Intrahepatic cholangiocarcinoma with FGFR alterations: A series of Chinese cases with an emphasis on their clinicopathologic and genetic features.

Intrahepatic Cholangiocarcinoma (iCCA) with FGFR alterations is relatively rare, and its identification is important in the era of targeted therapy. We collected a large series of FGFR-altered cases in the Chinese population and characterized their clinicopathological and genetic features. Among the 18 FGFR-altered cases out of 260 iCCAs, 10 were males and 8 were females, ranging in age from 35 to 74 years (mean, 57.3 years; median, 58 years). Pathologically, they include 9 cases of large duct (LD, 50 %) and small duct (SD, 50 %) types each. All of them (100 %, 18/18) showed microsatellite stable (MSS) and low tumor mutation burden (TMB). Genetically, FGFR alterations involved FGFR1 (20 %), FGFR2 (70 %), and FGFR3 (10 %), with FGFR2 rearrangement accounting for the most (11/18). The most frequently altered genes/biological processes were development/proliferation-related pathways (44 %), chromatin organization (20 %), and tumor suppressors (32 %). Our study further revealed the clinicopathological and genetic features of FGFR-altered iCCA and demonstrated that its occurrence may show regional or ethnic variability and is less common in the Chinese population. A significant number of LD-type iCCA cases also have FGFR alterations rather than the SD type.

Enantiodivergent kinetic resolution of 4-substituted 1,2,3,4-tetrahydroquinolines employing amine oxidase.

Optically pure 1,2,3,4-tetrahydroquinolines (THQs) represent a class of important motifs in many natural products and pharmaceutical agents. While recent advances on redox biocatalysis have demonstrated the great potential of amine oxidases, all the transformations focused on 2-substituted THQs. The corresponding biocatalytic method for the preparation of chiral 4-substituted THQs is still challenging due to the poor activity and stereoselectivity of the available enzyme. Herein, we developed a biocatalytic kinetic resolution approach for enantiodivergent synthesis of 4-phenyl- or alkyl-substituted THQs. Through structure-guided protein engineering of cyclohexylamine oxidase derived from Brevibacterium oxidans IH-35 A (CHAO), the variant of CHAO (Y215H/Y214S) displayed improved specific activity toward model substrate 4-phenyl substituted THQ (0.14 U/mg, 13-fold higher than wild-type CHAO) with superior (R)-stereoselectivity (E > 200). Molecular dynamics simulations show that CHAO Y215H/Y214S allows a suitable substrate positioning in the expanded binding pocket to be facilely accessed, enabling enhanced activity and stereoselectivity. Furthermore, a series of 4-alkyl-substituted THQs can be transformed by CHAO Y215H/Y214S, affording R-isomers with good yields (up to 50 %) and excellent enantioselectivity (up to ee > 99 %). Interestingly, the monoamine oxidase from Pseudomonas fluorescens Pf0-1 (PfMAO1) with opposite enantioselectivity was also mined. Together, this system enriches the kinetic resolution methods for the synthesis of chiral THQs.

Interfacial Metal-Support Interaction and Catalytic Performance of Perovskite LaCrO3-Supported Ru Catalyst.

Interfacial metal-support interaction (MSI) significantly affects the dispersion of active metals on the surface of the catalyst support and impacts catalyst performance. Understanding MSI is crucial for developing highly active and stable catalysts with a low metal loading, particularly for noble metal catalysts. In this work, we synthesized LaRuxCr1-xO3 catalysts with low Ru loading (x = 0.005, 0.01, and 0.02) using the sol-gel self-combustion method. We found that all of the Ru atoms immediately above or below the metal-support interface are closely bonded to the perovskite LaCrO3 surface lattice through Ru-O bonds, enhancing the MSI via interfacial reaction and charge transfer mechanisms. We identified a variety of Ru species, including small 3D Ru nanoparticles, 2D dispersed Ru surface atoms, and even 0D Ru single atoms. These highly dispersed Ru species exhibit high activity and stability under dry reforming of methane (DRM) conditions. The LaRu0.01Cr0.99O3 catalyst with very low Ru loading (0.42 wt %) was stable over a 50 h DRM test and the carbon deposition was negligible. The CH4 and CO2 conversions at 750 °C reached 83 and 86%, respectively, approaching the theoretical thermodynamic equilibrium values.

Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering.

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. KEY POINTS: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified.  • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer.

BACKGROUND: The purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC. MATERIALS AND METHODS: Blood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay. RESULTS: Risk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1ß, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects. CONCLUSION: In conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Reconstructing dynamics correlation network to simultaneously improve activity and stability of 2,3-butanediol dehydrogenase by design of distal interchain disulfide bonds.

The complete enzyme catalytic cycle includes substrate binding, chemical reaction and product release, in which different dynamic conformations are adopted. Due to the complex relationship among enzyme activity, stability and dynamics, the directed evolution of enzymes for improved activity or stability commonly leads to a trade-off in stability or activity. It hence remains a challenge to engineer an enzyme to have both enhanced activity and stability. Here, we have attempted to reconstruct the dynamics correlation network involved with active center to improve both activity and stability of a 2,3-butanediol dehydrogenase (2,3-BDH) by introducing inter-chain disulfide bonds. A computational strategy was first applied to evaluate the effect of introducing inter-chain disulfide bond on activity and stability of three 2,3-BDHs, and the N258C mutation of 2,3-BDH from Corynebacterium glutamicum (CgBDH) was proved to be effective in improving both activity and stability. In the results, CgBDH-N258C showed a different unfolding curve from the wild type, with two melting temperatures (Tm) of 68.3 °C and 50.8 °C, 19.7 °C and 2 °C higher than 48.6 °C of the wild type. Its half-life was also improved by 14.8-fold compared to the wild type. Catalytic efficiency (kcat/Km) of the mutant was increased by 7.9-fold toward native substrate diacetyl and 8.8-fold toward non-native substrate 2,5-hexanedione compared to the wild type. Molecular dynamics simulations revealed that an interaction network formed by Cys258, Arg162, Ala144 and the catalytic residues was reconstructed in the mutant and the dynamics change caused by the disulfide bond could be propagated through the interactions network. This improved the enzyme stability and activity by decreasing the flexibility and locking more "reactive" pose, respectively. Further construction of mutations including A144G showing a 44-fold improvement in catalytic efficiency toward meso-2,3-BD confirmed the role of modifying dynamics correlation network in tunning enzyme activity and selectivity. This study provided important insights into the relationship among dynamics, enzyme catalysis and stability, and will be useful in the designing new enzymes with co-evolution of stability, activity and selectivity.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Maternal RNA transcription in Dlk1-Dio3 domain is critical for proper development of the mouse placental vasculature.

The placenta is a unique organ for ensuring normal embryonic growth in the uterine. Here, we found that maternal RNA transcription in Dlk1-Dio3 imprinted domain is essential for placentation. PolyA signals were inserted into Gtl2 to establish a mouse model to prevent the expression of maternal RNAs in the domain. The maternal allele knock-in (MKI) and homozygous (HOMO) placentas showed an expanded junctional zone, reduced labyrinth and poor vasculature impacting both fetal and maternal blood spaces. The MKI and HOMO models displayed dysregulated gene expression in the Dlk1-Dio3 domain. In situ hybridization detected Dlk1, Gtl2, Rtl1, miR-127 and Rian dysregulated in the labyrinth vasculature. MKI and HOMO induced Dlk1 to lose imprinting, and DNA methylation changes of IG-DMR and Gtl2-DMR, leading to abnormal gene expression, while the above changes didn't occur in paternal allele knock-in placentas. These findings demonstrate that maternal RNAs in the Dlk1-Dio3 domain are involved in placental vasculature, regulating gene expression, imprinting status and DNA methylation.

The FTO Mediated N6-Methyladenosine Modification of DDIT4 Regulation with Tumorigenesis and Metastasis in Prostate Cancer.

