Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

The key technologies of Ganoderma lucidum liquid spawn preparation and scale expansion.

Ganoderma lucidum possesses a variety of valuable pharmacological activities, and it has long been used to prevent and treat various human diseases. Up to now, far too little attention has been paid to the liquid spawn of G. lucidum, and the development of the G. lucidum industry is constrained by them. This work aimed to study the key technologies and scale-up preparation of G. lucidum liquid spawn, to achieve large-scale preparation of liquid spawn and solve the problem of unstable quality of G. lucidum. The plate culture, primary shake flask culture, shake flask preparation, and fermentor preparation of G. lucidum liquid spawn were explored in the process of liquid fermentation. The results showed that plate broth volume significantly affected mycelial growth rate. Biomass in the primary shake flask culture is significantly influenced by the picking position of plate mycelium. An artificial neural network coupled with a genetic algorithm was used for carbon and nitrogen sources concentration optimization to increase biomass and substrate utilization. The optimized parameter combination is as follows: glucose, 14.5 g L-1; yeast extract powder, 8.5 g L-1. Under this condition, the biomass (9.82 g L-1) and biomass on reducing sugar (0.79 g g-1) increased by 18.03% and 27.41% compared to the control, respectively. The metabolic activity of liquid spawn prepared by different fermentation scales was diverse, and the liquid spawn prepared by the fermentor has better activity. Conceivably, the liquid spawn process can more conducive be applied to large-scale industrial production.

[Monoterpenoids from Paeoniae Radix Rubra based on UPLC-Q-TOF-MS].

The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 µm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 ℃. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5″-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.

Development a multiplex panel of AISNPs, multi-allelic InDels, microhaplotypes, and Y-SNP/InDel loci for multiple forensic purposes via the NGS.

Recently, next generation sequencing has shown the promising application value in forensic research. In this study, we constructed a multiplex amplification system of different molecular genetic markers based on the previous selected ancestry informative single nucleotide polymorphisms (SNPs), multi-allelic insertion/deletion (InDel) polymorphisms, microhaplotypes, and Y-chromosomal SNP/InDel loci, and evaluated forensic efficiencies of the system in Chinese Shaanxi Han, Chinese Hui, and Chinese Mongolian groups via the next generation sequencing platform. Ancestry information analyses of Shaanxi Han, Hui, and Mongolian groups revealed that most Mongolian individuals could be differentiated from Shaanxi Hans and Huis based on the selected ancestry informative SNPs. Multi-allelic InDels and microhaplotypes showed the multiple allele variations and possessed relatively high genetic polymorphisms in these three groups, indicating these loci could provide higher forensic efficiencies for individual identification and paternity testing. Based on Y-chromosomal SNPs, different haplogroup distributions were observed among Shaanxi Han, Hui, and Mongolian groups. In conclusion, the self-developed system could be used to simultaneously carry out the individual identification, paternity analysis, mixture deconvolution, forensic ancestry information analysis, and Y-chromosomal haplogroup inference, which could provide more valuable investigative clues in forensic practices.

Menisperdaurines A-W, structurally diverse isoquinoline alkaloids from Menispermum dauricum and their dopamine D1 receptor activities.

A total of 33 structurally diverse isoquinoline alkaloids were isolated from the rhizomes of Menispermum dauricum, including seventeen benzylisoquinoline analogues (menisperdaurines A-Q, 1-17), five protoberberine analogues (menisperdaurines R-V, 18-22), a quaternary phenanthrene alkaloid (menisperdaurine W, 23) and ten known compounds (24-33). Compound structures, including absolute configurations, were determined by extensive spectroscopic methods, quantum chemical calculations of chemical shifts, and calculated and experimental electronic circular dichroism (ECD) data. Compounds 1-5 were glycosidic benzylisoquinolines with glucose moieties attached at the C-12 position. Compound 8 was the first example that was isolated from the rhizomes of Menispermum dauricum, benzylisoquinoline and an aromatic unit connected by a sugar bridge. Compounds were evaluated for their inhibitory effects on the dopamine D1 receptor. Compounds 1, 8, 21, 24 and 29 showed potent D1 antagonistic activities, with IC50 values ranging from 1.0 to 4.5 µM. Compound 1 exhibited the highest antagonistic activity with an IC50 value of 1.0 ± 0.2 µM.

