Stibnite dissolution and Sb oxidation by Paraccocus versutus XT0.6 via direct and indirect contact.

In this study Paraccocus versutus XT0.6 was employed to address the mechanism of microbial dissolution and oxidation of stibnite. Results showed that with the growth of XT0.6, pH increased to 9.0 in both microbe-mineral contact (MM) and microbe-mineral non-contact groups (M[M]). Dissolved Sb(III) was released from stibnite, which was subsequently quickly oxidized to Sb(V) completely in MM and partially in M[M] groups. On the contrast, the final pH decreased to 6.5 and 4.9, respectviely, in system amended with extracellular secretion (EM) of XT0.6 and abiotic groups. Dissolution of stibnite and oxidation of Sb(III) were also observed in EM group, suggesting a potential contribution of extracellular enzyme in Sb(III) oxidation. The dissolution and oxidation rates were the highest in MM group, followed by those in M[M], EM and abiotic groups. To be noted, Sb(V) concentration decreased in MM group on the fifth day, which might indicate the formation of Sb(V)-bearing secondary mineral. Genome of XT0.6 consisted of two chromosomes and one plasmid, and most genes responsible for antimony oxidation and antimony resistance were located on the chromosomes. Proteomics analysis of the extracellular secretions indicated the up-regulated proteins were mainly related to electron-transfer, suggesting their potential role in Sb(III) oxidation.

Bioactivities and Mechanisms of Action of Sinomenine and Its Derivatives: A Comprehensive Review.

Sinomenine, an isoquinoline alkaloid extracted from the roots and stems of Sinomenium acutum, has been extensively studied for its derivatives as bioactive agents. This review concentrates on the research advancements in the biological activities and action mechanisms of sinomenine-related compounds until November 2023. The findings indicate a broad spectrum of pharmacological effects, including antitumor, anti-inflammation, neuroprotection, and immunosuppressive properties. These compounds are notably effective against breast, lung, liver, and prostate cancers, exhibiting IC50 values of approximately 121.4 nM against PC-3 and DU-145 cells, primarily through the PI3K/Akt/mTOR, NF-κB, MAPK, and JAK/STAT signaling pathways. Additionally, they manifest anti-inflammatory and analgesic effects predominantly via the NF-κB, MAPK, and Nrf2 signaling pathways. Utilized in treating rheumatic arthritis, these alkaloids also play a significant role in cardiovascular and cerebrovascular protection, as well as organ protection through the NF-κB, Nrf2, MAPK, and PI3K/Akt/mTOR signaling pathways. This review concludes with perspectives and insights on this topic, highlighting the potential of sinomenine-related compounds in clinical applications and the development of medications derived from natural products.

A comprehensive review on 3D tissue models: Biofabrication technologies and preclinical applications.

The limitations of traditional two-dimensional (2D) cultures and animal testing, when it comes to precisely foreseeing the toxicity and clinical effectiveness of potential drug candidates, have resulted in a notable increase in the rate of failure during the process of drug discovery and development. Three-dimensional (3D) in-vitro models have arisen as substitute platforms with the capacity to accurately depict in-vivo conditions and increasing the predictivity of clinical effects and toxicity of drug candidates. It has been found that 3D models can accurately represent complex tissue structure of human body and can be used for a wide range of disease modeling purposes. Recently, substantial progress in biomedicine, materials and engineering have been made to fabricate various 3D in-vitro models, which have been exhibited better disease progression predictivity and drug effects than convention models, suggesting a promising direction in pharmaceutics. This comprehensive review highlights the recent developments in 3D in-vitro tissue models for preclinical applications including drug screening and disease modeling targeting multiple organs and tissues, like liver, bone, gastrointestinal tract, kidney, heart, brain, and cartilage. We discuss current strategies for fabricating 3D models for specific organs with their strengths and pitfalls. We expand future considerations for establishing a physiologically-relevant microenvironment for growing 3D models and also provide readers with a perspective on intellectual property, industry, and regulatory landscape.

