P < 0.05). Compared with the model group, AI, MAD levels and Bax, iNOS protein expression levels in L-EPO, M-EPO, and H-EPO groups were significantly decreased, while T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly increased. The changes were dose-dependent. In summary, EPO can activate microglia transformation from M1 to M2 through Akt/mTOR/p70S6K signaling pathway.We investigated the mechanism of erythropoietin (EPO) in brain injury in premature mice based on Akt/mTOR/p70S6K signaling pathway. The brain injury model group of premature mice was obtained by intraperitoneal injection of lipopolysaccharide during pregnancy. Normal mice were taken as the control group. The model mice were divided into low-dose EPO , medium-dose EPO , and high-dose EPO groups  by intraperitoneal injection. The levels of malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were detected. TUNEL staining and Western blotting were used to detect the differences in neuronal apoptosis index (AI), microglial polarization marker protein, and Akt/mTOR/p70S6K-related protein expression levels in each group. Compared with the control group, the protein levels of AI, MDA, Bax, and iNOS in the model, L-EPO, and M-EPO groups were significantly increased, while the T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly decreased (  EPO inhibits neuron apoptosis in brain-injured premature mice that are dose dependent.EPO inhibits oxidative stress in brain-injured premature mice that are dose dependent.EPO inhibits microglia M1 to M2 in brain-injured premature mice that are dose dependent.EPO improves the degree of brain injury in preterm mice by Akt/mTOR/p70S6K pathway.1.Maternal inflammatory response during pregnancy, such as chorioamnionitis, is currently recognized as an influencing factor of fetal preterm birth . When thTo investigate the effect of EPO on the activation of Akt/mTOR/p70S6K signaling pathway and type M2 transformation of microglia in brain injury of preterm mice, the brain injury model of premature mice was induced by intraperitoneal injection of lipopolysaccharide. The transformation of M1 microglia to M2, the state of Akt/mTOR/p70S6K signal pathway, and the damage state of cell biofilm of mice were detected after EPO treatment. The purpose is to investigate the mechanism of EPO effect on brain injury in premature mice and analyze the effect of EPO on Akt/mTOR/p70S6K signaling pathway status, so as to provide theoretical guidance for reducing brain injury and neuronal biofilm autophagy injury in mice.2.2.1Male C57BL/6 mice and female BALB/c mice were purchased from Shanghai Model Organisms Center Inc. Lipopolysaccharide and TUNEL kits were purchased from Sigma, USA. In total, 10% chloral hydrate was purchased from Zhujiang Hospital of Southern Medical University. Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore, USA. Protein primary antibodies were purchased from cell signaling technology, USA. Protein secondary antibody and bicinchoninic acid (BCA) kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad, USA. EPO  was purchased from China resources Angde Biotech Pharma Co., Ltd. 4% paraformaldehyde was purchased from Guangzhou Chemical Reagent Factory (GCRF). Paraffin, xylene, and neutral gum were purchased in Beijing Solarbio Science & Technology Co., Ltd. SDS-PAGE gel preparation kit and RIPA lysate were purchased from Wuhan Servicebio Co., Ltd. Malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.2.2Clean-grade C57BL/6 male mice and BALB/c female mice were selected and reared in clean-grade environment for 7 weeks. After 7 days of adaptive feeding, breeding was conducted according to the male to female ratio of 2:1, and the vaginal plug was observed and detected. The day of vaginal plug was identified as the 0 day of pregnancy. On the 17th day of pregnancy, 125 was injected intraperitoneally μg/kg lipopolysaccharide solution, live offspring were born on the 18th day of pregnancy, so as to obtain preterm mice. All experiments were conducted in accordance with the guidelines of the International Council for Laboratory Animal Science and the relevant rules and regulations of the central laboratory animal experiment committee of the Shenzhen Second People’s Hospital and approved by the central laboratory animal experiment ethics committee of the Shenzhen Second People’s Hospital.2.3Normally born mice were used as the control group (Ctrl), a total of 6 mice were included in the control group, and then 24 preterm mice were randomly divided into preterm brain injury model group (Model) with 6 mice, EPO treatment group with 18 mice. EPO treatment mice were intraperitoneally injected with 1,000 IU/kg, 2,500 IU/kg, and 5,000 IU/kg EPO solutions on postnatal days 0, 1, 3, and 5. They were named as low-dose EPO group (L-EPO), medium-dose EPO group (M-EPO), and high-dose EPO group (H-EPO), with 6 mice in each group. The Ctrl and Model groups were injected with an equal volume of 9 g/L saline, respectively. The grouping is shown in 2.4.Paraffin sections of brain tissue of mice in each group were made. After dewaxing, protease K working solution was used and incubated at 37°C for 25 min. The slides were washed in neutral phosphate buffer for 3 times for tissue repair. After the tissue was covered with membrane breaking working solution, it was incubated at room temperature for 20 min, and the slides were washed for 3 times. The reagent was added according to the instructions of TUNEL kit, the slices were placed in a wet box and incubated in water bath at 37°C for 2 h. 3% hydrogen peroxide was added, the slices were incubated at room temperature in the dark for 15 min to block endogenous peroxidase, and washed for 3 times. DAB chromogenic solution was prepared for tissue staining, and hematoxylin was used for re-staining of nuclei. Alcohol solution was used to dehydrate the tissue and neutral gum seal was added. The staining state was observed under an optical microscope, and the apoptosis index was calculated by Image J software . The equ2.5The hippocampal tissue of mice was ground into 10% homogenate, measured according to the concentration of MDA and SOD, and heated in a water bath at 95°C for 30 min, centrifuged at 4,000 rpm for 10 min. The supernatant was taken and placed in the spectrophotometer, and the absorbance was detected at 532 nm and 550 nm, respectively. The activities of MDA and SOD were calculated .2.6Mice in each group were anesthetized by intraperitoneal injection of 0.2 mL of 100 g/L chloral hydrate. After exposing the chest, 0.1 mol/L phosphate buffer was injected into the heart until the liver turned white, and then the brain tissue of mice was quickly separated on ice. According to the ratio of 1:100, the PIPA lysate and brain tissue were placed in the tissue grinder for full lysis, the protein was extracted after centrifugation, and the protein concentration was quantitatively detected according to the instructions of BCA kit. After adjusting the protein concentration, one-fourth of the final volume of the loading buffer was added, and placed in a boiling water bath for denaturation treatment for 10 min. SDS-PAGE separation gel was prepared, sample protein was added and separated, and the target protein band was transferred to PVDF membrane. PVDF membranes were applied to rabbit anti-mouse iNOS primary antibody (ab_178945) (1:1000), rat anti-mouse ARG1 primary antibody (ab_283402) (1:1000), rabbit anti-mouse Akt primary antibody (ab_8805) (1:1000), rabbit anti-mouse p-Akt primary antibody (ab_8805) (1:1000), rabbit anti-mouse mTOR primary antibody ab_134903) (1:1000), rabbit anti-mouse p-mTOR primary antibody (ab_137133) (1:1000), rabbit anti-mouse p70S6K primary antibody (ab_283535) (1:1000), rabbit anti-mouse p-p70S6k primary antibody (ab_283535) (1:1000). Rabbit anti-mouse Bcl-2 primary antibody (ab_141523) (1:1000), rabbit anti-mouse Bax primary antibody (ab_32503) (1:1000), and rabbit anti-mouse GAPDH primary antibody (ab_8245) (1:1000) were incubated overnight in a refrigerator at 4°C. After washing, goat anti-rat IgG secondary antibody (ab_172730) (1:10000) labeled with horseradish peroxidase was added and incubated at room temperature for 2 h. After the membrane was washed by TBST, the strip was developed according to the ECL photoluminescence test kit. The relative expression level of the target strip was detected by Image Lab software in the gel imaging system [4903  was used for comparison between groups. It was considered that 3.The effect of different doses of EPO on neuronal apoptosis index, MDA, T-SOD, and Akt/mTOR/p70S6K signaling pathway status in premature mice and how EPO promoted the transition of microglia from M1 to M2 through Akt/mTOR/p70S6K signaling pathway and reduced brain injury in mice were analyzed. The results are as follows.3.1P > 0.05), and the neuronal apoptosis index in the Model group, L-EPO group, and M-EPO group was significantly increased, and the difference was statistically significant (P < 0.05). Compared with the Model group, the AI of neuron in the L-EPO, M-EPO, and H-EPO groups were significantly decreased (P < 0.05). Compared with the L-EPO group, the AI of neuron in the M-EPO and H-EPO groups were significantly decreased (P < 0.05). AI of neuron was significantly decreased in H-EPO group compared with M-EPO group (P < 0.05).The results of TUNEL staining of neurons in different regions are shown in 3.2P < 0.05). Compared with the model group, the expression level of MDA decreased significantly and the expression level of T-SOD increased significantly in L-EPO group, M-EPO group, and H-EPO group (P < 0.05). Compared with L-EPO group, the expression level of MDA decreased significantly and the expression level of T-SOD increased significantly in M-EPO group and H-EPO group (P < 0.05). Compared with M-EPO group, the expression level of MDA decreased significantly and the expression level of T-SOD increased in H-EPO group (P < 0.05). There was no significant difference in the expression of MDA and T-SOD between control group and H-EPO group (P > 0.05).Detect the difference between MDA and T-SOD activities in brain tissue of mice in each group. The results are shown in 3.3P < 0.05). Compared with the model group, the expression level of Bax decreased significantly and the expression level of Bcl-2 increased significantly in L-EPO group, M-EPO group, and H-EPO group (P < 0.05). Compared with L-EPO group, the expression level of Bax decreased significantly and the expression level of Bcl-2 increased significantly in M-EPO group and H-EPO group (P < 0.05). Compared with M-EPO group, the expression level of Bax decreased significantly and the expression level of Bcl-2 increased in H-EPO group (P < 0.05). There was no significant difference in the expression levels of Bax and Bcl-2 between control group and H-EPO group (P > 0.05).The difference of Bax and Bcl-2 protein expression levels in brain tissues of mice in each group was detected by Western blot. The results are shown in 3.4P < 0.05). Compared with the Ctrl, the expression level of ARG1 was significantly decreased in the Model group, and the iNOS expression level was significantly increased in the L-EPO, M-EPO, and H-EPO groups (P < 0.05). Compared with the Model group, the expression levels of iNOS were significantly decreased and the expression levels of ARG1 were significantly increased in the L-EPO, M-EPO, and H-EPO groups, and the differences were statistically significant (P < 0.05). Compared with the L-EPO group, the iNOS expression level was significantly decreased and the ARG1 expression level was significantly increased in the M-EPO and H-EPO groups, and the difference was statistically significant (P < 0.05).Western blotting was used to detect the differences in the expression levels of M1 (iNOS protein) and M2 (ARG1 protein) in microglia in the brain tissues of mice in each group, and the results are shown in 3.5P > 0.05). Compared with the control group, the expression levels of p-Akt, p-mTOR, and p-p70S6K in the model group, L-EPO group, and M-EPO group were significantly decreased, and the difference was statistically significant (P < 0.05). Compared with the model group, the expression levels of p-Akt, p-mTOR, and p-p70S6k in L-EPO group, M-EPO group, and H-EPO group were significantly lower (P < 0.05). Compared with L-EPO group, the expression levels of p-Akt, p-mTOR, and p-p70S6k in M-EPO group and H-EPO group were significantly lower (P < 0.05). Compared with M-EPO group, the expression levels of p-Akt, p-mTOR, and p-p70S6k in H-EPO group were significantly lower (P < 0.05). There was no significant difference in the expression levels of p-Akt, p-mTOR, and p-p70S6k between control group and H-EPO group (P > 0.05).Western blot was used to detect the differences in the expression levels of Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K proteins in the brain tissues of mice in each group, and the results are shown in 4.There are about 16 million premature infants in the world every year, which became a very serious world public health problem. The probability of abnormal brain development and injury caused by premature birth is high. About 50% of the children have short-term or long-term sequelae such as exercise and cognition, which will even affect their growth, development, and quality of life ,15. TherIt was found that EPO can reduce hypoxic-ischemic brain damage by reducing the expression of Fas and FasL, and the optimal therapeutic time window is 6–24 hours after brain injury . It was Biofilm is a state that is prone to lipid peroxidation in the life system, and its main product is MDA . When liMicroglia are very important immune effector cells in the brain, which play the functions of immune monitoring, defense, and clearance . The exp5.Based on Akt/mTOR/p70S6K pathway, it explored the effect of EPO on microglia polarization in brain injury model of preterm mice. The results suggested that the brain injury model of preterm mice had obvious neuronal apoptosis and cell biofilm injury, and most microglia were in M1 state, and Akt/mTOR/p70S6K pathway was inhibited. After EPO treatment, the neuronal apoptosis and biofilm damage state in the brain injury model of preterm mice decreased, the microglia transformed from M1 to M2, and the Akt/mTOR/p70S6K pathway was activated, and the treatment effect showed an obvious dose dependence. This study only explores the therapeutic effect of EPO on brain injury in preterm mice. In the follow-up, it is needed to combine transcriptome data to deeply explore the therapeutic mechanism of EPO and find potential new therapeutic targets. The results can provide experimental basis for EPO to improve the brain injury in preterm infants.


