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This tensile test is to mimic the worst-case scenario of the swab being caught on an obstruction when being pulled out of the nasopharyngeal space. It is to determine how much tensile force the swab can withstand without breaking.
Swabs Tested: 1. FAST Spiral NP Swab, designed and supplied by Abiogenix, printed from polyamide 11 (Nylon) using HP Multi Jet Fusion printers. 2. NP Swab designed by USF Health and Northwell Health in collaboration with Formlabs. Printed at LLNL using the Form 3B stereolithography printer in Formlabs Surgical Guide resin.
This report is an assessment of the availability, demand, and production capability of medical consumables needed to combat the COVID-19 pandemic. We focus here on the ability of the supply chain to increase production to meet demand during peak pandemic conditions. In addition, we report on domestic production efforts and supply chain issues, as well as the propensity of other countries to limit export of needed medical supplies to the United States. In brief, the domestic supply chain and production capability vary greatly depending on the consumable. Some consumables, such as N95 masks or face shields, can likely be nearly fully supplied by domestic production, while other consumables, such as gloves or surgical masks, have little domestic production and thus will depend on imports to meet demand. The assessment pulls primarily from published news sources and company press releases. As such, the demand and production numbers are not precisely known, and some degree of uncertainty exists in them. The total production capability is also often difficult to ascertain from these reports especially as other industries have begun to supplement existing supply chains. In addition, local conditions (including company financial decisions, local public health issues, and political aspects) can vary throughout the pandemic which will affect future production. The report also pulls from estimates of demand and domestic production of some consumables compiled by the White House COVID-19 Supply Chain Taskforce (SCTF) presented by Rear Admiral John Polowczyk before a June 9, 2020 hearing of the Senate Homeland Security and Governmental Affairs Committee. These numbers, where applicable, are likely to be more accurate than those found in public news sources since they are more directly tied to private companies’ actual orders and actual production estimates, as opposed to publicly released estimates. Finally, we note that other issues with the supply chain, including the production of raw materials, increase demand due to industries that have not previously used PPE now using it, and increased domestic production from non-traditional suppliers, make complete picture of a rapidly changing supply chain difficult to obtain. This report attempts to produce an accurate image of the supply and estimated demand, as understood by the authors who are not experts in the medical supply chain.
Recent advances in microphysiological systems (MPS) have made significant strides to include design features that reconstruct key elements found in the brain, and in parallel advanced technologies to detect the activity of electrogenic cells that form neural networks. In particular, three-dimensional multielectrode arrays (3D MEAs) are being developed with the increasing levels of spatial and temporal precision, difficult to achieve with current 2D MEAs, insertable MEA probes, and/or optical imaging of calcium dynamics. Thus, providing a means to monitor the flow of neural network activity within all three dimensions (X, Y, and Z) of the engineered tissue. In the last 6 years, 3D MEAs, using either “bottom-up” or “top-down” designs, have been developed to overcome the current technical challenges in monitoring the functionality of the in vitro systems. Herein, we will report on the design and application of novel 3D MEA prototypes for probing neural activity throughout the 3D neural tissue.
Engineered protein compartmentalization systems hold significant promise to enhance reaction efficiencies through co-localization, concentration, and sequestration of biosynthetic pathways. As such, they have the potential to enable the bioproduction of next generation bioproducts and biomaterials in genetically engineered microbes in support of DOE’s mission to build a strong bioeconomy. Among systems of particular interest are protein nanocompartment systems called encapsulins that are natively produced by a variety of bacteria including those with a high potential for bioproduction. This ECRP project is focused on understanding how encapsulins can be used to enhance the biosynthesis of next-generation biomaterials in Rhodococcusspecies. Specifically, we seek: (1) to probe the mechanistic basis for how these compartments are regulated, biosynthesized, and maintained, and (2) to engineer these systems to achieve new biosynthetic functions (e.g., CdS nanoparticle biosynthesis). We anticipate that this work will establish encapsulin compartmentalization systems as a means of improving yields and enabling biosynthetic routes toward new biomaterials, thus advancing the U.S. bioeconomy.
LLNL designed and built a rapid, RT-LAMP-based molecular diagnostics platform as a potential tool to quickly diagnose COVID-19 in under one hour. This point-of-care testing approach involves an initial high temperature swab sample inactivation step followed by amplification of viral RNA using up to 5 control and pathogen-specific assays. Results are determined based on a discreet reaction color change from red to yellow but can also be determined using fluorescence detection. Testing of this prototype platform was conducted with synthetic viral RNA and dried, stabilized reagents. Buffer systems, swab selection, and assay stabilization formulations were evaluated for performance. Limits of detection were determined using RNA; however, testing was not performed with viable SARS-CoV-2 virus or clinical samples.