The progression of numerous malignancies has been linked to N6-methyladenosine (m6A) alteration. However, the opposite trend of m6A levels in the development and metastasis of cancer has not been reported. This study aimed to evaluate the biological function and mechanism of fat mass and obesity-associated protein (FTO) in regulating m6A modification in prostate cancer development and epithelial-mesenchymal transition (EMT). An EMT model of LNCaP and PC-3 cells was established with transforming growth factor-ß treatment, and FTO knockout cell line was established in prostate cancer cells using the CRISPR/Cas9 gene editing technology. The level of m6A modification in tumor tissues was higher than that in normal prostate tissues; m6A levels were decreased after EMT. FTO deletion increased m6A expression and enhanced PC-3 cell motility, invasion, and EMT both in vitro and in vivo. RNA sequencing and functional investigations suggested that DDIT4, a novel EMT target gene, plays a role in m6A-regulated EMT, which was recognized and stabilized by the m6A effector IGF2BP2/3. Decreased FTO expression was an independent indicator of worse survival, and the level of DDIT4 was considerably elevated in patients with bone metastasis. Thus, this study revealed that the m6A demethylase FTO can play different roles in prostate cancer as a regulator of EMT and an inhibitor of m6A modification. Moreover, DDIT4 can be suggested as a possible biomarker for prostate cancer metastasis prediction.

Non-canonical amino acids uncover the significant impact of Tyr671 on Taq DNA polymerase catalytic activity.

Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus. Five non-canonical amino acids, namely l-3,4-dihydroxyphenylalanine (l-DOPA), p-aminophenylalanine (pAF), p-acetylphenylalanine (pAcF), p-cyanophenylalanine (pCNF) and p-nitrophenylalanine (pNTF), were individually incorporated at position 671. Strikingly, Y671pAF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671pAF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probe the role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.

Air and argon cold plasma effects on lipolytic enzymes inactivation, physicochemical properties and volatile profiles of lightly-milled rice.

This study investigated the effectiveness of cold-plasma treatment using air and argon as input gas on deactivation of lipolytic enzymes in lightly-milled-rice (LMR). The results showed no significant inactivation in lipase and lipoxygenase using air-plasma. However, using argon as input gas, the residual activities of lipase and lipoxygenase were reduced to 64.51 % and 29.15 % of initial levels, respectively. Argon plasma treatment resulted in more substantial augmentation in peak and breakdown viscosities of LMR starch, suggesting an enhancement in palatability of cooked LMR with increased stickiness and decreased hardness. In contrast to the decrease in volatile compounds in LMR following argon plasma treatment, the concentrations of several prevalent aroma compounds, including 1-hexanol, 1-hexanal, and 2-pentylfuran, exhibited significant increments, reaching 1489.70 ng/g, 3312.10 ng/g, and 58.80 ng/g, respectively. These findings suggest the potential for enhancing various facets of the commercial qualities of LMR by utilizing different input gases during plasma treatment.

A Comprehensive Study on the Influence of Superheated Steam Treatment on Lipolytic Enzymes, Physicochemical Characteristics, and Volatile Composition of Lightly Milled Rice.

Enzyme inactivation is crucial for enhancing the shelf life of lightly milled rice (LMR), yet the impact of diverse superheated steam (SS) treatment conditions on lipolytic enzyme efficiency, physicochemical properties, and volatile profiles of LMR remains unclear. This study investigated varying SS conditions, employing temperatures of 120 °C, 140 °C, and 160 °C and exposure times of 2, 4, 6, and 8 min. The research aimed to discern the influence of these conditions on enzyme activities, physicochemical characteristics, and quality attributes of LMR. Results indicated a significant rise in the inactivation rate with increased treatment temperature or duration, achieving a notable 70% reduction in enzyme activities at 120 °C for 6 min. Prolonged exposure to higher temperatures also induced pronounced fissures on LMR surfaces. Furthermore, intensive SS treatment led to a noteworthy 5.52% reduction in the relative crystallinity of LMR starch. GC/MS analysis revealed a consequential decrease, ranging from 44.7% to 65.7%, in undesirable odor ketones post-SS treatment. These findings underscore the potential of SS treatment in enhancing the commercial attributes of LMR.

