Sodium butyrate (SB) is a short-chain fatty acid and a safe antibiotic alternative. During 35 days, this study compared the impact of coated SB (Butirex C4) and lincomycin (Lincomix) on broiler growth, gut health, and litter hygiene in 1200 one-day-old Ross-308 broiler chicks that were randomly assigned into 5-dietary groups with 5-replications each. Groups divided as follows: T1: Basal diet (control), T2: Basal diet with buffered SB , T3: Basal diet with 100 g/ton lincomycin, T4: Basal diet with buffered SB  + 50 g/ton lincomycin, and T5: Basal diet with buffered SB  + 50 g/ton lincomycin. Birds were housed in a semi-closed deep litter house, where feed and water were available ad libitum. Results were statistically analyzed using ANOVA and Tukey’s post hoc tests.IGF-1 and TLR4 genes, the greatest villi length of the intestinal mucosa, and the lowest levels of litter moisture and nitrogen. Clostridia perfringens type A alpha-toxin gene was confirmed in birds’ caeca, with the lowest clostridial counts defined in T4.Combined dietary supplementation with SB and lincomycin (T4 and T5) significantly enhanced body weights, weight gains, feed conversion ratio, and profitability index. Also, carcasses in T4 and T5 exhibited the highest dressing, breast, thigh, and liver yields. T5 revealed the best blood biochemical indices, while T3 showed significantly elevated liver and kidney function indices. T4 and T5 exhibited the highest expression levels of Replacing half the dose of lincomycin (50 g/ton) with 0.5 or 1 kg/ton coated SB as a dietary supplement mixture showed the most efficient privileges concerning birds’ performance and health. Over the past decades, using antibiotics in subtherapeutic doses as feed additives for animal and poultry diets has achieved multiple benefits of promoting growth, minimizing mortalities, and reducing production costs although, numerous safety worries emerged due to that practice, as the persistent inclusion of antibiotics in the food animal industry caused injury to animal’s intestines, suppression of gut health, contamination of animal products with antibiotic residues, and polluting of the environment with antibiotic-resistant bacteria , 2. All The gut microbiome is a critical determinant of a bird’s performance and productivity , and recStreptomyces lincolnensis and is usually used against intestinal gram-positive bacterial infections. Lincomycin absorption through the gastrointestinal tract (GIT) was reported to be very weak, and its primary side effect is gastrointestinal inflammation, especially with long-term usage [Lincomycin is a natural lincosamide antibiotic that originates from rm usage , 7. The rm usage .Short-chain fatty acids (SCFAs) are vital metabolites in the intestinal microbiome and are essential as anti-inflammatory agents and for supporting gut health. In the Romick et al.  and ZhanTLR4) and insulin-like growth factor-1 (IGF-1) but decreases DNA fragmentation induced by pathogenic bacteria like Clostridium perfringens [Lactobacillus and Bifidobacterium beneficial bacteria [The buffered SB promotes intestinal mucosa modulation, regulates gene expression, augments SCFAs production, and improves protein synthesis –14. SB afringens –17. SB hfringens . Moreovebacteria . SB is abacteria , 20, 21.bacteria –25, as ibacteria .Previous studies indicated the application of coated SB at a rate of 600 to 1000 mg/kg of feed could enhance broiler chicken weights and feed conversion rates , 10. FurIGF-1 and TLR4), and litter microbial and chemical conditions.The current study examined the efficiency of dietary buffered SB 54% (Butirex C4®) supplement to replace lincomycin (Lincomix® 50) addition to broiler feed entirely or partly. The experimental design tested varying dosage regimens of both products. The evaluation included measuring a range of indices involving the weekly and overall performance parameters, carcass traits, blood biochemistry, intestinal histology and microbial counts, tissue gene expressions . The trial was conducted at the Animal and Poultry Research Center, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.A total of 1200 one-day-old Ross-308 broiler chicks from a commercial hatchery were weighed and randomly allocated into five groups with five replicates each (n = 45 birds/replicate). The bird grouping was as follows: T1; received basal diet (control), T2; basal diet with Butirex C4 (1 kg/ton in starter feed (0–15 d); and 0.5 kg/ton in grower and finisher feeds (16–35 d)), T3; basal diet with Lincomix at a level of 100 g/ton (the recommended dose) from 0 to 35 d, T4; basal diet with 50% of Butirex C4  and a half dose of Lincomix (50 g/ton), and T5; basal diet with Butirex C4  and a half dose of Lincomix (50 g/ton).Butirex C4®  is a novel feed additive of 54% SB coated with a physical-chemical matrix of buffer salts. Lincomix® 50  is a growth promotor and broad-spectrum antibiotic (lincomycin hydrochloride). The basal corn-soya bean meal-based diet was formulated to meet the nutrient requirements of Ross 308 broilers  during tad libitum in bell-shaped drinkers (4-litre capacity) and replaced with 8-litre drinkers in older age [The house with a semi-closed ventilation system was subdivided into 25 identical floor pens of 2.9 m × 1.6 m dimensions. The floor type was concrete covered with 7–10 cm wood shaving litter. Birds received similar managemental and hygienic conditions. After the chicks had arrived at the poultry house, they received 24 h of light for the first three days, then maintained under 23 L:1D for the remainder of the experiment. The temperature of the house was 32 ± 1 °C for the first 3-days, then gradually reduced by 0.5 °C per day until it declined to 24 °C . The humlder age . Feed walder age .Broilers received Newcastle disease virus (NDV) Hitchner B1 vaccination on the day 6. While on day 18, birds received NDV-Lasota vaccination through intraocular administration. Infectious Bursal Disease (IBD) and Avian Influenza (H5N1) vaccines were administered on day 14 (0.2 mL/bird) through S/C injection .Body weights (BW) of broilers were recorded weekly for each replicate on a pen basis at 1, 7, 14, 21, 28, and 35 days of age. The weekly body weight gain (BWG), average weekly feed intake (FI) and feed conversion ratio (FCR) for each week and the overall period of the experiment (from 1 to 35 days of age) was measured, as described previously . Daily bAt the end of the experiment day 35), 25 birds (5 birds/treatment) were slaughtered, defeathered, and eviscerated after 12 h of fasting . Carcass, 25 birdAt the end of the experiment (day 35), five blood samples per bird group were collected from the jugular vein after slaughter. After centrifuging for 15 min at 3000 rpm, sera were separated and stored at -20 °C until analysis. In serum samples, different biochemical parameters were measured by spectrophotometer  using spectrum diagnostics kits (Spectrum Diagnostics Egyptian Company for Biotechnology). Serum albumin , total p2 haemagglutination inhibition titers.Haemagglutination inhibition test was performed to assess NDV antibody titers, following  guidanceIGF-1 (growth-related gene) in both breast and thigh muscles and TLR4 (immune response-related gene) in the liver were evaluated at the mRNA level by qRT-PCR using RealMOD™ Green W² 2x qPCR mix  according to the manufacturer’s instructions. Each RT-PCR was performed in triplicate [β-actin gene [T) method (2−ΔΔCt) [https://primer3.ut.ee/) [About 100 mg of breast and thigh muscles and liver tissue were dissected from five birds per group. The samples were disrupted in a lysis buffer solution using a tissue homogenizer. Samples were processed for total RNA extraction according to the protocol of the easy-spin Total RNA Extraction Kit . The quantity and purity of RNA were assessed using Nanodrop . The cDNiplicate . Real-titin gene . The fol(2−ΔΔCt) . The pri.ut.ee/) –49. The After birds were slaughtered at day 35, five caecal contents and five deep-litter samples were collected from each group for microbiological examination. The upper 7 cm of deep litter were scraped from 3 different spots of each replicate pen and placed in sterile plastic bags , 51. All− 1 dilution was prepared by diluting and homogenizing 1 g of each sample in 9 ml sterile saline solution, followed by 10-fold serial dilutions till the 10− 10 dilution [− 1 dilution). Samples were kept at room temperature for 30–60 min and frequently shacked to allow the litter to mix well with the diluent [− 14 dilution. Then, 100 µl were taken from the last 3-dilutions and spread onto Nutrient Agar  plates to enumerate total aerobes and incubated for 24–48 h at 37 °C. Another 100 µL were spread onto Reinforced Clostridial Agar  plates to enumerate total Clostridia and incubated for 24–48 h at 37 °C under anaerobic conditions. Finally, the counts of bacterial colonies were reported as mean 10-logarithm colony-forming units (log10 CFU) for each gram of litter and caecal content.For microbial examination of caecal content, a 10dilution . For mic diluent , 51, folC. perfringens isolates was extracted using an extraction kit . The multiplex PCR assay was used to detect the presence of genes encoding alpha-toxin (cpa), beta-toxin (cpb), epsilon-toxin (etx), iotatoxin (iap) and CPE (cpe) [Genomic DNA of suspected PE (cpe) . Primer PE (cpe) . The PCRThe upper 7 cm of deep litter were scraped from 3 different spots of each replicate pen and placed in sterile plastic bags , 51. LitThe duodenum, jejunum, and ileum from five birds per group were collected and flushed with saline