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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Evidence of co-exposure with Brucella spp, Coxiella burnetii , and Rift Valley fever virus among various species of wildlife in Kenya

Co-infection, especially with pathogens of dissimilar genetic makeup, may result in a more devastating impact on the host. Investigations on co-infection with neglected zoonotic pathogens in wildlife are necessary to inform appropriate prevention and control strategies to reduce disease burden in wildlife and the potential transmission of these pathogens between wildlife, livestock and humans. This study assessed co-exposure of various Kenyan wildflife species with Brucella spp, Coxiella burnetii and Rift Valley fever virus (RVFV). A total of 363 sera from 16 different wildlife species, most of them (92.6%) herbivores, were analysed by Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against Brucella spp, C. burnetii and RVFV. Further, 280 of these were tested by PCR to identify Brucella species. Of the 16 wildlife species tested, 15 (93.8%) were seropositive for at least one of the pathogens. Mean seropositivities were 18.9% (95% CI: 15.0–23.3) for RVFV, 13.7% (95% CI: 10.3–17.7) for Brucella spp and 9.1% (95% CI: 6.3–12.5) for C. burnetii. Buffaloes (n = 269) had higher seropositivity for Brucella spp. (17.1%, 95% CI: 13.0–21.7%) and RVFV (23.4%, 95% CI: 18.6–28.6%), while giraffes (n = 36) had the highest seropositivity for C. burnetii (44.4%, 95% CI: 27.9–61.9%). Importantly, 23 of the 93 (24.7%) animals positive for at least one pathogen were co-exposed, with 25.4% (18/71) of the positive buffaloes positive for brucellosis and RVFV. On molecular analysis, Brucella DNA was detected in 46 (19.5%, CI: 14.9–24.7) samples, with 4 (8.6%, 95% CI: 2.2–15.8) being identified as B. melitensis. The Fisher’s Exact test indicated that seropositivity varied significantly within the different animal families, with Brucella (p = 0.013), C. burnetii (p = <0.001) and RVFV (p = 0.007). Location was also significantly associated (p = <0.001) with Brucella spp. and C. burnetii seropositivities. Of ~20% of Kenyan wildlife that are seropositive for Brucella spp, C. burnetii and RVFV, almost 25% indicate co-infections with the three pathogens, particularly with Brucella spp and RVFV.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Seroprevalence of Brucella spp. and Rift Valley fever virus among slaughterhouse workers in Isiolo County, northern Kenya

Brucella spp. and Rift Valley fever virus (RVFV) are classified as priority zoonotic agents in Kenya, based on their public health and socioeconomic impact on the country. Data on the pathogen-specific and co-exposure levels is scarce due to limited active surveillance. This study investigated seroprevalence and co-exposure of Brucella spp. and RVFV and associated risk factors among slaughterhouse workers in Isiolo County, northern Kenya. A cross-sectional serosurvey was done in all 19 slaughterhouses in Isiolo County, enrolling 378 participants into the study. The overall seroprevalences for Brucella spp. and RVFV were 40.2% (95% CI: 35.2–45.4) and 18.3% (95% CI: 14.5–22.5), respectively while 10.3% (95% CI 7.4%-13.8%) of individuals were positive for antibodies against both Brucella spp. and RVFV. Virus neutralisation tests (VNT) confirmed anti-RVFV antibodies in 85% of ELISA-positive samples. Our seroprevalence results were comparable to community-level seroprevalences previously reported in the area. Since most of the study participants were not from livestock-keeping households, our findings attribute most of the detected infections to occupational exposure. The high exposure levels indicate slaughterhouse workers are the most at-risk population and there is need for infection, prevention, and control programs among this high-risk group. This is the first VNT confirmation of virus-neutralising antibodies among slaughterhouse workers in Isiolo County and corroborates reports of the area being a high-risk RVFV area as occasioned by previously reported outbreaks. This necessitates sensitization campaigns to enhance awareness of the risks involved and appropriate mitigation measures.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