Iridoid Glycosides and Flavonoids Isolated from the Twigs and Leaves of Callicarpa nudiflora and Their Anti-Inflammatory Activities.

A new iridoid glycoside, named 6'-O-trans-feruloyl-8-epiloganic acid, together with fifteen known compounds were isolated from the twigs and leaves of Callicarpa nudiflora, a traditional Chinese medicine to treat inflammatory-related diseases. Their structures were identified by comprehensive spectroscopic analysis and comparison with reported data. Bioassay results revealed that twelve of the isolates could obviously inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cell lines with IC50 values from 0.64 to 38.72 µM. Among them, compounds 1 (3.27 µM), 6 (5.23 µM), 13 (1.56 µM) and 14 (0.64 µM) exhibited significantly higher activities than that of the positive control (27.13 µM). Additionally, it was supposed that the presence of the carboxy group at the C-4 position of iridoid glycosides and glycosylation at C-3 position of flavonoids might impact their inhibitory activities against NO production.

Theoretical Study of the Oxidation of Methane to Methanol by the [CuIICuII(µ-O)2CuIII(7- N-Etppz)]1+ Complex.

The reactivity patterns of a series of trivalent copper complexes have been studied to gain a better understanding of the chemical reactions occurring at the active site of particulate methane monooxygenase (pMMO). In this study, hybrid density functional theory is used to study the oxidation of methane to methanol mediated by the [CuIICuII(µ-O)2CuIII(7- N-Etppz)]1+ complex. Reaction mechanisms in different spin states were explored. Based on the calculated free-energy profile, a mechanism is suggested for the reaction of the oxidation of methane to methanol. The first step (1 → 2) is a hydrogen transfer to the bridged oxygen in the Cu2O2 core from the methane to form a methyl radical. The second step (2 → 3) is the radical recombination, in which the bridged hydroxyl rotates upward and exposes the oxygen moiety toward the methyl radical to form methanol. The radical recombination step is rate-limiting, with a calculated free-energy barrier of 19.6 kcal mol-1, which is in good agreement with the experimental value of 18.4 kcal mol-1. The mixed valent bis(µ-oxo)CuIICuIII species in the Cu3O4 core is directly responsible for the C-H activation of methane.

Self-Assembled Nano-Immunostimulant for Synergistic Immune Activation.

Immunotherapy has become one of the most promising therapies for the treatment of diseases. Synthetic immunostimulants and nanomaterial immunostimulant systems are indispensable for the activation of the immune system in cancer immunotherapy. Herein, a strategy for preparing self-assembled nano-immunostimulants (SANIs) for synergistic immune activation is reported. Three immunostimulants self-assemble into nanoparticles through electrostatic interactions. SANIs showed strong synergistic immunostimulation in macrophages. SANIs could also induce a strong antitumor immune response to inhibit tumor growth in mice and act as an efficient adjuvant of antitumor vaccines. Therefore, SANIs may be generally applied in cancer immunotherapy. This novel SANI strategy provides a new way for the development of both immunostimulants and -suppressants.

Nonadiabatic dynamics simulation of keto isocytosine: a comparison of dynamical performance of different electronic-structure methods.