solution (0.9% NaCl) to remove contents, then fixed in 10% neutral buffered formalin for 48 h for histological examination. After fixation, samples were dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and embedded in paraffin wax. Sections of 3–4 μm in thickness were obtained by rotatory microtome, deparaffinized, and stained with haematoxylin and eosin (H&E) stain for examination under the light microscope .H&E stained sections were used for the histomorphometry. Approximately five intestinal tissue sections were measured by a high-power lens (X 40). Parameters measured include villus height from the tip of the villus to the crypt and crypt depth from the base of the villi to the submucosa. A computerized microscopic image analyzer, attached to a full HD microscopic camera , was used to determine the histomorphometric parameters using statistical analysis.Data were checked for normality by the Shapiro-Wilk test. Then, the mean differences were compared between groups via analysis of variance (ANOVA) and Tukey’s post hoc tests using PASW Statistics 18.0 software . Results were reported as means and standard error of the mean (SEM). Charts were generated with R  using ggplot2 , ggpubr The results of the growth performance analysis Table  revealedThe inclusion of dietary buffered SB supplement with reducing lincomycin to half its dose T4 and T5) showed a significant increase (P < 0.05) in dressing yield (Table  showed aThere was a significant increase (P < 0.05) in the relative weight of the liver in groups that received SB and lincomycin combination , whereas; T3 reported significantly (P < 0.05) lowered liver %. No significant differences (P > 0.05) were observed in the relative weight of the gizzard, heart, and immune organs weights (spleen and Bursa of Fabricius) between different groups.The highest serum total protein and albumin concentrations were shown in T5 (P < 0.05), as presented in Table 2, respectively) than T1 and T2 . However, those levels did not exhibit significant differences (P = 0.430).Haemagglutination inhibition titers of NDV antibodies in birds’ sera, at day 35, showed that T3, T4, and T5 presented higher mean haemagglutination inhibition titers  in the breast muscle compared to the control . The transcript level of the TLR4 gene (immune response-related gene) in the liver was significantly higher in T3, T4, and T5 than in T1  increased the transcript level of the 10 CFU of total aerobic bacteria (P = 0.045) and 9.32 ± 0.53 log10 CFU of total Clostridia. The T3 and T5 groups did not demonstrate improvement from the control group (T1) but had bacterial counts higher by nearly 1.19 log10 CFU/g  displayed both C. perfringens type A alpha toxin and C. perfringens enterotoxin genes.The amplification of the Regarding the microbial quality of deep litter at day 35 Fig. , there wIn the duodenum, the intestinal villi and crypt depth in T2, T3, T4 and T5 were significantly greater than in control group T1 (P < 0.05). There were significantly higher increases in the length of duodenal villi and crypt depth in T4 and T5 compared to T2 and T3. In the jejunum, T5 showed the highest villi length, followed by T4, while T3 exhibited the lowest values ((P < 0.05). Jejunal crypt depth was the largest in T5. In the ileum, T5 recorded the highest villi length and crypt depth, followed by T4 (P < 0.05) Table ; Fig. 5.In the current study, performance data during the starter period revealed that BW, BWG, and FCR were not affected by either the dietary supplementation of SB or lincomycin, which, according to an earlier study , may be The combined supplementation of SB and lincomycin in T5 significantly reduced weekly FI at day 14. Other researchers have published similar findings; regarding the strong tendency of combined butyrate supplements to reduce feed intake and improve feed efficiency . In growTLR4) activation by lowering the activation of mitogen-activated protein kinase and nuclear factor B (NF-B) pathways, as well as the generation of proinflammatory cytokines [SB promotes nutrient digestibility and absorption due to the modification of gut microstructure and increased digestive enzymes’ activity, hence improving the productive efficiency of broilers , 75. SB ytokines , so imprytokines , 78 or lytokines .IGF-1), which promote growth and enhances feed utilization efficiency in broilers [In the present study, SB and lincomycin supplements enhanced dressing yield, especially in T4. Similar studies showed that organic acids and antibiotic combination supplements exhibited higher carcass yields . Furtherbroilers , resultiAccording to our results, no significant differences were observed in breast and thigh muscles between different groups. However, the percentage of the economically valuable parts, like drumstick muscle, was increased in SB and lincomycin-supplemented groups compared to the control, which could indicate faster maturity due to rapid growth. Like our findings, drumstick meat yields increased by organic acid supplementation compared to the control group . On the Combining dietary SB with lincomycin in T4 and T5 enhanced birds’ liver weights better than those supplemented with lincomycin (T3). That may be related to enhanced liver function indices via decreased ALT and AST levels in our biochemical results, as increased serum levels of AST demonstrated possible hepatocyte and liver dysfunction . SimilarIn this study, the mixture of SB and lincomycin in T5 significantly improved serum total protein concentrations, which indicated increased dietary protein utilization . The incHaemagglutination inhibition antibody titer is a marker of birds’ humoral immunity. In this study, the SB and lincomycin supplements did not enhance the humoral immunity of broilers at 35 days of age against NDV compared to the control. An earlier study  indicateIGF-1 gene is protein-encoded and produced by the liver under the stimulatory effect of growth hormone. It stimulates systemic body growth and has anabolic effects on different cells in the body, especially skeletal muscle, cartilage, bone, liver, kidney, and lung cells. In addition to the insulin-like activity, the IGF-1 gene can regulate cellular DNA synthesis [IGF-1 m-RNA in the breast muscle of T4 and T5 birds and the highest upregulation in the thigh muscle of the T5 group. These results come per the current results of breast and thigh relative weights. Formerly, the supplementation of broilers with SB was reported to increase the expression level of the IGF-1 gene [TLR4 gene is encoded by the TLR4 protein (a member of the toll-like receptor family). Its activation leads to an intracellular signaling pathway and inflammatory cytokine production, which stimulates the innate immune system [TLR4 gene in liver tissue was increased in T3, T4, and T5, while it was the highest in the T5 group. Smith et al. [TLR4 m-RNA in birds fed butyrate in their diet.The ynthesis . In thisF-1 gene . The TLRe system . In our h et al.  reportedAvBD9 gene in the caeca and caecal tonsil when supplementing birds with 0.1% butyrate compared to 0.2% butyrate [In the current study, the combination of SB and lincomycin at half their recommended doses significantly lowered the caecal bacterial and clostridial counts. These findings did not apply to litter. These results agreed with previous work that displayed that micro-encapsulated butyrate diminished the pathogen colonization in caeca . Notablybutyrate . They rebutyrate , 97, 98.butyrate .C. albicans. Those reports could interpret the significantly higher caecal bacterial and clostridial counts in lincomycin-supplemented birds T3 compared to T4, where birds received a diet with partial substitution of lincomycin with SB.The supplementation of dietary butyrate derivatives boosted butyrate concentration in the large intestine and the number of neutrophils in the colonic lamina propria, which indicated that butyrate is a powerful promoter of neutrophil activity during infection . LikewisRegarding litter quality parameters, the combined dietary supplementation with SB and lincomycin lowered the litter’s moisture and nitrogen contents in T4 and T5. Previous studies pointed out that SB reduced intestinal pH and increased the activity of digestive enzymes yet increasing the digestibility and absorption of protein and minerals –77. DietIn the current study, SB supplementation significantly increased the villi length and crypt depth of the duodenum and villi length of the ileum. These findings correlated with earlier experiments that reported improved villus length and crypt depth in the duodenum by 0.2, 0.4, and 0.6% dietary butyrate concentrations . SimilarIGF-1 and TLR4 genes exhibited the highest expression levels in SB + lincomycin-supplemented groups (T4 and T5). Caecal bacterial and clostridial counts were the lowest in T4. Litter hygiene became more satisfactory in T4 and T5 than in other groups. Dietary SB + lincomycin (T4 and T5) increased the villi length of the intestinal mucosa. Hence, supplementing broilers’ diets by SB with lincomycin in half their doses  positively impacted birds’ performance and functional indices.Combined dietary supplementation with buffered SB and lincomycin (T4 and T5) significantly enhanced body weights, weight gains, FCR, profitability index, and carcass yields. SB supplementation (in T4 and T5) mitigated the antibiotic-induced adverse effects of lincomycin on the intestine, liver, and kidney, which appeared in lowered caecal bacterial counts, improved intestinal histomorphology, and enhanced blood biochemistry indices compared to T3.


