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Results for “REGULATION”

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At least 253 records · Page 14

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DOE OSTI2014

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Epigenetic down-regulated DDX10 promotes cell proliferation through Akt/NF-κB pathway in ovarian cancer

Ovarian cancer contributes to the majority of ovarian cancer, while the molecular mechanisms remain elusive. Recently, some DEAD box protein 1 has been reported play a tumor suppressor role in ovarian cancer progression. However, the functions of DEAD box protein (DDX) members in ovarian cancer development remain largely unknown. In current study, we retrieved GEO databases and surprisingly found that DDX10 is significantly down-regulated in ovarian cancer tissues compared with normal ovary. These findings suggest that DDX10 might also play a suppressive role in ovarian cancer. We then validated the down-regulated expression pattern of DDX10 in fresh ovarian cancer tissues. Furthermore, both loss- and gain-functions assays reveal that the down-regulated DDX10 could promote ovarian cancer proliferation in vitro and the xenograft subcutaneous tumor formation assays confirmed these findings in vivo. In addition, we found that DDX10 is epigenetic silenced by miR-155-5p in ovarian cancer. Moreover, we further preliminary illustrated that down-regulated DDX10 promotes ovarian cancer cell proliferation through Akt/NF-κB pathway. Taken together, in current study, we found a novel tumor suppressor, DDX10, is epigenetic silenced by miR-155-5p in ovarian cancer, and the down-regulated expression pattern of DDX10 promotes ovarian cancer proliferation through Akt/NF-κB pathway. Our findings shed the light that DDX families might be a novel for ovarian cancer treatment. - Highlights: • A novel DEAD box protein, DDX10 is significantly down-regulated in ovarian cancer tissues. • Down-regulated DDX10 promotes ovarian cancer cell proliferation and growth both in vitro and in vivo. • miR-155-5p is highly expressed in ovarian cancer tissues and epigenetically targets DDX10. • DDX10 and miR-155-5p regulates Akt/p65 axis in ovarian cancer cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Nurr1 overexpression exerts neuroprotective and anti-inflammatory roles via down-regulating CCL2 expression in both in vivo and in vitro Parkinson's disease models

The abnormality of nuclear receptor-related protein 1 (Nurr1) in expression and function can contribute to neurodegeneration of dopaminergic neurons and occurrence of Parkinson's disease (PD). However, its related mechanism in PD is still unknown. In this study, we found that Nurr1 was down-regulated and CCL2 was up-regulated in PD patients and PD mice. CCL2 promoted apoptosis and secretion of TNF-α and IL-1β in SH-SY5Y cells and inhibited cell viability while knockdown of CCL2 exerted the opposite effects. Nurr1 overexpression inhibited apoptosis, the release of TNF-α and IL-1β and promoted viability in α-Syn-treated SH-SY5Y cells, which was markedly promoted by CCL2 antibody and dramatically reversed by CCL2. Nurr1 overexpression negatively regulated CCL2 expression in vivo and in vitro. Furthermore, Nurr1 overexpression remarkably relieved MPTP-induced movement disorder and spatial memory deficits and played neuroprotective and anti-inflammatory roles in MPTP-induced PD mice by down-regulating CCL2 in vivo. In conclusion, Nurr1 overexpression exerts neuroprotective and anti-inflammatory roles via down-regulating CCL2 in both in vivo and in vitro PD models, contributing to developing mechanism-based and neuroprotective strategies against PD. - Highlights: • Nurr1 was down-regulated and CCL2 was up-regulated in PD patients and PD mice. • Nurr1 overexpression inhibited apoptosis, release of TNF-α and IL-1β and promoted viability in α-Syn-treated SH-SY5Y cells. • CCL2 reversed the effect of Nurr1 overexpression on apoptosis, inflammatory cytokines secretion and viability. • Nurr1 overexpression negatively regulated CCL2 expression in vivo and in vitro. • Nurr1 overexpression remarkably relieved MPTP-induced movement disorder and spatial memory deficits.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Klotho down-regulates Egr-1 by inhibiting TGF-β1/Smad3 signaling in high glucose treated human mesangial cells

