Currently, there is no comparative study of how the disruptions of the five glycogen metabolism genes influence bacterial phenotypes, such as growth rate, biofilm formation, and environmental survival, etc. In this study, we systematically and comparatively studied five E. coli single-gene mutants  in terms of glycogen metabolism and explored their phenotype changes with a focus on environmental stress endurance, such as nutrient deprivation, low temperature, desiccation, and oxidation, etc. Biofilm formation in wild-type and mutant strains was also compared. E. coli wild-type stores the highest glycogen content after around 20-h culture while disruption of degradation genes  leads to continuous accumulation of glycogen. However, glycogen primary structure was abnormally changed in ΔglgP and ΔglgX. Meanwhile, increased accumulation of glycogen facilitates the growth of E. coli mutants but reduces glucose consumption in liquid culture and vice versa. Glycogen metabolism disruption also significantly and consistently increases biofilm formation in all the mutants. As for environmental stress endurance, glycogen over-accumulating mutants have enhanced starvation viability and reduced desiccation viability while all mutants showed decreased survival rate at low temperature. No consistent results were found for oxidative stress resistance in terms of glycogen metabolism disruptions, though ΔglgA shows highest resistance toward oxidation with unknown mechanisms. In sum, single gene disruptions in glgBXCAP operon significantly influence bacterial growth and glucose consumption during culture. Accumulation and structure of intracellular glycogen were also significantly altered. In addition, we observed significant changes in E. coli environmental viabilities due to the deletions of certain genes in the operon. Further investigations shall be focused on the molecular mechanisms behind these phenotype changes.Glycogen is a highly branched polysaccharide that is widely present in all life domains. It has been identified in many bacterial species and functions as an important energy storage compound. In addition, it plays important roles in bacterial transmission, pathogenicity, and environmental viability. There are five essential enzymes (coding genes) directly involved in bacterial glycogen metabolism, which forms a single operon  Streptomyces venezuelae and characterized in Escherichia coli ], glgA [glycogen synthase (GS)], glgB [glycogen branching enzyme (GBE)], glgP [glycogen phosphorylase (GP)], and glgX [glycogen debranching enzyme (GDE)]  revealed that 65 genes were involved in the glycogen accumulation in E. coli . In addimulation . Thus, bmulation . Among te (GDE)] . The fiv pathway . In partAzospirillum brasilense, V. cholera, and Corynebacterium glutamicum, etc.  and E. coli BW25113. In sum, this study provides a better understanding of glycogen functions in bacterial physiology through phenotypic characterization.Previous studies have investigated the influences of single gene mutants in the glycogen metabolism pathway on glycogen accumulation, glycogen structure, environmental persistence, and biofilm formation in different bacterial species, such as um, etc. . Single nditions . A recenysiology . HoweverE. coli mutants used in this study, ΔglgA, ΔglgB, ΔglgC, ΔglgP, and ΔglgX, together with the wide-type parent strain BW25113, were purchased from Horizon Discovery Ltd.  as a whole package of the commercial Keio Collection, a systematic, single-gene knockout mutant collection of E. coli non-essential genes via λ-Red Recombination System . Wild-type strain BL21(DE3) was purchased from Tiangen Biotech Co. . The presence and absence of the five genes  in the purchased wild-type and mutated E. coli strains were confirmed via PCR. For the results of agarose gel electrophoresis, please refer to 2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, and 0.5 g/L NaCl (Sigma-Aldrich) supplemented with 0.2, 0.4, 0.8, and 1.6% glucose (Sigma-Aldrich) was used for E. coli culture, respectively. Liquid and solid Luria-Bertani (LB) media  were also used for bacterial recovery from −80°C freezer and biofilm formation assay. Phosphate-buffered saline  was used for environmental stress endurance assay. All the strains were cultured at 37°C with shaking rate at 220 rpm when needed, until otherwise specified.All the five 600 values were measured and recorded by spectrophotometer (Thermo Fisher Scientific). OD600 readings, together with error bars, were drawn by correlating with corresponding time points for growth curves. For each time point, three independent replicates were repeated. As for the growth curves of the seven strains, that is, BL21(DE3), BW25113, ΔglgC, ΔglgA, ΔglgB, ΔglgP, and ΔglgX, cultured in 1 × M9 minimal medium supplemented with 0.8% glucose, the same procedure described above was followed.A single colony of BL21(DE3) was picked from LB agar plate and cultured in 5 ml LB broth for 5 h . 1 × M9 minimal medium supplemented with 0.2, 0.4, 0.8, and 1.6% glucose was inoculated with the starting culture at 1:20 ratio and cultured at 37°C with shaking rate at 220 rpm, respectively. At selected time points of 2, 4, 6, 8, 10, 12, 16, 18, 20, and 24 h, ODE. coli strains, we first measure the glucose content in wide-type BL21(DE3) 1 × M9 culture supplemented with 0.2, 0.4, 0.6, and 0.8% glucose at 0, 4, 6, 10, 12, 14, 16, 18, 20, and 24 h through GOPOD assay kit  according to the manufacturer’s instructions. 1 × M9 minimal medium with 0.8% glucose was then used as a standard culture, in which the glucose consumption amount for all the seven strains was measured by following the growth curves. All the experiments were repeated independently for three times.In order to explore the glucose consumption amount in different cultures by different Glycogen content was assayed for BL21(DE3) along growth curves at 4, 6, 10, 14, 16, 18, 20, and 24 h in 1 × M9 minimal medium supplemented with 0.2, 0.4, 0.8, and 1.6% glucose. As for BW25113 and the five single gene mutated strains, glycogen content and protein amount were assayed at the same time points supplemented with 0.8% glucose only. Three independent replicates were used for each measurement. For glycogen isolation and purification, sucrose gradient density ultracentrifugation (SGDU) was used. All the procedures were followed as previously described , 2019.Nde(X) were measured by standard FACE method. For details, please refer to Fluorophore-assisted carbohydrate electrophoresis (FACE) was used for characterizing glycogen primary structure (chain length distribution). In particular, 200 unit/mg isoamylase  was used to break down all α-1,6-glycosidic branching points in glycogen, which were then labeled with APTS . The distributions of the number of chains as a function of the degree of polymerization X of that chain after debranching, ® 96 well plates, polystyrene, flat bottom with lid, sterile) with 200 μl/well. Sterile media were added as blank control, correspondingly. The 96-well microplate was cultured statically at 30°C for 20 h. After that, liquid waste was discarded, and planktonic cells were washed out with distilled water for three times. Then, 200 μl 0.1% crystal violet was pipetted into each well, including blank control, for 10 min staining. After that, crystal violet liquid was shaken out of the plate over the waste tray and all plates were washed for three times with distilled water. Finally, all plates were air-dried. 200 μl of 20%/80% ethanol/acetone solution was added to each well for dissolving crystal violet in biofilm for 10 min, 150 μl of which at each well was transferred to a new 96-well plate and OD590 value was read and recorded in spectrophotometer.A single bacterial colony was picked up from LB agar to inoculate 5 ml LB broth, which was then cultured at 37°C overnight with 220 rpm shaking rate. 100 μl of the LB broth was inoculated into 10 ml 1 × M9 minimal medium (0.8% glucose) and LB broth, respectively. Each diluted culture was aliquoted to a 96-well microplate , centrifuged at 6000 × a method . All theg for 10 min, and the harvested cells were washed three times and resuspended in PBS buffer.For each studied strain, a single bacterial colony was picked up from LB agar plate for inoculating 5 ml LB medium at 37°C with 220 rpm shaking rate, which was then used to inoculate 100 ml 1 × M9 medium supplemented with 0.8% glucose. After the inoculated culture was incubated for 20 h , it was dispended into 1.5 ml Eppendorf tubes with 1 ml/tube and store at 4°C. At day 0, 3, 6, and 9, the suspension was serially and aseptically diluted in a hood by mixing 100 μl diluted culture with 900 μl PBS buffer. For each dilution, Miles and Misra method was used and three replicates were used . After i–2 to 10–6. For each dilution, 10 μl of the diluent was taken and dropped onto LB agar plate, which was incubate at 37°C for 24 h. The number of bacterial colonies was counted, and the number of viable cells in original culture was calculated as stated in section “Low Temperature Viability Assay.” The experiments were repeated three times for each strain.Bacterial cell culture was prepared as described in section “Starvation Viability Assay.” After cells were resuspended in 100 ul PBS buffer, 15 μl of concentrated bacterial solution was added to sterilized petri dish for air-dry inside hood. At 0, 1, 3, and 6 h, dried cells were re-suspended in 1.5 ml PBS buffer and continuously diluted from 102O2 was added onto a 6 mm sterilized round filter paper, which was then placed in the center of the agar plate. After culturing the plate statically at 37°C for 24 h, diameter of bacteriostatic ring from three different angles was measured and recorded. For each strain, the experiment was repeated independently three times.Cells were prepared in the same way as for starvation viability assay. After cultured for 20 h, 2 ml of bacterial solution was mixed with 18 ml LB broth containing 0.8% agarose agar and 1 × M9 minimal medium (0.8% glucose) containing 0.8% agarose agar for agar plate, respectively. 