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DOE OSTI2024

Biphasic response of human iPSC-derived neural network activity following exposure to a sarin-surrogate nerve agent

Organophosphorus nerve agents (OPNA) are hazardous environmental exposures to the civilian population and have been historically weaponized as chemical warfare agents (CWA). OPNA exposure can lead to several neurological, sensory, and motor symptoms that can manifest into chronic neurological illnesses later in life. There is still a large need for technological advancement to better understand changes in brain function following OPNA exposure. The human-relevant in vitro multi-electrode array (MEA) system, which combines the MEA technology with human stem cell technology, has the potential to monitor the acute, sub-chronic, and chronic consequences of OPNA exposure on brain activity. However, the application of this system to assess OPNA hazards and risks to human brain function remains to be investigated. In a concentration-response study, we have employed a human-relevant MEA system to monitor and detect changes in the electrical activity of engineered neural networks to increasing concentrations of the sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). We report a biphasic response in the spiking (but not bursting) activity of neurons exposed to low (i.e., 0.4 and 4 μM) versus high concentrations (i.e., 40 and 100 μM) of NIMP, which was monitored during the exposure period and up to 6 days post-exposure. Regardless of the NIMP concentration, at a network level, communication or coordination of neuronal activity decreased as early as 60 min and persisted at 24 h of NIMP exposure. Once NIMP was removed, coordinated activity was no different than control (0 μM of NIMP). Interestingly, only in the high concentration of NIMP did coordination of activity at a network level begin to decrease again at 2 days post-exposure and persisted on day 6 post-exposure. Notably, cell viability was not affected during or after NIMP exposure. Also, while the catalytic activity of AChE decreased during NIMP exposure, its activity recovered once NIMP was removed. Gene expression analysis suggests that human iPSC-derived neurons and primary human astrocytes resulted in altered genes related to the cell’s interaction with the extracellular environment, its intracellular calcium signaling pathways, and inflammation, which could have contributed to how neurons communicated at a network level.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2007

Toxicity of Lunar and Martian Dust Simulants to Alveolar Macrophages Isolated from Human Volunteers

NASA is planning to build a habitat on the Moon and use the Moon as a stepping stone to Mars. JSC-1, an Arizona volcanic ash that has mineral properties similar to lunar soil, is used to produce lunar environments for instrument and equipment testing. NASA is concerned about potential health risks to workers exposed to these fine dusts in test facilities. The potential toxicity of JSC-1 and a Martian soil simulant (JSC-Mars-1, a Hawaiian volcanic ash) was evaluated using human alveolar macrophages (HAM) isolated from volunteers; titanium dioxide and quartz were used as reference dusts. This investigation is a prerequisite to studies of actual lunar dust. HAM were treated in vitro with these test dusts for 24 h; assays of cell viability and apoptosis showed that JSC-1 and TiO2 were comparable, and more toxic than saline control, but less toxic than quartz. HAM treated with JSC-1 or JSC-Mars 1 showed a dose-dependent increase in cytotoxicity. To elucidate the mechanism by which these dusts induce apoptosis, we investigated the involvement of the scavenger receptor (SR). Pretreatment of cells with polyinosinic acid, an SR blocker, significantly inhibited both apoptosis and necrosis. These results suggest HAM cytotoxicity may be initiated by interaction of the dust particles with SR. Besides being cytotoxic, silica is known to induce shifting of HAM phenotypes to an immune active status. The immunomodulatory effect of the simulants was investigated. Treatment of HAM with either simulant caused preferential damage to the suppressor macrophage subpopulation, leading to a net increase in the ratio of activator (RFD1+) to suppressor (RFD1+7+) macrophages, a result similar to treatment with silica. It is recommended that appropriate precautions be used to minimize exposure to these fine dusts in large-scale engineering applications.

