Type I Cystatin Derived from Cysticercus pisiformis-Stefins, Suppresses LPS-Mediated Inflammatory Response in RAW264.7 Cells.

Cysticercus pisiformis is a kind of tapeworm larvae of Taenia pisiformis, which parasitizes the liver envelope, omentum, mesentery, and rectum of rodents such as rabbits. Cysteine protease inhibitors derived from helminth were immunoregulatory molecules of intermediate hosts and had an immunomodulatory function that regulates the production of inflammatory factors. Thus, in the present research, the recombinant Stefin of C. pisiformis was confirmed to have the potential to fight inflammation in LPS-Mediated RAW264.7 murine macrophages. CCK8 test showed that rCpStefin below 50 µg/mL concentration did not affect cellular viability. Moreover, the NO production level determined by the Griess test was decreased. In addition, the secretion levels of IL-1ß, IL-6, and TNF-α as measured by ELISA were decreased. Furthermore, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and proinflammatory mediators, including IL-1ß, IL-6, TNF-α, iNOS, and COX-2 at the gene transcription level, as measured by qRT-PCR. Therefore, Type I cystatin derived from C. pisiformis suppresses the LPS-Mediated inflammatory response of the intermediate host and is a potential candidate for the treatment of inflammatory diseases.

A retrospective study of an irradiation-based conditioning regimen and chidamide maintenance therapy in T-ALL/LBL.

OBJECTIVES: T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL) are highly malignant and aggressive hematologic tumors for which there is no standard first-line treatment. Chidamide, a novel histone deacetylase inhibitor, shows great promise. We assessed the efficacy and safety of an irradiation-containing conditioning regimen for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and post-transplantation chidamide maintenance in patients with T-ALL/LBL. METHODS: We retrospectively analyzed the clinical data of six patients with T-ALL/LBL who underwent allo-HSCT with a radiotherapy-containing pretreatment regimen and post-transplant chidamide maintenance therapy. The endpoints were relapse, graft-versus-host disease (GVHD), transplant-related mortality (TRM), progression-free survival (PFS), overall survival (OS), and adverse events (AEs). RESULTS: All of the patients had uneventful post-transplant hematopoietic reconstitution, and all achieved complete molecular remission within 30 days. All six patients survived, and two relapsed with a median relapse time of 828.5 (170-1335) days. The 1-year OS rate was 100%, the 2-year PFS rate was 66.7%, and the TRM rate was 0%. After transplantation, two patients developed grade I-II acute GVHD (2/6); grade III-IV acute and chronic GVHD were not observed. The most common AEs following chidamide administration were hematological AEs, which occurred to varying degrees in all patients; liver function abnormalities occurred in two patients (grade 2), and symptoms of malaise occurred in one patient (grade 1). CONCLUSION: Chidamide maintenance therapy after T-ALL/LBL transplantation is safe, but the efficacy needs to be further investigated.

Photochemical fate of ß-blocker pindolol in riverine and its downstream coastal waters.

Pindolol (PIN) is a commonly used ß-blocker drug and has been frequently detected in various natural waters. Comprehensive understanding of its environmental photochemical transformation is necessary to assess its environmental risk. In this study, the photodegradation kinetics and mechanisms of PIN in both freshwater and coastal water were investigated for the first time. The photodegradation experiments were carried out by steady-state photochemical experiment under simulated sunlight irradiation. The results showed that the photodegradation rate of PIN in the freshwater of the Pearl River estuary was significantly faster than that in its downstream coastal water. In river water, PIN can undergo both direct photolysis and indirect photolysis induced by riverine dissolved organic matter (DOM) mainly through excited triplet-state of DOM and singlet oxygen, while direct photolysis dominated its degradation in coastal water. The promotion effect was found to be much greater for Suwannee River Natural Organic Matter (SRNOM) than that of the sampled riverine DOM, due to its high steady-state concentrations of reactive species. Interestingly, coastal DOM in northern and southern China were found to have similar promotion effects on PIN photodegradation for the first time, but both less than that of riverine DOM. A total of seven degradation products of PIN resulting from hydroxylation, hydrogen abstraction and cleavage of ether bond were identified. Biological toxicity of one products were found to be higher than that of PIN. These results are of significance for knowing the persistence and ecological risk of PIN in natural waters.

Bone Morphogenetic Protein 2 Enhances Porcine Beige Adipogenesis via AKT/mTOR and MAPK Signaling Pathways.

