[Effects of urogenital mycoplasma and chlamydial infections on male fertility].

Objective： This study aimed to assess the impact of urogenital ureaplasma urealyticum (Uu), Mycoplasma hominis (Mh), and Chlamydia trachomatis (Ct) infections on semen quality in men.Methods： In this study, 1022 males were enrolled at the Department of Reproductive Medicine, Rizhao People's Hospital, Shandong Province from October 2014 to January 2023. The participants included 393 in the infertility group, 139 in the recurrent miscarriage group, and 490 in the control group. Based on age, 852 cases were < 36 years old, and 170 cases were ≥ 36 years old. All patients underwent routine semen analysis and tests for Uu, Mh, and Ct, with results statistically analyzed for their impact on semen quality and compared among different age groups. Results: Among the 1022 patients, 344 (33.6%) were Uu-positive, 49 (4.7%) were Mh-positive, and 31 (3.0%) were Ct positive. The sperm concentration, total sperm count, forward sperm motility rate (PR), sperm motility rate (PR+NP) and normal sperm morphology rate of Uu Mh and/or Ct-positive patients were significantly lower than those of the negative group, and the overall difference between the two groups was statistically significant (P<0.05). The positive rate of Uu infection was 41.7% in the infertility group, 30.2% in the recurrent miscarriage group and 28.2% in the control group, and the overall positive rate of the three groups was statistically significant(P<0.05). The positive rate of Mh infection was 6.9% in the infertility group, 8.6% in the recurrent miscarriage group and 2.0% in the control group, and the overall positive rate of the three groups was statistically significant (P<0.001). The positive rate of Ct infection was 6.1% in the infertility group, 2.9% in the recurrent miscarriage group and 0.6% in the control group, and the overall positive rate of the three groups was statistically significant (P<0.001). The positivity rate of Uu infection was 35.8% at the age of <36 years and 22.9% at the age ≥ 36 years, and there was a statistically significant difference in the positivity rate between the two groups (P<0.05). Conclusion: The prevalence of Uu infection in the male urogenital tract is significantly higher than that of Mh and Ct, which is the main pathogen of urogenital tract infection in men. Uu, Mh and Ct infections have adverse effects on sperm concentration, total sperm count, sperm forward motility rate (PR), sperm motility rate (PR+NP) and normal sperm morphology rate, which will lead to a decrease in semen quality and affect male fertility. Genital tract infections are closely related to age, and the prevalence of Uu infection is higher in the younger age group.

Efficient production of a cyclic dipeptide (cyclo-TA) using heterologous expression system of filamentous fungus Aspergillus oryzae.

BACKGROUND: Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. RESULTS: We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. CONCLUSIONS: In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.

Hierarchical porous carbon materials synthesized from the castor oil/MgO solids for high-performance supercapacitors.

A kind of biomass-based hierarchical porous carbons (HPCs) is easily synthesized by the direct pyrolysis of castor oil/MgO solids, in which the solids can be prepared by mixing appropriate amounts of MgO into castor oil. The morphology, microstructure, phase structure, textural property, surface element composition and thermal stability properties are studied for the achieved HPCs. It is demonstrated that the HPCs belong to a type of high-graphitization, graphene-like and foamed carbon materials with high specific surface area and wide pore size distribution. The HPC obtained at 900 °C (HPC-900) displays the highest specific surface area of 1013.17 m2g and more reasonable pore size distribution. Without the need of conductive agents, the HPC-900 exhibits a maximum capacitance (340 F g-1at 0.5 A g-1), excellent rate performance (70.1% of capacitance retention at high current density of 10 A g-1) and a remarkable long-term cycling stability (about 99% capacitance retention after 9000 cycles) in the aqueous electrolyte of 6 M KOH. Meanwhile, assembled as-prepared sample into symmetrical supercapacitor, the HPC-900 provides a high energy and power density, with 8.6 Wh kg-1and 426.7 W kg-1in 1 M Na2SO4, respectively. The HPCs prepared based on castor oil show high potential for the electrode materials of supercapacitors.

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes.

