Rationale: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by excessive immune response usually due to lung inflammation. Local immunosuppression is crucial for effective ARDS treatment. However, current methods are limited in their ability to target the lungs specifically.Methods: This study utilized lung-targeted lipid nanoparticles (LNPs) with 1,2-dioleoyl-3-trimethylammonium-propane (termed DOTAP-LNPs) to encapsulate chemically modified soluble programmed death ligand-1 (sPD-L1) mRNA and examined its physiological characteristics and therapeutic efficacy. A comparative analysis was performed between sPD-L1 mRNA delivered by DOTAP-LNPs, sPD-L1 mRNA delivered by regular LNPs (MC3-LNPs), and PD-L1-Fc recombinant protein administered systemically. Additionally, the survival rate of ARDS mice treated with different drugs was assessed.Results: Administration of sPD-L1 mRNA-LNPs to ARDS model mice significantly reduced leukocyte chemotaxis and protein accumulation in lung tissue, along with a decrease in pulmonary edema. Notably, in situ intervention using sPD-L1 mRNA-DOTAP-LNPs exhibited superior therapeutic effects compared to PD-L1-Fc recombinant protein and sPD-L1 mRNA encapsulated in MC3-LNPs. Importantly, treatment with sPD-L1 mRNA-DOTAP-LNPs improved the survival rate of ARDS model mice.Conclusion: This study demonstrates the feasibility of utilizing stable and reliable mRNA to express the immunosuppressive molecule sPD-L1 specifically in the lungs. The findings provide proof of concept for localized nanoparticle delivery and offer a novel therapeutic strategy for treating acute inflammation in ARDS. Acute respiratory distress syndrome (ARDS) is a severe respiratory disorder characterized by the presence of non-cardiogenic pulmonary edema, bilateral chest radiographic opacities, and severe hypoxemia with the etiology including pulmonary and extrapulmonary factors such as bacterial and viral pneumonia, aspiration, lung contusion, and severe systemic infections Recent studies have highlighted the crucial role of chemotaxis and activation of pro-inflammatory immune cells in the development of ARDS-associated lung injury in vitro transcribed (IVT) mRNA therapy has emerged as a promising protein replacement approach, garnering significant attention. This innovative method incorporates crucial structural elements like the 5' cap, poly(A) tail, and especially various chemical modifications, endowing it with remarkable advantages, including rapid preparation, low immunogenicity, and high expression efficiency Protein therapy is accompanied by limitations, such as high cost, short half-life, and the potential for antibody formation against exogenous proteins. However, in vivo delivery of mRNA and facilitates well expression. While achieving mRNA delivery, selective organ targeting (SORT) delivery platform implement specific delivery to lung tissue Due to the large molecular weight, negative charge, instability, and susceptibility to degradation by nucleases, mRNA molecules pose challenges for therapeutic applications. The use of lipid nanoparticles (LNPs) delivery platforms ensures stable In this study, we established a potent platform for tailored inflammation control via SORT LNP-based lung-targeted delivery of chemically modified sPD-L1 mRNA, which could be a promising strategy for ARDS treatment.2 concentration of 5%.The human embryonic kidney cell line (HEK293T) and mouse hepatic cell line (AML12) were utilized to assess the expression level of sPD-L1. The A549 human lung cancer cell line is utilized for investigating the co-localization of lysosomes and mRNA-LNPs uptake. All cells were obtained from the American Type Culture Collection (ATCC). AML12 cells were cultured in Dulbecco's modified eagle medium/nutrient mixture F-12 , supplemented with 0.45% insulin-transferrin-selenium  and 10% fetal bovine serum . HEK293T cells were maintained in high-glucose DMEM  and 10% FBS. A549 cells were cultured in RPMI 1640  and 10% FBS. All cells were incubated at 37 °C in a humidified environment with a COThe Ai6 (RCL-ZsGreen) mice were obtained from The Jackson Laboratory. 8-week-old male BALB/c and C57BL/6 mice were also utilized. All animal experiments were strictly conducted under pathogen-free conditions at the animal facility of the Shanghai Jiao Tong University School of Medicine. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committees of the School of Medicine, Shanghai Jiao Tong University.in vitro, a one-step method was employed. During PCR amplification with plasmid templates, poly(A) tails were added through primer extension. Following purification, the dsDNA products served as mRNA template fragments. The IVT mixture contained a DNA template, EZ-cap (B8177), ATP (K1043), CTP (K1045), or a chemically modified version, 5-methyl-CTP (B7967), GTP (K1044), UTP (K1048), or a chemically modified version, pseudo-UTP (B7972)/5-methoxy-UTP (B8061)/N1-methylpseudo-UTP (B8049), and T7 RNA Enzyme Mix (K1083). All these ingredients are purchased from APE×BIO. After incubating the mixture at 37 °C for 3 hours, the template was digested using DNase I . Subsequently, purification was performed using the RNA Clean and Concentrator Kit . Then, cellulose purification was conducted following a previously established protocol to eliminate dsRNA To synthesize sPD-L1/Luc/Cre mRNA The LNP formulations were prepared according to previously described methods The Cryo-TEM sample preparation and imaging procedures were conducted in the following manner: Firstly, a volume of 5 μL of the sample was meticulously applied onto a glow-discharged grid . Subsequently, the grids were subjected to blotting for a duration of 4 seconds under 100% humidity at a temperature of 4 °C. Without delay, the grids were rapidly submerged into liquid ethane by employing a Mark IV vitrobot (ThermoFisher). The imaging process was performed utilizing a Talos Glacios transmission electron microscope (ThermoFisher). The magnification level was set to 92,000×, with a pixel size of 1.57 Å.To assess the expression efficiency of mRNA, cells were seeded in a 6-well plate until reaching 50%-70% confluency. Subsequently, transfection was performed using 2 μg of sPD-L1 mRNA in 4 μL of Lipofectamine 2000 , or 2 μg of sPD-L1 mRNA-LNPs.Cells were lysed in mammalian cell lysis buffer (MCLB) containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, and supplemented with EDTA-free protease inhibitor cocktail  and 1 mM phenylmethyl sulfonyl fluoride . Protein concentrations were measured using the Bradford assay . Then, the protein was boiled in 5×sodium dodecyl sulfate sample buffer. The protein from each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Millipore). The membranes were blocked and incubated with primary antibodies (diluted 1:1000) in primary antibody dilution buffer (Beyotime Biotechnology). The primary antibodies used were anti-HA-Tag rabbit mAb , anti-GAPDH mAb , and anti-PD-L1 mAb . The membranes were then incubated with goat anti-rabbit IgG (H+L) secondary antibody  or goat anti-mouse IgG (H+L) secondary antibody . Images were acquired using the ChampChemi imaging system (Sage Creation Science).The cells were cultured until they reached 50%-70% confluence and then transfected with sPD-L1-HA mRNA. After 24 hours of transfection, the supernatant from the transfected cells was collected into a 15 mL centrifuge tube after passing through a 0.45-μm filter. To ensure sample integrity, an EDTA-free protease inhibitor cocktail  was added. Subsequently, HA magnetic beads  were added to the supernatant and incubated overnight at 4 °C with gentle rotation. On the following day, the immunocomplexes that precipitated onto the magnetic SampleRack were washed four times with ice cold MCLB. Finally, the samples were subjected to western blot for further analysis.in vitro transcription of sPD-L1 mRNA, 25% Cy3-UTP  was incorporated. Subsequently, the fluorescent mRNA was encapsulated within DOTAP-LNPs. After 18 hours of LNPs transfection in cells, LysoTracker Green DND-26  was added and incubated for 30 minutes. Live cell imaging was then performed using a Leica DMi8 S Live cell microscope.During the mRNA-LNPs (0.2 mg/kg) or recombinant mouse PD-L1-Fc chimera protein (0.8 mg/kg)  was administered via the tail vein. Blood samples were collected from the orbit at different time points using White's buffer (1:9) as an anticoagulant, and immediately centrifuged at 4,000 rpm for 10 minutes. The serum samples were then frozen at -80 °C. For detecting the abundance of PD-L1 protein in lung tissue, mouse lung tissue was collected at different time points after injection, placed in radioimmunoprecipitation assay lysis buffer , frozen, ground, and then centrifuged to obtain protein. The protein abundance was analyzed using western blot.To investigate the biodistribution of LNPs in mice, we administered mRNA-MC3-LNPs or mRNA-DOTAP-LNPs expressing firefly luciferase (Luc) through the tail vein of BALB/c mice. At 6, 24, and 48 hours after LNPs injection, the mice were intraperitoneally injected with 150 mg/kg D-luciferin . Ten minutes after the luciferin injection, IVIS Spectrum CT system  were used to image the mice. We quantified luminescence values using Living Image software (PerkinElmer).The nebulization delivery of mRNA-LNPs was carried out using the MicroSprayer Aerosolizer Model YAN 30012 (Yuyanbio) for targeted lung delivery. Each device was equipped with mounting quantitative columns, enabling precise measurement of 50 µL of the aerosolized solution. The aerosol droplets had a particle size of 30-50 μm. During the experimental procedure, mice were anesthetized with 4% isoflurane to ensure immobilization. The tongue was gently depressed using a tongue depressor to visualize the pharynx and facilitate the identification of the tracheal entrance. Subsequently, the nebulizer catheter was carefully inserted, and 50 µL of the aerosolized solution was rapidly delivered into the airway. To evaluate the nebulization efficiency, we conducted pre- and post-nebulization analysis of the mRNA encapsulated in LNPs using the Quant-iT RiboGreen RNA Assay . By comparing the measurements before and after nebulization, we accurately calculated the nebulization loss.The Cre mRNA-DOTAP-LNPs were prepared as described above and administered via intravenous (i.v.) injections