Archaea are better adapted to antimony stress than their bacterial counterparts in Xikuangshan groundwater.

Archaea are important ecological components of microbial communities in various environments, but are currently poorly investigated in antimony (Sb) contaminated groundwater particularly on their ecological differences in comparison with bacteria. To address this issue, groundwater samples were collected from Xikuangshan aquifer along an Sb gradient and subjected to 16S rRNA gene amplicon sequencing and bioinformatic analysis. The results demonstrated that bacterial communities were more susceptibly affected by elevated Sb concentration than their archaeal counterparts, and the positive stimulation of Sb concentration on bacterial diversity coincided with the intermediate disturbance hypothesis. Overall, the balance of environmental variables (Sb, pH, and EC), competitive interactions, and stochastic events jointly regulated bacterial and archaeal communities. Linear fitting analysis revealed that Sb significantly drove the deterministic process (heterogeneous selection) of bacterial communities, whereas stochastic process (dispersal limitation) contributed more to archaeal community assembly. In contract, the assembly of Sb-resistant bacteria and archaea was dominated by the stochastic process (undominated), which implied the important role of diversification and drift instead of selection. Compared with Sb-resistant microorganisms, bacterial and archaeal communities showed lower niche width, which may result from the constraints of Sb concentration and competitive interaction. Moreover, Sb-resistant archaea had a higher niche than that of Sb-resistant bacteria via investing on flexible metabolic pathways such as organic metabolism, ammonia oxidation; and carbon fixation to enhance their competitiveness. Our results offered new insights into the ecological adaptation mechanisms of bacteria and archaea in Sb-contaminated groundwater.

An integrated investigation of 16S rRNA gene sequencing and proteomics to elucidate the mechanism of Corydalis bungeana Turcz. on dextran sulfate sodium-induced colitis.

Corydalis bungeana Turcz. (CBT) is frequently used to treat inflammatory illnesses, the mechanisms underlying its use to ulcerative colitis (UC) remain unclear. A dextran sulfate sodium (DSS)-induced UC mice model was established. The disease activity index (DAI), colonic length, histological inspection by hematoxylin-eosin staining, the cytokines levels in the colon, proteomics and intestinal flora in mice were investigated to evaluate the effect of CBT. The results showed that CBT can significantly reduce the DAI, increase the length of colon, improve the pathological injury of colon tissue, decrease the level of TNF-α, IL-6, IL-1ß and increase the level of IL-10 in UC mice. Gut microbe sequencing showed that CBT could enhance the abundance of the intestinal microbiome, decrease possibly harmful bacteria and promote potentially helpful microbes. Proteomics investigation showed that 20 overlapping differentially expressed proteins (DEPs) were discovered in the control, model, and CBT administration groups. The DEPs in the CBT administration group were connected to biological procedures mainly involving detoxification. Extracellular matrix (ECM) receptor-associated proteins such as Col6a1 and CD36 may be important targets for CBT treatment of UC. Overall, this integrated methodology identified a comprehensive multi-omics network, composed of a certain set of gut microbiota and proteins, which may be potential targets for CBT treatment with UC.

Comparative genomics reveals intraspecific divergence of Acidithiobacillus ferrooxidans: insights from evolutionary adaptation.

Acidithiobacillus ferrooxidans serves as a model chemolithoautotrophic organism in extremely acidic environments, which has attracted much attention due to its unique metabolism and strong adaptability. However, little was known about the divergences along the evolutionary process based on whole genomes. Herein, we isolated six strains of A. ferrooxidans from mining areas in China and Zambia, and used comparative genomics to investigate the intra-species divergences. The results indicated that A. ferrooxidans diverged into three groups from a common ancestor, and the pan-genome is 'open'. The ancestral reconstruction of A. ferrooxidans indicated that genome sizes experienced a trend of increase in the very earliest days before a decreasing tendency during the evolutionary process, suggesting that both gene gain and gene loss played crucial roles in A. ferrooxidans genome flexibility. Meanwhile, 23 single-copy orthologous groups (OGs) were under positive selection. The differences of rusticyanin (Rus) sequences (the key protein in the iron oxidation pathway) and type IV secretion system (T4SS) composition in the A. ferrooxidans were both related to their group divergences, which contributed to their intraspecific diversity. This study improved our understanding of the divergent evolution and environmental adaptation of A. ferrooxidans at the genome level in extreme conditions, which provided theoretical support for the survival mechanism of living creatures at the extreme.

