Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells

Zhuo Zhou
Qinghua Xue
Yuli Wan
Yaowu Yang
Jianwei Wang
Tao Hung
Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells
R E S E A R C H Open Access
Background: Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virusencoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. Results: We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP) family, was significantly induced in human lung epithelial (A549) cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP) as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. Conclusions: LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

Background

Influenza viruses are a significant cause of morbidity and mortality in human as well as avian and animal species worldwide. Antiviral drugs targeting influenza viral proteins, such as oseltamivir, zanamivir, amantadine and rimantadine, have been licensed for treatment of influenza [1]. However, as influenza virus undergoes mutation very rapidly and can evolve drug resistance, the antiviral drugs can become ineffective during a flu outbreak [2,3]. Recently, host factors exploited by influenza virus have attracted increasing interest because therapeutics targeting these cellular factors may inhibit viral replication independent of the antigenic properties of the virus. Therefore, identification of host factors required for viral replication and development of antiviral agents targeting host factors is a promising strategy for reducing the viral resistance [4][5][6]. Recently, several research groups have identified a variety of host cell factors that are important for influenza virus replication by using genome-wide RNA interference screenings in conjunction with other integrative genomics strategies [7][8][9][10][11]. These findings provide a global view of the cellular processes that are exploited by influenza viruses and highlighted potential targets which may be used in antiviral research.

The influenza A virus genome is composed of eight segments of negative-sense, single-stranded RNA, which encodes 11 proteins [10]. Hemagglutinin (HA) and neuraminidase (NA) are critical for viral entry and release, respectively, and viral polymerase, composed of three subunits, PA, PB1, and PB2, is responsible for replication and transcription. Following initial interaction of its HA with its N-acetylneuraminic (sialic) acid receptor on the cell surface, the virus enters the cell by receptor-mediated endocytosis. Upon endosomal acidification, the HA protein undergoes conformational changes and mediates fusion between the viral envelope and endosomal membrane. The acidic environment of the endosome also triggers the disassembly of the viral core and the release of the viral ribonucleoprotein (vRNP) into the cytoplasm [12]. The vRNPs are then rapidly imported into the nucleus to catalyze viral genome replication and RNA transcription [13]. Subsequently, newly formed vRNPs, in association with other viral proteins are exported into the cytoplasm and transported to the cell membrane for budding and release. Transcriptional profiling reveals that a variety of host factors were induced upon influenza virus infection [14]. These viral inducible factors may play essential or inhibitory roles in the viral lifecycles, such as viral entry, vRNP trafficking, transcription, viral assembly and budding [9]. However, the function and regulatory mechanisms of these host factors remains largely unknown.

We here report that a host factor, lysosome-associated membrane glycoprotein 3 (LAMP3) is involved in the post-entry stages of influenza A virus infection. We found that LAMP3 was significantly up-regulated upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference inhibits viral replication in the early stage, suggesting that LAMP3 may play an important role in influenza life cycles.

Results

LAMP3 is induced upon influenza A virus infection

Influenza A virus infection induces multiple host gene expression. Interestingly, many of the up-regulated host factors appear to function by facilitating virus replication [9]. To investigate the dynamic host gene response profile upon influenza A virus infection, we utilized DNA microarray technology to determine global cellular mRNA levels at different time points post viral infection. Briefly, A549 cells were infected with influenza A/PR/8/ 34 virus at a multiplicity of infection (MOI) of 0.5. Then, total RNA were prepared at 4 h, 12 h, 24 h, and 48 h post-infection (p.i.), and were subjected to global gene expression analysis by microarray chips (Capitalbio human genome oligo array service, data not shown). Notably, LAMP3, also known as DC-LAMP, TSC403 or CD208 [15], was found to be among the most significantly upregulated genes. Upon A/PR/8/34 virus infection, LAMP3 mRNAs were stimulated a 45.2 fold at 24 h p.i., and a 34.7 fold at 48 h p.i., respectively ( Figure  1A). Nevertheless, the mRNA levels of two other LAMP protein family members, LAMP1 and LAMP2, were not altered at any time points indicated, suggesting that LAMP3 may be specifically stimulated by viral infection. To confirm the gene chip data, we used RT-PCR to detect the mRNA level of LAMP1, LAMP2, and LAMP3 at different time points post influenza A virus infection. Consistently, LAMP3, but not LAMP1 and LAMP2, is significantly induced upon influenza A virus infection ( Figure 1B). Finally, we determined the protein expression level of LAMP3 in A549 cells and IFN-deficient Vero cells [16], and the results demonstrated that, upon  18, or 24 h, total cellular RNA was extracted and subjected to RT-PCR with specific primers. GAPDH gene was used as an internal control. (C) Western blot analysis for LAMP3 expression. A549 or Vero cells were treated as indicated in (B) and were lysed with RIPA buffer. Cell lysates were subjected to Western blot analysis with antibodies against LAMP3. β-actin was used as a loading control. viral infection, LAMP3 protein was upregulated in A549 cells but not in Vero cells, indicating that LAMP3 induction may be interferon-dependent ( Figure 1C).

Knockdown of LAMP3 inhibits influenza A virus replication

LAMP3 is significantly and specifically induced upon influenza A virus infection, implying that LAMP3 may be involved in virus replication. To address the role of LAMP3 in virus replication, we silenced the expression of LAMP3 or LAMP1 in A549 cells by using specific siRNA oligonucleotides. Total amount of the LAMP3 or LAMP1 protein was detected at 72 h post transfection by Western blot analysis. In cells transfected with LAMP3 or LAMP1 siRNA, protein expression was substantially inhibited ( Figure 2A). We next examined if loss of LAMP3 or LAMP1 affects viral protein synthesis. A549 cells were infected withA/PR/8/34 virus at a MOI of 1 or 0.5 after transfection with either scrambled (control) or gene specific siRNA for 72 h. Twenty four hours post viral infection, cells were fixed and stained with anti-NP and anti-lamin A antibody simultaneously for in-cell western analysis [17]. The results showed that the amount of NP production in LAMP3 knockdown cells is remarkably lower than that in control cells and in LAMP1 knockdown cells ( Figure 2B), indicating that loss of LAMP3, but not LAMP1, affects virus protein synthesis. Further quantitative analysis for in-cell western results showed that NP production is reduced by nearly 50% in the LAMP3 knockdown cells ( Figure 2C). Then, we determined if knockdown of LAMP3 reduces virus titers. Briefly, A549 cells were transfected with LAMP3, LAMP1 or control siRNAs, and cells were infected with A/PR/8/34 virus at a MOI of 0.1 after 72 h. At 24 h p.i., viral supernatants were harvested and subjected to TCID 50 assay. Consistently, the virus titer was reduced by a 1.5 log10 in LAMP3 knockdown cells compared to the control cells or the LAMP1 knockdown cells ( Figure 2D), indicating that LAMP3 plays an important role in the viral replication. Since some siR-NAs could induce interferon response, we tested if the inhibition of viral replication by LAMP3 siRNA is related to siRNA induced interferons. The results suggested that transfection of LAMP3 or scrambled siRNAs could not induce IFN-β promoter activity ( Figure 2E), indicating that inhibition of viral replication could be attributed to LAMP3 depletion rather than siRNA induced IFN response.

