Molecular Insights into a Novel Cu(I)-Sensitive ArsR/SmtB Family Repressor in Extremophile Acidithiobacillus caldus.

Acidithiobacillus caldus is a common bioleaching bacterium that is inevitably exposed to extreme copper stress in leachates. The ArsR/SmtB family of metalloregulatory repressors regulates homeostasis and resistance in bacteria by specifically responding to metals. Here, we characterized A. caldus Cu(I)-sensitive repressor (AcsR) and gained molecular insights into this new member of the ArsR/SmtB family. Transcriptional analysis indicated that the promoter (PIII) of acsR was highly active in Escherichia coli but inhibited upon AcsR binding to the PIII-acsR region. Size exclusion chromatography and circular dichroism spectra revealed that CuI-AcsR shared an identical assembly state with apo-AcsR, as a dimer with fewer α helices, more extended strands, and more ß turns. Mutation of the cysteine site in AcsR did not affect its assembly state. Copper(I) titrations revealed that apo-AcsR bound two Cu(I) molecules per monomer in vitro with an average dissociation constant (KD) for bicinchoninic acid competition of 2.55 × 10-9 M. Site-directed mutation of putative Cu(I)-binding ligands in AcsR showed that replacing Cys64 with Ala reduces copper binding ability from two Cu(I) molecules per monomer to one, with an average KD of 6.05 × 10-9 M. Electrophoretic mobility shift assays revealed that apo-AcsR has high affinity for the 12-2-12 imperfect inverted repeats P2245 and P2270 in the acsR gene cluster and that Cu-loaded AcsR had lower affinity for DNA fragments than apo-AcsR. We developed a hypothetical working model of AcsR to better understand Cu resistance mechanisms in A. caldus. IMPORTANCE Copper (Cu) resistance among various microorganisms is attracting interest. The chemolithoautotrophic bacterium A. caldus, which can tolerate extreme copper stress (≥10 g/L Cu ions), is typically used to bioleach chalcopyrite (CuFeS2). Understanding of Cu resistance in A. caldus is limited due to scant investigation and the absence of efficient gene manipulation tools. Here, we characterized a new member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional proteins that repress operons linked to stress-inducing concentrations of heavy metal ions. This protein can bind two Cu(I) molecules per monomer and negatively regulate its gene cluster. Members of the ArsR/SmtB family have not been investigated in A. caldus until now. The discovery of this novel protein enriches understanding of Cu homeostasis in A. caldus.

The digestive behavior of pectin in human gastrointestinal tract: a review on fermentation characteristics and degradation mechanism.

Pectin is widely spread in nature and it develops an extremely complex structure in terms of monosaccharide composition, glycosidic linkage types, and non-glycosidic substituents. As a non-digestible polysaccharide, pectin exhibits resistance to human digestive enzymes, however, it is easily utilized by gut microbiota in the large intestine. Currently, pectin has been exploited as a novel functional component with numerous physiological benefits, and it shows a promising prospect in promoting human health. In this review, we introduce the regulatory effects of pectin on intestinal inflammation and metabolic syndromes. Subsequently, the digestive behavior of pectin in the upper gastrointestinal tract is summarized, and then it will be focused on pectin's fermentation characteristics in the large intestine. The fermentation selectivity of pectin by gut bacteria and the effects of pectin structure on intestinal microecology were discussed to highlight the interaction between pectin and bacterial community. Meanwhile, we also offer information on how gut bacteria orchestrate enzymes to degrade pectin. All of these findings provide insights into pectin digestion and advance the application of pectin in human health.

Enhancement of acid tolerance of Escherichia coli by introduction of molecule chaperone CbpA from extremophile.