at a dosage of 0.8 mg/kg. After 2 days, the mice were euthanized, and their lung, liver, and spleen tissues were collected for flow cytometry analysis. Additionally, the left lung tissue was collected specifically for DAPI staining of freezing sections.After euthanizing the mice, the lung tissue was immersed in 1640 medium, while the spleen and liver tissues were placed in high-glucose DMEM medium. For lung cell extraction, the lung tissue was minced and subjected to a 30-minute digestion at 37 °C, using type I collagenase  and DNase I . The digested tissue was then grinded and filtered through a 70-mesh nylon mesh to obtain a single-cell suspension. For spleen cell extraction, the minced spleen tissue was directly grinded and filtered through a 70-mesh nylon mesh. For liver cell extraction, the liver tissue was transferred into the type II collagenase buffer . It was then digested at 37 °C for 10 minutes and filtered through a 70 μm mesh filter  into a new tube.The collected tissue single-cell suspensions were treated with red blood cell lysis buffer after centrifuging . Following PBS resuspension, the cells were incubated with FVS780 viability dye  for 15 minutes. After one wash with Fluorescence-Activated Cell Sorting buffer , CD16/32 Fc block  was added and incubated for 20 minutes. Subsequently, flow cytometry antibodies were added and incubated at 4 °C for 30 minutes. After another wash with FACS buffer, the samples were analyzed using flow cytometry (Beckman Coulter CytoFLEX LX). The lung tissue was stained with PerCP/Cy5.5-CD45 antibody , BV421-F4/80 antibody , PE-CD31 antibody , and APC-CD326 antibody . The gating strategy and data analysis were performed using CytExpert software, as illustrated in The lung tissue was fixed by freezing it in optimal cutting temperature (OCT) compound , and thin sections were sliced and mounted onto glass slides. The frozen slices were then allowed to warm up to room temperature, and the drawing circles were organized. The slides were washed three times by shaking them in PBS. Subsequently, DAPI dye  was added and incubated at room temperature for 10 minutes, protected from light. The slides were then washed again. Finally, anti-fluorescence quenching sealing tablets  were applied to the slides to complete the staining process. The sections were imaged using the confocal microscopy (Leica STELLARIS 8 FALCON FLIM Microscope). Due to the pronounced green autofluorescence in lung tissue, we employed the fluorescence lifetime imaging module of the microscope to record the green fluorescence signal with a lifetime of 2.6 ns, aiming to eliminate the interference caused by autofluorescence.Serum samples were diluted 1/300 with reagent diluent and tested using the Mouse PD-L1 DuoSet ELISA kit  following the manufacturer's protocol. OD450 and OD570 were measured using a microplate reader . The standard sample data were recorded in Prism and fitted with a 4PL curve (x represents the concentration) to calculate the sample concentration based on the curve.Pseudomonas aeruginosa (PAO) was cultured in Luria-Bertani medium supplemented with spectinomycin (1:1000) and shaken for 12 hours at 37 °C and 220 rpm. The bacterial solution was then resuspended in PBS and adjusted to contain 2×106 bacterial colony-forming units (CFU) after centrifugation at 3000 rpm for 20 minutes. An intraperitoneal injection of 4% chloral hydrate (0.1 mg/10g) was used to anesthetize the mice and fix them on the surgical plate. Endotracheal intubation was performed using a guide wire, and 40 μL of the PAO bacterial solution was instilled into the lungs.Monoclonal To euthanize the mice, the cervical spine was severed, and the chest was opened to expose the lungs. The right lung was ligated, and the left lung was flushed with PBS to collect the bronchoalveolar lavage fluid (BALF). The BALF was centrifuged at 500×g for 5 minutes, and the supernatant was separated from the cell pellet. The cell pellet was then resuspended in red blood cell lysis buffer and subsequently resuspended in 0.2 mL of PBS for the BALF white blood cell count using a cell counter (JIMBIO). The protein concentration of the BALF supernatant was measured using the Bradford method, and the absorbance was read using a microplate reader . The lower lobe of the right lung was minced to obtain a single-cell suspension for flow cytometry, while the upper lobe was used for pathological sections or wet-to-dry weight ratio assay. For pathological sections, the lung tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned and stained with hematoxylin and eosin (H&E). The freshly obtained right upper lobe of the mouse lung was weighed immediately and recorded as the wet weight. The sample was then placed in a 50 °C oven and dried for 48 hours before being weighed again and recorded as the dry weight. The wet-to-dry weight ratio was used to measure the degree of lung tissue edema.The cell suspension of was collected as mentioned above. The cell pellet was dyed with FVS570  and resuspended in FACS, added with CD16/32 , and incubated at 4 °C for 15 minutes. Then we performed flow cytometry staining using BV421-CD279 antibody  and APC-CD45 antibody . We analyzed the cells using the Beckman CytoFlex S machine.To ensure a comprehensive evaluation of the severity of histological lung injuries, we adopted a systematic approach by randomly selecting two specific fields of view from each hematoxylin and eosin (HE)-stained histological section. Subsequently, these selected fields underwent a previously reported semi-quantitative assessment Table  34.Seventy-five C57BL/6 mice were divided into five groups, and four groups underwent ARDS modeling through intratracheal instillation of PAO. Four hours later, the groups were separately intravenously injected with the following agents in equal volumes: PBS, 0.8 mg/kg PD-L1-Fc, 0.2 mg/kg of sPD-L1 mRNA-MC3-LNPs, and 0.2 mg/kg of sPD-L1 mRNA-DOTAP-LNPs. The survival rates of the mice were monitored daily for a 7-day period after treatment with the respective agents.After establishing the PAO-induced ARDS mouse model, mice were administered tail vein injections of either PBS or sPD-L1 mRNA-DOTAP-LNPs (0.2 mg/kg) at 4 hours post-PAO. After a 12-hour interval, BALF was collected from the mice and diluted before being plated on agar plates. Subsequently, the agar plates were incubated at 37 °C for one day to facilitate the assessment of bacterial colony counts.Twelve mice were divided into four groups to receive different treatments via tail vein injection as follows: PBS, PD-L1-Fc (0.8 mg/kg), sPD-L1 mRNA-MC3-LNPs (0.2 mg/kg), and sPD-L1 mRNA-DOTAP-LNPs (0.2 mg/kg). After a 3-day period, the mice were euthanized, and their spleen tissues were immersed in 1640 culture medium, while the thymus tissues were immersed in high-glucose DMEM. The spleen tissues were processed to obtain cell suspensions, following the previously described method. Thymus cell extraction involved mincing the thymus tissues and treating them with type IV collagenase  and DNase I , followed by digestion at 37 °C for 20 minutes. Subsequently, single-cell suspensions were obtained by passing the thymus cells through a 70-mesh nylon mesh.The obtained cell suspensions were then treated with red blood cell lysis buffer and dyed with FVS510 viability dye  for 15 minutes. To prevent non-specific binding, CD16/32 Fc block  was used for blocking. Following that, the cell suspensions were incubated at 4 °C for 30 minutes with the following flow cytometry antibodies: APC-CD86 antibody , PerCP/Cy5.5-MHC II antibody , APC/Cy7-CD11c antibody , FITC-CD4 , PE/Cy7-CD8a antibody , and PE-CD25 antibody . After another wash with FACS buffer, the samples were analyzed using flow cytometry (Beckman Coulter CytoFLEX LX). The gating strategy and data analysis were performed using CytExpert software, as illustrated in All statistical analyses and graphs were performed using GraphPad Prism 8 software, and two-sided t-tests were used. The error bars represent the standard error of the mean (SEM), unless otherwise specified as the standard deviation (SD). Statistical significance was set at p < 0.05, and the level of significance in figures is represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.in vitro transcribed (IVT) mRNA, we conducted western blot assay to assess the expression efficiency of HA tagged sPD-L1 mRNA using five different chemical nucleotide modifications in HEK293T cells and a mouse liver cell line AML12. The five modifications tested were as follows: unmodified (unmod), 5-methoxyuridine (mo5U), 5-methylcytosine and pseudouridine (m5C/ψ), pseudouridine (ψ), and N1-methylpseudouridine (m1ψ). Our analysis revealed the presence of a 40 kDa precursor protein in the whole cell lysate, and a 50 kDa protein in the supernatant  exhibited the highest abundance in the cell culture medium . To determine the most efficient chemical modification of in situ expression with systemic expression of sPD-L1, we encapsulated sPD-L1 mRNA in both types of LNPs  technology, which facilitates precise engineering of nanoparticles for targeted mRNA delivery To assess the physicochemical properties of the LNPs, we conducted measurements of the encapsulation efficiency, size, and zeta potential of both types of mRNA-LNPs. The LNPs were subjected to morphological analysis using transmission electron microscopy (TEM), which provided insight into their distinct shapes and sizes Figure F. Determin vivo, Balb/c mice were administered luciferase mRNA, encapsulated within both types of LNPs, via intravenous (i.v.) injection at a dosage of 0.4 mg/kg. The expression of luciferase protein was assessed at various time points  after intraperitoneal administration of the substrate Luciferin, which generated a robust bioluminescent signal corresponding to the expression level. As anticipated, delivery of luciferase mRNA via mRNA-DOTAP-LNPs predominantly resulted in expression in the lungs, while mRNA-MC3-LNPs facilitated significant expression in the liver  of mouse immunoglobulin G1 (IgG1). The fusion of Fc segment can reduce complement activation effects and increase the protein's half-life to prolong circulation time in situ expression of sPD-L1 facilitated by mRNA-DOTAP-LNPs resulted in a 6-fold higher expression compared to endogenous PD-L1  to mice via intratracheal