Results for “intact proteins” — Page 7

NASA NTRS1983

Prevention of metabolic alterations caused by suspension hypokinesia in leg muscles of rats

Rats were subjected to tail-cast suspension hypokinesia for 6 days with one leg immobilized in dorsal flexion by casting. Control animals were also tail-casted. The soleus, gastrocnemius and plantaris muscles of uncasted hypokinetic legs were smaller than control muscles. Dorsal flexion prevented atrophy of these muscles and caused the soleus to hypertrophy. The anterior muscles were unaffected by hypokinesia. The smaller size of the soleus of the uncasted leg relative to the dorsal flexed and weight bearing limbs correlated with slower protein synthesis and faster proteolysis. The capacity of this muscle to synthesize glutamine (gln), which carries nitrogenous waste from muscle was also measured. Although tissue homogenates showed higher activities of gln synthetase, the rate of de novo synthesis was not altered in intact muscle but the tissue ratio of gln/glutamate was decreased. Glutamate and ATP were not limiting for gln synthesis, but availability of ammonia may be a limiting factor for this process in hypokinesia.

Tischler, M. E.↗

DOE OSTI2024

National User Resource for Biological Accelerator Mass Spectrometry

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2025

National User Resource for Biological Accelerator Mass Spectrometry (Final Report)

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions. Over the next five years, our goals are to: 1. Improve the efficiency of operation for AMS measurements through installation of new interfaces to our AMS systems, technical modifications to improve gas accepting ion source efficiency and upgrading our data analysis software for improved ease of use and data reporting. 2. Increase the accessibility and visibility of ultra-sensitive 14C measurements for the biomedical research community by training of new investigators and expanding our national user base. 3. Provide high throughput, ultra-sensitive 14C analysis for the NIGMS and NIH user community.

47 OTHER INSTRUMENTATION↗

NASA NTRS1999

Beta-Adrenergic Receptor Expression in Muscle Cells

beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

Young, Ronald B.↗

NASA NTRS1998

Beta-Adrenergic Receptor Population is Up-Regulated in Chicken Skeletal Muscle Cells Treated with Forskolin

Skeletal muscle hypertrophy is promoted by in vivo administration of beta-adrenergic receptor (betaAR) agonists. These compounds presumably exert their physiological action through the betaAR, and alterations in the population of betaAR could potentially change the ability of the cell to respond to the betaAR agonists. Since the intracellular chemical signal generated by the betaAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of functional betaAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 microM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the betaAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 microM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 50% at 10 microM. This increase in PAR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of betaAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 microM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

Bridge, K. Y.↗

NASA NTRS1997

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.

NASA Program Space Physiology and Countermeasures↗

NASA NTRS1993

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

NASA Discipline Cell Biology↗

DOE OSTI2023

Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor

Abstract Bacteriophage exclusion (‘BREX’) systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2001

Visualization of Microbial Biomarkers by Scanning Electron Microscopy

We are developing tools to link the biochemical structure of selected biomarkers with putative biogenic structures observed in mineralized samples. The detection of evidence of life on Mars and other planets will rely on methods that can discriminate compounds formed exclusively by living organisms. While biogenic compounds, such as amino acids and nucleotides have been discovered in extraterrestrial sources, such as meteorites and comets, their formation can be explained by abiotic means. The formation of cellular structures, or more elaborate organic molecules, such as complex lipids, proteins or nucleic acids, however, is strongly correlated to the presence of even the most primitive life processes. Recent evidence lends support to the hypothesis that life may have once existed on Mars. Carbonate globules and ppm concentrations of polycyclic aromatic hydrocarbons (PAHs) have been described in ALH84001, a meteorite originating from Mars ejecta captured by Earth over 13,000 years ago. The localized high concentration of PAHs that follow an increasing gradient from the intact fusion crust towards the interior corresponds to microgram quantities of hydrocarbon. Even though ALH84001 and other similar meteorites have withstood the forces capable of ejecting rock through Mars' escape velocity, upon entering Earth's atmosphere, their core temperatures are likely not to have been raised significantly, as evidenced by the survival of remanent magnetic signatures. Ideal biomarkers of ancient or modern biological life would include molecules that are (or were) pervasive and highly resistant to degradation. Also, requisite methods of detection should be simple, extremely sensitive and broadly inclusive (NASA SP-530). Lipopolysaccharide (LPS), peptidoglycan or pseudopeptidoglycan and beta-glucan are microbial cell wall components which together cover the entire microbial spectrum of eubacteria, archea and fungi. They are all remarkably resistant to thermal degradation. Fortunately, many antimicrobial defense systems of higher organisms require sensitive detection to combat microbial pathogens. We employ here the primitive immune system of the evolutionarily ancient horseshoe crab, Limulus polyphemus. This species relies on multi-enzyme signal amplification detection of cell wall molecules and they can be applied to the development of useful detectors of life. An extension of this work includes the visualization of microbial signatures by labeling LAL components with chromogenic or electron dense markers. The protein Limulus Anti-LPS Factor (LALF) has an extremely high affinity for LPS. By coupling LALF binding with colloidal gold labels we demonstrate a correlation of the structures visible by electron microscopy with biochemical evidence of microbial cell wall materials. Pure silica particles were mixed with cultures of E. coli (10(exp 6) cfu/mL). Samples were washed sequentially with buffered saline, LALF, antibody to LALF and finally colloidal gold-labeled Protein A. Negative controls were not exposed to E. coli but received identical treatment otherwise. Samples were coated with carbon and imaged on a JEOL JSM-840 scanning electron microscope with LaB6 source in the back scatter mode with the JEOL annular back scatter detector. 20 nm-scale black spots in this contrast-reversed image originate from electrons back-scattered by gold atoms. Negative controls did not give any signal. Future work will expand application of this technique to soil simulants and mineralized rock samples.