Livestock grazing modifies soil nematode body size structure in mosaic grassland habitats.

Body size is closely related to the trophic level and abundance of soil fauna, particularly nematodes. Therefore, size-based analyses are increasingly prominent in unveiling soil food web structure and its responses to anthropogenic disturbances, such as livestock grazing. Yet, little is known about the effects of different livestock on the body size structure of soil nematodes, especially in grasslands characterized by local habitat heterogeneity. A four-year field grazing experiment from 2017 to 2020 was conducted in a meadow steppe characterized by typical mosaics of degraded hypersaline patches and undegraded hyposaline patches to assess the impacts of cattle and sheep grazing on the body size structure of soil nematodes within and across trophic groups. Without grazing, the hypersaline patches harbored higher abundance of large-bodied nematodes in the community compared to the hyposaline patches. Livestock grazing decreased large-bodied nematodes within and across trophic groups mainly by reducing soil microbial biomass in the hypersaline patches, with sheep grazing resulting in more substantial reductions compared to cattle grazing. The reduction in large-bodied nematode individuals correspondingly resulted in decreases in nematode community-weighted mean (CWM) body size, nematode biomass, and size spectra slopes. However, both cattle and sheep grazing had minimal impacts on the CWM body size and size spectra of total nematodes in the hyposaline patches. Our findings suggest that livestock grazing, especially sheep grazing, has the potential to simplify soil food webs by reducing large-bodied nematodes in degraded habitats, which may aggravate soil degradation by weakening the bioturbation activities of soil fauna. In light of the widespread land use of grasslands by herbivores of various species and the ongoing global grassland degradation of mosaic patches, the recognition of the trends revealed by our findings is critical for developing appropriate strategies for grassland grazing management.

De Novo Design and Synthesis of Polypeptide Immunomodulators for Resetting Macrophage Polarization.

Modulating the extracellular matrix microenvironment is critical for achieving the desired macrophage phenotype in immune investigations or tumor therapy. Combining de novo protein design and biosynthesis techniques, herein, we designed a biomimetic polypeptide self-assembled nano-immunomodulator to trigger the activation of a specific macrophage phenotype. It was intended to be made up of (​GGS​GGP​GGG​PAS​AAA​NSA​SRA​TSN​SP)n, the RGD motif from collagen, and the IKVAV motif from laminin. The combination of these domains allows the biomimetic polypeptide to assemble into extracellular matrix-like nanofibrils, creating an extracellular matrix-like milieu for macrophages. Furthermore, changing the concentration further provides a facile route to fine-tune macrophage polarization, which enhances antitumor immune responses by precisely resetting tumor-associated macrophage immune responses into an M1-like phenotype, which is generally considered to be tumor-killing macrophages, primarily antitumor, and immune-promoting. Unlike metal or synthetic polymer-based nanoparticles, this polypeptide-based nanomaterial exhibits excellent biocompatibility, high efficacy, and precise tunability in immunomodulatory effectiveness. These encouraging findings motivate us to continue our research into cancer immunotherapy applications in the future.

Separation and purification of active ingredients in tobacco by free-flow electrophoresis.

The active ingredients from tobacco extracts were continuously separated and purified using a homemade free-flow electrophoresis apparatus. A rectangular free flow electrophoresis device was constructed for the continuous separation and preparation, and the operating conditions of the device were optimized. The fractions obtained from the free-flowing component collection unit were then detected by HPLC and GC-MS. The results showed that a 90% methanol-water solution could maximize the extraction of the active components from tobacco. Chlorogenic acid and nicotine were enriched in three and four of 24 fractions, respectively, after free-flow isoelectric focusing electrophoresis. 2-Hydroxy-2-cyclopentene-1-one, 1-(2-methyl-1,3-oxathiolan-2-yl) ethanone, nornicotine, cotinine, and scopolamine were separated and enriched synchronously. Overall, the use of free-flow electrophoresis technology for the separation and purification of the active substances in tobacco can improve the comprehensive utilization rate of tobacco.