instillation  to induce symptoms similar to those observed in human bacterial infection-induced ARDS. To assess the severity and acute response of lung injury, we measured white blood cell (WBC) counts and protein concentration in the bronchoalveolar lavage fluid (BALF), as well as evaluated the wet/dry weight (indicative of edema) of the mouse lung at different time points   Figure .To assess the therapeutic effectiveness of sPD-L1 mRNA-MC3-LNPs, we administered i.v. injections of PBS, PD-L1-Fc protein (0.8 mg/kg), Luc mRNA-MC3-LNPs (0.2 mg/kg), and sPD-L1 mRNA-MC3-LNPs (0.2 mg/kg) four hours after intratracheally administering PAO to induce ARDS. At the 12-hour time point, we measured crucial inflammatory indicators, including WBC and protein concentration in the BALF. This evaluation enabled us to evaluate the efficacy of sPD-L1 mRNA-MC3-LNPs during the peak of the inflammation response observed in prior experiments Figure A. RemarkIn subsequent experiments, we investigated the efficacy of lung-targeted delivery of sPD-L1 mRNA using mRNA-DOTAP-LNPs in attenuating acute immune response. ARDS mice were intravenously administered with the same dosage (0.2 mg/kg) of either sPD-L1 mRNA-MC3-LNPs or mRNA-DOTAP-LNPs. We measured various inflammatory indicators, including WBC counts, protein concentration in BALF, wet/dry weight ratio, and levels of inflammatory cytokines Figure A-D. AddiNext, we evaluated the therapeutic effect of sPD-L1 mRNA-DOTAP-LNPs (0.2 mg/kg) on ARDS, comparing it with PBS, PD-L1-Fc recombinant protein (0.8 mg/kg), and Luc mRNA-DOTAP-LNPs (0.2 mg/kg). In comparison to all these controls, the administration of sPD-L1 mRNA-DOTAP-LNPs demonstrated significant reductions in protein accumulation, WBC recruitment, edema, cytokine levels, and lung pathology Figure B. Based To confirm the specific immunosuppressive effects of sPD-L1 mRNA-DOTAP-LNPs in lung tissue, we conducted a comprehensive examination to validate the specific immunosuppressive effects of sPD-L1 mRNA-DOTAP-LNPs in the lungs while minimizing the impact on immune cells in other tissues. We analyzed changes in immune cell populations in the thymus and spleen of mice injected with a dosage of 0.2 mg/kg sPD-L1 mRNA-DOTAP-LNPs. Compared to the control group, the intervention with sPD-L1 mRNA-DOTAP-LNPs did not significantly affect the proportions of immune cells in the spleen and thymus. However, after the administration of 0.8 mg/kg PD-L1-Fc, dendritic cells in the spleen increased by approximately 2-fold, and after the injection of 0.2 mg/kg sPD-L1 mRNA-MC3-LNPs, a substantial number of macrophages were detected in the spleen. This result suggests that localized immune suppression by sPD-L1 mRNA-DOTAP-LNPs can reduce the impact on immune cells in other tissues in comparison to such effects of PD-L1-Fc protein and sPD-L1 mRNA-MC3-LNPs administration Figure .in situ pulmonary expression of sPD-L1 can effectively protect mice from death induced by acute inflammation.To confirm the impact of sPD-L1 intervention on the survival period of ARDS-modeled mice, we monitored the effect of a single dose of PD-L1-Fc (0.8 mg/kg), sPD-L1 mRNA-MC3-LNPs (0.2 mg/kg), and sPD-L1 mRNA-DOTAP-LNPs (0.2 mg/kg) on the 7-day survival rates of the ARDS mice Figure L. The PB in vivo safety profile of mRNA-DOTAP-LNPs by measuring changes in lung tissue bacterial colony-forming units (CFU) compare to the PBS-treated group in PAO-induced mice after administering sPD-L1 mRNA-DOTAP-LNPs (0.2 mg/kg). Notably, our findings indicated that the in-situ expression of sPD-L1 did not hinder bacterial clearance in lung tissue  mRNA delivered using DOTAP-added lipid nanoparticles (LNPs) can be expressed by lung tissue specifically, achieving In our study, we opted to express soluble PD-L1 (sPD-L1) to inhibit excessive immune responses, thereby highlighting the robust immunoregulatory potential of sPD-L1. This soluble form retains the extracellular domain of PD-L1 while lacking the transmembrane and intracellular domains. Here we reported that sPD-L1 can ameliorate immune cell accumulation, decrease inflammatory cytokine secretion, and even mitigate tissue swelling, indicating its potential as a therapeutic target. Furthermore, in a previous observational clinical study, we observed that low levels of serum sPD-L1 were associated with more severe disease and predicted a worse prognosis in patients with ARDS In addition to sPD-L1, numerous other immune inhibitory factors have been documented in vivo half-life and therapeutic effects of sPD-L1 mRNA-LNPs with a commercially available PD-L1-Fc recombinant protein in a murine model of ARDS. Our findings revealed that the mRNA-LNPs strategy exhibited superior pharmacokinetic characteristics and demonstrated a comparable half-life to PD-L1-Fc at lower doses. Importantly, it should be noted that while both protein therapy and mRNA-LNPs demonstrated therapeutic efficacy, the treatment with sPD-L1 mRNA-DOTAP-LNPs showed superior effectiveness in prolonging the survival of ARDS mice. These findings highlight the potential of mRNA-based therapy as a promising approach for the treatment of ARDS, offering a more robust and durable therapeutic effect compared to traditional protein-based therapies. Further research is warranted to explore and optimize the therapeutic potential of mRNA-based therapies in diverse disease contexts.Protein therapy has emerged as a valuable approach in the treatment of various diseases, with more than 100 protein and peptide-based therapeutic drugs currently approved for clinical use. However, the production of therapeutic proteins typically involves costly chemical synthesis or recombinant cell production methods. Fluctuations in external or internal factors such as temperature, pH, and chemical environment can lead to protein denaturation, aggregation, and precipitation, which may trigger immune responses in patients Western blot assay was performed to confirm the protein expression of the modified mRNA and we detected a 40 kDa precursor protein in the whole cell lysate, and a 50 kDa protein in the supernatant. Previous reports showed that the non-glycosylated form of PD-L1 is about 33 kDa, while the immunoblot band is 50 kDa after glycosylation is complete The development of tissue-specific delivery systems, like SORT LNPs, holds great promise for achieving localized immune suppression, avoiding disruptions to global immune balance while reducing excessive inflammatory responses. In this study, both systemic and lung tissue-specific immunosuppression have demonstrated promising inhibitory effects in treating ARDS, yet they entail distinct therapeutic strategies and considerations. Upon administering PD-L1-Fc and sPD-L1 mRNA-MC3-LNPs to mice, we observed changes in the proportions of immune cell subpopulations in the spleen. PD-L1-Fc led to an increase in regulatory T cells, while sPD-L1 mRNA-MC3-LNPs resulted in an elevated population of macrophages. Notably, lung-targeted sPD-L1 mRNA-DOTAP-LNPs administration successfully avoided these undesired changes in immune cell subpopulations. This feature underscores a key advantage of local immune suppression: its ability to preserve overall immune function. In contrast, systemic immune suppression often leads to a broad inhibition of immune system functions, including defense against pathogens and clearance of tumors. Our findings highlight the importance of optimizing delivery strategies to maximize the therapeutic potential of tissue-specific immune modulation.In addition to our exploration of lung tissue-specific delivery achieved through SORT LNPs, we also tested aerosol inhalation administration. However, we observed approximately 100-fold lower expression levels in lung tissue after 6 hours when compared to the intravenous administration of DOTAP-LNPs. Furthermore, the aerosolization process resulted in a 30% loss of LNPs. These limitations prompted us to refrain from further assessing the feasibility of aerosol inhalation as a potential therapeutic approach. The nebulizer we utilized is designed with an extended catheter inserted into the trachea of mice, thereby facilitating the immediate administration of the aerosolized solution and efficaciously delivering nebulized droplets directly to the lungs. This method indeed differentiates from the conventional human aerosol inhalation approach. We hold the perspective that the potential adoption of a direct nebulizer equipped with a nose mask to replicate human inhalation patterns could potentially offer a more advantageous method for aerosol administration. However, we acknowledge that this approach necessitates rigorous further evaluation and consideration in the times ahead.We observed that sPD-L1 mRNA-LNPs demonstrated therapeutic effects on protein concentration, leukocyte count, and lung edema in BALF, consistent with our previous research findings. In our previous study, sPD-L1 was shown to induce apoptosis in monocyte-derived macrophages (MDMs) Certainly, there is still significant work ahead before clinical translation can be achieved. Although we conducted histopathological analysis to assess the safety of sPD-L1 mRNA-DOTAP-LNPs in lungs and other vital tissues of mice, the safety of the drugs needs further assessed. Potential questions may include cell membrane damage caused by the cationic lipid DOTAP present in the LNP formulation and potential inhibition of the lung tissue's antibacterial properties due to sPD-L1 expression. To address the latter concern, exploring whether combination therapies involving antibiotics or antiviral agents could enhance the therapeutic efficacy is necessary. In fact, ARDS patients may receive various medications during the treatment process, including antibiotics for the prevention or treatment of potential bacterial infections, diuretics, vasopressors, and corticosteroids to maintain hemodynamic stability and fluid balance In conclusion, the significant potential of mRNA therapy has been demonstrated in this study, providing valuable insights into the effective application of tissue-specific targeted SORT LNPs-mediated mRNA therapy for localized immune suppression, opening up new avenues for the development of improved treatment strategies for ARDS and potentially other immune-related diseases.Supplementary figures.Click here for additional data file.


