Potyvirus, and that such resistance is counteracted by NUCLEAR INCLUSION B (NIb), the viral RNA-dependent RNA polymerase. We demonstrate that NIb binds to the SUMO-interacting motif 3 (SIM3) of NPR1 to prevent SUMO3 interaction and sumoylation, while sumoylation of NIb by SUMO3 is not essential but can intensify the NIb–NPR1 interaction. We discover that the interaction also impedes the phosphorylation of NPR1 at Ser11/Ser15. Moreover, we show that targeting NPR1 SIM3 is a conserved ability of NIb from diverse potyviruses. These data reveal a molecular “arms race” by which potyviruses deploy NIb to suppress NPR1-mediated resistance through disrupting NPR1 sumoylation.NONEXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1) is the master regulator of salicylic acid-mediated basal and systemic acquired resistance in plants. Here, we report that NPR1 plays a pivotal role in restricting compatible infection by turnip mosaic virus, a member of the largest plant RNA virus genus  Salicylic acid (SA) signaling pathway restricts the compatible infection of potyviruses. Here, Liu et al. show that potyviral NIb interacts with NPR1, the SA receptor in plants, preventing its sumoylation by SUMO3 and subsequent phosphorylation at Ser11/Ser15. This way, NPR1-mediated immunity is suppressed to promote virus infection. Innate immunity, which is further divided into pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI), plays critical roles in the “arms race” between plants and pathogens2. PTI is triggered by the perception of PAMPs via cell surface–localized pattern recognition receptors (PRRs), whereas ETI is activated by the recognition of pathogenic effectors through intracellular nucleotide-binding leucine-rich repeat receptors (NLRs)3. The perception of PAMPs or effectors causes a significant conformational alternation of the receptors and results in a series of downstream signaling events that lead to resistance responses, e.g., the expression of defense-related genes, the synthesis and deposition of callose at the plasmodesmata (PD), strengthening of the cell wall, and even a hypersensitive response (HR)4. Recent studies have shown that PTI and ETI work synergistically by potentiating each other to achieve stronger immune responses6.Plants are continually challenged by various phytopathogens, e.g., viruses, bacteria, fungi, nematodes, and oomycetes. To confront pathogen attacks, plants have evolved multilayered defense mechanisms, such as preformed physical barriers, toxic compounds, RNA silencing, nonhost resistance, and innate immunity8. In Arabidopsis, NPR1 is present in the cytoplasm as tetramers via intermolecular disulfide bonds under steady-state conditions9. SA rapidly accumulates upon the activation of PTI and ETI from both de novo biosynthesis and hydrolysis of inactivated forms10. The increased SA level alters the cellular redox state, which causes monomerization of cytoplasmic NPR1 via thioredoxins11. NPR1 is then phosphorylated by SNF1-RELATED PROTEIN KINASE 2.8 (SnRK2.8) at Ser589 and possibly Thr373, and these phosphorylations promote NPR1’s nuclear entry12. In the nucleus, NPR1 is further sumoylated by SMALL UBIQUITIN-LIKE MODIFIER 3 (SUMO3) and phosphorylated at Ser11/Ser15; then, it reprograms overall transcription via basic region/leucine zipper motif (bZIP) and WRKY transcription factors14. NPR1 is also subjected to ubiquitin modification through Cullin-RING E3 ligase and UBIQUITIN E4 LIGASE/MUTANT, SNC1-ENHANCING 3 (UBE4/MUSE3) and is degraded through the 26S ubiquitin‒proteasome system in the nucleus15. Interestingly, ubiquitination is also required for the full transcriptional activity of NPR1 during SAR16. In addition, NPR1 directly participates in ETI by forming SA-induced NPR1 condensates (SINCs) to modulate the turnover of stress response proteins in cells surrounding the HR sites to promote their survival17.Activation of PTI and ETI also induces resistance in distal tissues, a phenomenon known as systemic acquired resistance (SAR). SAR is mediated by the defense hormone salicylic acid (SA) through the function of NONEXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1)18. It is believed that ETI and RNA silencing are the two principle antiviral mechanisms in plants. However, how plants respond to infection by compatible viruses that spread systemically and cause disease is less well understood. Transcriptomic and proteomic studies have revealed extensive transcriptional reprogramming during compatible plant‒virus interactions, including upregulation of a group of defense-related genes19. Direct treatment with SA or its analog in wild-type (WT) plants but not npr1 mutants induces resistance to various phytopathogens, including viruses20. NPR1-knockout or NPR1-knockdown plants, SA biogenesis-deficient mutants, and transgenic plants expressing a salicylate hydroxylase gene (NahG) all show enhanced susceptibility to infection by adapted viruses25. These findings imply that the SA signaling cassette may play a role in compatible plant‒virus interactions. However, how the SA signaling cassette activated, its contribution to compatible plant‒virus interactions, and how plant viruses manipulate SA-mediated plant defenses are poorly understood.Viral pathogens are not generally viewed as PAMP-encoding intruders, since recognition of obligate intracellular parasites by extracellular PRRs is counterintuitivePotyviridae includes more than 30% of known plant-infecting RNA viruses, which are divided into at least 12 definitive genera and 3 unassigned species26. Potyvirus is the largest genus in the family, and includes many agriculturally important viruses, such as potato virus Y (PVY), turnip mosaic virus (TuMV), soybean mosaic virus (SMV), and papaya ringspot virus (PRSV)27. The genome of typical potyviruses consists of a positive-sense single-stranded RNA (+ssRNA) of ~9.6 K nucleotides (nt) that contains a single open reading frame (ORF) encoding a large polypeptide of ~350 kDa. In addition, a unique polymerase slippage motif within the P3 cistron enables the expression of an additional short polypeptide28. These two polypeptides are proteolytically processed by three viral proteases into 11 mature proteins and many precursors26. Although the replication of potyviruses takes place in the cytoplasm, several viral proteins, including NUCLEAR INCLUSION B (NIb), the viral RNA-dependent RNA polymerase (RdRp)29, localizes primarily in the nucleus for unknown reasons30. Previously, we reported that TuMV-encoded NIb interacts with the components of Arabidopsis sumoylation machinery, e.g., SUMO-CONJUGATING ENZYME 1 (SCE1) and SUMO3, in the nucleus and displays sumoylation-dependent immunosuppressive activity32, suggesting that nucleus-localized NIb may have an important function in inhibiting plant immunity.The family In this work, we further demonstrate that the SA signaling cassette plays a critical role in compatible plant‒virus interactions and that NIb directly targets NPR1 to suppress such resistance in the nucleus.32. SUMO3 also positively regulates Arabidopsis immunity, possibly by interacting with or modifying its components, such as NPR134. Since NIb displays sumoylation-dependent immunosuppressive activity32, we suspected that NIb may directly interact with SUMO3 substrates to compete for SUMO3. To test this idea, we performed a bimolecular fluorescence complementation (BiFC) assay between NIb and known SUMO3 substrates, e.g., NPR1, BRAMHMA (BRM), CYTOKININ RESPONSE FACTOR 6 (CRF6), several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4/FLORAL TRANSITION AT THE MERISTEM 6 (SPL4/FTM6), ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5 (ERF5), and FLOWERING BHLH 3 (FBH3)36. The signal of yellow fluorescent protein (YFP) was recorded in Nicotiana benthamiana epidermal cells expressing the C-terminal segment of YFP (YC)-fused NIb (NIb-YC) and the N-terminal segment of YFP (YN)-fused Arabidopsis NPR1, TCP3, TCP4, TCP23, BRM, SPL4/FTM6, ERF5, FBH3, and CRF6 -tagged NPR1 but not the free Trx-6×His tag was successfully pulled down by GST-NIb but not GST  assay was performed to confirm the interaction between NIb and NPR1 Fig. . Yeast crol Fig. . Moreoverol Fig. . We furtrol Fig. . Consist-YN Fig. . We furt-YN Fig. . We alsoGST Fig. . Very re7, namely, the N-terminal bric-à-brac, tram track, and broad complex/poxvirus and zinc finger (BTB/POZ) domain (amino acids 1-230), the central ankyrin repeat (ANK) domain (amino acids 231–465), and the C-terminal putative transcriptional activation (TAD) domain that binds SA  neighboring His334 may be essential for NIb interaction34. Indeed, we found that SIM3-mutated NPR1 mutant (sim3) failed to interact with NIb in both Y2H and BiFC assays . npr1-1 harbors H334Y7; npr1-0 (SALK_204100) is a null mutant that has a T-DNA insertion in the first exon of the NPR1 gene16; and npr1-6 (SAIL_708_F09) contains a T-DNA insertion in the third exons of the NPR1 gene, which allows the expression of amino acids 1-466 of NPR1 (NPR1ΔC)39. NPR1ΔC lacks the nuclear localization signal40. As a result, transiently expressed NPR1ΔC-YFP was located mostly in the cytoplasm of N. benthamiana epidermal cells  between the P1 and HC-Pro cistrons, to directly visualize virus infection40. At 14 days post-inoculation (dpi), we found that the ratios of TuMV-infected leaf area to total rosette leaf area of npr1-1, npr1-0, and npr1-6 were significantly higher than that of the WT, as indicated by the GFP fluorescence from TuMV-GFP , a marker gene of plant immunity41. Four-week-old seedlings of the WT and the three npr1 mutant strains were mechanically inoculated with TuMV-GFP to avoid the stimulation of agrobacteria. We found that TuMV-GFP infection induced the expression of PR1 in both Col-0 and npr1 mutants compared with mock-infected plants at 2 dpi  35S promoter in the npr1-1 background  in which the second SIM (SIM2) was mutated was also included, since NIb also interacts with and is sumoylated by SUMO3 via this motif32. We found that although NPR1 and SUMO3 were expressed at similar levels, the nuclear YFP signal and luciferase activity were significantly decreased by NIb compared with SUMO1  into 35S::NPR1-GFP-3 to produce XVE::NIb 35S::NPR1-GFP. Homozygous plants were treated with 1 mM SA alone or together with 17-β-estrogen to induce the expression of NIb. NPR1 was then immunoprecipitated by GFP-Trap agarose and detected with anti-SUMO3 antibodies. The results showed that the level of sumoylated NPR1 was significantly reduced by the presence of NIb  were produced to further evaluate the influence of NIb on NPR1 sumoylation. All homozygous seedlings displayed a normal phenotype similar to that of WT plants under steady-state conditions  was also included34. TuMV inoculation showed that the ratios of TuMV-infected to total rosette leaves of 35S::NPR1-GFP−3, sim3-GFP and 35S::npr1sim3 plants were comparable to that of the WT at 18 dpi, while 35S::NPR1-GFP−4 seedlings had a slightly reduced ratio of TuMV-infected to total rosette leaves  profiles of these transgenic seedlings by high-throughput sequencing. The results showed that no or less than 1.0 GFP-derived sRNA per million of total sRNAs was detected in 35S::NPR1-GFP-3, 35S::sim3-GFP and 35S::npr1sim3  but a significant growth enhancement of yeast cells that had been cotransformed with NIb and the phosphorylation-mimic mutant (S11/15D) -like phosphodegrons (Ser11/Ser15) orchestrate NPR1 activity5D) Fig. . Interes5D) Fig. . The BiF-YN Fig. . Moreove-YN Fig. . Togethenpr1-1 background. At least 5 independent lines per construct were