Influenza virus accumulates in LAMP3 positive vesicles

Previous studies suggested that LAMP3 is involved in the endosomal/lysosomal pathways [18]. To examine if LAMP3 is involved in influenza A virus trafficking, we determined the localization of LAMP3 and the virus NP by confocal microscopy. First we detected if LAMP3 is localized to the endosome compartments. Hela cells were transfected with GFP tagged LAMP3 and then labeled with EEA1, a marker of early endosomes [19]. Upon merging both channels, the LAMP3-GFP largely colocalize with the EEA1 labeled cytoplasmic compartments of early endosomes ( Figure 3A), indicating that LAMP3 may reside in the early endosomes, which is involved in the influenza endocytic pathway. We next determined if the localization of LAMP3 changes upon virus infection. Hela cells transfected with LAMP3-GFP were infected with A/PR/8/34 virus at a MOI of 10. After 1 h, cells were fixed and subjected to confocal microscopy analysis. In the virus infected cells, part of the LAMP3 translocated from the cytoplasm to the plasma membrane ( Figure 3B), suggesting that virus infection may regulate LAMP3 trafficking. Finally, we examined if LAMP3 is colocalized with influenza virus. LAMP3-GFP transfected cells were infected with A/PR/ 8/34 virus at a MOI of 10 for 1 h and stained with antibody against NP. By confocal microscopy, the cytoplasmic speckles of influenza NP colocalized within the LAMP3 positive vesicles ( Figure 3C), indicating that LAMP3 may be involved in influenza endocytosis or vRNP trafficking pathways.

LAMP3 regulates post-entry steps of influenza A virus replication

To evaluate which steps of viral life cycles LAMP3 may be involved in, we monitored the localization of the NP during influenza A virus infection. A549 cells were transfected with a LAMP3 specific siRNA or a control siRNA, and then infected with A/PR/8/34 virus at a MOI of 10. Cells were fixed at 1.5 h, 4 h, and 8 h, and then subjected to indirect immunofluorescence assays for detection of NP localization. As indicated in the Figure 4A, at 1.5 h p.i., viral NP were observed internalized in the cytoplasm in both control and LAMP3 knockdown cells, suggesting that depleting LAMP3 may not inhibit virus entry. Intriguingly, at 4 h p.i., about 70% of NP translocated to the cell nuclei in the control cells; whereas in the LAMP3 knockdown cells, NP largely remained in the cytoplasm ( Figure 4A and 4B). At 8 h p.i., the NP protein diffused in the control cells but accumulated as cytoplasmic puncta in the LAMP3 depleted cells, suggesting a role of LAMP3 in post-entry steps of influenza virus infection, such as viral uncoating or viral ribonucleoproteins (vRNP) trafficking. Consistent with the in-cell western results, at a MOI of 0.1, the production of NP is substantially decreased in the LAMP3 knockdown cells compared to the control cells ( Figure 4A), confirming the role of LAMP3 in virus replication.

Discussion

Influenza viruses have the ability to exploit or impede host cellular responses to support viral replication, and the interactions between viral and cellular factors determine host susceptibility to influenza infection [14,20]. Novel influenza A virus strains emerge periodically, resulting in global pandemics. Thus, identification of critical host factors for influenza virus replication will provide insights into the development of future anti-flu or broad-spectrum antiviral approaches.

LAMP family proteins have been implicated in the regulation of multiple cellular processes, such as cell growth, adhesion, and metastasis. Both LAMP1 and LAMP2 are located primarily in lysosomes, and shuttle between lysosomes, endosomes, and the plasma membrane [21][22][23][24][25][26][27]. LAMP3 was first cloned as a novel human lung-specific gene, and was subsequently found to be overexpressed in malignant cells [28,29]. Recent publications suggest that LAMP3 have metastatic potential in cancers [15]. However, the role of LAMP3 in virus-host interactions has not yet been clarified. Here we showed that the expression of LAMP3 is up-regulated upon influenza virus infection. Further we demonstrated that knockdown of LAMP3 inhibited nuclear accumulation of influenza NP protein at early stages of viral infection. These data indicated a critical role for LAMP3 in influenza virus replication.

LAMP3 bears significant similarity to LAMP1 and LAMP2 [28], and may promote metastasis in the same way as LAMP1 and LAMP2 [15]. Intriguingly, only LAMP3 was significantly up-regulated during influenza virus infection. It is not clear whether the expression of LAMP3 is stimulated by virus replication directly or by virus-induced interferon. Our results indicated that influenza A virus could not induce LAMP3 expression in the IFN-deficient Vero cells. Meanwhile, microarray data mining suggested that LAMP3 was significantly upregulated in interferon treated human blood monocytes [30]; implicating a mechanism by which LAMP3 expression may be regulated by virus-induced interferon.

Our results demonstrated that knockdown of LAMP3 did not affect virus entry while attenuated nuclear accumulation of NP at 4 h post-infection. These results suggest that LAMP3 may play a role in influenza virus post-entry events, such as virus uncoating, cytosolic transport, or nuclear import of viral components. Confocal microscopy analysis suggested that LAMP3 is localized to endosomal compartment, and virus particles are found to be accumulated in the LAMP3 positive vesicles, suggesting that it is more likely that LAMP3 plays a role in early post-entry stages of influenza infection rather than nuclear trafficking of vRNP. Further biochemical experiments, such as investigating vRNP-LAMP3 interaction by immunoprecipitation, will provide insights into the mechanism by which LAMP3 regulates influenza virus infection and replication.

In conclusion, we have identified LAMP3 as a specific host factor that is involved in influenza virus infection at post-entry stages. Further investigations of the roles for LAMP3 in influenza virus infection will provide insight into the network of host-virus interactions that control the early steps of influenza virus replication and new pharmacological targets for anti-viral therapies.

Methods

Cell culture and virus infection

The human lung carcinoma cell line A549, human cervical cancer cell line HeLa, Madin Darby Canine kidney cell line MDCK and African green monkey kidney cell line Vero were purchased from American Type Culture Collection (ATCC; Manassas, VA) and were maintained in the Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C. A influenza A virus strain A/PR/8/34 (H1N1) was used in this study. For virus infection, A549 cells or MDCK cells were inoculated with virus at the indicated multiplicities of infection (MOI). Two hours later, the inoculum was removed, and cells were washed twice with Hank's solution prior to the addition of serum-free DMEM containing 5% BSA and 1 μg/ml TPCK-trypsin (Sigma-Aldrich, St. Louis, MO) [16].