Bone morphogenetic protein 2 (BMP2) has been reported to regulate adipogenesis, but its role in porcine beige adipocyte formation remains unclear. Our data reveal that BMP2 is significantly induced at the early stages of porcine beige adipocyte differentiation. Additionally, supplementing rhBMP2 during the early stages, but not the late stages of differentiation, significantly enhances porcine SVF adipogenesis, thermogenesis, and proliferation. Furthermore, compared to the empty plasmid-transfected-SVFs, BMP2-overexpressed SVFs had the enhanced lipid accumulation and thermogenesis, while knockdown of BMP2 in SVFs exhibited the opposite effect. The RNA-seq of the above three types of cells revealed the enrichment of the annotation of thermogenesis, brown cell differentiation, etc. In addition, the analysis also highlights the significant enrichment of cell adhesion, the MAPK cascade, and PPARγ signaling. Mechanistically, BMP2 positively regulates the adipogenic and thermogenic capacities of porcine beige adipocytes by activating PPARγ expression through AKT/mTOR and MAPK signaling pathways.

Remodeling the polymer-binding cavity to improve the efficacy of PBAT-degrading enzyme.

Poly(butylene adipate-co-terephthalate) (PBAT) is among the most widely applied synthetic polyesters that are utilized in the packaging and agricultural industries, but the accumulation of PBAT wastes has posed a great burden to ecosystems. Using renewable enzymes to decompose PBAT is an eco-friendly solution to tackle this problem. Recently, we demonstrated that cutinase is the most effective PBAT-degrading enzyme and that an engineered cutinase termed TfCut-DM could completely decompose PBAT film to terephthalate (TPA). Here, we report crystal structures of a variant of leaf compost cutinase in complex with soluble fragments of PBAT, including BTa and TaBTa. In the TaBTa complex, one TPA moiety was located at a polymer-binding site distal to the catalytic center that has never been experimentally validated. Intriguingly, the composition of the distal TPA-binding site shows higher diversity relative to the one proximal to the catalytic center in various cutinases. We thus modified the distal TPA-binding site of TfCut-DM and obtained variants that exhibit higher activity. Notably, the time needed to completely degrade the PBAT film to TPA was shortened to within 24 h by TfCut-DM Q132Y (5813 mol per mol protein). Taken together, the structural information regarding the substrate-binding behavior of PBAT-degrading enzymes could be useful guidance for direct enzyme engineering.

Insights into the mechanisms of natural organic matter on the photodegradation of indomethacin under natural sunlight and simulated light irradiation.

Indomethacin (INDO) is an antipyretic and analgesic pharmaceutical that has been widely detected in the aquatic environment. Photodegradation is an essential pathway for removal of INDO in sunlit surface water, however the effect of dissolved organic matter (DOM) on its photodegradation and the ecotoxicity of photodegradation products are largely unknown. In this study, the effect of DOM on the photodegradation of INDO under both natural and simulated light irradiation was studied. The results showed that indirect photolysis is the main photodegradation pathway of INDO in presence of DOM where 3DOM* plays the most important promoting role. Compared to commercial DOM (SRNOM and SRFA), DOM extracted from local-lake water (SLDOM) promoted the photodegradation to the highest extent. Although the steady-state concentrations of 3DOM* of SRNOM and SRFA were higher than SLDOM, their inhibition effect surpassed SLDOM namely higher light screening effect and phenolic antioxidant concentrations. The photodegradation pathway in pure water is different from that in DOM system where the decarboxylation of acetic acid chain and the oxidative fracture of indole ring are the main degradation pathways. Density Functional Theory (DFT) calculation further supports the proposed degradation pathways of INDO. ECOSAR calculation showed that the toxicity of INDO photodegradation products to aquatic organisms may maintain or even exceed its parent compound. Therefore, comprehensive understanding of the impact of DOM on the photodegradation of INDO is of crucial significance for evaluating its ecological risk in the natural environment.

Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells.

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.

Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase.

