scholarly journals The Negative Regulative Roles of BdPGRPs in the Imd Signaling Pathway of Bactrocera dorsalis

Cells ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 152
Author(s):  
Ping Zhang ◽  
Zhichao Yao ◽  
Shuai Bai ◽  
Hongyu Zhang
Keyword(s):  
Signaling Pathway ◽  
Fat Body ◽  
Bactrocera Dorsalis ◽  
Bacterial Challenge ◽  
Negative Regulators ◽  
Amidase Activity ◽  
E Coli ◽  
Time Points ◽  
Imd Pathway ◽  
Exact Function

Peptidoglycan recognition proteins (PGRPs) are key regulators in insects’ immune response, functioning as sensors to detect invading pathogens and as scavengers of peptidoglycan (PGN) to reduce immune overreaction. However, the exact function of PGRPs in Bactrocera dorsalis is still unclear. In this study, we identified and functionally characterized the genes BdPGRP-LB, BdPGRP-SB1 and BdPGRP-SC2 in B. dorsalis. The results showed that BdPGRP-LB, BdPGRP-SB1 and BdPGRP-SC2 all have an amidase-2 domain, which has been shown to have N-Acetylmuramoyl-l-Alanine amidase activity. The transcriptional levels of BdPGRP-LB and BdPGRP-SC2 were both high in adult stages and midgut tissues; BdPGRP-SB1 was found most abundantly expressed in the 2nd instar larvae stage and adult fat body. The expression of BdPGRP-LB and BdPGRP-SB1 and AMPs were significantly up-regulated after injury infected with Escherichia coli at different time points; however, the expression of BdPGRP-SC2 was reduced at 9 h, 24 h and 48 h following inoculation with E. coli. By injection of dsRNA, BdPGRP-LB, BdPGRP-SB1 and BdPGRP-SC2 were knocked down by RNA-interference. Silencing of BdPGRP-LB, BdPGRP-SB1 and BdPGRP-SC2 separately in flies resulted in over-activation of the Imd signaling pathway after bacterial challenge. The survival rate of the ds-PGRPs group was significantly reduced compared with the ds-egfp group after bacterial infection. Taken together, our results demonstrated that three catalytic PGRPs family genes, BdPGRP-LB, BdPGRP-SB1 and BdPGRP-SC2, are important negative regulators of the Imd pathway in B. dorsalis.

scholarly journals Characterization of PGRP-LB and immune deficiency in the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae)

2021 ◽  
Author(s):  
Yaya Yu ◽  
Chunli Luo ◽  
Daowei Zhang ◽  
Jing Chen
Keyword(s):  
Immune Deficiency ◽  
Cell Walls ◽  
Secreted Protein ◽  
Sogatella Furcifera ◽  
Transcript Levels ◽  
Amidase Activity ◽  
E Coli ◽  
Imd Pathway ◽  
Insect Defense

AbstractPeptidoglycan recognition proteins (PGRPs) participate in insect defense against bacterial pathogens by recognizing bacterial cell wall peptidoglycans (PGNs). Here, we identified the PGRP-LB gene in the white-backed planthopper Sogatella furcifera (SfPGRP-LB). SfPGRP-LB is a secreted protein with a typical PGN-binding domain and five conserved amino acid (aa) residues required for amidase activity. Expression analysis showed that the SfPGRP-LB transcript levels were significantly higher in the midgut than in other tissues. Silencing SfPGRP-LB with dsRNA significantly downregulated the expression of Toll pathway genes Toll and Dorsal and Imd pathway genes Imd and Relish after Escherichia coli challenge. However, only Toll and Dorsal expressions were downregulated after Staphylococcus aureus challenge. E. coli and S. aureus challenges rapidly and strongly upregulated SfPGRP-LB expression. Recombinantly expressed SfPGRP-LB (rSfPGRP-LB) had strong affinities for E. coli Dap-type PGN and S. aureus Lys-type PGN and agglutinated the bacteria. However, rSfPGRP-LB inhibited S. aureus but not E. coli growth. Furthermore, rSfPGRP-LB had amidase activity, degraded Lys-type PGN, and destroyed S. aureus cell walls but had no such effects on E. coli Dap-type PGN. Thus, SfPGRP-LB recognizes and binds various bacterial PGNs but only has amidase activity against Lys-type PGN.

scholarly journals Characterization and Function of Two Short Peptidoglycan Recognition Proteins Involved in the Immunity of Bactrocera dorsalis (Hendel)

