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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 919 records · Page 51
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K{sub app} reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Impact of methionine oxidation on calmodulin structural dynamics

Highlights: • We measured the distance distribution between two spin labels on calmodulin by DEER. • Two structural states, open and closed, were resolved at both low and high Ca. • Ca shifted the equilibrium toward the open state by a factor of 13. • Methionine oxidation, simulated by glutamine substitution, decreased the Ca effect. • These results have important implications for aging in muscle and other tissues. - Abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron–electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4 nm (closed) and another at ∼6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1 to 3 nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM’s functional interaction with its target proteins by perturbing this Ca-dependent structural shift.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant and suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Effects of lysine residues on structural characteristics and stability of tau proteins

Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate

Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. - Highlights: • HIV-1 Vpr utilizes the microtubule network to traffic towards the nucleus. • Mechanism relies on interaction between Vpr and dynein light chain protein DYNLT1. • Long-term non-progressor derived mutation (F72L) impairs this interaction. • Key residues in the vicinity of F72 contribute to interaction with DYNLT1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Deacetylation of Ku70 by SIRT6 attenuates Bax-mediated apoptosis in hepatocellular carcinoma

SIRT6 is a class III histone deacetylase that has been implicated in HCC development. We previously reported that SIRT6 potentiated apoptosis evasion in hepatocellular carcinoma by inhibiting both Bax expression and mitochondrial translocalization. However, the mechanism underlying SIRT6-mediated inhibition of Bax mitochondrial localization remains elusive. In this study, we found that although SIRT6 had no effect on the expression level of Ku70, SIRT6 could interact with Ku70 and deacetylate it. The increased acetylation of Ku70 in SIRT6-depleted cells disrupt its interaction with Bax, which finally resulted in Bax mitochondrial translocalization. Furthermore, lysine K542 on Ku70 was the target for deacetylation by SIRT6. Ku70{sup K542Q} mutation abolished suppression of association between Ku70 and Bax and caused redistribution of Bax to the cytosol in SIRT6-depleted cells. Finally, Ku70{sup K542Q} mutation could reversed the inhibition of growth and apoptosis promotion mediated by SIRT6 silencing. Together, our findings revealed SIRT6 could block the mitochondrial translocation of Bax and decrease the apoptotic ratio of HCC cells by deacetylation of Ku70. SIRT6 may serve as a promising target for developing targeted therapies for HCC in the future. - Highlights: • SIRT6 could associate with and deacetylate Ku70 in HCC cells. • Deacetylated Ku70 could interact with Bax and block its translocation to mitochondria. • SIRT6 deacetylates Ku70 on the lysine 542. • Ku70{sup K542Q} mutation reverses the inhibition of proliferation and promotion of apoptosis which caused by SIRT6 suppression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Effect of sodium fluoride on the sperm mitochondrial DNA in mice

Previous studies demonstrated that fluoride, as a widespread environmental pollutant, induced the reproductive toxicity at high dose. Besides the decrease of sperm characteristics like concentration, survival, and sperm motility, it was found that high fluoride induced the destructive mitochondrial ultrastructure and decreased ATP production from mitochondrial respiration. However, whether fluoride exposure can damage the mitochondrial DNA (mtDNA) of sperm is still unknown. In this study, 100 male mice were randomly divided into four groups 25 each, which were administrated with the distilled water containing 0, 25, 50 and 100 mg L{sup −1} NaF, respectively for 60 days. After exposure, sperm mtDNA copy number was measured by real-time PCR, mtDNA integrity by acridine orange (AO) staining, and nuclear DNA (nDNA) integrity through long PCR amplification, as well as MTCYB and MTATP6 mutations by PCR and direct sequencing. The results showed that 100 mg L{sup −1} NaF significantly increased sperm mtDNA copy number, and reduced nDNA integrity. There were no changes observed in mtDNA integrity, and mutations of MTCYB and MTATP6 between fluoride groups and the control group. These results indicated that along with low sperm quality, sperm mtDNA copy number is also a sensitive biomarker to reflect the sperm toxicity of fluoride. - Highlights: • NaF significantly increased sperm mtDNA copy number, and reduced nDNA integrity. • NaF did not change sperm mtDNA integrity, and mutations of MTCYB and MTATP6. • Sperm mtDNA copy number can be a potential biomarker of fluoride-induced toxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CCN3 secretion is regulated by palmitoylation via ZDHHC22

Highlights: • The CCN3 thrombospondin type-1 (TSP1) domain is required for its secretion. • The TSP1 domain is also involved in CNN3 intracellular function. • Residue C241 is important for CNN3 secretion, but not its intracellular function. • Palmitoylation at C241 is necessary for CCN3 secretion. • Zinc finger DHHC-type containing 22 interacts with CCN3. Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Is There Pseudoprogression in Secondary Glioblastomas?

