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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 OSTI2025

In vitro maturation of fully active [FeFe]-hydrogenase in a defined system including the iron carrier NfuA

The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H 2 . A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that theEscherichia coliiron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H 2 production rates comparable to that of the in vivo-maturedChlamydomonas reinhardtii[FeFe]-hydrogenase (CrHydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [Fe II (cys)(CN)(CO) 2 ] – synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (Mb H64L ) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited H ox -CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and Mb H64L to the fully defined maturation system thus results in an in vitro [FeFe]-hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.

Science & Technology - Other Topics↗
DOE OSTI2021

New Approach Methodology for Assessing Inhalation Risks of a Contact Respiratory Cytotoxicant: Computational Fluid Dynamics-Based Aerosol Dosimetry Modeling for Cross-Species and In Vitro Comparisons

Regulatory agencies are considering alternative approaches to assessing inhalation toxicity that utilizes in vitro studies with human cells and in silico modeling in lieu of additional animal studies. In support of this goal, computational fluid-particle dynamics models were developed to estimate site-specific deposition of inhaled aerosols containing the fungicide, chlorothalonil, in the rat and human for comparisons to prior rat inhalation studies and new human in vitro studies. Under bioassay conditions, the deposition was predicted to be greatest at the front of the rat nose followed by the anterior transitional epithelium and larynx corresponding to regions most sensitive to local contact irritation and cytotoxicity. For humans, simulations of aerosol deposition covering potential occupational or residential exposures (1–50 µm diameter) were conducted using nasal and oral breathing. Aerosols in the 1–5 µm range readily penetrated the deep region of the human lung following both oral and nasal breathing. Under actual use conditions (aerosol formulations >10 µm), the majority of deposited doses were in the upper conducting airways. Beyond the nose or mouth, the greatest deposition in the pharynx, larynx, trachea, and bronchi was predicted for aerosols in the 10–20 µm size range. Only small amounts of aerosols >20 µm penetrated past the pharyngeal region. Using the ICRP clearance model, local retained tissue dose metrics including maximal concentrations and areas under the curve were calculated for each airway region following repeated occupational exposures. These results are directly comparable with benchmark doses from in vitro toxicity studies in human cells leading to estimated human equivalent concentrations that reduce the reliance on animals for risk assessments.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2024

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment

ABSTRACT Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998–2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infectionin vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancementin vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operatingin vivomay modulate ZIKV infection outcomes. IMPORTANCE We investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998–2020) to neutralize or enhance Zika virus infectionin vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, bothin vitroandin vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Microbiology↗
DOE OSTI2024

RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring

Abstract Background Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. Results We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO 2 and NaHCO 3 , and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO 2 were 5–7 times higher than the ones grown with 20 mM NaHCO 3 , at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO 3 − to CO 2 . The highest RuBisCO activity of 2.13 nmol of NAD + / µg of Chl-a/ min was observed with 50 µg of protein and 5% CO 2 . Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited ~ 3.4-fold slower reaction rate (0.37 min −1 ) than the biochemical assay when using 5% CO 2 . We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO 3 as compared to the cells that were grown without NaHCO 3 supplementation. Conclusions Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO 2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO 3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO 2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO 2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

An expedited screening platform for the discovery of anti-ageing compounds in vitro and in vivo

Background: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. Methods: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. Results: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. Conclusions: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Binding of adenovirus species C hexon to prothrombin and the influence of hexon on vector properties in vitro and in vivo

The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.