Hordeum vulgare L.). The experiment was carried out under controlled conditions in a growth chamber for 12 weeks. The sulphur-enriched manure showed the lowest manure pH and highest ammonium content. The co-fermentation of biochar and sulphur led to the highest sulphur content and an abundance of ammonium-oxidizing bacteria in manure. The biochar+sulphur-enriched manure led to the highest dry aboveground plant biomass in the amended soil, whose value was 98% higher compared to the unamended control, 38% higher compared to the variant with biochar-enriched manure and 23% higher compared to the manure-amended variant. Amendment of the sulphur-enriched manure types led to the highest enzyme activities and soil respirations . This innovative approach to improve the quality of organic fertilizers utilizes treated agricultural waste (biochar) and a biotechnological residual product (elementary sulphur from biogas desulphurization) and hence contributes to the circular economy.Improvement of manure by co-composting with other materials is beneficial to the quality of the amended soil. Therefore, the manure was supplied with either biochar, elemental sulphur or both prior to fermentation in 50 L barrels for a period of eight weeks. The manure products were subsequently analyzed and used as fertilizers in a short-term pot experiment with barley fodder ( Widely observed soil degradation is currently one of the main global concerns. It is caused by a combination of natural and anthropogenic detrimental processes, such as pollution, deforestation and consequences of poor land management and unsustainable agricultural practices: wind and water erosion, physicochemical changes as compaction, salinization, acidification, loss of soil organic matter (SOM) and nutrients . AdditioThe loss of SOM is closely related to the decline in soil fertility and the biological function of soils . ApplicaTherefore, amendment of soil with manure as the primary source of organic matter, enriched with biochar and elemental sulphur (S), has brought promising results in previous studies. Fermentation of biochar-enriched manure mitigated emissions of greenhouse gases , ammonia2) emissions, which have been reduced by 70–80% over the last 30 years . It was hypothesized that the acidifying effect of elemental S would counteract the alkalizing effect of biochar in the case of their co-fermentation in manure, and that it could modify the biological properties of manure via S oxidation-promoted nutrient mineralization, accompanied by reduced ammonia emission.The objectives of this study were to evaluate (I) the impact of manure enrichment (prior to fermentation) with biochar, elemental S and a combination of both on the fertilizing properties of produced manure types, (II) the effect of soil amendment with these various manure types on the chemical and biological properties (i.e. activity of nutrient-transforming soil enzymes), and biomass of a test crop, barley fodder (-1), [M+S] manure + elemental S (1.4 g·kg-1), [M+B+S] manure + biochar (40 g·kg-1) + elemental S (1.4 g·kg-1). Each variant was prepared in three replicates. Used biochar was produced from agricultural waste at 600°C , and its properties were according the analyses of manufacturer as follows: elements (in g·kg-1) - C 866, N 3.0, O 10.0, H 14.2; Ash550°C 11.7%, salts 0.42%, pH (CaCl2) 8.5. Elemental S was a waste product obtained during desulphurization of biogas at sugar factory biogas plant in THIOPAQ scrubber .Animal manure was collected from a cattle-breeding farm of Research Institute for Cattle Breeding Ltd., located in the village of Rapotin, Czech Republic, Central Europe . Experimental matured manure was prepared in the 50 L sealable containers (three containers per variant), filled with 10 kg of collected manure, which was  mixed with biochar and elemental S to create four experimental variants: [M] manure, [M+B] manure + biochar (40 g·kg2 was determined according to (ISO 10390:2005); total Kjeldahl nitrogen (TKN) was determined according to (ISO 11261:1995); and ammonium nitrogen (N-NH4) was measured according to (ISO 15476:2009). The available P was determined according to (org) was measured according to (EN 15936:2012). Total S was determined according to (EN 15749:2009), ammonium-oxidizing bacteria (AOB) according to (nirS) according to (dsr) according to (The activation process ran for eight weeks at a laboratory-controlled temperature (20 ± 2°C) at stable air humidity (measured weekly). At the end of the process, a mixed sample from each variant was taken and analyzed. Manure pH in CaClrding to ; dry matrding to ; and orgrding to , denitrirding to  and S-rerding to .Hordeum vulgare L.) as a test crop. All experimental pots (volume 5 L) were filled with soil substrate: fine quartz sand (0.1–1.0 mm) mixed with sieved (2.0 mm) topsoil (0–15 cm) from the rural area near the town of Troubsko, Czech Republic - 49°10’28”N 16°29’32”E in ratio 1:1, w/w. The soil was a silty clay loam , Haplic Luvisol [according to WRB soil classification (-1) - total C 14.00, total N 1.60 - available nutrients (mg·kg-1) - P 97, S 100, Ca 3259, Mg 236, K 231; mineral N forms (mg·kg-1) – Nmin 62.84, N-NO3 56.80, N-NH4 6.04; pH (CaCl2) 7.3.All four produced manure types were used as soil amendments in pot experiments with barley fodder . An unamended control contained only 5 kg of soil:sand blend. The treatments included (1) control, (2) manure (M), (3) manure + biochar (M+B), (4) manure + elemental S (M+S), (5) manure + biochar + elemental S (M+B+S). Each variant was prepared in five replicates. Each pot was sown with 16 barley seeds 2 cm under the soil surface and was watered with distilled water to achieve 65% water-holding capacity (WHC). This moisture level was maintained throughout the entire experiment. All pots were placed randomly into a growth chamber . Controlled conditions were set as follows: 12-hours photoperiod, light intensity 20 000 lx, temperature (day/night) 20/12°C, relative air humidity (day/night) 45%/70%. After 14 days, the number of plants was reduced to 12 in each pot. Moreover, the pots were randomly rotated every other day to ensure the homogeneity of the conditions for the treatments.The four pot experimental variants were made by thoroughly mixing a soil:sand blend (5 kg) with the particular manure type in amounts of 200 g per pot  – β-glucosidase (GLU), arylsulfatase (ARS), phosphatase (Phos), N-acetyl-β-D-glucosaminidase (NAG) and urease (Ure) – were freeze-dried. The samples stored at 4°C were used for determination of dehydrogenase activity (DHA), soil basal (BR) and substrate induced respirations : D-glucop-nitrophenol (PNP)-derivatives of the specific soil substrates were used for Vis spectrophotometric measurement  at Λ = 405 nm . Urease activity was determined as an amount of ammonium produced from the substrate urea, detected Vis spectrophotometrically by the reagent cyanurate (Λ = 650 nm). Other soil properties were determined by the standard methods and the data obtained was statistically analyzed as listed in -based method. The isted in Table 1.The Shapiro–Wilk and the Levene tests (at p ≤ 0.05) were performed for the verification of normality and homogeneity of variances. Principal component analysis (PCA), and one-way analysis of variance (ANOVA) type I  sum of squares at 5% significance level were used for characterization of relationship between the treatments and selected soil properties. Tukey’s HSD (honestly significant difference) test was used for detection the statistically significant difference among factor level means, and “treatment contrast” was calculated as factor level means for each treatment. The results were also graphically presented with Rohlf biplot for standardized PCA. Pearson correlation analysis was performed for measuring the linear dependence between soil properties. Pearson correlation coefficient was interpreted as follows: 0.0< r< 0.3 (negligible correlation), 0.3< r< 0.5 (low correlation), 0.5< r< 0.7 (moderate correlation), 0.7< r< 0.9 (high correlation), and 0.9< r< 1.0 (very high correlation).4 (by 83.1% as compared to the M) –  and increased N-NH4 (by 161%) content compared to the M+B.It was observed that both M+S and M+B+S exerted significantly lower pH values  compared to the M and M+B  – (org compared to the M (by 3.7%) and M+B+S. A similar decrease in the Corg content was detected in the M+S variant (by 5.5% compared to M) – (In both S-enriched variants (M+S and M+B+S), the available P was decreased compared to the M  – , and (at p ≤ 0.00) AOB (r = 0.76), nirS (r = -0.82), and to the N-NH4 nitrogen (r = 0.76)  was received, whereas the M+B+S variant was significantly the highest (41% higher than M) – ( = 0.76) Figure 2dsr (determinant of the S-reducing microorganisms) was found the in the M+S and M+B+S variants (supplied with S) compared to the M and M+B (non-supplied with S): the values were ∼10-fold and ∼7.7-fold higher (than M), respectively (dsr correlated positively (p ≤ 0.001) with AOB (r = 0.82) and N-NH4 (r = 0.93), whereas nirS correlated negatively (p ≤ 0.001) with N-NH4 (r = -0.81), S (r = -0.82), AOB (r = -0.64) and dsr (r = -0.56) (Significantly increased = -0.56) Figure 2The AOB was significantly increased in the M+S and M+B+S variants (by 102% and 169%) compared to the M and M+B; the significantly highest AOB value was detected in the M+B+S manure  showed a significant increase  of AGB compared to the AGB of the control  was significantly higher compared to the control: by 9%, 17%, 16%, 18% (TC) and by 13%, 18%, 17%, 19% (TN)  compared to the control: the values were higher by 35%, 53%, 67%, 50%  than in the non-enriched manure variant M (by 24% higher than control) –  than after the addition of the control sole manure M (nirS (r = 0.72), and negative with N-NH4 (r = -0.97), AOB (r = -0.85), dsr (r = -0.98) – these relations are also apparent from the PCA biplot.The lower pH of the M+S and M+B+S variants (compared to the M and M+B) was ascribed to the acidifying potential of the elemental S addition, which was already reported . Such bi4 content and decreased TKN compared to the M+B. The S-enriched variants  showed higher ammonium content than non-S-enriched ones, putatively due to increased acidity, which was coupled with the microbial production of H2SO4. Sulphuric acid may promote activity of proteolytic bacteria, neutralize and protonate NH3 and, thus, mitigate its release from the manure. This mechanism is in line with the findings of  was observed. Concurrent with the previously reported benefit of acid manure to nitrification, an acidic pH increased the formation of bicarbonate during the hydrolysis of uric acid and urea  or hydrogen sulphide (H2S) and increased access of protons from acidified ammonium org compared to the M and M+B+S. The TC content was close to the TN content. The access of biochar carbon putatively affected the Corg content in the variants M+B , which showed highest decomposition and C mineralization level. The highest composting rate could lead to increased C volatilization in the form of CO2 (or CH4) . A