Reverse transcription PCR

To determine the mRNA expression level of various LAMPs during influenza A virus infection, cells were infected with A/PR/8/34 virus at a MOI of 0.5. At different time points post infection, total cellular RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA samples were treated with DNase I (Pierce, Rockford, IL), and reversely transcribed using a Superscript cDNA synthesis kit (Invitrogen) following the manufacturer's protocols. cDNA samples were subjected to PCR amplification and

Western blot analysis

Cells were lysed in RIPA buffer containing 150 mM NaCl, 25 mM Tris (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA, and 1 mM EDTA with proteinase inhibitor cocktail (Roche, Indianapolis, IN), and total protein concentration was determined by the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL)before separation by SDS-PAGE. Expression of LAMP1 or LAMP3 protein was detected by the rabbit polyclonal anti-LAMP1 antibody (Sigma, L1418) or the rabbit polyclonal anti-LAMP3 antibody (AVIVA, ARP41598_P050), respectively.

RNA interference

siRNA against LAMP1 (M-013481-01), LAMP3 (M-004716-01), along with the non-targeting control (D-001206-13-05) were purchased from Dharmacon (Boulder, CO). A549 cells were transfected with the siR-NAs (50 nM) using Dharmafect1 (Dharmacon) according to the manufacturer's instructions.

Repoter assays

A549 cells were seeded in 24-well plates at a cell density of 1.5 × 10 5 cells per well. The next day, cells were left untransfected, or were transfected with pGL3-IFN-β-Luc and pRL-SV40, along with the scrambled siRNA, siRNA against LAMP3, or poly (I:C) using Lipofectamine 2000 (Invitrogen). At 48 h post transfection, cells were harvested, and cell lysates were subjected to luciferase activity assays (Promega, Madison, WI).

Indirect immunofluorescence assay

A549 cells either infected or mock infected by influenza

A virus were fixed in 4% paraformaldehyde and permeabilized with PBS containing 0.5% Triton X-100 (pH 7.4). Then, the cells were blocked with PBST (PBS containing 0.1% Tween-20; pH 7.4) containing 5% BSA for 50 min at room temperature. Further, the cells were incubated with mouse polyclonal anti-influenza A virus NP antibody (1:500; Millipore, Temecula, CA) at 4°C overnight. After three washes with PBST, cells were incubated with anti-mouse IgG/DyLight594-conjugated antibody (1:500; Zhongshanjinqiao Biotech, Beijing, China) for 1 h, and cell nuclei were labeled with Hoechst dye 33258 (Beyotime, Nantong, China) according to the manufacturer's instructions. For localizing LAMP3 and influenza virus, Hela cells were transfected with LAMP3-GFP (Origene, Rockville, MD), and were stained with NP antibody 18 h post-transfection. After extensive washes with PBST, cells were incubated with anti-mouse IgG/DyLight594conjugated antibody as indicated above. Fluorescence images were obtained by using a Leica TCS SP5 laser scanning confocal microscope.

In-cell western assay

The cells infected with influenza A virus or mock infected were immunostained with influenza A virus NP antibody together with a rabbit IgG antibody against Lamin A (1:1000; Sigma-Aldrich) as described above. Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody (1:500; LI-COR, Lincoln, NE) and goat anti-rabbit IgG IRDye 680 antibody (1:500; LI-COR). Protein expression was quantified using the Odyssey Imaging System. For statistical analysis, integrated intensities of fluorescence in wells were determined using software provided with the imager station (LI-COR). The relative amount of NP protein was obtained by normalizing to the endogenous lamin A.

TCID 50 assay

The TCID 50 assay was performed in MDCK cells according to the protocol recommended by the World Health Organization (http://www.who.int/csr/resources/ publications/influenza/whocdscsrncs20025rev.pdf), and data were analyzed according to the Reed and Muench method [31].

Figure 1
1LAMP3 is specifically induced upon influenza A virus infection. (A) Microarray results for LAMP1, LAMP2, and LAMP3 expression at different time points post influenza virus infection. A549 cells were infected with A/PR/8/34 virus at a MOI of 0.5. At indicated time points postinfection (p.i.), total cellular RNA was extracted and subjected to microarray analysis. (B) RT-PCR analysis for LAMP1, LAMP2, and LAMP3 expression. A549 cells were infected with A/PR/8/34 virus. After 12 h,

Figure 2
2Knockdown of LAMP3 inhibits influenza A virus replication. (A) Knockdown of LAMP1 or LAMP3 by RNAi. A549 cells were transfected with the siRNA against LAMP1, LAMP3 or a non-targeting control RNA (scrambled) (50 nM). Seventy two hours later, cells were lysed and subjected to Western blot analysis using antibody against LAMP1 or LAMP3. (B) In-cell western assay for NP production. A549 cells grown in 96-well plates were transfected siRNAs as indicated in (A). After 72 h, cells were either infected or mock infected by influenza virus, and were subjected to in-cell western assay at 24 h p.i.. NP was visualized via labeling with NP specific antibody, and endogenous lamin A was labeled as a control. (C) Quantitative analysis of in-cell western using Odyssey Imaging System. * Student's t test, p = 0.031.(D) A549 cells were transfected with LAMP3, LAMP1 or control siRNAs, cells were then infected with A/PR/8/34 virus at a MOI of 0.1 at 72 h post-transfection. Cell supernatants were harvested and subjected to TCID 50 assay 24 h p. i.(E) A549 cells were left untransfected, or were transfected with an IFN-β reporter plasmid, along with the scrambled siRNA, siRNA against LAMP3 (SiLAMP3), or poly (I:C) at 50 ng/ml (a positive control for IFN promoter activation), and analyzed 48 h later for luciferase assay.

Figure 3
3Influenza virus accumulates in LAMP3 positive vesicles. (A) Localization of LAMP3 and early endosome marker EEA1. Hela cells were transfected with a LAMP3-GFP plasmid. Eighteen hours later, cells were stained with EEA1 antibody and analyzed by confocal microscopy. (B) LAMP3 translocated to plasma membrane during influenza virus infection. Hela cells transfected with LAMP3-GFP were infected with A/PR/8/ 34 virus at a MOI of 10 for 1 h. Cells were then fixed and analyzed by confocal microscopy. (C) LAMP3 colocolized with influenza A virus. Hela cells were transfected with LAMP3-GFP for 18 h and then infected with influenza A virus at 10 MOI. After 1 h, cells were stained with the anti-NP antibody and were subjected to confocal microscopy analysis.

Figure 4
4LAMP3 regulates post-entry steps of influenza A virus replication. (A) NP localization during influenza A virus replication. A549 cells were transfected with a LAMP3 specific siRNA or a control siRNA (Scrambled), and then infected with A/PR/8/34 virus at a MOI of 10 or 0.1. Cells were fixed at indicated time points respectively, and then subjected to indirect immunofluorescence assays for NP localization. (B) Quantitative analysis for ratios of NP nuclear imported cells/total infected cells. Analyses were carried out for cells infected with A/PR/8/34 virus at a MOI of 10 for 4 h. * Student's t test, p = 0.016.
Zhou et al. Virology Journal 2011, 8:384 http://www.virologyj.com/content/8/1/384
Authors' contributions ZZ, JW and TH conceived the study and wrote the manuscript. ZZ, QX, YW, and YY carried out the experiments. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
Antiviral agents active against influenza A viruses. E De Clercq, Nat Rev Drug Discov. 5De Clercq E: Antiviral agents active against influenza A viruses. Nat Rev Drug Discov 2006, 5:1015-1025.

