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Liposomes were prepared by thin-film hydration (TFH) method, followed by extrusion and by microfluidic technique with Nanoassemblr apparatus. They were formulated with 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (Chol) with a molar ratio of DSPC: Chol 50:50, since preliminary experiments showed this was the more stable composition (see Supplementary data Table S4) by both preparation methods tested. The liposome compositions showing the best properties in terms of stability and particle size were selected for further encapsulation of either bovine serum albumin (BSA) or myoglobin (Myo). These proteins were used as models of protein drugs, such as growth factors (i.e., hepatocyte growth factor), with different molecular weights.
Liposome preparation by TFH was carried out as follows. The stock solutions of DPSC (10 mg/mL) and cholesterol (10 mg/mL) were prepared by solubilizing the lipids with a mixture of chloroform:methanol 9:1 volume ratio. Different volumes were taken from each stock solution to achieve the exact lipid DSPC:Chol 50:50 molar ratio and placed inside the round-bottom flask. The organic solvent was evaporated under vacuum using Heidolph^® Hei Vap Rotavapor (Heidolph, Germany) to obtain a homogeneous dry lipid film around the bottom flask wall. Then, the dry lipid film was rehydrated with phosphate-buffered saline (PBS) solution (pH 7.4) at 45 ± 3 °C to obtain placebo liposomes. Temperature set up (45 ± 3 °C) was chosen because it is close to DSPC (55 °C) and cholesterol (37 °C) main transition temperatures (Tm), making lipid chains more mobile and flexible. A round-bottom flask filled with PBS solution (1.3 mL) was vortexed (Advanced Vortex Mixer Zx3 Velp scientifica^®, Italy) for 3 min at 24,000 rpm to suspend the lipid film and, subsequently, it was sonicated (SONICA^® ultrasonic cleaner Soltec^®, Milan, Italy) for 3 min at 36–37 kHz to reduce the liposome size. Subsequently, the round-bottom flask with liposomes suspension was left 30 min in a warm water bath at 45 ± 3 °C to improve hydration and promote liposome formation. Finally, liposome suspension was manually extruded under mild heating (35 ± 3 °C) to produce a homogenous population of unilamellar liposomes through three polyvinylidene difluoride (PVDF) filter membranes of 0.45 µm, 0.22 µm and 0.1 µm. A total of 10 extrusion cycles were performed for each filter. Each filter was connected to two 5 mL (Luer-lock) syringes. The same procedure described above was applied to obtain myoglobin- and BSA-loaded liposomes, but, during rehydration phase, Myo solution in PBS buffer (0.15 mg/mL) or BSA solution in PBS buffer (10 mg/mL) were added. Figure 8a schematizes the thin-film hydration methods followed for the preparation methods of BSA- and Myo-loaded liposomes.
NanoAssemblrTM platform provided by Precision NanoSystems Inc. (Vancouver, Canada), characterized by a staggered herringbone micromixer (SHM), was employed for the liposomes’ preparation by microfluidic technique [29 (link)]. Micromixer cartridge dimensions are 6.6 × 5.5 × 0.8 cm (w × d × h); it is made of polypropylene, viton and cyclic olefin copolymer. The cartridge’s mixing channel is 200 × 79 μm (w × h) and the herringbone structure is 31 μm high and 50 μm thick. There is an angle of 45 between the ridges and the long axis of the channel. The microfluidic device consists of a Y-junction, known as a staggered herringbone, followed by a staggered mixing region. The staggered herringbone structures induce rapid mixing by chaotic advection [30 (link)]. Channel 1 was loaded with PBS (pH 7.4) or BSA solution in PBS (10 mg/mL) or Myo solution in PBS buffer (0.15 mg/mL), while DSPC:Chol 50:50 ethanol solution was loaded in channel 2 (see Figure 8b). Both inlet streams are controlled by syringe pumps connected to a computer that controls the process [31 (link)]. Stock solutions of DSPC (4.3 mg/mL) and cholesterol (2 mg/mL) were prepared by solubilizing lipids in ethanol (3 mL). Liposome preparation was carried out using a total flow rate (TFR) of 8 mL/min and a flow rate ratio (FRR) of aqueous phase: lipids phase 3:1 for 1 mL total volume. The preparation process was performed at room temperature (25 ± 3 °C). Preliminary experiments moving TFR from 8 mL/min to 12 mL/min and FRR between 5:1 and 3:1 were carried out to set up the process parameters (see Supplementary data Table S4).
In order to increase BSA EE%, BSA loading into liposomes prepared by microfluidic technique was also investigated, modifying the formulation and process parameters as follows:

Liposomes’ lipid molar ratio was changed from 50:50 to DSPC:Chol 70:30;

Total flow rate (TFR) was increased from 8 mL/min to 12 mL/min;

Different amount of trehalose (10% w/w, 20% w/w and 40% w/w) were added to BSA aqueous solution to increase ionic strength.