Insects ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 79 ◽  
Author(s):  
Dong Wei ◽  
Yu-Wei Liu ◽  
Ying-Xin Zhang ◽  
Jin-Jun Wang
Keyword(s):  
Immune Responses ◽  
Fat Body ◽  
Bactrocera Dorsalis ◽  
Amino Acid Residues ◽  
Transcriptional Expression ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
And Function

Peptidoglycans (PGNs) are major bacterial components recognized by the immune systems of insects and mammals. PGN recognition proteins (PGRPs) are widely distributed and highly conserved in vertebrates and invertebrates. PGRPs are a family of pattern recognition receptors that recognize peptidoglycan and regulate immune responses. In this study, we cloned two PGRP genes (BdPGRP-SA and BdPGRP-SD) from Bactrocera dorsalis (Hendel), which encode 192 and 196 amino acid residues, respectively. Both genes were highly expressed in adults, especially in the fat body and midgut. These two genes were up-regulated when challenged by the immune triggers, PGN-EB (Escherichia coli O111:B4) and PGN-SA (Staphylococcus aureus). The suppression of transcriptional expression of either gene by RNA interference (RNAi) resulted in increased sensitivities to Gram-negative E. coli and Gram-positive S. aureus PGNs. Suppression of BdPGRP-SA and -SD expression by RNAi resulted in weak expressions of four antimicrobial peptides (AMPs) upon injected with E. coli or S. aureus. BdPGRP-SA and -SD are involved in recognizing both Gram-negative and Gram-positive bacteria independently to activate the downstream AMP’s response to bacterial infection.

A New Fluorogenic Substrate Method for Assay of Bacterial Endotoxins Using Limulus Hemocyte Lysate

1979 ◽  
Author(s):  
T Harada ◽  
M Ohki ◽  
M Niwa ◽  
S Iwanaga
Keyword(s):  
Antithrombin Iii ◽  
Assay Method ◽  
Sensitive Assay ◽  
Fluorogenic Substrate ◽  
Toxin Concentration ◽  
Amidase Activity ◽  
E Coli ◽  
Bacterial Endotoxins ◽  
Plasmin Inhibitor ◽  
Limulus Test

Limulus hemocyte lysate contains a proclotting enzyme, which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Gly-Arg-4-methylcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidase activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5X10-6to 5xl0-2 µg endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.

scholarly journals Aedes aegypti (Diptera: Culicidae) Immune Responses with Different Feeding Regimes Following Infection by the Entomopathogenic Fungus Metarhizium anisopliae

Insects ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 95 ◽  
Author(s):  
Sara Cabral ◽  
Adriano de Paula ◽  
Richard Samuels ◽  
Rodrigo da Fonseca ◽  
Simone Gomes ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Immune Responses ◽  
Antimicrobial Peptides ◽  
Fungal Infection ◽  
Fat Body ◽  
Negative Regulator ◽  
Toll Pathway ◽  
Promising Alternative ◽  
Imd Pathway ◽  
Feeding Regimes

The mosquito Aedes aegypti is the most notorious vector of illness-causing viruses. The use of entomopathogenic fungi as bioinsecticides is a promising alternative for the development of novel mosquito control strategies. We investigate whether differences in immune responses could be responsible for modifications in survival rates of insects following different feeding regimes. Sucrose and blood-fed adult A. aegypti females were sprayed with M. anisopliae 1 × 106 conidia mL−1, and after 48 h, the midgut and fat body were dissected. We used RT-qPCR to monitor the expression of Cactus and REL1 (Toll pathway), IMD, REL2, and Caspar (IMD pathway), STAT and PIAS (JAK-STAT pathway), as well as the expression of antimicrobial peptides (Defensin A, Attacin and Cecropin G). REL1 and REL2 expression in both the midgut and fat body were higher in blood-fed fungus-challenged A. aegypti than in sucrose-fed counterparts. Interestingly, infection of sucrose-fed insects induced Cactus expression in the fat body, a negative regulator of the Toll pathway. The IMD gene was upregulated in the fat body in response to fungal infection after a blood meal. Additionally, we observed the induction of antimicrobial peptides in the blood-fed fungus-challenged insects. This study suggests that blood-fed A. aegypti are less susceptible to fungal infection due to the rapid induction of Toll and IMD immune pathways.

scholarly journals Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli.