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease worldwide and is associated with glomerular mesangial cell (MC) proliferation and excessive extracellular matrix (ECM) production. Klotho can attenuate renal fibrosis in part by inhibiting TGF-β1/Smad3 signaling in DKD. Early growth response factor 1 (Egr-1) has been shown to play a key role in renal fibrosis in part by facilitating the formation of a positive feedback loop involving TGF-β1. However, whether Klotho down-regulates Egr-1 by inhibiting TGF-β1/Smad3 signaling in DKD is unclear. In the present study, we assessed human MCs that were incubated under high-glucose conditions to mimic diabetes. Then, we transfected the cells with Klotho plasmid or siRNA to overexpress or knock down Klotho gene and protein expression. Klotho, Egr-1, fibronectin (FN), collagen type I (Col I), Smad3 and phosphorylated Smad3 (p-Smad3) gene and protein expression levels were determined by RT-qPCR and western blotting respectively. High glucose time-dependently down-regulated Klotho mRNA and protein expression in cultured human MCs. pcDNA3.1-Klotho transfection-mediated Klotho overexpression down-regulated Egr-1, FN and Col I expression and the p-Smad3/Smad3 ratio in human MCs. Conversely, siRNA-mediated Klotho silencing up-regulated Egr-1, FN, and Col I expression and the p-Smad3/Smad3 ratio. Moreover, the effects of si-Klotho on Egr-1 expression were abolished by the TGF-β1 inhibitor SB-431542. Klotho overexpression can prevent mesangial ECM production in high-glucose-treated human MCs, an effect that has been partially attributed to Egr-1 down-regulation facilitated by TGF-β1/Smad3 signaling inhibition. - Highlights: • High glucose time-dependently down-regulated Klotho mRNA and protein expression in cultured human MCs. • Klotho overexpression down-regulated Egr-1 and prevented mesangial ECM production in high-glucose-treated human MCs. • Klotho down-regulated Egr-1 by inhibiting TGF-β1/Smad3 signaling in high-glucose-treated human MCs.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Overexpression of eEF1A1 regulates G1-phase progression to promote HCC proliferation through the STAT1-cyclin D1 pathway

Highlights: • eEF1A1 influences HCC proliferation by regulating cell cycle G1 phase. • eEF1A1 influences cell cycle G1 phase by regulating cyclin D1 expression. • eEF1A1 influences cyclin D1 expression by STAT1 signaling. • STAT1 increases cyclin D1 expression by activating the transcriptional activity of cyclin D1. Hepatocellular carcinoma (HCC) is a common cancer worldwide with an aggressive and highly proliferative activity. Studies had confirmed that HCC cell proliferation is associated with the cell cycle's G1 phase, but the detailed molecular mechanism has not been thoroughly elucidated to date. Eukaryotic translation elongation factor 1A1 (eEF1A1) is an evolutionarily conserved elongation factor protein and is involved in tumor cell proliferation. However, which phase of the cell cycle is regulated by eEF1A1 to influence cell proliferation in HCC and its detailed molecular mechanism remain unclear. In this study, we observed that eEF1A1 influences HCC cell proliferation by regulating the cell cycle's G1 phase. In addition, eEF1A1 influences G1 phase by regulating cyclin D1 expression, promoting HCC cell proliferation both in vitro and in vivo. Moreover, our results indicated that eEF1A1 regulates cyclin D1 expression through STAT1 signaling. STAT1 increases the transcriptional activity of cyclin D1 by binding to the cyclin D1 promoter. Taken together, these findings enabled us to identify a novel mechanism by which eEF1A1 regulates the cell cycle's G1 phase to promote tumor proliferation by regulating cyclin D1 expression through STAT1 signaling in HCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2

Highlights: • LncRNA FTX inhibits apoptosis in cardiomyocytes. • miR-29b-1-5p regulates apoptosis by targeting Bcl2l2 in cardiomyocytes. • FTX sponges and regulates the activity of miR-29b-1-5p. • FTX regulates apoptosis by targeting miR-29b-1-5p and Bcl2l2. Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Rg1 inhibits high glucose-induced mesenchymal activation and fibrosis via regulating miR-2113/RP11-982M15.8/Zeb1 pathway