LLNL produced two lots of lyophilized LAMP assay containing cartridges, LNL022818 and LNL071818, for testing on Gen3 BioID systems. Thirty-nine cartridges were tested for lot LNL022818. All thirty-nine cartridges performed as expected regarding correct positive control (IPC) and negative control (NTC) reactions. Five different templates were tested on these 39 cartridges. The BioID system detected 79.1% true positives and 9.3% false positives along with 98.9% true negatives and 2.9% false negatives for cartridge lot LNL022818. Thirty cartridges were tested for lot LNL071818. It was noted early that due to a manufacturing issue with the cartridges related to incomplete sonic welding of the cartridge, a large fraction of lot LNL071818 cartridges leaked. Insufficient funds were available to replace this lot of cartridges. Twenty three of 30 cartridges performed as expected regarding correct positive control (IPC) and negative control (NTC) reactions. Four different templates were tested on these 30 cartridges. The BioID system detected 77% true positives and 23% false positives along with 96.9% true negatives and 3.1% false negatives for cartridge lot LNL071818. For all tested cartridges using DNA template, DNA was added at a final concentration of 25 pg/ul. All reactions contained positive control DNA at 2.5 pg/ul.
There is a critical need for new approaches to effectively counter emerging pathogens, especially those which do not respond to antibodies or antibiotics. T cells represent an essential element of native immune response in many of the deadliest pathogens: controlling T cell reactivity and behavior would allow for countermeasures for currently untreatable diseases from cancer to coronaviruses, especially if using a patient’s own T cells (autologous) where no host rejection will occur. However, current methods for modifying T cells, e.g., FDA-approved chimeric antigen receptor T cell (CAR) approaches, require genetic manipulation and expansion which can take weeks to generate.
Instead of genetic engineering, we sought to determine if a rapid method of membrane protein delivery could generate functional CAR-T cells in a rapid, safer, cost-effective manner. Using cell free protein synthesis, we generated CAR proteins embedded in a nanodisc, and thus effectively solubilized the protein. We demonstrated the first functional CAR proteins outside of a cellular context. We next tested uptake into immune cells and found extremely high uptake into T cells and multiple other populations of immune cells. We found some evidence of cytotoxicity in vitro conferred by the nanodisc delivered protein.
LLNL led two major Technical Areas (TAs) within the PEDOMETER program: TA3 focused on biocontainment and TA4 focused on developing electrochemical TNT degradation sensors and testbeds. A key takeaway from our work is the importance of chassis host strain choice for kill switch design and actuator choice. Genetic instability of the kill switch circuit is a major barrier toward establishing a kill switch, which is host dependent. Addition of a host down-selection step to regulator and actuator screening stages would be beneficial to hasten kill switch development.
The organization of genomic DNA into chromatin is a fundamental determinant of genome stability, regulation, and cellular function. Nucleosomes, the basic repeating units of chromatin, assemble into higher-order structures whose organization and heterogeneity remain difficult to characterize using conventional ensemble-averaged techniques. A key need in the field is the development of experimental approaches capable of directly visualizing nucleosome assemblies and their structural variability at the single-molecule level. This LDRD Lab-Wide project focused on establishing and evaluating atomic force microscopy (AFM)–based approaches for the characterization of nucleosome assemblies. The work emphasized experimental workflows for preparing, imaging, and assessing multi-nucleosome systems, rather than isolated single nucleosomes. Through method development and exploratory measurements, the project demonstrated the feasibility of applying scanning probe microscopy to investigate chromatin-relevant assemblies and provided preliminary insight into the strengths and limitations of this approach for future quantitative studies. Results and lessons learned from this effort were disseminated to the broader scientific community through multiple national conference presentations, helping to position LLNL for continued work in chromatin and genome organization research.
Acute care of patients with severe infections often relies on systemic administration of pharmaceuticals and monitoring of complex physiological symptoms to identify immune system dysfunction, which can lead to increased mortality. Furthermore, determining disease-specific treatment plans often leads to a delay in patient care. To address this, we proposed an immune modulation system that electrically detects and responds to a patient’s immune system status, creating an agnostic means of treating illness and infection. Two pieces of hardware were developed for this task: a minimally-invasive sensor and a vagus nerve stimulator. Stimulation of the vagus nerve is known to modulate the immune system. The sensor is a microfabricated, silicon-based microneedle array capable of interfacing with interstitial fluid to detect small molecules such as inflammatory proteins (cytokines) and pharmaceuticals (vancomycin). Process optimization to manufacture the needles refined the silicon etch process, creating needle patches long enough to penetrate skin and reach interstitial fluid. The needles were tested for mechanical strength and stability, and did not shatter when inserted into skin models. The needles are coated with a thin film metal, turning them into electrodes for electrochemical sensing of our target molecules. We hybridized aptamers to the surface of the electrode to act as the sensing layer and were able to detect changes in the conformation of the aptamer electrochemically in the presence of the target molecule. The stimulator was a cuff electrode that encircled the vagus nerve. Rodent studies were conducted in which rodents were exposed to an inflammatory event and vagus nerve stimulation (VNS) was applied. It was demonstrated that optimized electrical stimulation of the vagus nerve created measurably different levels of cytokines in blood samples, and certain cytokines released during the inflammatory event were either upregulated or downregulated. In sum, this project successfully developed new platforms and technologies that can, with further development, enable better temporal insight into biomarker changes in the body, letting healthcare providers know of possible immune system dysfunction before they are detected physiologically. We also demonstrated the value of VNS and its possible use in treating immune system response to inflammation and illness.