BSA was solubilized in purified water (pH 7.4 adjusted by NaOH 0.1 M) to prevent its dimerization and maintain Mw 66 kDa.

The batches produced are reported in Table 4.

Liposomes were produced by the thin-film hydration technique, followed by subsequent agitation and freeze-thaw cycles^{18 (link),36 (link)}. A lipid mix containing Ph-DPPC, DOPC, DSPG, and cholesterol in a 3:3:2:3 molar ratio was dissolved in a solution of 90% chloroform and 10% methanol. This mixture underwent evaporation at low pressure, after which the lipid content was reconstituted in tert-butanol and subjected to freeze-drying. The resulting lipid cake was then rehydrated with different payloads, including Sulforhodamine B (SRho), tetrodotoxin (TTX), polyethylene glycol (PEG), and albumin–fluorescein isothiocyanate conjugate (FITC-Ab) in PBS buffer (pH=7.4) or bupivacaine hydrochloride (Bup), or doxorubicin hydrochloride (Dox) in saline. After 10 cycles of freezing and thawing, the liposomal solution was subjected to a 48-h dialysis against either PBS or water. The molecular mass cut-off of the dialysis devices was 1000 kDa (Spectra/Por™ Float-A-Lyzer™ G2 Dialysis Devices). The dialysis fluid was routinely replaced with fresh PBS or saline approximately every 12 h. To determine the drug and dye content across different formulations, liposomes were disrupted using octyl-β-D-glucopyranoside (100 mM, twice the volume of liposomal solution) and analyzed.

The Caco-2 cell line was purchased from the Institute of Biochemistry and Cell Biology (SIBS, CAS, China). Caco-2 cell culture was performed using the method reported by Fernández et al. [32 (link)], with modifications. Briefly, Caco-2 cells were inoculated into Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% (v/v) antibiotics (100 U/mL penicillin sodium, 1.0 μg/mL streptomycin) and incubated at 37°C (5% CO₂). The culture medium was replaced the next day. After 15–18 days, the Caco-2 cells were transferred into six-well plates and grown to confluence. A monolayer of Caco-2 cells (5 × 10⁵ CFU/mL/cm²) was used for adhesion assays after washing twice with PBS (pH 7.2).
The wild-type and mutant strains of KLDS1.0391 were routinely grown overnight in MRS broth. Then, the cells were collected (8000 ×g, 4°C, 10 min), washed twice with PBS (pH 7.2), and resuspended in DMEM to the final concentration of 10⁸ CFU/mL. The suspensions (5 mL) were added to the abovementioned wells containing Caco-2 cells and incubated for 2 h (37°C, 5% CO₂). At the end of the incubation period, the Caco-2 cell cultures were washed twice with prewarmed PBS (37°C, pH 7.2) to remove the nonadherent cells and then treated with Triton X100 (1%, 10 min) to release the adhered bacterial cells. The adhesion ratio of bacterial cells was calculated by comparing the viable count on MRS agar plates before and after adhesion.

Caco-2 cell was obtained from Shanghai Zhongqiaoxinzhou Biotechnology Co.,Ltd.(Shanghai, China). Cells were cultured in DMEM (Basalmedia, Shanghai, China) supplemented with 20% (v/v) fetal bovine serum (FBS, NEWZERUM, China) and 5% antibiotics (penicillin and streptomycin, Basalmedia, Shanghai, China). Caco-2 cells were seeded at a concentration of 5 × 10⁴ cells per well in 24-well or 96-well cell culture plates to obtain a monolayer of differentiated cells.
The adhesion and invasion experiments refer to the previous studies with minor modifications (Schierack et al., 2011 (link)). After incubating A.hydrophila with EGCG for 24 hours, the mixture was centrifuged and resuspended in DMEM to adjust the bacterial concentration to 1×10⁸ CFU/mL. The bacterial suspension was then added to a monolayer of differentiated Caco-2 cells and incubated for 1 hour. In the adhesion experiments, the culture medium was discarded, and the cells were washed three times with PBS (pH 7.4) before adding 0.25% trypsin for digestion. After gradient dilution, bacterial counting was performed. In the invasion experiments, the culture medium was discarded, and 10% antibiotics (penicillin and streptomycin) were added for 1 hour before bacterial counting was performed. LDH release was tested according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

Human colorectal cancer cell lines (HCT 116 and RKO) were acquired from the Cell Bank of the Shanghai Institute of Cell Biology. The HCT 116 cells were cultured in McCoy’s 5A medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin mixture added to the above medium. The RKO cells were maintained in MEM with NEAA (Meilun Biotechnology, Dalian, China) supplemented with 10% FBS and 1% penicillin–streptomycin mixture. The cells were cultured in a 37 °C incubator (Thermo Fisher, Waltham, MA, USA) with 5% CO₂. The synthesis of small interfering RNA (siRNA) was entrusted to GenePharma (Shanghai, China). The cells were placed into 6-well plates (1.5 × 10⁵ cells/well), and the siNC group was transfected with the scrambled siRNA (negative control), and the siTMED1 group was transfected with the siRNA specific for TMED1 (Table S1). All siRNA transfections were performed with GP-transfect-Mate at a 75 nM final concentration following the manufacturer’s protocol. The transfected cells were collected for the following assays 48 h after transfection. Gene knockdown efficiency was determined by real-time quantitative PCR.