10 μl of 6.6 mol/L Ht-test was calculated for pairwise comparison wherever applicable, unless otherwise instructed. Significant difference is denoted with asterisk(s) when the P-value was less than 0.05 . The single-step multiple comparison procedure and statistical test, Tukey’s Honestly Significant Difference (HSD) test, was performed for biofilm formation ability and oxidative stress resistance ability among wild-type and mutated strains, which compared the means of every group to the means of every other group simultaneously. Means denoted by a different letter indicated significant differences between groups (P < 0.05).A two-tailed unequal variance Student E. coli, which involves the supplementation of complex trace elements and different concentrations of glucose (E. coli BL21(DE3). The results showed that the bacterium had a similar growth curve when cultured in 1 × M9 minimal medium with 0.8 and 1.6% glucose. In contrast, OD600 value decreased more rapidly after entering into stationary phase in 1 × M9 minimal medium with 0.2 and 0.4% glucose . However, by comparing OD600 values at each time point, statistical significance could be detected from 16 h. In particular, at 24 h, the OD600 value of BL21(DE3) in 1 × M9 minimal medium with 0.2% glucose was significantly lower than OD600 values of BL21(DE3) growing in other three media (P < 0.01) while OD600 values of BL21(DE3) in 1 × M9 minimal medium with 0.4% glucose were significantly lower than OD600 values of BL21(DE3) growing in 1 × M9 minimal medium with 0.8 and 1.6% glucose (P < 0.05). No significant difference was detected between OD600 values of BL21(DE3) growing in 1 × M9 minimal medium with 0.8 and 1.6% glucose. Thus, glucose concentration in the culture media has direct impact on E. coli growth pattern.Previous studies have already shown that glucose concentration is important in determining bacterial growth and glycogen accumulation . There a glucose . In this glucose . It is acultures . Two-taiglgC, ΔglgA, ΔglgB, ΔglgP, and ΔglgX) in 1 × M9 minimal medium supplemented with 0.8% glucose, and compared their growth curves . As for the two wild-type strains, BL21(DE3) and BW25113, they had similar growing pattern as ΔglgP until 20 h. Then, their OD600 values dropped evidently. No significant difference was detected for the two wild-type strains in terms of OD600 values at 24 h (P > 0.05). As for the other three mutants, ΔglgC, ΔglgA, ΔglgB, that were disrupted on glycogen biosynthesis pathway, they showed lowest OD600 values at 24 h. In addition, no significant difference was detected among the three strains. Interestingly, ΔglgC had similar growing pattern as ΔglgP until 14 h, after which its OD600 value decreased sharply to the lowest level at 24 h. Student’s t-test showed that OD600 values of ΔglgC mutant at 24 h was significantly lower than BW25113 (P < 0.05), ΔglgP (P < 0.01), and ΔglgX (P < 0.001). Thus, disruption of genes in glycogen metabolism pathway changed E. coli growth abilities. That is, growth of mutants with impaired glycogen degradation pathway was boosted while growth of mutants with compromised glycogen synthesis pathway was hindered.We then cultured wild-type strains BL21(DE3) and BW25113, together with the other five single-gene mutants (Δh curves . AccordiP < 0.05), except for the comparison between 0.2 and 0.4% glucose (P > 0.05). We then checked glucose consumption rates of different strains in 1 × M9 minimal medium with 0.8% glucose (P > 0.05). However, at 24 h, we noticed that glycogen over-accumulating mutants ΔglgP (0.27%) and ΔglgX (0.27%) had higher level of glucose left in the liquid culture while glucose left in ΔglgA culture (0.24%) is also comparatively high. Thus, disruption of glycogen metabolism pathway has impacts on the rate of glucose consumption in the culture. However, the specific mechanisms for each mutant might be different, which requires further experimental investigations.Glucose consumption rate for BL21(DE3) was monitored in 1 × M9 minimal medium with different concentrations of glucose . Accordi glucose . The res1.6h% glucose, 20  > glycogen0.8h% glucose, 20  > glycogen0.4h% glucose, 10  > glycogen0.2h% glucose, 6  (P < 0.05). In specificity, as for media supplemented with 0.8 and 1.6% glucose, glycogen accumulation starts from the beginning until 20 h, after which glycogen begins to degrade. In contrast, glycogen accumulation pattern is very different in media supplemented with 0.2 and 0.4% glucose, in which the peak glycogen amount arrives at 6 and 10 h, respectively. No glycogen could be detected at 14 and 16 h due to degradation processes.Glycogen is normally accumulated in bacteria under limited nitrogen source and abundant carbon source . It is aE. coli wild-type and single-gene mutants in 1 × M9 minimal medium with 0.8% glucose  disrupted in glycogen synthesis pathway showed non-detectable glycogen accumulation while two mutated strains (ΔglgP and ΔglgX) disrupted in glycogen degradation pathway accumulated abnormally higher amount of glycogen than that in wild-type strains BL21(DE3) and BW25113. The general trend of glycogen accumulation level in ΔglgP and ΔglgX mutants kept increasing during the 24 h culture. There is no statistical significance between the two mutants in terms of overall trend of glycogen accumulation (P > 0.05). As for the glycogen accumulation pattern in the two wild-type strains, both of them showed similar pattern, reaching peak value at 20 h and then starting to decline due to degradation. There is also no significant difference identified (P > 0.05). However, when comparing wild-type strains, BW25113 and BL21(DE3), and the mutated strains, ΔglgP and ΔglgX, pair-wisely, the difference is statistically significant (P < 0.001).We then studied how glycogen accumulation pattern changed in  glucose . AccordiglgBXCAP operon . When growing in LB broth, BL21(DE3) formed significantly less biofilm than BW25113 (P < 0.05). In addition, all mutated strain had significantly higher biofilm formation abilities than that of BW25113 (P < 0.05). In particular, ΔglgB had the highest biofilm formation potential (OD590 = 0.88). When growing in 1 × M9 minimal medium supplemented with 0.8% glucose, biofilm formation abilities of all the E. coli strains were significantly reduced to very low level (P < 0.05). In sum, 1 × M9 minimal medium (0.8% glucose) did not facilitate the formation of biofilms in E. coli while interruption of glycogen metabolism pathway in E. coli leads to increased biofilm formation. However, no specific molecular mechanisms are available to interpret such phenotype changes, which we aim to explore via transcriptomics analysis in follow-up studies.Biofilm formation abilities were compared among the two wild-type strains and five mutants in LB broth medium  and 1 × glgBXCAP operon influence E. coli survival under a variety of environmental stresses. Such studies could improve our understanding of glycogen functions further. Parental strain BW25113 and the five single-gene knock-out mutants were re-suspended for starvation in PBS buffer at room temperature for 12 days, during which viable cells in each strain were numerated every 2 days and the results are shown in glgX survives the best than all other strains under the starvation conditions for 12 days, which was tightly followed by ΔglgP (SΔglgX > SΔglgP). There is no significant difference between ΔglgX and ΔglgP in terms of the overall trend of starvation survival and when compared at each time point. Viability of the wild-type strain BW25113 is generally lower than ΔglgX and ΔglgP during the first 4-day starvation, but higher than the other three mutants with glycogen-deficient phenotype (SglgXΔ > SglgPΔ > SWT > SΔglgCor ΔglgBor ΔglgA). At day 6, viabilities of all the strains clustered together except for ΔglgC that had the lowest viability. In addition, wild-type strain survived better than ΔglgP with no statistical significance at day 6. Since it is already known that ΔglgX and ΔglgP store the highest and the second highest amount of glycogen, it is rational to conclude that glycogen accumulation improves bacterial survival under starvation conditions.Glycogen accumulation has been confirmed to facilitate bacterial environmental survival under abiotic stresses . In thisE. coli strains at low temperature, the wild-type BW25113 showed consistently higher viability than all mutated strains until day 9, after which no bacterial viability could be detected for all the strains . No significant difference was detected in terms of the overall trend of cold viability among E. coli BW25113 and the five mutants. We then tested the dual effects of starvation and low temperature on the survival rate of E. coli. According to the result, wild-type BW25113 had the best viability until day 6  decreased their survival abilities more rapidly when compared with the wild-type strain, among which ΔglgB performed the worst. As for the mutated strains ΔglgP and ΔglgX, they died off very quickly, which dropped from around 109 CFU/ml to 104 CFU/ml within only 3 h, and then to 0 CFU/ml within 6 h. The two mutants ΔglgP and ΔglgX formed an obviously separated cluster from other strains in terms of desiccation viabilities. However, no statistical significance was found among strains in terms of overall survival of desiccation stress.Desiccation is a strong selective stress on bacterial survival ability during their living in the environment. Due to the rapid death of thin 6 h . AccordiglgBXCAP operon could affect the growth of E. coli under the oxidative stress condition, bacteria were cultured in LB agar plates and 1 × M9 minimal medium supplemented with 0.8% glucose agar plate. A 6 mm sterilized paper disc contained 10 μl of 6.6 mol/L H2O2 was then placed in the center of each plate. The diameters of inhibition zones were recorded and compared (2O2 than wild-type BL21(DE3) (diameter = 3.81 cm) when cultured in 1 × M9 minimal medium (0.8% glucose) agar plate. As for the five single-gene mutants, oxidative stress resistance was consistently and significantly increased in ΔglgA, ΔglgB, and ΔglgP strains (P < 0.05) but insignificantly decreased in ΔglgC and ΔglgX strains (P > 0.05) in both LB and 1 × M9 minimal medium agar plates, when compared with wild-type strain BW25113.In order to determine whether the genes in compared . AccordiE. coli  and BW25113, and mutated strains, ΔglgC, ΔglgA, ΔglgB, ΔglgP, and ΔglgX, in LB broth medium and standardized 1 × M9 minimal medium, with a focus on the functions of the five genes in bacterial growth, glycogen