Double-layered N-S1 protein nanoparticle immunization elicits strong mobile immune and broad antibody responses towards SARS-CoV-2 | Journal of Nanobiotechnology

Animals, cells, and peptides

Feminine BALB/c mice aged 6–8 weeks have been procured from Henan Sikebesi Organic Expertise Co. Ltd (Henan, China) and housed in a particular pathogen-free (SPF) animal facility after approval by the Laboratory Animal Ethics Overview Committee.

E.coli. DH5α and BL21 (DE3) Chemically Competent Cells have been bought from TransGen Biotech Co., Ltd. (Beijing, China). Human embryonic kidney 293F (HEK293F) cells, cultured in SMM 293-TII Expression Medium (Sino Organic; M293TII), have been preserved by Henan Provincial Key Laboratory of Animal Immunology at Henan Academy of Agricultural Sciences (Henan, China). DC2.4 and RAW264.7 cells have been obtained from ATCC (Manassas, VA, USA).

14 overlapped artificial peptides pool of the SARS-CoV-2 N protein was used for ELISpot evaluation, with synthesis by GL Biochem (Shanghai, China), as proven in Further file 1: Desk S2.

Design, expression, and purification of S1 protein

The S1 protein gene sequence was retrieved from NCBI (Spike residues 319–668, GenBank: MN908947.3). The sign peptide encoding sequence of the SARS-CoV-2 spike protein was employed within the S1 protein constructs to facilitate secretion. The S1 protein plasmid was synthesized by Sangon Biotech (Shanghai, China). The merchandise have been ligated into the pcDNA3.1 (+) vector and subsequently launched into E.coli. DH5α competent cells via transformation. The recombinants have been amplified and abstracted using the EndoFree Plasmid Maxi Equipment (CWBIO; CW2104). The plasmid was transiently transfected into HEK293F utilizing PEI (Yeasen Biotechnology, China) [70]. The cell supernatant was collected and filtered to determine protein 72h after transfection. Then proteins have been purified by utilizing an immunoaffinity chromatography column based mostly on the SARS-CoV-2 Spike protein-specific mAb preserved in our laboratory. The eluted fractions have been pooled and exchanged to 0.01M PBS pH 7.4 by way of Sephadex G-25 (GE Healthcare, USA).

Design, expression, and purification of N protein

Primarily based on the unique SARS-CoV-2, we obtained the full-length sequence of N protein (GenBank: NC045512.2) from NCBI. The N protein recombinant plasmid was modified with a secretory expression sign peptide sequence, including a six-histidine-tag sequence to the C terminal of the protein. The modified plasmid was inserted into the pET28a vector and reworked into competent BL21 (DE3) E. coli, acquiring a single colony. Then PCR identification of the recombinant BL21 pressure was cultured into a ten mL LB liquid medium containing 50 μg/mL kanamycin for 12 h. The pre-culture was diluted 1:100 into 1 L LB medium for 3 h. After including Isopropyl β-D-1-thiogalactopyranoside (IPTG) with a remaining focus of 0.2 mM, persevering with to induce for 12 h at 16 °C. Bacterial cultures have been harvested by centrifugation and sonicated from collected supernatant, after which purified via binding into Ni Sepharose™ Excel affinity column (His Entice ™ Excel, GE) [21].

Fabrication of protein nanoparticles

The N core PNp was fashioned by desolvating with absolute ethanol, and the N-S1 protein double-layered nanoparticle was ready via crosslinking reactions. Particularly, a four-fold quantity of absolute ethanol was slowly dripped at 1 mL/min into N protein answer whereas fixed stirring at 600 rpm for 1 h, acquiring the PNps by discarding the supernatant after centrifugation at room temperature and 15,000 ×g for 15 min. The PNp was sufficiently resuspended in an answer of S1 protein by sonication. Then 5 mM DTSSP (Sigma-Aldrich; 803,200) was added and repeatedly stirred for 1.5 h at 4 °C earlier than being quenched with 30 mM Tris–HCl answer (pH 7.4) and stirred for an extra 15 min, centrifuged at 15 000 rpm for 30 min to discard the supernatant, resuspended the remaining in 0.5 mL PBS, stabilized by sonication on an ice tub. The fabrication of BSA double-layered PNp was analogous to the above-described methodology.

