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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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At least 541 records · Page 30
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

BST-2/Tetherin is involved in BAFF-enhanced proliferation and survival via canonical NF-κB signaling in neoplastic B-lymphoid cells

Highlights: • BST-2 doze dependently responded to BAFF in neoplastic B cells. • BST-2 inhibition defected the proliferation and survival of neoplastic B cells. • BST-2 facilitated BAFF-induced NF-κB signaling in neoplastic B-lymphoid cells. • BAFF-induced NF-κB signaling is required for the proliferation and survival of neoplastic B cells. The development of Sjögren's syndrome (SS) is accompanied by B cell hyperproliferation and mutation. Our previous study identified aberrant expression of BST-2 (also known as Tetherin/CD317) in B cells from either the peripheral blood or infiltrated salivary glands. However, the roles of BST-2 in the regulation of B cell activation remain unknown. In this study, we identified that BST-2 can respond to BAFF simulation but not to other B cell simulators in neoplastic B cell lines. A CCK-8 assay, an EdU assay and Annexin V/PI staining indicated that BST-2 inhibition attenuated BAFF-enhanced proliferation and survival in both Raji cells and Daudi cells. Screening of BAFF-related signaling in neoplastic B-lymphoid cells indicated that BST-2 was involved in the regulation of NF-κB signaling upon BAFF simulation. However, inhibition of NF-κB by JSH-23 significantly reduced the proliferation and survival of Raji and Daudi cells under both normal and BAFF-simulated conditions. Collectively, our results indicate that BST-2/Tetherin is a BAFF-responsive membrane factor involved in the regulation of NF-κB signaling, thereby assisting in the proliferation and survival of neoplastic B-lymphoid cells. Our study provides a potential molecular mechanism underlying aberrant overactivation of B cells upon SS development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

AFF4 facilitates melanoma cell progression by regulating c-Jun activity

Melanoma is characterized by high mortality and poor prognosis due to metastasis. AFF4 (AF4/FMR2 family member 4), as a scaffold protein, is a component of the super elongation complex (SEC), and is involved in the progression of tumors, e.g., leukemia, head and neck squamous cell carcinoma (HNSCC). However, few studies on AFF4 have focused on melanoma. Here, AFF4 expression levels and clinicopathological features were evaluated in melanoma tissue samples. Then, we performed cell proliferation, migration and invasion assays in A375 and A2058 cells lines in vitro to evaluate the role of AFF4 in melanoma. The effects of AFF4 knockdown in vivo were characterized via a xenograft mouse model. Finally, the correlation between c-Jun and AFF4 protein levels in melanoma was analyzed by rescue assay and immunohistochemistry (IHC). We found that AFF4 expression was upregulated in melanoma tumor tissues and that AFF4 protein expression was also closely related to the prognosis of patients with cutaneous melanoma. Moreover, AFF4 could promote the invasion and migration of melanoma cells by mediating epithelial to mesenchymal transition (EMT). AFF4 might regulate c-Jun activity to promote the invasion and migration of melanoma cells. Importantly, c-Jun was regulated by the AFF4 promoted melanoma tumorigenesis in vivo. Taken together, AFF4 may be a novel oncogene that promotes melanoma progression through regulation of c-Jun activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Growth differentiation factor-15 promotes immune escape of ovarian cancer via targeting CD44 in dendritic cells

Immune escape is the main cause of the low response rate to immunotherapy for cancer, including ovarian cancer. Growth differentiation factor-15 (GDF-15) inhibits immune cell function. However, only few reports described the mechanism. Therefore, the aim of this study was to investigate the mechanism of immune escape regulated by GDF-15 in ovarian cancer. Ovarian cancer patients and healthy women were enrolled in this study. Immunohistochemistry and ELISA were performed to measure GDF-15 expression. Immunoprecipitation combined with mass spectrometry, surface plasmon resonance, and co-immunoprecipitation assay were used to evaluate the interaction between GDF-15 and the surface molecules of DCs. Immunofluorescence analysis, flow cytometry and transwell assay were used to evaluate additional effects of GDF-15 on DCs. The results showed that GDF-15 expression was higher in the ovarian cancer patients compared to that in the healthy women. The TIMER algorithm revealed that highly GDF-15 expression is associated with immune DC infiltration in immunoreactive high-grade serous carcinoma. A further study showed that GDF-15 suppressed DCs maturation, as well as IL-12p40 and TNF-α secretion, the length and number of protrusions and the migration. More importantly, CD44 in the surface of DCs interacted with GDF-15. The overexpression of CD44 in DCs resulted in the suppression of the inhibitory effect of GDF-15 on the length and number of DC synapses. In DCs overexpressing CD44 the inhibition of GDF-15 on the expression of CD11c, CD83 and CD86 was decreased, while in DCs with a knockdown of CD44 the inhibition was further enhanced. Knockdown of CD44 in DCs enhanced the inhibitory effect of GDF-15 on DC migration, while the overexpression of CD44 inhibited the inhibitory effect of GDF-15 on DC migration. In conclusion, the present study suggested that GDF-15 might facilitate ovarian cancer immune escape by interacting with CD44 in DCs to inhibit their function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