significantly higher microbial activity in the M+B+S variant was presumed too and related to the evidence of increased aeration, which may cause desiccation similar as reported severe drying in the compost  and ammonium content . Both M+S and M+B+S exerted the available P content lower than the manure M. These results may be explained by the acidifying effect of either sulphuric acid (H studies  referred(or CH4) . Howeveractivity . Moreove compost .2S. Under insufficient aerobic conditions, one can expect a partial reduction of elemental S to H2S and its release into the environment.The M+B manure contained a significantly less total S compared to the unenriched manure, whereas the total S value of the M+B+S variant was significantly the highest. The M+B manure was supplied with the biochar, i.e. the material with significantly lower S content compared to the unmatured manure, whereas the M+B+S was enriched by the excessive dose of elemental S together with biochar. The pyrolyzed matter has the potential to adsorb and stabilize any form of S transformation  and mitidsr value in the M+S was attributed to the absence of the putative biochar-mediated adsorption (as assumed for M+B+S) of elemental S, as described by 2S  were contrary to the AOB values: the lowest value in the M+B+S variant (significantly decreased compared to the control manure) and significantly the highest value in the M+B variant. Denitrification is the biochar-mediated and stimulated process that may occur simultaneously with nitrification  positively also with DHA (r = 0.66) and Ala-SIR (r = 0.62). The study by ake of S , corroboorg-abundant; the soil M variant showed a significantly decreased TC compared to the M+B, M+S, M+B+S. However, both M+B+S and M+S manures showed higher ammonium nitrogen content and nitrification potential (AOB marker) compared to the M variant, and this indicated their higher N mineralization rate. Thus, enhanced C sequestration was presumably achieved due to application of manure with increased N conversion. The C:N ratio did not differ significantly between all variants. Both TC and TN correlated significantly (p ≤ 0.001) positively with DHA , Ure  and substrate-induced respirations, e.g. Tre-SIR , NAG-SIR , Lys-SIR  and Arg-SIR . These relations proved that the amendment of enriched manures  enhanced microbial activity and mineralization due to the derived higher nutrient content and availability  led to differences in soil properties compared to the control unamended soil, and between the variants amended with unenriched manure and the enriched manures. All manure variants, applied to the soil, significantly enhanced DHA activity compared to the control (4 and S-reducing microflora among the manures) that most enhanced the decomposing microbial activity in the amended soil. The higher access of S in the soil was reported to correlate with higher DHA  did not significantly change the soil ARS compared to the control, and the variants M and M+B showed even significantly lower ARS values. Our presumption of elemental S-stimulated enhancement of soil organic S mineralization , ascribed from , was denSignificantly increased Ure, Phos, NAG and GLU activities were observed in the variants M+S and M+B+S, compared to the control. The M+S variants also induced the Ure, NAG and GLU values compared to the M and M+B enzyme values. The Phos was the highest in M+B+S. Previously, elemental S amendment was referred to increase soil ARS, Phos and Ure . A signiS-enriched manure-treated variants (M+S and M+B+S) exerted higher basal respiration compared to both the control and the non-enriched manure variant M Figure 7The results of all types of substrates-induced respirations seemed to be close to the results of BR, as shown by the significant (p ≤ 0.001-0.05) moderate to high correlation (r up to 0.79). Nevertheless, it was ascribed that manure enriched with biochar tended to stimulate more respiration inducible by N-rich substrates, whereas the application of S-enriched manure promoted higher respiration inducibility by the (non-nitrous) sugars. These differences implied a variable impact of used types of manure on functional soil diversity with the final consequence in the changes in the nutrient and other soil properties that affected the plant growth and biomass yield.The sulphur-enriched manure showed the most lowered manure pH at the concurrent highest ammonium content. When manure, biochar and sulphur were co-fermented, the highest sulphur content and abundance of ammonium-oxidizing bacteria was observed. When added to soil, this biochar+sulphur-enriched manure promoted the highest dry aboveground plant biomass, the value was 98% higher compared to the unamended control, 38% higher compared to the amendment of biochar-enriched manure and 23% higher compared to the manure-amended variant. Sulphur-enriched manure types enhanced the most enzyme activities and soil respirations . Based on the results obtained, it was concluded that the co-fermentation of biological manures with bio-based materials, such as biochar and sulphur resulting as a by-product of biogas, is an attractive approach, not only to improve the enriched manure product but also to enhance soil fertility, health and crop productivity. This improvement of organic fertilizers may contribute to the circular economy and it will be further investigated by up-scaling on the field level.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Conceptualization, MB and TH; methodology, TH, AK, and OL; software, TB; validation, TB, PS, PR, and JH; formal analysis, TH; investigation, AM; resources, JH and OL; data curation, TH, OL, and AK; writing - original draft preparation, JH, TH, AM, and PS; writing - review and editing, TH, AK, AM, MN, PS, PR, and MB; visualization, TB and AM; supervision, MB, TH, and MN; project administration, MB and AK.; funding acquisition, JH, AK, and MB. All authors have read and agreed to the published version of the manuscript.The work was supported by the project of Technology Agency of the Czech Republic number TH04030142, by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO1218 and MZE-RO1722 and by Ministry of Education, Youth and Sports of the Czech Republic, grant number FCH-S-22-8001.The authors JH, OL and AK are employed by Agrovyzkum Rapotin, Ltd., Vyzkumniku 267, 788 13 Rapotin, Czech Republic and Agricultural Research, Ltd., Troubsko, Czech Republic.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E. coli phage ID52 lysis protein E was introduced to generating BGs for the first time. Above all, we compared the lysis activity of lysis protein of E. coli phage φX174 and E. coli phage ID52 as well as the effects of promoters on the lysis activity of ID52-E, which shown that the lysis activity and BGs formation rate of protein ID52-E was significantly higher than protein φX174-E. Further, the lysis activity of ID52-E was significantly improved under the control of L-arabinose inducible promoter which initial induction OD600 reached as high as 2.0. The applicability of lysis protein ID52-E induced by L-arabinose was proved by preparing probiotic E. coli Nissle 1917 BGs and pathogenic Salmonella typhimurium BGs in mass production. This paper introduced a novel and highly efficient method for BGs preparation depending on recombinant expression of E. coli phage ID52-E under eco-friendly and reasonable price inducer L-arabinose.Bacterial ghosts (BGs) are nonliving empty bacterial shells without cytoplasm retaining original morphology and identical antigenicity of natural bacteria, making them high potential and promising vaccine candidates and delivery vehicles. However, the low yield of commonly used BGs preparation methods limits its mass production and widely application. In order to improve BGs production,  Bacterial ghosts (BGs) mainly refer to the inactive hollow cell envelopes derived from Gram-negative bacteria without cytoplasmic contents, which retain the intact cellular morphology and natural surface antigenic structures such as pathogen-associated molecular patterns (PAMPs), which include lipopolysaccharides (LPS), peptidoglycan, monophosphoryl lipid A (MPL), flagella, adhesins and other immune-related stimulating elements of natural bacteria . TherefoE. coli phage phiX174 lysis protein gene E (φX174-E), which under the control of the temperature-inducible expression plasmid pBV220 without adding other chemicals. Contrary to lysis proteins derived from other phages, gene φX174-E has no inherent enzymatic function, which encodes a 91-amino acid membrane protein with the competence to fuse the inner and outer membranes of Gram-negative bacteria, giving rise to forming an E-specific transmembrane tunnel structure with a size in diameter between 40~400 nm [BGs have so many advantages that they have broad application prospect and great research value, while the low yield of BGs limits the large-scale production and application of BGs. In the short term, BGs were mainly prepared by inducing the expression of 0~400 nm  by oligo0~400 nm . Scannin0~400 nm ,20.600) approximated 0.2~0.6, host bacteria can be lysed effectively by a temperature elevation to 42 °C [600 can be improved during the production of BGs, not only can medium components be fully utilized, but also can the yield of BGs be significantly increased. In order to improve the yield of BGs, Yu et al. [600 was up to 1.1. Further, Zhu et al. [Staphylococcus aureus nuclease A gene, which could effectively induce host bacteria to lysis with the intracellular degradation of genetic material of host cells, and initial induced lysis OD600 could reach as high as 1.2.However, lysis activity of protein φX174-E depended on the growth phase of bacteria . In a geto 42 °C , resultiu et al.  obtainedu et al.  construcM by site-directed mutation of φX174-E, and the initial induced OD600 could reach 2.0 with the joint control of T7 promoter and pLysS plasmid, achieving efficient preparation of E. coli and Salmonella enteritidis. However, the inducer IPTG had a certain toxic effect, and the high price and cost of IPTG were not conducive to the large-scale production and application of BGs. Furthermore, Ma et al. [M without screening the mutant protein with high lysis activity. Therefore, it can be seen that the possibility of improving the yield of BGs by mutating lysis gene φX174-E was low, and this method has been in a bottleneck period. Some chemical agents such as NaOH, H2O2 and SDS have been proved to be an alternative method to commonly method involving protein E for producing BGs [However, the BGs yield of above preparation methods was still far from large scale production. Based on this, Ma et al.  screeneda et al.  continuecing BGs . But thecing BGs . TherefoE. coli Nissle 1917 (EcN), a widely used probiotic, not only possesses the special lipopolysaccharide (LPS) in outer membrane, which exhibits a good immunomodulating property, but also can colonize and replicate in tumors efficiently [Salmonella typhimurium (ST), an important food-borne non-host-adapted Gram-negative intracellular pathogen, has competence to