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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 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

Vinpocetine attenuates lipid accumulation and atherosclerosis formation

Highlights: •Vinpocetine attenuates hyperlipidemia-induced atherosclerosis in a mouse model. •Vinpocetine antagonizes ox-LDL uptake and accumulation in macrophages. •Vinpocetine blocks the induction of ox-LDL receptor LOX-1 in vitro and in vivo. -- Abstract: Atherosclerosis, the major cause of myocardial infarction and stroke, is a chronic arterial disease characterized by lipid deposition and inflammation in the vessel wall. Cholesterol, in low-density lipoprotein (LDL), plays a critical role in the pathogenesis of atherosclerosis. Vinpocetine, a derivative of the alkaloid vincamine, has long been used as a cerebral blood flow enhancer for treating cognitive impairment. Recent study indicated that vinpocetine is a potent anti-inflammatory agent. However, its role in the pathogenesis of atherosclerosis remains unexplored. In the present study, we show that vinpocetine significantly reduced atherosclerotic lesion formation in ApoE knockout mice fed with a high-fat diet. In cultured murine macrophage RAW264.7 cells, vinpocetine markedly attenuated oxidized LDL (ox-LDL) uptake and foam cell formation. Moreover, vinpocetine greatly blocked the induction of ox-LDL receptor 1 (LOX-1) in cultured macrophages as well as in the LOX-1 level in atherosclerotic lesions. Taken together, our data reveal a novel role of vinpocetine in reduction of pathogenesis of atherosclerosis, at least partially through suppressing LOX-1 signaling pathway. Given the excellent safety profile of vinpocetine, this study suggests vinpocetine may be a therapeutic candidate for treating atherosclerosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Trophoblast lineage cells derived from human induced pluripotent stem cells

Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

miR-137 attenuates Aβ-induced neurotoxicity through inactivation of NF-κB pathway by targeting TNFAIP1 in Neuro2a cells

Background: Accumulation of β-amyloid (Aβ) and neuroinflammation are implicated in the pathogenesis and development of Alzheimer's disease (AD). Neuron-enriched miR-137 was aberrantly downregulated and may be associated with the pathogenesis of AD. However, the detailed function of miR-137 in AD pathogenesis and the molecular mechanism have not been elucidated. Methods: The expressions of miR-137 and tumor necrosis factor alpha (TNFα)-induced protein 1 (TNFAIP1) at mRNA and protein levels in primary mouse cortical neurons and Neuro2a (N2a) cells exposed to different concentrations of Aβ{sub 25-35} were examined by qRT-PCR and western blot. Luciferase reporter assay was used to confirm the potential target of miR-137. MTT assay, flow cytometry analysis, caspase-3 activity assay, Enzyme-linked immunosorbent assay (ELISA), and western blot were used to detect cell viability, apoptosis, caspase-3 activity, Nuclear factor-kappa B (NF-κB) activity and level, respectively. Results: Aβ{sub 25-35} downregulated miR-137 and upregulated TNFAIP1 in primary mouse cortical neurons and N2a cells. In addition, miR-137 was found to directly target TNFAIP1 and suppress its mRNA and protein levels. Moreover, miR-137 restoration and TNFAIP1 knockdown facilitate Aβ{sub 25-35}-induced cell toxicity, apoptosis, caspase-3 activity, and activated NF-κB in N2a cells, which was partially abolished by TNFAIP1 overexpression. Conclusion: miR-137 attenuated Aβ-induced neurotoxicity through inactivation of NF-κB pathway by targeting TNFAIP1 in N2a cells, shedding light on the molecular mechanism of miR-137 underlying Aβ-induced neurotoxicity. - Highlights: • Aβ{sub 25-35} downregulated miR-137 and upregulated TNFAIP1 in cortical neurons and N2a cells. • miR-137 suppressed TNFAIP1 expression through direct binding at the 3′UTR. • miR-137 overexpression contributed to Aβ{sub 25-35}-induced cell toxicity and apoptosis. • miR-137 overexpression on Aβ{sub 25-35}-induced neurotoxicity activated NF-κB pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Increased circulating trimethylamine N-oxide contributes to endothelial dysfunction in a rat model of chronic kidney disease