Wainwright, Norman R.↗

DOE OSTI2024

Lanthanide binding peptide surfactants at air–aqueous interfaces for interfacial separation of rare earth elements

Rare earth elements (REEs) are critical materials to modern technologies. They are obtained by selective separation from mining feedstocks consisting of mixtures of their trivalent cation. We are developing an all-aqueous, bioinspired, interfacial separation using peptides as amphiphilic molecular extractants. Lanthanide binding tags (LBTs) are amphiphilic peptide sequences based on the EF-hand metal binding loops of calcium-binding proteins which complex selectively REEs. We study LBTs optimized for coordination to Tb 3+ using luminescence spectroscopy, surface tensiometry, X-ray reflectivity, and X-ray fluorescence near total reflection, and find that these LBTs capture Tb 3+ in bulk and adsorb the complex to the interface. Molecular dynamics show that the binding pocket remains intact upon adsorption. We find that, if the net negative charge on the peptide results in a negatively charged complex, excess cations are recruited to the interface by nonselective Coulombic interactions that compromise selective REE capture. If, however, the net negative charge on the peptide is −3, resulting in a neutral complex, a 1:1 surface ratio of cation to peptide is achieved. Surface adsorption of the neutral peptide complexes from an equimolar mixture of Tb 3+ and La 3+ demonstrates a switchable platform dictated by bulk and interfacial effects. The adsorption layer becomes enriched in the favored Tb 3+ when the bulk peptide is saturated, but selective to La 3+ for undersaturation due to a higher surface activity of the La 3+ complex.

Ortuno Macias, Luis E. (ORCID:0000000284342192)↗

DOE OSTI2022

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

NASA Discipline Plant Biology↗

DOE OSTI2024

Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Mapping the catalytic conformations of an assembly-line polyketide synthase module

Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large enzyme factories prized for their ability to produce specific and complex polyketide products. By channeling protein-tethered substrates across multiple active sites in a defined linear sequence, these enzymes facilitate programmed small-molecule syntheses that could theoretically be harnessed to access countless polyketide product structures. Additionally, using cryogenic electron microscopy to study DEBS module 1, we present a structural model describing this substrate-channeling phenomenon. Our 3.2- to 4.3-angstrom-resolution structures of the intact module reveal key domain-domain interfaces and highlight an unexpected module asymmetry. We also present the structure of a product-bound module that shines light on a recently described “turnstile” mechanism for transient gating of active sites along the assembly line.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1993

A Fiber Optic Probe for the Detection of Cataracts

A compact fiber optic probe developed for on-orbit science experiments was used to detect the onset of cataracts, a capability that could eliminate physicians' guesswork and result in new drugs to 'dissolve' or slow down the cataract formation before surgery is necessary. The probe is based upon dynamic light scattering (DLS) principles. It has no moving parts, no apertures, and requires no optical alignment. It is flexible and easy to use. Results are presented for excised but intact human eye lenses. In a clinical setting, the device can be easily incorporated into a slit-lamp apparatus (ophthalmoscope) for complete eye diagnostics. In this set-up, the integrated fiber optic probe, the size of a pencil, delivers a low power cone of laser light into the eye of a patient and guides the light which is backscattered by the protein molecules of the lens through a receiving optical fiber to a photo detector. The non-invasive DLS measurements provide rapid determination of protein crystalline size and its size distribution in the eye lens.

Ansari, Rafat R.↗

DOE OSTI2020

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Dense, continuous membrane labeling and expansion microscopy visualization of ultrastructure in tissues

Abstract Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines an innovative membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validate the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supports the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrate the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yields ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

Science & Technology - Other Topics↗

DOE OSTI2025

Continuity of Mitochondrial Budding: Insights from BS-C-1 Cells by In Situ Cryo-electron Tomography

Mitochondrial division is a fundamental biological process essensial for cellular functionality and vitality. The prevailing hypothesis that dynamin related protein 1 (Drp1) provides principal control in mitochondrial division, in which it also involves the endoplasmic reticulum (ER) and the cytoskeleton, does not account for all the observations. Therefore. the hypothesis may be incomplete. Our previous study in HeLa cells led to a new hypothesis of mitochondrial division by budding. To follow-up our previous study, we employed in situ cryo-electron tomography to visualize mitochondrial budding in the intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, consistent with our observations in HeLa cells. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, neither rings nor ring-like structures, nor the wrapping of ER tubes was observed in the budding regions, suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles that are distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. In conclusion, we propose that these interacting vesicles may have mitochondrial origins.

(Cryo-EM)↗