Highlights: • High glucose conditions up-regulate MMP-2, MMP-9 and down-regulate TIMP-2, and promote mesenchymal activation in Müller cells. • Inhibition of miR-2113 and over-expression of RP11-982M15.8 significantly restored the HG induced mesenchymal activasion. • Taken together, Rg1 inhibited HG-induced mesenchymal activation and fibrosis via regulating miR-2113/RP11-982M15.8/Zeb1 pathway. Recent study has showed that Ginsenoside Rg1, the mian active compound of Panax ginseng, could ameliorate oxidative stress and myocardial apoptosis in diabetes mellitus. However, the roles and mechanisms of Rg1 in proliferative diabetic retinopathy (PDR) are still unclear. In the present study, we aimed to investigate the effects of Rg1 on mesenchymal activation of high-glucose (HG) cultured müller cells. High glucose conditions up-regulate MMP-2, MMP-9 and down-regulate TIMP-2, and promote mesenchymal activation in Müller cells. And Rg1 inhibits the HG-induced mesenchymal activation and HG-increased MMP-2 and MMP-9 and HG-decreased TIMP-2 in Müller cells. HG up-regulates Zeb1 and lncRNA RP11-982M15.8, and down-regulates miR-2113, and Rg1 inhibits these effects of HG. Both inhibition of miR-2113 and over-expression of RP11-982M15.8 significantly restored the HG induced mesenchymal activasion. Taken together, our findings suggested that Rg1 inhibited HG-induced mesenchymal activation and fibrosis via regulating miR-2113/RP11-982M15.8/Zeb1 pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

The miR-31-SOX10 axis regulates tumor growth and chemotherapy resistance of melanoma via PI3K/AKT pathway

Highlights: • Firstly found up-regulation of SOX10 and down-regulation of miR-31 in MM. • miR-31 regulated SOX10 by binding to its 3′UTR. • SOX10 regulated tumor growth and chemotherapy resistance of melanoma in vitro and in vivo. • The miR-31-SOX10 axis mediated tumor growth and drug resistance via activation of PI3K/AKT pathway. MicroRNAs were thought to play a regulatory role through complementarity to target messenger RNA (mRNA). Our previous study revealed a miR-31-SOX10 axis that regulated tumor growth and resistance to chemotherapy of melanoma. Up-regulation of SOX10 and down-regulation of miR-31 were found in melanoma tissues. SOX10 was further identified as a target of miR-31. Overexpression of SOX10 dramatically promoted melanoma cell proliferation and chemotherapy resistance both in vitro and in vivo. While enforced miR-31 expression suppressed cell growth and enhanced the chemosensitivity of melanoma cells, the re-expression of SOX10 rescued these effects by activating PI3K/AKT signaling pathway. In conclusion, our results demonstrated that SOX10 acted as an oncogene and was negatively regulated by miR-31, which supports the potential therapeutic strategy against melanoma by targeting the miR-31-SOX10 axis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

microRNA-mediated regulation of splicing factors SRSF1, SRSF2 and hnRNP A1 in context of their alternatively spliced 3′UTRs

Highlights: • A group of microRNAs contributes to the regulation of SRSF1, SRSF2 and hnRNP A1. • microRNAs targeting SRSF1 and SRSF2 are involved in a regulatory feedback loop. • microRNAs that regulate SRSF2 affect also the expression of apoptotic genes. • 3′UTR variants of SRSF1 and SRSF2 differ by the presence of miRNA-binding sites. SRSF1, SRSF2 and hnRNP A1 are splicing factors that regulate the expression of oncogenes and tumor suppressors. SRSF1 and SRSF2 contribute to the carcinogenesis in the kidney. Despite their importance, the mechanisms regulating their expression in cancer are not entirely understood. Here, we investigated the microRNA-mediated regulation of SRSF1, SRSF2 and hnRNP A1 in renal cancer. The expression of microRNAs predicted to target SRSF1, SRSF2 and hnRNP A1 was disturbed in renal tumors compared with controls. Using qPCR, Western blot/ICC and luciferase reporter system assays we identified microRNAs that contribute to the regulation of expression of SRSF1 (miR-10b-5p, miR-203a-3p), SRSF2 (miR-183-5p, miR-200c-3p), and hnRNP A1 (miR-135a-5p, miR-149-5p). Silencing of SRSF1 and SRSF2 enhanced the expression of their targeting microRNAs. miR-183-5p and miR-200c-3p affected the expression of SRSF2-target genes, TNFRSF1B, TNFRSF9, CRADD and TP53. 3′UTR variants of SRSF1 and SRSF2 differed by the presence of miRNA-binding sites. In conclusion, we identified a group of microRNAs that contribute to the regulation of expression of SRSF1, SRSF2 and hnRNP A1. The microRNAs targeting SRSF1 and SRSF2 are involved in a regulatory feedback loop. microRNAs miR-183-5p and miR-200c-3p that target SRSF2, affect the expression of genes involved in apoptotic regulation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Chitosan oligosaccharides enhance lipid droplets via down-regulation of PCSK9 gene expression in HepG2 cells