Morphologically, diffuse cytoplasmic inclusions were significantly more prevalent in TDP-43 n/c-ir neurons than in c-ir neurons, while skein-like and round inclusions were less prevalent in n/c-ir neurons. The cytoplasmic inclusions of TDP-43 n/c-ir neurons were phosphorylated but poorly ubiquitylated when compared with those of c-ir neurons. TDP-43 n/c-ir neurons became less dominant than the c-ir neurons among cases with a prolonged disease duration. The expression level of nuclear TDP-43 was significantly lower in n/c-ir neurons than in normal neurons without cytoplasmic inclusions. Our results indicate that the maturation of cytoplasmic TDP-43 inclusions correlates with the depletion of nuclear TDP-43 in each affected neuron. This finding supports the view that an imbalance between nuclear and cytoplasmic TDP-43 may be an essential pathway to TDP-43 pathology.The transactive response DNA-binding protein of 43 kDa (TDP-43) is a pathological protein of amyotrophic lateral sclerosis (ALS). TDP-43 pathology is characterized by a combination of the cytoplasmic aggregation and nuclear clearance of this protein. However, the mechanisms underlying TDP-43 pathology have not been fully clarified. The aim of this study was to evaluate the relationships between the expression level of nuclear TDP-43 and the pathological properties of cytoplasmic aggregates in autopsied ALS cases. We included 22 consecutively autopsied cases with sporadic TDP-43-related ALS. The motor neuron systems were neuropathologically assessed. We identified 790 neurons with cytoplasmic TDP-43 inclusions from the lower motor neuron system of included cases. Nuclear TDP-43 disappeared in 84% ( Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that involves the upper and lower motor neuron systems. Clinical findings of ALS are characterized by a combination of upper and lower motor neuron signs. The pathological characteristics of ALS comprise the loss of upper and lower motor neurons in association with protein aggregation within neurons and glia. The transactive response DNA-binding protein of 43 kDa (TDP-43) is known as a pathological protein of ALS, and approximately 90% of autopsied ALS cases have TDP-43 aggregates ,2,3. CytTDP-43 gene mutation or aggregation models induced by patient-derived TDP-43 seed [TDP-43 is an RNA-binding protein that is localized within the nucleus in normal settings . In affe-43 seed ,22. This-43 seed ,24. It h-43 seed .The aim of this study was to clarify whether the depletion of nuclear TDP-43 is linked to the properties of TDP-43 cytoplasmic aggregates in autopsied cases with ALS. We undertook the research with the following processes: first, we identified neurons with cytoplasmic aggregates in combination with nuclear clearance of TDP-43 and those with cytoplasmic aggregates in presence of nuclear TDP-43 immunoreactivity; second, we compared the morphology and ubiquitylation of cytoplasmic TDP-43 aggregates between these groups of neurons. Consecutive autopsied cases with sporadic ALS-TDP were studied.The demographics of the included cases are summarized in The hypoglossal nerve nucleus and spinal cord were subjected to anti-full length TDP-43 (panTDP-43) immunohistochemistry that was able to immunolabel both cytoplasmic aggregates and intrinsic nuclear TDP-43. We defined cytoplasmic TDP-43 immunoreactivity in combination with nuclear clearance as ‘TDP-43 c-ir (cytoplasmic immunoreactivity)’. In contrast, cytoplasmic TDP-43 immunoreactivity in combination with nuclear expression was defined as ‘TDP-43 n/c-ir (nuclear and cytoplasmic immunoreactivity)’. All 22 cases with ALS-TDP showed TDP-43 c-ir neurons in the anterior horn of the spinal cord, and 16 cases also showed TDP-43 c-ir neurons in the hypoglossal nerve nucleus. In total, 15 out of the 22 ALS-TDP cases (68%) had TDP-43 n/c-ir neurons in the spinal cord anterior horn, and 5 of them also had n/c-ir neurons in the hypoglossal nerve nucleus . In contp < 0.00001, Fisher’s exact test, significance level was set at 0.016 after Bonferroni correction), whereas skein-like and round inclusions were more prevalent in TDP-43 c-ir neurons than in n/c-ir neurons . In particular, round inclusions were very rare in TDP-43 n/c-ir neurons.We identified 790 neurons with cytoplasmic TDP-43 aggregates from the lower motor neuron systems of the studied cases, with a total of 130 neurons with TDP-43 n/c-ir and 660 neurons with TDP-43 c-ir . Cytoplap = 0.0003, Mann-Whitney U test) (Double immunofluorescence toward panTDP-43 and phosphorylated TDP-43 (p-TDP-43) revealed that the cytoplasmic TDP-43 component of n/c-ir neurons was phosphorylated, whereas the nuclear TDP-43 component was not . Double  U test) .p < 0.00001, Mann-Whitney U test)  .In total, 16 out of the 22 studied ALS cases had neuronal TDP-43 aggregates in the primary motor cortex, and 4 of them showed TDP-43 n/c-ir. TDP-43 n/c-ir was observed in small pyramidal neurons and occasionally in Betz cells . These fIt is known that glia are often involved in TDP-43 pathology of ALS cases . Glial Tp = 0.0095, Spearman’s rank order) /(TDP-43 c-ir neurons)) in each case to assess the dominancy of these two types of TDP-43 abnormalities in lower motor neuron systems. As mentioned above, 790 neurons with TDP-43 aggregates (130 with n/c-ir and 660 with c-ir) in total were identified from the hypoglossal nerve nucleus and spinal cord of the studied cases, and this value was used in the calculation. The ratio ((TDP-43 n/c-ir neuron counts)/(TDP-43 c-ir neuron counts)) was negatively correlated with the disease duration of ALS  B. The cak order) . Similarly shown . In contly shown .The study results revealed that a subset of neurons with cytoplasmic TDP-43 aggregates showed the nuclear immunoreactivity of this protein, which was defined as n/c-ir, in the motor neuron system. For lower motor neuron system, this finding was observed in more than half of the studied cases. Moreover, a subset of them had TDP-43 n/c-ir neurons in the upper motor neuron system. The morphological characteristics and ubiquitylation level of cytoplasmic TDP-43 aggregates were clearly different between n/c-ir and c-ir neurons. Our results suggest that the process of cytoplasmic TDP-43 aggregation is linked to a reduction in nuclear TDP-43.It is known that aggregative TDP-43 is abnormally phosphorylated at several sites, and some hyperphosphorylated sites were included in C-terminal fragments . NNIs arMorphological observation clarified that diffuse inclusions were most prominent in TDP-43 n/c-ir neurons, followed by skein-like inclusions; round inclusions were very rare. This is in great contrast with neurons presenting TDP-43 aggregation with nuclear clearance (c-ir neurons), in which skein-like inclusions were most prevalent. Autopsy-based study findings have suggested that diffuse cytoplasmic inclusions are poorly ubiquitylated and are presumed to represent an early phase of aggregation, termed ‘preinclusions’ ,24. In cWe also found that the cytoplasmic inclusions of TDP-43 n/c-ir neurons were less ubiquitylated than those of c-ir neurons. We compared only skein-like inclusions in n/c-ir and c-ir neurons to avoid an effect of subtypes of inclusions; as mentioned above, diffuse cytoplasmic inclusions are known to be poorly ubiquitylated even in neurons with nuclear clearance of TDP-43 . ImportaOur clinicopathological assessment revealed that TDP-43 n/c-ir neurons became less dominant than c-ir neurons during prolonged disease durations. The fact indicates that the n/c-ir-dominant state is linked with a rapid progression of ALS. We speculate a possibility of high neurotoxicity demonstrated by poorly ubiquitylated, diffuse aggregates of TDP-43. Although studies have revealed that diffuse ‘preinclusions’ of TDP-43 lack amyloid nature detected by thioflavin S, an ultrastructural study clarified partial but definite formation of misfolded fibrils within the preinclusions ; at leasThe molecular basis of TDP-43 pathology includes many unclear components. An imbalance in the nuclear and cytoplasmic burden of TDP-43 has been hypothesized for the combination of cytoplasmic aggregation and nuclear clearance observed in ALS-TDP and FTLD-TDP. TDP-43 is actively and passively transported across the nuclear membrane in cells , and autTDP-43 is also abundantly expressed in the nuclei of glial cells. Glial cytoplasmic inclusion of TDP-43 is another finding in ALS-TDP and FTLD-TDP ,27. The As a limitation, our study design was cross-sectional and retrospective. We evaluated the correlation between the maturation of TDP-43 aggregates and the reduction in nuclear TDP-43, but it remains uncertain which of the cytoplasmic and nuclear events causes the other. Another unsolved question is why there were fewer TDP-43 n/c-ir neurons in the primary motor cortex than in the lower motor neuron system. Studies have hypothesized that the propagation of TDP-43 aggregates begins from the primary motor cortex and then spreads to the lower motor neurons ,12. A poIn conclusion, the morphology and ubiquitylation of cytoplasmic TDP-43 aggregates were clearly linked with the nuclear expression level of this protein; premature and poorly ubiquitylated TDP-43 inclusions were associated with nuclear expression, whereas mature and well-ubiquitylated inclusions were associated with nuclear clearance. Our results indicate that the pathogenesis of cytoplasmic aggregation progresses together with that of intranuclear reduction in TDP-43, which may be supportive of the hypothesis of TDP-43 imbalance inside and outside of the nucleus as being essential for TDP-43 pathology.n = 2), comorbid neuropathological changes of Alzheimer’s disease that were classified into high grade [n = 1), and comorbid neuropathological changes of limbic/diffuse neocortical stage of Lewy body disease [n = 1). We also evaluated 15 controls without any neurological disorders involving the spinal cord. The main disorders of the controls comprised muscle disease (n = 4), autoimmune disease (n = 4), and cardiovascular disease (n = 4), including normal pressure hydrocephalus (n = 1), cerebral contusion (n = 1), and chronic nephritis (n = 1). The age at death and sex of the controls were matched to those of the ALS cases.Signed informed consent for autopsy and tissue usage for research purposes was obtained from the family members of all cases, and research usage of autopsy-proven tissues was approved by the ethical committee of Aichi Medical University. We reviewed 26 pathologically diagnosed cases with ALS-TDP who were consecutively autopsied from Jan. 2015 to Aug. 2016 at Aichi Medical University. The pathological diagnosis of ALS-TDP was made according to the loss of upper and lower motor neurons in association with intraneuronal TDP-43 aggregations . We exclgh grade  (n = 1), disease  combinedClinical analyses are retrospective. Neurological findings, age at death, disease duration, and family history were obtained from clinical records.The left hemisphere of the brain and the spinal cord were fixed in 20% neutral-buffered formalin for at least two months, followed by paraffin embedding. The paraffin blocks were cut at a thickness of 5 μm for immunohistochemistry and 9 μm for hematoxylin–eosin and Klüver–Barrera methods.Segments of the spinal cord were identified with a classic method before cutting; the anterior roots became much thinner in the S2 segment than in the S1 segment or more rostral. The dorsal roots become much thinner in T2 than in T1 or more rostral ,42. ThenAnti-full-length TDP-43 (panTDP-43) immunohistochemistry was used to label intrinsic nuclear TDP-43 and pathological aggregates. Anti-phosphorylated TDP-43 (p-TDP-43) immunohistochemistry recognized aggregated TDP-43 but not intrinsic nuclear TDP-43. The primary antibodies that we used were as follows: anti-alpha-synuclein , anti-beta-amyloid , anti-cystatin-C , anti-hyperphosphorylated tau , anti-p62 , anti-panTDP-43 antibody , anti-phosphorylated TDP-43 , and anti-ubiquitin  antibodies. Secondary immunolabeling was performed using biotin-tagged immunoglobulins via the standard avidin–biotin method. We used 3,3′-diaminobenzidine (Wako) as the chromogen.Secondary antibodies were conjugated with Alexa Fluor 405, 488, or 568 . 