Highlights: • Circulating TMAO is elevated in chronic kidney disease. • Elevated TMAO in chronic kidney disease impairs endothelial function. • Elevated TMAO in chronic kidney disease reduces vascular eNOS activity. • Elevated TMAO in chronic kidney disease causes vascular oxidative stress. • Elevated TMAO in chronic kidney disease induces vascular inflammation. Chronic kidney disease (CKD) is strongly associated with increased cardiovascular risk. Impaired endothelial function, a key initiating step in the pathogenesis of cardiovascular disease, has been reported in patients with CKD, but the mechanisms responsible for endothelial dysfunction in CKD remain elusive. Emerging evidence reveals that trimethylamine-N-oxide (TMAO), a gut microbiota-generated metabolite, is involved in the pathogenesis of many cardiovascular diseases. Circulating TMAO is elevated in CKD. Here we tested the hypothesis that elevated TMAO plays a contributory role in the pathogenesis of endothelial dysfunction in CKD. Rats underwent 5/6 nephrectomy to induce CKD or sham operation, and were treated with 1.0% 3,3-Dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) or vehicle. Eight weeks after nephrectomy and DMB treatment, circulating TMAO levels were markedly elevated in CKD-vehicle rats compared with sham-vehicle rats, but were reduced in CKD-DMB rats. Acetylcholine-induced endothelium-dependent vasodilation was impaired in CKD-vehicle rats compared with sham-vehicle rats as indicated by reduced maximal relaxation (E{sub max}) and decreased area under the curve (AUC). E{sub max} and AUC were both normalized in CKD-DMB rats. No difference in sodium nitroprusside-induced endothelial-independent vasodilation was observed across groups. Molecular studies revealed that endothelial nitric-oxide synthase activity was decreased, while superoxide production and proinflammatory cytokine expression were increased in the aorta of CKD-vehicle rats compared with sham-vehicle rats. Of note, the abnormalities in above molecular parameters were completely restored in CKD-DMB rats. These results suggest that CKD elevates circulating TMAO levels, which may reduce eNOS-derived NO production by increasing vascular oxidative stress and inflammation, contributing to CKD-associated endothelial dysfunction and cardiovascular disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Mice overexpressing latent matrix metalloproteinase-2 develop lung emphysema after short-term exposure to cigarette smoke extract

Highlights: • Protease/anti-protease imbalance is an important event in the pathogenesis of lung emphysema. • Long time exposure to cigarette smoke is generally required to induce mouse model of lung emphysema. • Mice overexpressing matrix metalloproteinase-2 develop lung emphysema after short exposure to cigarette smoke extract. Chronic obstructive pulmonary disease is the major growing cause of mortality and morbidity worldwide, and it is going to become the third most common cause of death by 2020. Chronic obstructive pulmonary disease is pathologically characterized by lung emphysema and small airway inflammation. Animal models are very important to get insights into the disease pathogenesis but current models of chronic obstructive pulmonary disease take a long time to develop. The need of a new model is compelling. In the present study we focus on the role of matrix metalloproteinases in the pathogenesis of chronic obstructive pulmonary disease and hypothesized that lung overexpression of latent matrix metalloproteinases-2 would allow the development of emphysema after short-term exposure to cigarette smoke extract inhalation. Human latent matrix metalloproteinases-2 transgenic mouse expressing high level of the protein in the lungs and wild type mouse were exposed to aerosolized cigarette smoke extract for two weeks. Transgenic mice showed significant lung emphysematous changes, increased infiltration of inflammatory cells and enhanced lung concentrations of inflammatory cytokines in the lungs compared to their wild type counterparts after inhalation of cigarette smoke extract. This novel mouse model will be a very useful tool for evaluating the mechanistic pathways and for development of novel therapies in cigarette smoke-associated lung emphysema.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Identification of a rare homozygous c.790C>T variation in the TFB2M gene in Korean patients with autism spectrum disorder