A novel assay to measure low-density lipoproteins binding to proteoglycans

Background: The binding of low-density lipoprotein (LDL) to proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerosis. Although many techniques have been developed to assess this binding, most of the methods are labor-intensive and technically challenging to standardize across research laboratories. Thus, sensitive, and reproducible assay to detect LDL binding to PGs is needed to screen clinical populations for atherosclerosis risk. Objectives: The aim of this study was to develop a quantitative, and reproducible assay to evaluate the affinity of LDL towards PGs and to replicate previously published results on LDL-PG binding. Methods: Immunofluorescence microscopy was performed to visualize the binding of LDL to PGs using mouse vascular smooth muscle (MOVAS) cells. An in-cell ELISA (ICE) was also developed and optimized to quantitatively measure LDL-PG binding using fixed MOVAS cells cultured in a 96-well format. Results: We used the ICE assay to show that, despite equal APOB concentrations, LDL isolated from adults with cardiovascular disease bound to PG to a greater extent than LDL isolated from adults without cardiovascular disease (p<0.05). Conclusion: We have developed an LDL-PG binding assay that is capable of detecting differences in PG binding affinities despite equal APOB concentrations. Future work will focus on candidate apolipoproteins that enhance or diminish this interaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Two human antibodies to a meningococcal serogroup B vaccine antigen enhance binding of complement Factor H by stabilizing the Factor H binding site

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Developing a Vaccine Platform for a Balanced Mucosal Immune Response (CB11446), Project Report: Year 1

Mucosal vaccines can elicit protective immune responses at the infection site of respiratory or aerosolized pathogens. Achieving balanced mucosal and systemic responses is a key challenge in the design of mucosal vaccines, especially in terms of developing a broadly applicable vaccine delivery platform amenable to a wide range of subunit vaccine antigens. The overarching goal of the project entitled “Developing a Vaccine Platform for a Balanced Mucosal Immune Response” is to develop a pathogen-agnostic vaccine platform that elicits robust and balanced mucosal immune responses upon intranasal vaccination, in the context of a nanolipoprotein particle (NLP) platform. In year one, we investigated NLP-based vaccine formulations incorporating select immune adjuvants (Monophosphoryl Lipid A (MPLA), FSL-1, L18-muramyl dipeptide (MDP), and cholesterol-tagged ODN2006 (cCpG)) along with antigens relevant to plague (LcrV), tularemia (IglC), and Ebola virus (GP). We demonstrated the ability to prepare, characterize, and quantify these formulations. We then carried out studies in vivo using a BALB/c mouse model, comparing intranasal and intramuscular administration and systematically evaluated the resulting mucosal and systemic immune responses using ELISpot and ELISAs. All proposed tasks (Table 1) were completed within the twelve-month base period, and the established go/no go metric was successfully achieved. Numerous adjuvant:LcrV:NLP formulations were found to have statistically significant increases in IgA titers in serum and/or lung upon IN administration (compared to IM administration), while also producing robust IgG responses in serum. Some formulations also showed significant IFNγ responses using restimulated splenocytes. The data collected during the base period provide valuable insights for the future design of safe and effective mucosal subunit vaccines while also highlighting areas of research that merit further investigation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Development of high throughput and in vitro assays for analyzing RNA modifications