Latch, Judith N.↗

DOE OSTI2024

Simultaneous inhibition of ATM, ATR, and DNA-PK causes synergistic lethality

Here, in this paper, we report that simultaneous inhibition of the three primary DNA damage recognition PI3 kinase-like kinases (PIKKs) —ATM, ATR, and DNA-PK— induces severe combinatorial synthetic lethality in mammalian cells. Utilizing Chinese hamster cell lines CHO and V79 and their respective PIKK mutants, we evaluated effects of inhibiting these three kinases on cell viability, DNA damage response, and chromosomal integrity. Our results demonstrate that while single or dual kinase inhibition increased cytotoxicity, inhibition of all three PIKKs results in significantly higher synergistic lethality, chromosomal aberrations, and DNA double-strand break (DSB) induction as calculated by their synergy scores. These findings suggest that the overlapping redundancy of ATM, ATR, and DNA-PK functions is critical for cell survival, and their combined inhibition greatly disrupts DNA damage signaling and repair processes, leading to cell death. This study provides insights into the potential of multi-targeted DDR kinase inhibition as an effective anticancer strategy, necessitating further research to elucidate underlying mechanisms and therapeutic applications.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2012

Effects of High Dietary HEME Iron and Radiation on Cardiovascular Function

The radiation related health risks to astronauts is of particular concern to NASA. Data support that exposure to radiation is associated with a number of disorders including a heightened risk for cardiovascular diseases. Independent of radiation, altered nutrient status (e.g. high dietary iron) also increases ones risk for cardiovascular disease. However, it is unknown whether exposure to radiation in combination with high dietary iron further increases ones cardiovascular risk. The intent of our proposal is to generate compulsory data examining the combined effect of radiation exposure and iron overload on sensitivity to radiation injury to address HRP risks: 1) Risk Factor of Inadequate Nutrition; 2) Risk of Cardiac Rhythm Problems; and 3) Risk of Degenerative Tissue or other Health Effects from Space Radiation. Towards our goal we propose two distinct pilot studies using the following specific aims: Vascular Aim 1: To determine the short-term consequences of the independent and combined effects of exposure to gamma radiation and elevated body iron stores on measures of endothelial function and cell viability and integrity. We hypothesize that animals that have high body iron stores and are exposed to gamma radiation will show a greater reduction in endothelial dependent nitric oxid production and larger pathological changes in endothelial integrity than animals that have only 1 of those treatments (either high iron stores or exposure to gamma radiation). Vascular Aim 2: Identify and compare the effects of gamma radiation and elevated body iron stores on the genetic and epigenetic regulation of proteins associated with endothelial cell function. We hypothesize that modifications of epigenetic control and posttranslational expression of proteins associated with endothelial cell function will be differentially altered in rats with high body iron stores and exposed to gamma radiation compared to rats with only 1 type of treatment. Cardiac Aim 1: To determine the short-term consequences of the independent and combined effects of gamma radiation and elevated body iron stores on measures of cardiac structure. We hypothesize that modifications to cardiac structure and function will be greater in rats with high body iron stores and exposed to gamma radiation than in rats that have only 1 of those treatments. Cardiac Aim 2: Identify and compare the effects of gamma radiation and elevated body iron stores on the genetic and epigenetic regulation of proteins associated with cardiac structure and function. We hypothesize that modifications of epigenetic control and posttranslational expression of proteins associated with cardiac contractile function will be differentially altered in rats with high body iron stores and exposed to gamma radiation compared to rats with only 1 type of treatment.

Westby, Christian M.↗

DOE OSTI2024

The Impact of Sorbent Amendments for Mercury Remediation on the Viability of Soil Microorganisms

Mercury (Hg) remediation across contaminated environments in the United States is an ongoing project. As part of the Hg cleanup strategy at East Fork Poplar Creek (EFPC), located in Oak Ridge, TN, the deployment of sorbents is considered. However, the impact of sorbents on soil microorganisms is poorly understood. In this study, we investigated the effect of sorbents on soil microorganism viability and biofilm formation to assess soil health during sorbent application for Hg remediation. We specifically investigated the effect of two engineered sorbents, Organoclay PM-199 and Organoclay MRM (which are manufactured from clay minerals formulated for various remediation applications), on two gram-negative organisms ( Serratia marcescens and Burkholderia thailandensis ) isolated from the Hg-contaminated EFPC bank soil. Pure cultures of S. marcescens or B. thailandensis were amended with 5% (w/v) and 25% (w/v) PM-199 and MRM, respectively, for 9 days. The samples were harvested, and bacterial cell viability was determined using a BacLight staining kit. Results showed that the growth of sorbent-amended S. marcescens was inhibited in contrast to that of unamended control. Furthermore, biochemical assays were used to analyze bacterial biofilm formation and integral biofilm components. Our results suggest that biofilm formation by sorbent-amended S. marcescens was negatively affected. In contrast, B. thailandensis amended with low concentrations of MRM showed enhanced growth and notable differences in biofilm morphology. These results suggest that the use of organoclay PM-199 and MRM at higher concentrations in field studies may hinder the growth of specific soil microorganisms.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Pulsed laser ejection of single-crystalline III-V solar cells from GaAs substrates