infect a variety of animal hosts and humans around the world. Vaccination is one of the effective methods to reduce the infection of ST in animals [The iciently . Therefo animals , and it  animals . HoweverE. coli phage phiX174, E. coli phage ID52 also belongs to the Microvirus family, and it also contains lysis protein gene gpE (ID52-E), which encodes a 103-amino acid [E. coli phage ID52 lysis protein ID52-E to detect whether it can be applied to produce BGs and improve the yield of BGs. First, the lysis gene φX174-E and lysis gene ID52-E were cloned into pBV220-Chl plasmid to compare the lysis effects of two lysis proteins, whose results showed that the lysis effect of lysis protein ID52-E was significantly higher than that of φX174-E. In order to test whether the yield of BGs could be further improved, the temperature inducible promoter of pBV220 was replaced with L-arabinose inducible promoter and the results manifested that the OD600 initiation can reach as high as 2.0, while the minimum OD600 can be reduced to 0.3~0.4 after lysis. Finally, in order to test the feasibility of the efficient BGs preparation system by protein ID52-E under control of L-arabinose inducible promoter, the system was applied to prepare EcN ghosts and ST ghosts, respectively. And the results manifested that protein ID52-E could be applied to the high-efficiency and high-yield production of EcN ghosts And ST ghosts, whose initial induced OD600 could be as high as 2.5.Like ino acid . Accordi+), and other reagents used in this study were purchased from Sangon Biotech  unless otherwise specified. The final concentrations of chloramphenicol, kanamycin and ampicillin in LB were 25 μg/mL (Chl+), 50 μg/mL (Kan+) and 100 μg/mL (Amp+), respectively.The Bacterial strains, plasmids, and primers used in this research were listed in φX174-E and lysis gene ID52-E were amplified by PCR with primer pairs of φX174-E-F/φ174-E-R and ID52-E-F/ID52-E-R, respectively. Then PCR products were detected by 1% agarose gel and purified and recovered by DNA Gel Extraction Kit . Next, using the purified lysis gene φX174-E and the purified lysis gene ID52-E fragment as a pair of primers in a linear amplification reaction around a circular plasmid pBV220-sGFP-Chl, respectively. Later, the methylated parental plasmid was digested by treating the linear amplification reaction products with Dpn I enzyme. Then the reaction mixture was transformed into E. coli DH5α competent cells. Positive clones were confirmed by DNA sequencing . The resulting plasmids were designated as pBV220-φX174-E and pBV220-ID52-E.Plasmid pBV220-φX174-E and pBV220-ID52-E were constructed by restriction-free cloning technology (RF cloning) ,32 Figu. First, araC-ParaBAD was amplified from the plasmid pKD46 using primers “araC-ParaBAD-F/araC-ParaBAD-R”. Finally, seamless Assembly Cloning kit was used for seamless assembly cloning of linearized fragment pBV220-ID52-E and L-arabinose inducible promoter araC-ParaBAD. Then the reaction mixture was transformed into E. coli DH5α competent cells. Positive clones were confirmed by DNA sequencing . The resulting plasmids were designated as araC-ParaBAD-ID52-E. Primers required to the above vectors were listed in Plasmid araC-ParaBAD-ID52-E was constructed by seamless assembly cloning with Seamless Assembly Cloning kit  . FirstlyE. coli BL21(DE3) competent cells, respectively. Next, the recombinant strains containing different plasmid were cultured to different OD600 values, and the expression of lysis proteins in bacteria containing plasmid pBV220-φX174-E or pBV220-ID52-E was activated by shift the temperature to 42 °C, while L-arabinose with final concentration of 0.5 mg/mL was added to activate the expression of lysis protein ID52-E in bacteria containing plasmid araC-ParaBAD-ID52-E.The three aforementioned plasmids were transformed into 600 value is one of the indicators to evaluate the lysis activity of different lysis proteins E in E. coli BL21(DE3). The values of OD600 were detected at initial induction OD600 approximately 0.8, 1.2, 1.6, 2.0 and detected every 30 min after inducing the expression of lysis protein E. And the OD600 decrease degree was calculated by the following formula: OD600 decrease degree % = (1 − post-lysis OD600/pre-lysis OD600) × 100%. On the other hand, the lysis efficiency of different lysis proteins E was evaluated by detecting viable cell counts before and after lysis, which was conducted by serial dilution and plate count methods. And the lysis efficiency was calculated by the following formula: lysis efficiency % = (1 − post-lysis CFU/pre-lysis CFU) × 100%.On the one hand, the decrease degree of ODE. coli BL21(DE3) harboring pBV220-φX174-E, pBV220-ID52-E and araC-ParaBAD-ID52-E plasmid respectively was cultured to OD600 approximately 1.6 to induce the expression of lysis protein. Then 2 mL bacterial liquid before and after lysis were collected respectively and centrifuged at 5000 rpm for 20 min to collect the bacterial deposits whose genome were extracted by Rapid Bacterial Genomic DNA Isolation Kit  and the genome concentration was measured. The degree of genome leakage was calculated by the following formula: genome leakage degree % = (1 − post-lysis genome concentration/pre-lysis genome concentration) × 100%. All experiments were conducted with three biological replicates.Furthermore, the outflow of genome and intracellular protein after lysis were another indicator to evaluate the lysis activity of lysis protein. In order to detect the outflow of the genome after lysis, E. coli BL21(DE3) harboring pBV220-φX174-E, pBV220-ID52-E and araC-ParaBAD-ID52-E plasmid respectively was cultured to OD600 approximately 1.6 to induce the expression of lysis protein. Then 5 mL bacterial liquid before and after lysis were collected respectively and centrifuged at 5000 rpm for 20 min to collect the bacterial deposits and culture supernatant. The bacterial deposits were re-suspended with 500 μL PBS and ultrasonic crushing was performed. Then the bacterial deposits sample after crushing and the culture supernatant sample were detected by SDS-PAGE.At the same time, the protein leakage after lysis was detected by SDS-PAGE. E. coli BL21(DE3) harboring different plasmids respectively was cultured to OD600 approximately 1.6 to induce the expression of lysis protein Then 10 mL bacterial liquid was collected before lysis and after lysis and the bacteria cells were pelleted by centrifuging at 5000 rpm for 20 min at 4 °C, respectively. Then the precipitate was washed three times with sterile phosphate-buffered saline (PBS) to fully remove the medium. Next, the pellets were resuspended in 5 mL 2.5% glutaraldehyde special electron microscopic fixative and fixed at 4 °C for 8 h. After fixation, cells were washed 3 times with deionized water and eventually resuspended in 1 mL deionized water. Drop samples to the dried cell strip and embed them in filter paper. Subsequently, samples were dehydrated with 70%, 85% and 95% ethanol step by step for 15 min each time, and soaked with 100% ethanol for 15 min (repeat this step 3 times). Next, samples were dried with critical point dryer named Autosamdri-815 and following were coated by a gold sputter coater. Finally, the SEM images were acquired using Merlin SEM .E. coli BL21(DE3) harboring different plasmids respectively was cultured to OD600 approximately 1.6 to induce the expression of lysis protein Then 10 mL bacterial liquid was collected before lysis and after lysis and the bacteria cells were pelleted by centrifuging at 5000 rpm for 20 min at 4 °C, respectively. Same as SEM sample pretreatment, the pellets were washed three times with sterile PBS and fixed with 5 mL 2.5% glutaraldehyde special electron microscopic fixative at 4 °C for 8 h. Then samples were washed three times with deionized water to get rid of fixative. Eventually, 1 mL sterile ddH2O was used to resuspend the sample. Placed a clean piece of filter paper in the culture dish and placed the carbon coated copper mesh (face up) on top of the filter paper. Added 20 µL bacterial droplets to the carbon film copper mesh for 5~10 min at room temperature. Gently moved the copper mesh with tweezers so that the excess bacteria droplets on the copper net were absorbed by the filter paper. Then dropped 20 µL of 3% phosphotungstic acid staining solution to the carbon coated copper mesh with bacteria. Finally, the samples were visualized under a transmission electron microscope .g for 90 min at 4 °C to obtain supernatant and precipitation. Added 5× loading buffer to the whole liquid, supernatant and precipitation in proportion, and boiled for 10 min at 100 °C, respectively. 10 µL protein samples of E. coli BL21(DE3) was separated by 16% tricine-SDS-PAGE. After electrophoresis, proteins bands on the gel were blotted onto a nitrocellulose (NC) membrane  using the Bio-Rad Transblot Turbo system at 80 V for 1.5 h. Following washed with TBST buffer  for 10 min, the NC membrane was incubated with blocking buffer (5% (w/v) skimmed milk powder in TBST) for 2 h to block the reactive sites of the NC membrane. Then the NC membrane was washed with TBST for three times to get rid of superfluous milk, 10 min each time. Later, the NC membrane was incubated with Anti-Strep-Tag II Monoclonal Antibody 8C12 produced in mouse  at 1:5000 in TBST at 4 °C overnight. The following day, the NC membrane was washed with TBST for three times to remove surplus primary antibody, 10 min each time. The NC membrane was incubated subsequently with horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse IgG H&L  at 1:3000 in TBST at room temperature for 2 h. Following repeated washing with TBST, the Super Signal West Pico Chemiluminescent Substrate  was used to visualize the labeled bands on the NC membrane.Western blot analysis in this research was performed according to an established procedure with slight modifications . In brieE. coli Nissle 1917 and Salmonella Typhimurium engineering strain with araBAD gene deletion were constructed by using λ Red homologous recombination system. Firstly, the pKD46 plasmid was transformed into EcN or ST competent cells by electroporation with 2.0 kV and the recombinant positive clones were screened by ampicillin and colony PCR by P1/P2. Then the FRT-kanamycin-FRT linear fragment was amplified by using P3/P4 (EcN) and P5/P6 (ST) containing 56-nt homology arms targeting the araBAD gene as primers and pKD4 plasmid as PCR template following purified by DNA Gel Extraction Kit . The EcN or ST recombination clones containing pKD46 plasmid was inoculated into LB liquid medium with 100 µg/mL Amp+ at 30 °C. The next day, the cells were transferred to fresh LB broth and cultured at 30 °C and 220 rpm until OD reached 0.3~0.4, then L-arabinose with final concentration of 0.5 mg/mL was added to continue culturing until OD reached 0.5~0.6. Subsequently, the cells were made into electrocompetent cells and were transformed with 500 ng purified FRT-kanamycin-FRT linear fragment. Colony PCR was performed to verify whether araBAD gene was successfully replaced by kanamycin fragment by primers P7/P8 (EcN) and P9/P10 (ST) on the colonies grown on ampicillin and kanamycin double resistant plates, respectively. Nextly, the strain EcNΔaraBAD::kanamycin and the strain STΔaraBAD::kanamycin were obtained by culturing at LB broth at 42 °C for 12 h to remove the pKD46 plasmid. Then the plasmid pCP20 was transformed into the strain EcNΔaraBAD::kanamycin and STΔaraBAD::kanamycin competent cells by electroporation with 2.0 kV, respectively, which could express FLP recombinase at 42 °C to remove kanamycin resistance genes inserted into the genome and disappeared with the cell division and proliferation. The successful transfer of pCP20 plasmid was achieved by colony PCR using P11/P12 as primers. Finally, the EcN engineering strain and ST engineering strain with araBAD gene deletion named EcNΔaraBAD::FRT and STΔaraBAD::FRT were identified by PCR using primers P7/P8 and P9/P10 according to the nucleotide sequence of EcN and ST available in NCBI . The lysis curves of EcNΔaraBAD:FRT engineering bacteria and STΔaraBAD::FRT engineering bacteria were plotted by measuring OD600. The morphological observation and identification of EcNΔaraBAD:FRT BGs and STΔaraBAD::FRT BGs were performed by SEM and TEM.All statistical analyses were performed with GraphPad Prism 8.0.2 software and OriginPro 2021. The Data were expressed in the format of mean ± S.D, and the significance level was analyzed by ordinary one-way ANOVA with Tukey multiple comparison.The specific primers were used to conduct colony PCR on the positive clones obtained from the transformation products to verify whether the construction of lysis protein E expressing plasmid was successful. Bands of approximately 343 bp and bands of approximately 376 bp were visualized in positive clones harboring pBV220-φX174-E and pBV220-ID52-E by PCR using φX174-E-F/φX174-E-F and ID52-E-F/ID52-E-R primers, respectively . Bands o600 value decrease degree. Therefore, different initial induction OD600 values (low OD and high OD) were controlled to be the same before inducing the expression of lysis proteins E. When the initial induction OD600 value was low, there was no significant difference between lysis protein φX174-E and lysis protein ID52-E under the control of thermo-inducible lambda pL/pR-cI857 promoter, while there were significant differences between lysis protein ID52-E under the control of L-arabinose inducible promoter and the other two  containing araC-ParaBAD-ID52-E plasmid had the least residual amount of genome and the highest degree of genome leakage, while E. coli containing pBV220-φX174-E plasmid had the most residual amount of genome and the lowest degree of genome leakage. These results indicated that lysis protein ID52-E under the control of L-arabinose inducible promoter had the best lysis effect, which resulted in more genome flow to external media (E. coli BL21(DE3) containing araC-ParaBAD-ID52-E plasmid had the most intracellular protein leakage  showed uniform dark color due to containing DNA, protein and other cell contents  containing different plasmid (E. coli BL21(DE3) containing pBV220-φX174-E was significantly higher than E. coli BL21(DE3) containing pBV220-ID52-E or araC-ParaBAD-ID52-E plasmid, while the expression levels of different plasmids containing lysis protein ID52-E were almost the same. It was speculated that the molecular weight of the lysis protein φX174-E (11.64 kDa) was lower than that of ID52-E (12.77 kDa), so the expression quantity of lysis protein φX174-E was higher in the same induction time. Further, there is possible that the amount of protein required by lysis protein ID52-E to lyse host bacteria was far less than that required by lysis protein φX174-E to lyse host bacteria. When the number of lysis protein ID52-E synthesis reached the threshold of lysis host bacteria, lysis pores formed on the cell membrane, and the host bacteria began to lyse and die, and the lysis protein ID52-E synthesis was terminated, resulting that the expression of ID52-E was lower than φX174-E. And contrary to the expression levels of the two lysis proteins E, the lysis activity of protein ID52-E with lower expression level was significantly higher than those of φX174-E with higher expression level, indicating that the lysis activity of protein ID52-E was so stronger that lysis bacteria required less protein ID52-E.Western blot of the samples manifested that the expression levels of different lysis protein E were significantly different in  plasmid A–C. The Microviridae , EcN and ST have a faster consumption rate of inducer L-arabinose, so it was easy to cause insufficient supply of inducer and thus reduced the lysis rate. At the same time, a certain amount of L-arabinose can promote the growth of EcN and ST, and finally leaded to the phenomenon that the growth rate was faster than the lysis rate and the lysis curve OD600 value rised, resulting in a diminished output of BGs. Therefore, in order to reduce the use of inducers and increase the yield of BG, it was necessary to knock out araBAD gene related to L-arabinose consumption. And the λ Red homologous recombination system was used for gene knockout.In order to test the application scope of the recombinant expression of protein ID52-E under the control of L-arabinose inducible promoter to prepare BGs, the system was applied to EcN and ST. However, different from araBAD gene was amplified and then was identified by 1% agar electrophoresis analysis  ghosts, EcN ghosts and ST ghosts with high efficiency and high yield, but the immune effect was not performed, which still needs to be further expanded. And the lysis efficiency of protein ID52-E under control of L-arabinose inducible promoter was lower than that of protein φX174-E under control of temperature-inducible promoter, but the death of host bacteria didn’t represent the formation of BGs. As observed by TEM (And it has been reported that protein φX174-E mediated cell lysis mechanism was related to cell division, and its kinetics was species-specific, which was speculated to be the same for protein ID52-E ,24. Therd by TEM , proteind by TEM , but mosd by TEM . TherefoFinally, inspired by other scholars’ research on targeted tumor therapy with EcN BGs , becauseE. coli phage ID52-E under the control of L-arabinose inducible promoter, which provided the possibility for the efficient preparation of Enterobacteriaceae BGs and significantly improved the yield of BGs, laying a technical foundation for the large-scale production and application of BGs.In summary, this study not only described a novel and excellent lysis protein ID52-E for BGs preparation for the first time, but established an efficient high-yield BGs preparation system with the recombinant expression of


SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins—S-Alpha, S-Beta, S-Delta, and S-Omicron—were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2.IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease COVID-19) pandemic, continues to evolve. New variants with the potential for altered pathogenicity and transmissibility, as well as vaccine and therapeutic coverage are arising around the world 9 pandemi. The newEarly viral infection is mediated by the structural proteins present on the surface of the virus, most of which are glycoproteins . They meSpike gene affect S protein glycosylation, leading to further functional changes in S protein-mediated infection. So far, studies have shown that compared with the wild-type variant, the S-D614G variant shows a different distribution of glycans at about half of all N-glycosylation sites. The relative abundance of complex-type glycans in S-D614G is 45% lower, and that of high-mannose glycans is up to 33% higher , Helix pomation (HPA), and Dolichos biflorus agglutinin (DBA), which have a high affinity to galactose  derivatives such as GalNAc and present a strong binding signal. Different lectins with the same glycan-binding specificity were clustered into one group. For example, lectins binding to mannose, such as Galanthus nivalis (GNL), Calystega sepiem (CALSEPA), Allium sativum (ASA), Narcissus pseudonarcissus lectin (NPL), Arum maculatum (AMA), and Hippeastrum hybrid lectin (HHL), were grouped into the same cluster.The glycosylation pattern of each SARS-CoV-2 S protein was investigated using a lectin microarray. As shown in To compare the glycosylation patterns of SARS-CoV-2 VOC S proteins, the lectins were classified based on their binding affinity. Through fluorescence intensity analysis, a subset of lectins with differential fucose (Fuc)-binding abilities were detected among the S protein variants. In general, S-Delta and S-Omicron showed higher levels of non-core Fuc glycans (bound by the lectins Lotus tetragonolobus (LTL) and Ulex europaeus agglutinin I (UEA I)) than other variants. Conversely, S-Alpha and S-Beta showed higher levels of core Fuc glycans (Pisum sativum agglutinin (PSA), Aleuria aurantia lectin (AAL), and Lens Culinaris Agglutinin (LCA)) . RegardiFirst, the effect of S glycan pretreatment was verified based on band mobility shifts on SDS-PAGE. The molecular weight of the S protein was reduced to different extents after digestion with different glycosidases, as shown in Fig. S2.DK], 0.9 nM) was 2.2- and 1.4-fold stronger than that of S-OmicronFuc–  and S-OmicronGal– , respectively. However, the ACE2-binding affinity of S-OmicronSia– was found to be 7.1-fold higher than that of S-Omicron . However, the DK value of S-D614G treated with galactosidase  was ~2.5-fold lower than that of S-D614G . Together, these results indicated that the Fuc- and Gal-containing glycans on S-Omicron were key for mediating ACE2 interaction. The higher expression of Fuc-containing glycans on S-Omicron than on S-D614G facilitated ACE2 binding. However, a higher expression of Sia-containing glycans had the opposite effect.Next, to examine the relationship between ACE2 binding and the altered glycosylation patterns of S-Omicron, receptor-binding assays were performed. As shown in -Omicron . In contSurface glycans constitute an important viral defense mechanism because they mask neutralizing epitopes. Hence, we investigated the role of glycans differentially expressed on the S protein of the SARS-CoV-2 Omicron variant in protecting the virus against neutralizing antibodies. The binding of a commercially available broadly neutralizing SARS-CoV-2 Spike antibody (AM359b) to recombinant S-Omicron and S-D614G was tested using an enzyme-linked immunosorbent assay (ELISA). The S protein was treated with α1-2,4,6 fucosidase, β1-3,4 galactosidase, α2-3,6,8 neuraminidase, or Endo H. The corresponding deglycosylated S proteins were coated onto ELISA plates.AM359b could neutralize both S-D614G and S-Omicron, with no significant changes in binding after fucosidase treatment . MeanwhiThese results showed that S-Omicron was more susceptible to monoclonal antibody-mediated neutralization after neuraminidase or galactosidase treatment. Hence, the higher expression of both Sia- and Gal-containing glycans on S-Omicron potentially enhance its shielding effect against neutralization.Lectin microarrays