Brassica rapa subsp. chinensis). Mature Pak Choi plants were harvested and stored at two different temperatures, 4 °C and 25 °C. Senescence was tracked via chlorophyll content and leaf yellowing. Concurrently, alterations in the total nitrogen, nitrate, and protein content were quantified on days 0, 3, 6, and 9 in old, mid, and young leaves of Pak Choi plants. As expected, 4 °C alleviated chlorophyll degradation and delayed senescence of Pak Choi compared to 25 °C. Total nitrogen and protein contents were inversely correlated, while the nitrate content remained nearly constant across leaf groups at 25 °C. Additionally, the transcript levels of genes involved in nitrogen assimilation and remobilization revealed key candidate genes that were differentially expressed between 4 °C and 25 °C, which might be targeted to extend the shelf life of the leafy vegetables. Thus, this study provides pivotal insights into the molecular and physiological responses of Pak Choi to post-harvest storage conditions.In the agricultural industry, the post-harvest leafy vegetable quality and shelf life significantly influence market value and consumer acceptability. This study examined the effects of different storage temperatures on leaf senescence, nitrogen assimilation, and remobilization in Pak Choi ( Nitrogen (N) functions as a primary nutrient and serves a critical role in the life cycle of plants. It is essential for standard plant growth, development, and stress responses . N is praap2, aap3, and aap8 mutants of amino acid transporter genes exhibited delayed leaf senescence [NRT1.1/NPF6.3 led to delayed leaf yellowing under N-deficient conditions [Leaf senescence is the final phase of leaf development and is characterized by the loss of chlorophyll, leaf yellowing, and degradation of chloroplast proteins . Since cnescence ,10,11, tnditions . While t2 with high CO2 treatment enhanced carotenoid content without compromising the visual quality of lettuce, spinach, and corn salad [Leafy vegetables are highly perishable products and undergo significant nutritional loss during post-harvest storage. A comprehensive examination of the nutrient composition and visual quality of lettuce during post-harvest storage revealed a remarkable decrease in bioactive and antioxidant compounds, accompanied by deteriorated visual appeal . Thus farn salad . In anotrn salad . Exogenorn salad ,17,18. Irn salad .Brassica rapa subsp. chinensis) is one of the most important leafy vegetables in China and represents 30–40% of the total vegetable consumption [Pak Choi  were sterilized and sown on an agar medium containing Murashige and Skoog (MS) basal salts, 1% (w/v) sucrose, and 2.5 mM MES , pH 5.6. After undergoing imbibition at 4 °C in the dark for 2 days, seedlings were germinated in a tissue culture chamber at 22 °C with 60% relative humidity under 16 h light/8 h dark condition (100 µmol·m−2·s−1 light intensity). After 7 days, 42 seedlings were transplanted to seven hydroponic trays, each containing 3 L of Hoagland’s solution, and were placed in a growth chamber at 22 °C with 60% relative humidity under 16 h light/8 h dark condition (100 µmol·m−2·s−1 light intensity). The nutrient solution was renewed every 4 days.Seeds of Pak Choi . Twelve leaves from each plant were grouped as old , mid (leaves 5–8), and young (leaves 9–12) and used for all assays. The four leaves in each group were arranged on top of each other, and leaf discs were punched out and frozen in liquid nitrogen for qPCR analysis. Similarly, leaf discs were taken from multiple locations for total chlorophyll, nitrate, and total protein assays from old, mid, and young leaves. Leaf discs made from the four leaves were pooled as a single replicate. The remains of the leaves from each group were dried in individual glass beakers at 65 °C for total N analysis.Next, the remaining 36 plants were harvested, divided into two groups, and then stored under darkness at 4 °C and 25 °C, as shown in The total chlorophyll content in the leaves was determined as described previously ,22. Infr2, H2O, NO2, SO2, etc. These gases were then absorbed and separated using chromatography columns and analyzed with thermal conductivity detectors.For the measurement of total N content, we used the organic elemental analyzer vario EL cube . Briefly, the old, mid, and young leaves were dried at 65 °C and ground into a fine powder, and the analysis was based on combustion, during which the samples generated gases like COThe nitrate content in the leaves was measured using the method described previously ,24.To determine the total protein contents, the leaves were ground in liquid nitrogen into a fine powder, dissolved in a lysis buffer , and incubated on ice for 30 min. The tubes were then centrifuged at 14,000 rpm, 4 °C for 10 min; the supernatant was mixed with Bradford solution ; and the absorbance was recorded at 595 nm. The standard curve constructed using known concentrations of bovine serum albumin was used to calculate the total amount of protein in samples.TM Plant Total RNA Purification Kit  according to the manufacturer’s instructions. RNA samples were prepared DNA-free, and the quality was evaluated using an Agilent 2100 Bioanalyzer . RNA with an RIN value of >7.0 was then reverse transcribed to cDNA, using the iScript™ cDNA Synthesis Supermix , following the manufacturer’s instructions. PCR primers were designed using Primer3, and qRT-PCR was performed using a Bio-Rad CFX96 Connect real-time PCR instrument and SsoAdvanced Universal SYBR Green Supermix . Actin was used as the control. Primers are listed in The total RNA of Pak Choi leaves was extracted using the FavorPrept-test in Microsoft Excel.All results were expressed as the mean ± standard deviation (SD). Six biological replicates and three technical replicates were used for all experiments. The statistical analysis was performed using a two-tailed Leaf senescence is a critical developmental sequence in plants, as it is aligned with their resource management strategies. Plants have evolved to adopt programmed cell death and senescence to handle unpredictable nutrient shortages. Leaf senescence particularly focuses on the elimination of inefficient and aging photosynthetic organs, which constitute the plant’s nutrient storage ,26,27. TThe storage temperature plays a huge role in maintaining the quality of the leafy vegetables, which are highly perishable. The optimal temperature to achieve the best shelf life for many perishable produces is approximately 0 °C, and every 10 °C increase decreases the shelf life by 2-to-3-fold. In strawberries, low temperature storage delayed leaf aging and prolonged shelf life . The effN stands out as a pivotal element that is remobilized in massive quantities during natural senescence . To undeThe N remobilization from senescing leaves depends, to an uncertain extent, on inorganic N molecules such as nitrate, ammonium, and urea . The rolDuring leaf senescence, chloroplasts and proteins degrade, releasing nutrients like sugars, lipids, and amino acids, which are then remobilized. Previous studies have identified specific amino acids, notably glutamine, serine, asparagine, glutamate, and aspartate, as predominant in phloem sap; however, variations may occur depending on the plant species ,31,32,33NIA1 was highly expressed in the old and mid leaves on D0. However, its transcripts were completely suppressed in all leaves stored at 25 °C from D3  were not included in the qRT-PCR study, as the quality of the RNA isolated from those samples was poor. Differentially expressed genes between 4 °C and 25 °C are shown in  from D3 . In contnescence , whereasNiR was found to be downregulated in Arabidopsis [NiR transcripts were detected in the old leaves on D0 but were found reduced upon storage at 4 °C, similar to Arabidopsis natural senescence. However, at 25 °C storage, the expression levels of NiR increased from D6 in both old and mid leaves  and ferredoxin-dependent glutamate synthase (FD-GOGAT). However, as chloroplasts degrade during leaf senescence, the localization of glutamine synthetase shifts, with GS activity transitioning to the cytosol, where GS1 compensates for the reduced GS2 activity ,35. Whenin roots , was notnd 25 °C . The mRN at 4 °C . Additiobidopsis .GS1/GLN1-3.1 were found in low levels in the old leaves on D0 and were completely suppressed upon post-harvest storage at both 4 °C and 25 °C  and an E3 small ubiquitin-related modifier (SUMO) ligase SIZ1 regulate the leaf senescence process under N deficiency and N assimilation, respectively ,43. In ASIZ1 genes, SIZ1.1 was not detected on D3, whereas SIZ1.2 and SIZ1.3 showed low mRNA levels on the same day , the rate of senescence is accelerated, marked by the yellowing of leaves and a rapid decrease in chlorophyll content. At a lower temperature (4 °C), however, senescence is delayed, preserving the visual and textural quality of the leaves.Furthermore, our study illustrated the differential response of total nitrogen, nitrate, and protein content based on storage temperature. N remobilization appeared to be altered by storage conditions, impacting both young and old leaves differently. Key transporters and enzymes involved in N assimilation and remobilization were also impacted by temperature. Intriguingly, certain transporters  found in other species, like Arabidopsis, were absent in Pak Choi under post-harvest conditions, suggesting unique senescence pathways or potential redundancies in Pak Choi. The expression patterns of the nitrate transporters and associated genes, especially under 4 °C storage conditions, highlighted a deactivated nitrate remobilization mechanism, which contrasts with natural senescence conditions.Given the intricate orchestration of processes, we can expect that optimizing post-harvest storage conditions will significantly extend the shelf life of leafy vegetables, such as Pak Choi. Furthermore, understanding the molecular mechanisms underlying post-harvest senescence can inform agricultural practices and post-harvest management to minimize nutrient loss and maintain product quality. From our observations, it is also anticipated that different leafy vegetables might exhibit unique patterns of N remobilization and protein degradation, which can open avenues for species-specific post-harvest interventions.


SOCE-associated regulatory factor (SARAF) acts as an inhibitor of the SOCE response and, therefore, can be a potential antitumor factor. Herein, we generated a C-terminal SARAF fragment to evaluate the effect of overexpression of this peptide on the malignancy of triple-negative breast cancer cell lines. Using both in vitro and in vivo approaches, we showed that overexpression of the C-terminal SARAF fragment reduced proliferation, cell migration, and the invasion of murine and human breast cancer cells by decreasing the SOCE response. Our data suggest that regulating the activity of the SOCE response via SARAF activity might constitute the basis for further alternative therapeutic strategies for triple-negative breast cancer.Triple-negative breast cancer has a poor prognosis and is non-responsive to first-line therapies; hence, new therapeutic strategies are needed. Enhanced store-operated Ca Breast cancer is the most common type of cancer and the leading cause of death in women worldwide ,2. It is2+ signaling is deregulated in several types of cancers  was added to the supernatant samples, and then boiled for 5 min, followed by size fractionation on SDS–PAGE. Following SDS-PAGE, proteins were transferred to nitrocellulose membranes , which were then blocked for 1 h with BLOTTO [4% w/v nonfat dry milk/ 0.1% v/v Tween-20 in Tris-buffered saline ] followed by 2 h or overnight incubation with primary antibodies. After 3 washes with BLOTTO, the membranes were incubated with the appropriate HRP-conjugated secondary antibody for 1 h. After 3 washes for 10 min each with 0.1% v/v Tween-20/TBS, immunoblots were visualized using Pierce ECL Western Blotting Substrate . The images were acquired with a Mini HD9 imager  and quantified measuring the optical density of the bands with Image J2/FIJI v.2.9.0 software.Cells were lysed in lysis buffer containing: 50 mM Tris-HCl, 150 mM NaCl , 1 mM ethylenediaminetetraacetic acid , 1 mM sodium orthovanadate , 5 mM NaF , 1% v/v Triton X-100 , protease inhibitors 1 mM phenylmethylsulfonyl fluoride  and a protease inhibitor cocktail , adjusted to pH 7.4 for 30 min at 4 °C. The lysates were centrifuged at 12,000× v/v Triton X-100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 8.0), 1 mM NaVO4, 5 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail for 15 min at 4 °C, followed by centrifugation at 11,000× g for 10 min at 4 °C. The supernatants were incubated with anti-RFP antibodies   for 45 min. Cells were fixed with a fixative solution , and three images were obtained from each wound (t16h). The relative sizes of the wounds at t0 and t16h were obtained using a wound-healing size plugin of the Image J2/FIJI v.2.9.0 software and results were expressed as percentage of closing ([(Area t0h − Area t16h)/ Area t0h] × 100).MDA-MB-231 and 4T1 cells transfected with mScarlet or mScarlet-C-SARAF were seeded on coverslips pre-coated with 5 µg/mL of Human plasma Fibronectin , forming a monolayer. After 24 h, cells were depleted of serum for 4 h and then scratches were made with a sterile micropipette tip (p200). Detached cells were removed by washing three times with DPBS, and then the medium was replaced with DMEM 2% FBS and three images were obtained from each wound  [Stable cell lines of 4T1 that overexpress mScarlet or mScarlet-C-SARAF were injected to generate two animal models associated with the animal protocol 20382-MED-UCH, which was approved by the Bioethics Committee of the Faculty of Medicine, Universidad de Chile. Female Balb/c mice were orthotopically injected into the fatty tissue of the fourth mammary gland  with stable cell line 4T1. Cells were resuspended in Dulbecco’s modified phosphate-buffered saline (DPBS) with 0.04% trypan blue  . The gen, 50 µL) :VellipsoAdditionally, organs were removed, fixed, and processed for histological analysis and the evaluation of metastatic sites.w/v formaldehyde—freshly prepared from paraformaldehyde—4% w/v sucrose in DPBS, pH 7.4) for 24 h at 4 °C. Subsequently, the tissue was processed with increasing concentrations of alcohol, cleared with xylol and embedded in paraffin. Serial sections with a thickness of 3 μm were obtained and adhered to slides for histological hematoxylin and eosin and Masson’s trichrome staining. Images were acquired with a Leica DFC425C camera installed on a Leica DM2500 CCD microscope with 10× and 40× objectives .The main organs that were affected during cancer metastasis, either by indirect effect of primary tumor growth, were removed, washed with DPBS and fixed in fixative solution  tests and Dunnett’s post-test were applied. Analyses were carried out using GraphPad Prism v8.0 .


Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia.The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF.In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.The online version contains supplementary material available at 10.1186/s13062-023-00378-6. Giardia lamblia causes outbreaks of waterborne diarrhea disease known as giardiasis that predominately afflicts developing countries [ountries –3. It isountries . Variousountries . Followiountries . Antigenountries .G. lamblia is a unique model for differentiation of protozoan pathogens, as its two life-cycle stages can be reproduced in vitro [cwp1-3 genes are up-regulated with similar kinetics by transcription factors GARP1, ARID1, MYB2, WRKY, PAX1, E2F1, and MBF1 [Many protozoan pathogens of medical importance have a cyst form . G. lambin vitro , 8. Tropin vitro . Cyst, tin vitro , 9. Encyin vitro , 2. Syntin vitro –12. Duriand MBF1 –19.G. lamblia raises great biological interest for understanding eukaryotic evolution, because it has many primitive features, including the utilization of bacteria-like anaerobic metabolism and the lack of clear homologs of many known basal transcription factors, DNA replication proteins, and RNA processing factors [G. lamblia [G. lamblia [G. lamblia genome database [G. lamblia has a partial or unusual proteasome and autophagy pathway for protein metabolism [ factors , 20, 21. lamblia . In addidatabase , 24. Thetabolism , 24.Eukaryotic cells use autophagy to survive in various stresses, including starvation, oxidative stress, aging, and diseases –27. AutoG. lamblia and other protozoa, including Plasmodium, Trypanosoma, and Trichomonas [G. lamblia [Proteasome and autophagy systems involving in clearance of misfolded proteins, plays a positive role in differentiation of various cell types, such as neuronal, myocardial, and lens cells –33. Durichomonas , 35–39. Drosophila, the myeloid leukemia factor (mlf) gene products are implicated in the regulation of cell differentiation [Giardia [Giardia MLF is a positive driver for encystation using a CRISPR/Cas9 system [G. lamblia, we have used a mutant with a deletion of kinase domain, cyclin-dependent kinase 2 mutant 3 (CDK2m3), and double staining for visualization of potential factors with a role in protein metabolism pathway [G. lamblia, suggesting that MLF is important for aberrant protein substrates of the proteasome or autophagy [As has been found in mammals and ntiation , 41 To d[Giardia –43. Ther[Giardia , 45. Lik9 system . To stud pathway , 47. We utophagy . MLF alsutophagy , suggestG. lamblia because of the lack of some autophagy machineries, such as p62 and ATG5 [G. lamblia. However, treatment with an autophagy inhibitor, 3-methyl adenine (3-MA), resulted in a decrease in the levels of CDK2m3 and MLF proteins and the numbers of their vesicles. Further use of CRISPR/Cas9 system and complementation system suggest a positive role of MLF in survival from stress-induced cell death in the stress inducer treatment. We also found that MLF regulated stress-induced CDK2m3 formation, and that the human MLF2 had activity in inducting encystation, like Giardia MLF. Our results suggest that MLF is an evolutionarily conserved factor and functions in survival in stress and mutant protein metabolism, and that MLFVs, like autophagy compartments, have stress-induced characteristics and can be decreased by 3-MA.In this study, we will use a MLF-based assay to test the effect of autophagy-inducing agents on protein metabolism. Little is known of the mechanism of autophagy in and ATG5 , 24. To and ATG5 –64. We fGiardia MLF also can induce encystation for the differentiation into cysts [G. lamblia , indicating a high colocalization degree. There was a significant induction in cyst number and CWP1 protein level in the hMLF2- expressing cell lines compared with the Giardia MLF-expressing cell line  may function in maintaining protein stability and vesicle trafficking , 65. Likto cysts –42. We tlia Fig. A. Interelia Fig. B. In addion Fig. C, D. Peardia MLF . The aveGiardia MLF. It has been shown that Giardia MLF can induce the expression of cwp1-3 and myb2 genes [mlf, cwp1-3 and myb2 significantly decreased in the MLFm-expressing cell lines in comparison with the wild-type MLF-expressing cell line  template cassette with the neomycin phosphotransferase (neo) selectable marker  cell line was confirmed by PCR  inhibitor, which has been often used as an autophagy inhibitor as it can prevent the activation of autophagy and decrease autophagosome number in mammalian cells , 78. To G. lamblia, we also used the above stress inducers to test the drug sensitivity of the MLF KD cell line. All six stress inducers had growth-inhibiting effect on the control cells (obtained from ATCC), were cultured in modified TYI-S33 medium [5 cells/ml. In experiments exposing G. lamblia vegetative trophozoites to different drugs, WB clone C6 trophozoites were cultured in growth medium with 80μ M MG132, 36 μM rapamycin, 100 μM chloroquine, 5 μM nocodazole, 50 mM DTT, or 217 μM G418 for 24 h.Trophozoites of 3 medium . Encysta3 medium . BrieflyCyst count Cyst count was performed as previously described [escribed , 47.G. lamblia cell line during vegetative or encystation stages using TRIzol reagent (Invitrogen). For RT-PCR, 5 μg of DNase-treated total RNA was mixed with oligo (dT)12–18 and random hexamers and Superscript II RNase H− reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR. For quantitative real-time PCR, SYBR Green PCR master mixture was used (Kapa Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the mlf , cwp1 , cwp2 , ran , and 18S ribosomal RNA  genes: mlfrealF and mlfrealR; cwp1realF and cwp1realR; cwp2realF and cwp2realR; ranrealF and ranrealR; 18SrealF and 18SrealR. Each primer pairs were determined for amplification efficiency ~ 95% based on the slope of the standard curve. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as a relative expression level over control. Transcript levels were normalized to 18S ribosomal RNA levels. Results are expressed as the means ± 95% confidence intervals of at least three separate experiments. Student’s t-tests were used to determine statistical significance of differences between samples.Synthetic oligonucleotides used are shown in Additional file mlf gene was amplified using two primer pairs, mlfmF and MLFMR, and mlfmR and MLFNF. The two PCR products were purified and used as templates for a second PCR. The second PCR also included primers MLFNF and MLFMR, and the product was digested with NheI and MluI and cloned into the NheI and MluI digested pPop2NHA [Giardia mlf 5' untranslated region and hmlf2 gene flanked with NheI and MluI sites , includes a guide sequence targeting 20-nucleotide of the mlf gene (nt 61–80)[All constructs were verified by DNA sequencing with a BigDye Terminator 3.1 DNA Sequencing kit and an Applied Biosystems 3100 DNA Analyzer (Applied Biosystems). Plasmid 5’Δ5N-Pac was a gift from Dr. Steven Singer and Dr. Theodore Nash . PlasmidpPop2NHA . To makent 61–80).Escherichia coli and purified as previously described [The hMLF2 gene was amplified from hMLF2 synthetic fragment using oligonucleotides hMLF2F and hMLF2R. The product was cloned into the expression vector pET101/D-TOPO (Invitrogen) in frame with the C-terminal His and V5 tags to generate plasmid phMLF2. The phMLF2 plasmid was transformed into escribed . ProteinG. lamblia trophozoites were transfected with plasmid pNMLFtd and pgCas9, and then selected in 217 μM G418. The culture medium in the first replenishment contained 6 μM Scr7 and the same concentrations of G418 [G. lamblia WB trophozoites. Subsequent analysis was performed after the removal of the drug for 1 month. Stable transfectants were further analyzed by Western blotting, or DNA/RNA extraction as previously described [mlf gene with the neo gene was confirmed by PCR and sequencing as previously described [For CRISPR/Cas9 system in strategy 4,  of G418 . SCR7 is of G418 . The MLFescribed . The repescribed . Westernescribed , anti-CWescribed , anti-RAescribed , or preip < 0.05. **, p < 0.01. ***, p < 0.001.).The stable cell lines were cultured in growth medium under puromycin selection. Cells cultured in growth medium with or without indicated drugs, or encystation medium for 24 h were harvested and subjected to immunofluorescence assay as previously described , 107. CeG. lamblia trophozoites were determined as previously described [6 cells were harvested, washed in PBS, and incubated with 2’, 7’-dichlorodihydrofluorescein diacetate at a concentration of 25 μM in 200 μl cell suspensions. The cell suspensions were kept in dark at room temperature for 1 h. Data were collected from a Paradigm Multi-Mode Plate Reader (Beckman Coulter) at 530 nm after excitation at 488 nm.ROS levels in escribed , 109. WBThe stable cell lines were cultured in growth medium or inoculated into encystation medium with puromycin and harvested and lysed after 24 h as previously described . The celAdditional file 1. Supplementary figures and tables.