Modifications on RNAs play major roles in their stability, translation, and enzymatic activity. Despite its importance, the current techniques are insufficient to study the structure and function of RNA modifications. Indeed, the National Academies of Science, Engineering and Medicine indicate that developing new tools and further study the function of RNA modifications is strategically a high priority for advancing science in the coming years (https://www.nationalacademies.org/our-work/toward-sequencing-and-mapping-of-rna-modifications). RNA modifications occur in all domains of life controlling processes such as RNA turnover, translation regulation, cellular defenses and bioproduction. Our preliminary data indicated that the insulin mRNA might get ADP-ribosylated by the ADP-ribosyltransferase PARP12. RNA ADP-ribosylation has been described in Escherichia coli. Combined to the fact that ADP-ribosyltransferase (PARP) genes are conserved throughout evolution we hypothesize that this modification might play essential roles in cells. Therefore, we proposed to develop sequencing techniques and in vitro enzymatic assays to identify and validate ADP-ribosylation motifs and sites. Here we report the development of RNA-seq and qPCR assays to identify ADP-ribosylated RNAs, in addition to a nicotinamide adenosine dinucleotide (NAD – ADP-ribosylation donor) consumption assay and an enzyme-linked immunosorbent assay (ELISA) to measure ADP-ribosyltransferase activity. Testing these assays with the insulin mRNA confirmed that this transcript is ADP-ribosylated. These assays will not only enable studying the function of ADP-ribosylation but can be easily adapted for studying other RNA modifications. This will open opportunities to study RNA modifications in different model systems from bacteria to viruses to plants, bringing insights into their cellular functions and the possibility of targeting them for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16 + NK cell activation

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16 + NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16 + NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Discovering novel therapeutic V H Hs for emerging viruses: perspectives from VEEV selection strategies

Introduction: Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (V H Hs). Methods: In vitro selections offer a promising pathway for identification of therapeutic antibodies, here we explored isolation of V H Hs using phage and yeast display methodology with three antigen formats 1) recombinant E2, 2) linear peptides of E2, selected based on molecular dynamics analysis, and 3) UV inactivated virus. Results: Here we report four novel “human” V H Hs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These V H Hs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these V H Hs. Discussion: Though non-neutralizing, these V H Hs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these V H Hs to perform viral “sponging” which represents a novel therapeutic approach. The selection strategies we report may have applications to further antibody developments against other viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Functional analysis of Pogostemon cablin farnesyl pyrophosphate synthase gene and its binding transcription factor PcWRKY44 in regulating biosynthesis of patchouli alcohol

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in Pogostemon cablin . In this study, the full-length cDNA of the FPP synthase gene from P. cablin ( PcFPPS ) was cloned and characterized. The expressions of PcFPPS are different among different tissues (highly in P. cablin flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal FPPS deletion mutation in yeast CC25. Transient overexpression of PcFPPS in P. cablin leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of PcFPPS in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of PcFPPS can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of PcWRKY44 in P. cablin upregulated the expression levels of PcFPPS and patchoulol synthase gene ( PcPTS ), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the PcFPPS and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.

Wang, Xiaobing↗
DOE OSTI2024

Seroprevalence and risk factors for brucellosis amongst livestock and humans in a multi-herd ranch system in Kagera, Tanzania

Background Brucellosis remains a significant health and economic challenge for livestock and humans globally. Despite its public health implications, the factors driving the endemic persistence ofBrucellaat the human-livestock interface in Tanzania remain poorly elucidated. This study aimed to identify the seroprevalence ofBrucellainfection in livestock and humans within a ranching system and determine associated risk factors for disease endemicity. Methods A cross-sectional sero-epidemiological study was conducted in 2023 in Tanzania’s Karagwe District, involving 725 livestock (cattle, goats, sheep) from 10 herds and 112 humans from associated camps. Seroprevalence was assessed using competitive ELISA while epidemiological data were collected via questionnaires. Generalized Linear Models and Contrast Analysis were used to identify risk factors for infection. Results Overall seroprevalence was 34% in livestock and 41% in humans. Goats exhibited the highest prevalence (69.2%), while cattle had the lowest (22.6%). Mixed-species herds (Odds Ratio, OR = 2.96, CI [1.90–4.60]) and small ruminants-only herds (OR = 6.54, CI [3.65–11.72]) showed a significantly higher risk of seropositivity compared to cattle-only herds. Older cattle (OR = 5.23, CI [2.70–10.10]) and lactating females (OR = 2.87, CI [1.78–4.63]) represented significant risks for brucellosis in livestock. In humans, close contact with animals (OR = 7.20, CI [1.97–36.31]) and handling animals during parturition or aborted fetuses (OR = 2.37, CI [1.01–5.58]) were significant risk factors. Notably, no spatial association was found in seroprevalence between herds and nearby human communities. Conclusion The lack of spatial correlation between livestock and human seroprevalence suggests complex transmission dynamics, potentially involving endemic circulation in livestock and human infections from multiple sources of exposure to livestock. This study highlights the need for comprehensive zoonotic risk education and targeted intervention strategies. Further research is crucial to elucidate transmission pathways and improveBrucellainfection control. This includes developing robust methods for identifying infective species and implementing effective strategies to mitigateBrucellainfection in endemic regions.