have become a powerful tool for identifying differential glycan expression patterns among protein samples in an ultrasensitive and high-throughput manner. Unlike other conventional glycomics technologies, these microarrays can distinguish among diverse structural isomers and enable a comprehensive comparative analysis of all types of glycans , 18.Spike gene, resulting in different glycosylation patterns. These differences in glycosylation could be one reason the Beta variant has a higher binding affinity for receptors and shows stronger immune escape than the Alpha variant  was purchased from Acro Biosystems. The recombinant human ACE2 protein (mFc Tag) was purchased from Sino Biological. The 293-Free transfection reagent was purchased from Millipore, and Cy3-streptavidin was purchased from Thermo Fisher. EZ-Link sulfo-NHS-LC-Biotin was purchased from Sigma. Finally, α1-2,4,6 fucosidase O, β1-3,4 galactosidase, Endo H, and α2-3,6,8 neuraminidase were purchased from New England BioLabs.2 incubator. The cultures were transfected using the 293-Free transfection reagent when the cells grew to a density of 5 × 105 cells/mL. The supernatants were harvested after 72 h of cultivation, and cells were resuspended for another 72 h. Accordingly, two harvests were obtained.SARS-CoV-2 S constructs  were prepared using overlap PCR from a SARS-CoV-2 S ectodomain-containing plasmid. These were introduced into a pcDNA3.1(+) vector with an N-terminal signal peptide and a C-terminal hexa-histidine tag. Proline  and alanine substitutions (R683A and R685A) were introduced to stabilize the trimeric S protein in its prefusion state and remove the furin cleavage site, respectively. All the recombinant proteins were produced in HEK 293F cells grown in a suspension with 200 mL of FreeStyle 293 expression medium (Life Technologies) at 37°C and 130 rpm in a humidified 8% COg for 10 min at 4°C to separate the cells. The supernatant was transferred to fresh tubes without disturbing the cell pellet. The supernatant was further centrifuged at 10,000 × g for 20 min at 4°C to pellet down any debris. Then, 2 mL of Ni-NTA resin slurry (50% beads in 30% ethanol) was added to a Poly-Prep column (Bio-Rad). The resin was equilibrated with 5 mL Ni-NTA wash buffer (five column volumes). Then, the supernatant mixed with 10 mM imidazole was added to the column for protein binding. The column was washed with 5 mL Ni-NTA wash buffer (five column volumes). Then, 4 mL Ni-NTA elution buffer was added for protein elution, and the eluate was collected. The eluate was concentrated in a 50-kDa centrifugal filter unit at 4,000 × g for 5 min and 4°C. The samples were stored at −80°C, and purity was validated using SDS-PAGE.The purification of SARS-CoV-2 S protein mutants was performed using the protocols described by Schaub et al. . The culD614G and four VOC S protein samples were freeze-thawed, and the bicinchoninic acid (BCA) method was used for protein quantification. Samples were labeled with biotin according to the manufacturer’s instructions for the EZ-Link sulfo-NHS-LC-biotin labeling kit. A slide containing 56 lectins was placed at 4°C overnight and then blocked with 1× Tris-buffered saline (TBS) containing 10% bovine serum albumin (BSA) at 4°C for 3 h. The concentration of the samples was adjusted to 5 μg/mL with 200 μL probing buffer, and the mixture was added to the microarray and incubated with lectins at 4°C overnight. The slide was washed thrice at 25°C. Subsequently, it was placed on a side-swing shaker and incubated with a Cy3-streptavidin-containing probing buffer with a final concentration of 1 μg/mL at room temperature for 1 h in the dark. After washing with probing buffer, the slide was thoroughly dried, and the fluorescence signal was identified using a LuxScan 10K microarray chip scanner. Raw data were obtained from GenePix Pro 6.0 software, and the glycosylation pattern was analyzed. Each sample was examined in at least three biological replicates to exclude the influence of the microscopic heterogeneity of glycans.The protein samples were incubated with several glycosidases and the corresponding 1× Glycobuffer . Enzymatic digestion was performed based on the manufacturer’s protocol. After glycan pretreatment, solution exchange was performed via ultrafiltration to remove any enzymes and salts. The samples were redissolved in the appropriate buffer for subsequent assays. The extent of deglycosylation in each sample was assessed based on the mobility shift observed on SDS-PAGE.DK) was calculated from the koff to kon ratio.SPR measurements were performed on a Biacore 8K instrument (Cytiva) at 25°C using 1× PBS with Tween 20 (PBST) as the running buffer. The Protein A chip was used, and ACE2-Fc at a concentration of 10 μg/mL was used as the ligand. This ligand was immobilized (~150 Response Unit [RU]) on the chip surface through the Fc tag. The S protein, with or without glycosidase treatment, was used as the analyte flowing through the chip surface. A series of 2-fold analyte dilutions  was injected using a multicycle method. The flow rate of the S protein was 30 μL/min. The contact time between the analyte and the ligand was 180 s, and the dissociation time was 900 s. Eight cycles were repeated, with the concentration being increased from 0 to 200 nM. At the end of each analysis cycle, the surface of the sensor chip was completely regenerated with a 30-s pulse of 10 nM glycine . Biacore 8K control software 2.0.1 (Cytiva) was used to collect data, and Biacore X100 evaluation software 2.0.1 (Cytiva) was used to analyze data. The association (kon) and dissociation (koff) rate constants were calculated based on a 1:1 Langmuir binding model. The equilibrium dissociation constant (The enzyme-linked immunosorbent assay (ELISA) was employed to determine the antibody neutralizing ability. Briefly, recombinant SARS-CoV-2 variant D614G and Omicron (B.1.1.529) S protein (0.1 μg/well) were coated onto a 96-well plate and washed with washing buffer  four times. The wells were blocked with blocking buffer  at 37°C for 1 h. Then, an anti-SARS-CoV-2 RBD broadly neutralizing antibody (Acro Biosystems) with a concentration of 12 ng/mL was added and incubated at 37°C for 1 h. Subsequently, horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Abbkine) was added. After incubation with a tetramethylbenzidine (TMB) substrate, the reaction was terminated using a stop solution. The microplate was shaken to ensure uniform mixing, and the absorbance of each well was determined at 450 nm using the BioTek Synergy H1 microplate reader.post hoc test, with a P value adjustment for multiple comparisons by Tukey’s method using the studentized range distribution to maintain a family error rate of 0.05. All statistical analyses were performed using the SPSS Statistics 22 program .All results are expressed as the means ± standard deviations. The statistical significance of between-group differences was analyzed using one-way analysis of variance (ANOVA) along with a


Antisense noncoding RNA in the INK4 locus (ANRIL) is located on human chromosome 9p21, and modulation of ANRIL expression mediates susceptibility to some important human disease, including atherosclerosis (AS) and tumors, by affecting the cell cycle circANRIL and linear ANRIL are isoforms of ANRIL. However, it remains unclear whether these isoforms have distinct functions. In our research, we constructed a circANRIL overexpression plasmid, transfected it into HEK-293T cell line, and explored potential core genes and signaling pathways related to the important differential mechanisms between the circANRIL-overexpressing cell line and control cells through bioinformatics analysis.Stable circANRIL-overexpressing (circANRIL-OE) HEK-293T cells and control cells were generated by infection with the circANRIL-OE lentiviral vector or a negative control vector, and successful transfection was confirmed by conventional flurescence microscopy and quantitative real-time PCR (qRT-PCR). Next, differentially expressed genes (DEGs) between circANRIL-OE cells and control cells were detected. Subsequently, Gene Ontology (GO) biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore the principal functions of the significant DEGs. A protein–protein interaction (PPI) network and competing endogenous RNA (ceRNA) network were constructed in Cytoscape to determine circularRNA (circRNA)- microRNA(miRNA)-messenger RNA (mRNA) interactions and hub genes, and qRT-PCR was used to verify changes in the expression of these identified target genes.The successful construction of circANRIL-OE cells was confirmed by plasmid sequencing, visualization with fluorescence microscopy and qRT-PCR. A total of 1745 DEGs between the circANRIL-OE group and control were identified, GO BP analysis showed that these genes were mostly related to RNA biosynthesis and processing, regulation of transcription and signal transduction. The KEGG pathway analysis showed that the up regulated DEGs were mainly enriched in the MAPK signaling pathway. Five associated target genes were identified in the ceRNA network and biological function analyses. The mRNA levels of these five genes and ANRIL were detected by qRT-PCR, but only COL5A2 and WDR3 showed significantly different expression in circANRIL-OE cells. Antisense noncoding RNA in the INK4 locus (ANRIL) is located on human chromosome 9p21, within the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster. Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) at 9p21 that are linked to susceptibility to coronary artery disease, ischemic stroke and tumors . The cauMany researchers have demonstrated that circular RNA (circRNA) is a type of recently discovered endogenous noncoding RNA (ncRNA) that plays a vital role of posttranscription but has no ability to produce proteins . Some cihttp://www.cellbank.org.cn) and the cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin, in a 37 °C, 5% CO2 and saturated humidity incubator. To generate the circANRIL- OE cell line, sequcences from the circRNA hsa_circ_0008574 were obtained from http://www.circbase.org/, we constructed an expression plasmid using the following sequence: CGGAATTCTGAAATATGCTATCTTACAGTGTCCCTTTTGATGAGAAGAATAAGCCT CATTCTGATTCAACAGCAGAGATCAAAGAAAAGACTTCTGTTTTCTGGCCACCAGATAATGTTATCTGTGCTTAAAGAATTGAAAAACACACATCAAAGGAGAATTTTCTTGGAAA GAGAGGGTTCAAGCATCACTGTTAGGTGTGCTGGAATCCTTTCCCGAGTCA GCTTTCTAGAAGAAAACCGGGGAGATCTATTTGGAATGTATCTAACTCCAAAGAA ACCATCAGAGGTAACAGTAGAGACGGGGTTTCACCATGTTGGCCAGACTGGTCTT GAACTCCCGACCTCGTGATTCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTA CAGGTGTGAGACACCACGCCCGGCGGATAGAGAGAATTTTGACAGGTGAATATA TTTTTTCTTGAGGATCCCG. The restriction sites BamHI and EcoRI are underlined, and the splice sites were designed as donor sites (GT on the forward strand) and acceptor sites (AG on the reverse circularized sequence) which are shown in bold and italics, the other sites were target sequences. After digestion by the restriction enzymes EcoRI and BamHI, the PCR fragments were sequenced before cloning into the pUC57-Amp vector  to generate the pUC57-hsa_circ_0008574 construct, which