Pinostrobin (PI, 5-hydroxy-7-methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450-selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro. In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC50 > 100 µm). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.

Crocus genome reveals the evolutionary origin of crocin biosynthesis.

Crocus sativus (saffron) is a globally autumn-flowering plant, and its stigmas are the most expensive spice and valuable herb medicine. Crocus specialized metabolites, crocins, are biosynthesized in distant species, Gardenia (eudicot) and Crocus (monocot), and the evolution of crocin biosynthesis remains poorly understood. With the chromosome-level Crocus genome assembly, we revealed that two rounds of lineage-specific whole genome triplication occurred, contributing important roles in the production of carotenoids and apocarotenoids. According to the kingdom-wide identification, phylogenetic analysis, and functional assays of carotenoid cleavage dioxygenases (CCDs), we deduced that the duplication, site positive selection, and neofunctionalization of Crocus-specific CCD2 from CCD1 members are responsible for the crocin biosynthesis. In addition, site mutation of CsCCD2 revealed the key amino acids, including I143, L146, R161, E181, T259, and S292 related to the catalytic activity of zeaxanthin cleavage. Our study provides important insights into the origin and evolution of plant specialized metabolites, which are derived by duplication events of biosynthetic genes.

Unusual DNA structures formed on bare highly oriented pyrolytic graphite surfaces studied by atomic force microscopy.

It is important to know the detailed DNA structure on carbonaceous surfaces for further application of DNA-functionalized carbonaceous materials in diverse research areas. In this study, the topographic and structural characteristics of the separated single DNA molecules and their assembly on highly oriented pyrolytic graphite (HOPG) surfaces have been investigated by atomic force microscopy (AFM). AFM results indicate that both circular and linear DNA molecules tend to form hexagonal patterns along with some unusual structures that include node, protrusion, cruciform, parallel single-stranded DNA (ssDNA), and compact zigzag. Furthermore, parallel ssDNA patterns and their crossed structures have been obtained under high-temperature conditions. Our AFM results reveal that a bare HOPG surface can induce DNA molecules to form various unusual structures. This finding is helpful for understanding the adsorption behavior of DNA on other carbonaceous surfaces such as carbon nanotubes and graphene. In addition, the hexagonal DNA patterns in this study are similar to those formed on the alkylamine-modified HOPG surface, which implies that a bare HOPG, without any chemical modification, has a strong ability to align biomolecules. This study could expand our knowledge of the diversities of DNA structures and the aligning ability of carbonaceous surfaces.

Corrigendum: Research progress and trends in metabolomics of fruit trees.

[This corrects the article DOI: 10.3389/fpls.2022.881856.].

Research Progress and Trends in Metabolomics of Fruit Trees.

Metabolomics is an indispensable part of modern systems biotechnology, applied in the diseases' diagnosis, pharmacological mechanism, and quality monitoring of crops, vegetables, fruits, etc. Metabolomics of fruit trees has developed rapidly in recent years, and many important research results have been achieved in combination with transcriptomics, genomics, proteomics, quantitative trait locus (QTL), and genome-wide association study (GWAS). These research results mainly focus on the mechanism of fruit quality formation, metabolite markers of special quality or physiological period, the mechanism of fruit tree's response to biotic/abiotic stress and environment, and the genetics mechanism of fruit trait. According to different experimental purposes, different metabolomic strategies could be selected, such as targeted metabolomics, non-targeted metabolomics, pseudo-targeted metabolomics, and widely targeted metabolomics. This article presents metabolomics strategies, key techniques in metabolomics, main applications in fruit trees, and prospects for the future. With the improvement of instruments, analysis platforms, and metabolite databases and decrease in the cost of the experiment, metabolomics will prompt the fruit tree research to achieve more breakthrough results.

Adsorption of chitosan onto carbonaceous surfaces and its application: atomic force microscopy study.