The best III-V solar cells start out as single-crystalline multilayers on GaAs substrates. Separating these multilayers from their growth substrate enables higher performance and wafer reuse, which are both critical for terrestrial III-V solar cell viability. Here, we remove rigidly bonded, 16 mm 2 x 3.5μm thick devices from a GaAs substrate using an unfocused Nd:YAG laser pulse. The pulse is absorbed by a low-bandgap, lattice-matched layer below the device, driving an ablation event that ejected the crystalline multilayer from the substrate. Minutes of selective wet-chemical etching and device finishing yield a 0.1 cm 2 device with a 17.4% power conversion efficiency and open-circuit voltage of 1.07 V, using AM1.5 direct with no anti-reflection coating. We show that the performance is comparable to similar cells produced via conventional processes. We discuss unique process characteristics, such as the potential to separate wafer-sized solar cells per laser pulse.

14 SOLAR ENERGY↗

NASA NTRS2024

3D Printing and Surface Engineering of Ti6Al4V Scaffolds for Enhanced Osseointegration in an In Vitro Study

Ti6Al4V superalloy is recognized as a good candidate for bone implants owing to its biocompatibility, corrosion resistance, and high strength-to-weight ratio. While dense metal implants are associated with stress shielding issues due to the difference in densities, stiffness, and modulus of elasticity compared to bone tissues, the surface of the implant/scaffold should mimic the properties of the bone of interest to assure a good integration with a strong interface. In this study, we investigated the additive manufacturing of porous Ti6Al4V scaffolds and coating modification for enhanced osteoconduction using osteoblast cells. The results showed the successful fabrication of porous Ti6Al4V scaffolds with adequate strength. Additionally, the surface treatment with NaOH and Dopamine Hydrochloride (DOPA) promoted the formation of Dopamine Hydrochloride (DOPA) coating with an optimized coating process, providing an environment that supports higher cell viability and growth compared to the uncoated Ti6Al4V scaffolds, as demonstrated by the higher proliferation ratios observed from day 1 to day 29. These findings bring valuable insights into the surface modification of 3D-printed scaffolds for improved osteoconduction through the coating process in solutions.

metal additive manufacturing↗

DOE OSTI2021

Necrosulfonamide reverses pyroptosis-induced inhibition of proliferation and differentiation of osteoblasts through the NLRP3/caspase-1/GSDMD pathway

The acute inflammatory stimulation occurring after a bone fracture regulates the repair and healing of local bone injury; however, under certain conditions, pyroptosis may occur in osteoblasts, which affects osteoblast proliferation and differentiation, thereby affecting the growth, development and morphological changes of bone tissue. The aim of the present study was to examine the effect of the pyroptosis inhibitor necrosulfonamide (NSA) on the proliferation and differentiation of osteoblasts and elucidate the underlying mechanism. The results revealed that NSA reversed the effects of ATP/lipopolysaccharide (LPS) on cell viability and pyroptosis, and on the mRNA and protein expression of pyroptosis-related genes. It also suppressed the secretion of IL-6, TNF-α and IL-1β and reversed the effects of ATP/LPS on the activity of ALP and the mRNA expression of differentiation-related genes in osteoblasts. The fact that overexpression of caspase-1, gasdermin D (GSDMD) and NLRP3 abolished the effects of NSA on the viability and pyroptosis of osteoblasts, as well as the mRNA expression of differentiation-related genes and the activity of ALP in osteoblasts, indicated that NSA promoted the proliferation and differentiation of osteoblasts by inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway. The present study provides proof supporting the potential application of NSA for improving the function of osteoblasts in fracture repair and indicates the value of the NLRP3/caspase-1/GSDMD pyroptosis pathway as a pharmaceutical target.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2005

Influence of increased mechanical loading by hypergravity on the microtubule cytoskeleton and prostaglandin E2 release in primary osteoblasts

Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E2 (PGE2). In immature osteoblasts, a stimulus of 10x gravity (10 g) for 3 h increased PGE2 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (5-50 g) increases in PGE2 (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE2 release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts.