Effects of physical exercise on non-suicidal self-injury in adolescents: the chain mediating role of perceived social support and self-concept.

Objective: To study the effect of physical exercise on non-suicidal self-injury in adolescents and to verify the chain mediating role of perceived social support and self-concept. Methods: A survey study was conducted on 1,426 adolescents in Chengdu, Sichuan Province. A chain mediation model was used to verify whether perceived social support and self-concept played a mediating role. Results: Physical exercise was significantly negatively associated with non-suicidal self-injury in adolescents (ß = -0.53, p < 0.01) and significantly positively associated with perceived social support and self-concept (ß = 0.52, 0.54, p < 0.01), and perceived social support and self-concept were significantly negatively associated with non-suicidal self-injury (ß = -0.59, p < 0.01; ß = -0.64, p < 0.01), and perceived social support was able to significantly and positively associate self-concept (ß = 0.76, p < 0.01). Conclusion: Perceived social support and self-concept play a chain mediating role in the effect of physical exercise on non-suicidal self-injury in adolescents, and it is recommended that the development of perceived social support and self-concept be emphasized during adolescents' development, which has the potential to reduce the incidence of non-suicidal self-injurious behaviors in adolescents.

[New pretreatment method for detecting petroleum hydrocarbons in soil: silica-gel dehydration and cyclohexane extraction].

Oil is a primary source of energy worldwide. However, the use of oil produces large amounts of pollutants, which are detrimental to the environment. The presence of petroleum hydrocarbons in soil is a critical marker of environmental pollution and safety. Rapid on-site detection technology has been broadly used in emergency tracking, offering critical information support for effective reactions to environmental emergencies. Thus, it is expected to play an increasingly critical role in environmental remediation efforts. The current approach for petroleum hydrocarbon detection in soil mainly involves Soxhlet extraction with a combination of solvents, including acetone and n-hexane. The samples are then analyzed after rotary evaporation, dehydration with anhydrous sodium sulfate, and purification using a magnesium silica-type adsorbent. Unfortunately, this approach requires sample analysis to be performed in the laboratory, which is tedious and time consuming, and consumes large amounts of solvents. Moreover, the rotary evaporator is not portable. Therefore, this method is not appropriate for the rapid on-site detection of petroleum hydrocarbons. In this study, a rapid on-site detection method based on silica-gel dehydration and cyclohexane extraction was developed for the extraction and pretreatment of petroleum hydrocarbons (C10-C40) in soil. First, an appropriate amount of silica gel was added to the soil, and the mixture was completely ground to eliminate moisture. Next, petroleum hydrocarbons were extracted with 40 mL of cyclohexane, and the extract was cleaned by Florisil solid-phase extraction (SPE) column elution. Finally, the samples were analyzed by gas chromatography (GC) to evaluate the above method. The silica gel exhibited optimal adsorption properties compared with anhydrous sodium sulfate, calcium oxide, and molecular sieves, with recovery of 87.5%. The effects of different soil water content (5%, 10%, and 20%) and silica gel (1, 3, 5, and 10 times the moisture content) dosage on the extraction of petroleum hydrocarbons were investigated. The recoveries of petroleum hydrocarbons increased from 74.0% to 103.8% after 15 min of invasive extraction (relative standard deviation, RSD, <10.1%) when silica gel amounting to 10 times the moisture content was used. Five types of silica gels with different properties were purchased from four manufacturers, and the effects of these silica gels on the dehydration and extraction efficiency of petroleum hydrocarbons in soil were assessed. The results showed that amorphous silica gel led to low recoveries (<60%), spherical silica gel achieved extraction efficiencies of approximately 70%-90%, and alkaline silica gel produced recoveries with poor precision. Therefore, neutral spherical silica gel was used for further experiments. The fingerprints of petroleum hydrocarbons with different carbon numbers are an important reference for identifying pollution sources. Thus, ensuring good recoveries throughout the entire carbon range is necessary to ensure the accuracy of the fingerprint analysis results. The proposed method showed good recoveries for petroleum hydrocarbons of all carbon numbers (75%-101%). The findings above indicate that the developed method could be an efficient means to extract petroleum hydrocarbons from soil for both total quantity and fingerprint analyses. Compared with standard methods, the proposed method requires lower solvent dosages and features simpler processing steps. Another advantage of this method is that it does not require the use of highly toxic halogenated solvents; thus, it does not contribute to environmental pollution. It can be applied to the laboratory analysis of soil petroleum hydrocarbons and coupled with other rapid on-site detection techniques for soil petroleum hydrocarbons, such as infrared spectroscopy and portable GC. However, because it does not include a concentration process, the developed method exhibits relatively low sensitivity. In the future, we plan to develop a simple and flexible on-site sample-concentration system to further improve various indicators of this method.