1996 ◽  
Vol 183 (3) ◽  
pp. 1037-1044 ◽  
Author(s):  
M Hedlund ◽  
M Svensson ◽  
A Nilsson ◽  
R D Duan ◽  
C Svanborg
Keyword(s):  
Escherichia Coli ◽  
Signaling Pathway ◽  
Kinase Inhibitors ◽  
Human Kidney ◽  
Cytokine Response ◽  
Protein Kinase Inhibitors ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
Outer Leaflet

Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.

Endotoxin-Like Activities of Mycoplasmal Lipopolysaccharides (Lipoglycans)

1980 ◽  
Vol 29 (3) ◽  
pp. 990-994
Author(s):  
Robert C. Seid ◽  
Paul F. Smith ◽  
Gabriel Guevarra ◽  
H. Donald Hochstein ◽  
Michael F. Barile
Keyword(s):  
Escherichia Coli ◽  
Synthetic Peptide ◽  
Active Role ◽  
Peptide Bond ◽  
Febrile Response ◽  
Thermoplasma Acidophilum ◽  
Amidase Activity ◽  
E Coli

Lipoglycans (previously designated lipopolysaccharides) from several species of Acholeplasma and from Thermoplasma acidophilum were examined for endotoxin-like activities as measured by the standard rabbit fever test and the Limulus amoebocyte lysate assay. The lipoglycans from Acholeplasma granularum, Achloplasma laidlawii, Acholeplasma modicum , and Acholeplasma oculi caused a febrile response at concentrations of 1 ng/ml per kg or greater, whereas with control Escherichia coli EC-2 lipopolysaccharides, 6.25 ng/ml per kg was required. Similar results were obtained in the Limulus amoebocyte lysate test. The minimum concentrations in nanograms per milliliter required to stimulate formation of a solid clot were: Acholeplasma axanthum , 0.22; A. granularum , 0.85; A. modicum , 0.51; A. laidlawii , 1.05; A. oculi , 0.74. Standard E. coli 1B lipopolysaccharide required a concentration of 0.125 ng/ml. Thermoplasma lipoglycan was least active, requiring 4.25 ng/ml. Clotting of the Limulus lysate proceeds by the activation by lipopolysaccharide plus Ca 2+ of a proenzyme which cleaves an arginine-lysine peptide bond of the coagulogen. The clotting and amidase activities are inactivated by deoxycholate and can be reactivated by addition of lipopolysaccharide and Ca 2+ . As with E. coli 1B lipopolysaccharide, acholeplasmal lipoglycans were shown to restore both clotting and amidase activities of the deoxycholate-inactivated Limulus clotting enzyme. The degree of restoration of amidase activity by mycoplasmal lipoglycans relative to E. coli lipopolysaccharide (1.00) were: A. axanthum , 1.71; A. modicum , 1.22; A. granularum , 0.61; and Thermoplasma , 0.37. The coagulating enzyme, restored with either E. coli lipopolysaccharide or mycoplasmal lipoglycans, was able to react with the synthetic peptide benzoyl-Ile-Glu-(γ-OCH 3 )-Gly-p-nitroaniline (an analog of the coagulogen) or with the purified coagulogen itself to form the clot. The mycoplasmal lipoglycans alone were incapable of promoting these reactions when incubated with the synthetic peptide or with the purified coagulogen, thereby ruling out the contamination of these lipoglycans with proteases capable of cleaving the same Arg-Lys peptide bond of the coagulogen. These results show that acholeplasmal lipoglycans possess endotoxin-like activities. Their passive or active role in disease remains to be established.

scholarly journals Interaction of Salmonella with E. coli and Proteus spp. in Biofilm Formation

2021 ◽  
pp. 30-45
Author(s):  
S. U. Pathiranage ◽  
D. N. N. Madushanka ◽  
K. V. D. M. Hasintha ◽  
H. C. Nadishani ◽  
G. C. P. Fernando ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Broiler Chicken ◽  
Chicken Meat ◽  
Salmonella Spp ◽  
E Coli ◽  
Time Points ◽  
Dual Cultures ◽  
Microtitre Plate Assay ◽  
Biofilm Development ◽  
Numerous Time