Highlights: • TFB2M have been known to critical in mitochondrial DNA gene expression. • We identified a variation of TFB2M gene sequence in Korean autism patients. • The variation caused increased mitochondrial DNA gene expression. • Increased mitochondrial biogenesis resulted in ROS production and damaged cells. • Structural changes of variant TFB2M seems to delay unloading of DNA from TFB2M. Mitochondrial dysfunction and subsequent enhanced oxidative stress is implicated in the pathogenesis of autism spectrum disorder (ASD). Mitochondrial transcription factor B2 (TFB2M) is an essential protein in mitochondrial gene expression. No reports have described TFB2M mutations and variations involved in any human diseases. We identified a rare homozygous c.790C>T (His264Tyr) variation in TFB2M gene in two Korean siblings with ASD by whole-exome sequencing. The roles of the TFB2M variation in the pathogenesis of ASD were investigated. Patient fibroblasts revealed increased transcription of mitochondrial genes and mitochondrial function in terms of ATP, membrane potential, oxygen consumption, and reactive oxygen species (ROS). Overexpression of the TFB2M variant in primary-cultured fibroblasts demonstrated significantly increased transcription of mitochondrial genes and mitochondrial function compared with overexpression of wild-type TFB2M. Molecular dynamics simulation of the TFB2M variant protein suggested an increase in the rigidity of the hinge region, which may cause alterations in loading and/or unloading of TFB2M on target DNA. Our results suggest that augmentation of mitochondrial gene expression and subsequent enhancement of mitochondrial function may be associated with the pathogenesis of ASD in Korean patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our findings delineated the potential role of ultrafine particles alone and in combination with pesticide rotenone in the pathogenesis of PD. - Graphical abstract: Ultrafine particles and rotenone synergistically induce the assembly of active form NADPH oxidase complex in microglia inducing oxidative damage to dopamine neurons. - Highlights: • Ultrafine carbon black promotes dopaminergic neuronal loss induced by rotenone. • The role and underlying mechanism of ultrafine particles in the pathogenesis of PD • NADPH oxidase is a potential therapeutic target of Parkinson's disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Porcine reproductive and respiratory syndrome virus infection triggers HMGB1 release to promote inflammatory cytokine production

The high mobility group box 1 (HMGB1) protein is an endogenous damage-associated molecular pattern (DAMP) molecule involved in the pathogenesis of various infectious agents. Based on meta-analysis of all publicly available microarray datasets, HMGB1 has recently been proposed as the most significant immune modulator during the porcine response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, the function of HMGB1 in PRRSV pathogenesis is unclear. In this study, we found that PRRSV infection triggers the translocation of HMGB1 from the nucleus to the extracellular milieu in MARC-145 cells and porcine alveolar macrophages. Although HMGB1 has no effect on PRRSV replication, HMGB1 promotes PRRSV-induced NF-κB activation and subsequent expression of inflammatory cytokines through receptors RAGE, TLR2 and TLR4. Our findings show that HMGB1 release, triggered by PRRSV infection, enhances the efficiency of virus-induced inflammatory responses, thereby providing new insights into the pathogenesis of PRRSV infection. - Highlights: • PRRSV infection triggers HMGB1 release from MARC-145 cells and PAMs. • HMGB1 does not significantly affect PRRSV proliferation. • HMGB1 is involved in PRRSV-induced NF-κB activation and inflammatory responses. • HMGB1 promotes PRRSV-induced inflammatory responses through TLR2/4 and RAGE.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

ANS complex of St John’s wort PR-10 protein with 28 copies in the asymmetric unit: a fiendish combination of pseudosymmetry with tetartohedral twinning