Associations of Dietary Anthocyanidins Intake with Bone Health in Children: A Cross-Sectional Study.

PURPOSE: Bone health and body composition share several common mechanisms like oxidative stress and inflammation. Anthocyanins have antioxidant and anti-inflammatory properties. We have reported that anthocyanins are associated with better body composition in children, but the associations with bone health have not been elucidated. We aimed to explore the association of anthocyanins with bone mineral content (BMC) and bone mineral density (BMD) at multiple sites in children. METHODS: In this cross-sectional study, 452 Chinese children aged 6-9 years were recruited. A validated 79-item food frequency questionnaire was used to collect dietary information. BMC and BMD at multiple sites (whole body; whole body excluding head, WBEH; limbs; arms; legs) were measured by dual-energy X-ray. RESULTS: Higher dietary intake of total anthocyanidins (per one standard deviation increase) was associated with a 1.28-13.6 g (1.31-1.60%, compared to median) higher BMC at all sites and a 3.61-6.96 mg (0.65-0.90%) higher BMD at the whole body, WBEH, and arm sites after controlling for a number of possible covariates. The results were similar and more pronounced for cyanidin, but not for delphinidin and peonidin. Higher dietary intake of cyanidin (per one standard deviation increase) was associated with a 1.33-15.4 g (1.48-1.68%) higher BMC at all sites and a 4.15-7.77 mg (0.66-1.00%) higher BMD at all sites except the legs. No statistically significant associations with BMC or BMD were found for dietary intake of delphinidin and peonidin. CONCLUSIONS: Higher dietary intake of total anthocyanidins and cyanidins were associated with higher BMC and BMD in Chinese children.

Customized multiple sequence alignment as an effective strategy to improve performance of Taq DNA polymerase.

Engineering Taq DNA polymerase (TaqPol) for improved activity, stability and sensitivity was critical for its wide applications. Multiple sequence alignment (MSA) has been widely used in engineering enzymes for improved properties. Here, we first designed TaqPol mutations based on MSA of 2756 sequences from both thermophilic and non-thermophilic organisms. Two double mutations were generated including a variant H676F/R677G showing a decrease in both activity and stability, and a variant Y686R/E687K showing an improved activity, but a decreased stability. Mutations targeted on coevolutionary residues of Arg677 and Tyr686 were then applied to rescue stability or activity loss of the double mutants, which achieved a partial success. Sequence analysis revealed that the two mutations are abundant in non-thermophilic sequences but not in thermophilic homologues. Then, a small-scale MSA containing sequences from only thermophilic organisms was applied to predict 13 single variants and two of them, E507Q and E734N showed a simultaneous increase in both stability and activity, even in sensitivity. A customized MSA was hence more effective in engineering a thermophilic enzyme and could be used in engineering other enzymes. Molecular dynamics simulations revealed the impact of mutations on the protein dynamics and interactions between TaqPol and substrates. KEY POINTS: • The pool of sequence for alignment is critical to engineering Taq DNA polymerase. • The variants with low properties can be rescued by mutations in coevolving network. • Improving binding with DNA can improve DNA polymerase stability and activity.