The risk of seasonal and pandemic influenza: prospects for control. A S Monto, Clin Infect Dis. 481SupplMonto AS: The risk of seasonal and pandemic influenza: prospects for control. Clin Infect Dis 2009, 48(Suppl 1):S20-S25.

Pandemic influenza: an inconvenient mutation. S P Layne, A S Monto, J K Taubenberger, Science. 323Layne SP, Monto AS, Taubenberger JK: Pandemic influenza: an inconvenient mutation. Science 2009, 323:1560-1561.

Drugs in development for influenza. D A Boltz, J J Aldridge, R G Webster, E A Govorkova, Drugs. 70Boltz DA, Aldridge JJ, Webster RG, Govorkova EA: Drugs in development for influenza. Drugs 2010, 70:1349-1362.

. Zhou, Virology Journal. 8384Zhou et al. Virology Journal 2011, 8:384

New antivirals and drug resistance. P M Colman, Annu Rev Biochem. 78Colman PM: New antivirals and drug resistance. Annu Rev Biochem 2009, 78:95-118.

Influenza-virus-induced signaling cascades: targets for antiviral therapy?. S Ludwig, O Planz, S Pleschka, T Wolff, Trends Mol Med. 9Ludwig S, Planz O, Pleschka S, Wolff T: Influenza-virus-induced signaling cascades: targets for antiviral therapy? Trends Mol Med 2003, 9:46-52.

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. A Karlas, N Machuy, Y Shin, K P Pleissner, A Artarini, D Heuer, D Becker, H Khalil, L A Ogilvie, S Hess, Nature. 463Karlas A, Machuy N, Shin Y, Pleissner KP, Artarini A, Heuer D, Becker D, Khalil H, Ogilvie LA, Hess S, et al: Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 2010, 463:818-822.

Human host factors required for influenza virus replication. R Konig, S Stertz, Y Zhou, A Inoue, H H Hoffmann, S Bhattacharyya, J G Alamares, D M Tscherne, M B Ortigoza, Y Liang, Nature. 463Konig R, Stertz S, Zhou Y, Inoue A, Hoffmann HH, Bhattacharyya S, Alamares JG, Tscherne DM, Ortigoza MB, Liang Y, et al: Human host factors required for influenza virus replication. Nature 2010, 463:813-817.

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. A L Brass, I C Huang, Y Benita, S P John, M N Krishnan, E M Feeley, B J Ryan, J L Weyer, L Van Der Weyden, E Fikrig, Cell. 139Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, Ryan BJ, Weyer JL, van der Weyden L, Fikrig E, et al: The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 2009, 139:1243-1254.

A physical and regulatory map of hostinfluenza interactions reveals pathways in H1N1 infection. S D Shapira, I Gat-Viks, B O Shum, A Dricot, M M De Grace, L Wu, P B Gupta, T Hao, S J Silver, D E Root, Cell. 139Shapira SD, Gat-Viks I, Shum BO, Dricot A, de Grace MM, Wu L, Gupta PB, Hao T, Silver SJ, Root DE, et al: A physical and regulatory map of host- influenza interactions reveals pathways in H1N1 infection. Cell 2009, 139:1255-1267.

Drosophila RNAi screen identifies host genes important for influenza virus replication. L Hao, A Sakurai, T Watanabe, E Sorensen, C A Nidom, M A Newton, P Ahlquist, Y Kawaoka, Nature. 454Hao L, Sakurai A, Watanabe T, Sorensen E, Nidom CA, Newton MA, Ahlquist P, Kawaoka Y: Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 2008, 454:890-893.

Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. J J Skehel, D C Wiley, Annu Rev Biochem. 69Skehel JJ, Wiley DC: Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 2000, 69:531-569.

Uncoating of influenza virus in endosomes. A Yoshimura, S Ohnishi, J Virol. 51Yoshimura A, Ohnishi S: Uncoating of influenza virus in endosomes. J Virol 1984, 51:497-504.

Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. G K Geiss, M Salvatore, T M Tumpey, V S Carter, X Wang, C F Basler, J K Taubenberger, R E Bumgarner, P Palese, M G Katze, A Garcia-Sastre, Proc Natl Acad Sci. 99Geiss GK, Salvatore M, Tumpey TM, Carter VS, Wang X, Basler CF, Taubenberger JK, Bumgarner RE, Palese P, Katze MG, Garcia-Sastre A: Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci USA 2002, 99:10736-10741.

Overexpression of LAMP3/ TSC403/DC-LAMP promotes metastasis in uterine cervical cancer. H Kanao, T Enomoto, T Kimura, M Fujita, R Nakashima, Y Ueda, Y Ueno, T Miyatake, T Yoshizaki, G S Buzard, Cancer Res. 65Kanao H, Enomoto T, Kimura T, Fujita M, Nakashima R, Ueda Y, Ueno Y, Miyatake T, Yoshizaki T, Buzard GS, et al: Overexpression of LAMP3/ TSC403/DC-LAMP promotes metastasis in uterine cervical cancer. Cancer Res 2005, 65:8640-8645.

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero). J Desmyter, J L Melnick, W E Rawls, J Virol. 2Desmyter J, Melnick JL, Rawls WE: Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero). J Virol 1968, 2:955-961.

Application of an In-Cell Western assay for measurement of influenza A virus replication. Y Wan, Z Zhou, Y Yang, J Wang, T Hung, J Virol Methods. 169Wan Y, Zhou Z, Yang Y, Wang J, Hung T: Application of an In-Cell Western assay for measurement of influenza A virus replication. J Virol Methods 2010, 169:359-364.

Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes. L B Arruda, D Sim, P R Chikhlikar, M J Maciel, K Akasaki, J T August, E T Marques, J Immunol. 177Arruda LB, Sim D, Chikhlikar PR, Maciel MJ, Akasaki K, August JT, Marques ET: Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes. J Immunol 2006, 177:2265-2275.

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif. F T Mu, J M Callaghan, O Steele-Mortimer, H Stenmark, R G Parton, P L Campbell, J Mccluskey, J P Yeo, E P Tock, B H Toh, J Biol Chem. 270Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG, Campbell PL, McCluskey J, Yeo JP, Tock EP, Toh BH: EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif. J Biol Chem 1995, 270:13503-13511.

Functional association between viral and cellular transcription during influenza virus infection. O G Engelhardt, E Fodor, Rev Med Virol. 16Engelhardt OG, Fodor E: Functional association between viral and cellular transcription during influenza virus infection. Rev Med Virol 2006, 16:329-345.

Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. P Saftig, J Klumperman, Nat Rev Mol Cell Biol. 10Saftig P, Klumperman J: Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 2009, 10:623-635.

The effects of LAMP1 and LAMP3 on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein. L Xu, H Harada, A Taniguchi, J Biochem. 144Xu L, Harada H, Taniguchi A: The effects of LAMP1 and LAMP3 on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein. J Biochem 2008, 144:531-537.