Aims: Investigate the interaction of Salmonella spp. with E. coli and Proteus spp. in biofilm formation as mono and dual-species at different time durations Experimental Design: Salmonella, Proteus, and E. coli were isolated from Broiler chicken meat, and the biofilm-forming ability of these organisms were studied. Place and Duration of Study: The study was conducted at the Laboratory of Livestock Production, Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, from 2019 December to 2020 May. Methodology: This study investigated the biofilm-forming ability of Salmonella as a mono species and its interaction with E. coli and Proteus in the process of biofilm formation. Microorganisms used for this study were isolated from broiler chicken meat. Biofilm was quantified using a microtitre plate assay. The interaction effects were tested at the temperature of 280C in different time durations (up to 120 hours). Results: Salmonella 1 and Proteus monocultures showed significantly higher biofilm-forming ability than Salmonella 3 isolate at all tested time points. At 120 hr, additionally to the salmonella 1 and Proteus isolates E. coli also formed significantly higher biofilms than Salmonella 3. However, Salmonella 3 was the lowest biofilm former as mono biofilm at all tested time durations. Salmonella 1 interaction with Salmonella 3 isolates formed less biofilms than Salmonella 1 mono biofilm at 48hr and 72hr correspondingly. Salmonella 1 and its interactions with Salmonella 3, Proteus, E. coli showed similar biofilm-forming abilities without significant differences at all other tested time points. Specifically, Salmonella 3 interaction with Salmonella 1 as dual biofilm showed higher biofilm-forming ability than Salmonella 3 mono biofilm at all tested time points. Tested isolates and their interaction achieved the highest biofilm formation at numerous time points. In fact, at 48hr, Salmonella 3 isolates and its interaction of Proteus, E. coli, and Salmonella 1 interaction with Proteus attained their highest biofilm formation abilities. The highest biofilm formation was achieved by Salmonella 1 isolate as mono biofilm and Salmonella 1 interaction with E. coli as dual biofilm at 72hr. Biofilm-forming trend of respective isolates and interactions showed numerous patterns at tested time durations. Specifically, E. coli rapidly enhanced its biofilm-forming ability as monoculture from 24 hr to 120 hr. Proteus, Salmonella 3 as monocultures, Salmonella 3 interaction with Proteus and E. coli as dual cultures showed progressive biofilm development from 24 hr to 48 hr. Salmonella 1 monoculture and its interaction with Salmonella 3, E. coli as dual biofilm improved their biofilm-forming ability from 24 hr to 72 hr. Similar to Salmonella 3 interaction with Proteus, Salmonella 1 interaction with Proteus also increased its biofilm-forming ability from 24 hr to 48 hr. Conclusions: This study concluded that there is a variation among isolates and their combinations in forming the biofilms, where there is an enhancement of biofilm in dual-species over the mono-species in some interaction, and there is a reduction in biofilm formation by dual-species with some combinations. Further, this concluded that Salmonella is interacting with other commonly found bacteria such as Proteus and E. coli in biofilm formation.

scholarly journals Tissue-Specific Regulation of Drosophila NF-κB Pathway Activation by Peptidoglycan Recognition Protein SC

2015 ◽  
Vol 8 (1) ◽  
pp. 67-80 ◽  
Author(s):  
Denis Costechareyre ◽  
Florence Capo ◽  
Alexandre Fabre ◽  
Delphine Chaduli ◽  
Christine Kellenberger ◽  
...  
Keyword(s):  
Fat Body ◽  
Bacterial Load ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Peptidoglycan Recognition Protein ◽  
Imd Pathway ◽  
Pathway Activation ◽  
Recognition Protein ◽  
Specific Regulation

In Drosophila, peptidoglycan (PGN) is detected by PGN recognition proteins (PGRPs) that act as pattern recognition receptors. Some PGRPs such as PGRP-LB or PGRP-SCs are able to cleave PGN, therefore reducing the amount of immune elicitors and dampening immune deficiency (IMD) pathway activation. The precise role of PGRP-SC is less well defined because the PGRP-SC genes (PGRP-SC1a, PGRP-SC1b and PGRP-SC2) lie very close on the chromosome and have been studied using a deletion encompassing the three genes. By generating PGRP-SC-specific mutants, we reevaluated the roles of PGRP-LB, PGRP-SC1 and PGRP-SC2, respectively, during immune responses. We showed that these genes are expressed in different gut domains and that they follow distinct transcriptional regulation. Loss-of-function mutant analysis indicates that PGRP-LB is playing a major role in IMD pathway activation and bacterial load regulation in the gut, although PGRP-SCs are expressed at high levels in this organ. We also demonstrated that PGRP-SC2 is the main negative regulator of IMD pathway activation in the fat body. Accordingly, we showed that mutants for either PGRP-LB or PGRP-SC2 displayed a distinct susceptibility to bacteria depending on the infection route. Lastly, we demonstrated that PGRP-SC1 and PGRP-SC2 are required in vivo for full Toll pathway activation by Gram-positive bacteria.