Insertion sequence contributes to the evolution and environmental adaptation of Acidithiobacillus.

BACKGROUND: The genus Acidithiobacillus has been widely concerned due to its superior survival and oxidation ability in acid mine drainage (AMD). However, the contribution of insertion sequence (IS) to their biological evolution and environmental adaptation is very limited. ISs are the simplest kinds of mobile genetic elements (MGEs), capable of interrupting genes, operons, or regulating the expression of genes through transposition activity. ISs could be classified into different families with their own members, possessing different copies. RESULTS: In this study, the distribution and evolution of ISs, as well as the functions of the genes around ISs in 36 Acidithiobacillus genomes, were analyzed. The results showed that 248 members belonging to 23 IS families with a total of 10,652 copies were identified within the target genomes. The IS families and copy numbers among each species were significantly different, indicating that the IS distribution of Acidithiobacillus were not even. A. ferrooxidans had 166 IS members, which may develop more gene transposition strategies compared with other Acidithiobacillus spp. What's more, A. thiooxidans harbored the most IS copies, suggesting that their ISs were the most active and more likely to transpose. The ISs clustered in the phylogenetic tree approximately according to the family, which were mostly different from the evolutionary trends of their host genomes. Thus, it was suggested that the recent activity of ISs of Acidithiobacillus was not only determined by their genetic characteristics, but related with the environmental pressure. In addition, many ISs especially Tn3 and IS110 families were inserted around the regions whose functions were As/Hg/Cu/Co/Zn/Cd translocation and sulfur oxidation, implying that ISs could improve the adaptive capacities of Acidithiobacillus to the extremely acidic environment by enhancing their resistance to heavy metals and utilization of sulfur. CONCLUSIONS: This study provided the genomic evidence for the contribution of IS to evolution and adaptation of Acidithiobacillus, opening novel sights into the genome plasticity of those acidophiles.

Targeting CDK12 obviates the malignant phenotypes of colorectal cancer through the Wnt/ß-catenin signaling pathway.

Colorectal cancer (CRC) is the second most common cause of cancer-related mortality and lies third in terms of morbidity due to the limited number of effective druggable targets. Since cancer stem cells (CSCs) are considered to be one of the roots of tumorigenesis, outgrowth and metastasis, targeting CSCs may be a promising strategy to reverse the malignant phenotypes of CRC. Cyclin-dependent kinase 12 (CDK12) has been reported to be involved in the self-renewal of CSCs in various cancers, rendering it an attractive potential target against CSCs to consequently limit the malignant phenotypes in CRC. In the present study, we aimed to investigate whether CDK12 can be a potential therapeutic target for patients with CRC and clarify its underlying mechanism. We found that CDK12, but not CDK13 is required for CRC survival. CDK12 was found to drive tumor initiation according to the colitis-associated colorectal cancer mouse model. In addition, CDK12 promoted CRC outgrowth and hepatic metastasis in the subcutaneous allograft and liver metastasis mouse models, respectively. In particular, CDK12 was able to induce the self-renewal of CRC CSCs. Mechanistically, the activation of Wnt/ß-catenin signaling mediated by CDK12 was implicated in stemness regulation and malignant phenotype maintenance. These findings indicate that CDK12 is a candidate druggable target in CRC. Therefore, the CDK12 inhibitor SR-4835 warrants clinical trial testing in patients with CRC.