LAMP proteins are required for fusion of lysosomes with phagosomes. K K Huynh, E L Eskelinen, C C Scott, A Malevanets, P Saftig, S Grinstein, EMBO J. 26Huynh KK, Eskelinen EL, Scott CC, Malevanets A, Saftig P, Grinstein S: LAMP proteins are required for fusion of lysosomes with phagosomes. EMBO J 2007, 26:313-324.

Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. E L Eskelinen, Mol Aspects Med. 27Eskelinen EL: Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol Aspects Med 2006, 27:495-502.

Lysosomal membrane proteins. B G Winchester, Eur J Paediatr Neurol. 5Suppl AWinchester BG: Lysosomal membrane proteins. Eur J Paediatr Neurol 2001, 5(Suppl A):11-19.

Lysosome-associated membrane proteins h-LAMP1 (CD107a) and h-LAMP2 (CD107b) are activation-dependent cell surface glycoproteins in human peripheral blood mononuclear cells which mediate cell adhesion to vascular endothelium. K Kannan, R M Stewart, W Bounds, S R Carlsson, M Fukuda, K W Betzing, R F Holcombe, Cell Immunol. 171Kannan K, Stewart RM, Bounds W, Carlsson SR, Fukuda M, Betzing KW, Holcombe RF: Lysosome-associated membrane proteins h-LAMP1 (CD107a) and h-LAMP2 (CD107b) are activation-dependent cell surface glycoproteins in human peripheral blood mononuclear cells which mediate cell adhesion to vascular endothelium. Cell Immunol 1996, 171:10-19.

Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. S R Carlsson, J Roth, F Piller, M Fukuda, J Biol Chem. 263Carlsson SR, Roth J, Piller F, Fukuda M: Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem 1988, 263:18911-18919.

Isolation and characterization of a novel human lungspecific gene homologous to lysosomal membrane glycoproteins 1 and 2: significantly increased expression in cancers of various tissues. K Ozaki, M Nagata, M Suzuki, T Fujiwara, K Ueda, Y Miyoshi, E Takahashi, Y Nakamura, Cancer Res. 58Ozaki K, Nagata M, Suzuki M, Fujiwara T, Ueda K, Miyoshi Y, Takahashi E, Nakamura Y: Isolation and characterization of a novel human lung- specific gene homologous to lysosomal membrane glycoproteins 1 and 2: significantly increased expression in cancers of various tissues. Cancer Res 1998, 58:3499-3503.

Hypoxic regulation and prognostic value of LAMP3 expression in breast cancer. A Nagelkerke, H Mujcic, J Bussink, B G Wouters, H W Van Laarhoven, F C Sweep, P N Span, Cancer. Nagelkerke A, Mujcic H, Bussink J, Wouters BG, van Laarhoven HW, Sweep FC, Span PN: Hypoxic regulation and prognostic value of LAMP3 expression in breast cancer. Cancer 2011.

Amplification of IFN-alpha-induced STAT1 activation and inflammatory function by Syk and ITAM-containing adaptors. I Tassiulas, X Hu, H Ho, Y Kashyap, P Paik, Y Hu, C A Lowell, L B Ivashkiv, Nat Immunol. 5Tassiulas I, Hu X, Ho H, Kashyap Y, Paik P, Hu Y, Lowell CA, Ivashkiv LB: Amplification of IFN-alpha-induced STAT1 activation and inflammatory function by Syk and ITAM-containing adaptors. Nat Immunol 2004, 5:1181-1189.

A simple method of estimating fifty percent endpoints. L J Reed, H Muench, 10.1186/1743-422X-8-384Am J Hyg. 27Reed LJ, Muench H: A simple method of estimating fifty percent endpoints. Am J Hyg 1938, 27:493-497. doi:10.1186/1743-422X-8-384

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Zhou, Virology Journal. 8384Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Zhou et al.: Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells. Virology Journal 2011 8:384. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


Comparative study on Biochar salt absorption capacity in different saline concentrated solutions

Vanessa Mishell
Rocha Cabuyales
Vanessa Mishell
Rocha Cabuyales
Comparative study on Biochar salt absorption capacity in different saline concentrated solutions
10.21931/RB/2021.06.04.32150Biocharsalinityelectroconductivitysoilsalt
Salt-affected soils are caused by excess accumulation of salts. As soil salinity increases, salt effects can result in the degradation of soils. Previous studies have determined that biochar has the potential to reduce salt stress in soils. In this study, the electroconductive properties of biochar to adsorb salts were investigated in different saline-concentrated solutions. Pelletized, fragmented and powdered biochar were placed in solutions with concentrations of 0, 50, 500, 1000, and 2000 parts per million sodium chloride, respectively. Control treatments consisted of deionized water mixed with salt and no biochar addition. A week after setting the experiment, the electroconductivity measurements were significantly higher relative to the first day. Significant differences were observed among treatments for pelletized, fragmented, and powdered biochar treatments. Increases in electroconductivity values are attributed to ambient temperature changes and differences in particle size. However, pelletized biochar declined in electroconductive values, which is attributed to ions being retained inside the pores of bigger particles. Our study concludes that biochar can adsorb salts at lower sodium chloride concentrations; therefore, it may help mitigate soil salt stress.

Introduction

The accumulation of salts may cause soil degradation due to agricultural, industrial, or urban activities 1 . Soil salinization is a widespread limitation where over 397 million hectares of the world's soils are unproductive saline patches of land 2 . One of the most severe problems related to salinization is the progressive degradation of soil fertility 1 . The excessive concentration of soluble salts in soils inhibits beneficial microbiological activity, causes accumulation of toxic ions, limits plant growth, and accelerates organic matter loss due to wind erosion or leaching 1,3 . Because soil fertility is essential for nutrient cycling, structure, stability, and cation exchange processes, most management strategies aim to improve this property 1 . There is currently much research regarding the amelioration of saline soils 4 . For this reason, biochar has been widely proposed to improve soil quality and crop productivity 4 . Biochar can enhance soil organic matter quality by dealing with soil salinity problems because of its salt sorption capacity 5 .

Biochar is obtained from a process known as pyrolysis, which is the thermo-chemical decomposition of organic biomass at temperatures generally ranging from 300 to 700 °C in the absence of oxygen 6,7 . Pyrolysis processes stabilize the existing carbon in organic matter to be more resistant to chemical and biological decomposition 8 . When biochar is incorporated into soils, it degrades very slowly so that carbon is not emitted into the atmosphere, in contrast to non-pyrolyzed organic matter decomposition 9 . Moreover, biochar has the potential to decrease Na+ uptake by adsorbing salts 10 . These characteristics give biochar the capacity to improve the physical-chemical properties of soils, increase soil productivity, contribute to carbon sequestration, and alleviate salt stress in vegetation 8 .