Diterpene synthase VenA is responsible for assembling venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. VenA also demonstrates substrate promiscuity by accepting geranyl pyrophosphate and farnesyl pyrophosphate as alternative substrates. Herein, we report the crystal structures of VenA in both apo form and holo form in complex with a trinuclear magnesium cluster and pyrophosphate group. Functional and structural investigations on the atypical 115DSFVSD120 motif of VenA, versus the canonical Asp-rich motif of DDXX(X)D/E, reveal that the absent second Asp of canonical motif is functionally replaced by Ser116 and Gln83, together with bioinformatics analysis identifying a hidden subclass of type I microbial terpene synthases. Further structural analysis, multiscale computational simulations, and structure-directed mutagenesis provide significant mechanistic insights into the substrate selectivity and catalytic promiscuity of VenA. Finally, VenA is semi-rationally engineered into a sesterterpene synthase to recognize the larger substrate geranylfarnesyl pyrophosphate.

Occurrence and removal rate of typical pharmaceuticals and personal care products (PPCPs) in an urban wastewater treatment plant in Beijing, China.

The occurrence and removal rate of 52 typical pharmaceuticals and personal care products (PPCPs) were investigated in a wastewater treatment plant in Beijing, China. Thirty-three PPCPs were found in the influent, with caffeine (CF, 11387.0 ng L-1) being the most abundant, followed by N,N-diethyl-meta-toluamide (DEET, 9568.4 ng L-1), metoprolol (MTP, 930.2 ng L-1), and diclofenac (DF, 710.3 ng L-1). After treatment processes, the cumulative concentration of PPCPs decreased from 2.54 × 104 ng L-1 to 1.44 × 103 ng L-1, with the overall removal efficiency (RE) of 94.3%. Different treatment processes showed varying contributions in removing PPCPs. PPCPs were efficiently removed in sedimentation, anoxic, and ultraviolet units. For individual compounds, a great variation in RE (52.1-100%) was observed. Twenty-two PPCPs were removed by more than 90%. The highly detected PPCPs in the influent were almost completely removed. Aerated grit chamber removed nearly 50% of fluoroquinolone (FQs) and more than 60% of sulfonamides. Most PPCPs showed low or negative removals during anaerobic treatment, except for CF which was eliminated by 64.9%. Anoxic treatment demonstrated positive removals for most PPCPs, with the exceptions of DF, MTP, bisoprolol, carbamazepine (CBZ), and sibutramine. DEET and bezafibrate were efficiently removed during the secondary sedimentation. Denitrification biological filter and membrane filtration also showed positive effect on most PPCPs removals. The remaining compounds were oxidized by 16-100% in ozonation. DF, sulpiride, ofloxacin (OFL), trimethoprim, and phenolphthalein were not amenable to ultraviolet. After the treatment, the residue OFL, CBZ, and CF in receiving water were identified to pose high risk to aquatic organisms. Considering the complex mixtures emitted into the environment, therapeutic groups psychotropics, stimulant, and FQs were classified as high risk. These findings provide valuable insights into adopting appropriate measures for more efficient PPCPs removals, and emphasize the importance of continued monitoring specific PPCPs and mixtures thereof to safeguard the ecosystem.

An integrated approach to identify the source apportionment of potentially toxic metals in shale gas exploitation area soil, and the associated ecological and human health risks.

Public awareness of the potential environmental risks of shale gas extraction has increased in recent years. However, the status and environmental risks of potentially toxic metals (PTMs) in shale gas field soil remain unclear. A total of 96 topsoil samples were collected from the first shale gas exploitation area in China. The sources of nine PTMs in the soils were identified using positive matrix factorization and correlation analysis, and the ecological and human health risks of toxic metals from different sources under the two land use types were calculated. The results showed that mean pollution load index (PLI) values for farmland (1.18) and woodland (1.40) indicated moderate pollution, As, Cd and Ni were the most serious contaminants among all nine PTMs. The following four sources were identified: shale gas extraction activities (43.90%), nature sources (31.90%), agricultural and traffic activities (17.55%) and industrial activities (6.55%). For ecological risk, the mean ecological risk index (RI) values for farmlands (161.95) and woodlands (185.27) reaching considerable risk. The contribution ratio of shale gas extraction activities for farmlands and woodlands were 5.70% and 8.90%, respectively. Regarding human health risk, noncarcinogenic risks for adults in farmlands and woodlands were negligible. Industrial activities, agricultural and traffic activities were estimated to be the important sources of health risks. Overall, shale gas extraction activities had little impact on the ecological and human health risk. This study provides scientific evidence regarding the soil contamination potential of shale gas development activities.

PAHs in the monsoonal open ocean: Homogeneous spatial pattern and wind-driven significant seasonal variations.