Nitrate determines the bacterial habitat specialization and impacts microbial functions in a subsurface karst cave.

Karst caves are usually considered as natural laboratories to study pristine microbiomes in subsurface biosphere. However, effects of the increasingly detected nitrate in underground karst ecosystem due to the acid rain impact on microbiota and their functions in subsurface karst caves have remained largely unknown. In this study, samples of weathered rocks and sediments were collected from the Chang Cave, Hubei province and subjected to high-throughput sequencing of 16S rRNA genes. The results showed that nitrate significantly impacted bacterial compositions, interactions, and functions in different habitats. Bacterial communities clustered according to their habitats with distinguished indicator groups identified for each individual habitat. Nitrate shaped the overall bacterial communities across two habitats with a contribution of 27.2%, whereas the pH and TOC, respectively, structured bacterial communities in weathered rocks and sediments. Alpha and beta diversities of bacterial communities increased with nitrate concentration in both habitats, with nitrate directly affecting alpha diversity in sediments, but indirectly on weathered rocks by lowering pH. Nitrate impacted more on bacterial communities in weathered rocks at the genus level than in sediments because more genera significantly correlated with nitrate concentration in weathered rocks. Diverse keystone taxa involved in nitrogen cycling were identified in the co-occurrence networks such as nitrate reducers, ammonium-oxidizers, and N2-fixers. Tax4Fun2 analysis further confirmed the dominance of genes involved in nitrogen cycling. Genes of methane metabolism and carbon fixation were also dominant. The dominance of dissimilatory and assimilatory nitrate reduction in nitrogen cycling substantiated nitrate impact on bacterial functions. Our results for the first time revealed the impact of nitrate on subsurface karst ecosystem in terms of bacterial compositions, interactions, and functions, providing an important reference for further deciphering the disturbance of human activities on the subsurface biosphere.

Elevated antimony concentration stimulates rare taxa of potential autotrophic bacteria in the Xikuangshan groundwater.

Microbial communities composed of few abundant and many rare species are widely involved in the biogeochemical cycles of elements. Yet little is known about the ecological roles of rare taxa in antimony (Sb) contaminated groundwater. Groundwater samples were collected along an Sb concentration gradient in the Xikuangshan antimony mine area and subjected to high through-put sequencing of 16S rRNA genes to investigate the bacterial communities. Results suggested that both abundant and rare sub-communities were dominated by Betaproteobacteria, Gammaproteobacteria, and Alphaproteobacteria, whereas rare sub-communities showed higher alpha-diversities. Multivariate analysis showed that both the abundant and rare taxa were under the stress of Sb, but the impact on rare taxa was greater. Nitrate explained a large part for the variation of the abundant sub-communities, indicating the critical role of nitrate for their activities under anoxic conditions. In contrast, bicarbonate significantly impacted rare sub-communities, suggesting their potential autotrophic characteristics. To further explore the role of rare taxa in the communities and the mechanism of affecting the community composition, a network was constructed to display the co-occurrence pattern of bacterial communities. The rare taxa contributed most of the network nodes and served as keystone species to maintain the stability of community. Abiotic factors (mainly Sb and pH) and bacterial interspecific interactions (interactions between keystone species and other bacterial groups) jointly affect the community dynamics. Functional prediction was performed to further reveal the ecological function of rare taxa in the Sb-disturbed groundwater environment. The results indicated that the rare taxa harbored much more diverse functions than their abundant counterparts. Notably, elevated Sb concentration promoted some potential autotrophic functions in rare taxa such as the oxidation of S-, N-, and Fe(II)-compounds. These results offer new insights into the roles of rare species in elemental cycles in the Sb-impacted groundwater.

Transcriptome analysis of an arsenite-/antimonite-oxidizer, Bosea sp. AS-1 reveals the importance of the type 4 secretion system in antimony resistance.