obtained. All homozygous 35S::sim3|S11/15D-GFP and 35S::sim3|S11/15A-GFP plants with confirmed protein expression had phenotypes similar to those of WT plants, while most lines of 35S::S11/15D-GFP (5/9) plants displayed autoimmune phenotypes, e.g., a smaller size with curly rosette leaves under steady-state conditions . All homozygous npr1::sim3|S11/15D-Myc transgenic plants displayed a phenotype normal to that of WT plants under steady-state conditions , sugarcane mosaic virus (SCMV), and pepper veinal mottle virus (PVMV) were cloned, and their abilities to interact with Arabidopsis NPR1 or sim3 were analyzed. These potyviruses plus TuMV represent six major clades of the potyviral phylogeny43. Interestingly, we found that yeast cells transformed with NPR1 and either of the NIb proteins survived on the selective medium, while those cotransformed with sim3 failed  also interacts with soybean NPR1 (GmNPR1) but not Gmsim3  during ER stress can cause the translocation of NPR1 to the nucleus47. We are trying to confirm this possibility at present.The pivotal roles of SA signaling in PTI, ETI, and SAR have been well established; however, the contribution of SA signaling to compatible plant‒virus interactions have remained ambiguous. In this study, we found that loss-of-function of uMV Fig. . These r34. It is believed that SUMO3-mediated sumoylation and the phosphorylation of Ser11/Ser15 form a signal amplification cycle for robust immune reprogramming34. Interestingly, our results revealed that the overexpression of a phosphorylation-mimic NPR1 mutant (S11/15D) caused an autoimmunity phenotype , a structural mimic of jasmonates (JAs), to stimulate the transcription of JA-dependent genes and consequently attenuate SA-dependent genes expression48. AvrPtoB of Pseudomonas syringae, RxLR48 of Phytophthora capsica, and PUCCINIA NPR1 INTERACTOR (PNPi) of Puccinia striiformis have been found to target NPR151. RxLR48 promotes nuclear accumulation of NPR1 and inhibits NPR1 degradation; AvrPtoB mediates the degradation of NPR1 by the host 26S proteasome through its E3 ligase activity in the presence of SA; and PNPi interacts with the C-terminal domain of NPR1 and disrupts the NPR1–TGA2 interaction51. Our results show that NIb interacts with NPR1 exclusively in the nucleus and that the interaction is not affected by SA , cucumber mosaic virus , and oilseed rape mosaic virus 52. These results indicate that NPR1 is at the hub of the wrestling match between plants and viruses and that the function of NPR1 may have already been completely or almost completely suppressed by some of these viruses. It will be interesting to investigate how these viruses subvert NPR1-mediated defenses in the compatible interaction.It is well known that SA treatment provides broad-spectrum defense against many viruses, including both plant RNA and DNA viruses32. It is believed that cytoplasmic NIb is mainly involved in viral replication, as it is the only viral protein containing conserved motifs of RdRp. Indeed, several host proteins involved in RNA metabolism, e.g., EUKARYOTIC ELONGATION FACTOR 1A (eEF1A), POLY(A)-BINDING PROTEIN (PABP), HEAT-SHOCK PROTEIN 70-3 (Hsp70-3), and WHEAT LIGHT-INDUCED PROTEIN (TaLIP), have been found to be coopted by NIb for robust infection56. Moreover, cytoplasmic NIb is also targeted by the host surveillance system; e.g., NIb is targeted by beclin1 for degradation and is recognized by the broad-spectrum resistance gene Pvr4 in Capsicum annuum cv. CM33458. Previously, we discovered that NIb exhibits a key function of in the nucleus by interacting with sumoylation machinery to subvert host defenses32. Results of this study demonstrate that NIb directly targets SIM3 of NPR1 to compete for SUMO3 and suppress host defense. Sumoylated NIb may be exported from the nucleus by EXPORTIN 1 (XPO1) for infection59. In this study, we further showed that NIb likely interacts with all known SUMO3 substrates that are involved in varied pathways and biological processes. NIb may also influence their functions and the pathways they are involved, which further implies the versatile functions of NIb. Attachment of SUMO typically marks a protein for recognition by a SIM-containing protein60. Bioinformatic analyses have revealed that more than 40% of Arabidopsis proteins contain a sumoylation site  and/or a SIM motif (I/VDL/T)63. How NIb disarranges the plant SUMO interactome is another engaging subject for exploration in the future.Due to their small genome size and low encoding capacity, viruses have evolved various strategies for their survival. An important one is to make viral protein(s) multifunctional. NIb localizes both in the nucleus and cytoplasmnpr1-0, npr1-1, npr1-6, and 35S:npr1sim3 have previously been described64. Arabidopsis was transformed using the flower dip method65. Progeny seeds were screened by directly spraying 20 mg/L Basta aqueous solutions or on 1/2 Murashige and Skoog (MS) medium plates supplied with hygromycin B (50 ng/mL). Agroinfiltration and sap inoculation were performed as described earlier with seedlings of the same age and similar sizes32. To minimize artificial variations, the whole aerial part of seedlings was harvested and ground in liquid nitrogen, and equal amounts of plant material were used for subsequent analyses, e.g., total RNA extraction, immunoblotting, nucleocytoplasmic partitioning, and ELISA.Plants were grown in pots at 23°С in a growth chamber under a 16/8-h photoperiod with 50% humidity. NPR1 (At1g64280), TCP3 (At1g53230), TCP4 (At3g15030), TCP23 (At1g35560), BRM (At2g46020), SPL4/FTM6 (At1g53160), CRF6 (AT3G61630), TGA3 (At1g22070), ERF5 (At5g47230), FBH3 (At1g51140), mature form of SUMO3 (At5g55170) and NIb of TuMV (NC_002509), SMV (MN623290), PVY (MH933741), BCMV (KP903372), PVMV (MN082715), SCMV (KR108213), and PRSV (HQ424465) were amplified using the primers listed in Supplementary Table 66, and pBA-FLAG-4×Myc-DC was used to produce the N-terminal FLAG-4×Myc-tagged construct67. The gateway-compatible vectors pGBKT7-DEST and pGADT7-DEST were used to produce plasmids for the Y2H assay68, pEarleyGate201-YN, pEarleyGate202-YC, YC-pEarleyGate100, or YN-pEarleyGat100 were used to generate constructs for the BiFC assay68, and the Gateway compatible vectors pCAMBIA-NLuc-GW and pCAMBIA-CLuc-GW, which were modified from pCAMBIA1300-NLuc and pCAMBIA1300-CLuc, respectively69, were used for the split-luciferase assay. The vectors pET-32a (+)  and pGEX-4T-1  were used to express N-terminal Trx-6×His and GST-tagged recombinant proteins in Escherichia coli, respectively. All plasmids were verified by Sanger sequencing.Full-length or partial coding sequences of 32. All yeast selective media were purchased from Coolaber Technology (Beijing) Co., Ltd.Y2H assays were performed using the yeast strain Golden  as described previouslyN. benthamiana leaves were agroinfiltrated with agrobacteria harboring proper plasmids. The infiltrated leaves were integrally detached from the plant at 60 hpi and soaked in 10 μM d-luciferin  for ~20 min. The leaves were then visualized with a Tanon 5200CE Chemiluminescent Imaging System .To determine the luciferase activity, g for 10 min at 4 °C, and the supernatant was stored at 80 °C until use or directly used for SDS‒PAGE. For total yeast protein, a total volume of 5 mL of yeast overnight culture was used for total protein extraction using the yeast total protein extraction kit (Coolaber) according to the supplied protocol.For total plant protein, ~0.1 g of fine powder of the aerial parts of the plant or agroinfiltrated leaf tissues was resuspended in 100 µL of 1× sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) loading buffer . After boiling at 95 °C for 5 min, the crude lysate was centrifuged at 12,000×32. In brief, ~3 g leaf tissues were ground into fine powder in liquid nitrogen, mixed with 15 mL IP buffer I , and placed on ice for 15 min. After being filtered through two layers of Miracloth (Merch Millipore), the solution was sonicated 10 s at 10% power level eight times with a 1 min interval. The lysate was then centrifuged at 6000×g for 25 min at 4 °C. The resulting supernatant was added to 30 μL of Chromotek GFP-Trap  or anti-FLAG M2 affinity gel  and incubated for 3 h at 4 °C with gentle rotation. Agarose beads were collected by centrifugation, washed at least five times with IP Buffer and resuspended in 50 µL 1× SDS‒PAGE loading buffer. The solution was frozen at −80 °C until use or directly used for SDS-PAGE.Co-IP was performed as described previously with few modificationsSDS‒PAGE was performed using 10 or 12% homemade polyacrylamide gels. After SDS-PAGE, proteins were transferred to polyvinylidene fluoride (PVDF) membranes with a Trans-Blot Turbo Transfer System (Bio-Rad). The PVDF membranes were rinsed briefly in TBST buffer and then blocked with 5% nonfat dry milk (in TBST buffer) for 1 h at room temperature. The membranes were incubated with proper antibodies at the desired dilution at room temperature for 1 h or overnight at 4 °C. After washing six times with TBST, the membranes were incubated with the appropriate secondary antibodies (Sigma‒Aldrich) for 1 h at room temperature. After washing six times with TBST, the membranes were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Merch Millipore) following the manufacturer’s instructions with a Tanon 5200CE Chemiluminescent Imaging System. In each experiment, a parallel gel was stained with Coomassie Brilliant Blue as a loading control.The following antibodies were used: rabbit anti-GFP N-terminal (Sigma‒Aldrich) at a 1:5000 dilution, mouse monoclonal anti-GFP antibodies (Roche) at a 1:1000 dilution, rabbit anti-SUMO3  at a 1:2000 dilution, polyclonal anti-Myc (Abcam) at a 1:2000 dilution, mouse monoclonal anti-GAL4 AD [14-7E10G10] antibody (Abcam) at a 1:2000 dilution, Rabbit polyclonal to Histone H3 (Abcam) at a 1:5000 dilution, anti-Luciferase polyclonal antibody (Proteintech) at a 1:10,000 dilution, mouse monoclonal anti-RFP (Proteintech) at a 1:5000 dilution, HRP-conjugated anti-rabbit IgG secondary antibodies (Sigma-Aldrich) at a 1:10,000 dilution, and HRP-conjugated anti-rabbit IgG secondary antibodies (Sigma-Aldrich) at a 1:10,000 dilution. Rabbit polyclonal antibodies against TuMV CP (anti-CP), TuMV NIb (Anti-NIb), and Arabidopsis NPR1 (Anti-NPR1) were produced by ABclonal Biotechnology co., Ltd  using GST-tagged recombinant proteins of full-length CP, amino acids 165-488 of NIb, and amino acids 1-465 of NPR1, respectively. Both of these antibodies were used at a 1:1000 dilution. Validation data of anti-CP, anti-NIb, and anti-NPR1 have been provided as a Source Data file.3)2, 3.5 µM Phos-tag Biotin, and 0.15 µL/mL streptavidin-HRP) for 1 h at room temperature. After washing five times with TBST for 5 min each time at room temperature, the membrane was soaked in 500 µL of Immobilon Western chemiluminescent HRP substrate solution and visualized with a Tanon 5200CE Chemiluminescent Imaging System.The phosphorylated protein was detected on a PVDF membrane with the biotinylated Phos-tag  amino] propan-2-olato dizinc (II) complex (BTL-105) from APExBIO Technology LLC  and streptavidin-HRP . In brief, PVDF membranes that were blocked with 5% nonfat dry milk (in TBST buffer) for 1 h at room temperature were incubated in 10 mL of Phos-tag biotin solution . After being filtered through two layers of Miracloth, the lysate was centrifuged at 4000×g for 20 min at 4 °C. The resulting supernatant was added to 70 μL of 4× SDS‒PAGE loading buffer, mixed well, boiled for 5 min, and then directly used for SDS-PAGE. The