Nanoparticle characterization

The nanoparticle dimension of the N-S1 protein double-layered nanoparticle and the N core PNp have been assessed by dynamic mild scattering (DLS) evaluation. The samples within the quartz cuvette have been incubated for 3 min within the Malvern Zetasizer Nano ZS90 (Malvern, Worcestershire, UK), and measurements have been taken at 25℃ with 10 scans for 30 s every. The composition of the N-S1 protein double-layered nanoparticle was characterised by 12.5% and seven.5% SDS-PAGE, adopted by Coomassie blue staining and Western blotting. Anti-his-tag monoclonal antibody (Proteintech; 66,005–1-Ig) and anti-S1 mouse mab (ready and saved in our laboratory and developed in keeping with commonplace procedures) have been used to research the nanoparticles by Western blotting [71].Then the depth of S1 protein bands was analyzed utilizing Picture J software program. The protein focus of the N-S1 protein double-layered nanoparticle answer was decided utilizing the BCA package (Beyotime; P0012). A droplet of nanoparticle answer was utilized onto a 100 mesh carbon-coated TEM copper grid (Beijing Zhongjingkeyi Expertise Co., Ltd., China) and air-dried for two min. Subsequently, the surplus nanoparticle answer was faraway from the aspect of the TEM grid utilizing filter paper, adopted by speedy incubation with a drop of three% phosphotungstic acid damaging staining answer for two min on the identical grid. Earlier than the grid was subjected to air drying at room temperature for 4 h, preceded by one other spherical of wicking from its aspect with filter paper. TEM imaging with a Thermo Scientific™ Talos™ L120C transmission electron microscope at 120 kV.

The floor plasmon resonance (SPR) assays have been carried out utilizing the CM5 sensor Chip (Cytiva; 29,149,604). The binding affinity distinction of human ACE2 protein to soluble S1 protein or N-S1 PNp was decided utilizing a Biacore X100 system (GE Healthcare, USA) with the HBS-EP working buffer (Cytiva; BR100669). The human ACE2-His protein was diluted to a focus of 30 μg/mL in pH 4.0 10 mM sodium acetate buffer for covalent coupling to immobilization. The soluble S1 protein or N-S1 PNp was diluted to concentrations starting from 15.625 nM to 500 nM in HBS-EP buffer and flowed over the chip floor, respectively. Following an affiliation interval of 90 s, a dissociation interval of 90 s ensued, then a remaining regeneration step with glycine pH 1.5 regeneration buffer. The binding affinity worth (OkD) was decided.

Isolation and maturation of dendritic cells in bone marrow (BMDCs) assay

To acquire the BMDCs, bone marrow hematopoietic stem cells have been remoted from the femur of C57BL/6 mice. Bone marrow cell suspensions have been ready in RPMI Medium 1640 (Solarbio; 31,800) with 10% FBS via a 70 μm cell strainer. The cells have been then differentiated into BMDCs by the addition of GM-CSF (20 ng/mL; MCE; HY-P7361A) and IL-4 (10 ng/mL; Solarbio; P00196), adopted by incubation at 37 °C with 5% CO2. After 7 days of tradition, loosely adherent and floating cells have been harvested for subsequent experiments.

BMDCs have been straight plated with 1 × 106 per nicely into the 24-well plate, adopted by stimulation utilizing soluble protein (N or S1) or double-layered N-S1 PNps 5 μg/mL for twenty-four h, which the corresponding quantity of proteins in every group was the identical. Furthermore, the damaging and optimistic wells have been stimulated with PBS and LPS, respectively. Then, BMDCs have been harvested and centrifuged at 400 ×g for five min, earlier than being incubated with anti-CD11c-FITC, anti-CD86-PE, and anti-CD80-APC. Lastly, cells have been analyzed utilizing FCM as described in Strategies.

