Programmable Biosynthesis of Plant-Derived 4'-Deoxyflavone Glycosides by an Unconventional Yeast Consortium.

Previous data established 4'-deoxyflavone glycosides (4'-DFGs) as important pharmaceutical components in the roots of rare medical plants like Scutellaria baicalensis Georgi. Extracting these compounds from plants involves land occupation and is environmentally unfriendly. Therefore, a modular ("plug-and-play") yeast-consortium platform is developed to synthesize diverse 4'-DFGs de novo. By codon-optimizing glycosyltransferase genes from different organisms for Pichia pastoris, six site-specific glycosylation chassis are generated to be capable of biosynthesizing 18 different 4'-DFGs. Cellular factories showed increased 4'-DFG production (up to 18.6-fold) due to strengthened synthesis of UDP-sugar precursors and blocked hydrolysis of endogenous glycosides. Co-culturing upstream flavone-synthesis-module cells with downstream glycoside-transformation-module cells alleviated the toxicity of 4'-deoxyflavones and enabled high-level de novo synthesis of 4'-DFGs. Baicalin is produced at the highest level (1290.0 mg L-1 ) in a bioreactor by controlling the consortium through carbon-source shifting. These results provide a valuable reference for biosynthesizing plant-derived 4'-DFGs and other glycosides with potential therapeutic applications.

Qualitative and Quantitative Analysis of Ejiao-Related Animal Gelatins through Peptide Markers Using LC-QTOF-MS/MS and Scheduled Multiple Reaction Monitoring (MRM) by LC-QQQ-MS/MS.

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.

Dysregulation of Cytosolic c-di-GMP in Edwardsiella piscicida Promotes Cellular Non-Canonical Ferroptosis.

Programmed cell death plays an important role in modulating host immune defense and pathogen infection. Ferroptosis is a type of inflammatory cell death induced by intracellular iron-dependent accumulation of toxic lipid peroxides. Although ferroptosis has been associated with cancer and other sterile diseases, very little is known about the role of ferroptosis in modulating host-pathogen interactions. We show that accumulation of the secondary messenger bis-(3',5')-cyclic dimeric GMP (c-di-GMP) in the pathogenic bacterium Edwardsiella piscicida (E. piscicida) triggers a non-canonical ferroptosis pathway in infected HeLa cells. Moreover, we observed that the dysregulation of c-di-GMP in E. piscicida promotes iron accumulation, mitochondrial dysfunction, and production of reactive oxygen species, all of which that can be blocked by iron chelator. Importantly, unlike classical ferroptosis that is executed via excess lipid peroxidation, no lipid peroxidation was detected in the infected cells. Furthermore, lipoxygenases inhibitors and lipophilic antioxidants are not able to suppress morphological changes and cell death induced by E. piscicida mutant producing excess c-di-GMP, and this c-di-GMP dysregulation attenuates bacterial virulence in vivo. Collectively, our results reveal a novel non-canonical ferroptosis pathway mediated by bacterial c-di-GMP and provide evidence for a role of ferroptosis in the regulation of pathogen infection.

Transposon insertion mutation of Antarctic psychrotrophic fungus for red pigment production adaptive to normal temperature.

Polar regions are rich in microbial and product resources. Geomyces sp. WNF-15A is an Antarctic psy chrotrophic filamentous fungus producing high quality red pigment with potential for industrial use. However, efficient biosynthesis of red pigment can only realize at low temperature, which brings difficult control and high cost for the large-scale fermentation. This study aims to develop transposon insertion mutation method to improve cell growth and red pigment production adaptive to normal temperature. Genetic manipulation system of this fungus was firstly developed by antibiotic marker screening, protoplast preparation and transformation optimization, by which transformation efficiency of ∼50% was finally achieved. Then transposable insertion systems were established using Helitron, Fot1, and Impala transposons. The transposition efficiency reached 11.9%, 9.4%, and 4.6%, respectively. Mutant MP1 achieved the highest red pigment production (OD520 of 39) at 14°C, which was 40% higher than the wild-type strain. Mutant MP14 reached a maximum red pigment production (OD520 of 14.8) at 20°C, which was about twofold of the wild-type strain. Mutants MP2 and MP10 broke the repression mechanism of red pigment biosynthesis in the wild-type and allowed production at 25°C. For cell growth, eight mutants grew remarkably better (12%∼30% biomass higher) than the wild-type at 25°C. This study established an efficient genetic manipulation and transposon insertion mutation platform for polar filamentous fungus. It provides reference for genetic breeding of psychrotrophic fungi from polar and other regions.