Hypergravity↗

NASA NTRS1999

Three-Dimensional, Transgenic Cell Models to Quantify Space Genotoxic Effects

The space environment contains radiation and chemical agents known to be mutagenic and carcinogenic to humans. Additionally, microgravity is a complicating factor that may modify or synergize induced genotoxic effects. Most in vitro models fail to use human cells (making risk extrapolation to humans more difficult), overlook the dynamic effect of tissue intercellular interactions on genotoxic damage, and lack the sensitivity required to measure low-dose effects. Currently a need exists for a model test system that simulates cellular interactions present in tissue, and can be used to quantify genotoxic damage induced by low levels of radiation and chemicals, and extrapolate assessed risk to humans. A state-of-the-art, three-dimensional, multicellular tissue equivalent cell culture model will be presented. It consists of mammalian cells genetically engineered to contain multiple copies of defined target genes for genotoxic assessment,. NASA-designed bioreactors were used to coculture mammalian cells into spheroids, The cells used were human mammary epithelial cells (H184135) and Stratagene's (Austin, Texas) Big Blue(TM) Rat 2 lambda fibroblasts. The fibroblasts were genetically engineered to contain -a high-density target gene for mutagenesis (60 copies of lacl/LacZ per cell). Tissue equivalent spheroids were routinely produced by inoculation of 2 to 7 X 10(exp 5) fibroblasts with Cytodex 3 beads (150 micrometers in diameter). at a 20:1 cell:bead ratio, into 50-ml HARV bioreactors (Synthecon, Inc.). Fibroblasts were cultured for 5 days, an equivalent number of epithelial cells added, and the fibroblast/epithelial cell coculture continued for 21 days. Three-dimensional spheroids with diameters ranging from 400 to 600 micrometers were obtained. Histological and immunohistochemical Characterization revealed i) both cell types present in the spheroids, with fibroblasts located primarily in the center, surrounded by epithelial cells; ii) synthesis of extracellular matrix; and iii,, mitotic cells located throughout the spheroids. Spheroidal integrity and cell viability were retained for the 30-day test period after removal of spheroids from the bioreactor. Potential utility of this three-dimensional, transgenic model for genotoxicity was initially assessed by exposure of spheroids to 0-2 Gy neon at dose rates of 0.3 to 1.5 Gy/min (National Institute of Radiological Sciences, Chiba, Japan). Quantification of mutation at the lacl gene revealed a linear dose response for mutation induction. Limited sequencing analysis of mutant clones revealed higher frequencies of deletions and multiple base sequence changes with increasing dose. These results suggest that our three-dimensional, transgenic model is applicable to a wide variety of studies involving the quantification, identification, and characterization of genotoxicity incurred in space and on Earth. This model uniquely allows investigation of the interaction of relevant factors, namely cell-to-cell interactions and the mechanistic interaction of microgravity with radiation insults and DNA repair. Using this three-dimensional model will allow us to obtain dual genotoxic information (i.e., mutation rate plus chromosome aberration data) from the same system so that one endpoint can be used to reference the other, thereby increasing the fidelity of the data set. Moreover, the tissue-equivalent nature of the three-dimensional model provides high confidence for relevance of risk assessment, i.e., the establishment of quality factors directly applicable to the microgravity environment.

Gonda, S. R.↗

DOE OSTI2024

Rapid curing dynamics of PEG-thiol-ene resins allow facile 3D bioprinting and in-air cell-laden microgel fabrication

Thiol-norbornene photoclick hydrogels are highly efficient in tissue engineering applications due to their fast gelation, cytocompatibility, and tunability. In this work, we utilized the advantageous features of polyethylene glycol (PEG)-thiol-ene resins to enable fabrication of complex and heterogeneous tissue scaffolds using 3D bioprinting and in-air drop encapsulation techniques. We demonstrated that photoclickable PEG-thiol-ene resins could be tuned by varying the ratio of PEG-dithiol to PEG norbornene to generate a wide range of mechanical stiffness (0.5–12 kPa) and swelling ratios. Importantly, all formulations maintained a constant, rapid gelation time (<0.5 s). We used this resin in biological projection microstereolithography (BioPµSL) to print complex structures with geometric fidelity and demonstrated biocompatibility by printing cell-laden microgrids. Moreover, the rapid gelling kinetics of this resin permitted high-throughput fabrication of tunable, cell-laden microgels in air using a biological in-air drop encapsulation apparatus (BioIDEA). We demonstrated that these microgels could support cell viability and be assembled into a gradient structure. This PEG-thiol-ene resin, along with BioPµSL and BioIDEA technology, will allow rapid fabrication of complex and heterogeneous tissues that mimic native tissues with cellular and mechanical gradients. The engineered tissue scaffolds with a controlled microscale porosity could be utilized in applications including gradient tissue engineering, biosensing, and in vitro tissue models.