Highlights: • COS enhance lipid droplets in HepG2 cells. • COS down-regulate the expression of PCSK9 to maintain hepatic LDLR abundance and activity. • COS inhibit HNF1α and SREBP-2 activity leading to down-regulation of PCSK9. Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE{sup -/-} male mice showed that COS treatment of 500 mg kg{sup −1} d{sup −1} for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (p < 0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200 μg/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (p < 0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24 h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24 h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

GAS REGULATION OF GALAXIES: THE EVOLUTION OF THE COSMIC SPECIFIC STAR FORMATION RATE, THE METALLICITY-MASS-STAR-FORMATION RATE RELATION, AND THE STELLAR CONTENT OF HALOS

A very simple physical model of galaxies is one in which the formation of stars is instantaneously regulated by the mass of gas in a reservoir with mass loss scaling with the star-formation rate (SFR). This model links together three different aspects of the evolving galaxy population: (1) the cosmic time evolution of the specific star-formation rate (sSFR) relative to the growth of halos, (2) the gas-phase metallicities across the galaxy population and over cosmic time, and (3) the ratio of the stellar to dark matter mass of halos. The gas regulator is defined by the gas consumption timescale ({epsilon}{sup -1}) and the mass loading {lambda} of the wind outflow {lambda}{center_dot}SFR. The simplest regulator, in which {epsilon} and {lambda} are constant, sets the sSFR equal to exactly the specific accretion rate of the galaxy; more realistic situations lead to an sSFR that is perturbed from this precise relation. Because the gas consumption timescale is shorter than the timescale on which the system evolves, the metallicity Z is set primarily by the instantaneous operation of the regulator system rather than by the past history of the system. The metallicity of the gas reservoir depends on {epsilon}, {lambda}, and sSFR, and the regulator system therefore naturally produces a Z(m{sub star}, SFR) relation if {epsilon} and {lambda} depend on the stellar mass m{sub star}. Furthermore, this relation will be the same at all epochs unless the parameters {epsilon} and {lambda} themselves change with time. A so-called fundamental metallicity relation is naturally produced by these conditions. The overall mass-metallicity relation Z(m{sub star}) directly provides the fraction f{sub star}(m{sub star}) of incoming baryons that are being transformed into stars. The observed Z(m{sub star}) relation of Sloan Digital Sky Survey (SDSS) galaxies implies a strong dependence of stellar mass on halo mass that reconciles the different faint-end slopes of the stellar and halo mass functions in standard {Lambda}CDM models. The observed relation also boosts the sSFR relative to the specific accretion rate and produces a different dependence on mass, both of which are observed. The derived Z(m{sub star}, SFR) relation for the regulator system is fit to published Z(m{sub star}, SFR) data for the SDSS galaxy population, yielding {epsilon} and {lambda} as functions of m{sub star}. The fitted {epsilon} is consistent with observed molecular gas-depletion timescales in galaxies (allowing for the extra atomic gas), while the fitted {lambda} is also reasonable. The gas-regulator model also successfully reproduces the Z(m{sub star}) metallicities of star-forming galaxies at z {approx} 2. One consequence of this analysis is that it suggests that the m{sub star}-m{sub halo} relation is established by baryonic processes operating within galaxies, and that a significant fraction (40%) of baryons coming into the halos are being processed through the galaxies. This fraction may be more or less constant. The success of the gas-regulator model in simultaneously explaining many diverse observed relations over the 0 < z < 2 interval suggests that the evolution of galaxies is governed by simple physics that form the basis for this model.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2016