Minos and Restless transposon insertion mutagenesis of psychrotrophic fungus for red pigment synthesis adaptive to normal temperature.

The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD520 of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD520 of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.

High-level living cell production of cytidine-5'-diphosphocholine in metabolically engineered yeast.

Industrial production of neuroprotective drug CDP-choline is accomplished via permeabilized or lysed cell biotransformation because of the inefficient penetration of substrates into intact cells. We previously proposed a novel one-step living cell method for CDP-choline production by engineered yeast, but obtained low titer and molar yield. This study develops a high-production strain with improved molar yield by metabolic engineering strategies. The selective markers previously integrated into host cell were recovered for facilitating genetic modification, which however resulted a strain with improved CDP-choline titer and molar yield to CMP. Knockout of 5'-NT or CDA in CMP sinking pathway but not APY in CTP sinking pathway further improved CDP-choline titer and molar yield to CMP. However, overexpression of seven enzymes in CTP synthetic pathway showed no positive functions. Finally, optimization of CMP and choline phosphate levels for the optimized recombinant strains achieved a high-level CDP-choline of ~30 g/L, which was enhanced by 400% compared to the previous work. Also, the molar yield of CDP-choline to CMP increased from 40% to 84.7%. The titer and molar yield are comparable to the reported permeabilized or lysed cell based biotransformation methods. It represents a novel and competitive paradigm for the potential industrial production of CDP-choline.

Isolation of a Virulent Aeromonas salmonicida subsp. masoucida Bacteriophage and Its Application in Phage Therapy in Turbot (Scophthalmus maximus).

Aeromonas salmonicida is an aquatic pathogen that can infect a variety of fish. Phage therapy has been applied to treat bacterial infections. In this study, we obtained three A. salmonicida subsp. masoucida phage isolates from sewage, and one phage (vB_AsM_ZHF) exhibited the best antibacterial effect, based on in vitro kinetics experiments. Sequencing indicated that the vB_AsM_ZHF genome is 161,887 bp (41.24% C+G content) with 237 predicted open reading frames. No antibiotic resistance or virulence genes were detected in the complete genome, which is a requirement for phage therapy safety. Intraperitoneal injection of phage vB_AsM_ZHF into turbot at 8 × 104 PFU/fish rescued turbot from A. salmonicida subsp. masoucida injection and reduced the bacterial burden by 1 order of magnitude. Injection of vB_AsM_ZHF also decreased levels of inflammatory cell infiltration in muscle tissue, cytokines interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) in serum and the expression of the inflammatory factors IL-1ß, IL-6, IFN-γ, transforming growth factor ß, TNF-α, and hepcidin in the liver, spleen, and head kidney of turbot. Phage vB_AsM_ZHF demonstrated antibacterial ability in vitro and in vivo and significantly reduced mortality in turbot challenged by A. salmonicida subsp. masoucida. This study revealed that phage vB_AsM_ZHF can effectively treat the infection caused by A. salmonicida subsp. masoucida in turbot. IMPORTANCEA. salmonicida is an aquatic pathogen that can infect different fish and causes economic loss to the global aquaculture industry. Clinical strains of A. salmonicida have developed multidrug resistance, and phage therapy is being evaluated for controlling bacterial infections. Phages are biological antibacterial agents and have the potential to be therapeutic agents against multidrug-resistant bacteria. In this study, three A. salmonicida subsp. masoucida phages were isolated from sewage, and their biological behaviors were characterized. The newly isolated phage vB_AsM_ZHF could inhibit A. salmonicida subsp. masoucida infection in vitro and in vivo, suggesting that it may be an alternative strategy to antibiotics for protecting fish against multidrug-resistant A. salmonicida subsp. masoucida in the aquaculture industry.