accumulation and structure, biofilm formation and environmental survival abilities.Glycogen accumulation has been linked with bacterial growth, biofilm formation, and environmental survival abilities in a variety of species, some of which are important human pathogens . AccordiE. coli , 2010. Apression . Althoug600 value  and ΔglgX (OD600 = 0.76) have higher maximal OD600 values than glycogen-deficient strains ΔglgC (OD600 = 0.69), ΔglgA (OD600 = 0.43), and ΔglgB (OD600 = 0.61)  . In this mutants . It is we strain . The poscumulate . HoweverE. coli BL21(DE3) and BW25113, continuous glycogen accumulation was observed from 4 h until 20 h. Thus, glycogen can be accumulated at exponential phase in E. coli, which might be due to low nitrogen concentration in 1 × M9 minimal medium and the high concentration of glucose (0.8%) in the culture. After 20 h culture when glycogen accumulation reaches the peak, its amount starts to decrease sharply, though there was still sufficient glucose left in the medium  were found in the accumulated glycogen particles. However, it is interesting to notice that a certain portion of long chains (17 DP and beyond) also exists, which raises the questions of why these long chains are not degraded by GP and how these chains are organization spatially. Answering these questions would generate a better understanding of glycogen structure and metabolism in bacteria.Glycogen phosphorylase and GDE are responsible for glycogen degradation: GP works by removing glucose units from the non-reducing ends of outer chains in glycogen until four glucose residues left, which are then truncated enzymatically by GDE at α-1,6-glycosidic linkages . Glycogeendently . Here weSalmonella enteritidis revealed that increased glucose concentration in the media facilitated the biosynthesis of intracellular glycogen and biofilm formation (Enterobacteriaceae, such as E. coli (Biofilm formation is clinically important because around 80% of microbial infections in the body could be attributed to it . There iormation . However E. coli . In this E. coli .E. coli BW25113 and its single-gene mutants under starvation, low temperature, desiccation, and oxidative stress. As an important energy reserve, bacterial glycogen was able to provide both short-term benefits in changing environments (Glycogen accumulation facilitates bacterial environmental survival under a variety of harsh conditions . In addironments  and longronments . Our sturonments .E. coli at low temperatures (treS–pep2–glgE-glgB pathway (Propionibacterium freudenreichii (E. coli wild-type strain BW25113 and five mutated strains for nine consecutive days, the result of which showed that wild-type strain survived the best while other mutants had a similarly compromised survival. When coupling starvation with low temperature, the general trend was similar as the stress of low temperature alone except that all E. coli strains died off much faster. Thus, disruption of glycogen metabolism pathway compromised E. coli survival in cold environment, no matter whether glycogen was over- or less accumulated, which suggested that a complete glgBXCAP operon was necessary for E. coli to combat cold stress.Survival of temperature fluctuation is important for pathogens during prolonged transfer between hosts . Trehaloeratures . Meanwhi pathway . Previounreichii . In thisglgP deletion in A. brasilense showed significantly higher survival rate than wild-type in 1-h desiccation (glgB deletion in E. coli DH5α greatly reduced its survival rate when desiccated for 6 h (E. coli BW25113 and its five single-gene mutants in glgBXCAP operon, we found out that wild-type survives the best under the stress while glycogen over-accumulating strains ΔglgP and ΔglgX were more sensitive to desiccation than the other three mutants. However, there is currently no clear molecular mechanism to explain this difference. As for the oxidative stress, recent studies showed that glucose released from glycogen could be used for NADPH/glutathione reduction, which protects nematodes and human hepatocytes from oxidative stress; however, the accumulation of glycogen itself paradoxically shortens the lifespan of Caenorhabditis elegans (glgA unexpectedly showed the highest resistance toward oxidative stress, which requires further experimental investigation. For a complete summary of all these phenotypic changes influenced by the disruption of glycogen metabolism pathway, please refer to Desiccation is an environmental and food industry stress that bacteria commonly face, which significantly affects bacterial viability more than other stresses . glgP deiccation . However for 6 h . Through elegans . In bact elegans . In thisglgBXCAP operon influences the physiological activities of E. coli. Differences have been identified in terms of growth rate, glycogen accumulation and structure, biofilm formation, and environmental stress endurance, which confirms the importance of glycogen metabolism in bacteria. However, molecular basis for some of these phenotypic differences are still missing. Further investigation into the molecular mechanisms are needed, which could provide a better understanding of glycogen functions in bacteria.Taken together, this study systematically and comparatively investigates how single gene deletion in the important and classical All datasets presented in this study are included in the article/LW conceived and designed the experiments. LW, DT, and XZ contributed to project administration. LW, MW, QL, and XK carried out the experimental work. LW, DT, MW, QL, MG, YD, XZ, and XX wrote and revised the manuscript. LW, DT, XZ, ZZ, and HY provided platform, resources, and student supervision. All authors read and approved the final manuscript.The 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.


HSC activation is a key step in liver fibrogenesis and has a crucial role in collagen deposition, as it is primarily responsible for excessive ECM production. The effect of neratinib on HSC was evaluated in transforming growth factor (TGF-β)-incubated LX-2 cells and culture-activated primary human HSCs. In vivo study results indicated that neratinib inhibited the inflammatory response, HSC differentiation, and collagen accumulation induced by CCl4. Moreover, the anti-fibrotic effects of neratinib were not associated with the HER2 signaling pathways. Neratinib inhibited FGF2 expression in activated HSCs and serum FGF2 level in the model, suggesting that neratinib possessed therapeutic potency against liver fibrosis and the potential for application against other fibrotic diseases.Liver fibrosis, a common outcome of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), is a leading cause of mortality worldwide. The tyrosine kinase inhibitor neratinib is a human epidermal growth factor receptor 2 (HER2) inhibitor approved by the FDA for HER2-positive breast cancer treatment; however, it has not yet been evaluated for liver fibrosis treatment. We elucidated the anti-fibrotic effects of neratinib in hepatic stellate cells (HSCs) and in vivo models of CCl During the early stage of liver disease, the inflammatory response and hepatic injury are initiated, and persistent inflammation in the liver leads to pathological accumulation of ECM proteins, such as collagens, laminin, and fibronectin2, which are responsible for cell migration and proliferation. Accumulation of excessive ECM proteins results from the abnormal regulation of ECM-degrading matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs)3.The pathogenesis of liver fibrosis has been extensively studied for decades to identify effective therapies for this disease. The characteristic of liver fibrosis is the net accumulation of extracellular matrix (ECM) proteins caused by hepatic injuries, such as viral infection, alcohol, Nonalcoholic Steatohepatitis (NASH), autoimmune disorders, cholestatic disorders, and metabolic diseases5. Activated HSC (aHSC) consists of 90% ECM-producing MFBs resulting in the synthesis and secretion of matrix proteins that cause parenchymal and cholestatic liver damage9. In the healthy human liver, HSC represents 5–8% of total liver cells and has the function of vitamin A storage within cytoplasmic lipid droplets10. HSC is non-proliferative under normal conditions, but after chronic liver injury, it is transdifferentiated into the MFB phenotype to activate the immune response and angiogenesis11. aHSCs are characterized by cell proliferation, fibrogenesis, matrix degradation, contractility, and pro-inflammatory activity. Upon liver damage, aHSCs secrete platelet-derived growth factor (PDGF) and transforming growth factor (TGF-β), resulting in the activation of other hepatic cells, such as sinusoidal endothelial cells, Kupffer cells, and hepatocytes2. Type I, III collagen, α-smooth muscle actin (α-SMA), TGF-β, and tissue inhibitors of metalloproteinase (TIMP)-1 are considered well-characterized markers of HSC activation13.Hepatic stellate cells (HSCs) are the primary effector cells of liver fibrosis with an ability to be transdifferentiated into myofibroblasts-like (MFB) cell types by hepatic injury14. We determined whether neratinib mitigates liver fibrosis in a mouse model of carbon tetrachloride (CCl4)-induced fibrosis. To verify this hypothesis, we examined whether neratinib inhibits the profibrogenic activity of LX-2 cells and the activation of primary human stellate cells (hpHSCs). We also discussed the growth factor signaling to elucidate the underlying molecular mechanisms involved in HSC activation.This study aims to find druggable compounds targeting aHSCs to attenuate liver fibrosis without causing normal cell toxicity. Drug repurposing provides evidence for FDA-approved drugs that may also be effective in treating other diseases. To this end, neratinib was identified as a potential active candidate by cell-based screening using a kinase inhibitor library in HSCs. Neratinib  is an irreversible tyrosine kinase inhibitor that targets the human epidermal growth factor receptor (EGFR), HER2, and HER4. Currently, it has been approved by the FDA for early HER2-positive breast cancer in extended adjuvant treatment, and it is in ongoing clinical trials for lung, colorectal, and bladder cancers. Although diarrhea is considered an adverse effect, it is predictable and manageable with