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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 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Lycorine targets multiple myeloma stem cell‐like cells by inhibition of Wnt/β‐catenin pathway

Summary Multiple myeloma (MM) is characterised by the proliferation and accumulation of malignant plasma cells in the bone marrow. Despite the progress in treatment over the last few years, MM remains incurable and the majority of patients relapse. MM stem‐like cells (MMSCs) have been considered as the main reason for drug resistance and eventual relapse. Currently, therapeutic agents are not enough to eradicate MMSCs, and finding effective strategies to eradicate MMSCs may improve the outcome of patients. Here we showed that lycorine, a natural compound from the Amaryllidaceae species, effectively inhibits the proliferation of myeloma cells from cell lines or patients, mainly through decreasing ALDH1 + cells. Mechanistically, lycorine decreases the MMSC population through inhibition of the Wnt/β‐catenin pathway by reducing the β‐catenin protein level. Moreover, lycorine could overcome the increasing proportion of ALDH1 + cells caused by bortezomib (BTZ) treatment, and a combination BTZ and lycorine have a synergistic effect on anti‐myeloma cells. Furthermore, we found a similar reduction of MMSC characteristics by lycorine in BTZ‐resistant MM cells and primary CD138 + plasma cells. Collectively, our findings indicate lycorine as a promising agent to target MMSCs to overcome the drug resistance of BTZ, and that, alone or in combination with BTZ, lycorine is a potential therapeutic strategy for MM treatments.

Wang, Haiqin↗
DOE OSTI2023

Interchangeability of the Assays Used to Assess the Activity of Anti-SARS-CoV-2 Monoclonal Antibodies

The recent global COVID-19 pandemic caused by SARS-CoV-2 lasted for over three years. A key measure in combatting this pandemic involved the measurement of the monoclonal antibody (mAb)-mediated inhibition of binding between the spike receptor-binding domain (RBD) and hACE2 receptor. Potency assessments of therapeutic anti-SARS-CoV-2 mAbs typically include binding or cell-based neutralization assays. We assessed the inhibitory activity of five anti-SARS-CoV-2 mAbs using ELISA, surface plasmon resonance (SPR), and four cell-based neutralization assays using different pseudovirus particles and 293T or A549 cells expressing hACE2 with or without TMPRSS2. We assessed the interchangeability between cell-based and binding assays by applying the Bland–Altman method under certain assumptions. Our data demonstrated that the IC50 [nM] values determined by eight neutralization assays are independent of the cell line, presence of TMPRSS2 enzyme on the cell surface, and pseudovirus backbone used. Moreover, the Bland–Altman analysis showed that the IC50 [nM] and KD [nM] values determined by neutralization/ELISA or by SPR are equivalent and that the anti-spike mAb activity can be attributed to one variable directly related to its tertiary conformational structure conformation, rate dissociation constant Koff. This parameter is independent from the concentrations of the components of the mAb:RBD:hACE2 complexes and can be used for a comparison between the activities of the different mAbs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Probing the optical properties and toxicological profile of zinc tungstate nanorods

Zinc tungstate is a semiconductor known for its favorable photocatalytic, photoluminescence, and scintillation properties, coupled with its relatively low cost, reduced toxicity, and high stability in biological and catalytic environments. In particular, zinc tungstate evinces scintillation properties, namely the ability to emit visible light upon absorption of energetic radiation such as x rays, which has led to applications not only as radiation detectors but also for biomedical applications involving the delivery of optical light to deep tissue, such as photodynamic therapy and optogenetics. Here, we report on the synthesis of zinc tungstate nanorods generated via an optimized but facile method, which allows for synthetic control over the aspect ratio of the as-synthesized anisotropic motifs via rational variation of the solution pH. Additionally, we investigate the effect of aspect ratio on their resulting photoluminescent and radioluminescent properties. We further demonstrate the potential of these zinc tungstate nanorods for biomedical applications, such as photodynamic therapy for cancer treatment, by analyzing their toxicological profile within cell lines and neurons.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products’ efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells

Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2021

Three-dimensional spheroid culture induces apical-basal polarity and the original characteristics of immortalized human renal proximal tubule epithelial cells

