidate structured RNAs, encompassing a substantial number of putative tRNAs or tRNA-like structures, Rho-independent transcription terminators, and potentially cis-regulatory structures consistently positioned upstream of genes. In summary, our findings shed light on the extensive diversity of structured RNAs in viruses, offering a valuable resource for further investigations into their functional roles and implications in viral gene expression and pave the way for a deeper understanding of the intricate interplay between viruses and their hosts at the molecular level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

De novo transcriptome in roots of switchgrass ( Panicum virgatum L. ) reveals gene expression dynamic and act network under alkaline salt stress

Background: Soil salinization is a major limiting factor for crop cultivation. Switchgrass is a perennial rhizomatous bunchgrass that is considered an ideal plant for marginal lands, including sites with saline soil. Here we investigated the physiological responses and transcriptome changes in the roots of Alamo (alkaline-tolerant genotype) and AM314/MS-155 (alkaline-sensitive genotype) under alkaline salt stress. Results: Alkaline salt stress significantly affected the membrane, osmotic adjustment and antioxidant systems in switchgrass roots, and the ASTTI values between Alamo and AM-314/MS-155 were divergent at different time points. A total of 108,319 unigenes were obtained after reassembly, including 73,636 unigenes in AM-314/MS-155 and 65,492 unigenes in Alamo. A total of 10,219 DEGs were identified, and the number of upregulated genes in Alamo was much greater than that in AM-314/MS-155 in both the early and late stages of alkaline salt stress. The DEGs in AM-314/MS-155 were mainly concentrated in the early stage, while Alamo showed greater advantages in the late stage. These DEGs were mainly enriched in plant-pathogen interactions, ubiquitin-mediated proteolysis and glycolysis/gluconeogenesis pathways. We characterized 1480 TF genes into 64 TF families, and the most abundant TF family was the C2H2 family, followed by the bZIP and bHLH families. A total of 1718 PKs were predicted, including CaMK, CDPK, MAPK and RLK. WGCNA revealed that the DEGs in the blue, brown, dark magenta and light steel blue 1 modules were associated with the physiological changes in roots of switchgrass under alkaline salt stress. The consistency between the qRT-PCR and RNA-Seq results confirmed the reliability of the RNA-seq sequencing data. A molecular regulatory network of the switchgrass response to alkaline salt stress was preliminarily constructed on the basis of transcriptional regulation and functional genes. Conclusions: Alkaline salt tolerance of switchgrass may be achieved by the regulation of ion homeostasis, transport proteins, detoxification, heat shock proteins, dehydration and sugar metabolism. These findings provide a comprehensive analysis of gene expression dynamic and act network induced by alkaline salt stress in two switchgrass genotypes and contribute to the understanding of the alkaline salt tolerance mechanism of switchgrass and the improvement of switchgrass germplasm.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

COVID-19 biomarkers based on respiratory microbiome content

COVID-19 patient care management would greatly benefit from new tools that enable accurate assessment of disease severity and stage, potentially enabling a personalized medicine approach. Detection of the SARS-CoV-2 virus itself, or even quantitation of viral loads, is not sufficient for accurate assessment of disease state beyond diagnosis of infection [eg, doi:10.1093/cid/ciaa344]. Levels of usual suspect protein biomarkers associated with host response to infection [eg, C-reactive protein (CRP); cytokines like IL-6, TNF-alpha, and IL-10; complement proteins like C3a and C5a], and of individual blood cell types (eg, leukocytes, lymphocytes, and subsets thereof), show limited correlation with disease severity and stage, with high patient-to-patient and study-to-study variability [eg, doi:10.1093/cid/ciaa248]. High-dimensional panels of biomarkers should have greater predictive power and resilience to unavoidable sources of variability; however, their assembly from proteins and cell types is extremely difficult, due to technical limitations in analyte measurement, especially with regard to starting material requirements and detection sensitivity. Host response profiling through Next Generation Sequencing (NGS) of gene expression patterns (ie, RNA-Seq) is a promising approach, but at the time of this project there were only two publicly available datasets of relevance [doi:10.1093/cid/ciaa203, doi:10.1080/22221751.2020.1747363], and close inspection of them revealed that each had at least one major flaw that severely undermined its value in supporting robust analysis of host response to SARS-CoV- 2 infection. However, the first of these studies [doi:10.1093/cid/ciaa203] fortuitously collected NGS data not only from host cells, but also from bacteria present in bronchoalveolar lavage fluid (BALF) recovered from COVID-19 patients; and because the respiratory microbiome (in terms of bacterial species content) is far less complex than the human transcriptome, the NGS data collected were sufficient to provide coverage depth supporting robust analysis. Surprisingly, the authors of the study did not carry out a detailed analysis of these data and their potential for revealing important new information about COVID-19. Therefore, we carried out a meta-analysis of the dataset as a first step in evaluating the potential for profiling of respiratory microbiome dynamics as a means of accurately assessing COVID-19 disease state.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin’s role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