In vitro mobile uptake assay

To evaluate the mobile uptake of double-layered N-S1 PNps by antigen-presenting cells (APCs), oblique immunofluorescence (IFA) was carried out. Particularly, RAW264.7 and DC2.4 cells have been seeded into 96-well plates for in a single day tradition at a density of 8 × 104 cells/nicely. The cells have been incubated with 15 μg/mL soluble N protein, 35 μg/mL soluble S1 protein, 50 μg/mL N-S1 PNps, a protein combination of 35 µg S1 and 15 μg N, or PBS as a damaging management for two h. After eradicating the medium, the cells have been fastened with pre-cooled methanol for 10 min after which washed with PBS thrice. The cells have been blocked with 5% skim milk in PBST (PBS containing 0.05% Tween-20) for two h at 37 ℃. Subsequent, the cells have been incubated with mouse-specific S1 and N monoclonal antibodies towards SARS-CoV-2, respectively, then labeled with the Alexa Fluor488-conjugated goat-anti-mouse secondary antibody (Solarbio; A0428). After 1 h incubation, DAPI (Solarbio; C0065) was used to stain the cells and incubated for 4 min at room temperature at midnight. The cells have been washed thrice with PBS earlier than imaging by fluorescence microscopy (Zeiss, Germany).

In vitro cytotoxicity assay

To guage the cytotoxicity of double-layered N-S1 PNps, completely different concentrations of double-layered N-S1 PNps have been incubated with RAW264.7 and DC2.4 cells for twenty-four h. They have been plated into 96-well plates with a density of 1 × 104 cells/nicely 12 h upfront. After incubation, cytotoxicity was evaluated by Cell Counting Equipment-8 (Dakewe; 6,073,213). Briefly, the cell plate was added with DMEM medium containing 10% CCK-8 reagent and cultured for 4 h earlier than measuring the absorbance of OD 450 nm.

Immunization in mice research designs

Feminine mice (BALB/c pressure, 6–8 weeks, n = 8) have been randomly allotted to 6 teams and immunized intramuscularly with two doses of 100 μL vaccines in PBS containing aluminum hydroxide (Croda) and CPG 1018 (PARR BIO) adjuvants, spaced 21 days aside. The mice acquired the next vaccines respectively: 10 μg N-S1 protein double-layered nanoparticle, 7 µg soluble S1 protein, 3 μg soluble N protein, 10 μg BSA double-layered protein nanoparticles, or a formulation comprising a protein combination of seven µg soluble S1 protein and three μg soluble N protein (a dose of 10 µg whole soluble protein). A placebo group was acknowledged as a non-immunized management, which acquired 100 μL of PBS. The vaccine mixtures have been injected into the hind leg gastrocnemius muscle. Because the preliminary immunization, blood samples have been gathered 1 day earlier than prime immunization, and weekly after immunization for titer measurement. Moreover, immune sera have been collected bi-monthly following the enhance immunization to research its influence on long-term immunity.

ELISA binding antibodies assay

Serum samples from numerous teams of mice have been analyzed utilizing commonplace oblique Enzyme-linked immunosorbent assay (ELISA) to evaluate the IgG and its isotype-specific antibody ranges of S1 or N proteins. The purified S1 protein (WT, Beta, Delta, Lambda, and Omicron) was coated into ELISA plates by in a single day incubation at 4 ℃ for subsequent binding assay. The subsequent day, every nicely was blocked with 5% skim milk in PBST (PBS with 0.05% Tween-20) for two h at 37 ℃. Then, the serum was carried out as a major antibody and serially diluted with ELISA-blocking buffer was incubated for 1 h at 37 ℃. The S1-specific IgG, IgG1, and IgG2a antibodies have been titrated by incubating with the horseradish peroxidase (HRP)‐conjugated Goat Anti-Mouse IgG (Promega; W4021), IgG1 or IgG2a (Jackson ImmunoResearch Laboratories; 115–035-205, 115–035-206) as detection antibodies for 1 h at 37 ℃. Every nicely was developed with TMB Single-Part Substrate answer (Solarbio; PR1200) at midnight for 10 min. 2 M H2SO4 was used to cease coloration growth. Lastly, the absorbance at 450 nm was carried out instantly utilizing a multi-functional enzyme commonplace instrument (Omega, Germany). Much like the Technique described above, oblique ELISA was employed to determine the endpoint titers of N-protein-specific IgG, IgG2a, and IgG1.