Differential Regulation of the Two Ferrochelatase Paralogues in Shewanella loihica PV-4 in Response to Environmental Stresses

Determining the function and regulation of paralogues is important in understanding microbial functional genomics and environmental adaptation. Heme homeostasis is crucial for the survival of environmental microorganisms. Most Shewanella species encode two paralogues of ferrochelatase, the terminal enzyme in the heme biosynthesis pathway. The function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2 , were investigated in Shewanella loihica PV-4. The disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), the precursor to heme, and decreased intracellular heme levels. hemH1 was constitutively expressed, and the expression of hemH2 increased when hemH1 was disrupted. The transcription of hemH1 was regulated by the housekeeping sigma factor RpoD and potentially regulated by OxyR, while hemH2 appeared to be regulated by the oxidative stress-associated sigma factor RpoE2. When an oxidative stress condition was mimicked by adding H 2 O 2 to the medium or exposing the culture to light, PPIX accumulation was suppressed in the Δ hemH1 mutant. Consistently, transcriptome analysis indicated enhanced iron uptake and suppressed heme synthesis in the Δ hemH1 mutant. These data indicate that the two paralogues are functional in the heme synthesis pathway but regulated by environmental conditions, providing insights into the understanding of bacterial response to environmental stresses and a great potential to commercially produce porphyrin compounds. Shewanella is capable of utilizing a variety of electron acceptors for anaerobic respiration because of the existence of multiple c -type cytochromes in which heme is an essential component. The cytochrome-mediated electron transfer across cellular membranes could potentially be used for biotechnological purposes, such as electricity generation in microbial fuel cells and dye decolorization. However, the mechanism underlying the regulation of biosynthesis of heme and cytochromes is poorly understood. In conclusion, our study has demonstrated that two ferrochelatase genes involved in heme biosynthesis are differentially regulated in response to environmental stresses, including light and reactive oxygen species. This is an excellent example showing how bacteria have evolved to maintain cellular heme homeostasis. More interestingly, the high yields of extracellular protoporphyrin IX by the Shewanella loihica PV-4 mutants could be utilized for commercial production of this valuable chemical via bacterial fermentation.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

RRNPP-type quorum-sensing systems regulate solvent formation, sporulation and cell motility in Clostridium saccharoperbutylacetonicum

Abstract Background Clostridium saccharoperbutylacetonicum N1-4 (HMT) is a strictly anaerobic, spore-forming Gram-positive bacterium capable of hyper-butanol production through the well-known acetone–butanol–ethanol fermentation process. Recently, five putative RRNPP-type QSSs (here designated as QSS1 to QSS5) were predicted in this bacterial strain, each of which comprises a putative RRNPP-type regulator (QssR1 to QssR5) and a cognate signaling peptide precursor (QssP1 to QssP5). In addition, both proteins are encoded by the same operon. The functions of these multiple RRNPP-type QSSs are unknown. Results To elucidate the function of multiple RRNPP-type QSSs as related to cell metabolism and solvent production in N1-4 (HMT), we constructed qssR -deficient mutants ΔR1, ΔR2, ΔR3 and ΔR5 through gene deletion using CRISPR–Cas9 and N1-4-dcas9-R4 (with the QssR4 expression suppressed using CRISPR–dCas9). We also constructed complementation strains by overexpressing the corresponding regulator gene. Based on systematic characterization, results indicate that QSS1, QSS2, QSS3, and QSS5 positively regulate the sol operon expression and thus solvent production, but they likely negatively regulate cell motility. Consequently, QSS4 might not directly regulate solvent production, but positively affect cell migration. In addition, QSS3 and QSS5 appear to positively regulate sporulation efficiency. Conclusions Our study provides the first insights into the roles of multiple RRNPP-type QSSs of C. saccharoperbutylacetonicum for the regulation of solvent production, cell motility, and sporulation. Results of this study expand our knowledge of how multiple paralogous QSSs are involved in the regulation of essential bacterial metabolism pathways.