scholarly journals Comparative Transcriptome Analysis Provides Novel Insight into Morphologic and Metabolic Changes in the Fat Body during Silkworm Metamorphosis

2018 ◽  
Vol 19 (11) ◽  
pp. 3525 ◽  
Author(s):  
Jian Peng ◽  
Zheng Li ◽  
Yan Yang ◽  
Peng Wang ◽  
Xuan Zhou ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transcriptome Analysis ◽  
Time Course ◽  
Fat Body ◽  
Metabolic Changes ◽  
Bmp Signaling ◽  
Comparative Transcriptome ◽  
Lipid Mobilization ◽  
Comparative Transcriptome Analysis ◽  
Insight Into

The fat body plays key roles in energy storage and utilization as well as biosynthetic and metabolic activities in insects. During metamorphosis from larva to pupa, the fat body undergoes dramatic changes in morphology and metabolic processes. However, the genetic basis underlying these changes has not been completely understood. In this study, the authors performed a time-course transcriptome analysis of the fat body during silkworm metamorphosis using RNA-sequencing. A total of 5217 differentially expressed genes (DEGs) were identified in the fat body at different developmental time points. DEGs involved in lipid synthesis and degradation were highly expressed at the third day of the last larval instar and during the prepupal-pupal transition, respectively. DEGs involved in the ecdysone signaling and bone morphogenetic protein (BMP) signaling pathways that modulate organ development exhibited a high expression level during the fat body remodeling process from prepupa to pupa. Intriguingly, the RNA interference-mediated knockdown of either decapentaplegic (Dpp) or protein 60A (Gbb), two DEGs involved in the BMP signaling pathway, inhibited fat body dissociation but promoted lipid mobilization, suggesting that the BMP signaling pathway not only is required for fat body remodeling, but also moderately inhibits lipid mobilization to ensure an appropriate lipid supply during the pupal-adult transition. In conclusion, the comparative transcriptome analysis provides novel insight into morphologic and metabolic changes in the fat body during silkworm metamorphosis.

scholarly journals TmPGRP-SA regulates Antimicrobial Response to Bacteria and Fungi in the Fat Body and Gut of Tenebrio molitor

2020 ◽  
Vol 21 (6) ◽  
pp. 2113 ◽  
Author(s):  
Maryam Keshavarz ◽  
Yong Hun Jo ◽  
Tariku Tesfaye Edosa ◽  
Young Min Bae ◽  
Yeon Soo Han
Keyword(s):  
Escherichia Coli ◽  
Fat Body ◽  
Mrna Level ◽  
Tenebrio Molitor ◽  
Mortality Rates ◽  
E Coli ◽  
Peptidoglycan Recognition Protein ◽  
Recognition Protein ◽  
Bacteria And Fungi ◽  
Antimicrobial Immune Response

Antimicrobial immune response is mediated by a signal-transducing sensor, peptidoglycan recognition protein-SA (PGRP-SA), that can recognize non-self molecules. Although several studies have focused on the involvement of Drosophila PGRP-SA in antimicrobial peptide (AMP) expression in response to infections, studies on its role in Tenebrio molitor are lacking. Here, we present a functional analysis of T. molitor PGRP-SA (TmPGRP-SA). In the absence of microbes, TmPGRP-SA was highly expressed in the late-larval fat body, followed by hemocytes, and gut. Interestingly, following Escherichia coli, Staphylococcus aureus, and Candida albicans infections, the mRNA level of TmPGRP-SA was significantly upregulated in both the fat body and gut. TmPGRP-SA silencing had a significant effect on the mortality rates for all the microbes tested. Moreover, TmPGRP-SA is required for regulating the expression of eight AMP genes namely TmTenecin-1, -2, and -4; TmDefensin-1 and -2; TmColeoptericin-1; and TmAttacin-1b and -2 in the fat body in response to E. coli and S. aureus infections. TmPGRP-SA is essential for the transcription of TmTenecin-2, -4; TmDefensin-2; TmColeoptericin-1, -2; and TmAttacin-1a, -1b, and -2 in the gut upon E. coli and C. albicans infections. However, TmPGRP-SA does not regulate AMP expression in the hemocytes. Additionally, TmDorsal isoform X2, a downstream Toll transcription factor, was downregulated in TmPGRP-SA-silenced larval fat body following E. coli and S. aureus challenges, and in the gut following E. coli and C. albicans challenges.

Sign in / Sign up

Export Citation Format

Share Document