4,6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei. The labeled specimens were observed with a laser confocal microscope . Images were acquired under the same laser and detection settings for each combination of primary antibodies.2 visual field.We counted neurons with cytoplasmic TDP-43 inclusions in the primary motor cortex, hypoglossal nerve nucleus, and spinal cord anterior horn. We excluded neurons for which nuclei were not present in specimens because the study aimed to assess nuclear immunostaining of TDP-43. For the hypoglossal nerve nucleus and spinal cord anterior horn, the neurons were counted in the whole visual field of both sides. In contrast, for the primary motor cortex, we counted these in five randomly chosen visual fields with distances of 500 μm between each other. Counting was performed under ×200 magnification with a 3.8 mmIn this study, we defined the anterior horn of the spinal cord as follows: the anterior border of the anterior horn was defined by the boundary between the gray matter and anterior column, whereas the dorsal border of the anterior horn was defined by a line running between the most lateral point of the anterior gray matter and the most dorsal point of the medial anterior column .Neurons or glial cells with cytoplasmic TDP-43 aggregation were subclassified into two groups based on the expression or clearance of nuclear TDP-43. On the basis of DAB-labeled anti-panTDP-43 immunohistochemistry with hematoxylin counterstaining, we defined cytoplasmic TDP-43 immunoreactivity in combination with complete nuclear clearance as ‘TDP-43 c-ir (cytoplasmic immunoreactivity)’; in this setting, DAB-staining disappeared from the nucleus, and only hematoxylin staining was visible within the nucleus. In contrast, cytoplasmic immunoreactivity in association with nuclear expression of TDP-43 was defined as ‘TDP-43 n/c-ir (nuclear and cytoplasmic immunoreactivity)’; in this setting, TDP-43-immunostaining can be observed within the nucleus at all expression levels, ranging from almost normally dense staining to weak staining. The judgment of TDP-43 c-ir or n/c-ir was made by three observers . Nonspecific TDP-43 immunostaining, which shows coarse granules, amorphous staining, and lipofuscin staining , was excFor lower motor neurons, cytoplasmic inclusion bodies of TDP-43 were classified into diffuse, skein-like, and round . FTLD-reUsing double immunofluorescent slides, we computed the colocalization rate as /area of cytoplasmic inclusion (labeled red)). The computation was performed for each neuron. In brief, photomicrographs were captured under ×1000 magnification using a laser confocal microscope in the same settings of gain, pinhole value, photomultiplier voltage, and resolution. We automatically detected fluorescent signals using the Otsu-thresholding filter of ImageJ  for the red, green, and blue channels. Colocalized areas with yellow signals in merged images were also automatically detected using the AND-operation of the paste function of ImageJ .Using immunofluorescent slides, we measured fluorescent signal intensity in the neuronal nucleus. The nuclei were identified with DAPI labeling. Photomicrographs were captured under ×1000 magnification using a laser confocal microscope in the same setting. We generated a stacked image for each slide, and the measurement was undertaken at the point where the nucleus most largely appeared. Signal intensity along the longest diameter running neuronal nucleus was measured using the plot profile function of ImageJ. In this method, a value of 100 means the highest (saturated) intensity, whereas 0 means the lowest  intensity. Values obtained from pixels along the diameter were averaged, and the averaged values were defined as the nuclear signal intensity of a given neuron .p values obtained from multiple comparisons. All statistical analyses were two-sided and were performed using the SPSS 21.0 software program .The clinical and pathological findings were compared using the Mann-Whitney U test for quantitative variables and Fisher’s exact test for qualitative variables. We employed Spearman’s rank order to assess the correlations of nonparametric variables. The significance level was set at 0.05 for comparisons between two groups. Bonferroni correction was performed for


Sargassum vestitum to create habitat units of distinct morphotypes to test whether variation in the morphological traits  of S. vestitum or the interaction between these traits affects their value as habitat for associated communities in the presence and absence of predation. We found morphological traits did not interact, instead having independent effects on epifauna that were negligible in the absence of predation. However, when predators were present, habitat units with large fronds were found to host significantly lower epifaunal abundances than other morphotypes, suggesting that large frond alga provided low‐value refuge from predators. The presence of predators also influenced the size structure of epifaunal communities from habitat units of differing frond size, suggesting that the refuge value of S. vestitum was also related to epifauna body size. This suggests that habitat formers may chiefly structure associated communities by mediating size‐selective predation, and not through habitat provisioning. Furthermore, these results also highlight that habitat traits cannot be considered in isolation, for their interaction with biotic processes can have significant implications for associated communities.Habitat‐forming organisms provide three‐dimensional structure that supports abundant and diverse communities. Variation in the morphological traits of habitat formers will therefore likely influence how they facilitate associated communities, either via food and habitat provisioning, or by altering predator–prey interactions. These mechanisms, however, are typically studied in isolation, and thus, we know little of how they interact to affect associated communities. In response to this, we used naturally occurring morphological variability in the alga  Sargassum vestitum to create habitat units of specific morphotypes and found that intraspecific variation in frond size strongly affected the epifaunal communities associated with macroalgae. Experimentally excluding predators showed that these patterns were likely the result of predation, with epifaunal communities only varying in the presence of predators. This suggests that algal morphology affects the refuge value of macroalgae for associated epifauna.We used naturally occurring morphological variability in the alga  Agardh, to test hypotheses about this structural trait independent of species identity. Traits can, however, also interact, with their effects being either independent (i.e. no effect or additive effect) or dependent on each other (i.e. synergistic or antagonistic effect). In the case of habitat formers, morphological trait interactions could influence their value to the associated organisms as habitat. Despite this, multiple traits are rarely considered in trait‐based approaches, with the possible interaction between multiple traits considered even less frequently  but also within individual thalli   for habitat units was collected on snorkel from Shark Bay in Sydney Harbour, New South Wales, Australia  in November 2018. Eight large thalli were collected and once back on shore were placed into freshwater for ~10 min to remove all resident epifauna  and were therefore randomly allocated among all habitat unit morphology treatments. All habitat units was then kept in saltwater until deployment later that day.Algal material (n = five to six habitat units for each of the five morphotypes).To test whether habitat unit size (single‐ or double‐branched habitat units) interacted with frond size diversity  to affect the abundance and size structure of associated communities all five habitat unit morphotypes were deployed simultaneously and colonising epifaunal communities were compared (2 mesh size. The open cages had two openings (10 cm × 5 cm), on opposite sides of the cage.To test how variation in algal morphology and predation risk structure epifaunal communities, we used an orthogonal design with predation risk (three levels) and habitat unit morphology ; with six replicates for each combination level. The predation risk treatments employed full cages (excludes predators >1 cm wide), open cages  and uncaged . Cylindrical cages (18 cm high × 15 cm in diameter) were constructed of plastic gutter guard with 1 cmS. vestitum has naturally occurring vesicles (air sacks) to keep branches neutrally buoyant and upright therefore there was no need to attach floats to habitat units. Each patch of mesh was then floated out to the algal bed on a floatation device and was gently lowered to the seafloor. Patch locations were haphazardly selected; however, only flat surfaces were used to ensure algae remained upright and a minimum of 1 m between patches was maintained to ensure independence , was then attached with cable ties to one corner of a plastic mesh ‘patch’ (four treatments per patch). A patch consisted of a 50 cm × 50 cm square of plastic mesh with one concrete masonry brick  attached to the centre of the mesh patch with cable ties. To account for possible variation among patches, treatments were randomly assigned among different patches so that