Although prolonged liver damage exacerbated the fibrosis in mice of both sexes, activated HSCs and Ly6Chigh MoMφs were more numerous and active in the livers of female mice than those of male mice. After eight weeks of washout, only fibrotic females reported no activated HSCs, and a phenotype switching of Ly6Chigh MoMφs to anti-fibrogenic Ly6Clow MoMφs. The early stages of liver fibrosis mostly affected males rather than females, while long-term chronic liver damage was not influenced by sex, at least for liver fibrosis. Liver repair and regeneration were more efficient in females than in males.Liver fibrosis is a common and reversible feature of liver damage associated with many chronic liver diseases, and its onset is influenced by sex. In this study, we investigated the mechanisms of liver fibrosis and regeneration, focusing on understanding the mechanistic gaps between females and males. We injected increasing doses of carbon tetrachloride into female and male mice and maintained them for a washout period of eight weeks to allow for liver regeneration. We found that male mice were more prone to developing severe liver fibrosis as a consequence of early chronic liver damage, supported by the recruitment of a large number of Ly6C Chronic liver diseases (CLDs) usually develop from persistent liver injury of different etiologies, including viral, non-alcoholic, and alcoholic liver diseases. CLDs are a major global health issue, considering that, in 2021, they caused two million deaths ,4,5. Durhigh Gr-1high monocytes that orchestrate tissue response in mice with acute hepatic injury. Additionally, a greater expression of the IFNγ gene was recorded in female mice than in male mice during spontaneous recovery from acute damage . T. TPDGF-Aver time B 42]..PDGF-A ahigh decreased until it reached basal levels. However, by comparing the number of Ly6Chigh MoMφs to that of Ly6Clow MoMφs, we found that the number of anti-inflammatory Ly6Clow MoMφs was higher than the number of Ly6Chigh MoMφs in fibrotic mice after recovery. This finding suggested that a phenotype switching of these cells might have helped in sustaining liver regeneration  and reviewed by the University of Padova’s Animal Welfare and Etichal Review Board. Balb/C mice (eight weeks old) were maintained in a conventional temperature-controlled room and followed a 12 h/12 h light/dark cycle. They were housed in individually ventilated cages (IVC) and were provided free access to a standard rodent diet and tap water.4, cat. N. 289116, Sigma-Aldrich, Saint Louis, MO, USA) in corn oil (from 0.17 to 0.72 mL/kg × body weight) twice a week for a maximum of 12 weeks. Male and female mice were randomly divided into two groups, which included the CCl4-treated fibrotic mice (n = 5 per time point per gender) and control corn-oil-treated mice (n = 5 per time point per gender). The selected time points for suppression were baseline , 6 weeks, 12 weeks (end of CCl4 treatment), and 20 weeks . Then, the mice were sacrificed, and their livers were excised, weighed, and stored in 10% formalin  or snap-frozen with liquid nitrogen and stored at −80 °C until further analysis.Chronic liver injury was induced by intraperitoneally injecting increasing doses of carbon tetrachloride  in a Petri dish, and the tissue was smashed using the plunger of a syringe to gently pass the obtained cell suspension across the filter. The cell suspension was then transferred to a 15 mL conical tube, mixed with PBE buffer , and centrifuged twice at 300× g for 5 min. The cells were incubated for 15 min with 50 µL of Fc block (1:100) at room temperature  and then stained with a mixture of fluorochrome-conjugated antibodies. The samples were incubated with anti-mouse CD45-PE , anti-mouse CD3-PE/Cyanine7 , anti-mouse/human CD45R-B220-PE/Cyanine7 , anti-mouse NK1.1-PE/Cyanine7 , anti-mouse/human CD11b-BV711 , anti-mouse F4/80-APC , and anti-mouse Ly6C-SB436  for 15 min at room temperature. Then, the samples were washed twice with PBE and fixed in 2% formaldehyde. The cell samples were analyzed using a BD LSR II flow cytometer using the BD FACS Diva software . Single-stained samples and unstained samples were used as compensation controls, and the data were analyzed using the FlowJo software . Neutrophils were identified as CD45+ CD11b+ Ly6G+ cells, resident macrophages were identified as CD45+ Ly6G− CD3− NK1.1− B220− CD11b+ F4/80high cells, and recruited macrophages (Momφs) were found to be Ly6G− CD3− NK1.1− B220− CD11b+ F4/80int Ly6Chigh or Ly6Clow cells. The ratio of Ly6Chigh to Ly6Clow in the fibrotic mice that were administered CCl4 for 12 weeks was compared to the ratio recorded in fibrotic mice after the washout period to assess phenotype switching from Ly6Chigh to Ly6Clow macrophages.A flow cytometry analysis was performed to identify neutrophils and resident and recruited macrophages. After perfusion with saline, the livers were collected in 0.1% PBS-EDTA buffer and incubated at 37 °C for 45 min with a digestion mixture of 20 mg/mL Collagenase IV and 0.005 µg/mL DNAse I in PBS. The digested liver tissue was filtered with 40 µm Falcong for 3 min, the supernatant was transferred to a 96-well plate and incubated with the reagent mixture. The absorbance was recorded at 560 nm using the VICTOR Nivo Multimode Microplate Reader , and the hydroxyproline content was calculated according to a linear regression curve.The levels of hydroxyproline, which can be used in liver tissue as an index of fibrosis, were detected using a commercial Hydroxyproline Assay Kit , following the manufacturer’s instructions. Briefly, 10 mg of hepatic tissue was homogenized with a pellet pestle in 200 µL of a solution containing distilled water and hydrochloric acid (1:1). The samples were then heated at 120 °C for 3 h using a multi-block heater. After centrifugation at 10,000 ® . The images of the stained slices were acquired using Nikon Eclipse Ti-S .To perform the histological analysis, the hepatic tissue sample was fixed in 10% formalin and processed for paraffin embedding. After deparaffinization and rehydration, 5 µm thick sections were stained with the Masson-Goldner kit . After dehydration, the slides were mounted with Eukitt2O2 for 20 min to block endogenous peroxidase. The sections were incubated for 1 h at 37 °C with the following secondary antibodies: goat anti-rat HRP-conjugated IgG (1:200)  and goat anti-mouse HRP-conjugated IgG (1:500) . Finally, the slices were incubated with the DAB-Substrate  for 8 min at room temperature, counterstained with hematoxylin  for 30 s, dehydrated, and a coverslip was placed with Eukitt® . Images were captured using a Nikon Eclipse Ti-S microscope and analyzed with the ImageJ software .First, tissue sections (5 µm thick) were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed in citrate (pH 6.0) or Tris-EDTA (pH 9.0) buffer for 20 min. Permeabilization for intracellular epitopes was performed using 0.2% Triton-X-100 buffer for 10 min. Blocking was performed by incubating the slices in PBS containing 5% fetal bovine serum (FBS) and 1% bovine albumin serum (BSA) for 30 min at room temperature. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal αSMA antibody (1:500) , mouse monoclonal MMP-9 antibody (1:200) , and rat monoclonal TIMP-1 antibody (1:100) . After washing with a solution of phosphate-buffered saline (PBS), the tissues were exposed to 3% H®, SYBR® Green RT-PCR Kit . All samples were analyzed in triplicate with 200 ng of RNA in each well. The real-time cycling conditions were set as follows: the reverse transcription was performed at 50 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C for the real-time polymerase chain reaction, and 15 s at 95 °C, 15 s at 55 °C, and 15 s at 95 °C for the elaboration of the melting curve. The primer sequences used to assess gene expression are presented in −∆∆Ct method, as described in another study [Total RNA was extracted from the liver tissue using a commercial animal tissue RNA purification kit , following the manufacturer’s instructions. Briefly, 10 mg of hepatic tissue was homogenized using a manual pestle in lysis buffer and loaded into a spin column to purify the RNA. After the DNA was digested and washed, RNA was eluted from the spin column, and its final volume was 100 µL. The total extracted RNA was quantified using a NanoDrop 2000/2000c spectrophotometer  and stored at –80 °C until further use. The qRT-PCR analysis was conducted using the commercial QuantiNovaer study .Plasma concentrations of CCL2 and IL-6 were determined using 25 µL of mouse plasma using a multiplexed-bead-based immunoassay, following the manufacturer’s instructions . The plate was read using a Luminex 200 Bioanalyzer , and the concentration of the two analytes (pg/mL) was calculated using a logistic model with five parameters.p < 0.05.All statistical analyses were performed using the GraphPad Prism software, ver. 8.0 . The differences among or between experimental groups were determined by performing one-way or two-way ANOVA, whichever was appropriate. All data were expressed as the mean ± SEM unless stated otherwise. All differences among and between groups were considered to be statistically significant at high MoMφs, which, in turn, contributed to the activation of HSC, probably via TGF-β-mediated signaling (high MoMφs were more numerous and active in the livers of female mice than those of male mice, which matched the levels of CCL2 mRNA in the liver (TNF-α, PDGF-A, and TGF-β, might promote pro-fibrogenic processes in males during the advanced stages of liver injury. Therefore, we highlighted that the early and late stages of liver fibrosis are differently regulated between females and males.The liver exhibits high sexual dimorphism, and although it is well known that liver fibrosis predominantly affects men, the mechanisms driving these differences remain poorly understood. Our in vivo study provided an overview of how the progression and regression of liver fibrosis differ between the sexes . Our resignaling C. Althouhe liver C. Howeve4 was stopped, fibrosis regression and liver regeneration occurred in mice of both sexes. Repair and regeneration were more efficient in females than in males since no activated HSCs were detected, while Ly6Chigh MoMφs underwent phenotype switching to anti-fibrogenic Ly6Clow MoMφs (When the administration of CClow MoMφs D. These ow MoMφs D.4 model is commonly used to induce liver fibrosis, this model does not reflect the eventual role of the etiology of liver fibrosis in humans. Second, we provided a valid description of how a fibrotic liver from males and females might appear, and based on our findings, we hypothesized that the triggering mechanisms of liver fibrosis might be differentially driven in males and females. However, further investigation, including an analysis of the role of hormones in the regulation of fibrogenic and regenerative mechanisms, is needed to comprehensively understand this complex issue.Although our study provided in vivo evidence that some pro-fibrogenic and regenerative mechanisms might differ between males and females, it had some limitations. First, although the CCl


Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-β1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.  According to the World Health Organisation, cardiovascular diseases cause the deaths of 17.9 million people each year, which is 31% of all deaths worldwide . For pulApart from biomechanical properties, it is important to improve bioactivity and bioconductivity of collagen gels. In various studies, the incorporation of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin-1 or conditioned medium, obtained from dermal fibroblast, into the collagen scaffold supported cell ingrowth, migration, differentiation, and the healing process ,29,30,31Autologous stem cells, such as bone marrow- or adipose tissue-derived MSCs, are relatively easily available for the colonisation of collagen gels/composites intended as vascular patches, and these cells are able to differentiate into SMCs, as shown in our previous studies and studies by other authors  . For stetunica media of the physiological vascular wall. For inducing this differentiation pathway, we developed an external drug delivery system, represented by nanofibrous PVA meshes releasing human platelet lysate into the culture medium, or we supplemented the culture medium with a combination of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-4 (BMP-4), which has been proven as highly efficient for differentiation of ASCs towards SMCs [tunica intima, to mimic a physiological blood vessel wall, and to render the constructs applicable for creating vascular patches and blood vessel replacements. For these purposes, the construct incubated with the platelet lysate-loaded PVA meshes seemed to be the most promising, as indicated by a relatively high degree of ASCs differentiation towards SMCs, a relatively high mechanical stability, low shrinkage of the construct, and its homogeneous and confluent endothelialisation. The novelty of this study therefore lies in preparation of a fully biodegradable nature-derived collagen-based composite, i.e., collagen hydrogel reinforced with collagen particles, incorporated with human adipose tissue-derived stem cells (ASCs) with the aim to differentiate these cells towards SMCs phenotype and to reconstruct the rds SMCs ,38. The 2α2(I) [−1 representing N–H stretching and amide B at ∼3100 cm−1 representing stretching vibration of the N–H bonds in secondary amides. The amide I band (∼1650 cm−1) originates from C=O stretching vibrations coupled with N–H bending vibration, while the amide II band (∼1550 cm−1) arises from N–H bending vibrations coupled with C–N stretching vibrations [−1 and a triple band at ∼1205, 1240, and 1280 cm−1 (amide III) [−1) was deconvoluted into six distinct bands with maxima at ∼1695, 1675, 1660, 1650, 1635, and 1620 cm−1. The most dominant components are bands ∼1660 cm−1 assigned to the triple helix with contribution from α-helix, and the band at ∼1650 cm−1, which corresponds to random coils of α and β chains. Band 1635 cm−1 represents a denaturated state. Bands at 1675 cm−1 (β-turn), at 1695 cm−1 (β-sheet) [−1 (imide residues) representing other structural states can be associated with the existence of LMP proved by SDS-PAGE. The ATR-FTIR spectrum also contains bands of impurities such as lipids , and low intensity bands in spectral region 900–1100 cm−1 relating to GAGs. These residual impurities remain in the collagen after the isolation procedure [Composition of isolated COL is shown in 2α2(I) . The sim2α2(I) . In addibrations . Anotheride III) . The amiβ-sheet)  and bandrocedure . Analysirocedure . n = 100). The average length of particles was 363 ± 160 µm (n = 100). These measurements confirmed that the collagen fibers were of submicrometer dimensions, but the collagen particles contained both amorphous and fibrous structures. The produced nanofibrous materials from PVA with incorporated proteins  have a fiber diameter 455 ± 159 nm with a wide range fiber diameter distribution A,B. The We developed novel composite scaffolds based on a collagen gel reinforced with embedded collagen particles and compared them with collagen gels without reinforcement. During the first 6 days, the ASCs in collagen without particles were proliferating and reached the highest densities in TGF + BMP and PVA_PL samples . The TGFTM Green CMFDA dye was relatively fast, and thus this prevented cells sedimentation and allowed for initial homogeneous cell distribution in the collagen gel. Collagen particles had a weak autofluorescence, and therefore they were partially visible on confocal images. The prepared samples showed a similar morphology and population density of ASCs on day 1 . Cultivation of cells in gels in standard DMEM media and in media with PVA mats without PL served as negative controls (DMEM_part and PVA_part samples). Conversely, the addition of TGF-β1 and BMP-4 to the media represented a positive control for the cell differentiation towards SMCs (TGF + BMP_part). The process of gel preparation with ASCs previously stained with CellTrackeron day 1 A–D,A`–D`on day 1 A. Seedinon day 1 A. Collagen particles hindered the measurement of the gel depth via microscopic evaluation. Thus, in order to evaluate the gel shrinkage, the gel area was measured inside cell culture dishes. There was no shrinkage of gels on day 7 B. On dayASCs differentiation towards SMCs, which are an essential cellular component of vascular wall, was evaluated by immunofluorescence staining of alpha-actin and calponin, an early marker and a mid-term marker of differentiation into smooth muscle cells. On day 6, the DMEM gel without particles contained cells positive for alpha-actin, but the cells were negative for calponin . The PVAThe collagen gels with particles seeded with ASCs were also evaluated for ECM proteins fibronectin and type I collagen by immunofluorescence staining and SHG microscopy . FibroneEndothelialisation of the collagen gels reinforced with particles and incorporated with ASCs was performed with ECs from day 8 to day 14 of the culture. The DMEM_part, PVA_part, and PVA_PL_part samples were homogeneously endothelialised with almost confluent EC layer, as it was proved by staining of the von Willebrand factor A–C. In cA total of five groups, namely collagen gels with ASCs immediately after preparation (D0), and collagen samples after 12 days (D12) in culture, i.e., DMEM_part, PVA_part, PVA_PL_part, and TGF + BMP_part containing ASCs and ECs, were analysed by mass spectrometry according to their protein concentration. There was a high number of significant changes detected between the control group (D0) and the samples after a 12-day cultivation, i.e., DMEM_part/D0 = 731 significantly upregulated proteins (s.u.p.) for a 12-day cultivation group; PVA_part/D0 = 718 s.u.p.; PVA_PL_part/D0 = 756 s.u.p.; TGF + BMP_part/D0 = 895 s.u.p.). Because the number of significantly altered proteins was too high, we selected three protein groups, i.e., proteins involved in cell differentiation , ECM Ta and protThe increased concentration of caldesmon was in both PVA_PL_part and TGF + BMP_part samples compared to PVA_part sample. The TGF + BMP_part samples contained significantly increased concentrations of myosin phosphatase Rho-interacting protein, transgelin, and tropomyosin alpha-1 chain. On the contrary, higher concentrations of both nexilin and fascin were found in the PVA_part compared to TGF + BMP_part samples. There were detected 19 significantly upregulated proteins between day 12 and control D0 sample in the ECM group . The ECMIn both PVA-part and PVA_PL_part, the cells expressed more extracellular matrix protein 1, galectin-3-binding protein, laminin subunit alpha-4, nidogen-1 and thrombospondin-4 compared to the TGF + BMP_part. Among ECM remodelling proteins, there were detected 13 significantly upregulated proteins (D12/D0 cultivations) in DMEM_part, PVA_part, PVA_PL_part and TGF + BMP_part between day 12 and control D0 group . This reiniE) was higher in all collagen samples after 5 days in culture compared to the DMEM sample immediately after preparation  were observed for DMEM, PVA and PVA_PL samples. The stiffness of the samples increased by more than 39% after 5 days of cultivation. This result suggests remodelling of the gel by ASCs. Other biomechanical parameters, i.e., total deformation energy (cU) and ratio of elastic energy (el/UcU), were similar for all samples . Moreover, for co-culture of ASCs inside the material with ECs on its surface, the cell culture medium contained growth factors, such as VEGF, FGF-2, insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF) and heparin, from which VEGF with heparin were observed to stimulate the differentiation of ASCs towards SMCs in our earlier study . In our In addition, ASCs have the capacity to mediate wound healing through mitochondrial transfer and the paracrine secretion of various growth, antiapoptotic, angiogenic, antioxidant, anti-inflammatory and immunomodulatory factors, contained in exosomes and extracellular vesicles . FurtheIn samples exposed to the external drug delivery system consisting of PVA meshes slowly releasing platelet lysate, we achieved not only the differentiation of ASCs towards SMCs, but also a homogeneous and almost confluent endothelialisation. Platelet α-granules contain