Purpose: Pseudoprogression (PP) during adjuvant treatment of glioblastoma (GBM) is frequent and is a clinically and radiologically challenging problem. While there are several reports of the frequency of PP in GBM cohorts including mainly patients with primary GBM, there are few data on the incidence of PP in patients with secondary glioblastomas (sGBM). Therefore, the goal of this study was to evaluate the frequency of PP in sGBM. Methods and Materials: We retrospectively evaluated the incidence of PP in adult patients with sGBM treated with chemoradiation therapy (CRTx) using temozolomide (TMZ) and sought to assess if there was an association between PP and MGMT promoter methylation status, IDH mutations status, or 1p/19q codeletion. The definition of PP according to the Response Assessment in Neuro-Oncology Working Group was used. Results: None of the evaluable 15 sGBM patients in our series demonstrated a PP. Of the 9 sGBM patients who received concomitant CRTx with TMZ, 6 patients had the methylated MGMT promoter, and 6 patients had IDH mutations. There also was no PP identified in sGBM patients who received sequential CRTx, irrespective of MGMT or IDH status. The median time of follow-up was 3.4 years after diagnosis of an sGBM, and the median overall survival was 18.2 months (range, 14.3-45.2 months). Three of 15 patients had previously received radiation therapy for their World Health Organization low-grade 2 glioma, while none of them had received chemotherapy at that stage. Conclusions: Based on this small series of sGBM patients treated with CRTx (concomitantly or sequentially) the frequency of PP appears to be very low in sGBM, even in those patients with methylated MGMT promoter or IDH mutations. Our results highlight the differences between primary glioblastomas and sGBM in particular as they relate to PP.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2021

Current Status of Clinical Trials for Cervical and Uterine Cancer Using Immunotherapy Combined With Radiation

Novel therapies combined with radiation continue to be of significant interest in the developmental treatment paradigm of gynecologic cancers. Clinical implementation of immunotherapy in oncology has rapidly changed the treatment landscape, options, paradigm, and outcomes through clinical trials. Immunotherapy has emerged as a therapeutic pillar in the treatment of solid tumors with demonstrable synergistic activity when combined with radiation therapy and chemoradiotherapy by an alteration or enhancement of the immune system. In solid tumors, radiation therapy induces migration of dendritic cells, T cell activation, and proliferation, and increases in tumor-infiltrating lymphocytes. These immunomodulatory effects in conjunction with immune checkpoint blockade are currently under active investigation in the adjuvant, definitive, and metastatic settings. Results from early phase trials demonstrate promising efficacy and overall tolerable toxicity profiles of combined modality treatment. There is significant interest in optimizing the treatment for patients with locally advanced cervical cancer beyond the standard of care—chemoradiation—which has been in place for the last 30 years. The majority of cervical cancer emerges after persistent infection with a high-risk subtype of the human papillomavirus, where viral oncoproteins lead to cellular changes and immortalization. As a result, immune tolerance can develop, resulting in cancer. Knowledge of the mechanism of human papillomavirus–related oncogenesis suggests that immune therapy or checkpoint blockade can reinvigorate an antitumor immune response. Current clinical trials are exploring the therapeutic potential of these approaches. Uterine cancers have been grouped into 4 molecular subclasses by their driver mutations, mutational burden, and copy-number alterations. Of these subgroups, the polymerase epsilon–mutated and microsatellite-unstable may represent up to 40% of endometrial cancers, and they have been shown to be immunogenic. Because of the inherent immunogenicity of these MSI-high tumors, combined immune modulation strategies, including chemotherapy, radiation, and immunotherapy and immune checkpoint inhibitor therapy, are being explored to improve treatment outcomes. In this review, we explore current immunomodulatory and multimodality therapeutic approaches in the treatment of cervical and uterine cancer through ongoing clinical trials investigating the combination of immunotherapy and radiation therapy.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2022

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%–5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Finally, our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Alteration of intersubunit acid–base pair interactions at the quasi-threefold axis of symmetry of Cucumber mosaic virus disrupts aphid vector transmission

In the atomic model of Cucumber mosaic virus (CMV), six amino acid residues form stabilizing salt bridges between subunits of the asymmetric unit at the quasi-threefold axis of symmetry. To evaluate the effects of these positions on virion stability and aphid vector transmissibility, six charged amino acid residues were individually mutated to alanine. All of the six engineered viruses were viable and exhibited near wild type levels of virion stability in the presence of urea. Aphid vector transmissibility was nearly or completely eliminated in the case of four of the mutants; two mutants demonstrated intermediate aphid transmissibility. For the majority of the engineered mutants, second-site mutations were observed following aphid transmission and/or mechanical passaging, and one restored transmission rates to that of the wild type. CMV capsids tolerate disruption of acid–base pairing interactions at the quasi-threefold axis of symmetry, but these interactions are essential for maintaining aphid vector transmissibility. - Highlights: ► Amino acids between structural subunits of Cucumber mosaic virus affect vector transmission. ► Mutant structural stability was retained, while aphid vector transmissibility was disrupted. ► Spontaneous, second-site mutations restored aphid vector transmissibility.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

The West Nile virus strain Kunjin virus (WNV{sub KUN}) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV{sub KUN} replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV{sub KUN} replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PAX5 haploinsufficiency induce cancer cell dormancy in Raji cells

PAX5 mutations have important role in leukemogenesis and leukemia relapse, cancer cell dormancy participates in cancer relapse, but there was no report about PAX5 mutation inducing cancer cell dormancy. we constructed the PAX5 deletion Raji cell lines using gene editing technology, evaluated dormancy biological characteristics of cell lines. Our results showed PAX5 haploinsufficiency restrained the proliferation of Raji cells, induced G0/G1 arrest of Raji cells, reduced chemotherapy sensitivity. The tumor formation rate reduced in PAX5 mutation Raji cells. Our results showed PAX5 insufficiency induced cancer cell dormancy in Raji cell.

60 APPLIED LIFE SCIENCES↗