Highlights: • Immortalized proximal tubule cells (HK-2 cells) formed a tubule-like structure. • The 3D spheroid culture enhanced apical-basal polarity of HK-2 cells. • The 3D spheroid culture induced Na transport function of HK-2 cells. • The 3D spheroid culture promoted up-regulation of kidney development-related genes. The proximal tubules, which are part of the kidney, maintain blood homeostasis by absorbing amino acids, glucose, water, and ions such as sodium (Na), potassium, and bicarbonate. Proximal tubule dysfunction is associated with the pathogenesis of many kidney diseases. Renal proximal tubular epithelial cells (RPTECs) are responsible for the main functions of the proximal tubules. Therefore, in vitro experiments using RPTECs would greatly enhance our understanding of nephron physiology and pathobiology. It is preferable to use immortalized cell lines, such as human kidney-2 (HK-2) cells, because they are derived from humans and maintain growth indefinitely. However, tissue-specific RPTEC phenotypes, including apical-basal polarization, are frequently lost in conventional two-dimensional culture methods in part due to microenvironmental deficiencies. To overcome this limitation, we developed a three-dimensional (3D) spheroid culture method for HK-2 cells using an extracellular matrix. HK-2 spheroids in 3D culture formed a tubule-like architecture with cellular polarity and showed markedly restored Na transport function. 3D culture of HK-2 cells also increased expression of kidney development–related genes, including WNT9B. Models of human renal tubules using HK-2 spheroids will greatly improve our understanding of the physiology and pathobiology of the kidney.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Transient histone deacetylase inhibition reveals cell type invariant and specific effects of chromatin decondensation on irradiation response

Radiation therapy plays a prominent role in breast cancer treatment, but the high doses of radiation damage both healthy and cancerous cells. Therefore, additional research is needed into combination therapies that could preferentially radiosensitize cancer cells compared to surrounding healthy tissue without causing deleterious side effects. Histone deacetylase inhibitor drugs (HDACis) have been tested as radiosensitizers in both basic research and clinical trials, but the long exposure time typically used in these treatments and the lack of matched healthy cell controls often leave aspects of their mechanism of action unclear. Here, we show that transient (2 h) trichostatin A (TSA) treatment of cancerous and non-tumorigenic breast epithelial cell lines increases immediate DNA damage and decreases long term cell viability in both cell types at high radiation doses. Transient TSA treatment also causes an increase in DNA damage signals after 5 Gy X-rays in other cancer and healthy cell types: A375 melanoma cells and BJ5-ta fibroblasts. This suggests that chromatin decompaction acts to increase cellular vulnerability to initial DNA damage from high doses of radiation in a cell type independent manner that does not rely on changes to DNA repair pathways caused by longer TSA treatment. However, responses to lower doses of radiation and long term survival are more cell type specific: only MCF7 cells experience an effect of TSA on DNA damage after 1 Gy X-ray radiation while MCF10a cells experience somewhat more evident cell viability effects of combined TSA and radiation treatment long term.

Li, Heng [Biochemistry & Cellular and Molecular Bi↗
DOE OSTI2021

Vimentin loss promotes cancer proliferation through up-regulating Rictor/AKT/β-catenin signaling pathway

Vimentin protein is one of the main cytoskeleton and plays an important role in cell motility and metastasis. Nowadays, vimentin is widely studied as an epithelial-mesenchymal transition (EMT) marker of cancer cells while its involvement in cancer proliferation is poorly understood. In this study, we investigated the participation of vimentin in regulating cancer proliferation by silencing VIM gene in four cancer cell lines. Our results demonstrated that vimentin loss significantly induced cancer cell proliferation both in vitro and in vivo, which has not been reported so far. Mechanistically, knockdown of vimentin expression activated AKT phosphorylation and its downstream β-catenin signaling. Nuclear translocation and transcriptional activity of β-catenin was enhanced after silencing vimentin expression. Furthermore, vimentin loss could prevent Rictor from autophagy-dependent degradation via reducing AMPK-mediated autophagy signaling. AICAR, an AMPK activator, down-regulated Rictor and p-AKT levels while vimentin knockdown could rescue the effects. In vivo, it was also found that Ki67 expression and p-AKT/β-catenin signaling pathway were obviously up-regulated in the tumor tissues in which vimentin was silenced compared to control groups. Taken together, these data showed the novel function of vimentin in regulating cancer proliferation via Rictor/AKT/β-catenin signaling pathway, which suggested that it need more careful consideration before inhibiting metastatic cancers through targeting vimentin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Anticancer effect of AZD2461 PARP inhibitor against colon cancer cells carrying wt or dysfunctional p53

Colon cancer is one of the most common cancers, currently treated with traditional chemotherapies or alternative therapies. However, these treatments are still not enough effective and induce several side effects, so that the search of new therapeutic strategies is needed. The use of Poly-(ADP-ribose)-polymerase (PARP) inhibitors, although originally approved against BRCA-1 or BRCA-2 mutated cancers, has been extended, particularly in combination with other treatments, to cure cancers that do not display defects in DNA repair signaling pathways. The role of p53 oncosuppressor in the regulating the outcome of PARP inhibitor treatment remains an open issue. In this study, we addressed this topic by using a well-tolerated PARP 1/2/3 inhibitor, namely AZD2461, against colon cancer cell lines with different p53 status. We found that AZD2461 reduced cell proliferation in wtp53 and p53−/− cancer cells by increasing ROS and DNA damage, while R273H mutant (mut) p53 counteracted these effects. Moreover, AZD2461 improved the reduction of cell proliferation by low dose radiation (IR) in wtp53 cancer cells, in which a down-regulation of BRCA-1 occurred. AZD2461 did not affect cell proliferation of mutp53 colon cancer cells also in combination with low dose radiation, suggesting that only wt p53 or p53 null colon cancer cells could benefit AZD2461 treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Serum amyloid A1: Innocent bystander or active participant in cell migration in triple-negative breast cancer?