Total genomic DNAs of each population were extracted with a modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle., 1987 ). To confirm the species, rbcL genes were determined before genome sequencing. The rbcL marker, which encodes the ribulose-1,5-bisphosphate carboxylase large subunit, is the most widely used in species identification in the red algae (Saunders and Moore, 2013 (link)). The rbcL primers were F57 and rbcLrevNew described in Freshwater and Rueness (1994) (link) and Saunders and Moore (2013) (link). Amplified DNAs were purified using the LaboPass™ PCR Purification Kit (COSMO Genetech, Seoul, Republic of Korea). PCR products were sent for sequencing to Macrogen (Seoul, Republic of Korea). After a quality check, the forward and reverse sequences were merged using Geneious prime 2020.0.4 (https://www.geneious.com). For phylogenetic analysis, published rbcL sequences of Rhodymeniophycidae and Ahnfeltia were obtained from NCBI (see accession number of each taxon in Supplementary Table S1), and alignments for all sequences were conducted with MAFFT v7.310 (Katoh and Standley, 2013 (link)). A maximum likelihood (ML) phylogenetic tree was reconstructed by IQ-TREE v.1.6.8 with 1,000 ultrafast bootstrap replications (-bb 1000) and model test option (-m TEST) (Nguyen et al., 2015 (link)).

Neural differentiation medium: DMEM/F12, 2% serum replacement, 1% N2 supplement, 1× Glutamax, 50 unit/ml penicillin-streptomycin, 80 ng/mL Noggin, 10 $\mathsf{\mu }$ M SB431542, 2 $\mathsf{\mu }$ M dorsomorphin (instead combination of 10 $\mathsf{\mu }$ M SB431542, 2 $\mathsf{\mu }$ M dorsomorphin and 0.5 $\mathsf{\mu }$ M LDN-193189 can be used).
Neuronal progenitor medium: DMEM/F12, 2% B27 supplement, 1× Glutamax, 50 unit/mL penicillin-streptomycin, 100 ng/mL Shh, 100 ng/mL FGF8 and 2 $\mathsf{\mu }$ M purmorphamine.
NB maturation medium: Neurobasal, 2% B27 supplement, 1× Glutamax, 50 unit/mL penicillin-streptomycin, 20 ng/mL BDNF, 20 ng/mL GDNF, 200 $\mathsf{\mu }$ M ascorbic acid and 4 $\mathsf{\mu }$ M forskolin.
NBA maturation medium: Neurobasal-A, 2% B27 supplement, 1× Glutamax, 50 unit/mL penicillin-streptomycin, 20 ng/mL BDNF, 20 ng/mL GDNF, 200 $\mathsf{\mu }$ M ascorbic acid and 10 $\mathsf{\mu }$ M forskolin.

For immunophenotyping using flow cytometry, cells were harvested, centrifuged for 10 min at 400× g, washed once with DPBS/EDTA containing 0.05% bovine serum albumin (BSA, Sigma-Aldrich), and stained with different panels of monoclonal antibodies. In this study, CD4+ T cells were stained with mouse anti-chicken CD4-phycoerythrin (PE) or CD4-SpectralRed (SPRD) (CT-4, SouthernBiotech, Birmingham, Alabama, USA). CD8+ T cells were labeled with CD8-allophycocyanin (APC) (CT-8, ThermoFisher Scientific). In addition, human anti-chicken CD25-FITC (AbD13504, Bio-Rad Laboratories, Hercules, California, USA) and CD28-PE (AV7, SouthernBiotech) were used to investigate the activation of CD4+ T-helper cells (CD4+CD25+, CD4+CD28+) and CD8+ cytotoxic T cells (CD8+CD25+, CD8+CD28+). All CD3+ T cells were labeled with the pan-T-cell marker CD3-APC (CT-3, ThermoFisher Scientific). Bu-1+ B cells were stained with Bu-1-FITC (AV20, ThermoFisher Scientific). As a viability marker, 1.5 µL of 1 mg/mL 4′, 6-diamino-2-phenylindole (DAPI, ThermoFisher Scientific) was used. 20,000 vital PBMCs were recorded on a BD FACSCanto™ II (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer.