PAHs enter the ocean via surface runoff and atmospheric transport pathways, and the distribution of PAHs is highly variable in coastal seas due to the influence of direct human activities, inputs of surface runoff, and strong biological activities. However, highly temporal variability of PAHs has also been widely observed in the open oligotrophic tropical and subtropical oceans without the influence of three types of factors mentioned above. This study developed a method to quantify the variability of oceanic PAHs based on wind frequency and wind-speed-weighted wind frequency using in-situ survey data from three cruises in the Philippine Sea, and tested the validity of this method using publicly available data from other monsoonal open oceans. The result showed that the wind frequency could better explain the variation of dissolved PAHs and particulate PAHs in the surface ocean, while the wind-speed-weighted wind frequency could better explain the variation of particulate PAHs. This study suggests that the influence of seasonal atmospheric transport cannot be ignored when describing and interpreting the distribution patterns of PAHs in the monsoon-influenced low and mid-latitude open oceans and also provides a reference method for a better understanding of the global-scale distribution patterns of PAHs in the ocean.

[Migration, Transformation, and Toxicity of Quaternary Ammonium Antimicrobial Agents in the Environment].

Quaternary ammonium compounds (QACs) are one type of widely used cationic biocide, and their usage amount is growing rapidly due to the flu and COVID-19 pandemic. Many QACs were released into the environment in or after the course of their use, and thus they were widely detected in water, sediment, soil, and other environmental media. QACs have stronger surface activity and non-specific biotoxicity, which poses a potential threat to the ecosystem. In this study, the environmental fate and potential toxicity of QACs were documented in terms of their migration and transformation process, biological toxicity effects, and the main mechanisms of bacterial resistance to QACs. Aerobic biodegradation was the main natural way of eliminating QACs in the environment, and the reaction was mainly initiated by the hydroxylation of C atoms at different positions of QACs and finally mineralized to CO2and H2O through decarboxylation, demethylation, and ß-oxidation reaction. Toxicological studies showed that QACs at environmental concentrations could not pose acute toxicity to the selected biotas but threatened the growth and reproduction of aquatic organisms like Daphnia magna. Their toxicity effects depended on their molecular structure, the tested species, and the exposed durations. Additionally, our team first investigated the toxicity effects and mechanisms of QACs toward Microcystis aeruginosa, which showed that QACs depressed the algae growth through the denaturation of photosynthetic organelles, suppression of electron transport, and then induction of cell membrane damage. In the environment, the concentrations of QACs were always lower than their bactericidal concentrations, and their degradation could induce the formation of a concentration gradient, which facilitated microbes resistant to QACs. The known resistance mechanisms of bacteria to QACs mainly included the change in cell membrane structure and composition, formation of biofilm, overexpression of the efflux pump gene, and acquisition of resistance genes. Due to the similar targets and mechanisms, QACs could also induce the occurrence of antibiotic resistance, mainly through co-resistance and cross-resistance. Based on the existing data, future research should emphasize the toxicity effect and the potential QACs resistance mechanism of microorganisms in real environmental conditions.

Fecal Microbiota Was Reshaped in UCP1 Knock-In Pigs via the Adipose-Liver-Gut Axis and Contributed to Less Fat Deposition.

The relationship between the host gut microbiota and obesity has been well documented in humans and mice; however, few studies reported the association between the gut microbiota and fat deposition in pigs. In a previous study, we generated uncoupling protein 1 (UCP1) knock-in pigs (UCP1 pigs), which exhibited a lower fat deposition phenotype. Whether the gut microbiota was reshaped in these pigs and whether the reshaped gut microbiota contributes to the lower fat content remain unknown. Here, we revealed that the fecal microbiota composition and metabolites were significantly altered under both chow diet (CD) and high-fat/high-cholesterol (HFHC) diet conditions in UCP1 pigs compared to those in wild-type (WT) pigs. The abundance of Oscillospira and Coprococcus and the level of metabolite hyodeoxycholic acid (HDCA) from feces were observed to be significantly increased in UCP1 pigs. An association analysis revealed that Oscillospira and Coprococcus were significantly negatively related to backfat thickness. In addition, after fecal microbiota transplantation (FMT), the mice that were orally gavaged with feces from UCP1 pigs exhibited less fat deposition under both CD and high-fat diet (HFD) conditions, suggesting that the fecal microbes of UCP1 pigs participate in regulating host lipid metabolism. Consistently, HDCA-treated mice also exhibited reduced fat content. Mechanistically, we found that UCP1 expression in white adipose tissue alters the gut microbiota via the adipose-liver-gut axis in pigs. Our study provides new data concerning the cross talk between host genetic variations and the gut microbiota and paves the way for the potential application of microbes or their metabolites in the regulation of fat deposition in pigs. IMPORTANCE This article investigated the effect of the ectopic expression of UCP1 on the regulation of fecal microbiota composition and metabolites and which alters the fat deposition phenotype. Bacteria, including Oscillospira and Coprococcus, and the metabolite HDCA were found to be significantly increased in feces of UCP1 pigs and had a negative relationship with backfat thickness. Mice with fecal microbiota transplantation phenocopied the UCP1 pigs under both CD and HFD conditions, suggesting that the fecal microbes of UCP1 pigs participate in regulating host lipid metabolism. Our study provides new data regarding the cross talk between host genetic variations and the gut microbiota and paves the way for the potential application of microbes or their metabolic production in the regulation of fat deposition in pigs.