a wide variety of bioactive molecules that are released upon activation via physical or physiological stimuli and participate during wound healing, such as coagulation factors , adhesion molecules , platelet factor 4 (PF-4), protease inhibitors , plasma proteins , proteoglycans, EGF, heparin-binding EGF-like growth factor, IGF-1, transforming growth factors , platelet-derived growth factors , soluble CD40L, immunoglobulins such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), chemokine (C-C) ligand 5 and chemokine (C-X-C) ligand 1/2/3 . The groTM medium supplemented with 1% human platelet lysate enhanced proliferative capacity by about 70% compared to supplementation with 10% of FS. The ability to form colonies  was, however, reduced with human PL [Similarly, in studies by other authors, ASCs cultured in a medium supplemented with 5% of platelet lysate and without FS created more homogeneous phenotype, had a high proliferative capacity, secreted various proteins, such as basic fibroblast growth factor (bFGF), interferon-γ (IFN-γ), and IGF-1, and had immunomodulatory effects . ASCs cuhuman PL . In our 6 ASCs cells/mL of the gel. Camasão and colleagues [6 cells/mL, increased the initial collagen gel compaction/shrinkage, but did not negatively influence the long-term collagen gel compaction. In the samples with high cell densities, the cells produced a higher amount of collagen than the samples seeded with a lower cell density (0.5 × 106 cells/mL). The initial (E0) and equilibrium elastic modulus (EE) for the construct with 4.5 × 106 cells/mL were 220 ± 18 kPa and 58 ± 4 kPa, respectively, and these values were about two- and threefold higher compared to the gel with the lowest cell seeding density. At the same time, on day 7, the EE/E0 ratio, which expresses an elastic component, was greater in the high cell seeding density sample [Mechanical properties of cardiovascular patches are important for further clinical use. Biomechanical evaluation of our samples was performed on day 5 of static culture, as the construct had a proper volume for testing via an unconfined compression test. Despite this short time interval, there was an increase in the initial Young modulus of elasticity in all samples compared to the D0 time interval, although there were no differences among the groups. Our gels contained 0.5 × 10lleagues  have obsy sample . For comy sample . TGF + By sample . TGF + By sample . The elay sample . The gely sample , the addy sample . The tuby sample , loaded y sample , TGF-β1 y sample . Static y sample .v/v) solution , washed 3× by water and subsequently exposed to an acetic acid solution in ratio 1:1000 (v/v), 1 g of COL/20 mL for 48 hrs). COL in collected supernatant was precipitated using 0.1 M NaOH solution in ratio 6:1 (v/v) up to slightly neutral pH 6–7. The obtained pellets were then dissolved again in acetic acid solution 1:1000 (v/v), frozen to −30 °C and lyophilised. All isolates were stored in a freezer at −20 °C.The type I collagen (COL), used for preparing the hydrogel matrix, was isolated from porcine skin  using the following protocol: incubation in 70% ethanol (N-ethyl-N′-(3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at a weight ratio of 4:1 , for 24 h at 37 °C. Cross-linked layers were further washed in 0.1 M Na2HPO4 (2 × 45 min), and by rinsed using deionised water (30 min). In this step, PEO was fully leached out [Collagen fibers were used as a reinforcement for optimisation of mechanical properties of the collagen hydrogels. Fibers based on collagen  were prepared via the electrospinning  of an 8 wt% collagen PBS/ethanol solution modified by 8 wt% (to collagen) polyethylene oxide . The stability of all the collagen layers was enhanced by means of crosslinking with a 95% ethanol solution containing ched out . The mato-phthaldialdehyde [Composition of isolated COL lyophilisates was studied by various analytical methods. The determination of interstitial water (directly bound to the triple-helix) was performed according to the standard ISO 6496:1983 , i.e., by drying to 160 ± 2 °C for 4 h. Amino acid analysis was performed using an Ingos AAA 400 analyser . The hydroxyproline (Hyp) content was determined according to the ISO 3496:1994(E) standard (Meat and Meat products—the determination of hydroxyproline content). Content of lipids was assessed by the Schmidt-Bondzyński–Ratzlaff method, according to Czech technical standard EN ISO 1735:2004. Total content of glycosaminoglycans (GAGs), i.e., long unbranched polysaccharides, was quantified by high-performance liquid chromatography (HPLC) method based on hexosamines; i.e., glucosamine and galaktosamine, due to the formation of fluorescent N-acetylated hexosamine derivatives by reaction with a specific derivatisation agents—aldehyde . Sodium aldehyde .http://imagej.nih.gov/ij/ (accessed on 30 August 2022).The morphology of electrospun submicrometer-scale fibers and particles were characterised using scanning electron microscopy (SEM) . Randomly selected images representative of the entire electrospun fibers  and homogenised particles (mag. 100×) were used to measure the diameters of 100 individual fibers and/or particles using the ImageJ software . Then, the cell lysate was centrifuged , and the supernatant after centrifugation (PL) was stored at 4 °C until use (not longer than 24 h) [Platelet lysate (PL) was prepared from platelet-rich solution, which was obtained from healthy blood donors , and all blood donors signed the informed consent. Concentration of platelets was 843 × 10an 24 h) .Nanofibrous mats were prepared from 10% solution of PVA  in water: ethanol (9:1) solvent system. In case of protein loaded mats, the 10 g of platelet lysate was added into 90 g pre-PVA solution 30 min before electrospinning and gently mixed at room temperature until use . MateriThe protein release was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) after incubation of PVA mats containing proteins in phosphate-buffered saline . Cumulative analysis was performed placing 10 mg of materials into 1 mL of PBS; subsequently, 200 µL of solution was taken for analysis at certain time intervals, and 200 µL of fresh PBS was added to keep the volume of 1 mL. Proteins were analysed after incubation at 37 °C for 1, 2, 4, 6, 8, 24, 72, and 168 h. Solutions after protein release , of the prepared nanofibrous mats. Ten milligrams of samples was analysed using a differential scanning calorimeter DSC1/700 . Aluminium pots with samples were cooled to 0 °C and then heated to 300 °C at a rate of 10 °C/min. Heating was performed under inert conditions (nitrogen), and crystallinity was calculated from the recorded DSC curves. α1.2 radiation (40 kV and 40 mA), 0.02 mm Ni Kβ absorber, 5–50° 2θ range and a 0.02° step scan with a sample rotation speed of 30 RPM. The relative proportion of crystalline regions in the samples was calculated using the following formula:α is the degree of crystallinity, cI is the sum of the intensities below the crystalline peaks, and aI is the sum of the intensities below the amorphous sections of the spectra.In addition, the crystallinity of the nanofibrous PVA materials was measured by X-ray diffraction (XRD) on a Bruker D8 Advance diffractometer equipped with a LynxeyePSD detector  and Cu K2, TPP, Trasadingen, Switzerland) in a density of 0.16 mL of original lipoaspirate/cm2. The ASCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) of fetal bovine serum , gentamicin  and human recombinant basic fibroblast growth factor . The cells were passaged, when they reached 70–80% subconfluence. Flow cytometry analysis  revealed that the ASCs were positive for cell surface antigens CD73 , CD105 (endoglin) and CD29 (fibronectin receptor), all >95%, while they were almost negative for CD31 , CD34 (antigen of hematopoietic progenitor cells), CD45 (protein tyrosine phosphatase receptor type C), and CD146 . For experiments, the ASCs were seeded at the passages 4–5. Human umbilical vein endothelial cells (ECs) were purchased from LONZA  and were seeded at the passages 3–4. Lipoaspirates were harvested by liposuction from subcutaneous fat of tight regions of human female donors using a negative pressure of −700 mmHg. The procedure was accomplished in compliance with the tenets of the Declaration of Helsinki and under ethical approval issued by the Ethics Committee of the Bulovka Hospital in Prague  and under Informed Consent of the patients. Adipose tissue-derived stem cells (ASCs) were isolated using the enzymatic digestion method first described by Estes and colleagues  and furtv/v/v), respectively. Collagen suspension with the medium with cells was left to polymerise for at least 10–15 min at 37 °C, in a humidified atmosphere with 5% CO2, to reach pH around 7.4, and then 1.5 mL of cell culture medium was added above the gel. The change in pH from acidic to neutral and 37 °C caused the COL to polymerise into a hydrogel. Final COL concentration in gels was 3.3 mg/mL, and the final ASCs density was 500,000 cells/mL. The lyophilised COL I was dissolved in 0.02 M acetic acid in the concentrations of 6 mg/mL and was stored at 4 °C for 5 days. The COL suspension was homogenised by means of a disintegrator . Before collagen gel preparation, 32 mg of collagen particles per 1 mL of the gel were added into each well of a 24-well glass bottom plate . The hydrogel was prepared by mixing the COL suspension with the ASCs suspension in DMEM , supplemented with 2% of FS (Cat. No. 10270-106 GIBCO), gentamicin , 2-phospho-L-ascorbic acid trisodium salt  and with sodium bicarbonate (7.5 wt.