Hyp-1, a pathogenesis-related class 10 (PR-10) protein from H. perforatum, was crystallized in complex with the fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS). The asymmetric unit of the tetartohedrally twinned crystal contains 28 copies of the protein arranged in columns with noncrystallographic sevenfold translational symmetry and with additional pseudotetragonal rotational NCS. Hyp-1, a pathogenesis-related class 10 (PR-10) protein from St John’s wort (Hypericum perforatum), was crystallized in complex with the fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS). The highly pseudosymmetric crystal has 28 unique protein molecules arranged in columns with sevenfold translational noncrystallographic symmetry (tNCS) along c and modulated X-ray diffraction with intensity crests at l = 7n and l = 7n ± 3. The translational NCS is combined with pseudotetragonal rotational NCS. The crystal was a perfect tetartohedral twin, although detection of twinning was severely hindered by the pseudosymmetry. The structure determined at 2.4 Å resolution reveals that the Hyp-1 molecules (packed as β-sheet dimers) have three novel ligand-binding sites (two internal and one in a surface pocket), which was confirmed by solution studies. In addition to 60 Hyp-1-docked ligands, there are 29 interstitial ANS molecules distributed in a pattern that violates the arrangement of the protein molecules and is likely to be the generator of the structural modulation. In particular, whenever the stacked Hyp-1 molecules are found closer together there is an ANS molecule bridging them.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2025

Dysregulation of lung epithelial cell homeostasis and immunity contributes to Middle East respiratory syndrome coronavirus disease severity

Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoVs from the past 20 years (SARS-CoV, SARS-CoV-2, and MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers, and acute lung injury among lethal and sub-lethal virus doses. Virus-induced apoptosis of type I and II alveolar epithelial cells suggests that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness.

MERS-CoV↗
DOE OSTI2018

IFN-γ, IL-17A, or zonulin rapidly increase the permeability of the blood-brain and small intestinal epithelial barriers: Relevance for neuro-inflammatory diseases

Highlights: • Zonulin rapidly (within 60 min) increases the permeability of the IEB and BBB in vitro. • Both IL-17A and IFN-γ enhance the permeability of the BBB and IEB within 1 h. • All three pro-inflammatory mediators rapidly modify IEB TJs, by causing mislocalization of ZO-1, claudin-5, and occludin. • Rapid TJ disassembly by all mediators was accompanied by dramatic depolymerization of the peri-junctional actin cytoskeleton. Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24 h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to zonulin, and that zonulin increased BBB permeability to a 40 kDa dextran tracer within 1 h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to zonulin, IFN-γ, or IL-17A in vitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or zonulin can increase the permeability of the IEB and BBB rapidly in vitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2'O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2'O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2'O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2'O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2'O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2'O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.

Evolview, evolgenius, SARS-CoV, NSP14 exonuclease↗
DOE OSTI2013

Methamphetamine Enhances Cryptococcus neoformans Pulmonary Infection and Dissemination to the Brain

Abstract: Methamphetamine (METH) is a major addictive drug of abuse in the United States and worldwide, and its use is linked to HIV acquisition. The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis in patients with AIDS. In addition to functioning as a central nervous system stimulant, METH has diverse effects on host immunity. Using a systemic mouse model of infection and in vitro assays in order to critically assess the impact of METH on C. neoformans pathogenesis, we demonstrate that METH stimulates fungal adhesion, glucuronoxylomannan (GXM) release, and biofilm formation in the lungs. Interestingly, structural analysis of the capsular polysaccharide of METH-exposed cryptococci revealed that METH alters the carbohydrate composition of this virulence factor, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Additionally, we show that METH promotes C. neoformans dissemination from the respiratory tract into the brain parenchyma. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms. Importance: Methamphetamine (METH) is a major health threat to our society, as it adversely changes people’s behavior, as well as increases the risk for the acquisition of diverse infectious diseases, particularly those that enter through the respiratory tract or skin. This report investigates the effects of METH use on pulmonary infection by the AIDS-related fungus Cryptococcus neoformans. This drug of abuse stimulates colonization and biofilm formation in the lungs, followed by dissemination of the fungus to the central nervous system. Notably, C. neoformans modifies its capsular polysaccharide after METH exposure, highlighting the fungus’s ability to adapt to environmental stimuli, a possible explanation for its pathogenesis. The findings may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating complications of METH abuse.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Differential Gene Expression in Host Ubiquitination Processes in Childhood Malarial Anemia