resulting pellets were mixed with 200 µL of nuclear extraction buffer  and 70 μL 4× SDS‒PAGE loading buffer. After vortex mixing, the mixture was boiled for 5 min and then directly used for SDS-PAGE. Equal amounts of supernatant and pellets were used for immunoblotting with exactly the same treatments and conditions.Approximately 0.1 g fine powder of the aerial parts of the plant was resuspended in 200 µL of extract buffer  was used as the substrate for color development.ELISA was performed using polyclonal antiserum to the TuMV CP. Goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase  was used as a secondary antibody, and N. benthamiana leaves at the indicated OD values. The fluorescence was visualized with a TCS SP8 LIGHTNING Confocal Microscope  as described previously70. Sequential mode was used when multiple fluorescent proteins were recorded.Agrobacteria harboring the proper plasmid were infiltrated into E. coli BL21 (DE3) cells with 0.5 mM isoprophyl-β-D-thiogalactoside (IPTG) at 16 °C overnight. Trx-His and GST-tagged proteins were purified with Ni–nitrilotriacetic acid (Ni-NTA) agarose (Thermo Fisher Scientific) and glutathione agarose (Thermo Fisher Scientific), respectively. For the GST pulldown assay, equal amounts of recombinant GST and GST-NIb were incubated with Trx-His or Trx-His-NPR1 and GST beads at 4 °C for 2 h. After washing with wash buffer  six times, the GST beads were boiled for 5 min in 2× SDS‒PAGE loading buffer. Equal amounts of supernatants were loaded onto a 10% acrylamide gel, separated by electrophoresis, and detected with antibodies against GST or 6×Histidine.Recombinant proteins were expressed in http://www.gelanalyzer.com/) according to the instructions provided.Relative quantification of protein bands in Western blotting was performed by densitometric analysis using GelAnalyzer 19.1 software  in the nuclei was then determined using the particle analysis function in Fiji with default parameters and the average value of the nuclear fluorescence of the micrograph was calculated.For quantitative analyses of confocal microscopic images, the fluorescence of target protein(s) was excited and recorded at minimized intensity  to avoid possible overexposure, while the signal of marker protein was recorded at optimized intensity . Fluorescence quantitative analyses were performed using Fiji, a version of ImageJ2 for scientific image analysis71. The total leaf area was determined by the red channel pixels, while the virus-infected area was assessed by the green channel pixels using the Analyze Particles function with size 100-infinity and circularity 0.0–1.0. A pseudocolor image was also produced with the ROI manager function.Images of Arabidopsis plants were taken using a Nikon DH-SLR D5200 camera. Images under UV light were split into red, green, and blue channels using Fiji20 or random hexamers using a HiScript III 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme) as instructed. RT-qPCR was performed using a Roche LightCycler 96 System in a 20 µL volume system containing 4 µL of 10-fold-diluted cDNA, 5 µM of each primer, and 1× AceQ® Universal SYBR qPCR Master Mix (Vazyme). The genomic RNA of TuMV was determined by amplification of a 257 bp fragment of the TuMV CP gene. The Arabidopsis PR1 (AT2G14610), UBQ5 (AT3G62250), GAPDH (AT3G26650), and ACTIN II (AT3G18780) were amplified with primers listed in Supplementary Table Total RNA was isolated using the Eastep® Super Total RNA Extraction Kit  according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized by Oligo (dT)72. The confidence of the phylogenetic tree was tested by bootstrap method with 1000 replicates. Multiple alignment was performed using the ClustalW version 2.073 and rendered using the ESPript374.The phylogenetic tree was constructed using MEGA 11 software with the neighbor-joining methodt test with Office Excel 2016 or GraphPad Prism 8.0.2. All data are represented as the mean ± SD. Source data are provided as a Source Data file.All statistical analyses were performed by two-tailed Student’s Further information on research design is available in the Supplementary inforationPeer Review FileReporting summary


Serious Exergames (SEGs) have been little concerned with flexibility/equivalence, complementarity, and monitoring . These functionalities are necessary for health SEGs due to the variety of treatments and measuring requirements. No known SEG architectures include these three functionalities altogether. In this paper, we present the 123-SGR software architecture for the creation of an SEG that is appropriate to the needs of professionals and patients in the area of rehabilitation. An existing SEG was adapted and therapy-related sensor devices  were built to help the patient interact with the SEG. The architecture allows the most varied input combinations, with and without fusion, and these combinations are possible for both conscious and unconscious signals. Health and Technology professionals have assessed the SEG and found that it had the functionalities of flexibility/equivalence, complementarity, and monitoring, and that these are really important and necessary functionalities. The 123-SGR architecture can be used as a blueprint for future SEG development. Serious games (SGs) are digital games with a specific purpose that extends beyond entertainment  and can One of the health aspects is rehabilitation, which aims to recover patients’ lost abilities. In recent decades, studies have emerged that promote rehabilitation through the use of SEGs as a driving force, among them are SGs for motor rehabilitation. Serious Exergames (SEG) help relieve symptoms resulting from stroke , ParkinsAmong SEGs, there are those that are focused on rehabilitation (SGRs—Serious Games for Rehabilitation) and those focused on education, for instance. SEGs offer a compelling and valuable alternative for rehabilitation techniques, capitalizing on engaging, motivating, and immersive strategies to facilitate interventions. To further enhance the efficacy of such approaches, the integration of Multimodal Interaction Systems becomes paramount. These systems encompass the use of at least two distinct input and/or output modalities , holding the potential to elevate the user experience and improve therapeutic outcomes. However, despite the promising capabilities of Multimodal Interaction Systems, certain critical functionalities in rehabilitation games remain underutilized in current SEG implementations , such asFlexibility/Equivalence: Without flexibility, the execution of an SEG for rehabilitation would be limited to a single controller device, restricting the therapy’s applicability. For instance, inspiratory flow or pressure could be triggered by some game action but they require different devices to be used, otherwise there is no such flexibility. To enhance this scenario, the architecture allows for multiple interaction modalities (forms of control for the SEG), providing therapists with diverse options for patient interaction [eraction . For insComplementarity (more complete information): Through the combination of different sources of information about a phenomenon, one can reach the best understanding of it [ng of it . The acqng of it . For exang of it .Monitoring: When using an SEG for rehabilitation, or for health promotion in general, where some kind of physical activity is required, it is possible that excessive effort may occur, or another factor could cause patient discomfort or even something more harmful that hinders their therapy. As an example, during prolonged exercise it is possible to experience hyperventilation, which is a condition in which one begins to breathe very fast. Severe hyperventilation can lead to loss of consciousness or result in underlying problems [problems . Anotherproblems . Also, wproblems  (a) warnproblems .Within the domain of Serious Exergames (SEG) for health, the significance of these three essential functionalities becomes evident. To address and enhance these critical aspects, we have thoroughly explored the integration of Multimodal Interaction Systems. Notably, one of the key advantages of multimodal SEGs lies in their emphasis on safety and the potential for long-term benefits. By incorporating multiple modalities, these systems create a safer and more immersive gaming experience, fostering increased user engagement. Moreover, the utilization of various modalities allows for a comprehensive and personalized approach to rehabilitation, ultimately contributing to improved long-term therapeutic outcomes. The exploration of Multimodal Interaction Systems within the realm of SEGs demonstrates their potential to revolutionize rehabilitation strategies, and their capacity to offer considerable advantages in terms of safety and sustained benefits for individuals undergoing therapy . Using aTherefore, this work focuses on the following general research question: “How to create an SEG compliant with the needs of rehabilitation with various interaction devices?”The objective of this paper is to introduce a multimodal software architecture for SEGs, called “123-SGR Architecture”, where the number “1” represents a single solution for multimodal interactive software architecture; the number “2” emphasizes a dual directionality of the interaction flow and two distinct flows of information (conscious/voluntary and unconscious/involuntary); and the number “3” highlights achieving all three functionalities . “SGR” stands for Serious Game for Rehabilitation. The primary focus of this architecture is to effectively address the specific needs of therapists and patients in the domain of physical rehabilitation. Additionally, a proof of concept will be provided through its application in respiratory rehabilitation (RR) therapy. By implementing the proposed approach within the context of RR therapy, the aim is to demonstrate the practicality and effectiveness of the multimodal SEG architecture, while also validating its applicability and advantages in real-world rehabilitation settings.This section presents the 123-SGR architecture, a pivotal development in the context of Serious Games for Rehabilitation (SGRs). It encompasses essential features that play a significant role in enhancing SEGs for rehabilitation purposes.The motivation behind focusing on specific aspects of the architecture stems from the objective of creating a robust and versatile framework for SEGs in rehabilitation. The incorporation of Decision, Action, Perception, and Interpretation states  ensures The architecture prioritizes patient engagement and motivation by intercepting and adapting game controller signals based on physiological inputs . FollowiBy incorporating fusion, fission, flexibility/equivalence, and complementarity functionalities , the arcLeveraging elements from game design, such as mechanics, dynamics, and aesthetics , enricheThe 123-SGR architecture for multimodal SEGs encompasses three interaction flows: conscious flow, unconscious flow, and feedback flow see .Conscious flow is what characterizes the purposeful manipulation of the modalities, performed by the player to generate a result in the exergame. Conscious flow goes to the following states:Decision: The player consciously creates a message.Action: The player performs the action that represents the thought message.Perception: Messages are perceived by one, or multiple devices.Signal Deaggregator: this core separates information so that it can be used in the game.Signal Treatment: this determines how the signal is collected , whether there is a need for signal