Pronounced temporal changes in soil microbial community and nitrogen transformation caused by benzalkonium chloride.

As one typical cationic disinfectant, quaternary ammonium compounds (QACs) were approved for surface disinfection in the coronavirus disease 2019 pandemic and then unintentionally or intentionally released into the surrounding environment. Concerningly, it is still unclear how the soil microbial community succession happens and the nitrogen (N) cycling processes alter when exposed to QACs. In this study, one common QAC (benzalkonium chloride (BAC) was selected as the target contaminant, and its effects on the temporal changes in soil microbial community structure and nitrogen transformation processes were determined by qPCR and 16S rRNA sequencing-based methods. The results showed that the aerobic microbial degradation of BAC in the two different soils followed first-order kinetics with a half-life (4.92 vs. 17.33 days) highly dependent on the properties of the soil. BAC activated the abundance of N fixation gene (nifH) and nitrification genes (AOA and AOB) in the soil and inhibited that of denitrification gene (narG). BAC exposure resulted in the decrease of the alpha diversity of soil microbial community and the enrichment of Crenarchaeota and Proteobacteria. This study demonstrates that BAC degradation is accompanied by changes in soil microbial community structure and N transformation capacity.

Interlayer adsorption of cationic dye on cationic surfactant-modified and unmodified montmorillonite.

Water pollution by toxic organic dyes is one of the most critical health and environmental problems worldwide. By means of molecular dynamics method, the present work aims to evaluate the applicability of montmorillonite (Mt) modified by hexadecyltrimethylammonium cations (HDTMA+) compared to unmodified Na-Mt for the adsorption of cationic methylene blue (MB) dye. The results showed that the adsorption energy of MB on both HDTMA-Mt and Na-Mt absorbent ranged from - 100 to - 250 kJ/mol, indicating the effectiveness of two types of adsorbents in dye water treatment. The highest adsorption energy was found at w = 50% in each adsorbent system. Adsorption mechanisms of MB depend on molecular orientations, which is influenced by the surfactant and water content. The adsorption mechanism of MB is chemisorption dominated by strong electrostatic interaction between CH3 groups of MB and oxygen atoms of Mt surfaces. Besides, physisorption also plays a minor role in MB orientations. It is found that the existence of cationic surfactants can slightly improve the adsorption capacity of MB only at higher water content through enlarging the interlayer space of Mt and reducing mobility of MB. However, there will be a negative impact on the reduction of adsorption sites for dyes especially at low water content. Our results provide a possible application for swelling clay minerals being a promising adsorbent for dyes-surfactants co-existing wastewater treatment.

Considering zooplankton as a black box in determining PAH concentrations could result in misjudging their bioaccumulation.

