Characterization of a mouse-adapted strain of bat severe acute respiratory syndrome-related coronavirus.

Bats carry genetically diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Some of them utilize human angiotensin-converting enzyme 2 (hACE2) as a receptor and cannot efficiently replicate in wild-type mice. Our previous study demonstrated that the bat SARSr-CoV rRsSHC014S induces respiratory infection and lung damage in hACE2 transgenic mice but not wild-type mice. In this study, we generated a mouse-adapted strain of rRsSHC014S, which we named SMA1901, by serial passaging of wild-type virus in BALB/c mice. SMA1901 showed increased infectivity in mouse lungs and induced interstitial lung pneumonia in both young and aged mice after intranasal inoculation. Genome sequencing revealed mutations in not only the spike protein but the whole genome, which may be responsible for the enhanced pathogenicity of SMA1901 in wild-type BALB/c mice. SMA1901 induced age-related mortality similar to that observed in SARS and COVID-19. Drug testing using antibodies and antiviral molecules indicated that this mouse-adapted virus strain can be used to test prophylactic and therapeutic drug candidates against SARSr-CoVs. IMPORTANCE The genetic diversity of SARSr-CoVs in wildlife and their potential risk of cross-species infection highlights the importance of developing a powerful animal model to evaluate the antibodies and antiviral drugs. We acquired the mouse-adapted strain of a bat-origin coronavirus named SMA1901 by natural serial passaging of rRsSHC014S in BALB/c mice. The SMA1901 infection caused interstitial pneumonia and inflammatory immune responses in both young and aged BALB/c mice after intranasal inoculation. Our model exhibited age-related mortality similar to SARS and COVID-19. Therefore, our model will be of high value for investigating the pathogenesis of bat SARSr-CoVs and could serve as a prospective test platform for prophylactic and therapeutic candidates.

Protective effect of fermented Diospyros lotus L. extracts against the high glucose-induced apoptosis of MIN6 cells.

Date plum persimmon (Diospyros lotus L.) is a fruit crop from the Ebenaceae family. Its microorganism-fermented extract (DPEML) was shown to exhibit a hypoglycemic effect in our previous work. Here, we investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF, and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. In summary, our study suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus. PRACTICAL APPLICATIONS: We investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. We suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus.

Separation, identification, and molecular docking of tyrosinase inhibitory peptides from the hydrolysates of defatted walnut (Juglans regia L.) meal.

Defatted walnut meal protein was hydrolyzed using alcalase to yield tyrosinase inhibitory peptides. After separation by ultrafiltration and Sephadex G-25, the fraction with the highest tyrosinase inhibitory activity was identified using liquid chromatography-tandem mass spectrometry and 606 peptides were obtained. Then, molecular docking was used to screen for tyrosinase inhibitory peptides and to clarify the theoretical interaction mechanism between the peptides and tyrosinase. A peptide with the sequence Phe-Pro-Tyr (FPY, MW: 425.2 Da) was identified and the synthesized peptide inhibited tyrosine monophenolase and diphenolase with IC50 values of 1.11 ± 0.05 and 3.22 ± 0.09 mM, respectively. The inhibition of tyrosinase by FPY was competitive and reversible. Good stability of FPY toward digestion was observed in an in vitro gastrointestinal digestion simulation experiment. These results indicated that FPY can be used as a potential tyrosinase inhibitor in the food, medicine, and cosmetics industries.

Preparation and characterization of selenium nanoparticles decorated by Spirulina platensis polysaccharide.

Selenium nanoparticles (SeNPs) have attracted lots of attention recently owing to their excellent bioavailability and low toxicity. However, the stability of SeNPs needs to be improved. To enhance the stability of SeNPs, we used Spirulina platensis polysaccharides (SPs) as stabilizers to prepare SPs-SeNPs. The SPs-SeNPs were spherical, with a mean particle size of 73.42 ± 0.69 nm. The optimal preparation conditions for the SPs-SeNPs were a SPs concentration of 100 mg/L, ascorbic acid/sodium selenite concentration ratio of 3:1 and reaction time of 6 hr. The prepared SPs-SeNPs were stable for 75 d at 4°C. Furthermore, MTT assay showed that the median toxic concentration (TC50 ) of SPs-SeNPs was approximately 22,000 µg/L. In contrast, the TC50 value of selenite was approximately 400 µg/L, which confirmed that the cytotoxicity of SPs-SeNPs on RAW264.7 cells was significantly reduced compared with sodium selenite. In conclusion, SPs can improve the stability of SeNPs. The toxicity of SPs-SeNPs is lower than that of sodium selenite. PRACTICAL APPLICATIONS: Spirulina platensis polysaccharides can improve the stability and reduce the toxicity of selenium nanoparticles. It can lay the foundation for the comprehensive utilization of S. platensis and the development and research of polysaccharide nano-selenium complexes.