Bosea sp. AS-1 is an arsenite [As(III)] and antimonite [Sb(III)] oxidizer previously isolated by our group from the Xikuangshan Antimony (Sb) Mine area. Our previous study showed that Bosea sp. AS-1 had a preference for oxidizing As(III) or Sb(III) with different carbon sources, which suggested that different metabolic mechanisms may be utilized by the bacteria to survive in As(III)- or Sb(III)-contaminated environments. Here, we conducted whole-genome and transcriptome sequencing to reveal the molecular mechanisms utilized by Bosea sp. AS-1 to resist As(III) or Sb(III). We discovered that AS-1 acquired various As- and Sb-resistant genes in its genome and might resist As(III) or Sb(III) through the regulation of multiple pathways, such as As and Sb metabolism, the bacterial secretion system, oxidative phosphorylation, the TCA cycle and bacterial flagellar motility. Interestingly, we discovered that genes of the type IV secretion system (T4SS) were activated in response to Sb(III), and inhibiting T4SS activity in AS-1 dramatically reduced its oxidation efficiency and tolerance to Sb(III). To our knowledge, this is the first study showing the activation of T4SS genes by Sb and a direct involvement of T4SS in bacterial Sb resistance. Our findings establish the T4SS as an important Sb resistance factor in bacteria and may help us understand the spread of Sb resistance genes in the environment.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Antimony transformation and mobilization from stibnite by an antimonite oxidizing bacterium Bosea sp. AS-1.

Soils and waters are heavily contaminated by antimony in Xikuangshan (XKS) mine area. It is widely accepted that oxidative dissolution of sulfide minerals and aqueous dissolution are the most prevalent geochemical mechanisms for the release of Sb to the environment. Bosea sp. AS-1 is an antimonite-oxidizer isolated from the mine slag in Xikuangshan Sb mine. Whole genome sequencing revealed the presence of multiple sulfur-oxidizing genes, antimony (Sb) metabolism genes and carbon fixation genes in AS-1's genome. We therefore hypothesized that under oxic conditions, AS-1 could mediate the oxidation of sulfide and Sb(III) in stibnite (Sb2S3) and lead to the release of Sb. Indeed, strain AS-1 was discovered as an autotrophic Sb(III)-oxidizer. Antimony mobilization studies conducted with strain AS-1 showed significantly enhanced mobilization of Sb, and complete oxidation of released Sb and sulfur to Sb(V) and sulfate. In addition, AS-1 induced a faster release of Sb under heterotrophic condition, and new acicular minerals might form. These findings support the hypothesis that microorganisms play an important role in the mobilization and transformation of Sb in XKS mine area and may contribute to our further understanding of the Sb biogeochemical redox cycle in natural environment.

Microbial Interactions Drive Distinct Taxonomic and Potential Metabolic Responses to Habitats in Karst Cave Ecosystem.

The geological role of microorganisms has been widely studied in the karst cave ecosystem. However, microbial interactions and ecological functions in such a dark, humid, and oligotrophic habitat have received far less attention, which is crucial to understanding cave biogeochemistry. Herein, microorganisms from weathered rock and sediment along the Heshang Cave depth were analyzed by random matrix theory-based network and Tax4Fun functional prediction. The results showed that although the cave microbial communities have spatial heterogeneity, differential habitats drove the community structure and diversity. Actinobacteria were predominant in weathered rock, whereas Proteobacteria dominated the sediment. The sediment communities presented significantly higher alpha diversities due to the relatively abundant nutrition from the outside by the intermittent stream. Consistently, microbial interactions in sediment were more complex, as visualized by more nodes and links. The abundant taxa presented more positive correlations with other community members in both of the two networks, indicating that they relied on promotion effects to adapt to the extreme environment. The keystones in weathered rock were mainly involved in the biodegradation of organic compounds, whereas the keystone Nitrospira in sediment contributed to carbon/nitrogen fixation. Collectively, these findings suggest that microbial interactions may lead to distinct taxonomic and functional communities in weathered rock and sediment in the subsurface Heshang Cave. IMPORTANCE In general, the constant physicochemical conditions and limited nutrient sources over long periods in the subsurface support a stable ecosystem in karst cave. Previous studies on cave microbial ecology were mostly focused on community composition, diversity, and the relationship with local environmental factors. There are still many unknowns about the microbial interactions and functions in such a dark environment with little human interference. Two representative habitats, including weathered rock and sediment in Heshang Cave, were selected to give an integrated insight into microbial interactions and potential functions. The cooccurrence network, especially the subnetwork, was used to characterize the cave microbial interactions in detail. We demonstrated that abundant taxa primarily relied on promotion effects rather than inhibition effects to survive in Heshang Cave. Keystone species may play important metabolic roles in sustaining ecological functions. Our study provides improved understanding of microbial interaction patterns and community ecological functions in the karst cave ecosystem.