NPRL2 reduces the niraparib sensitivity of castration-resistant prostate cancer via interacting with UBE2M and enhancing neddylation

In this study, we explored the regulatory effects of nitrogen permease regulator 2-like (NPRL2) on niraparib sensitivity, a PARP inhibitor (PARPi) in castrate-resistant prostate cancer (CRPC). Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) program were retrospectively examined. Gene-set enrichment analysis (GSEA) was conducted between high and low NRPL2 expression prostate adenocarcinoma (PRAD) cases in TCGA. CCK-8 assay, Western blot analysis of apoptotic proteins, and flow cytometric analysis of apoptosis were applied to test niraparib sensitivity. Immunofluorescent (IF) staining and co-immunoprecipitation (co-IP) were conducted to explore the proteins interacting with NPRL2. Results showed that the upregulation of a canonical protein-coding transcript of NPRL2 (ENST00000232501.7) is associated with an unfavorable prognosis. Bioinformatic analysis predicts a physical interaction between NPRL2 and UBE2M, which is validated by a following Co-IP assay. This interaction increases NPRL2 stability by reducing polyubiquitination and proteasomal degradation. Depletion of NPRL2 or UBE2M significantly increases the niraparib sensitivity of CRPC cells and enhances niraparib-induced tumor growth inhibition in vivo. NPRL2 cooperatively enhances UBE2M-mediated neddylation and facilitates the degradation of multiple substrates of Cullin-RING E3 ubiquitin ligases (CRLs). In conclusion, this study identified a novel NPRL2-UBE2M complex in modulating neddylation and niraparib sensitivity of CRPC cells. Therefore, targeting NPRL2 might be considered as an adjuvant strategy for PARPi therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Transcription factor FOXC1 positively regulates SFRP1 expression in androgenetic alopecia

Androgenetic alopecia (AGA) is the most common type of hair loss dysfunction. Secreted frizzled related protein 1 (SFRP1) is found to be associated with hair loss, but its role in AGA and the regulation mechanism of its transcription level is unclear. The aim of our study is to explore the expression of SFRP1 in AGA samples and its transcriptional mechanism. Male frontal and occipital scalp hair follicles from AGA patients were collected, and human dermal papilla cells (DPCs) were isolated and cultured. SFRP1 gene was cloned and constructed into recombinant plasmids to perform dual-luciferase reporter assay. Transcription factor binding sites were predicted through the Jaspar website and further confirmed by the chromatin immunoprecipitation (ChIP) assay. Expression of genes in DPCs was determined by immunofluorescence (IF) staining, quantitative real-time PCR (qRT-PCR) and western blotting. Our findings showed that SFRP1 was highly expressed in DPCs of AGA patients. The core promoter region of SFRP1 was from −100 to +50 bp and was found to be positively regulated by forkhead box C1 (FOXC1), a transcription factor related to hair growth, both at mRNA and protein level in DPCs. Our study suggests that FOXC1 plays an important role in regulating SFRP1 transcription, which may provide new insights into the development of therapeutic strategies for the treatment of AGA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes

Highlights: • Cortical granule exocytosis is sensitive to tetanus toxin in mouse oocytes. • VAMP1 and VAMP3 proteins participate in cortical granule exocytosis. • This work confirms that cortical granule exocytosis is mediated by SNARE complex. Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

YAP/STAT3 promotes the immune escape of larynx carcinoma by activating VEGFR1-TGFβ signaling to facilitate PD-L1 expression in M2-like TAMs