it was possible for any combination of treatments to occur on each patch. After 5 days, all algae were retrieved by snorkelling—a time period that allows colonisation of mobile epifauna to close to natural densities at this study site Poore, . Each brA generalised linear model (GLM) was used to test for differences in the abundance of epifauna that colonised habitat units across all five morphology treatments. In these models, algal biomass was included as an offset to account for differences in the biomasses of habitat units. A Poisson error distribution was used. Similar analyses were run with mean body size and variance in body size as response variables. Mean body size was, however, analysed using a linear model, while variance in body size was analysed using a GLM with a Gamma log link error distribution. Another GLM which used a factorial design was used to test for an interaction between frond size and habitat unit size on the abundance of epifauna by excluding the mixed frond double‐branched habitat units. For all these analyses, one entire habitat unit was the smallest unit of measurement (i.e. both branches were included for double‐branched habitat units).p = .01, Table Suspected herbivory during these experiments resulted in only one replicate of the large frond habitat units remaining in open‐cage treatments. Analyses showed no significant differences between open‐cage and uncaged treatment for small frond double branch habitat units and only slight differences between mixed frond double branch habitat units for the abundance of epifauna . Models were visually assessed for heteroscedasticity , which had higher densities than large frond habitat units , suggesting that there was no interactive effect of frond size traits on the epifauna abundance. There were no significant differences between single‐ and double‐branched habitat units of the same frond size . The results from the orthogonal analyses supported this, with no significant interaction between frond size and habitat unit size  and small (p = .001) frond habitat units    Figure , Table 5) Figure , Table 5There was no significant effect of predation on the mean body length or variance in body length of epifaunal communities Table . Epifaun4S. vestitum to create habitat units of specific morphotypes and found that intraspecific variation in frond size, a key morphological trait, strongly affected the epifaunal communities associated with macroalgae, irrespective of the size of habitat units. Contrary to our hypothesis, however, frond size traits did not interact, meaning that higher morphological diversity in the habitat  did not lead to higher abundances of colonising epifauna. Experimentally excluding predators showed that these patterns were likely the result of predation, with little effect of variation in habitat structure on epifauna in the absence of predation. In comparison, epifaunal communities varied strongly with habitat structure when subject to predation.Previous studies demonstrate that habitat structure and predation interact to influence associated community structure. However, how these factors are meditated by morphological traits of the habitat are poorly known. We used naturally occurring morphological variability in the alga S. vestitum is the result of size‐selective predation of communities from macroalgae with large fronds, rather than epifauna preferentially selecting for macroalgae with small fronds. These findings highlight the significant impact habitat‐former traits can have on associated communities, but that these impacts occur only in the presence of predators and also vary with prey body size.Comparisons of epifaunal community body size distributions supported these findings, with little variation found among caging treatments for both small and mixed frond habitat units. Predator access to communities on large frond habitat units, however, resulted in epifaunal communities with a lower proportion of small and more large individuals than those that were protected from predators. These results support our hypothesis that morphology affects the refuge value of macroalgae, suggesting that the structure of epifaunal communities associated with 4.1In terrestrial systems, there is growing evidence that individual traits do not act in isolation. For example, floral traits such as colour, orientation and nectar quality are known to synergistically interact to affect pollinators  if we are to fully understand the mechanisms that structure the communities associated with habitat‐forming organisms.By harnessing the natural morphological variability that occurs among individuals of Talia P. Stelling‐Wood: Conceptualization ; formal analysis (lead); investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Alistair G. B. Poore: Conceptualization ; methodology ; supervision ; writing – review and editing . A. Randall Hughes: Conceptualization (supporting); writing – review and editing . Jason D. Everett: Data curation (supporting); formal analysis (supporting); writing – review and editing . Paul E. Gribben: Conceptualization ; methodology ; supervision ; writing – review and editing .The authors declare no conflicts of interest.Collections were made with permission from permit P13/0007 issued by the Department of Primary Industries, New South Wales.


Amyotrophic lateral sclerosis (ALS) is a disease that affects motor neurons and has a poor prognosis. We focused on TAR DNA-binding protein 43 kDa (TDP-43), which is a common component of neuronal inclusions in many ALS patients. To analyze the contribution of TDP-43 mutations to ALS in human cells, we first introduced TDP-43 mutations into healthy human iPSCs using CRISPR/Cas9 gene editing technology, induced the differentiation of these cells into motor and sensory neurons, and analyzed factors that are assumed to be altered in or associated with ALS . We aimed to clarify the pathological alterations caused solely by TDP-43 mutation, i.e., the changes in human iPSC-derived neurons with TDP-43 mutation compared with those with the same genetic background except TDP-43 mutation. Oxidative stress induced by hydrogen peroxide administration caused the death of TDP-43 mutant-expressing motor neurons but not in sensory neurons, indicating the specific vulnerability of human iPSC-derived motor neurons with TDP-43 mutation to oxidative stress. In our model, we observed aggregate formation in a small fraction of TDP-43 mutant-expressing motor neurons, suggesting that aggregate formation seems to be related to ALS pathology but not the direct cause of cell death. This study provides basic knowledge for elucidating the pathogenesis of ALS and developing treatments for the disease.The online version contains supplementary material available at 10.1186/s13041-023-01050-w. Amyotrophic lateral sclerosis (ALS) is a progressive disease characterized by motor neuron dysfunction, muscle weakness in the extremities, dysphagia, and respiratory impairment. ALS has a poor prognosis, with an average time from onset to death of approximately 3.5 years . The cauTARDBP gene encoding TDP-43 have been reported. Interestingly, TDP-43-containing intraneuronal inclusions form in many ALS patients with and without TARDBP mutations, suggesting that determining the molecular mechanism of TDP-43 is key to elucidating the pathogenesis of ALS [The majority of ALS cases are sporadic, and approximately 10% are familial cases , 3. It iSeven TDP-43 mutants  were previously used to examine the relationship between TDP-43 half-life and age at onset of ALS, and the findings revealed that mutants with a longer half-life resulted in an earlier age at onset, i.e., there was a negative correlation between half-life and age at onset . Based oTransgenic and knockout mice have been used as animal models in the study of many neurodegenerative diseases. TDP-43 knockout mice are embryonic lethal . OverexpThis study elucidates the pathogenesis of ALS caused solely by TDP-43 mutation using human iPSC-derived motor and sensory neurons carrying the TDP-43 A382T mutation (introduced by CRISPR CAS9 gene editing technology) with the same background genes as healthy controls except for TDP-43..The direct relationship between TDP-43-containing intraneuronal inclusions and motor neuron cell death in ALS patients is controversial –16, and Using CRISPR CAS9 gene editing technology, we established a cell line with the A382T mutation. A single A382T allele was introduced into healthy human iPSC cells. Since FRT sequences are inserted during gene editing, we established a WT line expressing WT TDP-43 as a control for gene editing Fig. . The estIn this study, iPSCs were differentiated into motor and sensory neurons to verify whether the phenotypes caused by TDP-43 mutation are restricted to motor neurons. For motor neuron differentiation, iPSCs were maintained in Stem Fit medium containing CHIR99021, SB431542 and dorsomorphin for 6 days. We dissociated iPSCs into single cells and cultured these cells in neurosphere medium containing KBM, B27, CHIR99021, SB431542, retinoic acid, purmorphamine, bFGF and hLIF for 12 days to induce sphere formation see “”. The ceThree lines of unedited iPSCs (control) and gene-edited iPSCs WT and A382T lines) were induced to differentiate into motor and sensory neurons. We evaluated the neurons at approximately 60 days after seeding. There were no differences in the morphology of motor and sensory neuron bodies, axons, or dendrites among the three groups. In ALS patients, TDP-43 is localized in the cytoplasm instead of the nucleus and forms aggregates in the cytoplasm. Immunostaining of cultured neurons showed that TDP-43 was localized to the nucleus, and no aggregates were observed , indicating hypersensitivity to stress. No significant difference in the ratio of G3BP1 granules in sensory neurons was found among the three groups , a marker of apoptosis. After exposure to 25 μM hydrogen peroxide for 1.5 h, the expression of CC3 was significantly increased in motor neurons expressing the A382T mutant compared to control and WT motor neurons . No significant difference in the expression of CC3 was observed in sensory neurons among the three groups , TDP-43 localization tended to shift from the nucleus to the cytoplasm in motor neurons expressing the A382T mutant compared to WT motor neurons Fig. B. TDP-43Increasing the concentration of hydrogen peroxide or the duration of exposure resulted in cell death in all three groups  increases, and the proportion of BCL2L variants (BCL2L-L/BCL2L-S) decreases. In this study, we evaluated the alternative splicing function of TDP-43 by collecting RNA the day after and 5 days after prolonged exposure to low concentrations of hydrogen peroxide. Evaluation of the splicing patterns of target RNAs revealed that TDP-43 function was