The adsorption of chitosan onto highly ordered pyrolytic graphite(HOPG) surfaces and its applications have been studied by atomic force microscopy (AFM). The results indicated that chitosan topography formed on the HOPG surface significantly depends on the pH conditions and its concentration for the incubation. Under strongly acidic conditions (pH < 3.5) and at a concentration of 1 mg ml⁻¹, chitosan formed into uniform network structures composed of fine chains. When the solution pH was changed from 3.5 to 6.5, chitosan tends to form a thicker film. Under neutral and basic conditions, chitosan changed into spherical nanoparticles, and their sizes were increased with increasing pH. Dendritic structures have been observed when the chitosan concentration was increased up to 5 mg ml⁻¹. In addition, the chitosan topography can also be influenced by ionic strength and the addition of different metal ions. When 0.1 M metal ions Na+, Mg²+, Ca²+ and Cu²+ were added into the chitosan solution at pH 3.0 for the incubation, network structures, branched chains, block structures and dense networks attached with many small particles were observed, respectively. The potential applications of these chitosan structures on HOPG have been explored. Preliminary results characterized by AFM and XPS indicated that the chitosan network formed on the HOPG surface can be used for AFM lithography, selective adsorption of gold nanoparticles and DNA molecules.

Adsorption of humic acid onto carbonaceous surfaces: atomic force microscopy study.

The adsorption of humic acid (HA) onto highly ordered pyrolytic graphite (HOPG) surfaces at different concentrations has been studied by atomic force microscopy. When HA concentration was increased from 10 to 1,000 mg/L, HA can sequentially form spherical particles, layered structures, and connected blocks on HOPG surfaces. The findings of the layer structures and small amount of fine chains have been verified and discussed. When HA was acidified by addition of acetic acid, it changed into small rigid particles. These results indicated that HA can be considered as supramolecular associations of self-assembling heterogeneous and relatively small molecules, and a small amount of polymers. The present results are important for understanding HA molecular structures and their adsorption characteristic on carbonaceous surfaces.

Cajanin Stilbene Acid Inhibited Vancomycin-Resistant Enterococcus by Inhibiting Phosphotransferase System.

Antimicrobial resistance has become a serious threat to human and animal health, and vancomycin-resistant Enterococcus has become an important nosocomial infection pathogen, causing thousands of deaths each year. In this study, after screening a variety of natural products, we found that cajanin stilbene acid (CSA) had significant inhibitory effect on sensitive and vancomycin-resistant Enterococcus (VRE) in vitro. And we also confirmed that CSA had significant anti-VRE infection ability in vivo. Subsequently, we studied the antibacterial mechanism of CSA through proteomics experiments, and the results showed that CSA killed Enterococcus by inhibiting the phosphotransferase system of Enterococcus, thus hinders the normal growth and metabolic functions of bacteria. The results of this study provided evidence for the in-depth study on the mechanism of the antibacterial action of CSA and also provided a candidate for the development of anti-VRE drugs.

Tumor-Targeting Peptide for Redox-Responsive Pt Prodrug and Gene Codelivery and Synergistic Cancer Chemotherapy.

A new codelivery system combining prodrug strategy, siRNA/BAplatin @CRGDK NPs, to overcome cisplatin (CDDP) resistance in human breast cancer was designed and researched. Negatively charged siRNA was deposited onto the surface of tumor-targeting peptide-functionalized BAplatin@CRGDK NPs. SiRNA/BAplatin@CRGDK NPs could facilitate cellular uptake of BAplatin and increase the cell proliferation suppression effect of Pt against MDA-MB-231/DDP cells. The tumor-to-kidney uptake ratio of the siRNA/BAplatin@CRGDK NPs in MB-231/DDP tumors is 2.4-fold higher than that of cisplatin in MB-231/DDP tumors, thus giving rise to more significant antitumor efficacy. It demonstrated that the siRNA/BAplatin@CRGDK NPs is a potential, safe, and efficient therapeutic agent for cancer therapy.

Synthesis and characterization of proanthocyanidins-functionalized Ag nanoparticles.