%). The ratio of COL, cell suspension and sodium bicarbonate solution was 1.766:1:0.0294 (The ASCs incorporated in the COL hydrogel without COL particles and in COL hydrogel reinforced with COL particles (part) were then cultured by the following manners: (1) In DMEM medium supplemented with 2% of FS, 40 µg/mL of gentamicin and 50 µg/mL of ascorbic acid , mentioned above (samples labelled as DMEM_part);2; samples labelled as PVA_part);(2) In the same medium with the addition of pure PVA nanomats (2 × 1 cm2) representing an external drug delivery system (samples labelled as PVA_PL_part), and(3) In the same medium with addition of PVA nanomats loaded with PL (2 × 1 cm(4) In the same medium supplemented with transforming growth factor β1  and bone morphogenetic protein 4  at the concentration of 2.5 ng/mL, representing a culture medium proven for cell differentiation towards SMCs (samples labelled as TGF + BMP_part).2 at 37 °C. The scheme of the whole study is in In all four groups of samples, the medium was replaced twice per week during a 14-day culture. For endothelialisation of the gels reinforced with particles, ECs were seeded on the top of all four groups of samples at the density of 100,000 cells/well on day 8 after ASC incorporation, and cultured either in a pure EGM-2 medium  or in an EGM-2 medium with PVA nanomats, with PVA_PL namomats or with TGF + BMP until day 12 (for mass spectrometry) or until day 14 . For measurement of biomechanical properties, the prepared gels with collagen particles were as thick as 1 cm, and the samples with ASCs cells were cultured for 5 days or were analysed immediately after preparation (day 0). The samples were incubated in a humidified atmosphere with 5% of COTM Red CMTPX dye or with CellTrackerTM Green CMFDA  in DMEM with 10% of FS for 45 min. After washing with PBS, the cells were kept in a standard cell culture medium until the stained cells’ suspension was used for collagen gel preparation. The gels with embedded cells were vizualised using Andor Dragonfly 503—a spinning disk confocal microscope on day 14 of culture. The 3D and 2D projections were created, and cell densities were calculated via IMARIS software. The ASCs cells cultured in TPP flasks were incubated with CellTrackerThe samples were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT), washed twice with PBS, and permeabilised with 1% BSA in PBS containing 0.1% Triton X-100 for 20 min. The samples were then washed with PBS, treated with 1% Tween for 20 min at RT and washed again with PBS.The samples were incubated with a rabbit anti-calponin antibody , dilution 1:200) at 4 °C overnight; then, they were washed twice with PBS and incubated with a mouse anti-alpha smooth muscle actin antibody  at RT for 3.5 h. Then, the samples were washed twice with PBS and were incubated in a goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488  for 90 min, washed with PBS and incubated in a goat anti-mouse secondary antibody conjugated with Alexa Fluor 546  for 90 min. Finally, the samples were washed twice with PBS. For immunocytochemical staining of the co-culture of ASCs and ECs, mouse anti-alpha smooth muscle actin antibody  was applied at 4 °C overnight, washed twice with PBS, and then rabbit anti-von Willebrand factor antibody  for 3 h were applied. After washing twice with PBS, the anti-mouse and anti-rabbit secondary antibodies, mentioned above, were then applied sequentially at RT for 120 min each. The cell nuclei dye DAPI was added into the last secondary antibody solution (1 µg/mL). TM 488 , which was applied at RT for 90 min and a mixture of AlexaFluor546 goat anti-rabbit IgG, cross-adsorbed secondary antibody  and DAPI (1 µg/mL), applied at RT for 90 min. Quantification of the fluorescence signal of von Willebrand factor, fibronectin and type I collagen was performed by ImageJ software, and the data were normalised to the cell nuclei signal. Similarly, immunofluorescence staining of fibronectin and type I collagen in reinforced collagen gels with ASCs on day 14 was performed using the primary antibodies as follows: the mouse monoclonal anti-human fibronectin antibody, clone IST-3 , applied at RT for 3 h, and the rabbit polyclonal anti-type I collagen antibody , applied at RT for 90 min. The secondary antibodies were F(ab’)2-goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorImages were obtained by the Dragonfly 503 spinning disk confocal microscope using software Fusion version 2.1.0.80 , equipped with objectives HC PL APO 10×/0.40 DRY and camera Zyla 4.2 PLUS sCMOS—2048 × 2048 pixels. Images were acquired with a voxel edge size of 6.5 µm and a z-step of 5 µm using a 25 μm pinhole size. Cells stained by CellTracker Red CMTPX or CellTracker Green CMFDA  were excited by a 561 nm laser; emissions were collected in a range of 575–625 nm for the red tracker, excited by a 488 nm laser, and collected in a range of 500–550 nm for the green tracker.Lightsheet microscopy 3D images were acquired with a Zeiss Z.1 lightsheet microscope using the 10×/0.2 NA excitation and 20×/1.0 water immersion detection objective lenses, 488 nm and 561 nm excitation and the respective green (505–545 nm) and red (575–615 nm) emission. Image processing and visualisation were performed in ZEN  and Arivis Vision 4D  software, respectively.Second Harmonic Generation (SHG), able to visualize mature collagen fibers , was perSamples were measured using multiphoton microscope Bruker Ultima  using the CFI75 Apo 25XC W 1300 objective (1.1 NA). Images of samples were captured as 12-bit images of different z-planes 2 µm apart at 1024 × 1024 pixel resolution with 525.45 × 525.45 µm using Prairie View . The excitation was carried out with two different wavelengths, 940 nm and 1100 nm (or 760 nm). The emission signal was filtered through bandpass filters ET525/70m-2P and ET595/50m-2P (or 450/50m-2P)  and detected with GaAsP photosensors sensitive in the visible light region . Three-dimensional projections were created using ImageJ and IMARIS software .The two-channel lightsheet data were processed in Arivis Vision 4D version 3.1.3. After stitching, data were denoised with a 2D median filter, and background correction was applied. Objects were segmented using the global intensity threshold, and their morphological and intensity features were exported for further statistical analysis.Biomechanical testing of the collagen hydrogel was performed using an unconfined compression test. The MTS Mini Bionix 858.02 testing system  equipped with 10 N force sensor (sensitivity 0.001 N) was used for the compression tests in quasi-static mode at a constant cross head loading speed of 2.0 mm/min. A total of 5 cycles were realised, i.e., 5 times loaded to 1 N and 5 times unloaded to 0.05 N. The hydrogel disks for this test were placed between two stainless steel cylinders at room temperature (24 °C). The force and the deformation data were recalculated into the stress–stain curves. The mechanical properties of the composite material were characterised by the initial Young modulus of elasticity, total deformation energy and the ratio of elastic energy, i.e., the ratio between the elastic deformation energy and the total deformation energy. The data were acquired by the MTS Mini Bionix testing system and were analysed in Matlab 2020b . The environmental conditions were recorded by a COMET  digital thermometer hygrometer.g; 5 min), the supernatant was extracted and used for subsequent analysis (both pellet and supernatant were stored at −25 °C). Supernatant samples were heated at 105 °C for 5 min., digested with trypsin and desalted on Empore C18 columns, dried in Speedvac . Samples were dissolved in 0.1% TFA + 2% acetonitrile. About 0.5 µg of peptide digests were separated on 50 cm C18 column using 2.5 h elution gradient and analysed in a DDA mode on Orbitrap Exploris 480 mass spectrometer . The resulting raw files were processed in MaxQuant (v. 1.6.17.0) with label-free quantification (LFQ) algorithm MaxLFQ. The search was performed at 0.01 false discovery rate (FDR) levels using FASTA files from UniProt (release 2021_01). Downstream analysis and visualisation were performed in Perseus (v. 1.6.15.0.). The p-value returned was corrected by FDR based on a frequency histogram. An FDR adjusted p-value threshold was less than 0.01.Biological samples (n = 4 in each group) were harvested, and MS LFQ analysis was performed. Briefly, each sample was lyophilisated and 10 mg of dry weight was taken for analyses. Samples were homogenised in liquid nitrogen, and 0.3 mL of 0.1 M ammonium bicarbonate (pH = 7.8) was added. After 30 min of sonication and centrifugation  was used. Biomechanical data were analysed using a Student’s Collagen gels reinforced with collagen particles, incorporated with ASCs and endothelialised with HUVECs were prepared and cultured either in a medium with TGF-β1 and BMP-4 (TGF + BMP_part sample), or in a medium with a newly developed external drug delivery system, represented by a nanofibrous PVA mesh releasing platelet lysate (PVA_PL_part sample). Cell-material constructs incubated only in a standard cell culture medium (DMEM_part) or in this medium with pure PVA nanomats (PVA_part) served as negative controls. Gel reinforcement with collagen particles prevented the planar shrinkage of all gels until day 7. On day 14, significant gel shrinkage was observed with TGF + BMP_part gels either with or without endothelial cells. On the contrary, the minimum shrinkage was observed with PVA_PL_part gel regardless of its endothelialisation. Endothelial cells formed a confluent layer on all gels; however, TGF + BMP_part gels evinced less homogeneous coverage with endothelial cells. The differentiation of ASCs towards SMCs, assessed by the intensity of calponin immunofluorescence staining as well as proteomic analysis, was strongest in TGF + BMP_part samples, moderate in PVA_PL_part and PVA_part samples, and the lowest in DMEM_part samples.The highest concentrations of proteins involved in ECM synthesis and remodelling were found in TGF + BMP_part samples, lower in PVA_PL_part and PVA_part samples, and the lowest in DMEM_part samples.In all samples, we observed improved mechanical properties on day 5 after seeding compared to day 0. The PVA_PL samples seem to have a slightly higher ratio of elastic energy, and TGF + BMP_part a slightly higher total deformation energy value.PVA_PL_part samples with an external drug delivery system, releasing platelet bioactive components into the culture medium, represent a more natural way of stimulating ASCs proliferation, differentiation, ECM production and remodelling than the direct addition of TGF-β1 and BMP-4 into the medium. The slower effect of the external PVA_PL system, probably limited by the amount of released PL components, was balanced with homogeneous endothelialisation and minimum gel shrinkage.