A modERN resource: identification of Drosophila transcription factor candidate target genes using RNAi

Transcription factors (TFs) play a key role in development and in cellular responses to the environment by activating or repressing the transcription of target genes in precise spatial and temporal patterns. In order to develop a catalog of target genes of Drosophila melanogaster TFs, the modERN consortium systematically knocked down the expression of TFs using RNAi in whole embryos followed by RNA-seq. We generated data for 45 TFs which have 18 different DNA-binding domains and are expressed in 15 of the 16 organ systems. The range of inactivation of the targeted TFs by RNAi ranged from log2fold change -3.52 to +0.49. The TFs also showed remarkable heterogeneity in the numbers of candidate target genes identified, with some generating thousands of candidates and others only tens. We present detailed analysis from five experiments, including those for three TFs that have been the focus of previous functional studies (ERR, sens, and zfh2) and two previously uncharacterized TFs (sens-2 and CG32006), as well as short vignettes for selected additional experiments to illustrate the utility of this resource. The RNA-seq datasets are available through the ENCODE DCC (http://encodeproject.org) and the Sequence Read Archive (SRA). TF and target gene expression patterns can be found here: https://insitu.fruitfly.org. These studies provide data that facilitate scientific inquiries into the functions of individual TFs in key developmental, metabolic, defensive, and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks during embryogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped young patient cohort

The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 “critical” (in the intensive care unit under mechanical ventilation) and 25 “non-critical” (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2023

High throughput analysis of B cell dynamics and neutralizing antibody development during immunization with a novel clade C HIV-1 envelope

A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (>90% viability at 48 h), good (>50%), or unusable (<50%). We evaluated drug response to “robust” or “good” microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Phaseolus vulgaris SUT1.1 is a high affinity sucrose–proton co–transporter

Plant sucrose transporters are required for phloem loading, and therefore are essential for plant growth and development. In common beans (Phaseolus vulgaris) there are only two sucrose transporters functionally characterized. Through a previous RNA-seq study, we identified a putative sucrose transporter in common bean, which we hypothesize to function in import of sucrose into plant cells. In silico analysis revealed that PvSUT1.1 is a putative sucrose-proton co-transporter distinct from other characterized sucrose transporters in common bean indicating that this is a previously undescribed transporter protein in beans. Further analysis revealed that PvSUT1.1 shares high protein sequence homology to the phloem loader Arabidopsis SUC2; both have 12 transmembrane domains, a typical characteristic of plant sucrose transporters. Heterologous expression in yeast further showed PvSUT1.1 to be functional and it imported sucrose into yeast cells with a K m of 0.7 mM sucrose. Import of sucrose through PvSUT1.1 is also pH-dependent with highest uptake at pH 4.0, and activity is lost in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Consistent with identification of PvSUT1.1 as a Type I transporter, PvSUT1.1 also transports esculin. Finally, PvSUT1.1 showed expression in multiple tissues and the protein was localized to the plasma membrane. The results show that PvSUT1.1 is a sucrose transporter that is probably involved in the uptake of sucrose into source and sink cells. The potential role of PvSUT1.1 in leaf phloem loading of sucrose in common beans and its importance in heat tolerance of reproductive tissues are further discussed.

59 BASIC BIOLOGICAL SCIENCES↗