antidiarrheal medicine15. After incubation with TGF-β (5 ng/mL) for 48 h, the cells were treated with 100 nM neratinib (a non-cytotoxic concentration) at various times up to 48 h , Col1a2 (collagen I), TIMP-1 , TNF-a (tumor necrosis factor α), Il-6 (interleukin 6), and Mcp-1 (monocyte chemoattractant protein 1)  was administered orally (P.O.) for 2 weeks daily after induction of fibrosis with CCl4, as shown in the treatment schedule in Fig. 4 promotes exacerbation of the damaged area with signs of hepatic fibrosis. Next, immunohistochemistry (IHC) analysis was performed to investigate the pathophysiological efficacy of neratinib in the liver tissues. IHC and quantified digital analyses showed that the positive area of α-SMA staining and collagen deposition (Sirius Red) in the liver of neratinib-treated mice were significantly reduced compared to that of the vehicle-treated group , TNF-a, IL-6, MCP-1, and CCR-2 (C–C chemokine receptor 2) mRNA. However, neratinib treatment strongly suppressed the levels of mRNA, except for TNF-a and CCR-2 , TIMP-1, TGF-b, and PDGFR-b (platelet-derived growth factor β)  pathways via the ERK/CREB pathway. As expected, Smad2 and Smad3 were highly phosphorylated in LX-2 cells exposed to TGF-β. However, pre-treatment with neratinib did not affect this phenomenon , hepatitis C virus (HCV), and metabolic disorders, identified as significant causes of liver fibrosis3. Prolonged liver fibrosis leads to liver cirrhosis, liver failure, hepatocellular carcinoma, and death20. Recently, several drugs, including antiviral drugs, have been approved by the FDA for liver fibrosis. However, these drugs are only useful in some circumstances, and there is currently no successful liver-specific fibrosis therapies21. Also, there are no FDA-approved small molecules targeted anti-fibrotic therapy except Mulpleta (lusutrombopag), which stimulates the production of platelets. Therefore, the development of an anti-fibrotic medication is indispensable for the regression of advanced fibrosis through inhibition of the source of liver fibrosis.The accumulation of ECM proteins is a result of the wound healing process that occurs after chronic inflammation induced by liver injury4-induced liver fibrosis in the early and advanced stages by inhibiting HSC activation. aHSCs are the primary effector cells in liver fibrogenesis, contributing approximately 90% of ECM-producing myofibroblasts22. Inhibition of HSC activation and removal of aHSCs have been studied as primary approaches for liver fibrosis25. It would be a logical strategy to develop the small molecule targeting aHSC in chronic liver diseases to improve the treatment efficacy. Neratinib was selected as a drug with potent inhibitory effects against fibrogenesis in hpHSCs and LX-2 cells after kinase inhibitor library screening.Our results show that neratinib ameliorates CCl+ early-stage breast cancer that does not cause severe adverse effects, except diarrhea26. In addition, neratinib shows anticancer effects in non-small cell lung cancer, colorectal cancer, and glioblastoma through the EGFR/HER-2 pathway28. Several studies reported that EGFR/HER-2 inhibitors attenuated liver fibrosis through inhibition of HSCs30. However, we found that activated hpHSCs do not express HER-2 protein  significantly increased at the early stage of liver fibrosis, and the expression of these markers was higher at the advanced stage. In contrast, the expression of inflammatory cytokines was much lower at the advanced stage than at the early stage. Our results show that neratinib treatment significantly ameliorates both stages of liver fibrosis mice, as confirmed by the reductions in α-SMA expression, collagen deposition, and other fibrogenic markers  in liver tissues. The levels of mouse IL-6 partially blocked when we conducted an additional study using isolated primary mouse Kupffer cells after co-treatment with Neratinib/LPS for 4 h, indicating that neratinib may also directly target immune cells  were purchased from Charles River . The mice were randomly divided into four groups: (1) vehicle-treated healthy group, in which mice received olive oil and vehicle treatment (0.5% methylcellulose-0.2% Tween 80), (2) neratinib-treated healthy group, (3) vehicle-treated liver fibrosis group, and (4) neratinib-treated liver fibrosis group.4  three times per week i.p. or with olive oil (as a control) for a total of 6 weeks. On day 15, mice were treated with 10 mg/kg neratinib or vehicle p.o. daily for 14 days. Mice were anesthetized on day 28, and blood and liver tissue samples were collected for analysis. In the long-term treatment of liver fibrosis, the mice were treated with 2 mL/kg CCl4 three times per week for 4 weeks before neratinib treatment. On day 29, the mice were treated with 10 mg/kg of neratinib or vehicle p.o. daily for 42 days. Mice were anesthetized on day 70, and blood and liver tissue samples were collected for analysis.To evaluate short-term treatment of liver fibrosis, the mice were treated with 2 mL/kg CClBlood samples were collected by cardiac puncture, stored at 20–25 °C for 2 h, and then centrifuged at 10,000 rpm for 10 min. The levels of the liver enzymes AST and ALT in serum were analyzed as a measure of liver function. Serum FGF level was analyzed by using an FGF2 Quantikine ELISA kit  following the manufacturer's protocol.Liver tissues were fixed in 10% buffered formalin, embedded in paraffin, and cut into 4 µm-thick sections. The sections were then stained with H&E and Sirius Red by standard protocols. The amount of collagen was quantified from Sirius Red staining by using an image processing software . For IHC staining, anti-α-SMA  antibodies were used to detect activated HSCs. IHC was performed following a standard protocol, and the stained tissues were quantified from 20 randomly selected images from each section.Collagen level in the liver was measured by using a hydroxyproline assay kit  following the manufacturer's protocol. Briefly, approximately 10 mg of liver tissues were homogenized, hydrolyzed at 120 °C for 3 h in 12 N HCl, incubated with 100 μL Chloramine T/Oxidation buffer mixture for 5 min, and then incubated with DMAB reagent for 90 min at 60 °C. The absorbance at 560 nm was measured by using a microplate reader .41, LX-2 cells were maintained in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. hpHSC was purchased from ScienCell Research Laboratories , and cultured in stellate cell medium supplemented with 2% FBS, 1% stellate cell growth supplement, and 1% penicillin/streptomycin solution in poly-l-Lysine-coated plates. The hpHSCs were seeded in a six-well culture plate for 1, 3, 5, and 7 days and harvested for analysis. The expression of the activation marker α-SMA in cultured stellate cells was determined by western blotting and qPCR analyses. Cells were cultured at 37 °C in an atmosphere of 5% CO2/95% air under saturating humidity.As described our previous report42. Briefly, mouse liver was perfused via the portal vein with a 1X EGTA and then HBSS (Ca2+/Mg2+-free HBSS) containing 0.75 mg/mL of type I collagenase . The liver was treated with a digestion buffer (0.08 mg/mL type I collagenase) and incubated for 15 min at 37 °C. The tissue went through 100 μm nylon cell strainer  followed by three times of washing. 5 × 104 cell/well of resuspended cells were plated at collagen-coated 96 well plates (Corning) in DMEM  with 10% of FBS and 1% antibiotics.Seven weeks of age mice were used for primary hepatocytes isolation as previously describedCytotoxicity assay was performed using a CellTiter-Glo Luminescent Cell Viability Assay kit. After incubation with chemicals, cells were lyzed with lysis reagent, and an equal volume of CellTiter-Glo reagent was added to the cells. After incubation for 10 min at room temperature, luminescence was measured by using a luminometer .41, total RNA from cells and liver tissues was extracted by TRIzol reagent  following the manufacturer's protocol. RNA concentration was measured by using a NanoDrop 2000 . cDNA was synthesized from 1–2 µg of total RNA by using a High-Capacity cDNA Reverse Transcription System (Life Technologies). Comparative qPCR was performed in duplicate for each sample by using SYBR Green Master Mix (Life Technologies) and StepOnePlus Real-Time PCR System (Life Technologies). mRNA expression was quantified using the threshold cycle (Ct) method. Ct values for each gene of interest were normalized to that of GAPDH. The name and sequences of primers and primers for humans and mice are summarized in Supplementary Tables As described our previous report41. The protein concentration of the lysates was quantified by using a BCA Protein Assay Kit  Denatured proteins were loaded into SDS–polyacrylamide gel, resolved by electrophoresis, and transferred to a nitrocellulose membrane, blocked by incubation with 3% bovine serum albumin (Sigma) for 1 h at room temperature, and the membrane was then incubated with each primary antibody overnight. Incubation with the appropriate secondary antibodies conjugated to HRP was performed for 1 h. Protein bands were developed onto an X-ray film (Fujifilm) after incubation with enhanced chemiluminescence.Anti-alpha SMA. , anti-collagen , anti-TGF-β , anti-PDGFR-β , anti-GAPDH , anti-p-MEK1/2 , anti-MEK1/2 , anti-p-ERK1/2(p44/42) , anti-ERK , and p-CREB  were used for western blotting. Cell or tissues were lysed with RIPA buffer  with a protease inhibitor cocktail followed by western blotting as previously describedTNF-α (SMTA00B) and IL-6 (SM6000B) were determined using Quantikine ELISA (R&D Systems). Neratinib was pretreated for 30 min followed by LPS  for 4 h in mouse Kupffer. The collected supernatant was processed according to the manufacturer’s protocol.41. Differences between groups were assessed by one-way analysis of variance followed by Tukey’s post-hoc test or Student’s t-test, as appropriate. Error bars represent the standard deviation (SD) or standard error of the mean (SEM), as indicated. Statistical significance was accepted at P < 0.05.The GraphPad Prism 7 software  and Excel 2016  were used to analyze the data as previously describedSupplementary information