Zebrafish gasdermin E cleavage-engaged pyroptosis by inflammatory and apoptotic caspases.

As the executor of pyroptosis known to date, gasdermins (GSDMs), consists of GSDMA, GSDMB, GSDMC, GSDMD, GSDME and pejvakin, might play critical roles in anti-bacterial infection as well as inflammatory diseases. However, zebrafish only harbors a pair of Gsdme (Gsdmea/b), and their activation mechanisms remain largely unknown. Herein, we investigate the activation mechanism of Gsdmea/b cleaved by inflammatory and apoptotic caspases in zebrafish，and found that Gsdmea/b are equally cleaved by Caspase 19b, a sister of Caspy2, but not Caspy. Moreover, the zebrafish apoptotic effector caspases, including Caspase 3a/b and Caspase 7, also can cleave Gsdmea/b at the same sites as inflammatory caspases recognized. Importantly, our results reveal that Caspase 8a/b can cleave Gsdmeb, but only Caspase 8a can cleave Gsdmea. Taken together, these findings suggest that zebrafish Gsdmea/b can concurrently function as GSDMD and GSDME in mammals, which will contribute to better understanding the mechanism of pyroptosis activation in teleost, as well as provide a clue for drug screening model against inflammatory diseases.

Estimation of leaf water content from hyperspectral data of different plant species by using three new spectral absorption indices.

The leaf equivalent water thickness (EWT, g cm-2) and fuel moisture content (FMC, %) are key variables in ecological and environmental monitoring. Although a variety of hyperspectral vegetation indices have been developed to estimate the leaf EWT and FMC, most of these indices are defined considered two or three specific bands for a specific plant species, which limits their applicability. In this study, we proposed three new spectral absorption indices (SAI970, SAI1200, and SAI1660) for various plant types by considering the symmetry of the spectral absorption at 970 nm, 1200 nm and 1660 nm and spectral heterogeneity of different leaves. The indices were calculated considering the absorption peak and shoulder bands of each leaf instead of the same specific bands for all leaves. A pooled dataset of three tree species (camphor (VX), capricorn (VJ), and red-leaf plum (VL)) was used to test the performance of the SAIs in terms of the leaf EWT and FMC estimation. The results indicated that, first, SAI1200 was more suitable for estimating the EWT than FMC, whereas SAI970 and SAI1660 were more suitable for estimating the FMC. Second, SAI1200 achieved the most accurate estimation of the EWT with a cross-validation coefficient of determination (Rcv2) of 0.845 and relative cross-validation root mean square error (rRMSEcv) of 8.90%. Third, SAI1660 outperformed the other indices in estimating the FMC at the leaf level, with an Rcv2 of 0.637 and rRMSEcv of 8.56%. Fourth, SAI970 achieved a moderate accuracy in estimating the EWT (Rcv2 of 0.25 and rRMSEcv of 19.68%) and FMC (Rcv2 of 0.275 and rRMSEcv of 12.10%) at the leaf level. These results can enrich the application of the SAIs and demonstrate the potential of using SAI1200 to determine the leaf EWT and SAI1660 to obtain the leaf FMC among various plant types.

Practical protocols for timed artificial insemination of jennies using cooled or frozen donkey semen.