Highlights: • SAA1 knockdown reduced cell viability in triple-negative breast cancer cells. • SAA1 knockdown induced a S to G{sub 2}/M cell cycle shift in MDA-MB-231 cells. • SAA1 silencing inhibited cell migration in MDA-MB-231 and HCC70 cells. • SAA1 silencing altered epithelial-to-mesenchymal transition markers in MDA-MB-231 cells. The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G{sub 2}/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1β, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

The Gene Fitness Atlas: A Roadmap for Predicting Evolution

We developed a novel, high-throughput microfluidic device design containing “interaction zones” where progeny cell lines compete against each other allowing for accurate analysis of bacterial cell fitness. The goal of the project was to use the device for two applications: 1) gene knockout screening and 2) antibiotic resistance screening. The microfluidic platform was fabricated using photolithography and soft lithography in polydimethylsiloxane (PDMS). E.coli Keio mutants and fluorescent wildtype parent were chosen for the study. Cells were grown overnight and their loading into the devices and seeding in mother machines was optimized. For mutant screening, the least fit mutant and wildtype parent were cultured individually and then added to the microfluidic device. The mother machines which were seeded with mutant and wildtype were imaged through time lapse microscopy and the growth of cells was observed. For antibiotic screening, wildtype E.coli cells which were grown overnight were added to the device and washed with media containing the antibiotic ampicillin. The growth pattern in presence and absence of ampicillin was observed through time lapse microscopy. It was observed that over a period of four hours, both the mutant and the wildtype divided in the mother machine and pushed daughter cells out into the interaction zone. In case of the antibiotic screening experiment, the fluorescent wildtype divided both in the absence and presence of sublethal concentration of ampicillin. This study is a proof of concept demonstration of high- throughput single cell analysis of cells using a novel microfluidics device.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A human cell type similar to murine central nervous system perivascular fibroblasts

Highlights: • Human diploid cell lines were established from tumor and non-tumor brain tissue. • Human cells share a common transcriptomic profile with murine perivascular fibroblasts. • Human cells resemble commercially available cell of presumed pericyte origin. The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g. for treatment of brain tumors. In the murine brain, perivascular fibroblasts were recently identified as a novel potential constituent of the BBB. Here we present the existence of human cells that could be the equivalent to the murine brain perivascular fibroblasts. Using RNA sequencing, we show a similar transcriptomic profile of cultured human brain cells and murine perivascular fibroblasts. These data open up a window for new hypotheses on cell types involved in human CNS diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

EIF3H knockdown inhibits malignant melanoma through regulating cell proliferation, apoptosis and cell cycle

Highlights: • EIF3H is highly expressed in malignant melanoma and correlated with tumor stage and risk of lymphatic metastasis. • Knockdown of EIF3H inhibits cell proliferation, cell migration and promotes cell apoptosis of melanoma in vitro. • Knockdown of EIF3H suppresses tumor growth in vivo. Malignant melanoma (MM) causes 80% of skin cancer-related deaths and becomes the most lethal type of skin cancer. The molecular mechanism of MM is still not clear. This study aimed to reveal the relationship between MM and EIF3H. Clinical specimens were collected to preliminarily explore the role of EIF3H in MM. MM cell lines with EIF3H knockdown were constructed for investigating the effects of EIF3H on cell proliferation, apoptosis, cell cycle and cell motility. Mice xenograft model was constructed for verification in vivo. We found that EIF3H was obviously upregulated in MM tissues compared with normal skin tissues, which was correlated with tumor stage and risk of lymphatic metastasis. The in vitro results indicated that silencing EIF3H in MM cells could significantly suppress cell proliferation, promote cell apoptosis and induce cell cycle arrest. Moreover, EIF3H knockdown significantly restrained cell motility through regulating EMT-related proteins. The effects of EIF3H knockdown were also verified in mice xenograft model, which were represented by slower growth rate, smaller volume and lighter weight of tumors. Therefore, EIF3H was identified as a critical factor in the development and progression of MM which may be used as a novel therapeutic target in the treatment of MM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin β3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Unique features and emerging in vitro models of human placental development