amplification via software, how valid data are extracted, and how these valid data are used in the game.Combination: This receives the signals from the devices and determines how each of them will proceed: (i) if flexible ; (ii) with different assignments ; (iii) as a fusion of redundant modalities (one and one); (iv) as a fusion of complementary modalities (one and the other); (v) or directly . At the end of the combination of the signals, they are sent to the Adaptation Grid core.Adaptation Grid: this is responsible for performing the tests, parameterized by a therapist, to generate adaptations that can affect the mechanics, dynamics, and aesthetics of the SEG. It deals only with adaptations for flow purposes (maintaining player interest) and for physical evaluation (monitoring), but this core allows the addition of as many tests and adaptations as necessary, accounting for possible conflicts among triggers .Fission: this analyzes the feedback message from the core game and delivers it to the player via the devices available, for example, a monitor and a speaker sound box.Mixer Module: The signal management module consists of five cores: Signal Deaggregator, Signal Treatment, Combination, Adaptation Grid, and fission (the latter used only in the feedback flow):Mechanics: this is responsible for the actions to be performed, such as jumping or walking, based on the information received from the Adaptation Grid.Dynamics: this is responsible for the difficulty of the game; it controls parameters such as speed, number of repetitions, size of obstacle collision areas, height of target collision areas, score formula, etc.Aesthetics: this determines the visual aspects of the game, such as colors, shapes, and sizes of objects.Game: where the mechanical, dynamic, and aesthetic aspects are applied.Storage: this is where game data as well as device signals are recorded, separated by player and timeline.Profile: this is a core that serves the Adaptation Grid and the mechanics, dynamics, and aesthetics cores, so that the adaptations are made in accordance with users’ capabilities and pathologies.Interaction Module: This is responsible for applying the adaptations and the interaction into the SEG. This module has 6 distinct cores:Serious Game: The multimodal SEG interprets the data and their meaning by using the Mixer and Interaction modules:Unconscious flow captures the patient’s unconscious or unintentional physiological characteristics (sometimes beyond the desirable limit). For example, changes in heart rate and oxygen saturation. This interaction flow allows functionality to be monitored and is described through the states below.Decision: in unconscious flow, there is no decision being made on purpose, but a reaction provoked by the conscious actions of the first flow; for this reason, the state “Decision” is not evidenced in this flow.Action: the player’s physiological state is captured by the system regardless of the player’s interactions.Perception: the same as the conscious flow behavior; however, signals (messages) are captured by other specific devices, or other signals from existing devices are reasoned with.Serious Game: at this state, the unconscious flow goes through the same modules with the same functionalities as the conscious flow, but they are concerned with unintentional signals for safety monitoring goals.Upon receiving signals from the conscious and unconscious flows, the necessary modifications to the game are applied, and to complete the interaction cycle, the game returns a response, showing the patient what changed in the game. The feedback flow is described below.Game: this core sends a response message to the Mixer module.Profile: this is a core that helps the Mixer module to send messages consistent with the player’s profile. For instance, if the player has a hearing impairment condition the Mixer module should not send a response on an audio channel or send it at a specific audible frequency.Mixer Module: this now triggers the fifth core, fission, which analyzes the possibilities and selects the devices to deliver the game response.Serious Game: This is responsible for analyzing game occurrences and producing feedback to the player:Action: the fissioned response is then passed to available devices.Perception: the player’s senses capture the response.Interpretation: the player interprets what the response given by the game meant and the multimodal interaction cycle repeats until the end of the game execution.The definitions of the Mixer and Interaction modules assume a crucial role within the 123-SGR architecture. The Mixer module consists of five cores and is responsible for signal management, combination, and adaptation processes, ensuring an efficient integration of diverse modalities to enhance gameplay. Conversely, the Interaction module, with its six distinct cores, governs game mechanics, dynamics, aesthetics, and interactions, resulting in a comprehensive and personalized gaming experience. These modules collaboratively enable seamless interaction between players and the SEG, thereby empowering effective rehabilitation interventions and monitoring functionalities throughout the conscious, unconscious, and feedback flows.At the signal level, where the voltage and current in analogic or digital forms are manipulated.At the data level, where the signal level is made available as a sequence of values in the computer.At the feature level, where elements of game design that refer to parameters such as target height, size of obstacles, space between objects, and game speed, for instance are manipulated.At the decision level, where the signals from patient interaction are applied to modifications in the game.The 123-SGR can be used in various areas of physical rehabilitation, particularly in respiratory rehabilitation. Processing multimodal inputs can be performed at various levels of the information:123-SGR architecture works at the data level upwards. The following existing game and architectural requirements were incorporated :This section explores quality attribute scenarios . QualityQuality attribute scenarios measure the architecture’s effectiveness. By creating an immersive and engaging gaming experience through rigorous evaluation, patients are motivated to actively participate in their rehabilitation journey. By quantifying the impact of these design elements on user engagement, optimization of the architecture to achieve the desired therapeutic outcomes is facilitated.Justification: The therapy is important for patients’ recovery. System disruptions can hinder this progress.Uptime: “I Blue It” targets a minimum 95.0% uptime.Rapid Recovery: system recovery is achieved within minutes.Proactive Notifications: users are alerted about potential disruptions.Redundancy: key components, like devices and databases, have redundancy.Success Criteria:Continuously monitor for availability.Use the “123-SGR” architecture for resilience and adaptability.Set clear maintenance protocols.Strategies:Source: an external device.Stimulus: a failure (crash).Artifact: communication channels.Environment: normal operation.○Log the failure.○Notify relevant entities.○Address the failure and mitigate.Response:○The failure is addressed within minutes.Response measure:Example of Quality Attribute Scenario for Availability:The Decision, Action, Perception, and Interpretation states contribute to the accuracy and responsiveness.Definition: “I Blue It” processes user inputs accurately.Justification: precise processing is key for user satisfaction.Input Processing: system accurately interprets inputs.Objective Metrics: system accuracy is quantifiable.Alignment: system accuracy aligns with user expectations.Success Criteria:Emphasize algorithm accuracy.Use quality attribute scenarios.Adjust based on feedback.Strategies:Source: patient interacting with the system.Stimulus: user input during a session.Artifact: “I Blue It” SEG.Environment: active user interaction session.○Processes input using Decision, Action, Perception, and Interpretation.○Provide accurate feedback.Response:○Process inputs with 95% accuracy.Response Measure:Example of an Accuracy Scenario:Quality attribute scenarios assess the architecture’s adaptability to devices like PITACO, MANO-BD, and extensor belts. Adaptability ensures tailored user experiences and signal integration from diverse modalities.Definition: “I Blue It” adapts to various interaction modalities.Justification: adaptation enhances personalized rehabilitation.Modal Adaptability: the system transitions between modalities.User Experience: seamless interactions.Signal Integration: modalities are integrated for comprehensive data.Success Criteria:Recognize and adapt to different devices.Integrate diverse signals.Optimize user experience across different devices.Strategies:Source: patient using a specific device, e.g., PITACO.Stimulus: device interaction.Artifact: “I Blue It” SEG.Environment: rehabilitation session.○The system detects and adapts to the device.○Integrates device signals.○Provides tailored interaction.Response:○System adapts according to measures detected by the devices.Response Measure:Example of an Adaptation and Flexibility Scenario:Monitoring of accurate physiological data, like heart rate, is vital during rehabilitation sessions. Quality attribute scenarios ensure data precision and reliability.Definition: “I Blue It” monitors physiological data accurately.Justification: accurate monitoring, such as heart rate tracking, is essential for patient safety.Data Accuracy: Precise physiological measurements are provided.Real-Time Monitoring: game features adapt according to physiological data.Reliability: data acquisition and processing are consistent.Patient Safety: patient well-being is prioritized.Success Criteria:Validate data for accuracy.Monitor and adjust based on safety.Strategies:Source: physiological sensors worn by the patient.Stimulus: data capture, e.g., heart rate.Artifact: Mixer module of the game’s architecture.Environment: rehabilitation session.○Capture and process data.