36 MATERIALS SCIENCE↗

DOE OSTI2023

Immunomodulation by cannabidiol in bovine primary ruminal epithelial cells

Ruminant livestock experience a number of challenges, including high concentrate diets, weaning and transport, which can increase their risk of disorders such as ruminal acidosis, and the associated inflammation of the ruminal epithelium. Cannabidiol (CBD), a phytochemical from hemp (Cannabis sativa), is a promising target as a therapy for gastrointestinal inflammation, and may be extremely valuable as either a treatment or prophylactic. However, the effects of CBD in the the ruminant gastrointestinal tract have not been explored, in part due to the restrictions on feeding hemp to livestock. Therefore, the objective of this study was to investigate the immunomodulatory properties of CBD using a model of inflammation in primary ruminal epithelial cells (REC). In addition, CBD dose was evaluated for possible cytotoxic effects. Negative effects on cell viability were not observed when REC were exposed to 10 μM CBD. However, when the dose was increased to 50 μM for 24 h, there was a significant cytotoxic effect. When 10 μM CBD was added to culture media as treatment for inflammation induced with lipopolysaccharide (LPS), expression of genes encoding for pro-inflammatory cytokine IL1B was less compared to LPS exposure alone, and CBD resulted in a down-regulation of IL6. As a pre-treatment, prior to LPS exposure, REC had decreased expression of IL6 and CXCL10 while CBD was present in the media, but not when it was removed prior to addition of LPS. Results suggest that CBD may reduce cytokine transcription both during LPS-induced inflammation and when used preventatively, although these effects were dependent on its continued presence in the culture media. Overall, these experiments provide evidence of an immunomodulatory effect by CBD during a pro-inflammatory response in primary REC in culture.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

N -Terminal Octylated Peptoid Hydrogels as 3D-Printable Cell Scaffolds and Proteolytically Robust Cargo Depots

Supramolecular hydrogels that mimic the extracellular matrix (ECM) represent promising materials for tissue engineering and drug delivery. However, conventional hydrogels formed via the self-assembly of natural or synthetic building blocks often face a trade-off between biological functionality and biochemical stability, limiting their utility in long-term or protease-rich environments. Peptoids, a class of peptide-inspired, sequence-defined polymers, offer a compelling alternative due to their exceptional proteolytic resistance and bioactivity. Despite this potential, the development of supramolecular peptoid hydrogels has been hindered by the absence of backbone hydrogen bond donors, which limits long-range ordering necessary for efficient hydrogel formation. This work describes a short peptoid functionalized at the N-terminus with an octyl chain that readily self-assembles into hydrogels. Hydrophobic interactions among pendant octyl groups promote directional peptoid packing into highly ordered nanosheets, which interconnect to form a porous hydrogel network. These hydrogels exhibit tunable viscoelasticity, shear-thinning, and self-healing properties, enabling their use as inks for extrusion-based 3D printing. They support NIH-3T3 fibroblast adhesion, spreading, and proliferation, maintaining greater than 95% cell viability over 4 days. Moreover, the hydrogels retain their macroscopic integrity under protease-rich conditions, enabling sustained cargo release and uniform cellular uptake. Together, this study demonstrates a class of supramolecular peptoid hydrogelators that integrate biocompatibility, 3D printability, and proteolytic stability, providing a versatile platform for ECMmimetic scaffolds in regenerative medicine and long-term therapeutic delivery.

cargo delivery↗

NASA NTRS2002

Pulmonary toxicity of simulated lunar and Martian dusts in mice: II. Biomarkers of acute responses after intratracheal instillation