Larynx carcinoma (LC) is the most prevalent head and neck cancer among adults. LC xenograft mouse model was generated to verify the effect of VEGF on macrophage polarization and tumor growth in vivo. EdU assay was performed to measure the cell proliferation. Transwell assay was applied to assess cell migration. The expression of YAP and STAT3 was also significantly increased in LC tumor tissues. Moreover, both YAP and STAT3 overexpression in LC cells promoted the proliferation, migration, as well as the secretion of PD-L1 in M2-like TAMs. Mechanistically, the interaction between YAP and STAT3 facilitated the transcription of VEGF. Moreover, with a co-culture system, VEGF secretion in LC cells enhanced PD-L1 expression in M2-like TAMs via activating VEGFR1-TGFβ signaling pathway. Furthermore, VEGF secreted from LC cells also promoted the tumor growth of LC in vivo. We revealed that dysregulated YAP/STAT3 activity in LC cells could enhance the secretion of VEGF, which then functioned on M2-like TAMs via activating VEGFR1-TGFββ pathway to promote the expression of PD-L1 and immunosuppressive function of M2-like TAMs. Therefore, VEGF and PD-L1 might have a pivotal crosstalk between M2-like TAMs and LC cells, which provided a novel therapeutic target in regulating the metastasis of LC in future.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Chorioallantoic membrane vascularization. A meta-analysis

The CAM is a widely used experimental assay to study angiogenesis, wound healing, tumor growth and metastatic process. In this study, we have analyzed and compared the existent literature data concerning the growth of the CAM. Moreover, we have analyzed the data concerning the development of the vascular system and the expression of the most important pro-angiogenic and anti-angiogenic factors. The availability of these data and their comparative evaluation allow to better analyze the experimental data concerning the testing of different pro-angiogenic and anti-angiogenic molecules, as well as biomaterials in the CAM assay. Moreover, the dynamic of the angiogenic response to different tumor cell lines and or tumor bioptic specimens, may be also better evaluated and estimated.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Aberrant FBXW7-mediated ubiquitination and degradation of ZMYND8 enhances tumor progression and stemness in bladder cancer

ZMYND8, an epigenetic regulator, was identified as a common oncogene across various tumors. However, little was reported about the association between ZMYND8 and bladder cancer. Besides, aberrant mechanisms that contribute to abnormal ZMYND8 expressions still remain unclear. In the current study, we first found that ZMYND8 protein levels were significantly elevated in Bca samples versus normal tissues, but not the mRNA levels. We then utilized the Cell Counting Kit-8 (CCK-8) assay, clone formation assay and transwell analysis to confirm that ZMYND8 could remarkably promote the tumor progression in vitro, including growth capacity and migration. Bioinformatic predictive analysis revealed that E3 ubiquitin ligase FBXW7 interacts directly with ZMYND8 and degrades ZMYND8 in a polyubiquitination manner. Low FBXW7 was a hazard factor for promoting and depending on accumulated ZMYND8 proteins to promote Bca progression. Gene set enrichment analysis (GSEA) further indicated that ZMYND8 was notably associated with stemness process, which was well functionally validated. Lastly, ZMYND8 deficiency was observed to inhibit tumor growth of Bca in vivo, revealing a promising translational significance in Bca treatment. In conclusion, our study for the first time provided evidence for a novel mechanism of FBXW7/ZMYND8 axis in Bca, providing therapeutic vulnerability for individualized cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Incomplete radiofrequency ablation induced chemoresistance by up-regulating heat shock protein 70 in hepatocellular carcinoma

Highlights: • iRFA treatment inhibited cisplatin-induced pyroptosis of HCC cells. • Heat shock protein 70 was involved in the suppression of NLRP3 activation. • iRFA treatment decreased the cisplatin sensitivity of HCC through regulating HSP70. Radiofrequency ablation (RFA) is a common minimally invasive treatment for hepatocellular carcinoma (HCC). Incomplete RFA (iRFA) due to the sub-lethal heat shock challenge of some cell populations leads to the generation of transformed survivor cells with enhanced chemoresistance. However, the underlying mechanism of iRFA on HCCs chemoresistance remains unknown. In the present study, we investigated the effect of iRFA on HCCs sensitivity to cisplatin. Cells treated with the sub-lethal heat shock challenge were used to mimic iRFA treatment in vitro. An orthotopic implantation HCC model was established and also performed iRFA treatment. Flow cytometry, transwell assay, and cell counting kit-8 assay were used to determine the effect of iRFA treatment on cisplatin-induced HCC cell apoptosis, invasion, and cell viability. ELISA and Western blot were used to detect the effect of iRFA treatment on cisplatin-induced HCC cell pyroptosis. We found that iRFA treatment increased the HCC cell proliferation and invasion ability, and inhibited cisplatin-induced pyroptosis. Further experiments showed that iRFA treatment induced upregulation of HSP70, which inhibited the cisplatin-induced NLRP3 inflammasome activation, leading to inhibition of pyroptosis. HSP70 knockdown or NLRP3 overexpression could reverse the effect of iRFA treatment in vitro. In vivo, HSP70 knockdown reversed the chemosensitivity of HCC to cisplatin, which was decreased by iRFA. In conclusion, we demonstrated that iRFA induced drug resistance by HSP70-mediated inhibition of cell pyroptosis in HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. Furthermore, the mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14 C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p -coumarate