not impaired by hydrogen peroxide, and there were no significant differences among the three groups  in Stem Fit medium (Ajinomoto) under feeder-free conditions and passaged every 4–7 days by 0.5× TrypLE Select Solution ; FUJIFILM WAKO) .The flow chart of the CRISPR Cas9-mediated gene editing protocol is shown in Fig. Left arm-F 5′–3′ GCCACCACGCCTGGCTGATTTTTTTGLeft arm-R 5′–3′ GCAGCAGTGATATTTCAGTCGATTTTCRight arm-F 5′–3′ TTTAAATGAAATGAGTGTTCATTGCRight arm-R 5′–3′ CAGTTCACTTTCAACCACTCGuide RNA5′–3′ CACCGaaaactaaaagctgtattgg5′–3′ AAACccaatacagcttttagttttCIntroduction of mutant TDP-43 (A382T) into a healthy control iPSC line (201B7) was carried out by CRISPR‒Cas9 genome editing. iPSCs were passaged, and px330 and pFNF were transfected into the cells. The cells were sorted with neomycin (f.c. 50 µg/ml) because the cells acquired neomycin resistance after successful transfection. At the next passage, pCAGGS-flpE-puro was transfected into the cells, and neomycin resistance was removed using the Flp-FRT system. Puromycin (f.c. 2 µg/ml) was used to sort the cells because successful expression of the Flp-FRT system caused the cells to transiently acquire puromycin resistance. Surviving colonies were selected, and cell lines with mutations in one allele were established by target region PCR. We established two cell lines, the WT cell line, in which WT TDP-43 was knocked in, and the A382T cell line, in which the mutant sequence was knocked in. Genomic DNA was extracted from the established cell lines using the QIAamp DNA Mini Kit (QIAGEN), and the insertion of the correct sequence was confirmed as follows.Off target 1-F 5′–3′ GGGAAACAAGAAACTGAGGTGTGGOff target 1-R 5′–3′ CAGCTTGTTTTGCCTTCAGGGCTTOff target 2-F 5′–3′ GCAAATCTGTCCTTTGTGGGGGOff target 2-R 5′–3′ GCCAAGCAGCAAGACAAAAAGGGOff target 3-F 5′–3′ GAGACTTTCTCCCTGTCAGACAGCOff target 3-R 5′–3′ TGTCACCCTCTTTTACCATACGCCOff target 4-F 5′–3′ GGATTGCCTTTTTGATTTGGTCATCAGCTAGOff target 4-R 5′–3′ CAGCCAACATCACACTTAGTGGAGOff target 5-F 5′–3′ AGCAGCCTTAGGTAAGAGGTAGTGOff target 5-R 5′–3′ CCTCAAATTCCTGGGCTCAAGTGBy using the COSMID web tool , we seleIn addition, karyotyping analysis was performed by Nihon Gene Research Laboratories, Inc., Japan.l-ornithine (Sigma-Aldrich) and laminin (Invitrogen) at a density of approximately 0.5 to 1 × 105 cells/cm2 and incubated with adenovirus vector (AdV) carrying three transcription factors, Lhx3, Ngn2 and Isl1, at a multiplicity of infection (MOI) of approximately 15. The Ngn2-, Isl1- and Lhx3-expressing adenovirus vector was constructed using the cosmid cassette pAxEFwit2, which has a cloning site SwaI flanked by the EF1 alpha promoter and rabbit beta-globin polyA signal in the E1-deleted region of the adenovirus genome. Each cDNA was ligated with 2A sequences derived from porcine Teschovirus for expression in the same vector. The AdV was prepared as previously described [iPSCs were maintained in Stem Fit medium containing 3 μM CHIR99021 , 3 μM SB431542 (Sigma‒Aldrich), and 3 μM dorsomorphin (Santa Cruz) for 6 days from the day after passage. The cells were dissociated into single cells with TrypLE Select (Gibco) and passaged in a flask in neurosphere medium consisting of KBM neural stem cell medium (KOHJIN Bio) containing 2% B27 supplement (Gibco), 3 µM CHIR99021, 2 µM SB431542, 1 µM retinoic acid (Wako), 1 µM purmorphamine , 10 ng/ml basic fibroblast growth factor (Wako), 10 ng/ml human leukemia inhibitory factor (Millipore), and 10 µM Y-27632 (Wako) to induce sphere formation. The medium was changed on day 3. Neurospheres were passaged on day 6, and the medium was replaced on day 9 with neurosphere medium containing all of the components described above except Y-27632. On day 12, cells in neurospheres were dissociated into single cells with TrypLE Select, replated on plates coated with poly-escribed . The AdVescribed . Brieflyl-ornithine and laminin at a density of approximately 0.5 × 104 cells/cm2 to induce differentiation into sensory neurons. After seeding, the sensory neurons were cultured with neuron medium consisting of KBM neural stem cell medium containing 2% B27, 10 ng/ml neurotrophin-3 (R&D Systems), 20 ng/ml NGF (R&D Systems), 20 ng/ml BDNF, and 20 ng/ml GDNF. The medium was changed every 2–3 days.For sensory neuron differentiation, a previously reported method was used with modifications . iPSCs wCells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 5 min at room temperature. After permeabilization and blocking with 0.3% Triton X-100 and 5% bovine serum albumin (BSA) for 60 min, the cells were incubated with primary antibodies at 4 °C overnight. Primary antibodies against the following were used for analyses: ChAT , SMI32 , Brn3a , Peripherin , MAP2 , MAP2 , Cleaved Caspase 3 , G3BP1 , TDP-43(N) . The following day, the cells were washed with PBS and incubated with secondary antibodies for 60 min at room temperature. The secondary antibodies used were goat or donkey antibodies conjugated to Alexa 488, 546, 555, 568, 594, 633, or 647 . Nuclear staining was performed with Hoechst 33342  with secondary antibodies. Images were obtained using a confocal laser microscope .2O2, 30% w/v, Wako) was administered on day 21 of neuron culture. The expression of CC3, a marker of apoptosis, was assessed after 5 days of exposure to 25 μM H2O2 for 1.5 h. The expression of G3BP1, a marker of stress granules, was assessed after exposure to 12.5 μM H2O2 for 10 h. TDP-43 localization was evaluated after 5 days of exposure to 12.5 μM H2O2 for 10 h. RNA was extracted using TRIzol (Life Technologies) and an RNeasy Plus Mini Kit (QIAGEN) on the day after and 5 days after exposure to 12.5 μM H2O2 for 10 h. A total of 3 independent experiments were carried out.Hydrogen peroxide . Numerous primers were designed against alternative splicing targets of TDP-43 , 58. TheAs controls for the evaluation of splicing function, control siRNA and siRNA targeting TDP-43 were transfected into HEK293T cells. Lipofectamine RNAiMAX (Life Technologies) was used for transfection of siRNAs. The interference efficiency was evaluated by PCR amplification of cDNAs in the target region. The sequences of the siRNAs were previously reported .TDP-43 TaqMan probe design 5′–3′ AAAAAGGAAGAGCTAAAGGATDP-43-F 5′–3′ TTTGTTCAGTGTGGAGTATATTCAGCATDP-43-R 5′–3′ AACCACTCAATATTTCAACCTTTCATGReal-time RT-PCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) with a Quant Studio 5 Real-time PCR system (Thermo Fisher Scientific). TDP-43 mRNA levels in the samples were normalized using beta-actin (TaqMan Gene Expression Assay: Assay ID: Hs01060665_g1). Probes and primers for TDP-43 were designed. The sequences were as follows:3VO4, 1 mM NaF, 20 mM Na4P2O7, and 0.02% NaN3, which was added to each well on ice. The samples were collected in tubes, vortexed for 2 s, and then sonicated for 20 s. Centrifugation was performed at 15,000 rpm for 5 min at 4 °C, and the lysates were stored at − 80 °C. The samples were diluted with equal volumes of Laemmli sample buffer (Bio-Rad) containing 62.5 mM Tris–HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% bromophenol blue, and 5% 2-mercaptoethanol and then incubated for 3 min at 95 °C. Protein samples were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 1% nonfat skim milk for 30 min at room temperature (RT) and incubated in primary antibody for 16 h at 4 °C. Thereafter, the membranes were incubated with secondary antibodies for 2 h at RT. Signals were detected using Chemiluminescence HRP Substrate (Takara Bio) with a Lumino Graph I system (ATTO). Semiquantitative analysis of the signals was performed using ImageJ software .Protein extraction from cultured iPSCs and neurons was performed using a mixture of Complete Mini (Sigma-Aldrich), phosphatase inhibitor  and Tissue Extraction Reagent I (Invitrogen) consisting of 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 2 mM NaStatistical analyses were performed using a commercially available software package (GraphPad Prism 9). The data are presented as the mean ± SEM or SD as indicated. Statistical significance was assessed using Student’s t test or one-way ANOVA . The number of independent experiments (n) is indicated in each figure legend.Additional file 1: Figure S1. (A) Off-target analysis of WT KI and A382T KI iPSCs. (B) Karyotype analysis of WT KI and A382T KI iPSCs.Additional file 2: Figure S2. Evaluation of neurite length analysis in motor (A) and sensory (B) neurons derived from control and gene-edited iPSCs. Using the same hydrogen peroxide loading conditions as those used to evaluate CC3, we evaluated the total length of neurites/cell count of motor and sensory neurons as seen by SMI32 or MAP2 immunostaining at DIV45 and 34, respectively. The cells were evaluated separately as cleaved caspase3 negative or positive cells and normalized by the data of cleaved caspase 3 negative control neuron.Additional file 3: Figure S3. Evaluation of TDP-43 localization following oxidative stress. TDP-43 aggregation in the cytoplasm was observed only in A382T-expressing motor neurons, but only to a small extent. Scale bars, 20 μm.Additional file 4: Figure S4. Evaluation of TDP-43 RNA levels in sensory neurons following oxidative stress. There were no significant differences in TDP-43 RNA levels among the three groups.Additional file 5: Figure S5. Evaluation of changes induced by severe oxidative stress. Increasing the concentration hydrogen peroxide and the duration of exposure to hydrogen peroxide resulted in detachment from the dish and cell death in all three groups of motor and sensory neurons, making them unassessable. Scale bars, 200 μm.Additional file 6: Figure S6.  Evaluation of the alternative splicing function of TDP-43. PDP1 and BCL2L are known TDP-43 alternative splicing targets, and when TDP-43 function is reduced, the proportion of PDP1 splicing variants (PDP1-L/PDP1-S) increases, and the proportion of BCL2L splicing variants (BCL2L-L/BCL2L-S) decreases. In this study, we evaluated the alternative splicing function of TDP-43 by collecting RNA the day after and 5 days after prolonged exposure to low concentrations of hydrogen peroxide. TDP-43 function was not impaired by hydrogen peroxide, and there were no significant differences among the three groups.Additional file 7: Figure S7. (A) TDP-43 RNA sequence in A382T-expressing iPSCs and motor neurons. The presence of mutations was confirmed in the genome but not at the RNA level. (B) Schematic diagram of the gene editing protocol; control, WT (heterozygous), WT (homozygous), and A382T cells. (C) Relative TDP-43 RNA and protein levels in iPSCs. There were no significant differences in TDP-43 RNA and protein levels among the four groups. (C); n = 3 in each of three groups. The data are presented as the mean ± SEM. n.s., not significant. Statistical analyses were performed by one-way ANOVA.