Tian, Jie↗
DOE OSTI2022

Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform

Three-dimensional (3D) culture systems are increasingly being used for genotoxicity studies due to improved cell-to-cell interactions and tissue-like structures that are limited or lacking in 2D cultures. The present study optimized a 3D culture system using metabolically competent HepaRG cells for in vitro genotoxicity testing. 3D HepaRG spheroids, formed in 96- or 384-well ultra-low attachment plates, were exposed to various concentrations of 34 test articles, including 8 direct-acting and 11 indirect-acting genotoxicants/carcinogens as well as 15 compounds that show different genotoxic responses in vitro and in vivo. DNA damage was evaluated using the high-throughput CometChip assay with concurrent cytotoxicity assessment by the ATP assay in both 2D and 3D cultures. 3D HepaRG spheroids maintained a stable phenotype for up to 30 days with higher levels of albumin secretion, cytochrome P450 gene expression, and enzyme activities compared to 2D cultures. 3D spheroids also demonstrated a higher sensitivity than 2D cultures for detecting both direct- and indirect-acting genotoxicants/carcinogens, indicating a better prediction of in vivo genotoxicity responses. When DNA damage dose-response data were quantified using PROAST software, 3D spheroids generally had lower or similar benchmark dose values compared to 2D HepaRG cells and were more comparable with primary human hepatocytes. These results demonstrate that 3D models can be adapted to the CometChip technology for high-throughput genotoxicity testing and that 3D HepaRG spheroids may be used as a reliable and pragmatic in vitro approach to better support the hazard identification and risk assessment of potential human genotoxic carcinogens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

Tsang, Joyce↗
NASA NTRS1993

Evolution in vitro: analysis of a lineage of ribozymes

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

Non-NASA Center↗
DOE OSTI2021

In vitro assembly of bacterial microcompartments

The present disclosure is related to a BMC fusion protein that is capable of in vitro assembly, comprising a constituent BMC shell protein subunit and a sterically hindering protein domain that is cleavable. The BMC fusion protein is capable of in vitro assembly triggered by removal of the fused sterically hindering domain. The present disclosure is also related to a means to produce BMC shells in vitro, triggered by removal of a fused sterically hindering domain from one or more constituent BMC shell protein subunits. The BMC fusion protein enables encapsulation of broad classes of materials and biophysical studies of shell assembly, encapsulation, and permeability that would otherwise be unavailable from BMCs assembled in vivo.

Hagen, Andrew Ronald↗
DOE OSTI2023

System and method for three-dimensional in vitro flexible microelectrode array

The present disclosure relates to a biocompatible, in vitro probe system. The probe system may have a substrate and a culture well supported on the substrate. The culture well defines a three-dimensional volume for containing in vitro cultures of electroactive cells. The probe system has at least one probe subsystem supported on the substrate. The probe subsystem has at least one probe having an array of electrodes, with the probe being disposed within the culture well for in vitro electrically communicating with the electroactive cells. The probe subsystem is adapted to be interfaced to an external instrumentation/recording device.

Soscia, David↗
DOE OSTI2024

SIVB's 2024 In Vitro Biology Meeting Proceedings

SIVB's 2024 World Congress on In Vitro Biology took place in Saint Louis, Missouri, from June 8th to 12th, 2024. The conference featured renowned speakers from academic and non-academic institutions who will present recent advancements in critical areas like plant transformation, genome editing, synthetic biology, advanced breeding technologies, cellular agriculture, future food sources, chromosome engineering, epigenetics, artificial intelligence, and machine learning. The Society for In Vitro Biology (SIVB) has always considered the education and professional development of young researchers as an integral component of its mission. The 2024 World Congress program, along with SIVB’s student initiatives, was customized to foster scientific growth and professional development among students and young scientists empowering them in their professional journeys. The recording of the DOE supported "Single Cell RNA Sequencing" workshop was made publicly available at https://youtu.be/A0UnuYwefwg for easy retrieval and reference of all information shared during the live event, thereby increasing accessibility and knowledge transfer. Their are 14 articles in the proceedings and the full list of files is located at https://link.springer.com/journal/11626/volumes-and-issues/60-1/supplement.

10 SYNTHETIC FUELS↗
DOE OSTI2023

The effect of food vehicles on in vitro performance of pantoprazole sodium delayed release sprinkle formulation