To enhance the understanding of the salt adsorption properties of biochar, it is necessary to measure its ion electroconductivity (EC). The analysis of biochar's electroconductive properties allows measuring the concentration of salts present in a solution based on a conductivity meter, and this can be used to determine whether biochar might help mitigate salt stress in plants and soils 11,4 . In addition, the effects of salinity amelioration on soils may vary depending on the biochar's phy-sical characteristics 12 . Biochars in this study range from pelleted, powdered, and fragmented particle types and at a range of sizes. It is essential to consider the use of different biochar morphologies because of their varied porosity. Porosity determines the conductivity and ability of biochar to retain ions on its surface 13 . It is predicted that biochar can reduce the electroconductivity of saline treatments because of its salt sorption capacity. Since biochar's salt adsorption potential has no tests, this study conducted electroconductivity measurements to understand biochar's salt adsorption capacity and identify the physical-chemical properties of biochar's particle morphology. Biochar salt adsorption analysis can be used to enhance the limited studies on biochar's amendment of salt stress on soils and vegetation. The objective of this study is to understand which variables affect biochar's salt adsorption capacity. This research paper focuses on the following specific objectives:

Measure the salt adsorption capacity of biochar through electroconductivity.

2.

Evaluate the salt adsorption function of different biochar morphologies under different salinity conditions.

Methods

Biochar and salt stock solutions: Different types of biochar were analyzed based on particle morphology, including pelletized, fragmented, and powdered biochar. We used biochar pellets in the range of 2.8-2.00mm. These pellets were processed from highly compacted wood and manufactured by BioForest. We also used biochar fragments processed from sugar maple (Acer saccharum Marshall) and conifer wood. The size of the biochar fragments ranged from 0.5mm to 4mm. These sugar maple biochar fragments were manufactured at Haliburton Forest and Wildlife Reserve Ltd, whereas the conifer fragments and biochar powder were processed at Titan Carbon Smart Technologies. The titan fragments and titan powdered biochar were dried at 100°C for 24 hours.

Universidad de Toronto , Canadá

Corresponding author: email@example.com In this study, five different salinity stock solutions were prepared with 99% pure reagent-grade sodium chloride (NaCl) and deionized water (DI) on a weight-in-volume basis. The choice of deionized water as solvent was motivated by its neutral electron conductance potential (5.5μS/m at 23°C). Deionized water is characterized by a lack of minerals and contaminants usually present in tap water, which could otherwise affect the experiment. The addition of sodium chloride was motivated by the specific salt concentration concerning seawater, which known measure acted as our reference (35ppt or 35g/L) 14 . Thus, 0.5g of sodium chloride was weighted in a mechanical balance and dissolved in a 250mL bulb flask to obtain a stock solution with 500ppm Na+ solution per every 15mL of deionized water. We assumed that a more concentrated solution would require double the number of salts, so we prepared a series of stock solutions with 1000ppm and 2000ppm and a negligible salt concentration stock solution with 50ppm Na+. The saline solutions consisted of low (50mmol NaCl), medium (500mmol NaCl), high (1000mmol NaCl), and saturated (2000mmol NaCl) sodium chloride concentrations in a fixed amount of 15 milliliters of deionized water. All the stock solutions were prepared 24 hours to let the sodium chloride dissolve and come to equilibrium before mixing them with biochar.

Experimental procedure: Preliminary tests were conducted on biochar and salt solutions at room temperature (23 -25°C) to learn when equilibrium could be reached. The preparation of the preliminary samples took at least five days, so it was decided to conduct preliminary samples by adding solutions to biochar at 1:20 biochar: solution ratio. This ratio reflects the usual mix of 1g of solid biochar per every 20mL of liquid. Because of flask size, we adjusted the ratio to 15mL; thus, the fixed amount of biochar added was 0.75g (w:v). Biochar was weighted on a mechanical balance, and 0.75 grams were immersed in 25mL glass containers. Then, a plastic pipette was used to transfer 15 mL of each stock solution into lidded glass containers with moderate stirring. All of the treatments were performed in triplicate as was the control group. For the control samples, we mixed sodium chloride with deionized water without the addition of biochar. All the prepared samples were shaken at a speed of 80 rpm on a WWR DS-500E Orbital Shaker for an hour at room temperature. Initial electron conductivity measurements were taken after one hour of shaking the samples. Electrical conductivity was measured at room temperature (23°C) using a bench-top conductivity meter (Orion Star A112, Thermo Electron Co., Mass., U.S.A.) coupled to a conductivity cell (Orion 013005MD). The conductivity cell was calibrated to two standard solutions at 1413um and 12.0mS. After every measurement, the conductivity cell was washed with deionized water to remove any adhering sample and was blotted before using it in the subsequent measurement. Once the results of the initial measurements were recorded, all the samples were placed in the shaker for seven days, and electroconductivity values were taken at room temperature (25°C ± 0.5°C). At the end of the experiment, we dried all biochar samples in small aluminum sample holders so they could be used to measure salt content later on. After drying, the samples were transferred back to the identical washed vials, where the measurements were taken initially and stored with lids.

A detailed experimental design chart is depicted in Table 1. Statistical data and graphs were analyzed with R studio version 1.1.463 (R Core Team 2018). Data for each dependent variable were first analyzed for normal distribution by the Shapiro-Wilks normality test and equality of variances among biochar and salinity treatments by the Levene test. Data meeting these assumptions were further tested on the influence of biochar type and salinity level on the variability of electroconductivity by a two-way analysis of variance test or ANOVA. Statistics reported for each test are F-values, P-values, and standard errors. Microsoft Excel was used to show tables corresponding to the means and standard errors ( Table 2). A line graph of electroconductivity grouped by salinity treatment is presented in Figure 1.

Results

Contrary to our initial hypotheses, biochar particles did not consistently result in decreased salinity. Electroconductivity values showed a considerable increase in response to biochar added to saline treatments except for the low concentration solutions, while powder biochar did not show any decrease in EC values at any saline level. A two-way analysis of variance was conducted on the influence of biochar type and salinity level on the variability of electroconductivity values. For day one, the ANOVA test yields an F-value of 4.528 and a P-value of 2.2*10-16, indicating that the ANOVA is statistically significant according to the standard significance level of p<0.05. This result demonstrates that EC does differ from one sali- Table 1. Detailed experimental procedure and design. Table 2. Average (mean ± standard error N=3) for EC salinity treatments by biochar type (1:20 w/v ratio). nity treatment to another. For day seven, where biochar and salinity are the independent variables, and EC values are the dependent variable, a significant relationship is found, supported by a p-value of 0.00191 (no log transformation). In other words, biochar type and salt concentration are the factors that influence EC values. Results of the variability in the EC measurements between saline solutions of the biochar samples are listed in Table 2. The results are discussed as the interaction of biochar on the different saline concentration treatments over time, and a comparison is made between the four different biochar types.

Discussion

Electrical conductivity is a way to determine the concentration of ions present in an aqueous solution 16 . In an ionic solution, the specific conductivity measurement depends on the concentration and, therefore, on the number of ions (cations and anions) 15 . The more ions that are present, the higher the electroconductivity of the aqueous solutions 16 . These conductive ions include dissolved salts (NaCl) and organic and inorganic matter that composes biochar 16 . The duration of biochar's exposure to saline solutions had a pronounced effect on electroconductivity. As shown in Table 2, day-seven EC values were higher than day-one saline treatments and across the four types of biochar.