Background: Malaria remains one of the leading global causes of childhood morbidity and mortality. In holoendemic Plasmodium falciparum transmission regions, such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dl] is the primary form of severe disease. Ubiquitination is essential for regulating intracellular processes involved in innate and adaptive immunity. Although dysregulation in ubiquitin molecular processes is central to the pathogenesis of multiple human diseases, the expression patterns of ubiquitination genes in SMA remain unexplored. Methods: To examine the role of the ubiquitination processes in pathogenesis of SMA, differential gene expression profiles were determined in Kenyan children ( n = 44, aged <48 mos) with either mild malarial anemia (M l MA; Hb ≥9.0 g/dl; n = 23) or SMA (Hb <6.0 g/dl; n = 21) using the Qiagen Human Ubiquitination Pathway RT 2 Profiler PCR Array containing a set of 84 human ubiquitination genes. Results: In children with SMA, 10 genes were down-regulated ( BRCC3 , FBXO3 , MARCH5 , RFWD2 , SMURF2 , UBA6 , UBE2A , UBE2D1 , UBE2L3 , UBR1 ), and five genes were up-regulated ( MDM2 , PARK2 , STUB1 , UBE2E3 , UBE2M ). Enrichment analyses revealed Ubiquitin-Proteasomal Proteolysis as the top disrupted process, along with altered sub-networks involved in proteasomal, protein, and ubiquitin-dependent catabolic processes. Conclusion: Collectively, these novel results show that protein coding genes of the ubiquitination processes are involved in the pathogenesis of SMA.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1997

Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor

The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Tissue Culture/instrumentation/methods↗
DOE OSTI2011

The Crystal Structure of GXGD Membrane Protease FlaK

The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Interrelations of Apoptotic and Cellular Senescence Genes Methylation in Inflammatory Bowel Disease Subtypes and Colorectal Carcinoma in Egyptians Patients

Ras-related domain family member 1 transcript variant A (RASSF1A) controls apoptosis and cell proliferation while p14/ARF gene has a regulatory role in cellular senescence. Failure of apoptosis and cellular senescence occurs during inflammatory bowel disease (IBD) and colorectal cancer (CRC). To reveal the role of peripheral leukocyte promoter methylation of RASSF1A and p14/ARF in the pathogenesis of IBD subtypes and CRC we investigated the methylation state of the two genes by methylation-specific polymerase chain reaction (MSP-PCR) in 60 CRC patients, 60 patients with IBD; 27 with ulcerative colitis and 33 had Crohn’s disease and also in 30 healthy subjects. Methylated RASSF1A and p14/ARF genes were detected in 55% and 60% of CRC, while the frequency of the methylated RASSF1A and p14/ARF genes was 23.3% and 43.3% in IBD patients and 3.3% and 13.3% in the control group (P = 0.000 each). Also, the frequency of methylated RASSF1A gene was significantly higher in ulcerative colitis than in Crohn’s disease, while a non-significant frequency of methylated p14/ARF was detected between ulcerative colitis and Crohn’s disease. Furthermore, methylated RASSF1A and p14/ARF were associated with the grade of CRC but not associated with the age of patients, family history, or tumor location. Results suggest that methylated RASSF1A and p14/ARF are related to CRC and IBD pathogenesis and may be used as molecular biomarkers for early detection of CRC and IBD.

60 APPLIED LIFE SCIENCES↗