Certain patient populations, including children, the elderly or people with dysphagia, find swallowing whole medications such as tablets and capsules difficult. To facilitate oral administration of drugs in such patients, a common practice is to sprinkle the drug products (e.g., usually after crushing the tablet or opening the capsule) on food vehicles before consumption which improves swallowability. Thus, evaluation of the impact of food vehicles on the potency and stability of the administered drug product is important. Here, the aim of the current study was to evaluate the physicochemical properties (viscosity, pH, and water content) of common food vehicles used for sprinkle administration (e.g., apple juice, applesauce, pudding, yogurt, and milk) and their impacts on the in vitro performance (i.e., dissolution) of pantoprazole sodium delayed release (DR) drug products. The food vehicles evaluated exhibited marked difference in viscosity, pH and water content. Notably, the pH of the food as well as the interaction between food vehicle pH and drug-food contact time were the most significant factors affecting the in vitro performance of pantoprazole sodium DR granules. For example, the dissolution of pantoprazole sodium DR granules sprinkled on food vehicles of low pH (e.g., apple juice or applesauce) for short durations remained unchanged compared with the control group (i.e., without mixing with food vehicles). However, use of high pH food vehicles (e.g., milk) with prolonged contact time (e.g., 2h) resulted in accelerated pantoprazole release, drug degradation and loss of potency. Overall, a thorough assessment of physicochemical properties of food vehicles and formulation characteristics are a necessary part of the development of sprinkle formulations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Novel method for effectively amplifying human peripheral blood T cells in vitro

The use of chimeric antigen receptor-modified T cells (CAR T cells) is an effective therapy for advanced cancer, especially hematological malignancies, and this method has attracted widespread attention in the last several years. The type, number and vitality of the effector cells clearly play important roles in this approach. In this study, to expand the possibility of curing cancer through adoptive cell therapy (ACT), we developed a novel method for effectively obtaining abundant T cells in vitro. The fusion proteins of three cytokines, SA-hIL-2, SA-hIL-7 and SA-hIL-21, were anchored onto biotin magnetic beads to increase the number of cytokines on the surface of the magnetic beads, which increased the local concentration of cytokines and thus promoted the binding of cytokines to T cells. Next, we examined the effects of these modified magnetic beads on the proliferation rate of T cells and CD19 CAR T cells. In this study, we report the expression and purification of the active bifunctional fusion proteins SA-hIL-2, SA-hIL-7 and SA-hIL-21, which were bound to biotin magnetic beads to develop a platform that was employed to increase the local concentration of cytokines. When the cells had been cultured for 14 days, the proliferation rate of the CD3{sup +} T cells in the group that received cytokine-coupled biotin magnetic beads (Beads-SA-CK) was higher than that of the cells in the groups that received soluble cytokines (Soluble-SA-CK) and that of the cells in the standard group (Standard-CK). We speculate that this difference may be the result of the increased expression of Bcl-2 and the increased phosphorylation of Stat5. Moreover, our results preliminarily indicate that compared with the other two treatments, Soluble-SA-CK and Standard-CK, adding cytokine-coupled biotin magnetic beads more effectively increases the proliferation rate of CD19 CAR-T cells. As expected, the CD19 CAR-T cells stimulated by Beads-SA-CK had a stronger anticancer effect than the cells stimulated by the other two treatments. An effective method of preparing abundant T cells in vitro was developed, and it may provide a novel strategy for ACT.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Knockdown of NLE1 inhibits development of malignant melanoma in vitro and in vivo NLE1 promotes development of malignant melanoma

Melanoma, which originates from neural crest derived melanocytes, causes severe pain and even death to numerous patients. Previous studies reported that Notchless Homolog 1 (NLE1) plays an important role in cell proliferation, transcription and signal transduction. However, the clinical significance and biological behavior of NLE1 in melanoma remain a mystery. Thus, the role of NLE1 in melanoma was investigated in vitro and in vivo. The expression of NLE1 in melanoma was elevated and the expression level was positively correlated with lymphatic metastasis and tumor stage. In addition, NLE1 knockdown by shRNA specifically inhibited proliferation, enhanced the apoptotic sensitivity and hindered migration of melanoma cells in vitro. Mice xenograft model further showed that NLE1 knockdown could inhibit the tumor formation of melanoma in vivo. Additionally, the induction of apoptosis of melanoma cells by NLE1 knockdown required the participation of a series of apoptosis-related proteins. Besides, NLE1 can activate the PI3K/AKT signaling pathway. In summary, NLE1 was involved in the development and progression of melanoma, which may be a novel potential target for molecular therapy of melanoma.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Chaotrope-Based Approach for Rapid In Vitro Assembly and Loading of Bacterial Microcompartment Shells

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here, we describe the development of an efficient and rapid method for the in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

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

Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation

Abstract Background Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Results Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca 2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Conclusions Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

59 BASIC BIOLOGICAL SCIENCES↗