The NSs were systematically characterized by using XPS, EDS, TEM, FTIR, UV/vis, and XRD. The fabricated NSs onto the glassy carbon electrode (GCE) was successfully applied for the selective and sensitive detection of 3-MPHyd in the phosphate buffer system (PBS), which displayed the highest sensitivity, good selectivity with ultra-trace detection limit, high stability, good reproducibility, and quick response time. The real environmental samples were tested for validation from stand point of the ternary doped nanomaterials for sensing in the practical applications using by electrochemical method.In this approach, thin spikes (NSs) of ternary nano-formulated mixed CuO/MnO Due to exposure of these chemicals by human even at trace amount, may cause the hostile effect to human such as liver and kidney injury, haemolytic anaemia, skin irritation and dermatitis, etc. and the phenyl hydrazine and their derivatives are considered as a carcinogenic4. Then it is necessary to capture of 3-MPHyd at ultra-trace level to safe the human, environment, and the eco-system. The literatures have been described the detection methods of phenyl hydrazine and their derivatives such as fluorimetry, capillary electrophoresis, chromatography, spectrophotometry, photometry and photometric titrations, electrochemical technique7. But most of them, the electrochemical methods are widely implemented technique due to its high sensitivity, lower detection limit, greater simplicity, lower cost and long-term stability in chemical environment. Except few articles, the most of reported studies have been suffering in sensitivity, reproducibility and stability. To the phenyl hydrazine and their derivatives, the higher potential is required for GCE. Therefore, the modified GCE needs easy to fabricate for increasing the electrons transferring-rate as well as improving the sensitivity with lower detection limit8.As strong reducing agents, generally the phenyl hydrazine and their derivatives are well-known, and they have various industrial applications such as blooming agent for plastics, corrosion inhibitor insecticides, and oxygen scavenger in the boiler as rocket fuel, photographic chemicals, aerospace fuel and explosives9. Recently, the un-doped metal oxides  such as iron oxide, tungsten oxide, manganese oxide, and copper oxide are also studied as a detecting material of hydrazine and the derivatives of hydrazine10. Among them, MnO2 is the potential electron arbitrator as exhibited the high catalytic activity, precise surface area, lower resistances and pleasant electrochemical properties11. The CuO is a good electro mediator with better electro-chemical property with large surface area, which can enhance the electron transferring12. Generally, nanostructure materials have given significant attention for various organic as well as inorganic components, which is attracted owing to the binding affinity at optimum working conditions16.To reduce the over potential and increase the electron transfer rate, the various transition material such as copper oxides are used as detecting or capturing materials to the phenyl hydrazine and their derivatives17. Another hydrazine derivate material based on silver-doped zinc oxide was reported, which was successful with high sensitivity and detection limit18. It is already established that CuO and MnO2 are efficient sensing elements to monitor the toxins for the safety of environment. Therefore, the ternary combination of CuO/MnO2/Gd2O3 NSs may be an effective detection material to detect environmental toxins due to the large active surface area in their interior or exterior surfaces. Various toxic analytes such as phenols and their derivatives, alcohol, acetone, ammonium hydroxide, dichloromethane, heavy-metal ions, hydrazine, aliphatic and aromatic compounds have been detected by various compositions of metal oxide/sulphides with the doped or un-doped, mixed/dual combinations19. The nanostructure materials prepared by sol–gel method have been shown elsewhere with the higher surface area for good adsorption of chemicals and ions23. From the specific advantage of the active surface area with ternary metal oxides compared to single or dual/doped combination, it is highly demanded to introduce new doped materials with metal oxides.A successful hydrazine derivative material with zinc oxide nano-urchins is reported and exhibited the good sensitivity as well as lower detection limit2/Gd2O3 NS nanostructure material by converting it to a doped mixed oxides with other materials is also cost-effective. Here, CuO/MnO2/Gd2O3 NS nanostructured materials have employed a great deal of consideration due to their chemical, structural, physical, and optical properties in terms of large-active surface area, high-stability, and high porosity. These properties are directly depend on the structural morphology prepared by reactant precursors for making the CuO/MnO2/Gd2O3 porous materials in the basic medium at low-temperature. This CuO/MnO2/Gd2O3 NSs material was synthesized by a facile solution method using NaOH solution. This technique has several advantages including facile preparation, accurate control of reactant temperature, easy to handle, one-step reaction. Optical, morphological, electrical, and chemical properties of the CuO/MnO2/Gd2O3 NS nanomaterials are of huge significance from the scientific aspect, compared to other undoped materials. The non-stoichiometry, mostly oxygen vacancies, Gd2O3 makes it conducting nature in the doped nanostructured materials29. The formation energy of oxygen vacancies and metal interstitials in the semiconductor is very low and thus these defects form eagerly, resulting the increased conductivity of CuO/MnO2/Gd2O3 NS materials compared to other undoped materials. CuO/MnO2/Gd2O3 NS materials have also attracted considerable interest due to their potential applications in fabricating optoelectronic, electro-analytical, selective detection of assays, chemical sensor devices, hybrid-composites, electron-field emission sources for emission exhibits, biochemical detections, and surface-enhanced Raman properties, etc. CuO/MnO2/Gd2O3 NS material offers improved performance due to the large-active surface area, which increased conductivity and current responses of the CuO/MnO2/Gd2O3 NSs/Nafion/GCE assembly during the electrochemical investigation.Generally, mixed doped metal oxide exhibits promising results for the detection and quantification of hazardous chemicals by various detection approaches. Besides this, as enhancing of electrochemical properties of CuO/MnO2/Gd2O3 NSs. The fabricated NSs were practice for the coating onto a GCE, and the binding properties between these were enhanced by the addition of nafion solution. The resulted GCE was investigated for efficient capturing of 3-MPHyd in the aqueous media. The analytical capturing performances were studies very carefully, and the outcome of the 3-MPHyd chemical material was high sensitivity with the low detection limits. Here, it is introduced a significant material in this approach for selective and sensitive monitoring and capturing of selective 3-MPHyd with CuO/MnO2/Gd2O3 NSs sensor probe.The most reliable hydrothermal process was applied to synthesize CuO/MnO2), manganese(II) chloride (MnCl2), gadolinium(III) chloride, and ammonium hydroxide (NH4OH) were purchased from well-known Sigma-Aldrich, which was deployed to prepare ternary CuO/MnO2/Gd2O3 NSs. As a part of these studies, the analytical grade chemicals 2,4-DNP , 3,4-DAT , pyridine, BH , 3-chlorophenol, THF (tetrahydrofuran), methanol, 3-MPHyd (3-methoxyphenylhydrazine), AH (ammonium-hydroxide), coating agent nafion (in 5% ethanol), NaH2PO4, and Na2HPO4 were also bought from the Sigma-Aldrich and deployed as-received. For the details characterization of synthesized CuO/MnO2/Gd2O3 NSs, conventional XRD, XPS, FTIR, UV/Vis and FESEM were implemented to evaluate the structural, crystalline, functional, optical, morphological and elemental analyses. The reliable current versus potential  was used to determine 3-MPHyd with active CuO/MnO2/Gd2O3 NSs material by using Keithley electrometer  at room conditions.In this research work, the required chemicals were used as received without further purification. The inorganic salts of transition metals such as copper(II) chloride (CuCl2), manganese chloride (MnCl2), gadolinium chloride (GdCl3) and alkali ammonium hydroxide (NH4OH) were used to prepare CuO/MnO2/Gd2O3 NSs by solvo/hydrothermal method at low temperature. The solvothermal process was widely used efficient method to fabricate nanomaterials of metal oxides, and the resultant guest or doped metal oxides are smaller in size as well as phase formation. Following this method, 100.0 mL of 0.10 M CuCl2, 100.0 mL of 0.10 M MnCl2, 100.0 mL of 0.10 M GdCl3 and 100.0 mL of 0.10 M NH4OH were prepared in a different four 200.0 mL beaker with de-ionized water and resultant solutions were kept with continuous magnetic stirring. Another 250.0 mL of conical flask was taken and 50.0 mL of each prepared metallic salt solution was added. Then the mixture was shacked with continuous magnetic stirring onto the hot plate. To obtain the co-precipitation of metal hydroxides, the prepared 0.10 M NH4OH was added slowly and at the pH value 10.5, all metal hydroxides were precipitated out in conical flask. Then the total solution was kept at 80 °C on the hot plate with continuous magnetic stirring around 6 h. As-prepared participate of metal hydroxides were washed thoroughly by de-ionized water and kept it to dry at room condition for overnight. Consequently, the powdered sample was heated for calcination at 510 °C for 6 h. Under higher temperature, the metal oxides is transform to crystalline metal oxide i.e., CuO/MnO2/Gd2O3 nanostructure shapes, which contains the higher metallic-ions. The prepared material was properly grained into fine powder of nano-sized materials for details characterization. The following reactions may happen:The inorganic salts copper chloride (CuClIn the aqueous medium:In furnace:Ks was low (Ks = 2.2 × 10–20 in Cu(OH)2, 2.0 × 10–13 for Mn(OH)2 and 1.88 × 10–23 for Gd(OH)330. Metal ions were precipitated out quantitatively as various oxides. The crystal formation was happened initially, where an aggregation to the Gd(OH)3 was started. In the reaction system, pH was continued to enhance, then the Cu(OH)2 was started to precipitate, which was re-aggregated onto the Gd(OH)3 crystallites. Further increasing of pH, Mn(OH)2 is