Binding kinetics of antigen-specific IgGs via SPR assays

Binding kinetics of N-S1 PNps vaccinated-murine S1-specific serum IgG was decided utilizing a Biacore X100 gear (GE Healthcare, USA) and Amine coupling package (GE Healthcare; BR-1000–50) with the Collection S CM5 sensor Chip (Cytiva; 29,149,604). After injecting a combination of EDC/NHS (1:1) to activate the CM5 Sensor Chip, Anti-Mouse antibodies (Cytiva; BR100838) have been diluted to 30 μg/mL in 10 mM sodium acetate buffer pH 5.0 for immobilization utilizing HBS-EP working buffer (Cytiva; BR100826). Residual-free NHS-esters have been rendered inactive by the injection of 1 M ethanolamine-HCl, pH 8.5. Sera at 21 days post-boost immunization have been diluted 1:100 in 1 × HBS-EP buffer and utilized to the energetic move cell for seize by the immobilized antibody for 60 s. S1 proteins of SARS-CoV-2 WT, beta, delta, lambda, and omicron have been diluted with concentrations starting from 0 to 25 nM in 1 × HBS-EP (pH, 7.4) buffer, respectively, which have been utilized a multi-cycle kinetic methodology for analyzing the binding of captured murine IgG antibodies. Lastly, Glycine–HCl of pH 1.7 was used for the regeneration step. Binding kinetics have been decided with a world kinetic match mannequin (1: 1 Langmuir, Biacore X100 analysis software program, Cytiva) and confirmed by OkD values.

ACE2-receptor-blocking antibodies assay

ACE2-receptor-blocking antibodies have been analyzed utilizing ELISA. Ninety-six-well ELISA plates have been coated with S1 protein in a single day incubated at 4 °C. The serum obtained from immunized mice was serially diluted and incubated at 37 °C for 1 h. PBS was added into wells as a management concurrently. After 1 h, histidine-tagged hACE2 was launched to plates and left at 37 °C for an additional hour, adopted by washing thrice by PBST (PBS with 0.05% Tween-20). HRP-conjugated mouse anti-histidine IgG (Proteintech; HRP-66005) was used because the secondary antibody, incubated for 1 h at 37 °C. The inhibition fee was decided utilizing the next equation: [(OD value of control—OD value of samples/OD value of control) × 100%]. The titer was calculated utilizing GraphPad Prism 8.0.2 based mostly on the serum dilution that prompted a 50% inhibition of receptor binding.

Pseudovirus neutralization assay

A recombinant replication-deficient vesicular stomatitis virus (VSV) spine, during which the G gene was changed with the firefly luciferase (Fluc) reporter gene (G*ΔG-VSV), was pseudotyped with the SARS-CoV-2 Prototype, Delta, Omicron, and Omicron sub-variant lineages (BA.1, BA.2, BA.5, and BQ.1.1) S protein in keeping with revealed pseudotyping protocols [72,73,74]. Huh-7 cells with steady expression of hACE2 (Huh-7/hACE2) and enough pseudotyped virus suspension have been ready upfront. Mouse serum samples have been subjected to serial dilutions and pre-incubated with equal volumes of various SARS-CoV-2 pseudoviruses suspension together with the Prototype, Delta, Omicron, and Omicron sub-variant lineages (BA.1, BA.2, BA.5, and BQ.1.1) (Beijing Tiantan Pharmaceutical Biotechnology Growth Firm, China) in 96 wells plates for 1 h at 37 °C earlier than transferring the combination to Huh-7/hACE2 cells. Inoculated Huh-7/hACE2 cells have been incubated for an additional 24 h at 37 °C to specific the reporter gene luciferase. In every plate, the cell management (CC) containing solely cells and the virus management (VC) comprising virus in addition to cells have been established. Lastly, the supernatant was eliminated and discarded, and 100 µL lysis reagent with luciferase substrate (Beyotime, China) was added to every nicely. A multi-mode microplate reader (Molecular Units, USA) was used to research the relative mild unit (RLU). The inhibition charges of an infection for every dilution of the pattern have been decided based mostly on the RLU values as follows: inhibition fee = [1–(average RLU of sample–average RLU of CC)/(average RLU of VC–average RLU of CC)] × 100%. The 50% pseudovirus neutralizing titers (pVNT50) have been decided because the reciprocal of the best serum dilution that prompted 50% inhibitory in comparison with the typical of the virus management wells.