BACKGROUND: With the expansion of the donkey industry, timed artificial insemination (TAI) is becoming increasingly important in the reproductive management of jennies, however, TAI has not been widely investigated in donkeys. OBJECTIVES: To develop efficient TAI protocols for cooled or frozen semen in jennies, based around ovulation induction with a GnRH analogue. STUDY DESIGN: Experimental exploratory study. METHODS AND RESULTS: In experiment 1, the effects of different GnRH analogue (deslorelin) doses, follicle diameter (FD) at induction, repeated use of a GnRH analogue, and the influence of season on induction efficiency, as well as distribution of ovulations over time after induction were investigated. Induction efficiency was sufficient with 2.2 mg deslorelin (≥90% ovulation within 48 hours of treatment). Ovulation rate between 24 and 48 hours was highest when the FD at treatment was 31-35 mm, as compared to 25-30 mm or 36-40 mm. Repeated use of deslorelin or treatment during different seasons had no effect on induction efficiency. About 70% of ovulations occurred between 32 and 48 hours, and highest incidence of ovulation was at 36-38 hours after induction. In experiment 2, TAI using cooled semen (1 × 109 motile sperm in a 10 mL volume) was performed once at 8 hours after induction (n = 59). Pregnancy rate after TAI with cooled semen was 49.2% (29/59). In experiment 3, jennies were inseminated twice with 10 (n = 23), 5 (n = 31), 3 (n = 32), 2 (n = 82) and 1 (n = 66) straws (more than 50 × 106 motile spermatozoa in each 0.5 mL straw) of frozen semen at 34 and 42 hours after induction. The pregnancy rates were 30.4%, 35.5%, 34.4%, 29.3% and 28.8%, respectively (P > 0.05). MAIN LIMITATIONS: In the frozen semen trial, 22.5% (68/302) jennies were excluded after failure to ovulate during the appropriate time interval. In addition, there were no control groups for the AI trials. CONCLUSION: When FD reaches 31-35 mm, a donkey jenny can be inseminated once using cooled semen at 8 hours or twice using frozen semen at 34 and 42 hours after deslorelin treatment. The frozen semen TAI protocol resulted in acceptable pregnancy rates using 1 × 108 motile spermatozoa per cycle.

Characteristics of follicular dynamics and reproductive hormone profiles during oestrous cycles of jennies over an entire year.

Although donkeys have been domesticated for over 6,000 years, limited information is available concerning their reproductive physiology, especially under intensive rearing conditions. The aims of this experiment were to study follicular dynamics and reproductive hormone variation in jennies during the inter-ovulatory interval in different seasons. A total of 12 continuous cycles of six Dezhou Black (DB) donkey jennies were examined in four different seasons. The diameters of the six largest follicles of each jenny were measured daily by ultrasonography, and blood samples were collected at fixed times for reproductive hormone assays. The results demonstrated that most jennies displayed regular oestrous cycles in all seasons. The follicular dynamics were similar in Spring, Summer and Winter, while the jennies had longer oestrous cycles with delayed follicular deviation and dominant selection in Autumn. At least two follicular waves were observed in each oestrous cycle, throughout the study, but two jennies presented oestrous cycles with three follicular waves in the Autumn. The numbers of follicular waves were consistent with the numbers of FSH surges. Oestrous characteristics of the jennies in a large herd were also analysed. The results showed that the rates of regular oestrous cycles were 83.1% (265/319), 89.6% (215/240), 80.2% (235/293) and 77.1% (178/231), with 26.4% (70/265), 19.5% (42/215), 22.1% (52/235) and 23.0% (41/178) double ovulation rates in Spring, Summer, Autumn and Winter, respectively. The results presented may be useful for donkey farms in the design of breeding strategies.

Donkey genomes provide new insights into domestication and selection for coat color.