Volcanic ashes from Arizona and Hawaii, with chemical and mineral properties similar to those of lunar and Martian soils, respectively, are used by the National Aeronautics and Space Administration (NASA) to simulate lunar and Martian environments for instrument tests. NASA needs toxicity data on these volcanic soils to assess health risks from potential exposures of workers in facilities where these soil simulants are used. In this study we investigated the acute effects of lunar soil simulant (LSS) and Martian soil simulant (MSS), as a complement to a histopathological study assessing their subchronic effects (Lam et al., 2002). Fine dust of LSS, MSS, TiO(2), or quartz suspended in saline was intratracheally instilled into C57Bl/6J mice (4/group) in single doses of 0.1 mg/mouse or 1 mg/mouse. The mice were euthanized 4 or 24 h after the dust treatment, and bronchoalveolar lavage fluid (BALF) was obtained. Statistically significant lower cell viability and higher total protein concentration in the BALF were seen only in mice treated with the high dose of quartz for 4 h and with the high dose of MSS or quartz for 24 h, compared to mice treated only with saline. A significant increase in the percentage of neutrophils was not observed with any dust-treated group at 4 h after the instillation, but was observed after 24 h in all the dust-treated groups. This observation indicates that these dusts were not acutely toxic and the effects were gradual; it took some time for neutrophils to be recruited into and accumulate significantly in the lung. A statistically significant increase in apoptosis of lavaged macrophages from mice 4 h after treatment was found only in the high-dose silica group. The overall results of this study on the acute effects of these dusts in the lung indicate that LSS is slightly more toxic than TiO(2), and that MSS is comparable to quartz. These results were consistent with the subchronic histopathological findings in that the order of severity of lung toxicity was TiO(2) < LSS < MSS < quartz.

Non-programmatic↗

DOE OSTI2021

Head on Comparison of Self- and Nano-Assemblies of Gamma Peptide Nucleic Acid Amphiphiles

Peptide nucleic acids (PNAs) are nucleic acid analogs with hybridization properties and enzymatic stability superior to that of DNA. In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymers due to the Watson–Crick-based molecular recognition and flexibility of synthesis. Here, in this work, PNA amphiphiles are engineered using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA ($\gamma$PNA) amphiphiles self-assemble into spherical vesicles. Further, nano-assemblies (NA) are formulated using the amphiphilic $\gamma$PNA as a polymer via ethanol injection-based protocols. Comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and NA is performed. Small-angle neutron and X-ray scattering analysis reveal ellipsoidal morphology of $\gamma$PNA NA that results in superior cellular delivery compate to the spherical self-assembly. Next, the functional activities of $\gamma$PNA self-and NA in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays are compared. Overall, it is established that $\gamma$PNA amphiphile is a functionally active bio-polymer to formulate NA for a wide range of biomedical applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

A Pro‐Angiogenic Immunoprotective Membrane for Cell Therapies

Abstract Immunoisolation strategies that rely on porous membranes play an important role in cell transplantation therapies to protect cells from the host's immune system. These membranes must possess immunoprotective properties while facilitating the transport of nutrients and cell products to maintain the functional integrity of encapsulated cells. An easy and scalable process is described to fabricate a dual function porous polymeric membrane that shields cells against immune cell attack and promotes vascularization to address the nutritional and oxygen requirements of transplanted cells. The fabrication process results in a membrane cross‐section with a gradient of nanopores to micropores that support cell immunoisolation and interfacial vascularization requirements, respectively. The membranes demonstrate excellent cell compatibility and effectively prevent T cell transmigration without compromising glucose diffusion and oxygen permeability. In a murine subcutaneous implantation model, membranes are stable for 60 days and exhibit significantly reduced fibrous capsules, with enhanced vascularization near the membrane. These porous polymeric membranes can potentially be used as pro‐angiogenic immunoprotective membranes for cell transplantation applications where maximizing cell viability and function is of critical importance.

Engineering↗

DOE OSTI2021

Fed-batch production of deuterated protein in Escherichia coli for neutron scattering experimentation

Neutron scattering is a powerful technique for determining the structure and dynamics of biological materials in a variety of environmental conditions. A distinguishing property of the neutron is its sensitivity to detecting hydrogen and distinguishing it from its isotope deuterium. This enables unique types of experiments that take advantage of this differential sensitivity called isotopic contrast variation. Using this approach, the chemistry of the system is not changed, but the visibility of individual sample components can be tuned by varying the deuterium content of the system under investigation. Deuterated proteins are commonly produced in bacterial systems that are adapted to growth in D2O minimal media. To maximize the yield of deuterium-labeled protein and efficiently utilize D2O and occasionally the deuterated substrate, fed-batch processes are routinely used to maximize biomass production without compromising cell viability. A step-by-step procedure will be described along with a case study of the production of deuterated green fluorescent protein. Limitations of the process will also be discussed.

Weiss, Kevin L.↗

DOE OSTI2023

A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

59 BASIC BIOLOGICAL SCIENCES↗