Salinity was not measured directly but is instead derived from electrical conductivity measurements 15 . For biochar pellets, the relationship between EC and salt is statistically significant (p< 0.0026). As shown in Figure 1a-b, low (50 ppm), medium (500ppm), and high (1000ppm) saline concentrations of Na+ presented a slight decrease in electroconductivity, with values ranging from 1% to 5% variation over a week. In contrast to other biochar types, pellets have a low surface area to volume ratio. For this reason, we believe that biochar pellets might be able to retain and hold more ions on their surface. However, the saturated saline concentration (2000ppm) presented an increment of 8% on the EC measurements. We attribute the observed EC variation to the fact that once biochar reaches equilibrium, it adsorbs salts, but these salts could also be released in the presence of saturated saline solutions because the particle might no longer be able to hold more ions.

EC values increased at the 500, 1000, and 2000 salinity levels ( Table 2). The relationship between salinity level and EC measurements is supported by a significant p-value of 0.0025 for sugar maple fragments and a significant p-value of 0.0026 for biochar conifer fragments (Figure 1c-d). The electroconductivity measurements show that the values increased significantly across all five salinity treatments (p<0.0029). In contrast to pellets, values for powdered and fragmented biochar increased at each salinity level. Biochar powder particles have a higher surface area to volume ratio; such characteristics might allow the biochar to adsorb and de-adsorb Na+ salts at a different rate 16,17 . Therefore, salt ions are prevented from holding onto the powder's particle surface and are subsequently leached into the salinity solution, and since these ions are leached into the solution, electroconductivity values tend to increase.

Biochar particle size, shape, and internal structure likely play essential roles in controlling sodium-ion storage because they might alter pore characteristics. For instance, biochar has pores inside of its particles. These pores are called intrapores and might provide additional space for ion storage beyond the pore space between particles or interpores 16 . As a result, electroconductivity values tend to decrease. A larger particle size, which is the case of pelletized and fragmented biochar, may increase sodium adsorption capacity, whereas a smaller particle size might not. Bigger biochar particles can store more ions inside their internal structure (Figure 3a).

In contrast, smaller biochar particles, such as powdered and fragmented biochar, might have limited ion storage capacity to hold ions on their internal structures (Figure 3b-c-d). Therefore, finely powdered biochar particles will not adsorb many ions, unlike bigger biochar particles like pellets. This addition of biochar grains with different shapes and sizes will eventually change the adsorption of minerals in the salinity solutions. According to the Food and Agriculture Organization 19 , the higher EC variability of the salinity treatments may be attributed to increments in temperature. As shown in Table 2, the temperature of the saline solutions increased from 23°C ± 0.3°C in day one to 25°C ± 0.5°C on day seven. Unlike metallic conductivity, electrical conductivity increases at approximately 1.9% per Celsius degree increase in temperature 19 . When sodium electrolytes dissolve in water, they produce positive cation and harmful anion particles; this means that the electron conductance of the solutions increases with added ions 10 . An increase in EC may also be attributed to including substantial amounts of biochar ions and minerals into salinity solutions 19 . The presence of phenolic, carboxylic, pyrones, and ketone groups in biochar samples due to pyritization may in part explain the higher variability with the EC values between day one and seven 10 . An additional finding supporting this assumption is the increased electroconductivity presented by the control group, consisting of pure deionized water and different saline concentrations without biochar addition ( Figure 2). Electroconductivity increased by approximately 0.5 to 12% from day one to day seven. It is important to note that temperature for day one was recorded to be 23°C ± 0.5°C whereas temperature for day seven was 25°C ± 0.5°C instead. Therefore, we believe that temperature played a significant role in rising EC values as higher temperatures might evaporate water, leaving saline ions behind.

Even though most of the biochar treatments showed increased EC values, low salt concentration treatments (50ppm) tend to progress toward a decrease in electroconductivity. All biochar types, except for powder, showed a decrease of 0.2-1.0 mS/cm in EC values at the 50ppm salinity concentration level. This finding suggests that biochar can adsorb salts in solutions with a small number of dissolved ions in contrast to solutions with a saturated number of ions 20,21 .

Conclusions

This study concludes that biochar can adsorb salts at lower sodium chloride concentrations; therefore, it may help mitigate salt stress in soils. Under a chemical-biological approach, the findings of this research will help select the best Canadian wood biochar options for different soil saline conditions in the environment. The presented information can serve as a guide to develop conservation strategies that aim to mitigate the adverse impacts of salt on vegetation and soils. However, further research still needs to be conducted to analyse a more precise interaction between biochar type and sodium sorption capacity. These analyses should implement different techniques to measure salinity values to get the exact sodium sorption levels.

Figure 1 .
1The graph shows the EC values obtained for the control group (deionized water with NaCl without biochar). The x-axis represents salinity levels in grams, whereas the y axis represents the EC of deionized water.

Figure 2 .
2The figure illustrates the electroconductivity values for the four types of biochar tested: pellets, fragments (sugar maple and conifer) and powder. Graph a) shows biochar pellets, b) biochar powder, c) sugar maple fragments and d) conifer biochar fragments. For all four graphs, the x axis shows the salinity level concentration in grams whereas the y axis shows the electroconductivity values for the respective biochar in saline treatments.

Figure 3 .
3The four different biochar types were used in the study. From left to right: Biochar pellets, Sugar Maple fragments, Conifer fragments, and Biochar Powder.
AcknowledgementsForemost, I would like to express my sincere gratitude to my advisor Prof. Sandy Smith, my research labmate Ph.D. candidate Melanie Sifton for the continuous support of this research, for their patience, motivation, enthusiasm, and immense knowledge. Their guidance helped me in all the time of research and writing this paper. Besides them, I would like to thank Prof. Sean Thomas Laboratory for providing me with all the necessary facilities and the University of Toronto for supporting this research project.Bibliographic references
Effects of irrigation-induced salinity and sodicity on soil microbial activity. D N Rietz, R J Haynes, Soil Biology and Biochemistry. 356Rietz, D. N., and Haynes, R. J. Effects of irrigation-induced salinity and sodicity on soil microbial activity. Soil Biology and Biochem- istry, 35(6), 845-854.

Extent and causes of salt-affected soils in participating countries. Global network on integrated soil management for sustainable use of salt-affected soils. FAO 2000. FAO-AGL websiteFAO 2000. Extent and causes of salt-affected soils in participat- ing countries. Global network on integrated soil management for sustainable use of salt-affected soils. FAO-AGL website.

Salt-affected soils, reclamation, carbon dynamics, and biochar: a review. S Amini, H Ghadiri, C Chen, P Marschner, Journal of Soils and Sediments. 16Amini, S., Ghadiri, H., Chen, C., and Marschner, P. Salt-affected soils, reclamation, carbon dynamics, and biochar: a review. Jour- nal of Soils and Sediments, 2016,16:939-953.