Current knowledge about the evolutionary history of donkeys is still incomplete due to the lack of archeological and whole-genome diversity data. To fill this gap, we have de novo assembled a chromosome-level reference genome of one male Dezhou donkey and analyzed the genomes of 126 domestic donkeys and seven wild asses. Population genomics analyses indicate that donkeys were domesticated in Africa and conclusively show reduced levels of Y chromosome variability and discordant paternal and maternal histories, possibly reflecting the consequences of reproductive management. We also investigate the genetic basis of coat color. While wild asses show diluted gray pigmentation (Dun phenotype), domestic donkeys display non-diluted black or chestnut coat colors (non-Dun) that were probably established during domestication. Here, we show that the non-Dun phenotype is caused by a 1 bp deletion downstream of the TBX3 gene, which decreases the expression of this gene and its inhibitory effect on pigment deposition.

A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii.

Malonyl-CoA is a key metabolic molecule that participates in a diverse range of physiological responses and can act as a building block for a variety of value-added pharmaceuticals and chemicals. The cytosolic malonyl-CoA concentration is usually very low, and thus dynamic metabolic control of malonyl-CoA variation will aid its stable formation and efficient consumption. We developed a synthetic malonyl-CoA metabolic oscillator in yeast. A synthetic regulatory protein, Prm1-FapR, was constructed by fusing a yeast transcriptional activator, Prm1, with a bacterial malonyl-CoA-sensitive transcription repressor, FapR. Two oppositely regulated biosensors were then engineered. A total of 18 hybrid promoter variants were designed, each carrying the operator sequence (fapO) of FapR and the core promoter of PAOX1 (cPAOX1), which is naturally regulated by Prm1. The promoter activities of these variants, regulated by Prm1-FapR, were tested. Through this process, a sensor for Prm1-FapR/(-52)fapO-PAOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of PGAP with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/PGAP-(+22) fapO that contained a repression/derepression regulation module. Both sensors were subsequently integrated into a single cell, which exhibited correct metabolic switching of eGFP and mCherry reporters following manipulation of cytosolic malonyl-CoA levels. The Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were also used to control the malonyl-CoA source and sink pathways, respectively, for the synthesis of 6-methylsalicylic acid. This finally led to an oscillatory metabolic mode of cytosolic malonyl-CoA. Such a metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.

Engineering substrate and energy metabolism for living cell production of cytidine-5'-diphosphocholine.

Cytidine-5'-diphosphocholine (CDP-choline) is a widely used neuroprotective drug for multiple indications. In industry, CDP-choline is synthesized by a two-step cell culture/permeabilized cell biotransformation method because substrates often do not enter cells in an efficient manner. This study develops a novel one-step living cell fermentation method for CDP-choline production. For this purpose, the feasibility of Pichia pastoris as a chassis was demonstrated by substrate feeding and CDP-choline production. Overexpression of choline phosphate cytidylyltransferase and choline kinase enhanced the choline transformation pathway and improved the biosynthesis of CDP-choline. Furthermore, co-overexpression of ScHnm1, which is a heterologous choline transporter, highly improved the utilization of choline substrates, despite its easy degradation in cells. This strategy increased CDP-choline titer by 55-folds comparing with the wild-type (WT). Overexpression of cytidine-5'-monophosphate (CMP) kinase and CDP kinase in the CMP transformation pathway showed no positive effects. An increase in the ATP production by citrate stimulation or metabolic pathway modification further improved CDP-choline biosynthesis by 120%. Finally, the orthogonal optimization of key substrates and pH was carried out, and the resulting CDP-choline titer (6.0 g/L) at optimum conditions increased 88 times the original titer in the WT. This study provides a new paradigm for CDP-choline bioproduction by living cells.

RNA-sequencing based bone marrow cell transcriptome analysis reveals the potential mechanisms of E'jiao against blood-deficiency in mice.