○Therapists monitor.Response:○Game parameters change immediately after reasoning.Response Measure:Example of Interoperability Scenario:Incorporating quality attribute scenarios strengthens the architecture’s foundation, ensuring its applicability in real-world rehabilitation settings. These scenarios validate the architecture’s patient-centric, adaptive, and engaging nature.The 123-SGR architecture was utilized in the adaptation of the Serious Exergame (SEG) titled “I Blue It” ,28, it p® engine-based.It is an open-source project freely available in C# and is UnityThe assembly of the ID is also available and detached freely.It benefits greatly from the ability to use other IDs because not all respiratory dysfunctions deal with airflow, but also with air pressure and other aspects. Thus, it benefits from multimodal flexibility.It benefits greatly from the ability to use other IDs because respiratory exercises can make someone faint; this can be identified through peripheral oxygen saturation or heart rate, for instance. Thus, it benefits from multimodal monitoring.It benefits greatly from the ability to use IDs alongside others because some exercises aim to change patients’ breathing, and not only the amount of airflow one can produce but also how muscles, postures, and other measures are adjusted. Thus, it benefits from multimodal complementarity.The “I Blue It” respiratory rehabilitation SEG see  is desigThe latest version of I Blue It complies with the 123-SGR architecture, incorporating various interaction devices such as the Pneumotachograph Device (PITACO), Manovacuometer Device (MANO-BD), Extensor Belt Device, and Pulse Oximeter Device. These devices measure and monitor essential respiratory parameters, encouraging specific respiratory actions, including expiratory flow, inspiratory flow, and maintaining a steady flow, all of which are crucial for effective respiratory rehabilitation.Using the PITACO construction manual  as a basBased on the PITACO ID, we created the MANO-BD , an ID that captures the air pressure blown into the device. A MANO-BD ID is composed of an absolute pressure sensor, model MPX5700; an Arduino for electronic prototyping, model Uno Revision 3; a connection between the sensor and Arduino, which is made using flat cables; a PVC tube into which the player blows; and a PVC cap to prevent the air from escaping.A Pressure Belt ID was created to measure the pressure exerted by the chest or abdomen of the player against the belt while using the game. The Pressure Belt is composed of a resistive force sensor, model FSR402; an Arduino, model Uno Revision 3; and a connection between the sensor and Arduino, which is made using flat cables located inside a rigid metal can. The Pressure Belt uses an adjustable nylon strap with Velcro around the chest holding the ID in place to measure respiratory effort and respiratory rate.An Oximeter ID was built to measure the blood oxygen saturation and the heart rate of the player while using the game. The Oximeter ID is composed of an oximetry sensor, model MAX30102; a connection bar to weld the sensor; an Arduino, Model Nano Revision 3; and a connection between the sensor and Arduino, which is made with flat cables on a 70-point mini protoboard.To achieve all required functionalities, we needed a variety of multimodal IDs useful for respiratory rehabilitation:It must be noted that the validation of the IDs with commercially available medical instruments is beyond the scope of this paper. ID for conscious actions can be seen as providers of flexibility/equivalence or complementarity functionalities, and IDs for unconscious actions can be seen as providers of monitoring functionality, as they can indirectly show indications of tiredness and dizziness. IDs were analyzed to see which ones can provide specific actions that can be used in gameplay see .By analyzing Some of the many possible modality combinations to generate complementarity are shown in Combination 01: can estimate whether a player is commanding the game through predominantly thoracic or predominantly diaphragmatic breathing and determines the volume of the player’s respiratory flow.Combination 02: this can also estimate whether a player is commanding the game through predominantly thoracic breathing, or diaphragmatic, and provides the measure of the pressure exerted by the musculature of the player’s respiratory tract.The player thinks about and performs a breathing maneuver to control the dolphin, Blue, and this maneuver is perceived by one ID or a combination of IDs , as shown in The following describes the analysis regarding the unconscious flow for the multimodal I Blue It SEG:Decision: there is no conscious decision being made by the player in this flow.Action: the player’s blood oxygen saturation can vary depending on the respiratory disease or breathing pattern.Perception: oxygen saturation in the blood is perceived by the Oximeter ID.Signal Deaggregator: used in the Oximeter, whose signal is composed of the player’s arterial oxygen saturation (SpO2) and the heart rate (HR) see .Signal Treatment: Amplification: not required. Sampling: 98/min. In-game use: speed parameter .Adaptation Grid: The SEG adapts according to the percentage of the player’s SpO2. If it falls in a given range, the speed of the game is decreased; if it falls below a second safety threshold, the game is interrupted to avoid implications to the player’s health.Mixer Module:Mechanics: responsible for changing the speed of the game.Dynamics: generates an easier level or an interruption.Aesthetics: not used.Game: multimodal I Blue It receives and applies the elements of the previous three cores.Storage: game data and ID signals are all stored in a player-separated record chronologically.Interaction ModuleMultimodal I Blue It: interpretation is performed through the components below:The feedback flow is explained below:Game: sends a reply message to the Mixer module’s fission core.Mixer Module: this now triggers the fifth core, fission, which combines the message returned by the game and the player profile, and delivers it to the player through the available and appropriate devices, such as a monitor and a sound box.Action: the computed response is then passed to the selected devices.Perception: the player’s senses capture the response.Interpretation: the player interprets the response given by the game and the interaction cycle repeats until the end of the game execution.Within the Mixer module implemented in the multimodal I Blue It, there is the Combination core. As soon as the signals enter this core, several forms of automatic combination are possible: individual, flexibility/equivalence, assignment, complementarity, and redundancy.Individual: if only one signal arrives , a combination does not occur and the signal exits the same way it entered.Flexibility: if two or more equivalent signals arrive, a combination may occur that allows only one of the signals to go forward.Assignment: With two or more non-equivalent signals , the signal of each of them can be used for different purposes/assignments, i.e., signals come and go independently of the core, each one with a different purpose. Another example of an assignment is when a single ID has more than one signal, such as the Oximeter that generates a heart rate signal (HR) and a blood oxygen saturation (SpO2) signal. The two signals enter the Combination core and the two come out, each with a different assignment.The association of the signals of both is performed, and the resulting signal is the junction of 75% of PITACO’s and 25% of the Pressure Belt’s signals, making PITACO the preferred ID .The association of the signals of both is performed, and the resulting signal is the junction of 25% of PITACO’s and 75% of the Pressure Belt’s signals, making the belt the preferred ID .The negative values of PITACO and the positive values of the Pressure Belt are used.The positive values of PITACO and the negative values of the Pressure Belt are used.Complementarity: If two or more non-equivalent signals arrive at the core, besides being able to record both of them to correlate one to another, a merge may occur that generates only one output signal as a result. As an example, the combination of the PITACO ID and the Pressure Belt ID can be parameterized to occur in the following ways :Redundancy: Finally, if two or more “equal” signals reach the core, a fusion may occur, generating only one resulting signal . The only type of multimodal combination “with signal fusion” applied in multimodal I Blue It is complementarity. The possibility of merging for data redundancy has not been considered.Fission Process for the Multimodal I Blue It SEG: This feedback is triggered via game mechanics as a message and the core divides the message consistently into each type of feedback requested and sends each fragment to appropriate devices. For example, if the message is “The target has been hit”.Visual feedback: the image of the collected target is sent to a visual output device, such as a monitor.Sound feedback: the collected target sound is sent to a sound output device, such as a loudspeaker.To assess the utility of all multimodal functionalities and of the 123-SGR architecture, a questionnaire was sent to 399 professionals who have a health background  or technical background .Twenty-seven (27) professionals answered the questionnaire (6.77%), which had questions regarding the importance, necessity, and practical criteria on a five (5)-point Likert Scale. The questionnaire had five demographic questions; three (3) questions regarding the importance, necessity, and practical issues of the multimodal concept; three (3) questions for each functionality regarding some criteria ; and two (2) open questions on the positives/advantages and negatives/disadvantages of such multimodal approach. The questionnaire was applied remotely (online) and presented nine demonstrative videos to illustrate the IDs and functionalities. The best mean value results for each criterion are highlighted in green and the overall worst result is highlighted in red . From It can be seen from When dividing the objective responses among health see  and techFor health professionals, complementarity is the second most important and necessary feature, perhaps because it represents a general concept of interaction, whose applicability is not shown in practice but is crucial to the professionals in this area. On the other hand, professionals from the technology area considered the general concept of multimodal interaction as the most important feature, perhaps because it is closely linked to computer systems in general and that may have led these professionals to better understand their potential.For health professionals, the complementarity feature is the least practical one, perhaps because it usually means a second device must be worn by the patient, which could be distracting, limiting, or cumbersome.The least important and least necessary functionality for technology professionals is also complementarity, possibly because they do not see how important it is to correlate meaningful signals.According to the mean scores, the professionals of the two groups reached a consensus when assessing that complementarity is the least-practical functionality since it requires synchronization among more than one ID, which can lead to difficulties in control and adaptation and extra costs, and present a hurdle to the player.The greatest standard deviation among the responses of health professionals is related to the practicality of complementarity, possibly because it is subdivided into several areas, such as respiratory rehab, motor rehab, physical educators, speech therapists, etc. The greatest standard deviation among the responses of the professionals in the technology area is related to the practicality of monitoring, which may be due to a more accurate view of the sensor used in the Oximeter ID and its best reading condition, which requires the player’s finger to be strictly still during the game, which can represent difficulty in practice.Dividing the objective responses among professionals “with” and “without” experience in respiratory rehabilitation (RR), all average evaluations were above 3.8, also suggesting a positive agreement on all functionalities. Experienced professionals consider the general concept of multimodal interaction to be the most important, that is, it is important in the application of RR techniques. For the professionals without experience in RR, the most important functionality is flexibility/equivalence, which may reflect that the explanation of functionality in the questionnaire was successful in demonstrating its importance to professionals outside the focus area of the multimodal I Blue It SG.The least-important functionality for experienced professionals is flexibility/equivalence, and monitoring is the most important, which strengthens the need for players to take careful measures during rehabilitation. For professionals without experience in RR, the concept of complementarity is the least important and