Preparation of Ag nanoparticles with high antibacterial activities and low adverse effects is urgently needed to apply to the medical and consumer products. In this study, a facile, one-step hydrothermal method has been developed to synthesize proanthocyanidins-functionalized Ag nanoparticles in aqueous solution. UV absorption spectra measurement indicated that the obtained Ag nanomaterials are mainly spherical in shape. FTIR results implied that the poly flavonoids moiety of proanthocyanidins have specific interactions with the surface of Ag nanoparticles. HR-TEM and XRD determination revealed that the Ag nanoparticles had a shell-core structure and the Ag core possessed a highly crystalline structure. Antibacterial tests demonstrated that the proanthocyanidins-functionalized-Ag nanoparticles have high bactericidal efficiency against both bacterial and fungus. Further taking into account their excellent stability, the proanthocyanidins-functionalized Ag nanoparticles in present study have the potential to be applied to the products required for the long-acting antibacterial action.

Green Synthesis, Characterization and Application of Proanthocyanidins-Functionalized Gold Nanoparticles.

Green synthesis of gold nanoparticles using plant extracts is one of the more promising approaches for obtaining environmentally friendly nanomaterials for biological applications and environmental remediation. In this study, proanthocyanidins-functionalized gold nanoparticles were synthesized via a hydrothermal method. The obtained gold nanoparticles were characterized by ultraviolet and visible spectrophotometry (UV-Vis), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and X-ray diffraction (XRD) measurements. UV-Vis and FTIR results indicated that the obtained products were mainly spherical in shape, and that the phenolic hydroxyl of proanthocyanidins had strong interactions with the gold surface. TEM and XRD determination revealed that the synthesized gold nanoparticles had a highly crystalline structure and good monodispersity. The application of proanthocyanidins-functionalized gold nanoparticles for the removal of dyes and heavy metal ions Ni2+, Cu2+, Cd2+ and Pb2+ in an aqueous solution was investigated. The primary results indicate that proanthocyanidins-functionalized gold nanoparticles had high removal rates for the heavy metal ions and dye, which implies that they have potential applications as a new kind of adsorbent for the removal of contaminants in aqueous solution.

Magnetically triggered drug release from nanoparticles and its applications in anti-tumor treatment.

The objective of this study was to describe the magnetic nanoparticle-drug conjugates for improved control of drug delivery and drug release. The widely used anticancer agent Doxorubicin (DOX) was successfully conjugated via amine groups to the carboxylic functional groups coating magnetic nanoparticles (fluidMAG-CMX). Following purification of the nanoparticles, the conjugation of DOX on fluidMAG-CMX was confirmed using FTIR spectroscopy and confocal microscopy. The observed drug loading capacity of DOX was 22.3%. Studies of magnetically triggered release were performed under an oscillating magnetic field (OMF). DOX exhibited a significant release percentage of 70% under an OMF, as compared with the release in enzyme. A magnetic field turn-on and turn-off experiment was also conducted to confirm the control of drug release using this triggered system. In vivo experiments indicated that the tumor-inhibitory rate of CMX-DOX NPs under a magnetic field was higher than the other control groups. According to the toxicity assessments, CMX-DOX NPs were not noticeably toxic to mice at our tested dose.

Synthesis, characterization and antibacterial study on the chitosan-functionalized Ag nanoparticles.

This study provided a facile, one-step hydrothermal method to synthesize stable Ag colloid in aqueous solution by utilizing chitosan as both reductant and stabilizer. The formation of chitosan-functionalized Ag nanoparticles was verified by UV-Vis, FTIR, TEM, AFM and XRD measurements. FTIR results revealed that the primary amine groups and amide groups of chitosan have specific interactions with the surface of Ag nanoparticles. The average diameter of the Ag nanoparticles is 10.0±5.4nm as determined by TEM. Ag nanoparticles are highly crystalline as revealed by HR-TEM and XRD measurements. The size and shape of Ag nanoparticles are also found to depend on the pH condition in the synthesis. Ag nanoparticles were the main products at pH5.0 whereas large Ag nanotriangle and truncated triangular nanoplate were dominant at pH4.0 in the synthesis. Due to its monodispersity and good stability, the chitosan-functionalized Ag colloid synthesized at pH5.0 was further tested for its antibacterial activities against gram-positive bacteria, gram-negative bacteria and fungus. The results of zone of inhibition, inhibition ratio and SEM characterization revealed that chitosan-functionalized Ag nanoparticles have great bactericidal efficiency against both bacteria and fungus.