Methanol (>99.9% pure) and dipotassium hydrogen phosphate (anhydrous) were purchased from Fisher Chemical (Fair Lawn, NJ, USA). Sodium 2,2-dimethyl- 2-silapentane-5-sulfonate (DSS-d6, 98%) and deuterium oxide (D₂O, 98%) were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA).

LC‒MS/MS analysis was supported by Jingjie PTM BioLabs (Hangzhou, China). In brief, the tryptic peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile/in water) and separated by a homemade reversed-phase analytical column (25 cm length, 100 μm i.d.) on a nanoElute UHPLC system (Bruker Daltonics). After being subjected to a capillary source, the peptides were subjected to timsTOF Pro (Bruker Daltonics) mass spectrometry for further analysis in parallel accumulation serial fragmentation (PASEF) mode. The MS/MS data were processed by the MaxQuant search engine (v.1.6.6.0). Tandem mass spectra were searched using the human SwissProt database (20,366 entries) and reverse decoy database. FDR < 1%. The relative quantitative values of modified peptides in different samples were obtained by centralizing the signal intensity values in different samples. After filtering lysine lactylation sites (localization probability > 0.75), the relative quantitative values of each sample were obtained by two experiments. All the ratios of quantified lysine lactylation peptides were normalized according to their corresponding protein expression levels. The protein pathways were annotated using the KEGG database. The STRING database was used to identify protein–protein interactions of the DELPs.

TEM was performed as described previously with some modifications (Yuan et al., 2013 (link)). Briefly, small pieces of WT and miR-dKO testes were fixed in 0.1 M cacodylate buffer (pH 7.4) containing 3% paraformaldehyde and 3% glutaraldehyde plus 0.2% picric acid for 2 h in 4°C, then for 1 h at RT. Following washes with 0.1 M cacodylate buffer, the samples were post-fixed with 1% OsO₄ for 1 h at RT. Dehydration was performed using 30%, 50%, 70%, 90% and 100% ethanol solutions sequentially, followed by infiltration of propylene oxide and Eponate with BDMA overnight at RT. After infiltration, samples were embedded in Eponate mixture (Electron Microscopy Sciences, Hatfield, PA, USA) and polymerized at 60°C for 24 h. Ultrathin sections (60–70 nm in thickness) were cut with a diamond knife using an ultra-microtome (Leica). The sections were collected on collodion covered electron microscope nickel grids and stained with uranyl acetate and lead citrate. The ultrastructure of the samples was observed and photographed using a transmission electron microscope (Phillips CM10) at 80 kV.

JTP-74057, a highly specific and potent MEK1/2 inhibitor [35 (link)], and GDC-0994, a highly selective ERK1/2 inhibitor [36 (link)], were used to prevent the activation of the MEK/ERK pathway. Cells were treated with 1 nmol/L JTP-74057 or GDC-0994 in a humidified incubator for 24 h at 37 °C with 5% CO₂.
Doxorubicin is a chemotherapy medication used to treat cancer. Cells were treated with 5 μmol/L doxorubicin for 24 h, followed by the cell counting kit-8 (CCK-8) assay and western blotting.

Cells were plated in a 24 well plate (Corning, Corning, NY) and were allowed to adhere over night, at 37°C and 5% CO2. The following day, apoptosis was induced by several methods: Staurosporine- cells were incubated with 0.5 µM Staurosporine (Sigma-Aldrich) for 1.5 h and then washed with fresh media. γ-Irradiation- cells were irradiated (10Gy in GammaCell 220 Excel, MDS Nordion). Peroxide treatment- medium was aspirated and cells were washed with PBS with Ca2⁺ and Mg2⁺ (Biological Industries). Cells were incubated with H₂O₂ (30%, Sigma, Steinheim, Germany) diluted in PBS to a final concentration of 200 µM, at 37°C. After 1 h, cells were washed and fresh medium was added. Anti-FAS mAb- cells were treated with 1 µg/ml of anti-human FAS mAb (MBL, Naka-ku Nagoya, Japan). The number of apoptotic cells in the cultures was determined at different time points following apoptosis induction using Annexin/PI kit (MBL) according to manufacturer instructions.

IHC was performed on 4 µm formalin-fixed, paraffin-embedded tissue sections using BRG1 (SMARCA4), AR, FOXA1, ERG and C-Myc. The IHC process was performed on the Ventana ULTRA automated slide staining system using the Omnimap and Ultraview Universal DAB detection kit. The antibody details are provided in SI Appendix, Table S1. The following commercial kits from Roche-Ventana Medical System were used: Discovery CC1 (Cat No. 950-500), Discovery CC2 (Cat No. 950-123), OptiView Universal DAB Detection Kit (Cat No. 760-700), and OmniMap Universal DAB Detection Kit (Cat No. 760-149).