also participated out to form aggregation with other two metal hydroxides. The formation of NSs is similar with the previously reported article31. The synthesized NSs were characterized in terms of elemental composition, crystallinity, optical property, morphology, structure, and functional properties. Later, CuO/MnO2/Gd2O3 NSs were applied to detect 3-MPHyd by reliable electrochemical method at room conditions. This is the first time, the produced CuO/MnO2/Gd2O3 NSs were implemented for the selective determination of 3-MPHyd for environmental safety by electrochemical method.The 2/Gd2O3 was successively implemented to determine the target environmentally unsafe 3-MPHyd in reaction medium. To prepare the working electrode for 3-MPHyd detection, the ethanolic slurry of CuO/MnO2/Gd2O3 NSs was put onto the GCE. Platinum wire (Pt-wire) was used as a counter electrode. The dispersed materials was attached between NSs of ternary metal oxides and GCE by air dry initially. It was fabricated on the flat GCE and dried in air for complete thin-film formation. Later, after drying completely, 1.0 μL of 5.0% Nafion (ethanolic) was dropped onto the fabricated electrode surface and waited until dry it completely. Here, nafion is used as a chemical glue for the stable attachment of ternary materials onto the surface of flat-GCE. Then the dried fabricated electrode was used as working electrode in this investigation. The electrochemical cell was composed by CuO/MnO2/Gd2O3/binders/GCE sensor probe as working electrode. The fabrication scheme is presented in the Fig. 2). The other analytical properties of 3-MPHyd chemical sensor such as LDR and LOD were estimated according to ratio of 3 N/S.The ternary doped materials based on the NSs of CuO/MnO2/Gd2O3 NSs were carried out by the implementation of XRD. As shown in Fig. 2 and Gd2O3. The resultant XRD spectra is represented the Bragg planes of CuO indices as θ such as (110), (111), (200), (202), (113), and (022), which has the similarities with JCPDS No. 72-0629 and earlier published articles33. The other diffracted peaks created from MnO2 indices as ß are (101), (110), (111), (200), (210), (211), (002) and (310). This XRD pattern of MnO2 is agreed with the JCPDS Card, No. 24-0735 and literatures35. The additional sharpest peaks of Gd2O3 indices as λ are (211), (222), (411), (442), (611) and (622), which are agreed with those reported for gadolinium oxide nanoparticles36 and JCPDS No. 43-1015. There is a provision for measuring the crystallinity by the XRD pattern through Scherer’s expectation (vii).37. Here, the determined crystallinity was 43.31 nm. Additionally, it is also compared the XRD spectrum of individual CuO, MnO2, and CuO/MnO2/Gd2O3 NSs and presented in the Fig. 2/Gd2O3 NSs is depicted in Fig. −1. The main characteristic absorption peak is at 539 cm−1 which is corresponding to Mn–O or Cu–O stretching modes38 and the identical peak at 1402 cm−1 is responsible for C−O stretching39. The other two peaks at 3370 and 3622 are corresponding to the OH group40. The visual spectra of CuO/MnO2/Gd2O3 NSs were completed at the range between 200.0 and 800.0 nm wavelengths. As judged from Fig. 2/Gd2O3 NSs41. According to the Eq. (bg) was 4.05 eV of the CuO/MnO2/Gd2O3 NSs.bg = energy band-gap and λ = maxima absorbed area.The crystallographic studies of prepared CuO/MnOgth 1.541 Å and β 2/Gd2O3 NSs from higher to lower magnifying images are depicted in Fig. 2/Gd2O3 nanospikes. From Fig. 2/Gd2O3 demonstrated the existence of Cu, Mn, Gd and O and the elemental arrangements of calcined co-doped metal oxides are nanospikes in shape. The atomic compositions (wt%) of CuO/MnO2/Gd2O3 NSs are as O 3.58%, Cu 2.17%, Gd 84.1% and Mn 10.15%. Any additional peaks are not detected, which is associated with impurities. Therefore, the synthesized NSs are consisted only Cu, Mn, Gd and O.The structural analyses of the fabricated NSs were evaluated by FESEM images. The resultant FESEM of CuO/MnO2/Gd2O3 NSs. As it is shown the full spectrum . The distance between these Cu 2p main peaks positions is 21.6 eV, which agrees well with previous reports about CuO spectrum. It is also denoted to the existence of Cu2+ chemical state as an indication of the formation of CuO, which is matched to the reported literatures52. Moreover, additional confirmation of CuO state was seen with the broad satellite peaks at a higher binding energy than the main peaks. The main peak of Cu 2p3/2 at 930.5 eV was accompanied by satellite peaks on the higher binding energy side at 939.2 eV, 941.5 eV and 943.1 eV, which suggests the existence of CuO. From the full spectrum, we can clearly see that the main peak of Cu 2p1/2 at 952.1 eV, which also confirms the presence of CuO. The XPS spectrum is also exhibited the two major peaks of Mn2p orbital and the resultant spectrum of Mn2p is represented in Fig. p3/2 (641.2 eV) and Mn2p1/2 (653.1 eV) are also the adjacent position with the reported data for MnO246, which is presented separately in Fig. d spectra , 3,4-DAT, pyridine, BH, 3-CP, THF, methanol, 3-MPHyd, and AH. As it is depicted in Fig. The synthesized CuO/MnO2/Gd2O3/binder/GCE is 9.25 s. It was evaluated under the certain amount of 0.1 nM of 3-MPHyd solution, which is shown in Fig. 2/Gd2O3/binder/GCE sensor probe by electrochemical method. After the 9.25 s, signal become sensor response become stable and flat, due to the saturation of contact surface with target analyte. The fabricated materials CuO/MnO2/Gd2O3/binder/GCE was studied and compared in presence of various hydrazine derivatives  is 1.10%, which is measured at + 1.0 V. The response time with the fabricated working electrode based on CuO/MnOves Fig. a. It wasves Fig. a compare2/Gd2O3/binder/GCE sensor probe intra-day reproducibility  and electrochemical measurement was carried out in the range from 0.0 to + 1.5 V. The linearity (r2: 0.9919) of the calibration plot , LOD (0.4 ± 0.02 pM at SNR of 3), LDR (1.0 pM to 0.1 mM), and Response time (9.25 s). Sensitivity was calculated from the slope of the calibration plot. It was calculated from the slope of the calibration plot by considering the active surface area of fabricated electrode (0.0316 cm2). During the sensing performances of 3-MPHyd chemical, the electrochemical current responses with CuO/MnO2/Gd2O3 NSs were increased with increasing of the target 3-MPHyd concentration. In presence of higher concentration of target analyte, the resultant current is gradually increased due to the oxidation of 3-MPHyd during electrochemical process with CuO/MnO2/Gd2O3 NSs. During the addition of analyte into the electrochemical solution, the oxidation current is increased gradually until 0.1 nM. After that the current response is found stable until 1.0 pM. No significant increase of current is occurred. A comparison between the electrodes fabricated on binary MnO/CuO, and ternary combinations of CuO/MnO2/Gd2O3 NSs were studied, and it was found that CuO/MnO2/Gd2O3 NSs/binder/GCE electrode was exhibited the highest electrochemical responses  and extracted samples. The collected industrial effluent was initially filtered to remove the floating large particles and then filtered sample was directly used for analysis. The extracted sample from plastic baby bottle, plastic water bottle, and PVC food packaging bags were also filtered and analysed with CuO/MnO2/Gd2O3 NSs/Nafion/GCE sensor probe by electrochemical method. The analysis report is presented in the Table The ternary CuO/MnO2/Gd2O3 spike was fabricated and reported in details. The NSs of transition metal oxides were prepared by the hydrothermal approach in alkaline phase at low-temperature. The fabricated nanospikes were totally characterized by using FTIR, UV–Vis, XRD, XPS and FESEM. The slurry of ternary CuO/MnO2/Gd2O3 NSs was coated onto GCE as a layer of thin-film with conducting binder of nafion solution for selective and sensitive electrochemical detection of 3-MPHyd toxic compounds. The important experimental parameters such as sensitivity, low limit of detection, quantification, reaction time, sensitivity and reusability were performed systematically. The obtain results were good and satisfactorily enough to determine the target 3-MPHyd in short response time by electro-chemical approach. Here, CuO/MnO2/Gd2O3 NSs materials are an effective and potential for the selective detection of 3-MPHyd. Thus the materials can be used in broad scales for the efficient detection of selective 3-MPHyd by electrochemical method at room conditions for environmental remediation.In this study, selective 3-MPHyd chemical material based on nano-formulated CuO/MnO


Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease. Blood plasma is an easily accessible biological fluid that has contact with all tissues in the body. Plasma has been used for diagnostic ,2 and thMajor components of plasma include water, gases, ions, proteins, lipids, carbohydrates, lipoproteins, and extracellular vesicles (EVs)  12,13,1,113,14. Glycobiology is an expanding field that has promising implications for improving the understanding of health and disease . PreviouDifferences in glycan expression in healthy donor-matched whole plasma and plasma-derived EVs were assessed by glycan node analysis (GNA), a medium-throughput method for analyzing carbohydrates. Introduced and analytically validated in detail in 2013 , GNA is To date, the GNA of whole blood plasma or serum from over 1000 of patients has proven effective at detecting and predicting progression, reoccurrence, and/or survival in lung cancer ,32 and bN-morpholinopropanesulfonic acid (MOPS) buffer, NuPAGE 12% bis-tris protein gels, Teflon-lined piercable caps, and cluster of differentiation 9 (CD9) antibody (Cat. No. 10626D) from Thermo Fisher Scientific ; dimethylsulfoxide (DMSO), iodomethane , chloroform, trifluoroacetic acid (TFA), ammonium hydroxide, sodium borohydride, acetic anhydride, sodium acetate and sodium hydroxide beads  from Sigma-Aldrich ; GC consumables from Agilent ; MS consumables from Waters ; clinical grade sucrose buffer  from Lonza ; ultrapure sterile water from Rocky Mountain Biologicals ; calnexin antibody (Cat. No. 2433S) and rabbit secondary antibody (Cat. No. 7074S) from Cell Signaling Technology ; nitrocellulose transfer membrane from Abcam ; HyClone phosphate buffered saline (PBS) from GE Healthcare  