The nonadiabatic dynamics of keto isocytosine in the gas phase has been investigated using the on-the-fly trajectory surface hopping method based on two electronic-structure methods: SA-CASSCF and ADC(2). The results estimate an excited-state lifetime of around 1000 fs at the SA-CASSCF level, while a much shorter lifetime of 250-350 fs is obtained at the ADC(2) level. Although three conical intersections (CIs) (Ethyl. I, Ethyl. II and C[double bond, length as m-dash]O stretching) are relevant to the nonadiabatic decay of keto isocytosine, their contributions to the nonadiabatic decay are highly dependent on the electronic-structure methods employed in the dynamics simulation. The Ethyl. II CI is the main channel in the dynamics simulations at the SA-CASSCF level, while the C[double bond, length as m-dash]O stretching CI becomes dominant at the ADC(2) levels. Other high-level electronic-structure methods (MR-CISD and MS-CASPT2) are involved to benchmark our dynamics results. Through the analysis of the reaction pathways from the ground state minimum to the relevant CIs, we expect that the excited-state dynamical features obtained at the MR-CISD and MS-CASPT2 levels should be very similar to those at the SA-CASSCF level. The comparison of results obtained using different excited-state electronic-structure methods could provide guidance for further studies of similar systems.

Theoretical Investigations of the Chiral Transition of α-Amino Acid Confined in Various Sized Armchair Boron-Nitride Nanotubes.

We computationally study the chiral transition process of the α-Ala molecule under confined different sizes of armchair SWBNNTs to explore the confinement effect. We find that the influence of a confinement environment (in armchair SWBNNTs) on the α-Ala molecule would lead to different reaction pathways. Meanwhile, the preferred reaction pathway is also different in various sizes of armchair SWBNNTs, and their energy barriers for the rate-limiting step decrease rapidly with the decreasing of the diameters of the nanotubes. It is obvious that significant decrease of the chiral transition energy barrier occurs compared with the isolated α-Ala molecule chirality conversion mechanism, by ∼15.6 kcal mol-1, highlighting the improvement in the activity the enantiomers of α-Ala molecule. We concluded that the confinement environment has a significant impact at the nanoscale on the enantiomer transformation process of the chiral molecule.

[Cloning, prokaryotic expression and function of the Bufo bufo gargarizans tryptophan hydroxylase I gene].

Tryptophan hydroxylase I（TPH1） catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1 984 bp (GenBank accession No. JQ768313) with a 1 443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 k Da and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L(-1) IPTG at 20 ℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.

Glycopeptide Nanoconjugates Based on Multilayer Self-Assembly as an Antitumor Vaccine.

Antitumor vaccine, which is promising for tumor therapy, has been extensively studied. Some encouraging results of chemically synthetic vaccine designs based on the tumor-associated antigen mucin 1 have been achieved. However, some shortcomings such as low efficiency and difficult purification restrict their clinical application. To overcome these difficulties, we designed a novel antitumor vaccine of glycopeptide nanoconjugates based on the multilayer self-assembly through the interaction of positive and negative charges. This vaccine formed the spherical structure and effectively activated the macrophage in vitro. Besides, it also induced high titer of antibodies against mucin 1 glycopeptide. The induced antibodies could highly bind to the tumor cells and effectively kill them by activation of the complement dependent cytotoxicity complex. This novel strategy provides a new way for the development of simple and effective antitumor vaccine.

MSLoc-DT: a new method for predicting the protein subcellular location of multispecies based on decision templates.