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Altogether, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.

09 BIOMASS FUELS↗
DOE OSTI2024

Untargeted Spatial Metabolomics and Spatial Proteomics on the Same Tissue Section

An increasing number of spatial multiomic workflows have been recently developed. Some of these approaches have leveraged initial mass spectrometry imaging (MSI)-based spatial metabolomics to inform region of interest (ROI) selection for downstream spatial proteomics. However, these workflows have been limited by varied substrate requirements between modalities or have required analyzing serial sections (i.e., one section per modality). To mitigate these issues, we present a novel multiomic workflow that uses desorption electrospray ionization (DESI)-MSI to identify representative spatial metabolite patterns on-tissue prior to spatial proteomic analyses on the same tissue section. Further, this workflow is demonstrated here with a model mammalian tissue (coronal rat brain section) mounted on a polyethylene naphthalate-membrane slide. Initial DESI-MSI resulted in 160 annotations (SwissLipids) within to the METASPACE platform (≤20% false discovery rate). A segmentation map from the annotated ion images informed downstream ROI selection for spatial proteomics characterization from the same sample. The unspecific substrate requirements and minimal sample disruption inherent to DESI-MSI allowed for an optimized, downstream spatial proteomics assay, resulting in 3888 ± 240 to 4717 ± 48 proteins being confidently directed per ROI (200 µm x 200 µm). Finally, we demonstrate the integration of multiomic information, where we found ceramide localization to be correlated with SMPD3 abundance (ceramide synthesis protein), and we also utilized protein abundance to resolve metabolite isomeric ambiguity. Overall, the integration of DESI-MSI into the multiomic workflow allows for complementary spatial and molecular-level information to be achieved from optimized implementations of each MS assay inherent to the workflow itself.

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

Role of Ammonium Hydroxide on Glucose Oxidase Immobilized in Metal–Azolate Framework-7 Enzyme Activity

Enzymes are nature’s catalysts, but their instability outside native environments limits practical applications. Metal–organic frameworks (MOFs) offer a promising platform for enzyme immobilization, enhancing stability, and reusability through their tunable porosity and crystallinity. While ZIF-based MOFs have been extensively studied, metal azolate framework-7 (MAF-7) remains largely unexplored for biomimetic mineralization. Its hydrophilic character makes it a promising alternative for enzyme encapsulation; however, the role of essential basic modulators such as ammonium hydroxide (NH 4 OH) on enzyme structure and function has not been investigated. Here, in this study, we examine the NH 4 OH mediated synthesis of glucose oxidase within MAF-7 using powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and enzyme assays. Our results show that NH 4 OH modulates crystal morphology, enzyme activity, and nucleation behavior, with higher concentrations leading to partial enzyme denaturation and reduced catalytic performance. By integrating time-resolved cryo-TEM with activity assays, we uncover how modulator concentration impacts early stage crystallization and enzyme folding, a mechanistic insight not previously demonstrated in MOF systems. These findings highlight modulator chemistry as a critical, underexplored variable in MOF synthesis, advancing rational design strategies for enzyme@MOFs (E@MOF) beyond ZIFs and toward more tunable, biocompatible frameworks like MAF-7.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Enzymatic Biocontrol of Fire Blight ( Erwinia amylovora ) Using an Engineered Glycosyl Hydrolase