necessary, which may reflect the lack of experience in RR.In the positive points/advantages question, 11 professionals highlighted monitoring as the most valuable point, which stresses the importance of this functionality and also stresses the need for a multimodal architecture such as 123-SGR. Practicality was also highlighted, even though this was a least-popular issue among the objective questions; this is a situation that exemplifies the disagreement between the professionals as shown by the standard deviation, which could be mitigated if the professionals had obtained the ID in person and did not evaluate it only through demonstrative videos.In summary, there seems to be a consensus that all functionalities are important and necessary, but run into practical issues. There is also an agreement among the professionals on the importance and need for multimodal interactions within the scope of RR.Among the answers to the open questions, one of the professionals noticed the absence of adaptations related to the monitoring of the player’s heart rate, a fact that was described in the questionnaire as information directed only to the therapist’s visualization.As for the worry that the ID can allow air to escape through the mouth, both the PITACO and MANO-BD IDs use filters with proper inlets. The need for a nasal clip to prevent players from breathing through the nose can be a significant contribution, which can be directly incorporated by the therapists who may use the multimodal I Blue It SG.It should be stressed that all IDs are prototype versions and, for this reason, the comfort, design, and proportions may not be the most fit for use. However, they are sufficient to confirm the potential of the architecture and as prototypes that will inspire improved IDs. Another point is that, as specified before, ID validation against medical instruments available in the market is out of the scope of this paper.These comments were all related to the practicalities of using the SG, which proves that professionals can foresee using the multimodal SG in their practice.Multimodal SEGs for rehabilitation require a diversity of inputs that includes movements of limbs, speech, and breathing among others that are captured using unusual devices (other than a mouse and keyboard). Multimodal features such as flexibility/equivalence, and unconventional ways of controlling a game, particularly for some people with disabling pathologies, are the only possible methods of game/therapy access. This can therefore be seen as a limitation that Multimodal Interaction Systems help to solve.The 123-SGR architecture can also serve as a guide, a checklist of elements to be planned and developed. Each population is to be rehabilitated , and each focus of the physical rehabilitation  has its particular characteristics. The 123-SGR architecture allows the SEG to be adjustable because it leaves room for appropriate devices to be chosen, and for combinations of appropriate devices to be used in addition to all parameterization of the adaptation thresholds that are available for a therapist to customize.The flexibility functionality is seen not only as the equivalence functionality (CARE), which selects between two or more modalities. This functionality in 123-SGR also determines that the application can be flexible enough to lose or gain modalities at run time.The complementarity functionality not only allows the fusion of different modalities , but determines that it can be produced in different ways, according to mathematical equations  or by giving parts of the same task to each modality  can also be achieved via the 123-SGR architecture. However, they were not used in the SG for the area of physical rehabilitation, because the assignment  is usually implemented in software in general and does not allow a diversity of possibilities (since it does not perform fusion). A typical example of an assignment that occurs on multiple systems is use of a mouse device, typically used to click buttons/menus and a keyboard device to generate text in writing fields. Redundancy, on the other hand, has been interpreted as a CARE functionality to increase software reliability (higher availability) so that it merges but no information is supplemented. There is no extra information beyond that achieved with individual modalities/devices. For this reason, it did not gain relevance to the present SEG proof of concept.Monitoring is a functionality designed particularly for physical rehabilitation with SEGs because while requiring physical exercise, damage resulting from effort may occur that is not noticeable without the use of physiological sensors. Considered the single most necessary functionality according to the professionals , monitoring can be achieved through any of the four CARE functionalities: via equivalence, if choosing between one device  with the same sensory ability; via assignment, using more than one device, each with a different task; via redundancy, using equal devices with data united contingently (reliability); and via complementarity, in which different devices complement each other to achieve more complete information about the physiological state of the player.The functionalities highlighted in the architecture are representations of CARE 15  functionalities with a specific application in SEGs for physical rehabilitation. However, there are some features not described by the CARE functionalities but present in 123-SGR, such as:All CARE functionalities were achieved using the 123-SGR architecture, both in the conscious and unconscious interaction flows.The parameterization of optimal and acceptable oxygenation rates requires analysis by a therapist. On the other hand, it was noted that low oxygenation saturation always creates negative feedback for the player , while positive feedback could be implemented when rates are at good levels.The merging of input modalities used in the multimodal I Blue It was performed only at the data level. Fusions could also be created at the characteristic level, such as to detect abdominal diaphragmatic breathing, through a complementary fusion of characteristics (such as from PITACO or MANO-BD) plus the Pressure Belt (coupled to the abdomen of the player), as shown in ® engine, producing visual and audible feedback.In the multimodal I Blue It, the fission was managed only via the UnityWhen answering the questionnaire, no controls were used that required the professionals  to start the videos, nor to watch them to the end, which may have influenced the emergence of some doubts/comments already explained in the video. Thus, it would be of great help to use technologies that allow this control. As a counterpoint, the use of remote questionaries allowed us to reach more professionals than in a face-to-face evaluation.In general, the data show that the I Blue It SEG and its new functionalities (including monitoring) have been highly appreciated by professionals from all areas. This finding indicates that multimodal SEGs for physical rehabilitation should be further explored and, for this, an architecture such as 123-SGR can facilitate the design and development of new SEGs.The present I Blue It SEG was evaluated by professionals due to the need to know if such functionalities could somehow improve therapy . The data show a positive response on all accounts. However, patients’ views and feelings must also be evaluated.Traditional physical rehabilitation can become tiring and monotonous. Serious Exergames (SEGs) can help maintain a patient’s interest and achieve effectiveness in treatment. However, current SEGs have not given due attention to three functionalities seen as important for therapists and patients: flexibility/equivalence (more than one control option); complementarity (combining information to be more clear about how rehabilitation exercises are being performed); and monitoring . To achieve these three functionalities, it was observed that several devices need to be worn by the patient.Multimodal Interactive Systems have been dealing with a variety of ways to convey intentional commands to the system, but for SEGs we have demonstrated that intentional as well as unintentional signals are important, necessary, and practical, particularly when applied to Serious Games for Respiratory Rehabilitation (SGR).Architectures were found for the construction of SEGs and Multimodal Interaction Systems; however, none of them aimed to build an SEG with all three mentioned functionalities. A new software architecture was created, the 123-SGR architecture, which allows the creation/adaptation of SEGs with a focus on physical rehabilitation so that they become multimodal SEGs and achieve all functionalities.The 123-SGR architecture was used to adapt an existing SEG towards a multimodal one. Software elements were added and adapted, and new interaction devices (ID) were designed and built  for sensing the respiratory process, which provided multimodal interaction.The multimodal SEG, I Blue It, was evaluated by health and technology professionals. We found that all scores were above the center of the scale and thus all evaluators positively agreed with all features; there are issues with game design and ID usability that must be taken care of; the multimodal I Blue It SEG has potential utility  in respiratory rehabilitation (RR); I Blue It was able to acquire the flexibility/equivalence, complementarity and monitoring functionalities; and; multimodalities are really important and necessary functionalities for a respiratory rehabilitation SEG.The main contribution of this research is an architecture for multimodal SEG systems that is appropriate to the needs of the area of physical rehabilitation; is compliant with, but goes beyond, CARE functionalities; and that includes a flow to meet the monitoring functionality . The 123-SGR architecture emphasizes flexible design aspects of Multimodal Interaction Systems, such as the various ways to combine input information  and these combinations are possible for both conscious and unconscious signals coming from the player. Further, the 123-SGR architecture also emphasizes the relationship of input information with the game elements  of a digital SEG for rehabilitation.Adopting the 123-SGR architecture will facilitate the development of new multimodal SEGs for physical rehabilitation, making them flexible, information-rich, and safe to use. Although tested on an SEG for respiratory rehabilitation, we argue that 123-SGR can be a useful architecture for all SEGs.We expect that 123-SGR would require some coding for every new ID that is added as an input to the SEG. Other multimodal architectures present some high-level description language to describe and facilitate new devices being added, but we found that there is no standard or widely accepted description language for this, and they would also require some sort of coding. We also acknowledge that promoting the adaptations to just one SEG does not allow us to show all features and benefits of 123-SGR, but the chosen SEG was the one that we had free and easy access to.Future work should consider handling all input signals and data to make the resulting SEG a more intelligent one. That is, the core module of 123-SGR architecture—the Mixer module—could incorporate Artificial Intelligence reasoning.In addition, future research should investigate if all extra IDs could become a nuisance to the patient and compromise rehabilitation somehow. We suggest that unconscious flow should be made more evident to patients as a way to better conduct the treatment.