Achieving decarbonization and electrification goals will require expanded production of critical minerals (CM), including Li, Co, Cu, rare earths, Ni, and graphite, whose supply chains are geopolitically vulnerable. Problematically, current extraction and separation processes pose severe environmental burdens that impede the development of a diversified domestic supply chain and undercut the environmental benefits of energy technologies [1, 2]. The development of efficient, economical, and environmentally sustainable processing technologies is thus important for meeting the CM demand of the emerging energy technology market. To this end, we have recently developed an all-aqueous protein-based process for rare earth element (REE) extraction and separation. To extend our protein-based approach to critical metals beyond REEs, the goal of this project was to develop a protein discovery and engineering pipeline to generate a panel of proteins that selectively bind target critical metals.
Engineered protein compartmentalization systems hold significant promise to enhance reaction efficiencies through co-localization, concentration, and sequestration of biosynthetic pathways. As such, they have the potential to enable the bioproduction of next generation bioproducts and biomaterials in genetically engineered microbes in support of DOE’s mission to build a strong bioeconomy. Among systems of particular interest are protein nanocompartment systems called encapsulins that are natively produced by a variety of bacteria including those with a high potential for bioproduction. This ECRP project is focused on understanding how encapsulins can be used to enhance the biosynthesis of next-generation biomaterials in Rhodococcus species. Specifically, we seek: (1) to probe the mechanistic basis for how these compartments are regulated, biosynthesized, and maintained, and (2) to engineer these systems to achieve new biosynthetic functions (e.g., alkene, inorganic nanoparticle biosynthesis). We anticipate that this work will establish encapsulin compartmentalization systems as a means of improving yields and enabling biosynthetic routes toward new biomaterials, thus advancing the U.S. bioeconomy.
Please cite as : Josué A. Rodríguez-Ramos, Amy E. Zimmerman, Ruonan Wu, Sheryl Bell, Trinidad Alfaro, Kirsten Hofmockel, William C. Nelson. 2025. Inter-Kingdom Viral Interactions. [Data Set] PNNL DataHub. This data is published under a CC0 license. The authors encourage data reuse and request attribution by referencing the above citations for the data package and associated manuscript. Deciphering viral ecology in soils is challenging due to their high physiochemical and community complexity. To enhance detection of sub-communities of DNA and RNA viruses, we applied fractionation approaches to soils collected across a moisture gradient from a grassland field experiment. Analyses included metagenomics and metatranscriptomics of size-fractionated extracellular viruses (i.e., DNA and RNA viromes), metagenomics of bacteria/archaea- or eukaryote-enriched samples, and whole soil metatranscriptomes with rRNA-depletion or polyadenylation enrichment. While RNA virome and whole soil RNA methods captured similar viral diversity, RNA viromes identified longer, higher-quality genomes. Further, we showed that significantly more DNA viruses were active in higher moisture than lower moisture samples, whereas responses by overall diversity vary by genome type (DNA versus RNA genomes). Finally, we demonstrate the power of fractionation approaches for identifying distinct viral communities that infect unique hosts, which has significant implications for ecological investigations, particularly related to interkingdom interactions.
We tested 294 domestic pet dogs in Mexico for neutralizing antibodies for mosquito-borne flaviviruses. We found high (42.6%) exposure to West Nile virus in Reynosa (northern Mexico) and low (1.2%) exposure in Tuxtla Gutierrez (southern Mexico) but very limited exposure to Aedes-borne flaviviruses. In conclusion, domestic dogs may be useful sentinels for West Nile virus.
The terrestrial serpentinite-hosted ecosystem known as “The Cedars” is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars (‘Paeni-Cedars’), Alishewanella sp. BS5-314 (‘Ali-BS5-314’), and Anaerobacillus sp. CMMVII (‘Anaero-CMMVII’). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7–10 (minimum pH tested was 7), temperatures 20–40°C, and 0–3% NaCl concentration. The addition of 10–20 mM CaCl 2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10–12 and temperatures 10–40°C, with limited growth observed 1–5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9–12, with the addition of 0.01–1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.