was transformed into Stbl3 competent cells . The status of individual colonies selected from plates was also confirmed by sequencing.The human cell line HEK-293T was purchased from the Cell Bank, Chinese Academy of Sciences .The infection efficiency was determined under a fluorescence microscope after 48 h. Stably transfected cell lines were selected using puromycin  and the cells were divided into three groups: circANRIL-OE group (transfected with the constructs), blank control group (untransfected cells) and negative control group . Each group contained three samples, and each sample contained approximately 1 × 10To validate the specificity of the HEK-293T cell transduction, total RNA was extracted from HEK-293T cells by using TRIzol reagent  according the manufacturer’s protocol. mircoRNA (mRNA) levels of circANRIL and lincANRIL in these groups were analyzed with a qRT-PCR kit  following the manufacturer’s instruction.https://cran.r-project.org/bin/windows/contrib/3.4/ggfortify_0.4.6.zip).According to the manufacturer’s protocol, a cDNA library was established by using the RNA Sequencing Library Preparation Kit (Illumina). The libraries were applied to a flowcell on a cBOT instrument (Illumina) to generate clusters, and then subjected to Illumina high-throughput RNA sequencing platform . To obtain clean data, the sequencing data were subjected to quality control, and the clean reads were mapped to the human reference genome (GRCh38) using TopHat (v2.1.0), The R function cor was utilized to obtain the Pearson correlation coefficient (Pearson’s r), the correlation coefficient ranged from 0 to 1, and higher scores indicate highly similar expression patterns among samples. Principal component analysis (PCA) was performed by R function prcomp, and the results were visualized using the ggfortify package version: 0.4.5 (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) to screen for DEGs between the circANRIL-OE group and control groups. Raw counts were normalized with the TMM method, and then presented as log2 counts per million (LogCPM) values. We obtained the logFC and P value of each gene after assessing the data, and the adjusted P value  was determined after multiple test correction with the Benjamini & Hochberg method, FDR < 0.05 and —logFC— > 1 were used as thresholds for DEG identification.Differential expression analysis was performed using the edgeR language package version 3.4 (https://david-d.ncifcrf.gov/) functional annotation tools. GO biological process (BP) analysis was included in this study to explore the key signaling pathways. GO BP terms and KEGG pathway terms with a P value of < 0.05 and an enrichment score of >5 were defined as significantly enriched. Default DAVID database setting with medium stringency and Homo sapiens was defined as background.All the DEGs were subjected to GO and KEGG pathway enrichment analyses. The analyses were performed using Database for Annotation, Visualization, and Integrated Discovery . The species selected was “Homo sapiens”. The PPI score was set at 0.9 (highest confidence), Cytoscape  was used to determine the PPI network pairs. The key topological properties of the PPI network were analyzed with the Cytoscape plug-in Network Analyzer  in Cytoscape (version 3.4.0). The parameter was set to ‘without weight’. All PPI pairs with a combined score >0.9 were extracted,Three parameters were chosen to evaluate every node in the network, including degree (the number of nodes directly linked to one node), betweenness (the relative importance of a vertex within the network) and closeness (the proximity of a network node to other nodes). the final combined scores were used to sequence and evaluate central proteins.To construct the PPI network, the DEGs were input for PPI analysis into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database  was used to identify RNA- binding proteins (RBPs) related to circANRIL and to determine whether any RBPs differentially expressed between the two groups. The TargetScan database (http://www.targetscan.org) was used to predict miRNAs within the 90th percentile. We then predicted the target genes of these miRNAs with miRWalk2.0 , and the results of the following 6 databases were comprehensively considered to confirm the target genes: miRWalk , MicroT4 4.0 , miRanda (https://anaconda.org/bioconda/miranda), miRDB (http://mirdb.org/miRDB/), PITA version 6 (https://web.archive.org/web/20200529050843/https://omictools.com/pita-tool) and RNA22 databases (https://cm.jefferson.edu/rna22/). Only those miRNA target genes included in all the gene sets were used for further analysis. Here, we reasoned that if circANRIL is upregulated, the same miRNA-regulated target genes should also be upregulated. Therefore, the potential miRNA-target gene pairs were obtained by overlapping the previously predicted target genes of the PPI and the upregulated DEGs. The circRNA-miRNA and miRNA-mRNA were combined for the construction of the circRNA-miRNA-mRNA regulatory network using Cytoscape 3.4.0. miRNAs and mRNAs with FDR <1%, absolute log2FC >2 and P < 0.01 were retained to establish the ceRNA network. Hub genes were selected according to the node degrees of the ceRNA network by calculating the number of circRNA-miRNA and miRNA-mRNA pairs. qRT-PCR was used for further validation of the selected genes. The overall procedure is summarized in the flow diagram in To explore the underlying mechanism of circANRIL, the online database circInteractome . All experiments were performed at least three times.Data in each group were assessed using SPSS 22.0 software (SPSS Inc.) and are presented as the mean ± SD. Differences between two groups were analyzed by the chi-square test and the Mann−Whitney U test. Comparisons between multiple groups were carried out using one-way analysis of variance or the Kruskal −Wallis H-test. P < 0.05), while linear ANRIL showed a trend of decreased expression in the circANRIL-OE group compared to the control groups . After filtering out low quality reads, a total of 20,313 genes were further analyzed. All the raw sequencing data have been deposited into the SRA (accession number: PRJNA756043). The Pearson correlations(r) which ranged from 0.921 to 1, and P values are shown in  by TopHat. GENCODE annotation of the human genes was performed by htseqcount (shown in . The medshown in . Accordishown in -sheet 2.P < 0.05) by the upregulated DEGs (P < 0.05) and 127 terms were enriched by the downregulated DEGs.The top 20 enriched GO BP terms are shown in In enrichment analysis, 26 GO BP terms were significantly enriched  (The RBPs of 13 circRNAs were identified via circBase (se.org/) . The seahttp://www.targetscan.org). A total of 197 circRNA-miRNA pairs including 13 circRNAs and 56 miRNAs were obtained, and 2456 miRNA-targets of these 56 miRNAs were predicted by miRWalk2.0 and six other databases. Based on the previously mentioned methods, the overlapping genes of these predicted mRNAs and genes encoding the upregulated proteins of the PPI network were determined for 36 miRNA-target pairs, including 22 miRNAs and 20 target genes. Furthermore, 71 circRNA-miRNA-mRNA pairs were obtained by identifying miRNAs and target genes regulated by the same miRNAs through intergration of the circRNA-miRNA and miRNA-mRNA relationships. The circRNA-miRNA-mRNA network contained 31 circRNA-miRNA pairs, 69 miRNA-mRNA coexpression relationships, and 18 miRNA-mRNA pairs. There were a total of 34 nodes, including 11 circRNAs, 10 miRNAs and 13 mRNAs. Among them, miR-1305 had the most miRNA target sites (six) while miR-1264 had two miRNA target sites, indicating that miR-1305 and miR-1264 serve as crucial miRNAs regulating the ceRNA network  were identified as core protein in the PPI network, but their change trend differ from that predicted for circANRIL. In the ceRNA network, five hub genes with a high degree of interaction, COL5A2, DOK6, ABCA1, GOLGA4 and WDR3 were identified as hub genes of circANRIL, which was further confirmed by qRT-PCR validation, The results demonstrated that COL5A2 and WDR3 mRNA expression was markedly increased in the circANRIL- OE cell line compared to the control cell lines, as predicted. while ANRIL, ABCA1, DOK6 and GOLG4 mRNA expression was remarkably decreased. COL5A2 is a subtype of collagen V involved in the pathogenesis of fibrosis as a regulatory fibril-forming collagen. The COL5A2 gene encodes a 46 kDa protein that inhibits nuclear localization and suppresses transcriptional activity. It is the main structural element of extracellular matrices in connective tissue and can be found in all organisms. It was also found to be enriched in the protein digestion and absorption and platelet activation processes, and to be related to extracellular matrix remodeling. Few reports have indicated its role in pathological process in multiple cancers, the immune system and angiogenesis . COL5A2 The WDR3 gene is a protein-coding gene located on chromosome 1p12-p13 (25) in a region that is related to tumors . As a nuNotably, COL5A2 and WDR3 are related to cell proliferation, as previously reported. To the best of our knowledge, no previous studies have investigated the affects of these two genes on circANRIL or ANRIL expression, However, their known mechanisms in regulating cell proliferation and signaling pathways in other diseases may indirectly explain their effects on circANRIL.in vivo and in vitro. Encouragingly, the development of laboratory techniques will provide new methods for determining the biological features and roles of circANRIL in the pathophysiological of processes of multiple diseases.The limitation of our study is that we only assessed one dataset from circANRIL-OE cells. Although our results cannot be directly applied to humans, they will be of help to our further research. Based on our analysis, we cannot infer the potential mechanism of the hub genes since molecular biological studies are needed to verify the potential mechanism In conclusion, 179 up-regulated DEGs, one KEGG pathway, and two hub genes and their signaling pathways were identified in this research. Only COL5A2 and WDR3 were identified to circANRIL function, warranting further studies to explore their application in the clinical setting. However, molecular biological experiments are required to confirm the function of the identified genes in regulating circANRIL expression.10.7717/peerj.13135/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13135/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13135/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.13135/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.13135/supp-5Supplemental Information 5cirRNA-miRNA pairs extracted by TargetscanClick here for additional data file.10.7717/peerj.13135/supp-6Supplemental Information 6miRNA-target gene pairs predicted by miRwalk 2.0 et al.Click here for additional data file.10.7717/peerj.13135/supp-7Supplemental Information 7ceRNA network constructed by cytoscapeClick here for additional data file.10.7717/peerj.13135/supp-8Supplemental Information 8Click here for additional data file.


AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca2+]i-alternans. Myofilaments are known to play a role as intracellular Ca2+ buffers and here we show increased Ca2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca2+. Similarly, EMD57033, a myofilament Ca2+ sensitiser, replicated the abnormal [Ca2+]i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca2+ dynamics, normalising Ca2+ transient profile and minimising the occurrence of Ca2+ alternans at physiological frequencies. This finding suggests that increased Ca2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([CaThe online version contains supplementary material available at 10.1007/s00395-022-00912-z. Dilated cardiomyopathy (DCM) represents the most common cardiomyopathy and is a major contributor to heart failure and sudden cardiac death . Roughly2+) handling and reduced contractility are key features of patient-specific iPSC-CM carrying TnT-R173W i), Fluo-3 was excited at 488 nm and emitted light (> 520 nm) converted to [Ca2+]i, assumingkd is the dissociation constant of Fluo-3 (864 nM), F is the Fluo-3 fluorescence; Fmax is the Ca2+-saturated fluorescence obtained at the end of each experiment , as determined during the decay of the caffeine-induced Ca2+ transient i at which buffers are half saturated, was significantly lower in DCM-TnT-R173W, compared to Ctrl , allowing estimation of the time course of changes of total cytosolic Ca2+ during ICa,L-triggered Ca2+ transient  as a function of the free cytosolic Ca2+ level  plotted against the free [Ca2+]i with unaltered slope in DCM-TnT-R173W. The unaltered slope shows that the slowed decay of the systolic free Ca2+ transient in DCM-TnT-R173W  showed no difference between groups, confirming unaltered Ca2+-dependence of NCX function. Since [Ca2+]i dependence of decay rate of total Ca2+  was unaltered during systolic and caffeine-induced Ca2+ transients, it can be concluded that [Ca2+]i dependence of SERCA activity is unaltered in DCM-TnT-R173W, which has been estimated based on the difference between the two respective slopes  was unaltered  and time constant of decay was greater in EMD57033-treated iPSC-CM, compared to Ctrl  i separately from alterations in the Ca2+ binding affinity of myofilaments. The latter represent the major Ca2+ buffers in cardiac myocytes and alterations in binding affinity are thought to have relevant impact on cellular Ca2+ homeostasis i as opposed to direct activation by ICa,L channels [2+ handling and contractile function [In the present study, we used iPSC-CM from DCM patients harbouring the TnT-R173 mutation. iPSC-CM represent myocytes at an immature developmental stage. iPSC-CM exhibit poor co-localisation between Iand RYR2 , resultichannels , 78. Nevfunction , 23. In function , 13, makfunction , 44, 77.Ca,L in DCM-TnT-R173W iPSC-CM. In contrast, SR Ca2+ content was unchanged in DCM-TnT-R173W iPSC-CM . In addition, Ca2+ removal by forward mode NCX was increased in DCM-TnT-R173W iPSC-CM to compensate for the higher Ca2+ influx through upregulated ICa,L. The mechanisms underlying increased ICa,L are beyond the scope of the present study. Nevertheless, unaltered mRNA expression of the underlying Ca2+ channel subunit, Cav1.2, renders intrinsic differences of its expression levels between DCM-TnT-R173W and Ctrl iPSC-CM unlikely . Further analysis of the biphasic ICa,L inactivation revealed unaltered time course of fast ICa,L decay , which is thought to be mainly due to Ca2+ dependent inactivation of ICa,L [Ca,L in DCM-TnT-R173W is unlikely due to reduced Ca2+-dependent inhibition of ICa,L in response to reduced free cytosolic Ca2+ levels. In accordance, increased cytosolic buffering due to EMD57033 leads to a similar decrease in Ca2+ transient amplitude but had no effect on ICa,L. ICa,L is regulated by various post-transcriptional and post-translational mechanisms including miRNA-dependent inhibition, phosphorylation and expression of accessory units [Ca,L alterations in DCM patients in general, but also those harbouring the TnT-R173W mutation.Our data show significant upregulation of I+ efflux , 67. Accry units , 56. FutCa,L, the triggered Ca2+ transient amplitude was smaller in DCM-TnT-R173W compared to Ctrl. Based on our experiments, we conclude that this is largely due to increased cytosolic Ca2+ buffering. In accordance, the total amount of Ca2+ released from the SR during ICa,L-triggered Ca2+ transients was unaltered in DCM-TnT-R173W . Therefore, impaired interaction between L-type Ca2+ channel and RyR2 may additionally contribute to reduced free ICa,L-triggered Ca2+ transient amplitude.Despite greater I2+ buffering, we employed a method that allows investigation of Ca2+ homeostasis in intact cardiomyocytes [2+ has been quantified by the fluorescent Ca2+ indicator Fluo-3, which represents an intracellular Ca2+ buffer itself. We assume that the contribution of Fluo-3 to intracellular Ca2+ buffering is comparable between experimental groups and does not contribute to the differences observed in the present study. Based on the dissociation constant (Kd = 0.864 µM) and an estimated buffer concentration (bmax) of 100 µM, the contribution of Fluo-3 to the calculated buffer curves is illustrated in Supplementary Fig. 5. In addition, the employed techniques do only allow indirect conclusions on altered Ca2+-binding to the troponin complex. Direct quantification of Ca2+ binding to troponin C will require further biochemical analysis that are beyond the scope of the present study [To quantify cytosolic Camyocytes . Intracent study .2+ handling abnormalities in iPSC-CM carrying a specific mutation in cardiac TnT that has been associated with the occurrence of DCM. Given the multifactorial aetiology of DCM, it is unclear whether reduced Ca2+ uptake by SERCA, secondary to increased Ca2+ buffering by myofilaments may represent a “final common pathway” underlying arrhythmogenesis in DCM patients [2+ reuptake into the SR [2+ sensitivity of contraction has also been found in patients and a dog model of pacing induced DCM [2+ sensitivity were abrogated after treatment with the catalytic subunit of PKA, suggesting that, as in DCM caused by TnT-R173W mutations, the increased Ca2+ sensitivity of myofilaments may be due to a reduction in PKA-mediated phosphorylation of myofibrillar regulatory proteins.In the present study, we investigated Capatients . Nevertho the SR . Furtheruced DCM , 75. The2+ may contribute to arrhythmogenesis in various cardiac diseases, modulation of myofilament Ca2+ sensitivity may represent an important novel concept to prevent cardiac arrhythmias [Based on our findings and given the fact that increased myofilament affinity for Cahythmias , 59.2+ binding of myofilaments is a classical therapeutic concept to improve contractile dysfunction in heart failure patients. Levosimendan and omecamtiv represent traditional drugs aiming to improve contractile force by increasing Ca2+ sensitivity and Ca2+-myofilament binding [2+ [Targeting Ca binding , 49. Nevding [2+ , 20.2+ alternans in mouse hearts harbouring Ca2+-sensitising TnT mutations in vitro [2+ alternans in DCM-TnT-R173W iPSC-CM. Similar to blebbistatin, mavacamten, a small molecule modulator of β-cardiac myosin, which has been recently evaluated in patients with hypertrophic cardiomyopathy, has also been shown to reduce Ca2+ affinity of myofilaments [Blebbistatin is an inhibitor of the myosin ATPase and has been shown to prevent the occurrence of Cain vitro . Accordiilaments . Both blSupplementary file1 (DOCX 2314 KB)Below is the link to the electronic supplementary material.


CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C− cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c−/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c Non-alcoholic fatty liver disease (NAFLD) is primarily a manifestation of metabolic syndrome in the liver. NAFLD is a disease that encompasses a wide range of liver pathologies, from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocarcinoma . The eti+ M1 macrophages in adipose tissue are pro-inflammatory, whereas CD206+ M2 macrophages are anti-inflammatory  and and63] acDNA synthesis and amplification from 1 ng total RNA were performed by using the SMART-Seq HT kit (Takara Bio Inc.), followed by the tagmentation using the Nextera XT DNA library prep kit . Nextera PCR Master Mix (Illumina) and Nextera-DNB Conversion Primer  were useTrimming of the low-quality reads and sequence adaptor (NNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG or NNGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG) from raw sequence reads were performed by using Trim Galore Ver 0.6.6, followed by the mapping of the processed sequenced reads to the mouse genome  with HISAT2 Ver 2.1.0. Samtools Ver 1.9 were used for the conversion of the obtained SAM files to BAM format followed by gene expression profiling by using Strand NGS software Ver4.0. For global normalization, raw sequence read counts were processed using Trimmed means of M value (TMM) algorithm. Principal component analysis (PCA) was performed using R Version 3.6.0. Gene network analysis was performed using Ingenuity Pathway Analysis tools , following the extraction of genes differentially expressed by >5.0- or >10.0-fold.t-test for two groups comparison. When we analyzed Student’s t-tests, we first performed an F-test on GraphPad Prism 9 software  to confirm that the p-value of the F test was >0.05 and equally distributed. Statistical analysis was performed using GraphPad Prism 9 software. p < 0.05 was considered statistically significant. The results were presented as the mean ± SD.Statistical significance was evaluated by two-way ANOVA followed by post hoc Sidak test for multiple comparison, and Student’s