USCγ Dominated Community Composition and Cooccurrence Network of Methanotrophs and Bacteria in Subterranean Karst Caves.

Karst caves have recently been demonstrated to act as a sink for atmospheric methane, due in part to consumption by microbes residing in caves that can oxidize methane at atmospheric levels. However, our knowledge about the responsible atmospheric methane-oxidizing bacteria (atmMOB) in this vast habitat remains limited to date. To address this issue, weathered rock samples from three karst caves were collected in Guilin City and subjected to high-throughput sequencing of pmoA and 16S rRNA genes. The results showed that members of the high-affinity upland soil cluster (USC), especially upland soil cluster gamma (USCγ), with absolute abundances of 104 to 109 copies · g-1 dry sample, dominated the atmMOB communities, while Proteobacteria and Actinobacteria dominated the overall bacterial communities. Moreover, USCγ was a keystone taxon in cooccurrence networks of both the atmMOB and the total bacterial community, whereas keystone taxa in the bacterial network also included Gaiella and Aciditerrimonas. Positive links overwhelmingly dominated the cooccurrence networks of both atmMOB and the total bacterial community, indicating a consistent response to environmental disturbances. Our study shed new insights on the diversity and abundances underlining atmMOB and total bacterial communities and on microbial interactions in subterranean karst caves, which increased our understanding about USC and supported karst caves as a methane sink. IMPORTANCE Karst caves have recently been demonstrated to be a potential atmospheric methane sink, presumably due to consumption by methane-oxidizing bacteria. However, the sparse knowledge about the diversity, distribution, and community interactions of methanotrophs requires us to seek further understanding of the ecological significance of methane oxidation in these ecosystems. Our pmoA high-throughput results from weathered rock samples from three karst caves in Guilin City confirm the wide occurrence of atmospheric methane-oxidizing bacteria in this habitat, especially those affiliated with the upland soil cluster, with a gene copy number of 104 to 109 copies per gram dry sample. Methanotrophs and the total bacterial communities had more positive than negative interactions with each other as indicated by the cooccurrence network, suggesting their consistent response to environmental disturbance. Our results solidly support caves as an atmospheric methane sink, and they contribute to a comprehensive understanding of the diversity, distribution, and interactions of microbial communities in subsurface karst caves.