Public, Environmental & Occupational Health↗
DOE OSTI2020

Microbiological, Physicochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt

Nut-based milks and yogurts are gaining popularity, but may not offer the same benefits as dairy yogurts to consumers. Cashew nuts often cause severe allergic reactions, and cashew nut allergens are stable to several types of processing. To compare its characteristics to dairy yogurt and characterize the effects of fermentation on the Ana o 1–3 cashew nut allergens, a commercial yogurt made from cashew nuts (Cashewgurt) was evaluated for microbiological, physiochemical, and immunological properties. Average counts for lactobacilli and Streptococcus thermophilus were greater than 10 million colony forming units per milliliter, indicating the capacity to provide a health benefit. Cashewgurt pH and viscosity values were comparable to cow milk yogurts, and it was off white in color. SDS-PAGE analysis indicated a clear reduction in Ana o 1 and 2, and immuno-assay with polyclonal anti-cashew IgG antibody and cashew-allergic IgE indicated an overall reduction in allergen content. In contrast, SDS-PAGE, mass spectrometry, immunoblot, and ELISA all revealed that Ana o 3 was relatively unaffected by the fermentation process. In conclusion, Ana o 1 and Ana o 2 are sensitive to degradation, while Ana o 3 survives lactic acid bacterial fermentation during yogurt production. The analysis presented here indicates that cashew nut yogurt is not suitable for those with cashew nut allergy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-ΔI177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals

The African swine fever virus (ASFV) mutant ASFV-G-ΔI177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-ΔI177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-ΔI177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-ΔI177L/ΔEP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-ΔI177L. Domestic pigs intramuscularly (IM) inoculated with either 10 2 or 10 6 HAD 50 of ASFV-G-ΔI177L/ΔEP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 10 2 HAD 50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-ΔI177L/ΔEP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-ΔI177L/ΔEP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-ΔI177L, ASFV-G-ΔMGF, or ASFV-G-Δ9GL/ΔUK. Therefore, ASFV-G-ΔI177L/ΔEP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Endemic Seroprevalence and Farmer Control Measures of Anthrax Among Ruminate Animals in Pakistan

Anthrax, caused by Bacillus anthracis (B. anthracis), is an important zoonotic disease of livestock that poses a threat to public safety. The present study aimed to determine the seroprevalence of Bacillus anthracis in sheep and cattle in endemic areas (Punjab and Balochistan provinces) of Pakistan by enzyme-linked immunosorbent assay (ELISA). Crosssectional survey was also done to assess the control measures of the farmers. Out of 401 serum samples from sheep and cattle, a total of 68 (16.9%) samples were seropositive for anti-PA antibodies; seroprevalence of B. anthracis in sheep and cattle was 22.11% (48/217) and 10.86% (20/184), respectively. Seroprevalence was significantly higher (p<0.05) in Baluchistan province (33.3%, 44/132) compared to Punjab province (8.92%, 24/269). Most farmers (65.90%, 29/44) were not disposing carcasses as recommended. Around 77.28%, 17/22 of the veterinary officials reported the nonavailability of vaccine against anthrax and 63.63% (14/22) stated the lack of cold chain for the vaccine storage and transportation. We concluded that seroprevalence of B. anthracis in unvaccinated sheep and cattle is evidence that the pathogen is circulating in Baluchistan and Punjab provinces. So must applied an active surveillance and, strengthened veterinary practices vaccination programs to control anthrax outbreaks in endemic areas.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs

Abstract The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a K d of ∼9.8 μM and competitively inhibits its activity with an inhibition constant (K i) of ∼5.8 μM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure–activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Immunology↗
NASA NTRS1982