Molecular chaperone CbpA from extreme acidophile Acidithiobacillus caldus was applied to improve acid tolerance of Escherichia coli via CRISPR/Cas9. Cell growth and viability of plasmid complementary strain indicated the importance of cbpAAc for bacteria acid tolerance. With in situ gene replacement by CRISPR/Cas9 system, colony formation unit (CFU) of genome recombinant strain BL21-ΔcbpA/AccbpA showed 7.7 times higher cell viability than deficient strain BL21-ΔcbpA and 2.3 times higher than wild type. Cell morphology observation using Field Emission Scanning Electron Microscopy (FESEM) revealed cell breakage of BL21-ΔcbpA and significant recovery of BL21-ΔcbpA/AccbpA. The intracellular ATP level of all strains gradually decreased along with the increased stress time. Particularly, the value of recombinant strain was 56.0% lower than that of deficient strain after 5 h, indicating that the recombinant strain consumed a lot of energy to resist acid stress. The arginine concentration in BL21-ΔcbpA/AccbpA was double that of BL21-ΔcbpA, while the aspartate and glutamate contents were 14.8% and 6.2% higher, respectively, compared to that of wild type. Moreover, RNA-Seq analysis examined 93 genes down-regulated in BL21-ΔcbpA compared to wild type strain, while 123 genes were up-regulated in BL21-ΔcbpA/AccbpA compared to BL21-ΔcbpA, with an emphasis on energy metabolism, transport, and cell components. Finally, the working model in response to acid stress of cbpA from A. caldus was developed. This study constructed a recombinant strain resistant to acid stress and also provided a reference for enhancing microorganisms' robustness to various conditions.

EpsRAc is a copper-sensing MarR family transcriptional repressor from Acidithiobacillus caldus.

The MarR family, as multiple antibiotic resistance regulators, is associated with the resistance of organisms to unfavorable conditions. MarR family extracellular polymeric substances (EPS)-associated transcriptional regulator (EpsRAc) was closely associated with copper resistance in Acidithiobacillus caldus (A. caldus). Transcriptional analysis showed high activity of the epsR promoter (PI) in Escherichia coli and differential response to metal ions. The copper content and UV absorption spectrum of the co-purified protein did not increase, but a stoichiometry of 0.667 mol Cu(I) per EpsRAc monomer was observed in vitro in copper titration experiments, suggesting that Cu(II) acts with low affinity in binding to the EpsRAc protein. Electrophoretic mobility shift assays (EMSA) demonstrated that EpsRAc could bind to its own promoter in vitro, and the binding region was the palindrome sequence TGTTCATCGTGTGTGAGCACACA. EpsRAc negatively regulated its own gene expression, whereas Cu(II) mitigates this negative effect. EpsRAc did not bind to other neighboring gene promoters. Finally, we developed a working model to illustrate the regulatory mechanism of A. caldus in response to extreme copper stress. KEY POINTS: • Identification of a MarR family EPS-associated transcriptional regulator, named EpsRAc. • Cu(I) can bind to the EpsRAc protein with low affinity. • EpsRAc negatively regulates the expression of epsR, and Cu(II) can alleviate this negative regulation.

Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

Simultaneously enhance iron/sulfur metabolism in column bioleaching of chalcocite by pyrite and sulfur oxidizers based on joint utilization of waste resource.

In chalcocite (Cu2S) bioleaching, the lack of iron metabolism is a key restricting factor. As the most common sulfide mineral, pyrite (FeS2) can release Fe(Ⅱ) and compensate for the iron metabolism deficiency in chalcocite bioleaching. The bioleaching of chalcocite in an imitated industrial system was improved by enhancing the iron-sulfur metabolism simultaneously using pyrite and sulfur oxidizers based on the joint utilization of waste resources, while the bioleaching performance and community structure in the leachate were systematically investigated. Due to the active sulfur/iron metabolism, the pH reached 1.2, and Fe3+ was increased by 77.78%, while the biomass of planktonic cells was improved to 2.19 × 107 cells/mL. Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) analysis results showed that more iron-sulfur crystals were produced due to more active iron-sulfur metabolism. Scanning electron microscopy (SEM) revealed that many derivative particles and corrosion marks appeared on the surface of the ore, implying that the mineral-microbe interaction was strengthened. Confocal laser scanning microscopy (CLSM) showed the accumulation of cells and extracellular polymeric substances (EPS) on the ore surface, indicating a stronger contact leaching mechanism. Furthermore, the community structure and canonical correspondence analysis (CCA) demonstrated that the introduction of sulfur-oxidizing bacteria and pyrite could maintain the diversity of dominant leaching microorganisms at a high level. Sulfobacillus (27.75%) and Leptospirllillum (20.26%) were the dominant sulfur-oxidizing and iron-oxidizing bacteria during the bioleaching process. With the accumulation of multiple positive effects, the copper ion leaching rate was improved by 44.8%. In general, this new type of multiple intervention strategy can provide an important guide for the bioleaching of low-grade ores.