Matrix factorization is a long-established method employed for analyzing and extracting valuable insight recommendations from complex networks containing user ratings. The execution time and computational resources demanded by these algorithms pose limitations when confronted with large datasets. Community detection algorithms play a crucial role in identifying groups and communities within intricate networks. To overcome the challenge of extensive computing resources with matrix factorization techniques, we present a novel framework that utilizes the inherent community information of the rating network. Our proposed approach, named Community-Based Matrix Factorization (CBMF), has the following steps: (1) Model the rating network as a complex bipartite network. (2) Divide the network into communities. (3) Extract the rating matrices pertaining only to those communities and apply MF on these matrices in parallel. (4) Merge the predicted rating matrices belonging to communities and evaluate the root mean square error (RMSE). In our experimentation, we use basic MF, SVD++, and FANMF for matrix factorization, and the Louvain algorithm is used for community division. The experimental evaluation on six datasets shows that the proposed CBMF enhances the quality of recommendations in each case. In the MovieLens 100K dataset, RMSE has been reduced to 0.21 from 1.26 using SVD++ by dividing the network into 25 communities. A similar reduction in RMSE is observed for the datasets of FilmTrust, Jester, Wikilens, Good Books, and Cell Phone. To predict recommendations to the users based on past behavior and their preferences, a technique in recommender systems named collaborative filtering is used . The maiMatrix factorization (MF) is a technique utilized in collaborative filtering to decompose a matrix of user-item ratings into lower-rank matrices capturing the latent factors underlying the data ,4. The uIn network analysis, community detection stands as a fundamental task, endeavoring to identify cohesive subsets of nodes, referred to as communities or modules within a network . These cMatrix factorization techniques have been widely applied across diverse domains, demonstrating their versatility and effectiveness. Some of the notable applications of the matrix factorization techniques include1.Natural Language Processing (NLP): Within the field of NLP, techniques for matrix factorization have been used in a variety of tasks in topic modeling, text classification, etc. . approach.In any type of MF method, the rating matrix is constructed by using users and items. The predicted rating matrix is obtained by using different update rules for the latent feature matrices in each kind of MF. The variation between the original and the predicted rating matrix is evaluated using root mean square error. The matrix factorization method predicts a rating for every user item combination. However, there are some cases such that a user may never consume a set of products. Because MF predicts a rating for them, they are recommended to users. This creates a lot of false recommendations. There are only a limited group of items that a user may consume. We propose that by modeling the matrix into a bipartite network containing user and item nodes, users and items can be clustered into tight communities using community detection algorithms. By limiting MF to only the communities, the prediction of false recommendations will significantly reduce. This approach also reduces the time complexity of MF methods.Algorithm 1: Community-Based Matrix Factorization (CBMF)With this motivation, we propose a Community-Based Matrix Factorization (CBMF) approach, which is given in Algorithm 1.The proposed CBMF constructs a bipartite graph a users, b items, and k latent features. The iterations are the same for every method and are taken as constant. The time complexity required for calculating the MF method is typically considered to be n is the number of nodes of graph [For the MF method, let e O(abk) . The Louof graph .The bipartite graph Hence, the overall time complexity for the MF method using the Louvain community detection method is Similarly for the SVD++ and FANMF methods, the time complexity is We have evaluated the approach of CBMF on different datasets from different domains. Datasets, namely MovieLens 100K, Film Trust, Jester, Good Books, Wikilens, and Cell Phone Recommendation, are taken. The dataset statistics of all these datasets are shown in All of these datasets’ distribution plots are displayed in In the dataset Jester, the users are the people, and the items are jokes having a rating distribution from The Wikilens dataset is shown in As community detection is a major part of CBMF, we evaluate the modularity of different datasets using the Louvain algorithm for different communities. The modularity of six different datasets using 25 communities is shown in The specifics regarding the hardware and software specifications employed in our implementation are as follows: The system boasts impressive specifications, featuring an 11th Gen Intel Core i9 processor clocked at 2.5 GHz, a formidable 64 GB of RAM, and a 64-bit operating system. Furthermore, Python is the programming language, renowned for its simplicity and effectiveness in a wide array of programming and data analysis tasks.This section presents a series of experiments designed to demonstrate the validity of the hypothesis proposed in the paper. Using the Louvain community detection algorithm, three different matrix factorization methods  are employed to predict the missing entries in the rating matrix across six distinct datasets as shown in The RMSE results of CBMF against their non-community variations for three MF techniques on six datasets are tabulated in Because the best number of communities is not uniform for all networks, k as k should be the minimum of number of users and items. Therefore, the iteration for k is limited to 30.The computation of the root mean square error (RMSE) as shown in Equation  involvesk values on a set of 25 communities. A notable observation from the graph is that, in all datasets, the absence of the Louvain algorithm results in the highest RMSE value when c is 1. However, as the number of communities increases through the utilization of the Louvain algorithm, i.e., c values range from k values. Conversely, in the Film Trust and Jester datasets, the RMSE value stabilizes after reaching a certain number of communities, regardless of the k value. Furthermore, in the Wikilens dataset, there exhibit a constant relationship between the RMSE value for different communities as well as the k values. Overall, it is evident that the MF method consistently yields better RMSE values with lower k values across all datasets.k values across a set of 25 communities. Across all datasets, a noticeable observation is that the computation time for the MF method, without the utilization of community division, exceeds that of the method with community division. As the number of communities increases, a reduction in computational time is observed. Interestingly, the evaluation time of the method remains constant as the number of communities expands, regardless of the specific k values employed.k value 2 latent features, the RMSE calculation takes approximately k value 2), the time required is significantly reduced to a mere k values on a set of 25 communities. Notably, at community 1, a significantly high RMSE value is observed across all datasets. However, as the communities are divided, a decline in the RMSE value is observed, reaching a stable state after a certain number of communities. It is worth noting that in the SVD++ method, subtle variations in the RMSE value can be observed for different k values across all datasets.k values across a set of 25 communities. Noteworthy observations can be made across various datasets. In the MovieLens 100K and Good Books datasets, an increase in the number of communities leads to a decrease in the evaluation time of the method. The maximum evaluation time is observed at community 1. Conversely, in the Jester dataset, the maximum time is taken to evaluate the SVD++ method at c value 1, with slight fluctuations in time as the communities increase. Furthermore, for the Film Trust, Wikilens, and Cell Phone Recommendation datasets, it is observed that initially, evaluating the method without dividing into communities requires less time compared to the division into communities. However, after reaching a certain number of community divisions , the evaluation time becomes less than that at the time taken without community division and remains constant. Similarly, in the case of the Cell Phone Recommendation dataset, the evaluation time becomes less than that at c value 1, and starting from c value 7 constant time is maintained for evaluation.k across a set of 25 communities, are illustrated in k value results in a decrease in the RMSE, regardless of the specific communities. Significantly, in the Film Trust, Wikilens, and Cell Phone Recommendation datasets, it is notable that the RMSE values stabilize after a certain number of communities. This stability is observed across all k values, indicating a consistent RMSE value. Moreover, across all datasets, it can be observed that a higher number of latent features leads to lower RMSE values when employing the FANMF method.The obtained RMSE values from applying the FANMF method to six distinct datasets, using different values of k values over a set of 25 communities is shown in The computational time needed to run the FANMF method on six different datasets using various In this study, we have delved into the realm of parallel computation. We proposed Community-Based Matrix Factorization (CBMF), which parallelizes matrix factorization utilizing community information. The experimental results obtained from our study provide compelling evidence of the scalability and speedup attained through CBMF, surpassing the performance of sequential implementations. These findings underscore the immense potential of parallel computation in effectively tackling the resources in the distributed environment. Looking ahead, the introduction of our parallel computation framework unlocks fresh opportunities for the efficient processing of large datasets. It empowers researchers and practitioners to extract valuable insights from intricate networks. This framework serves as a stepping stone for future advancements in providing accurate recommendations across diverse domains. It lays the foundation for future innovations and breakthroughs in the realm of data-driven solutions.