Despite pharmacological advances such as lenvatinib approval, therapeutic failure of hepatocellular carcinoma (HCC) remains a big challenge due to the complexity of its underlying molecular mechanisms. Neuropilin-1 (NRP1) is a co-receptor involved in several cellular processes associated to chemoresistance development. Since both the double-edged process of autophagy and hypoxia-derived response play crucial roles in the loss of therapeutic effectiveness, herein we investigated the interplay among NRP1, autophagy and hypoxia in development of lenvatinib resistance in HCC cell lines. We first analyzed NRP1 expression levels in human HCC samples from public databases, found significantly increased NRP1 expression in human HCC samples as well as its correlation with advanced tumor and metastasis stages. Among 3 HCC cell lines , Hep3B and Huh-7 cells showed significantly increased NRP1 expression levels and cell migration ability together with higher susceptibility to lenvatinib. We demonstrated that NRP1 gene silencing significantly enhanced the anticancer effects of lenvatinib on Hep3B and Huh-7 cells. Furthermore, lenvatinib suppressed NRP1 expression through promoting autophagy in Hep3B and Huh-7 cells; co-treatment with bafilomycin A1 attenuated the antitumor effects of lenvatinib, and NRP1 silencing prevented this loss of in vitro effectiveness of lenvatinib even in the presence of bafilomycin A1. In addition, exposure to a hypoxic microenvironment significantly decreased NRP1 expression through autophagy in Hep3B and Huh-7 cells. Under hypoxia, HIF-1α directly modulated NRP1 expression; HIF-1α silencing not only enhanced the anticancer effects of combined lenvatinib and hypoxia, but also prevented the loss of effectiveness caused by bafilomycin A1, highlighting the potential role of HIF-1α-derived hypoxia response in the adaptive cellular response to lenvatinib and promoting resistance acquisition by autophagy modulation. Overall, NRP1 may constitute a potential therapeutic target to prevent lenvatinib failure derived from a hypoxia-associated modulation of autophagy in advanced HCC. Hepatocellular carcinoma (HCC) is the most frequent type of liver cancer and is placed as the third cause of cancer-related death worldwide, after lung and colorectal cancers . HCC conEven though the tyrosine kinase inhibitor (TKI) sorafenib was the only drug approved against advanced HCC for a decade, lenvatinib antitumor properties have shown an increased median overall survival along with better second outcomes in HCC patients, becoming the first therapeutic alternative to sorafenib . TKIs arNeuropilin-1 (NRP1) is a transmembrane glycoprotein which acts as a co-receptor of semaphorins (SEMAs), axon guidance molecules, and different growth factors involved in key oncogenic signaling pathways , 8. NRP1Hypoxia conditions have been described to be a relevant process in resistance acquisition in HCC , 5, 10. Although numerous processes are involved in the loss of drug sensitivity in HCC , 10, autIn the current study, we evaluated the role of NRP1 on the loss of therapeutic effectiveness of lenvatinib and the modulation exerted by the HIF-1α-mediated response to hypoxia. We determined the potential interest of NRP1 as a molecular target preventing autophagy-associated resistance development in an in vitro model of human HCC complemented with human HCC patient data from available databases.https://www.proteinatlas.org/)  × 100) .™ medium  following the manufacturer’s protocol. At 8 h post-transfection, medium was replaced by complete fresh DMEM-high glucose medium and cells were re-seeded according to the different experiments. After 24 h from transfection, treatments were performed and maintained during 24 h, and the corresponding assays were performed 48 h post-transfection and 24 h post-treatments.For specific gene silencing, cells were initially seeded in 6-well plates and, after 24 h, ON-TARGETplus Human HIF1A siRNA SMARTPool (a mixture of 4 siRNA targeting HIF1A), ON-TARGETplus Human NRP1 siRNA SMARTPool (a mixture of 4 siRNA targeting NRP1) or ON-TARGETplus Non-targeting Control Pool (a negative control pool of 4 siRNA) were introduced into cells using the DharmaFECT 4 Transfection Reagent  and serum-reduced Opti-MEMP-values from human data analysis were provided directly for the corresponding databases, regardless of GEO database and the GSE14520 dataset, in which NRP1 overexpression was analyzed and represented with the software GraphPad Prism 8. Likewise, correlation significance between NRP1 and the above-mentioned genes in human HCC samples, together with the in vitro results , were analyzed with the statistical package GraphPad Prism 8 . For each corresponding experiment, unpaired t-test, one-way or two-way ANOVA followed by Tukey, Dunnett or Sidak post-hoc tests were used. Statistical significance was considered when P < 0.05.The statistical To determine the potential interest of NRP1 in the development and progression of HCC, we analyzed the NRP1 expression levels in different HCC datasets from human databases Fig. . RepreseAssociation of NRP1 expression levels with tumor stages in human HCC was also evaluated, finding a significant increased expression of NRP1 in advanced HCC stages from UALCAN and GEPIA databases Fig. . InteresTo confirm the results obtained from the clinical databases and select a suitable in vitro model, we analyzed the NRP1 expression and the migration ability of different HCC cell lines Fig. , with di50 were selected, being higher for the HepG2 line. As previously observed, lenvatinib was more effective in Hep3B and Huh-7 cells, showing a significant inhibition of colony formation ability and reduction of Ki67 proliferation index with both 2.5 and 5 µM lenvatinib  in Hep3B and Huh-7 after 48 h of lenvatinib treatment Fig. . Interesnib Fig. . Howevernib Fig. .Fig. 2AnConsidering these findings and the close association of NRP1 and lenvatinib with cell migration and angiogenesis in cancer , 29, we In order to elucidate the association between NRP1 and lenvatinib efficacy in the human HCC cell lines, we analyzed the effects of lenvatinib treatment on NRP1 mRNA and protein levels Fig. . Remarka50 was only reached by 50 µM EG after 48 h in the Huh-7 cell line  and protein degradation through proteasome  or autophagy  Fig. . ResultsTherefore, these findings indicated that autophagy may be the mechanism that underlies the NRP1 downregulation exerted by lenvatinib in the HCC cells.P < 0.0001) was reached for all these correlations observed.Autophagy has shown to be a key process in the modulating actions of lenvatinib on NRP1 expression. Considering the double-edged role of autophagy in cancer  and the Based on these findings, we further analyzed the relationship between NRP1, autophagy and lenvatinib efficacy in our in vitro models of human HCC. As previously observed, combination of bafilomycin A1 with lenvatinib partially restored NRP1 protein levels; however, when NRP1 was silenced autophagy blockade did not prevent NRP1 downregulation Fig. . Cell prRegarding cell migration, autophagy inhibition also reduced the lenvatinib inhibitory effects, increasing the wound closure ability of the HCC cells in the presence of the drug Fig. . AlthougAltogether, autophagy-dependent degradation of NRP1 seems to be a crucial mechanism in the loss of lenvatinib efficacy, which could be modulated by HCC cells during drug resistance development in order to avoid antitumor actions of lenvatinib. Therefore, NRP1 could be supposed as an interesting molecular target in human HCC in order to prevent autophagy-related lenvatinib resistance.2 as hypoximimetic . We further performed an in vitro analysis by specifically silencing HIF-1α. Interestingly, we observed that an effective HIF-1α silencing under hypoxia led to a significant downregulation of NRP1 protein levels (Fig. The hypoxic microenvironment has shown to be a relevant mechanism on cancer progression and is mainly modulated by the HIF-1α, which has been closely related to development of drug resistance in HCC , 6. Baseels Fig. . Additioels Fig. . On the els Fig. . We alsoels Fig. .Fig. 7StSince autophagy inhibition could act as a relevant mechanism in the loss of lenvatinib efficacy through NRP1 modulation, as well as in the HIF-1α-associated response to hypoxia, crucial in the adaptive cellular response to chemotherapy, we decided to elucidate the interesting relationship among them in both HCC cell lines. Lenvatinib treatment under hypoxia increased HIF-1α protein expression, while augmented the NRP1 downregulation exerted by hypoxia Fig. . RemarkaAltogether, these results suggest that NRP1 is directly modulated by HIF-1α under hypoxia and that autophagy plays a crucial role in lenvatinib efficacy and cell response to hypoxia through NRP1 modulation. Therefore, not only NRP1, but also HIF-1α, could act as potential targets in order to prevent therapeutic failure overpassing an adaptive cellular response through autophagy modulation.HCC remains a global health problem  and, desAlthough NRP1 expression was firstly identified in the central nervous system, this receptor is expressed in different tissues , being oAutophagy is a widely studied process with an interesting role in tumor progression and chemotherapeutic efficacy . In thisIn order to avoid an autophagy-associated mechanism of adaptation to lenvatinib treatment, we tested the effects of targeting NRP1 when autophagy was blocked. We observed that NRP1 silencing prevented the loss of lenvatinib antitumor actions derived from autophagy inhibition Fig. . LikewisOverall, these results suggest that NRP1 contributes to a higher cell survival and migration-associated abilities of HCC cells, highlighting the crucial modulation exerted by the autophagy-dependent degradation of NRP1 as a potential mechanism associated with the loss of lenvatinib effectiveness.2 as a hypoximimetic to induce a hypoxia-derived response. CoCl2 is an agent that acts by inactivating the enzymes responsible for the degradation of the HIFs and, therefore, promoting the stabilization of these factors [2 has been chosen for inducing a representative hypoxic response for several years, due to the hypoxic chamber conditions are usually different depending on the cell line analyzed. Our results indicated that hypoxia induction markedly reduced protein expression of NRP1 and, as observed by lenvatinib treatment, autophagy was the main mechanism responsible (Fig. 50 for lenvatinib increased under hypoxia and that HCC cells exhibited a lenvatinib resistance phenotype under hypoxia conditions that could be mediated by changes observed in the extracellular matrix, mainly associated to fibronectin [The hypoxia microenvironment has been closely related to the development of resistance mechanisms by tumor cells, including liver cancer cells , 6, 51.  factors . The useble Fig. . In thisronectin . Finallyronectin . These fHIF-1α has been widely associated with resistance acquisition in several solid tumors, including HCC, through cellular adaptation to hypoxia –12. For Evidences of the autophagy and hypoxia interplay have placed them as key mechanisms in therapeutic failure in HCC , 17. TheThis study provides novel findings on the molecular mechanisms associated with NRP1-dependent survival and migration of human HCC cells underlying the loss of lenvatinib effectiveness. Overall, these results displayed the key role of NRP1 in antitumor actions of lenvatinib in human HCC, where autophagy acts as a crucial mechanism involved in cell adaptation to lenvatinib treatment through NRP1 modulation, thus promoting cell resistance development, in which HIF-1α-associated hypoxia response seems to be a potential mediator. Therefore, NRP1 stands as a valuable target in advanced HCC to prevent therapeutic failure through autophagy-related lenvatinib resistance.Supplementary Fig. S1Supplementary Fig. S2Supplementary Fig. S3Supplementary Fig. S4Supplementary Fig. S5Supplementary Fig. S6Supplementary Fig. S7Supplementary Fig. S8Supplementary Fig. S9Supplementary Fig. S10Supplementary Fig. S11Supplementary Fig. S12Supplementary Fig. S13Supplementary Fig. S14Supplementary Fig. S15Supplementary Fig. S16Supplementary Fig. S17Supplementary Fig. S18Supplementary Fig. S19Supplementary figure legends