The adaptation mechanisms of Acidithiobacillus caldus CCTCC M 2018054 to extreme acid stress: Bioleaching performance, physiology, and transcriptomics.

To understand the acid-resistant mechanism of bioleaching microorganism Acidithiobacillus caldus CCTCC M 2018054, its physiology and metabolic changes at the transcriptional level under extreme acid stress were systemically studied. Scanning electron microscopy (SEM), Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) showed that with an increase in acidity, the absorption peak of sulfur oxidation-related functional groups such as S-O decreased significantly, and a dense sulfur passivation film appeared on the surface of the ore. Confocal laser scanning microscopy (CLSM) revealed that coverage scale of extracellular polymeric substance (EPS) and biofilm fluctuated accordingly along with the increasing acid stress (pH-stat 1.5, 1.2 0.9 and 0.6) during the bioleaching process. In response to acid stress, the increased levels of intracellular glutamic acid, alanine, cysteine, and proline contributed to the maintenance of intracellular pH homeostasis via decarboxylation and alkaline neutralization. Higher unsaturated fatty acid content was closely related to membrane fluidity. Up to 490 and 447 differentially expressed genes (DEGs) were identified at pH 1.5 vs pH 1.2 and pH 1.2 vs pH 0.9, respectively, and 177 common DEGs were associated with two-component system (TCS) regulation, transporter regulation, energy metabolism, and stress response. The upregulation of kdpB helped cells defend against proton invasion, whereas the downregulation of cysB and cbl implied stronger oxidation of sulfur compounds. The transcriptional level of sqr, sor, and soxA was significantly increased and consolidated the energy supply needed for resisting acid stress. Furthermore, eight of the identified DEGs (sor, cbl, ompA, atpF, nuoH, nuoC, sqr, grxB) were verified as being related to the acid stress response process. This study contributes toward expanding the application of these acidophiles in industrial bioleaching.

Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity.

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis-fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126-fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.

Simultaneous denitrification and desulfurization-S0 recovery of wastewater in trickling filters by bioaugmentation intervention based on avoiding collapse critical points.

In order to better achieve efficiently simultaneous desulfurization and denitrification/S0 recovery of wastewater, the intervention of sulfur oxidizing bacteria (SOB) and denitrifying bacteria (DNB) was employed to avoid the collapse critical points (the dramatically decrease of S/N removal efficiency) under the fluctuated load. With the assistance of DNB and SOB, collapse critical point of trickling filter (TF) was delayed from the P8 (105-114 d) to P10 stage (129-138 d). The treatment efficiency of nitrogen and sulfur was the highest with the S/N ratio of 3:1. The bioaugmentation of DNB and SOB at collapse critical point could effectively regulated collapse situation, which further increased the maximum system utilization/elimination capacity to 4.50 kg S m-3·h-1 and 0.90 kg N m-3·h-1 (increased by 56.89% and 65.56% in comparison to control). High-throughput sequencing analysis indicated that Proteobacteria (average 78.59%) and Bacteroidetes (average 9.30%) were dominant bacteria in the reactor at all stages. As the reaction proceeds, the microbial community was gradually dominated by some functional genera such as Chryseobacterium (average 2.97%), Halothiobacillus (average 22.71%), Rhodanobacter (average 14.02%), Thiobacillus (average 9.01%), Thiomonas (average 16.70%) and Metallibacterium (average 21.63%), which could remove nitrate or sulfide. Both of Principal Component Analysis (PCA) and Canonical Correlation Analysis (CCA) demonstrated the important role of DNB/SOB during the long-term run in the trickling filters (TFs).

Metabolic transcriptional analysis on copper tolerance in moderate thermophilic bioleaching microorganism Acidithiobacillus caldus.