Zooplankton play an important role in energy transfer in the marine food web and form the dietary basis for the size of important fish stocks and the maintenance of their resources. Although zooplankton include numerous taxa with significantly different ecological characteristics and the interspecific differences in optimum body size and taxonomic specificity in fish feeding on zooplankton are remarkable, they are always considered as a whole (like a "black box") in current studies about the transport of persistent organic pollutants through the food chain. This approach might result in misjudgment of their bioaccumulation. In this study, the distribution properties of each taxa of zooplankton community were discerned using data from two cruise surveys conducted in the northern South China Sea. Twelve groups of zooplankton were identified, all of which had distinct ecological and functional characteristics. The carbon-based community structure of zooplankton could explain their variability with respect to polycyclic aromatic hydrocarbons (PAHs). Smaller-sized zooplankton (smaller calanoids and cyclopoids) were more likely to accumulate low molecular weight PAHs (LMW-PAHs), while larger-sized zooplankton (larger calanoids) were more likely to accumulate high molecular weight PAHs (HMW-PAHs). The bioaccumulation capacity of the zooplankton community for LMW-PAHs was negatively correlated with the proportion of omnivores and carnivores, while the opposite was true for HMW-PAHs. These results suggested that the effects of complex community structure within plankton communities should be taken into account when assessing the transfer and bioaccumulation effects of PAHs in the marine food chain.

Functional tailoring of a PET hydrolytic enzyme expressed in Pichia pastoris.

Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future.

A Structural and Bioinformatics Investigation of a Fungal Squalene Synthase and Comparisons with Other Membrane Proteins.

There is interest in the development of drugs to treat fungal infections due to the increasing threat of drug resistance, and here, we report the first crystallographic structure of the catalytic domain of a fungal squalene synthase (SQS), Aspergillus flavus SQS (AfSQS), a potential drug target, together with a bioinformatics study of fungal, human, and protozoal SQSs. Our X-ray results show strong structural similarities between the catalytic domains in these proteins, but, remarkably, using bioinformatics, we find that there is also a large, highly polar helix in the fungal proteins that connects the catalytic and membrane-anchoring transmembrane domains. This polar helix is absent in squalene synthases from all other lifeforms. We show that the transmembrane domain in AfSQS and in other SQSs, stannin, and steryl sulfatase, have very similar properties (% polar residues, hydrophobicity, and hydrophobic moment) to those found in the "penultimate" C-terminal helical domain in squalene epoxidase, while the final C-terminal domain in squalene epoxidase is more polar and may be monotopic. We also propose structural models for full-length AfSQS based on the bioinformatics results as well as a deep learning program that indicate that the C-terminus region may also be membrane surface-associated. Taken together, our results are of general interest given the unique nature of the polar helical domain in fungi that may be involved in protein-protein interactions as well as being a future target for antifungals.

Structural insights to a bi-functional isoprenyl diphosphate synthase that can catalyze head-to-tail and head-to-middle condensation.

Isoprenoids represent the largest group of natural products, whose basal skeletons are synthesized by various isoprenyl diphosphate synthases (IDSs). As majority of IDSs catalyze head-to-tail reaction to produce linear form isoprenoids, some catalyze head-to-middle reaction to produce branched form products. In a previous study, an IDS termed MA1831 from Methanosarcina acetivorans was found to be capable of catalyzing both types of reaction. In addition to the canonical linear product of C35 in length, MA1831 also catalyzes head-to-middle condensation of farnesyl diphosphate (FPP) and dimethylallyl diphosphate (DMAPP) to produce geranyllavandulyl diphosphate. In order to investigate the mechanism of action of MA1831, we determined its crystal structures in apo-form and in complex with substrates and analogues. The complex structures that contain isopentenyl S-thiolodiphosphate and DMAPP as homoallylic substrates were also reported, which should represent the reaction modes of MA1831-mediated head-to-tail and head-to-middle reaction, respectively. Based on the structural information, the mechanism of MA1831 catalyze head-to-tail and head-to-middle condensation reaction was proposed.

Learning Chinese Classical Music with the Aid of Soundscape by Using Intelligent Network.

A soundscape is a sound environment of the awareness of auditory perception and social or cultural understanding. To improve the subjective initiative of the Chinese classical music students, this study explores new learning modes and methods using soundscape and investigates its learning effect using intelligent music software. To examine the emotional experience of players in playing before and after learning, 50 students from music majors and 50 from non-music majors were selected. Results show that in the positive and negative emotional classical music experiment, most music majors and non-music majors have weak emotional experience at the beginning, and only a few have a strong emotional experience, which could reach 6 points. In the second scoring, most majors have a score of about 7 points, indicating strong emotional experience and a few have a score of about 4 points and 9 points, representing that there were relatively few majors with weak emotional experience and strong emotional experience. The overall emotional experience score is low in the comparison between non-music majors and music majors, and the second score in the entire experiment is significantly higher than the first score, signifying that the learning effect of players is obvious, and intelligent music software and soundscape play a role in the exploration of Chinese classical music.