Anti-atopic effect of Viola yedoensis ethanol extract against 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Viola yedoensis Makiho (VY, Violaceae) is a well-known medicinal herb in Chinese medicine, which is traditionally used to treat inflammation-related disorders, including allergic skin reactions. Although studies have uncovered its anti-inflammatory effects and corresponding bioactive constituents, the exact mechanism of action is still unclear in treating allergic skin reactions. OBJECTIVE: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe pruritus, dry, edema and inflamed skin. It affects people's quality of life seriously and causes huge economic losses to society. This study proposes VY as a possible remedy for atopic dermatitis since its traditional usage and superior anti-inflammatory effects. MATERIALS AND METHODS: Atopic dermatitis-like skin lesion was induced by topical application of 2,4-dinitrochlorobenzene (DNCB) in ICR mice. After treatment with Viola yedoensis Makiho ethanol extract (VYE) or dexamethasone (positive control) for 3 weeks, skin pathological observation and the molecular biological index were performed for therapeutic evaluation, including visual inspection in the change of the stimulated skin, scar formation, pathological morphology by hematoxylin and eosin (HE) staining, the measurement of interleukin IL-1ß, IL-6 and tumor necrosis factor-alpha (TNF-α) levels in serum as well as spleen index. The expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by western blot. The ratio of CD4+/CD8+ T lymphocyte in the spleen was detected by flow cytometry. Meanwhile, immunohistochemistry staining for CD68 identified the number of activated macrophages in skin lesions. Additionally, a reliable ultrahigh-performance liquid chromatography coupled with a Q exactive hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) method was established for the systematic identification and characterization of main components in VYE. RESULTS: VYE alleviated DNCB-stimulated AD-like lesions symptoms as evidenced by a significant decrease in hypertrophy, hyperkeratosis, and infiltration of inflammatory cells in dorsal skin. The levels of IL-1ß, IL-6, and TNF-α in serum were suppressed in mice treated with VYE as compared to the DNCB-induced model group. Also, the administration of VYE reduced the ratio of CD4+/CD8+ T lymphocyte in the spleen and the number of activated macrophages stimulated by DNCB. Besides, the expression of iNOS and COX-2 were down-regulated in the dorsal skin. CONCLUSIONS: VYE showed therapeutic effects on atopic dermatitis in DNCB-induced AD-like lesion mouse models by inhibiting the T cell-mediated allergic immune response. Our results indicated that VY could act as a potential remedy for atopic dermatitis.

Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli.

BACKGROUND: Methanol has attracted increased attention as a non-food alternative carbon source to sugar for biological production of chemicals and fuels. Moreover, the high degree of reduction of methanol offers some advantages in increasing the production yields of NAD(P)H-dependent metabolites. Here, we demonstrate an example of methanol bioconversion with the aim of improving production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli. RESULTS: A synthetic methylotrophic E. coli was engineered with a nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (MDH) and ribulose monophosphate (RuMP) pathway. Regarding the limited MDH activity, the role of activator proteins in vivo was investigated, and the NudF protein was identified capable of improving MDH activity and triggering increased methanol metabolism. Using 13C-methanol-labeling experiments, we confirmed methanol assimilation in the methylotrophic E. coli. A cycling RuMP pathway for methanol assimilation was also demonstrated by detecting multiple labeled carbons for several compounds. Finally, using the NAD(P)H-dependent metabolite lysine as a test, the potential of methanol bioconversion to generate value-added metabolites was determined. To further characterize the benefit of methanol as the carbon source, extra NADH from methanol oxidation was engineered to generate NADPH to improve lysine biosynthesis by expression of the POS5 gene from Saccharomyces cerevisiae, which resulted in a twofold improvement of lysine production. Moreover, this new sink further pulled upstream methanol utilization. CONCLUSION: Through engineering methanol metabolism, lysine biosynthesis, and NADPH regeneration pathway from NADH, the bioconversion of methanol to improve chemical synthesis was successfully achieved in methylotrophic E. coli.

Urinary proteomics analysis based on mass spectrometry and identification of therapeutic targets of Shenkangling interventions in rats with adriamycin nephropathy using iTRAQ.