Revealing the subcellular location of newly discovered protein sequences can bring insight to their function and guide research at the cellular level. The rapidly increasing number of sequences entering the genome databanks has called for the development of automated analysis methods. Currently, most existing methods used to predict protein subcellular locations cover only one, or a very limited number of species. Therefore, it is necessary to develop reliable and effective computational approaches to further improve the performance of protein subcellular prediction and, at the same time, cover more species. The current study reports the development of a novel predictor called MSLoc-DT to predict the protein subcellular locations of human, animal, plant, bacteria, virus, fungi, and archaea by introducing a novel feature extraction approach termed Amino Acid Index Distribution (AAID) and then fusing gene ontology information, sequential evolutionary information, and sequence statistical information through four different modes of pseudo amino acid composition (PseAAC) with a decision template rule. Using the jackknife test, MSLoc-DT can achieve 86.5, 98.3, 90.3, 98.5, 95.9, 98.1, and 99.3% overall accuracy for human, animal, plant, bacteria, virus, fungi, and archaea, respectively, on seven stringent benchmark datasets. Compared with other predictors (e.g., Gpos-PLoc, Gneg-PLoc, Virus-PLoc, Plant-PLoc, Plant-mPLoc, ProLoc-Go, Hum-PLoc, GOASVM) on the gram-positive, gram-negative, virus, plant, eukaryotic, and human datasets, the new MSLoc-DT predictor is much more effective and robust. Although the MSLoc-DT predictor is designed to predict the single location of proteins, our method can be extended to multiple locations of proteins by introducing multilabel machine learning approaches, such as the support vector machine and deep learning, as substitutes for the K-nearest neighbor (KNN) method. As a user-friendly web server, MSLoc-DT is freely accessible at http://bioinfo.ibp.ac.cn/MSLOC_DT/index.html.

[Dynamic studies on photosynthetic and growth characteristics of Angecila dahurcia].

Photosynthetic and growth characteristics of Angelica dahurica were studied in order to clarity the relations of photosynthesis, growth and root dry weight, and provide a theoretical basis for its cultivation. Photosynthesis and growth indexes were meas- ured every 25 days. The contents of chlorophyll a, b, a + b, soluble protein and the activities of Hill reaction, Ca(2+)-ATPase, Mg(2+)-ATPase had an increasing trend; They had the highest value in leaf high-speed growth period. Then, they were decreased in root high- speed growth period. The root dry weight showed negative corelation with photosynthetic characteristics indexes except stomatal con- ductance, however, the negative corelation only from net photosynthetic rate and Ca(2+)-ATPase were significant. The vegetative growth period of spring sowing A. dahuricia was divided into three phases: seedling period, leaf high-speed growth period and root high-speed growth period. The root dry weight showed a significantly positive corelation with the root diameter, leaf dry weight, shoot dry weight, aboveground dry weight. There was the competitive relation between aboveground and underground, so underground growth could be es- timated from leaf area and shoot dimeter.

Quantifying peripapillary vessel density and retinal nerve fibre layer in type 1 diabetic children without clinically detectable retinopathy using OCTA.

AIM: To quantify changes in radial peripapillary capillary vessel density (ppVD) and the peripapillary retinal nerve fiber layer (pRNFL) in children with type 1 diabetes without clinical diabetic retinopathy by optical coherence tomography angiography (OCTA), providing a basis for early retinopathy in children with type 1 diabetes. METHODS: This was a retrospective study. A total of 30 patients (3-14y) with type 1 diabetes without clinical diabetic retinopathy (NDR group) were included. A total of 30 age-matched healthy subjects were included as the normal control group (CON group). The HbA1c level in the last 3mo was measured once in the NDR group. The pRNFL thickness and ppVD were automatically measured, and the mean pRNFL and ppVD were calculated in the nasal, inferior, temporal, and superior quadrants. The changes in ppVD and pRNFL in the two groups were analyzed. RESULTS: Compared with CON group, the nasal and superior ppVDs decreased in the NDR group (all P<0.01). The thickness of the nasal pRNFL decreased significantly (P<0.01), while the inferior, temporal and superior pRNFLs slightly decreased but not significant in the NDR group (all P>0.05). Person and Spearman correlation analysis of ppVD and pRNFL thickness in each quadrant of the NDR group showed a positive correlation between nasal and superior (all P<0.01), while inferior and temporal had no significant correlation (all P>0.05). There was no significant correlation between the HbA1c level and ppVD and pRNFL in any quadrant (all P>0.05). There was no significant correlation between the course of diabetes mellitus and ppVD and pRNFL in any quadrant (all P>0.05). CONCLUSION: ppVD and pRNFL decrease in eyes of children with type 1 diabetes before clinically detectable retinopathy and OCTA is helpful for early monitoring．.