Bioleaching, an alternative environmental smelting technology, typically uses high concentrations of heavy metal ions, especially in the subsequent phase, due to metal ion accumulation from the mineral. In this study, we analyzed the overall response of the bioleaching microorganism Acidithiobacillus caldus to copper stress through physiological and transcriptomic analyses. Scanning electron microscopy results showed higher extracellular polymeric substances secretion and cell aggregation under copper stress. Intracellular levels of glutamic acid, glycine and cysteine increased, favoring the synthesis of glutathione for maintenance of the oxidation-reduction state. GSH, during copper stress conditions, the activity of GSH-PX and CAT increased, resulting in reduced oxidative damage while maintaining stable intracellular pH. Higher unsaturated and cyclopropane fatty acid levels resulted in increased membrane fluidity and compactness and decreased ATP levels to support the energy requirements for stress resistance. Initially, H+-ATPase activity increased to provide energy for proton output and decreased later at higher copper ion stress. From transcriptome analysis, 140 genes were differentially expressed under low copper stress (1 g/L), while 250 genes exhibited altered transcriptional levels at higher copper stress (3 g/L). These differentially expressed genes were involved primarily in metabolic pathways such as energy metabolism, two-component systems, amino acid metabolism, and signal transduction. The Sox family cluster gene cluster involved in the conversion of thiosulfate to sulfate was upregulated in the sulfur metabolism pathway. In the oxidative phosphorylation pathway, genes participating in the synthesis of NADH oxidoreductase and cytochrome c oxidase, nuoL, cyoABD (cyoA, cyoB and cyoD) and cydAB (cydA and cydB), were downregulated. The TCS element ompR, closely associated with the osmotic pressure, exhibited active response, while Cu2+ efflux system gene cusRS was upregulated. In the amino acid metabolism, the glnA involved in nitrogen fixation was upregulated and promoted the synthesis of glutamine synthetase for reducing excessive oxidative stress. This study provides new insights into the mechanism underlying A. caldus response to heavy-metal ion stress under harsh bioleaching conditions.

Adaptive mechanism of Acidithiobacillus thiooxidans CCTCC M 2012104 under stress during bioleaching of low-grade chalcopyrite based on physiological and comparative transcriptomic analysis.

Acidithiobacillus thiooxidans (A. thiooxidans) is often used for sulfur-bearing ores bioleaching, but its adaptive mechanism to harsh environments remains unclear. Here, we explored the adaptive mechanism of A. thiooxidans in the process of low-grade chalcopyrite bioleaching based on the physiology and comparative transcriptome analysis. It was indicated that A. thiooxidans maintains intracellular pH homeostasis by regulating unsaturated fatty acids, especially cyclopropane fatty acids, intracellular ATP, amino acid metabolism, and antioxidant factors. Comparative transcriptome analysis indicated that the key genes involved in sulfur oxidation, sor and soxABXYZ, were significantly up-regulated, generating more energy to resist extreme environmental stress by more active sulfur metabolism. Confocal laser scanning microscope analysis found that down-regulation of flagellar-related genes was likely to promote the biofilm formation. System-level understanding of leaching microorganisms under extreme stress can contribute to the evolution of these extremophiles via genetic engineering modification work, which further improves bioleaching in future.

Enhanced "contact mechanism" for interaction of extracellular polymeric substances with low-grade copper-bearing sulfide ore in bioleaching by moderately thermophilic Acidithiobacillus caldus.

In order to enhance the "contact mechanism" governing the interaction of extracellular polymeric substances (EPS) with low-grade copper-bearing sulfide ore for the bioleaching of copper, moderately thermophilic Acidithiobacillus caldus was subjected to exogenous intervention with iron and sulfur. The enhancement of the contact mechanism was systematically investigated by evaluating the attached cells/EPS dynamics, intracellular adenosine triphosphate (ATP), cell functional groups, gene transcriptional level, and ore characteristics. Confocal laser scanning microscopy (CLSM) revealed that exogenous intervention with iron and sulfur led to the production of a denser EPS layer and faster adsorption of the attached cells to the ore based on differential fluorescence staining, which indicated enhancement of the "contact mechanism". The increased intracellular ATP content of the attached cells in the exogenous substrate system provided the required energy for the adsorption processes associated with the "contact mechanism". Fourier-transform infrared spectroscopic (FTIR) analysis of the attached cells and the ore showed a dramatic shift of the NH and COS peaks (associated with EPS formation), whereas the FTIR peaks of SO and SO42- associated with sulfur metabolism were also significantly influenced. Moreover, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the expression of genes related to cellular energy metabolism (nuoB, nuoC, atpE, atpF), sulfur metabolism (sor, sqr, sdo, soxA), biofilm formation (pgaA, pgaB), and cell colonization (acfA, acfB, acfC, acfD) was up-regulated after exogenous intervention, verifying enhancement of the "contact mechanism" at the transcriptional level. In addition, scanning electron microscopy (SEM) indicated more obvious adsorption traces on the ore surface. X-ray diffraction (XRD) indicated the presence of more complex derivatives, such as Fe3(SO4)4, FeSO4, Fe2(SO4)3, and Cu2S, which is suggestive of more active iron/sulfur metabolism with addition of the exogenous iron and sulfur. Overall, a model for bioleaching of low-grade copper-bearing sulfide ore by moderately thermophilic A. caldus was constructed. The results of this investigation should provide a guide for similar industrial bioleaching processes.