OBJECTIVE: The isobaric tags for relative and absolute quantification (iTRAQ) technique for proteomic analysis was employed to identify diagnostic markers and therapeutic targets of Shenkangling intervention or prednisone tablets in rats with adriamycin nephropathy (AN). METHODS: Fifty healthy, clean-grade Sprague-Dawley rats were selected, with 10 rats in the normal group and the remaining 40 rats receiving a tail vein injection of 5.5 mg/kg of adriamycin (ADR) to induce AN. Treatment began 1 week later. The normal group received gastric administration of normal saline. Forty rats with induced AN were further randomly divided into the AN modeling group (n = 10), AN modeling + prednisone treatment group (n = 10), AN modeling + Shenkangling intervention group (n = 10), and AN modeling + prednisone + Shenkangling intervention group (n = 10). iTRAQ was employed in combination with mass spectrometry to analyze the differentially expressed proteins in the urine after 3 weeks of treatment (in the fourth week of the experiment). RESULTS: Compared with normal rats, AN rats had 6 down-regulated proteins and 1 upregulated protein. Compared with AN rats, prednisone rats had 2 down-regulated and 6 upregulated proteins. Compared with AN rats, combined treatment rats had 2 down-regulated and 8 upregulated proteins. Compared with the AN model group, the Shenkangling treatment group had 3 down-regulated and 9 upregulated proteins. Gro, Afamin, Cystatin-related protein 2, Afamin, and isoform CRA_a were considered diagnostic markers of primary nephrotic syndrome (PNS). Telomerase was considered the therapeutic target of prednisone. Urinary protein 2, Apolipoprotein A-II, 45 kDa calcium-binding protein, Vitronectin, and Osteopontin were the therapeutic targets of the Shenkangling intervention. Afamin, isoform CRA_a, Apolipoprotein A-IV, Coagulation factor XII, Prolactin-induced protein, and Coagulation factor XII were the therapeutic targets of the Shenkangling intervention combined with prednisone. CONCLUSION: The feasibility of urinary proteomics analysis in rats using a large number of proteins with finite molecular weights is controversial. The markers screened in this study may be of clinical value for the diagnosis and treatment of nephropathy. However, these findings should be confirmed in future cohort studies.

Characterization of the antimonite- and arsenite-oxidizing bacterium Bosea sp. AS-1 and its potential application in arsenic removal.

Arsenic (As) and antinomy (Sb) usually coexist in natural environments where both of them pollute soils and water. Microorganisms that oxidize arsenite [As(III)] and tolerate Sb have great potential in As and Sb bioremediation, In this study, a Gram-negative bacterial strain, Bosea sp. AS-1, was isolated from a mine slag sample collected in Xikuangshan Sb mine in China. AS-1 could tolerate 120 mM of As(III) and 50 mM of antimonite [Sb(III)]. It could also oxidize 2 mM of As(III) or Sb(III) completely under heterotrophic and aerobic conditions. Interestingly, strain AS-1 preferred to oxidize As(III) with yeast extract as the carbon source, whereas Sb(III) oxidation was favored with lactate in the medium. Genomic analysis of AS-1 confirmed the presence of several gene islands for As resistance and oxidation. Notably, a system of AS-1 and goethite was found to be able to remove 99% of the As with the initial concentration of 500 µg/L As(III) and 500 µg/L Sb(III), which suggests the potential of this approach for As removal in environments especially with the presence of high Sb.

A Novel Process for Cadaverine Bio-Production Using a Consortium of Two Engineered Escherichia coli.

Bio-production of cadaverine from cheap carbon sources for synthesizing bio-based polyamides is becoming more common. Here, a novel fermentation process for cadaverine bio-production from glucose was implemented by using a microbial consortium of two engineered Escherichia coli strains to relieve the toxic effect of cadaverine on fermentation efficiency. To achieve controllable growth of strains in the microbial consortium, two engineered E. coli strains grown separately on different carbon sources were first constructed. The strains were, an L-lysine-producing E. coli NT1004 with glucose as carbon source, and a cadaverine-producing E. coli CAD03 with glucose metabolism deficiency generated by modifying the PTSGlc system with CRISPR-Cas9 technology and inactivating cadaverine degradation pathways. Co-culturing these two engineered E. coli strains with a mixture of glucose and glycerol led to successful production of cadaverine. After optimizing cultivation conditions, a cadaverine titer of 28.5 g/L was achieved with a multi-stage constant-speed feeding strategy.