Quantikine enzyme-linked immunosorbent assay (ELISA) for apolipoprotein B (apoB) (Cat. No. DAPD00) from R&D Systems .Materials were acquired from the following sources: acetone from Avantor Performance Materials ; methanol from Honeywell Burdick & Jackson ; acetonitrile, methylene chloride, pierce spin columns , GC-MS autosampler vials, mouse secondary antibody (Cat. No. 31450), high performance liquid chromatography (HPLC)-grade water, SuperSignal West-Femto maximum sensitivity substrate , Pierce bicinchoninic acid assay (BCA) Protein Assay Kit, Tween 80, 3-The Mayo Clinic Biospecimens Review Group (ID: 17-010290) provided approval for the pre-clinical research use of human de-identified residual plasma. Plasma was collected in blood bags containing citrate phosphate dextrose-adenine-1 (CPCD-1), an anticoagulant, from healthy donors, for routine clinical use. Approximately 200 mL of plasma was frozen in transfusion bags at −20 °C, for up to one year before being considered expired. Plasma was thawed at 37 °C, stored at 4 °C, and if not used within five days of thawing, it was considered expired and repurposed for research use. Donor 1 and donor 3 were O+ blood type, while donor 2 was O-; all other donor demographics were unknown. Plasma samples were processed through SEC within one month of storage at −80 °C.g for 10 min, to remove any remaining large particles after storage, followed by a final centrifugation step at 10,000× g for 10 min. These columns have a target particle size range of 70–1000 nm. Elution fractions were counted in 0.5 mL intervals, beginning after the void volume of 3 mL had passed through the column. Elution fractions seven through nine were pooled for analysis as ‘fraction 1′  and elution fractions ten and eleven were pooled for analysis as ‘fraction 2′ . These two size exclusion fractions were isolated from each of three different blood plasma donors. For GNA, this process was repeated for each donor plasma sample in quadruplicate . GNA was performed on samples isolated in water (without cryoprotectant), as preservation of EV structure upon storage (−80 °C) prior to analysis is not required. For non-GNA based characterization, this process was repeated once for each donor in a previously published sucrose-based cryoprotectant  [Izon qEV original 70 nm SeriesTM SEC columns were used to collect EV fractions from 500 µL of plasma according to the manufacturer’s instructions, including centrifugation at 1500× mM MgCl) , to presA NanoSight LM10  was used to assess EV size distribution profiles and concentration. Fractions 1 and 2 were diluted 1:50 in HPLC grade water and three measurements were taken for a duration of one minute. Each replicate was measured under a continuous syringe pump flow rate of 40 µL/min. After adjusting the camera level to 12 and detection threshold to four for the first sample, only the focus was changed for the remaining samples, not the analytical software parameters.AFM imaging was performed in tapping mode, using the dimension V Bioscope , equipped with PPP-FMR-SPL silicon tips . Briefly, EVs in sucrose buffer (50 µL) were diluted 1:20 in cell and molecular ultrapure sterile water. A fixed amount of properly diluted EV samples (50 μL) was then spin cast onto freshly cleaved mica sheets , using a model WS-650SZ-6NPP/LITE spin coater , at an acceleration of 600 rpm/s, up to a speed of 2000 rpm for 15 s. Imaging was performed using the Nanoscope Software 7.3 . Scan size ranged from 2.5 to 25 μm, and the scan speed ranged from 5 to 10 μm per second.The Zetasizer Nano ZS series ZEN 3600  was used to determine the zeta potential, measured by laser Doppler micro-electrophoresis (Smoluchowski’s theory). Fractions 1 and 2 from all three donors were diluted 1:50 in PBS, for a final volume of 1 mL. Each dilution was placed into a plastic, disposable folded capillary cell, and any air bubbles were removed from the cuvette through gentle tapping prior to recording. Three sets of 20 measurements were taken for each sample.A BCA was performed to determine the protein concentration of each sample according to the manufacturer’s instructions.All samples were normalized to protein content. Sodium dodecyl-sulfate (6×) was added to a final concentration of 1×, and samples were boiled for five minutes at 90 °C. The equivalent of 1200 µg of protein was loaded in each well of a 12% polyacrylamide gel in MOPS buffer, and electrophoresis occurred for 1.5–2 h at 120 V. Transfer from the gel to a nitrocellulose membrane was completed at 200 mA for 1.5 h. The membrane was blocked at room temperature with 5% milk in TBS + 0.1% Tween (TBST) for two hours. Primary antibodies in 1% milk in TBST (1:500 dilution) were incubated overnight at 4 °C. After three ten-minute washes with TBST, secondary antibody in 1% milk in TBST (1:3000 dilution) was incubated for two hours at room temperature before three final ten minute washes with TBST and developing the membrane in an ECL solution for five minutes at room temperature in the dark. Images were collected using an Amersham 600 imager .7 EVs per well and analyzed in triplicate. Best fit curve yielded an R2 value of 0.9989.Standards and samples were incubated with kit reagents, as per the manufacturer’s instructions. All samples were diluted to 2 × 10The following glycan node analysis procedure was adapted from Borges et al.’s ,38 EV saBlood plasma or concentrated EV samples (10 µL) were added to 1.5 mL snap-cap polypropylene tubes, followed by the addition of DMSO (270 µL). Sodium hydroxide beads (~0.7 g) were collected in a ~1 mL Pierce spin column and washed with acetonitrile , followed by two rinses with DMSO (350 µL). The sample was combined with DMSO (270 µL) and iodomethane (105 μL), followed by immediate vortexing. The sample was then added to the pre-conditioned NaOH beads in the plugged microfuge spin column. The sample was allowed to sit in contact with the NaOH beads for 11 min, with occasional gentle stirring. The microfuge spin column was then unplugged and spun into a 2 mL sample collection tube for 30 s at 5000 rpm (1000 g in a fixed-angle rotor). The collected sample solution was quickly transferred into a silanized 13 × 100 mm glass test tube, containing 0.5 M NaCl in 0.2 M sodium phosphate buffer (pH 7) (3.5 mL). To maximize glycan recovery, the NaOH beads were washed twice with ACN (300 µL), with all spun-through liquid immediately transferred into the same silanized glass test tube. Liquid/liquid (L/L) extraction was then carried out by adding chloroform (1.2 mL) to each test tube, which was then capped and shaken well. After brief centrifugation to separate the layers, the upper aqueous layer was discarded and replaced with 0.5 M NaCl solution in 0.2 M sodium phosphate buffer (pH 7) (3.5 mL). After three L/L extraction rounds, the chloroform layer was recovered and dried under a gentle stream of nitrogen in a heater block set to 74 °C.To perform acid hydrolysis, 2 M TFA (325 µL) was added to each sample, followed by heating at 121 °C for two hours. Samples were then dried under a gentle stream of nitrogen in a heater block set to 74 °C. To reduce the sugar aldehydes, freshly made 10 mg/mL sodium borohydride in 1 M ammonium hydroxide (475 µL) was added to dissolve each sample, followed by incubation at room temperature for one hour. To remove excess borate, methanol  was added, mixed well and dried under nitrogen, followed by adding 9:1 (v/v) MeOH:acetic acid (125 µL), which was dried in like fashion. Samples were then fully dried in a vacuum desiccator for 20 min. To acetylate hydroxyl groups introduced by the acid hydrolysis step, deionized water (18 µL) was added to each test tube, to dissolve any precipitates. This was followed by the addition of acetic anhydride (250 µL) and sonication in a water bath for two minutes. Each sample was then incubated for ten minutes at 60 °C, followed by mixing with concentrated TFA (230 µL) and incubation again at 60 °C for ten minutes. To clean up the sample mixture prior to GC-MS, L/L extraction was performed twice, after adding dichloromethane (1.8 mL) and deionized water (2 mL) to each sample. The upper aqueous layer was discarded each round and the organic layer was then transferred to a silanized autosampler vial, and dried under nitrogen. Each sample was reconstituted in acetone (120 µL) and then capped in preparation for injection onto the GC-MS.Samples were analyzed on an Agilent A7890 gas chromatograph equipped with a CTC PAL autosampler , coupled to a Waters GCT (time-of-flight: TOF) mass spectrometer . For each sample, 1 µL of the 120 µL total volume was injected at a split ratio of 20:1 onto a hot (280 °C), silanized glass liner , containing a small plug of silanized glass wool. Volatilized sample components were separated on a 30-m DB-5ms GC column using helium as the carrier gas, at a constant flow rate of 0.8 mL/minute. The GC oven temperature was initially kept at 165 °C for 0.5 min, then increased to 265 °C at a rate of 10 °C/minute, followed by immediate ramping to 325 °C at a rate of 30 °C/minute, and finally held at 325 °C for 3 min. As sample components entered the mass spectrometer, they were subjected to electron ionization . Positive-ion mode mass spectra from individual TOF pulses over a m/z range of 40–800 were summed every 0.1 s. The mass spectrometer was tuned and calibrated daily with perfluorotributylamine, to ensure reproducible relative abundances of electron ionization (EI) ions and mass accuracy within 10 ppm.Summed extracted-ion chromatogram (XIC) peak areas for all glycan nodes were integrated with Quanlynx 4.1 software. A list of ions corresponding to each glycan node can be found elsewhere . Peak arIndividual hexoses were normalized to the sum of all endogenous hexoses, and individual N-acetylhexosamines (HexNAcs) were normalized to the sum of all endogenous HexNAcs. This approach provided relative glycan node profiling, and therefore direct comparison of glycan node profiles between EV and plasma samples. This does not facilitate quantitative comparisons between different glycan nodes within the same sample. Notably, terminal mannose is not chromatographically or mass spectrally resolved from terminal glucose (which is mostly derived from free blood glucose). If specific hexoses or HexNAcs were not reported as