In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 µM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 µM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA.

PA-1, a novel synthesized pyrrolizidine alkaloid, inhibits the growth of Escherichia coli and Staphylococcus aureus by damaging the cell membrane.

In the present study, antimicrobial activity and mode of a novel synthesized pyrrolizidine alkaloid (PA-1) were investigated. PA-1 exhibited predominantly strong antibacterial activity toward six bacteria tested with minimal inhibitory concentration (MIC) values ranging from 0.0039 to 0.025 mg ml(-1). The time-kill assay indicated that PA-1 killed Escherichia coli and Staphylococcus aureus completely at 2MIC (minimum bactericidal concentration) within 8 h. Besides, PA-1-induced death rates of most sensitive strains (E. coli, 97.80% and S. aureus, 96.24%) were analyzed by flow cytometry. A combination of approaches was used to verify the membrane damage of E. coli and S. aureus. Results showed that release of 260 nm absorbing materials quickly increased after PA-1 treatment. PA-1 also rapidly promoted the uptake of crystal violet from 24.52 to 97.12% for E. coli and from 19.68 to 97.63% for S. aureus when the concentrations were changed from MIC to 4MIC. Furthermore, the cellular membrane damages were testified by the significant increase of fluorescence intensity and decrease of membrane potential. Finally, lecithin and phosphate groups were applied to search the possibly targets on the cytoplasmic membrane. Results showed that PA-1 acted on cytoplasmic membrane phospholipids and phosphate groups of S. aureus but not of E. coli. In conclusion, the novel synthesized PA-1 exerted its antibacterial activity by acting on membrane phospholipids and phosphate groups and then damaging the structures of cellular membrane, which finally led to cell death.

Externally controlled triggered-release of drug from PLGA micro and nanoparticles.

Biofilm infections are extremely hard to eradicate and controlled, triggered and controlled drug release properties may prolong drug release time. In this study, the ability to externally control drug release from micro and nanoparticles was investigated. We prepared micro/nanoparticles containing ciprofloxacin (CIP) and magnetic nanoparticles encapsulated in poly (lactic-co-glycolic acid) PLGA. Both micro/nanoparticles were observed to have narrow size distributions. We investigated and compared their passive and externally triggered drug release properties based on their different encapsulation structures for the nano and micro systems. In passive release studies, CIP demonstrated a fast rate of release in first 2 days which then slowed and sustained release for approximately 4 weeks. Significantly, magnetic nanoparticles containing systems all showed ability to have triggered drug release when exposed to an external oscillating magnetic field (OMF). An experiment where the OMF was turned on and off also confirmed the ability to control the drug release in a pulsatile manner. The magnetically triggered release resulted in a 2-fold drug release increase compared with normal passive release. To confirm drug integrity following release, the antibacterial activity of released drug was evaluated in Pseudomonas aeruginosa biofilms in vitro. CIP maintained its antimicrobial activity after encapsulation and triggered release.

Efficient production of isoflavonoids by Astragalus membranaceus hairy root cultures and evaluation of antioxidant activities of extracts.

In this study, Astragalus membranaceus hairy root cultures (AMHRCs) were established as an attractive alternative source for the efficient production of isoflavonoids (IF). A. membranaceus hairy root line II was screened as the most efficient line and was confirmed by PCR amplification of rolB, rolC and aux1 genes. Culture parameters of AMHRCs were systematically optimized, and five main IF constituents were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total IF accumulation of 34 day old AMHRCs was 234.77 µg/g dry weight (DW). This yield was significantly higher compared to that of 3 year old field grown roots (187.38 µg/g DW). Additionally, in vitro antioxidant assays demonstrated that AMHRC extracts exhibited antioxidant activities with lower IC50 values (1.40 and 1.73 mg/mL) as compared to those of field grown roots (1.96 and 2.17 mg/mL). Overall, AMHRCs may offer a promising and continuous product platform for naturally derived, high quality and valuable nutraceuticals.