Mouse splenocyte isolation, IFN-γ, and IL-4 enzyme-linked immunospot assay

On day twenty first after the enhance immunization, splenocytes of vaccinated mice have been collected from all immunization teams as single-cell suspensions utilizing the mouse spleen lymphocyte isolation package (Solarbio; P8860). Freshly remoted splenocytes have been plated straight at a density of 4 × 105 per nicely in RPMI Medium 1640 (Solarbio; 31,800) supplemented with 10% FBS for restimulation. Splenocyte of every nicely was stimulated with the N peptide pool (consisting of equal quantities of 14 overlapping amino acids artificial peptides of SARS-CoV-2 N protein, at a remaining focus of 8 µg/mL per peptide), purified S1 protein (A remaining focus of 14 µg/mL), or mock-restimulation in wells pre-coated with anti-IFN-γ or anti-IL-4 antibodies for corresponding lymphocyte ex vivo stimulation in a quantity of 20 µL/nicely. Incubated at 37 °C in a 5% CO2. Every stimulation situation was employed in triplicate wells. After 36 h, AEC substrate options (Dakewe; 2,210,002 and a pair of,210,402) have been added to the plates for 20 min, then completely rinsed with purified water and subsequently air-dried previous to counting. The spots have been enumerated and analyzed with an ELISpot plate reader (ImmunoSpot® S6 Extremely Analyzer [CTL], US). Spot numbers have been assessed and normalized to spots/106 splenocytes.

Cytokine secretion ranges measurement assay

Lymphocytes have been separated from spleens and re-stimulated with an artificial N peptides pool or purified S1 protein as described in Strategies. After 60 h, concentrations of IFN-γ, IL-2, and IL-4 within the splenocyte tradition supernatants (1 × 106 cells/mL) have been decided utilizing industrial Mouse IFN-γ ELISA KIT (Solarbio; SEKM-0031), Mouse IL-4 ELISA KIT (Solarbio; SEKM-0005), Mouse IL-2 ELISA KIT (Solarbio; SEKM-0004) in keeping with the directions for makes use of, respectively. Alerts have been measured at 450 nm and 630 nm wavelengths inside 5 min by the multi-functional enzyme commonplace instrument (Omega, Germany).

Lymphocyte proliferative exercise assay

Lymphocytes have been remoted and re-stimulated from immunized mice 3 weeks after the ultimate immunization as described in Strategies. The splenocytes (1 × 106 cells/mL) have been ready and stimulated for 60 h at 37 °C. Furthermore, the damaging wells have been handled with the medium as a stimulant. The proliferation of lymphocytes was assessed with Cell Counting Equipment-8 (CCK-8) (Dakewe; 6,073,213). The cell proliferation fee was decided utilizing the stimulation index (SI). SI = (the imply of OD 450 nm values of the N peptide pool or S1 protein stimulated wells)/(the imply of OD 450 nm values of damaging wells).

Circulation cytometry assay

Spleens of mice have been excised in 3 weeks after the enhance vaccination and splenocytes suspension (1 × 106 cells/mL) was ready as described in Strategies. Purple blood cells have been first lysed with ACK Lysis Buffer (Beyotime; C3702) earlier than cell floor markers have been stained utilizing numerous antibodies. Then, the lymphocytes have been stained and incubated with anti-CD3-FITC, anti-CD4-PerCP-Cy5.5, and anti-CD8-APC at midnight for 25 min at 4 °C. To guage GCs formation in spleens, Tfh cells have been stained with anti-CD4-PerCP-Cy5.5, anti-CXCR5-BV421, anti-ICOS-APC, and anti-PD-1-PE. As well as, GCs B cells have been stained with anti-B220-BV510, anti-CD95-APC, and anti-GL7-Alexa Fluor 647. To evaluate the maturation of DCs, the cells have been stained with anti-CD11c-FITC, anti-MHC class II-BV421, and anti-CD80-APC. To measure the proportion of central reminiscence of T cells, the cells have been stained with anti-CD3-FITC, anti-CD62L-PerCP-Cy5.5. All fluorochrome-conjugated antibodies have been bought from Biolegend (BioLegend, USA). Furthermore, stained cells have been washed and centrifuged twice (at 300 ×g for five min every time) earlier than move cytometry evaluation. The resuspended cells have been detected using the CytoFLEX (Beckman Coulter, USA) and the acquired information have been analyzed with FlowJo software program (BD Biosciences).