09 BIOMASS FUELS↗

DOE OSTI2019

LurR is a regulator of the central lactate oxidation pathway in sulfate-reducing Desulfovibrio species

The central carbon/lactate utilization pathway in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, is encoded by the highly conserved operon DVU3025-3033. Our earlier in vitro genome-wide study had suggested a network of four two-component system regulators that target this large operon; however, how these four regulators control this operon was not known. Here, we probe the regulation of the lactate utilization operon with mutant strains and DNA-protein binding assays. We show that the LurR response regulator is required for optimal growth and complete lactate utilization, and that it activates the DVU3025-3033 lactate oxidation operon as well as DVU2451, a lactate permease gene, in the presence of lactate. We show by electrophoretic mobility shift assays that LurR binds to three sites in the upstream region of DVU3025, the first gene of the operon. NrfR, a response regulator that is activated under nitrite stress, and LurR share similar binding site motifs and bind the same sites upstream of DVU3025. The DVU3025 promoter also has a binding site motif (Pho box) that is bound by PhoB, a two-component response regulator activated under phosphate limitation. The lactate utilization operon, the regulator LurR, and LurR binding sites are conserved across the order Desulfovibrionales whereas possible modulation of the lactate utilization genes by additional regulators such as NrfR and PhoB appears to be limited to D. vulgaris .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Systems Level Regulation of Rhythmic Growth Rate and Biomass Accumulation in Grasses

Objectives: Several breakthroughs have been recently made in our understanding of plant growth and biomass accumulation. It was found that plant growth is rhythmically controlled throughout the day by the circadian clock through a complex interplay of light and phytohormone signaling pathways. While plants such as the C4 energy crop sorghum (Sorghum bicolor (L.) Moench) and possibly the C3 grass Brachypodium distachyon also exhibit daily rhythms in growth rate, the molecular details of its regulation remain to be explored. A better understanding of diurnally regulated growth behavior in grasses may lead to species-specific mechanisms highly relevant to future strategies to optimize energy crop biomass yield. Here we propose to devise a systems approach to identify, in parallel, regulatory hubs associated with rhythmic growth in C3 and C4 plants. We propose to use rhythmicity in daily growth patterns to drive the discovery of regulatory network modules controlling biomass accumulation. Description: The project is divided in three main parts: 1) Performing time-lapse imaging and growth measurement in B. distachyon and S. bicolor to determine growth rate dynamic during the day/night cycle. Identifying growth-associated genes whose expression patterns follow the observed growth dynamics using deep sequencing technology, 2) identifying regulators of these genes by screening for DNA-binding proteins interacting with the growth-associated gene promoters identified in Aim 1. Screens will be performed using a validated yeast-one hybrid strategy paired with a specifically designed B. distachyon and S. bicolor transcription factor libraries (1000 clones each), and 3) Selecting 50 potential growth regulators from the screen for downstream characterization. The selection will be made by using a sytems biology approach by calculating the connectivity between growth rate, rhythmic gene expression profiles and TF expression profile and determine which TF is likely part of a hub and a potential regulator of plant growth. We will confirme the 50 DNA-protein interactions using transient transcriptional assays in B. distachyon and S. bicolor, and perform further testing in vivo by measuring growth parameters in transgenic loss- or gain-of-function lines for the selected transcription factors (25 B. distachyon and 3 S. bicolor lines). In 2016 the Principal Investigator relocated the laboratory to The Scripps Research Institute (TSRI) where the project has continued with an end date of 08/31/17. Accomplishments: We successfully collected large datasets of gene expression form the model grass Brachypodium. We used and developed bioinformatics analysis tools to investigate the structure, dynamics and robustness of circadian regulated gene expression in Brachypodium. Relevant Discoveries: We were able to determine that the endogenous circadian clock appears to play a much more subdued role in growth regulation in Brachypodium, that has been demonstrated in either Arabidopsis, or crop plants like Rice, Corn and Soybean. This led to our conclusion that Brachypodium unfortunately is unlikely to serve as an informative model for understanding how growth regulation in plants is under the control of circadian network circuitry. However, we were able to leverage our datasets in Brachypodium to inform us and reinforce a large collaborative study on gene networks governing cell wall deposition in Arabidopsis. This led to a major and highly cited publication relevant to improving biomass production.