Enterococcus feacium DB01, Staphylococcus aureus ATCC 13565, Listeria monocytogenes ATCC 33090, Escherichia coli O157:H7 ATCC 23514, Aeromonas hydrophila ATCC 7966, and Pseudomonas aeruginosa ATCC 27583. The SGNPs also exhibited promising tyrosinase inhibition potential, with 32.83% inhibition at 100 μg/mL concentration as compared to 52.4% by Kojic acid, taken as a reference standard compound. The SGNPs also displayed significant photocatalytic degradation effects, with 44.87% methylene blue dye degradation after 5 h of incubation. Moreover, the antibacterial mode of action of the SGNPs was also investigated against E. coli and E. feacium, and the results show that due to the small size of the nanomaterials, they could have adhered to the surface of the bacterial pathogens, and could have released more ions and dispersed in the bacterial cell wall surrounding environment, thereby disrupting the cell membrane and ROS production, and subsequently penetrating the bacterial cells, resulting in lysis or damage to the cell by the process of structural damage to the membrane, oxidative stress, and damage to the DNA and bacterial proteins. The overall outcome of the current investigation concludes the positive effects of the obtained SGNPs and their prospective applications as a natural antibacterial agent in cosmetics, environmental, and foodstuff industries, and for the management of environmental contagion.In recent times, numerous natural materials have been used for the fabrication of gold nanoparticles (AuNPs). Natural resources used for the synthesis of AuNPs are more environment friendly than chemical resources. Sericin is a silk protein that is discarded during the degumming process for obtaining silk. The current research used sericin silk protein waste materials as the reducing agent for the manufacture of gold nanoparticles (SGNPs) by a one-pot green synthesis method. Further, the antibacterial effect and antibacterial mechanism of action, tyrosinase inhibition, and photocatalytic degradation potential of these SGNPs were evaluated. The SGNPs displayed positive antibacterial activity (8.45–9.58 mm zone of inhibition at 50 μg/disc) against all six tested foodborne pathogenic bacteria, namely,  Presently, bacteria in different forms are resistant to existing antibiotics, and the number of antibiotic resistant organisms is rising progressively, which is a severe risk to public health in numerous parts of the world, affecting both developed and developing nations ,2. The cIn the last two decades, the study of nanotechnology has developed as an encouraging field of interdisciplinary study because of its enormous application across a varied field of scientific research . PresentNanoparticles are extensively used, and play a major role in a variety of fields, including biology, medicine, physics, chemistry, and sensing, due to their exclusive potential ,17. ManyAmong the nanoparticles, gold nanoparticles (AuNPs) are one of the most highly important nanoparticles, and have attracted much interest from researchers due to their unique properties, such as small size, ionic neutrality, and precise targeting ,23,24,25In the current times, the environmental pollution caused by the textile, paper, food, and cosmetics industry dyes such as methylene blue and methyl orange is a major concern throughout the world ,33. ThesSericin is a globular protein, usually discarded in the process of manufacturing silk from the silk cocoon by the sericulture and textile industries ,42,43. SE. feacium, S. aureus, L. monocytogenes, E. coli, P. aeruginosa, and A. hydrophila, and the results are displayed in E. coli and P. aeruginosa pathogens completely ceases at the concentrations of 100 μg/mL and 150 μg/mL, respectively. Further, in another study, De Lima Oliveira et al. [S. aureus, and found that the MBC value was 12.5 mg/mL. In a different study, the authors discussed the antibacterial effect of sericin against many pathogenic bacteria, namely, Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis [The antibacterial effectiveness of SGNPs was estimated for six (three Gram-positive and three Gram-negative) bacterial pathogens, namely, a et al.  discussedermidis . Since tE. coli and E. feacium). The outcome is displayed in The antibacterial mode of action effect of SGNPs was evaluated by using two selected pathogens  of sodium chloride. When the NA plates of the SGNP-treated microbes were observed, it was found that at 0% and 2.5% salt content, there is much less of a decline in the bacterial count (cfu) . HoweverE. coli and E. feacium (MIC concentrations) were treated with SGNPs, there was a persistent rise in the concentration of cellular materials absorbed at 260 nm in a spectrophotometer during the period of incubation  to the outer atmosphere because of cell membrane leakage [For the discharge cellular materials (detected at 260 nm), when both cubation . Howevercubation . Specifi leakage . Noticea leakage . Several leakage . The abo leakage . These aE. coli pathogen [Some reports show that the possible mode of antimicrobial action by nanoparticles could occur in several ways, such as inducing oxidative stress in the bacterial cells, releasing the metal ions into the cell membrane, and other non-oxidative actions, and it has been stated that due to the small size of the nanomaterials, they could have adhered to the surface of the pathogens, and could have released more ions and dispersed in the bacterial cell wall surrounding environment, thereby disrupting the cell membrane and ROS production, and subsequently penetrating the bacterial cells, resulting in lysis or damage to the cell by the process of structural damage to the membrane, oxidative stress, and damage to the DNA and bacterial proteins ,3,59. Inpathogen ; in anotpathogen . These fOverall, the possible mode of antibacterial action of the SGNPs can be explained in many different ways. It is usually seen that when the metal ions dissolved in solvents come into contact with a bacterial cell, they become mostly detached from the surrounding environment of the bacterial cell . HoweverThe results of tyrosinase enzyme inhibition are displayed in Melanin is a factor in skin coloration, and is produced by the melanogenesis pathway in the melanocyte cells situated in the lower epidermis . MelaninThere is remarkable interest among the cosmetic industries in the formulation of natural products with anti-tyrosinase activity for the manufacture of a low-cost, herbal, and highly effective anti-tyrosinase product for bleaching off exceedingly pigmented lesions . TherefoThe SGNPs facilitated the photocatalytic degradation of dyes (methylene blue and methyl orange) with light exposure. The results depict that with an increase in the light exposure time, the OD values declined for MB in the presence of SGNPs . At diveThe current results specify that the cationic dyes (methylene blue and methyl orange) demonstrated a decrease in the absorbance with the increase in the incubation time; this suggests the selective interaction of the cationic dyes with the SGNPs that facilitates their degradation in the reaction solution. As reported by Singh et al., the elimination of dyes from the solution is affected by several aspects, such as electrostatic interaction, pore filling, or hydrogen bonding, and the variation of the surface charge is influenced by the pH of the solution . Thus, iEnterococcus feacium DB01, Staphylococcus aureus ATCC 13565, Listeria monocytogenes ATCC 33090, Escherichia coli O157:H7 ATCC 23514, Aeromonas hydrophila ATCC 7966, and Pseudomonas aeruginosa ATCC 27583.The extraction, purification, and synthesis of sericin-based gold nanoparticles (SGNPs), and synthesized SGNP characterization is detailed in our earlier published research . A schem8 cfu/mL (0.5 McFarland standard). The disc diffusion assay was used for the antibacterial assay. A paper disc of 50 μg SGNPs/disc was prepared for the disc diffusion assay. On the prepared agar plates, the freshly subcultured pathogens were spread evenly. Next, the filter paper disc with SGNPs was placed onto the agar plates and stored for 24 h at 37 °C for incubation. After the incubation time, the inhibition consequence of SGNPs was measured by the zone of inhibition diameter. For the antibacterial assay, we have used the standard antibiotic cephalexin as the positive control and the 5% DMOS as the negative control. The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) were also determined as per previously standardized two-fold serial dilution and plate count methods [The antibacterial effect of SGNPs was assessed against both Gram-positive and Gram-negative pathogenic bacteria, as earlier described . The bac methods .E. coli and E. feacium, were selected from among the six tested pathogens due to the better inhibition activity of the SGNPs. Initially, the pathogenic bacterial culture was used at the MIC concentration. The bacterial culture without any treatment was taken as the control. The culture solution was 10 mL (107 cfu/mL), and the nanoparticles were kept for 10 h at 37 °C for incubation. The bacterial colony was counted as cfu/mL.The antibacterial mode of action of SGNPs on the tested Gram-positive and Gram-negative microbes was determined following the time–kill assay, as discussed in our previous study . For theThe salt tolerance potential of the investigated microbes, used to find out the capability of the microbes to osmoregulate, was investigated by following the standard procedure . The pat3 rpm for 10 min), followed by the recording of the optical density (OD) at 260 nm at constant time intervals. In this experiment, peptone water mixed with microbes and SGNPs, separately, was taken as the negative and positive control, respectively.The release of cellular materials from the microbial cells was determined using standard procedures . Before The evaluation of the tyrosinase inhibition effect of SGNPs was conducted by a spectrophotometric technique . ConcisesC” is the OD of control and “sT” is the OD of the test.“The photocatalytic degradation effect of SGNPs was evaluated on methyl orange (MO) dye and methylene blue (MB) dye by following the standard method described by ,82 with p < 0.05 using SPSS statistical software (version 27.0).The experimental outcomes are detailed as the mean with SD, and were analyzed via one-way ANOVA and Duncan’s multiple range test at There are many challenges faced by the people of the world due to the harmful effects of different types of microbial infections and other related diseases concerning pathogenic microbes. Despite the availability of several advanced techniques and medications, it sometimes becomes difficult to tackle these pathogenic bacteria due to their multiresistant nature. The unique features of the nanomaterials make them an ideal candidate to apply in treatment against these multiresistant pathogenic microbes and eradicate them. The small particle sizes and shapes of the nanomaterials, along with their charged surfaces, make the nanoparticles an ideal path by which to enter the cell membrane of the pathogenic bacteria, thereby causing cellular lysis in many ways, including the rupture of the cell membrane and destruction of the DNA and protein, and also initiating apoptosis. Additionally, nano-based antibiotic treatment is now being considered as one of the most advanced systematic approaches to tackle harmful microbial pathogenicity. In the present research, the SGNPs exhibited promising antibacterial activity, along with the tyrosinase inhibition photocatalytic degradation potentials. As a consequence of their multifunctional perspective, they could be explored for their future applications as an antibacterial agent, in cosmetics trades as sunscreen, and in the food sector as a coating/film material in the packaging of food, owing to its antibacterial and tyrosinase inhibitory abilities. Furthermore, it can be used in wastewater contamination management of textile dyes. However, a more detailed investigation of the mode of antibacterial action is needed under various conditions and against more pathogenic microbes, including fungi and viruses. Next, detailed studies on the application of the SGNPs in multiple dye degradation scenarios and their kinetics are needed for their potential future applications in the waste toxic dye treatment analysis.