Abstract Background The placenta is an essential organ for the normal development of mammalian fetuses. Most of our knowledge on the molecular mechanisms of placental development has come from the analyses of mice, especially histopathological examination of knockout mice. Choriocarcinoma and immortalized cell lines have also been used for basic research on the human placenta. However, these cells are quite different from normal trophoblast cells. Methods In this review, we first provide an overview of mouse and human placental development with particular focus on the differences in the anatomy, transcription factor networks, and epigenetic characteristics between these species. Next, we discuss pregnancy complications associated with abnormal placentation. Finally, we introduce emerging in vitro models to study the human placenta, including human trophoblast stem (TS) cells, trophoblast and endometrium organoids, and artificial embryos. Main findings The placental structure and development differ greatly between humans and mice. The recent establishment of human TS cells and trophoblast and endometrial organoids enhances our understanding of the mechanisms underlying human placental development. Conclusion These in vitro models will greatly advance our understanding of human placental development and potentially contribute to the elucidation of the causes of infertility and other pregnancy complications.

Shibata, Shun↗
DOE OSTI2020

Synthesis, Characterization, and Biological Activity of Hybrid Thiosemicarbazone–Alkylthiocarbamate Metal Complexes

A series of hybrid ligands (H 2 L 1 –H 2 L 3 ) derived from 4-methyl-3-thiosemicarbazide and hydrazinecarbothioic acid O-alkyl esters were synthesized and characterized by NMR. The ligands were chelated with copper (4–6), nickel (7–9), and zinc (10–12) and characterized by spectroscopy, electrochemistry, and single crystal X-ray crystallography. The chelated metals displayed substantial anodic shifts in the Cu II/I reduction potential of ~160 mV relative to their bis(thiosemicarbazone) analogues. The metal chelates 4–12 were evaluated for potential anticancer activity by MTT assays, and selected results were confirmed by clonogenic and trypan blue assays. The copper derivatives 4 and 6 were found to have potent and cancer-selective antiproliferative effects, with GI 50 values less than 100 nM in A549 lung adenocarcinoma cells compared with at least 20-fold less activity in IMR90 nonmalignant lung fibroblasts. In comparison, the nickel complexes were much less active and had little cancer-selectivity. Varying by ligand, the zinc complexes were less potent or had comparable activity compared to that of the corresponding copper complex. UV–visible spectroscopy indicated that zinc complex 10 was transmetalated in the presence of equimolar copper, whereas nickel complex 7 was not. Copper complexes 4 and 6 were also assessed in the NCI60 screen and were found to have cytotoxic activity against most solid tumor cell lines. In MTT assays, 4 and 6 were substantially more active against A549 cancer cells than Cu(ATSM) and were more cancer-selective (for A549 compared to IMR-90) than Cu(GTSM). Finally, our results suggest that hybrid thiosemicarbazone–alkylthiocarbamate copper complexes have potential for development as new anticancer agents.

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

19-LW-045 Full Length Final Report. Molecular Mechanisms of Bacterial Pathogenesis: Waging the Arms Race with Superbugs

As the current global pandemic makes abundantly clear, we need a better understanding of infectious disease to safeguard human health, the economy and global security. Modern omics techniques hold the promise of providing a comprehensive understanding of the molecular mechanisms of life, including causes of pathogenesis from infectious disease at the molecular level, but we there is a serious gap in annotation of gene function. For as much as half of the genes and gene products encoded in genomes the molecular and/or cellular function is unknown or only partially understood. Recent innovations in fluorescence microscopy for live cell imaging and genetic engineering make it possible to determine the temporal correlation between molecular events, such as a gene being expressed due to host-pathogen interaction, and cellular events, such as bacterial invasion of immune cells. This is turn allows us to gain new insight as to the molecular and cellular role of individual genes and will enable the discovery and validation of new molecular mechanisms essential for infectious disease. Knowing the molecular mechanisms of disease processes will provide new therapeutic targets or novel countermeasure strategies. We aimed to develop a lattice light sheet fluorescence microscope as a unique resource at LLNL for long time course live cell imaging experiments; to develop the reagents and cell lines needed to monitor molecular events during the course pathogenic bacteria infecting mammalian immune cells; and to demonstrate that we could capture molecular events during an infection. We fully commissioned the LLNL lattice light sheet microscope and conducted initial proof of principle imaging experiments on mammalian immune cells and pathogenic bacteria. It is clear from the experience gained that long time course live cell imaging has tremendous potential to help elucidate molecular mechanisms of host-pathogen interactions and to help annotate gene function, which would establish a basis for new countermeasures. It is also clear that if live cell imaging is to realize its full potential new data processing and analysis tools will need to be developed to facilitate analysis of molecular events within cells; new sample chambers and stages could facilitate studies with a wider range of cell and tissue types; and alternative molecular tagging methods need to be explored to enable more facile engineering of cells labeled with molecular specificity.

59 BASIC BIOLOGICAL SCIENCES↗