[Cloning and Expression of Tibetan Sheep-origin Echinococcus granulosus Antigen B Gene and Protein Identification Using Immunological Method].

Objective: To clone and express the Tibetan Sheep-origin Echinococcus granulosus Antigen B8/2 Gene, and immunologically identify the encoded protein. Methods: The cDNA of EgAgB8/2 gene was amplified by RT-PCR. The prokaryotic expression vector pET-EgAgB8/2 was constructed and transformed into E. coli BL21（DE3） for expression. Proteins were extracted, separated in SDS-PAGE and identified by Western blotting. Results: The cloned EgAgB8/2 gene was 335 bp in length, and had a 98%-100% sequence homology with the reported cDNA sequence of EgAgB8/2, indicating the successful construction of the pET-EgAgB8/2 vector. SDS-PAGE revealed large amount of proteins in supernatant. Western blotting further confirmed the expression of the target protein. Conclusion: The EgAgB8/2 gene of Tibetan Sheep-origin in Qinghai is successfully cloned, and the constructed pET-EgAgB8/2 vector can be used to express the target protein.

Affinity purification of aprotinin from bovine lung.

An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg.

Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli.

Sarcosine oxidase (SOX) was an important diagnostic enzyme in the renal function examination. An integrated strategy coupling codon and fermentation optimization was firstly proposed for improving SOX production from recombinant E. coli in 3-L fermentor. The expression suppression (gene phase) and poor balance between SOX expression and cell growth (fermentation phase) in the traditional SOX production were respectively improved by the multiple strategies. Based on the codon bias, the expression suppression was weakened via codon optimization and SOX activity reached 1,521 U/L. The induction toxicity was reduced with the optimal induction condition and SOX production increased to 4,015 U/L. Based on the kinetic analysis of µ x and µ p , a better balance between cell growth and expression was achieved by the two-stage pH-stat control strategy. The SOX activity was further improved to 8,490 U/L and fermentation cycle was also significantly shortened from 44 to 32 h. The substrate inhibition was weakened with a constant feeding fed-batch. With the assistance of integrated strategy, the activity and productivity reached 12,466 U/L and 389.6 U/(L h), respectively, or 3.1-fold and 4.3-fold of the uncontrolled fermentation. The strategy would be also useful in the industrial application of other similar enzymes.

Thermal inactivation of xanthine oxidase from Arthrobacter M3: mechanism and the corresponding thermostabilization strategy.

The mechanism of thermal inactivation about xanthine oxidase (XOD) from Arthrobacter M3 was investigated. Results of reducing SDS-PAGE indicated that the inactivation of XOD was not related to the peptide degradation. Meanwhile, fluorimetry and circular dichroism spectroscopy suggested that XOD inactivation might be associated with the exposure of hydrophobic residues to surface and partial loss of secondary structure. Specific formation of soluble aggregates of XOD was detected by size exclusion chromatography. In addition, the thermal-dynamic analysis showed that the inactivation kinetics of XOD followed the first-order model. Therefore, trehalose (cosolute) and betaine (osmolyte) were accordingly employed to attenuate the inactivation of this enzyme. The results associated with these two reagents further confirmed that the loss of XOD activity was mainly due to the exposure of hydrophobic residues and formation of aggregation. Owing to the added trehalose and betaine, half-life could be significantly increased, and the inactivation rate constant (k) was detected as decreased.

Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer ß-sheets, and ß-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 µM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5'-ACCCCAATTACGAAAATTTTT-3'), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

AUY922 improves sensitivity to sunitinib in clear cell renal cell carcinoma based on network pharmacology and in vitro experiments.