As a health-care food and traditional Chinese medicine, E'jiao, from the skin of Equus animus L, has been used to nourish blood in China for more than 2000 years. In modern medicine, there are also evidences indicate it has a beneficial effect on chemotherapy-caused blood deficiency. However, its mechanism of action for blood invigoration remains unclear. In the present study, we investigated the hematopoietic effect of E'jiao in 5-Fluorouracil-treated mice. In addition to the counting of bone marrow nucleated cells (BMNCs), flow cytometry was used to detect the population of hematopoietic stem cells (HSCs), and colony-forming unit (CFU) was used to assay the differentiation ability of hematopoietic progenitor cells (HPCs). Gene expression profiles of bone marrow cells were obtained from RNA sequencing (RNA-seq) and differentially expressed genes (DEGs) were analyzed with an emphasis on hematopoiesis-related pathways. The results show that E'jiao promotes the proliferation of both BMNCs and HSCs, as well as the differentiation of HPCs. By providing a hematopoiesis-related molecular regulatory network of E'jiao, we point out that the mechanism of E'jiao is associated with pathways including ECM-receptor interaction, Wnt signaling pathway, PI3K-Akt signaling pathway, TGF-beta signaling pathway, Hematopoietic cell lineage and Osteoclast differentiation, in which Ibsp, Col1a1, Col1a2, Notum, Sost, Dkk1, Irx5, Irx3 and Dcn are the key regulatory molecules. These findings provide valuable molecular basis for the mechanism of action of E'jiao.

CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris.

BACKGROUND: Pichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris. RESULTS: A non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris. CONCLUSIONS: A CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

Donkey milk oligosaccharides influence the growth-related characteristics of intestinal cells and induce G2/M growth arrest via the p38 pathway in HT-29 cells.

Donkey milk is considered to be a valuable nutritional source. Deeper knowledge of the constituents of donkey milk is necessary. As multifunctional components of milk, oligosaccharides have been reported to have the potential to support intestine development. We studied the composition and content of donkey milk oligosaccharides (DMOs). Sialylated oligosaccharides were found to be the primary oligosaccharides in DMOs, consisting of 3'-sialyllactose (SL) and 6'-SL. The amount of 3'-SL and 6'-SL in donkey milk was 18.3 ± 0.7 mg L-1 and 33.1 ± 0.7 mg L-1, respectively. Moreover, we found that DMOs induced differentiation, promoted apoptosis and inhibited proliferation in HT-29, Caco-2 and HIEC cells in a concentration-dependent manner, suggesting that DMOs promote maturation of intestinal epithelial cells. The mechanism of the DMOs' effects on HT-29 cells was associated with activation of the p38 pathway and cell cycle arrest at the G2/M phase. Our research will help understand the biological functions of DMOs and assess their potential roles in infant nutrition.

[Research progress on quality control methods of traditional Chinese medicine glues].

Traditional Chinese medicine( TCM) glues,including leather glues,horn glues,nail glues and bone glues,have a long application history and unique characteristics. In recent years,their market demand has increased year by year because of their remarkable curative efficacy and nourishing effects,which leads to insufficient supply of raw material resources,and widespread use of fake and inferior products,seriously affecting the reputation of TCM glues and drug safety. In this context,the establishment of a more specific quality detection method for the TCM glues according to their specific characteristics can effectively improve the quality control level,promote rational use,and have a far-reaching impact on the industrial development of TCM glues. In this paper,the classification of TCM glues,as well as the production and application status of their representative( Ejiao) were briefly introduced; the papers on quality control technologies of TCM glues,including traditional identification experience,authenticity identification,physical property determination,protein,peptide and amino acid contents determination,element analysis,biological evaluation,and brand protection technology of TCM glues,were reviewed,and their advantages and disadvantages were summarized and analyzed comprehensively.Based on the specific characteristics of TCM glues,such as complex material basis,unclear pharmacodynamic components and different production processes,it was proposed in this paper to research and develop information-rich,convenient,fast,and non-destructive analytical techniques for the quality control of TCM glues and brand protection of famous products,thus promoting the healthy development of TCM glues industry.