09 BIOMASS FUELS↗

DOE OSTI2019

Rainwater Harvesting State Regulations and Technical Resources

Pacific Northwest National Laboratory (PNNL) conducted in-depth research of state-level rainwater harvesting regulations for the Federal Energy Management Program (FEMP) to help federal agencies strategically identify locations conducive to rainwater harvesting projects. Currently, rainwater harvesting is not regulated by the federal government; instead it is up to individual states to regulate the collection and use of rainwater. There is no centralized information about state-level regulations on rainwater harvesting maintained by a federal agency or outside organization. To fill this information gap, PNNL performed detailed internet searches for each state, which included researching regulations associated with state agencies, universities, Cooperative Extension Offices, city governments, and related organizations. The state-by-state information about rainwater harvesting regulations was compiled and assembled into an interactive map that is color coded by state regulations. The map provides a visual representation of the general types of rainwater harvesting policies across the country as well as general information about the state programs if applicable. The map allows the user to quickly discern where rainwater harvesting is supported and regulated by the state. This map was created in June 2015 and the data were updated in June 2019.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1973

Design for pressure regulating components

The design development for Pressure Regulating Components included a regulator component trade-off study with analog computer performance verification to arrive at a final optimized regulator configuration for the Space Storable Propulsion Module, under development for a Jupiter Orbiter mission. This application requires the pressure regulator to be capable of long-term fluorine exposure. In addition, individual but basically identical (for purposes of commonality) units are required for separate oxidizer and fuel pressurization. The need for dual units requires improvement in the regulation accuracy over present designs. An advanced regulator concept was prepared featuring redundant bellows, all metallic/ceramic construction, friction-free guidance of moving parts, gas damping, and the elimination of coil springs normally used for reference forces. The activities included testing of actual size seat/poppet components to determine actual discharge coefficients and flow forces. The resulting data was inserted into the computer model of the regulator. Computer simulation of the propulsion module performance over two mission profiles indicated satisfactory minimization of propellant residual requirements imposed by regulator performance uncertainties.

Wichmann, H.↗

NASA NTRS2004

The Effects of Protein Regulators on the Vascular Remodeling of Japanese Quail Chorioallantoic Membrane

Contributing to NASA s mission, the Microgravity Fluid Physics research program conducts experiments to promote space exploration and improvement of processes and products on Earth. One of the projects through this program deals with the affect of regulators on vascular remodeling and angiogenesis. This project is being led by Dr. Patricia Parsons-Wingerter. To perform the experiments, protein regulators are tested on the chorioallantoic membrane (CAM) of the Japanese quail embryos. The different types of regulators used can be broken down into two major groups of stimulators, and inhibitors. Stimulators increase the rate of blood vessel growth and inhibitors decrease of blood vessel growth. The specified regulator proteins include thrombospondin 1 (TSP-1) and a novel vessel tortuosity factor (TF), these are just the ones used in this specific experiment; other various protein regulators can also be used. The novel vessel tortuosity factor (TF) is a special kind of stimulator because it stimulates vessel tortuosity and curvature, rather than actual blood vessel growth. These regulators are being tested on Japanese quail embryos. The Japanese quail embryos naturally form a chorioallantoic membrane (CAM) from which blood flow, vascular remodeling, and angiogenesis can be observed. Chorioallantoic membranes are also easier to use because they are two dimensional when mounted onto a slide for examination. The analysis of the affect of the regulators on the CAM can be studied through PIVPROC; the program is used to analyze the altered blood flow in response to application of TF. Regulators are being thoroughly studied because cardiovascular alterations are the second highest, NASA-defined, risk categories in human space exploration. This research done on the quail is extending to even more projects that will be done on lab animals such as mice and also in human clinical studies like the diabetic retina. Not only will this research be beneficial to further space exploration, but it will also help life here on Earth. The higher understanding of the formation of blood vessels can also help further research in health problems such as diabetes and heart disease.

Deshpande, Arati↗