Liquid-Phase Adsorption Behavior of ß-D-Glucooligosaccharides When Using Activated Carbon for Separation, and the Antioxidant Stress Activity of Purified Fractions.

The adsorption characteristics of ß-glucooligosaccharides on activated carbon and the purification were systematically investigated. The maximum adsorption capacity of activated carbon reached 0.419 g/g in the optimal conditions. The adsorption behavior was described to be monolayer, spontaneous, and exothermic based on several models' fitting results. Five fractions with different degrees of polymerization (DPs) and structures of ß-glucooligosaccharides were obtained by gradient ethanol elution. 10E mainly contained disaccharides with dp2a (G1→6G) and dp2b (G1→3G). 20E possessed trisaccharides with dp3a (G1→6G1→3G) and dp3b (G1→3G1→3G). 30E mainly consisted of dp3a and dp4a (G1→3G1→3(G1→6)G), dp4b (G1→6G1→3G1→3G), and dp4c (G1→3G1→3G1→3G). In addition to tetrasaccharides, 40E and 50E also contained pentasaccharides and hexasaccharides with ß-(1→3)-linked or ß-(1→6)-linked glucose residues. All fractions could inhibit the accumulation of intracellular reactive oxygen species (ROS) in H2O2-induced Caco-2 cells, and they could improve oxidative stress damage by increasing the activity of superoxide dismutase (SOD) and reduced glutathione (GSH), which were related to their DPs and structures. 50E with high DPs showed better anti-oxidative stress activity.

[Development of a Prognostic Model for Overall Survival Adult Patients with Core Binding Factor Acute Myeloid Leukaemia].

OBJECTIVE: To analyze the factors affecting overall survival (OS) of adult patients with core-binding factor acute myeloid leukemia (CBF-AML) and establish a prediction model. METHODS: A total of 216 newly diagnosed patients with CBF-AML in the First Affiliated Hospital of Zhengzhou University from May 2015 to July 2021 were retrospectively analyzed. The 216 CBF-AML patients were divided into the training and the validation cohort at 7∶3 ratio. The Cox regression model was used to analyze the clinical factors affecting OS. Stepwise regression was used to establish the optimal model and the nomogram. Receiver operating characteristic (ROC) curve, calibration curve and decision curve analysis (DCA) were used to evaluate the model performance. RESULTS: Age(≥55 years old), peripheral blood blast(≥80%), fusion gene (AML1-ETO), KIT mutations were identified as independent adverse factors for OS. The area under the ROC curve at 3-year was 0.772 and 0.722 in the training cohort and validation cohort, respectively. The predicted value of the calibration curve is in good agreement with the measured value. DCA shows that this model performs better than a single factor. CONCLUSION: This prediction model is simple and feasible, and can effectively predict the OS of CBF-AML, and provide a basis for treatment decision.

LncRNA SNHG26 promotes gastric cancer progression and metastasis by inducing c-Myc protein translation and an energy metabolism positive feedback loop.

Metastasis is a bottleneck in cancer treatment. Studies have shown the pivotal roles of long noncoding RNAs (lncRNAs) in regulating cancer metastasis; however, our understanding of lncRNAs in gastric cancer (GC) remains limited. RNA-seq was performed on metastasis-inclined GC tissues to uncover metastasis-associated lncRNAs, revealing upregulated small nucleolar RNA host gene 26 (SNHG26) expression, which predicted poor GC patient prognosis. Functional experiments revealed that SNHG26 promoted cellular epithelial-mesenchymal transition and proliferation in vitro and in vivo. Mechanistically, SNHG26 was found to interact with nucleolin (NCL), thereby modulating c-Myc expression by increasing its translation, and in turn promoting energy metabolism via hexokinase 2 (HK2), which facilitates GC malignancy. The increase in energy metabolism supplies sufficient energy to promote c-Myc translation and expression, forming a positive feedback loop. In addition, metabolic and translation inhibitors can block this loop, thus inhibiting cell proliferation and mobility, indicating potential therapeutic prospects in GC.