Rapid diagnostic methods for influenza virus in clinical specimens - A comparative study

A comparison of five rapid viral diagnostic techniques for identifying influenza virus in nasopharyngeal aspirates has been made on patients with influenza-like illnesses. Initial results with immune electron microscopy were positive in only one of 11 specimens from which virus was isolated and further work abandoned. Four other rapid tests were carried out on 39 specimens from which influenza virus had been isolated in tissue culture in 28. Of these 28 specimens yielding virus, 24 (85.7 percent) were positive by an indirect fluorescent antibody test (IFAT) on nasopharyngeal cells, 18 (64.3 percent) by enzyme-linked immunosorbent assay (ELISA), 19 (67.8 percent) by enzyme-linked fluorescent assay (ELFA), and 26 (92.8 percent) by a rapid tissue culture amplification method (TCA) in a continuous Rhesus monkey kidney line (LLC-MK2) with identification of virus by fluorescent antibody. In terms of sensitivity, simplicity, and rapidity, a combination of the IFAT and TCA methods seems to be very useful.

Evans, A. S.↗
NASA NTRS1996

Dextroamphetamine: a pharmacologic countermeasure for space motion sickness and orthostatic dysfunction

Dextroamphetamine has potential as a pharmacologic agent for the alleviation of two common health effects associated with microgravity. As an adjuvant to Space Motion Sickness (SMS) medication, dextroamphetamine can enhance treatment efficacy by reducing undesirable Central Nervous System (CNS) side effects of SMS medications. Secondly, dextroamphetamine may be useful for the prevention of symptoms of post-mission orthostatic intolerance caused by cardiovascular deconditioning during spaceflight. There is interest in developing an intranasal delivery form of dextroamphetanmine for use as a countermeasure in microgravity conditions. Development of this dosage form will require an analytical detection method with sensitivity in the low ng range (1 to 100 ng/mL). During the 1995 Summer Faculty Fellowship Program, two analytical methods were developed and evaluated for their suitability as quantitative procedures for dextroamphetamine in studies of product stability, bioavailability assessment, and pharmacokinetic evaluation. In developing some of the analytical methods, beta-phenylethylamine, a primary amine structurally similar to dextroamphetamine, was used. The first analytical procedure to be evaluated involved hexane extraction and subsequent fluorescamine labeling of beta-phenylethylamine. The second analytical procedure to be evaluated involved quantitation of dextroamphetamine by an Enzyme-Linked ImmunoSorbent Assay (ELISA).

Snow, L. Dale↗
NASA NTRS2002

Osteoblast Differentiation Decreases Hypergravity-Stimulated Release of PGE(sub 2)

We determined if progressive differentiation of osteoblasts influences their sensitivity to gravitational loading. Osteoblasts were cultured for 4 days (confluent monolayer), 6 days (prenodules), 9 days (nodules) and 19 days (mineralized nodules), then centrifuged at 10 times gravity (g) or 50-g for 3 hours using the NASA Ames 1-ft. Diameter Centrifuge. Stationary controls were placed in an adjacent incubator. Following centrifugation, conditioned media were collected and analyzed for PGE, by ELISA. Microtubules were fluorescently labeled and analyzed by confocal microscopy to determine microtubule network morphology and height. Centrifugation at 10-g reduced microtubule network height by 15% on d4 and 10% on d6, with variable changes in more mature cultures. No major changes in microtubule morphology were observed. PGE(sub 2) release by d4 cultures increased in a dose-dependent fashion (3-fold at 10-g and 6-fold at 50-g relative to controls). D6 cultures produced a 5-fold increase for both 10-g and 50-g. PGE(sub 2) increased only 1.5-fold by d9, and by d19, PGE(sub 2) was not delectable in either the control or hypergravity-stimulated cells. Thus, as osteoblasts differentiate in culture, responsiveness of the microtubule cytoskeleton and the PGE(sub 2) pathway to hypergravity declines.

Searby, Nancy D.↗