Reverse-transcription quantitative polymerase chain response (RT-qPCR) assay

The spleen lymphocytes of mice in numerous immunization teams have been harvested, then extracted the entire RNA by the RNAiso Plus (Takara; 9108) methodology and reverse-transcripted into cDNA using PrimeScript™ RT Grasp Combine (Takara; RR036A). The qRT-PCR was carried out utilizing the ChamQ Common SYBR qPCR Grasp Combine (Vazyme Biotech; Q711-02) and Utilized Biosystems™ 7500 Quick Actual-Time PCR System (Thermofisher, USA). Primer sequences utilized are proven in Further file 1: Desk S1, whereas the relative gene expression values have been decided via the two−ΔΔCt methodology with β-actin as management.

H&E and immunofluorescence staining assay

Briefly, inguinal lymph node samples have been harvested from immunized mice 3 weeks after enhance immunization, fastened in formalin, and embedded in paraffin (FFPE). Then paraffin-embedded tissue blocks have been sectioned and incubated with hematoxylin for five min. The sections have been subjected to a 30-s therapy with 1% hydrochloric acid alcohol earlier than additional therapy with eosin for two min. The outcomes have been seen via an orthotopic optical microscope (Nikon Eclipse CI, Japan).

The immunofluorescence staining was carried out on four-micron sections following commonplace procedures, which included deparaffinization, antigen retrieval, circle of the spontaneous fluorescence quenching, BSA blocking, major and secondary antibody incubation, in addition to counterstaining with DAPI. The first antibody was the Rabbit Anti-CD45 antibody (Bioss; bs-4819R). Lastly, pattern sections have been seen on an upright fluorescence microscope (Nikon Eclipse Ci, Japan).

RNA-Seq assay

Whole RNA extracted from 15 to 30 μg spleen tissue on day 21 post-boost immunization. The standard of RNA was inspected utilizing a NanoDrop One spectrophotometer (NanoDrop Applied sciences, Wilmington, DE), Qubit 3.0 Fluorometer (Life Applied sciences, Carlsbad, CA, USA), and agarose gel electrophoresis. The RNA pattern preparations have been carried out with 2 μg of whole RNA per pattern as enter materials. mRNA was enriched utilizing magnetic beads earlier than synthesizing double-stranded cDNA to generate Sequencing libraries. Subsequent, the procedures of terminal restore, A-tailing, and adapter added have been applied, and following fragment screening and PCR, obtained the ultimate library, which was generated utilizing MGIEasy RNA Library Prep Equipment for BGI®. Lastly, the libraries have been sequenced utilizing a BGI DNBSEQ platform (BGI Genomics, Shenzhen, China) supplied by Wuhan Benagen Expertise Co., Ltd.

Adaptors and low-quality sequences that negatively influence subsequent meeting have been filtered out from the uncooked information by FastQC (v0.11.9, tasks/fastqc/). Then, the trimmed clear reads have been mapped to the Mus musculus reference genome ( utilizing the Star aligner (v2.7.9a, To match the extent of similarity among the many samples, pattern correlation evaluation was carried out (Further file 1: Fig. S1A). Differential expression genes (DEG) have been carried out by DESeq2 (v1.34.0, The DEGs discuss with a major false discovery rate-adjusted P worth (FDR) < 0.05 based mostly on three organic replicates. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for the DEGs utilizing the clusterProfiler model 3.14.3 ( As well as, the pathways with a P-value < 0.05 have been chosen and the highest 20 are proven.

In vivo security and serum chemistry

BALB/c mice have been intramuscularly injected with 20 μg of double-layer N-S1PNp, whereas soluble S1 or N proteins, double-layer BSA PNp, and PBS management teams have been arrange. Report the limb accidents and weight adjustments on the injection website after immunization, and gather blood on the 14th day for testing the entire blood cell depend parameters (WBC, RBC, PLT, and HGB ranges) and biochemical indexes (Crea, ALT, URA, and ALP ranges) to judge acute poisonous results.

Statistical evaluation

All figures have been ready with GraphPad Prism 8.0 and FlowJo 10.6.2 Software program. The P values have been obtained via one-way ANOVA evaluation adopted by a number of comparisons submit hoc assessments utilizing the IBM SPSS Statistics 21.0 for group comparisons. Variations with a P worth of < 0.05 have been thought-about vital: ***p-value < 0.001, **p-value < 0.01, *p-value < 0.05.

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