present, it was because their summed extraction ion chromatogram signal-to-noise ratio was less than ten.Isolation of intact EVs from biological samples containing many components can be performed with high purity using SEC, which outperforms ultracentrifugation (UC), the most commonly used method in the field . A majorThere was a lack of statistically significant differences in GNA data from four SEC technical replicates from each donor, and data were statistically pooled to facilitate comparisons. As expected, numerous significant differences were observed between EV glycan node profiles (fraction 1 and fraction 2 combined) and those from plasma of the same donor . MoreoveCompared to plasma EVs, whole plasma was relatively enriched in 4-linked glucose (4-Glc), 6-linked glucose or mannose , 3,4-linked N-acetylglucosamine , and 3,4-linked galactose . The most likely sources of 4-Glc and 6-Glc in plasma are lactose (4-Glc), glycogen fragments (4- and 6-Glc) glycosylphosphatidylinositol (GPI)-anchored proteins. Furthermore, 3,4-GlcNAc represents antennary fucosylation and 3,4-Gal is most commonly found in glycolipids .A lack of statistically significant differences was observed between healthy donor EV glycans , indicatThe findings from this study confirm the expectation that glycan profiles of plasma-derived EVs are distinct from donor-matched whole plasma. The results also reveal that EV glycans do not substantially differ among a small set of healthy donors. Moreover, this study demonstrates that plasma EV fractions from SEC can be pooled for analysis, as glycan expression does not significantly differ among fractions. The results also reveal that glycan nodes corresponding to those observed in the GAG residues of proteoglycans such as chondroitin sulfate, dermatan sulfate, type I keratan sulfate, or type II keratan sulfate are relatively enriched on healthy plasma EVs. Chondroitin sulfate-decorated serglycin has previously been observed in serum-derived EVs, and found to play an important role in tumor-derived EV protein cargo loading . HeparanIt is worth noting that the glycan features reported in this study may include those present in the EV protein corona (biomolecular surface absorption). Currently, the EV protein corona remains a largely unexplored area of research ,17,52,53Overall, identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. The GNA of EVs is likely to be an important, cost effective, and time-efficient future method to assess glycan-dependent health from a simple blood draw, as EVs have been associated with multiple disease conditions ,20,25,55


We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics . Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO The key carbon source—carbon monoxide (CO)—accounts for a significant portion of steel mill off-gas and synthesis gas (syngas), which can be generated from multiple high-volume, non-gaseous waste feedstocks   represents a more diverse and plentiful waste stream compared to CO   is the most recognized energy source for CO2 utilization—as metabolism of sugars or nitrates cause shifts in metabolism that result in lower CO2 or H2 utilization , and Mock et al.  was used in all experiments and stored as glycerol stocks at −80°C. This non-commercial strain was grown on CO2/H2  and CO/CO2/H2  in chemically defined medium . Chemostat continuous culture achieved steady-states at dilution rates (D) = 0.47 ± 0.01  = 0.0196 ± 0.0004 [average ± standard deviation]), 0.5 ± 0.01, and 1 ± 0.01 day−1 . See A derivate of Biomass concentration (gDCW/L) was estimated, and extracellular metabolome analysis carried out as specified in Valgepea et al. .2, CO, ethanol, H2S, Ar, and CO2 at 2, 14, 31, 34, 40, and 44 atomic mass units (amu), respectively, in the bioreactor off-gas. These masses were chosen so that each target compound would be represented by a unique signal. This was determined to be essential to achieve the highest confidence in quantification using preliminary experiments, as interferences from other compounds at a shared mass could not be reliably accounted for . Gas from the cylinder was used as the calibration gas for each MS-cycle  to achieve reliable off-gas analysis  and production (ethanol) were determined using “online calibration” of the MS by analyzing the respective feed gas directly from the cylinder after each analysis cycle of the bioreactors. Specific rates (mmol/gDCW/h) were calculated by taking into account the exact composition of the respective gas, bioreactor liquid working volume, feed gas flow rate, off-gas flow rate , the ideal gas molar volume, and the steady-state biomass concentration.Gas uptake , as specified in Valgepea et al.  , ATP dissipation flux calculated above, and maximization of biomass yield as the objective function. Complete simulation results identified as SIMx  in the text are in 2 reduction to formate was forced from the formate dehydrogenase (FdhA) reaction scheme (Reaction ID: rxn00103_c0) in SIM42-48, to the correct reaction scheme in SIM56-62, as identified by Mock et al.  iCLAU786 of k et al. —FdhA/HydClostridium autoethanogenum cells reached steady-state when growing on CO2/H2 in chemostats at dilution rate (D) ~0.5 day−1 [specific growth rate (μ) ~0.02 h−1] with a biomass concentration of 0.18 ± 0.02 g dry cell weight (gDCW)/L /L . It is ir et al.  showed c−1, cells reached steady-state at dilution rate = 0.5 day−1, with a specific rate of carbon incorporation  of 480 ± 80 mmol/gDCW/day  showed that higher H2 uptake rates are achievable (compared to old datasets). These results show that higher carbon yields are possible  to find candidate mechanisms for improving CO2/H2 fermentation using the metabolic model iCLAU786.Despite the different dilution rate, the COcultures ,D. Howeves  under autotrophic conditions, with the HytABCDE (leq000001) and Nfn complex (leq000002) likely facilitating cofactor production via electron bifurcation   showed rurcation   was detailed by Haas et al.  flux was not significantly in the direction of pyruvate  Valgepe. Under as et al. .Clostridium autoethanogenum was cultured with a low concentration of carbon monoxide in addition to CO2 and H2 (CO/CO2/H2) in chemostats. A steady-state was reached at D = 0.5 day−1 (μ ~0.02 h−1), and at D =1 day−1  and at a D = 0.5 day−1 (CO/CO2/−1) showed simultaneous uptake of CO  and CO2  (2/2/H2 in this work). This also resulted in significant improvements to culture performance compared to CO2/H2 fermentations.To validate our modeling hypothesis, ctively) . The co-a et al.  or CO2/H2/H2, CO/CO2/2/2 fermentation achieved a higher ethanol titer (11.6 ± 0.4 g/L), while CO and syngas fermentations were similar  fermentation to C≥2 products  fermenting syngas and fructose. A. woodii generates a sodium ion (Na+) gradient  gradient for membranous ATP generation  is also important for reducing AOR product inhibition. In this way, NADH facilitates fast metabolism of acetaldehyde to ethanol via alcohol dehydrogenase (Adh(E); rxn00543_c0). Decreasing the acetate concentration reduces acidification and the ATP cost for excreting acetate . Therefore, CO consumption decreases the intracellular H+ pool, and following Le Chatelier's principle, drives HytABCDE activity. Indeed, the change in specific H2 uptake relative to specific CO2 uptake is greater than that of CO ; 2 was roughly half of that of CO/CO2/2/H2 culture seems to facilitate this as (H2/carbon)feed–(H2/carbon)flux was ~0 mol/mol for CO/CO2/H2 fermentations only  where characterized. Industry is actively developing gas fermentation to valorize CO2 (Tizard and Sechrist, 2 valorization is foreseeable, and CO supplementation may play a role in the continuing diversification of industrial gas fermentation.We established a dataset quantifying steady-state of the model acetogen All datasets generated for this study are included in the article/All authors viewed and approved the manuscript and contributed significantly to the work. KV, EM, and LN conceived the project. JH, KV, and EM designed the experiments and analyzed the results. JH and KV performed experiments, supported by RS, IC, MP, and EM. JH wrote the manuscript with the help of KV, EM, RT, SS, MK, and LN.LanzaTech has interest in commercializing gas fermentation with C. autoethanogenum. RT, SS, and MK are employees of LanzaTech.


Phyllodiaptomus diaptomus was sequenced and assembled via Next-Generation Sequencing (NGS) and iterative assembly with a reference seed. The genome is 16446 bp long, A + T biased (69.4%), with 13 protein-coding genes, 22 tRNAs, and 2 rRNA. All protein-coding genes are initiated by a typical “ATN” codon. ND1, ND2, ATP6 genes are terminated with “TAG”, the other 10 genes are terminated with “TAA”. This is the first complete mitogenome published in the Diaptomidae. It provides molecular information useful to a better understanding of the phylogeny of calanoids.The mitochondrial genome of  Phyllodiaptomus tunguidus is a Chinese endemic and was first described by Shen and Tai  and fixed with absolute ethanol. The specimens (accession number COZOOP03003A) and extracted DNA were preserved at −20 °C in the Aquatic Collection of Institute of Hydrobiology, Jinan University, Guangzhou, China. We used the TIANamp Marine Animals DNA Kit  to extract the genome. Genomic DNA was sequenced using a next-generation method. The assembly and annotation procedure followed Xu et al.  with theP. tunguidus mitogenome is 16446 bp , with A + T biased composition: A (34.6%), C (12.3%), G (18.2%), T (34.8%). Thirty-seven genes were annotated: 13 protein coding genes, 22 tRNA genes and 2 rRNA genes. All protein-coding genes are initiated by a typical “ATN” codon. COX1, COX2, COX3, ND2, ND3, ND4, ND5, ND6, ND4L, CYTB are end with TAA; ND2, ND1 and ATP6 end with TAG; tRNA genes have lengths ranging from 56 to 68bp, and they are folded into a typical cloverleaf structure. The Phylogenetic tree shows that copepods are split into three orders-level clades, namely Calanoida, Cyclopoida and Harpacticoida (Phyllodiaptomus tunguidus is monophyletic with L. raynerae and the two calanoids have a sister taxon relationship with Calanus hyperboreus. Compared with the mitogenome of L. raynerae, a different arrangement of tRNAs was found, but no shift of PCGs was discovered.The total length of the cticoida . Phyllod