Current management of fire blight, caused by Erwinia amylovora , relies heavily on streptomycin a practice that has contributed to the emergence of antibiotic-resistant strains and raised environmental and regulatory concerns. Enzyme-based biocontrol agents offer a promising antibiotic-free approach that combines target specificity with environmental compatibility. This study evaluates CAase, a bacteriophage-derived glycosyl hydrolase, for its ability to disrupt E. amylovora biofilms and reduce disease severity. Biochemical assays and scanning electron microscopy confirmed that CAase efficiently degraded the extracellular polysaccharide (EPS) matrix, releasing cells from biofilms. Gas chromatography–mass spectrometry (GC–MS) linkage analysis of EPS isolated from two Erwinia amylovora strains demonstrated that CAase preferentially cleaves galactan-rich amylovoran produced by strain EA273, while exhibiting only limited activity toward the levan-rich EPS from strain EA1430. Functional assays revealed that CAase reduced bacterial viability by nearly 2 orders of magnitude at higher enzyme concentrations, strongly suppressed surface motility, and induced ultrastructural damage visible by transmission electron microscopy. Importantly, field trials showed that CAase significantly lowered blossom and shoot blight incidence under orchard conditions. These results highlight CAase as a potent enzyme-based strategy for reducing the virulence of E. amylovora and demonstrate its potential as a sustainable alternative to antibiotics in fire blight management.

antibiotic resistance↗
DOE OSTI2022

Bimetallic Metal–Organic Framework Fe/Co-MIL-88(NH 2 ) Exhibiting High Peroxidase-like Activity and Its Application in Detection of Extracellular Vesicles

Metal–organic frameworks (MOFs) have many attractive features, including tunable composition, rigid structure, controllable pore size, and large specific surface area, and thus are highly applicable in molecular analysis. Depending on the MOF structure, a high number of un-saturated metal sites can be exposed to catalyze chemical reactions. In the present work, we report that by using both Co(II) and Fe(III) to prepare the MIL-88(NH 2 ) MOF, we can produce the bimetallic MOF that can catalyze the conversion of 3,3', 5,5"-tetramethylbenzidine (TMB) to a color product through reaction with H 2 O 2 at a higher reaction rate than the monometallic Fe-MIL-88(NH 2 ). The Michaelis constants (K m ) of the catalytic reaction for TMB and H 2 O 2 are 3-5 times smaller, and the catalytic constants (k cat ) are 5-10 times higher than those of the horse-radish peroxidase (HRP), supporting ultrahigh peroxidase-like activity. These values are also much more superior to those of the HRP-mimicking MOFs reported previously. Interestingly, the bimetallic MOF can be coupled with glucose oxidase (GOx) to trigger the cascade enzymatic reaction for highly sensitive detection of extracellular vesicles (EVs), a family of important biomarkers. Through conjugation to the aptamer that recognizes the marker protein on EV surface, the MOF can help isolate the EVs from biological matrices, which are subsequently labeled by GOx via antibody recognition. The cascade enzymatic reaction between MOF and GOx enables detection of EVs at a concentration as low as 7.8 × 10 4 particles/ml. Further, the assay can be applied to monitor EV secretion by cultured cells, and also can successfully detect the different EV quantities in the sera samples collected from cancer patients and healthy controls. Overall, we prove that the bimetallic Fe/Co-MIL-88(NH 2 ) MOF, with its high peroxidase-activity and high biocompatibility, is a valuable tool deployable in clinical assays to facility disease diagnosis and prognosis.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Biocatalytic Synthesis of α-Amino Esters via Nitrene C–H Insertion

α-Amino esters are precursors to noncanonical amino acids used in developing small-molecule therapeutics, biologics, and tools in chemical biology. α-C–H amination of abundant and inexpensive carboxylic acid esters through nitrene transfer presents a direct approach to α-amino esters. Methods for nitrene-mediated amination of the protic α-C–H bonds in carboxylic acid esters, however, are underdeveloped. This gap arises because hydrogen atom abstraction (HAA) of protic C–H bonds by electrophilic metal-nitrenoids is slow: metal-nitrenoids preferentially react with polarity-matched, hydridic C–H bonds, even when weaker protic C–H bonds are present. This study describes the discovery and evolution of highly stable protoglobin nitrene transferases that catalyze the enantioselective intermolecular amination of the α-C–H bonds in carboxylic acid esters. We developed a high-throughput assay to evaluate the activity and enantioselectivity of mutant enzymes together with their sequences using the Every Variant Sequencing (evSeq) method. Furthermore, the assay enabled the identification of enantiodivergent enzymes that function at ambient conditions in Escherichia coli whole cells and whose activities can be enhanced by directed evolution for the amination of a range of substrates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells

Abstract The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34 + progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.

60 APPLIED LIFE SCIENCES↗