Clear Cell Renal Cell Carcinoma (ccRCC), the most prevalent form of renal cell carcinoma (RCC), poses a significant threat to human health due to its rising morbidity and mortality rates. Sunitinib, a pivotal targeted drug for the treatment of ccRCC, presents a significant challenge due to the high susceptibility of ccRCC to resistance. HSP90 inhibitor AUY922 has demonstrated anti-tumor activity in a range of cancer types. However, its efficacy in combination with sunitinib for ccRCC treatment has not been evaluated. In this study, we employed bioinformatics, network pharmacology, and in vitro assays to verify that AUY922 inhibits cell viability, proliferation, and migration of ccRCC cell lines 786-O and ACHN, with IC50s of 91.86 µM for 786-O and 115.5 µM for ACHN. The effect of AUY922 enhancing the inhibitory effect of sunitinib on ccRCC was further confirmed. The CCK-8 assay demonstrated that the IC50 of sunitinib was reduced from 15.10 µM to 11.91 µM for 786-O and from 17.65 µM to 13.66 µM for ACHN, after the combined application of AUY922. The EdU assay and wound healing assay indicated that AUY922 augmented the inhibitory impact of sunitinib on the proliferation and migration of ccRCC cells. Western blot and RT-PCR analyses demonstrated that AUY922 increased the sensitivity of ccRCC cells to sunitinib by targeting the HIF-1α/VEGFA/VEGFR pathway. Our study represents the first investigation into the role and mechanism of AUY922 in enhancing the sensitivity of ccRCC to sunitinib. In conclusion, the findings indicate the potential for AUY922 to enhance the therapeutic efficacy of sunitinib and overcome sunitinib resistance in ccRCC.

Revealing the Mechanism of Esculin in Treating Renal Cell Carcinoma Based on Network Pharmacology and Experimental Validation.

PURPOSE: This study aims to explore the potential mechanisms of esculin in the treatment of renal cell carcinoma (RCC). METHODS: We employed network pharmacology to predict the potential mechanisms and targets of esculin in RCC. Molecular docking techniques were then employed to validate the predicted targets. Additionally, a series of in vitro experiments were conducted to verify the anticancer effects of esculin on RCC cells, including the CCK-8 assay, EdU assay, wound healing assay, apoptosis assay, and Western blot. RESULTS: Network pharmacology and molecular docking results identified GAPDH, TNF, GSK3B, CCND1, MCL1, IL2, and CDK2 as core targets. GO and KEGG analyses suggested that esculin may influence apoptotic processes and target the PI3K/Akt pathway in RCC. Furthermore, the CCK-8 assay demonstrated that esculin inhibited RCC cell viability. Microscopic observations revealed that following esculin treatment, there was an increase in cell crumpling, a reduction in cell density, and an accumulation of floating dead cells. Additionally, with increasing esculin concentrations, the proportion of EdU-positive cells decreased, the wound closure ratio decreased, the proportion of PI-positive cells increased, the expression levels of BAX and cleaved-caspase-3 proteins increased, and the expression level of Bcl2 protein decreased. These findings suggested that esculin inhibits the proliferation and migration of RCC cells while promoting apoptosis. Moreover, esculin was found to target GAPDH and inhibit the PI3K/Akt pathway. CONCLUSIONS: This study is the first to elucidate the therapeutic effects of esculin on RCC cells. The results provide evidence supporting the clinical application of esculin and introduce a promising new candidate for RCC treatment.

Enhancing column bioleaching of chalcocite by isolated iron metabolism partners Leptospirillum ferriphilum/Acidiphilium sp. coupling with systematically utilizing cellulosic waste.

The iron metabolism partners Leptospirillum ferriphilum and Acidiphilium sp. were screened from industrial bioheap site. An integrated multi-stage strategy was proposed to improve chalcolite column bioleaching coupling with synergistical utilization of cellulosic waste such as acid hydrolysate of aquatic plants. L. ferriphilum was used to accelerate the initial iron metabolism, and Acidithiobacillus caldus maintained a lower pH in the middle stage, while Acidiphilium sp. greatly inhibited jarosite passivation in the later stage. Meanwhile, L. ferriphilum (38.3 %) and Acidiphilium sp. (37.0 %) dominated the middle stage, while the abundance of Acidiphilium sp. reached 63.5 % in the later stage. The ferrous, sulfate ion and biomass were improved and the transcriptional levels of some biofilm and morphology related genes were significantly up-regulated. The final Cu2+ concentration reached 325.5 mg·L-1, improved by 43.8 %. Moreover, Canonical Correlation Analysis (CCA) analysis between bioleaching performance, iron/sulfur metabolism and community verified the important role of iron metabolism partners.