Does biochar improve establishment of tree seedlings in saline sodic soils? Land Degradation & Development. J A Drake, T R Cavagnaro, S C Cunningham, W R Jackson, Patti , A F , 27Drake, J. A., Cavagnaro, T. R., Cunningham, S. C., Jackson, W. R., and Patti, A. F. Does biochar improve establishment of tree seedlings in sa- line sodic soils? Land Degradation & Development, 2016, 27(1), 52-59.

Biochar mitigates salinity stress in potato. S Akhtar, Andersen, F Liu, Journal Agronomy and Crop Science. 201Akhtar, S., Andersen, M and Liu, F. Biochar mitigates salinity stress in potato. Journal Agronomy and Crop Science, 2015, 201: 368-378.

Influence of pyrolysis temperature on production and nutrient properties of wastewater sludge biochar. M K Hossain, V Strezov, K Y Chan, A Ziolkowski, P F Nelson, Environmental Management. 921Hossain, M. K., Strezov, V., Chan, K. Y., Ziolkowski, A., and Nel- son, P. F. Influence of pyrolysis temperature on production and nutrient properties of wastewater sludge biochar. Environmental Management, 2011, 92(1), 223-228.

Biochar for environmental management: science, technology and implementation. S J Lehmann, Taylor & Francis GroupLondon2nd edition. RoutledgeLehmann, S. J. Biochar for environmental management: science, technology and implementation. 2nd edition. Routledge, Taylor & Francis Group, London, 2015.

Biochar mitigates negative effects of salt additions on two herbaceous plant species. S C Thomas, S Frye, N Gale, M Garmon, R Launchbury, Machado, C Winsborough, Journal of Environmental Management. 62Thomas, S. C., Frye, S., Gale, N., Garmon, M., Launchbury, R., Machado, N and Winsborough, C. Biochar mitigates negative ef- fects of salt additions on two herbaceous plant species. Journal of Environmental Management, 2013, 129, 62.

Biochar as a viable carbon sequestration option: Global and Canadian perspective. D Matovic, Energy. 436Matovic, D. Biochar as a viable carbon sequestration option: Glob- al and Canadian perspective. Energy, 2016, 36(4).

Biochar soil amendment on alleviation of drought and salt stress in plants: A critical review. S Ali, M Rizwan, M F Qayyum, Y S Ok, M Ibrahim, M Riaz, A Shahzad, Environmental Science and Pollution Research International. 2414Ali, S., Rizwan, M., Qayyum, M. F., Ok, Y. S., Ibrahim, M., Riaz, M., and Shahzad, A. Biochar soil amendment on alleviation of drought and salt stress in plants: A critical review. Environmental Science and Pollution Research International, 2017, 24(14), 12700-12712.

The role of biochar porosity and surface functionality in augmenting hydrologic properties of a sandy soil. W Suliman, J B Harsh, N I Abu-Lail, A Fortuna, I Dallmeyer, M Garcia-Pérez, Science of the Total Environment. 574Suliman, W., Harsh, J. B., Abu-Lail, N. I., Fortuna, A., Dallmeyer, I., and Garcia-Pérez, M. The role of biochar porosity and surface functionality in augmenting hydrologic properties of a sandy soil. Science of the Total Environment, 2017, 574(Complete), 139-147.

High surface area biochar negatively impacts herbicide efficacy. E Graber, L Tsechansky, Z Gerstl, Lew , B , Plant and Soil. 3531-2Graber, E., Tsechansky, L., Gerstl, Z., and Lew, B. High surface area biochar negatively impacts herbicide efficacy. Plant and Soil, 2012, 353(1-2), 95-106.

Pelleted biochar: Chemical and physical properties show potential use as a substrate in container nurseries. R K Dumroese, J Heiskanen, K Englund, A Tervahauta, Biomass and Bioenergy. 355Dumroese, R. K., Heiskanen, J., Englund, K., and Tervahauta, A. Pelleted biochar: Chemical and physical properties show poten- tial use as a substrate in container nurseries. Biomass and Bioen- ergy, 2011, 35(5), 2018-2027.

UNESCO. Joint Panel on Oceanographic Tables and Standards, 11th. Sidney, CanadaUNESCO. Joint Panel on Oceanographic Tables and Standards, 11th, Sidney, Canada 1980.

Synthesis, structure, and electroconductivity of new radical cation salt. L A Kushch, E B Yagubskii, S V Konovalikhin, Russian Chemical Bulletin. 48Kushch, L. A., Yagubskii, E. B., Konovalikhin, S. V. Synthesis, struc- ture, and electroconductivity of new radical cation salt. Russian Chemical Bulletin, 1999, 48, 1513-1515.

Comparative study on Biochar salt absorption capacity in different saline concentrated solutions 17. R Sinmyo, H Keppler, Z Liu, B Dugan, C A Masiello, H M Gonnermann, Journal of Contributions to Mineral Petrol. 172179079Plos one JournalSinmyo, R., and Keppler, H. Electrical conductivity of NaCl-bear- ing aqueous fluids to 600 °C and 1 GPa. Journal of Contributions to Mineral Petrol, 2017, 172, Comparative study on Biochar salt absorption capacity in different saline concentrated solutions 17. Liu, Z., Dugan, B., Masiello, C. A., Gonnermann, H. M. Biochar par- ticle size, shape, and porosity act together to influence soil water properties. Plos one Journal, 2017, 12:0179079.

Investigating the Influence of Biochar Particle Size and Depth of Placement on Nitrous Oxide (N2O) Emissions from Simulated Urine Patches. A F Mahmud, M Camps-Arbestain, M Hedley, Journal of Agriculture. 8175Mahmud, A. F., Camps-Arbestain, M., and Hedley, M. Investigating the Influence of Biochar Particle Size and Depth of Placement on Nitrous Oxide (N2O) Emissions from Simulated Urine Patches. Journal of Agriculture, 2018, 8:175.

Soil Salinity assessment: Methods and interpretation of electrical conductivity measurements. FAO Irrigation and Drainage Paper. Fao, FAO. Soil Salinity assessment: Methods and interpretation of electrical conductivity measurements. FAO Irrigation and Drain- age Paper 1999.

Electrical conductivity of NaCl-H2O fluid in the crust. H Akuma, M Ichiki, Journal of Geophysic Resources Solid Earth. 121Akuma, H., and Ichiki, M. Electrical conductivity of NaCl-H2O flu- id in the crust. Journal of Geophysic Resources Solid Earth, 2016, 121, 577-594.

An investigation of drum granulation of biochar powder. B Bowden-Green, L Briens, Journal of Powder Technology. 288Bowden-Green, B., and Briens, L. An investigation of drum gran- ulation of biochar powder. Journal of Powder Technology, 2016, 288(Complete), 249-254.

Environmental benefits of biochar. J A Ippolito, D A Laird, W J Busscher, Journal of Environmental Quality. 41Ippolito, J. A., Laird D. A., and Busscher, W. J. Environmental benefits of biochar. Journal of Environmental Quality, 2012, 41, 967-972.