[Quality evaluation of Salvianolate for Injection by quantitative analysis of multi-components with single-marker].

Developing high-quality standard is useful for promoting the quality of traditional Chinese medicine injections, which could be evaluated by establishing the comprehensive quality control method. A method for simultaneous determination of salvianolic acid B, rosmarinic acid and lithospermic acid in Salvianolate for Injection was developed for quantitative analysis of multi-components with single-marker(QAMS). ZORBAX Eclipse Plus C_(18) chromatographic column was adopted, with 0.1% phosphoric acid and acetonitrile as mobile phase. The flow rate was set at 1 mL·min~(-1). The column temperature was set at 20 ℃, and the detection wavelength was 286 nm. Salvianolic acid B was used as internal reference. The relative correction factors of rosmarinic acid and lithospermic acid(f_(s/i)) were 0.58 and 0.94, respectively. About 85% of substances in Salvianolate for Injection were quantified by the established QAMS method. The analysis of different batches of intermediates and preparations during four years showed that the contents of salvianolic acid B were 77.1%-81.5% in intermediates and 70.5%-80.1% in preparations; The total content of rosmarinic acid and lithospermic acid was about 6%. The ratio of rosmarinic acid to lithospermic acid was(3.4∶1-10∶1) and(2.5∶1-5∶1), respectively, which showed that the ratio was more stable in preparation. The QAMS method established is feasible for comprehensive quality control of multiple components of in Salvianolate for Injection.

SP94 Peptide-Functionalized PEG-PLGA Nanoparticle Loading with Cryptotanshinone for Targeting Therapy of Hepatocellular Carcinoma.

To achieve improved drug delivery efficiency to hepatocellular carcinoma (HCC), biodegradable poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NP), surface-modified with SP94 peptide, were designed for the efficient delivery of cryptotanshinone to the tumor for the treatment of HCC. Cryptotanshinone NP and SP94-NP were prepared by using nanoprecipitation. The physicochemical and pharmaceutical properties of the NP and SP94-NP were characterized, and the release kinetics suggested that both NP and SP94-NP provided continuous, slow release of cryptotanshinone for 48 h. The in vitro cellular experiment demonstrated that SP94-NP significantly enhanced the cellular uptake of cryptotanshinone and induced high cytotoxicity and cellular apoptosis of hepatocellular carcinoma (HepG2) cells. The in vivo detecting results of targeting effect using the Cy5.5 probe evidenced that SP94-NP showed an accumulation in tumor more efficiently than that of unconjugated ones. Meanwhile, SP94-NP exhibited the smallest tumor size than other groups and showed no toxicity to body. The results of this study provide a promising nanoplatform for the targeting of HCC.

Potato Spindle Tuber Viroid Modulates Its Replication through a Direct Interaction with a Splicing Regulator.

Viroids are circular noncoding RNAs (ncRNAs) that infect plants. Despite differences in the genetic makeup and biogenesis, viroids and various long ncRNAs all rely on RNA structure-based interactions with cellular factors for function. Viroids replicating in the nucleus utilize DNA-dependent RNA polymerase II for transcription, a process that involves a unique splicing form of transcription factor IIIA (TFIIIA-7ZF). Here, we provide evidence showing that potato spindle tuber viroid (PSTVd) interacts with a TFIIIA splicing regulator (ribosomal protein L5 [RPL5]) in vitro and in vivo PSTVd infection compromises the regulatory role of RPL5 over splicing of TFIIIA transcripts, while ectopic expression of RPL5 reduces TFIIIA-7ZF expression and attenuates PSTVd accumulation. Furthermore, we illustrate that the RPL5 binding site on the PSTVd genome resides in the central conserved region critical for replication. Together, our data suggest that viroids can regulate their own replication and modulate specific regulatory factors leading to splicing changes in only one or a few genes. This study also has implications for understanding the functional mechanisms of ncRNAs and elucidating the global splicing changes in various host-pathogen interactions.IMPORTANCE Viroids are the smallest replicons among all living entities. As circular noncoding RNAs, viroids can replicate and spread in plants, often resulting in disease symptoms. Potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids, requires a unique splicing form of transcription factor IIIA (TFIIIA-7ZF) for its propagation. Here, we provide evidence showing that PSTVd directly interacts with a splicing regulator, RPL5, to favor the expression of TFIIIA-7ZF, thereby promoting viroid replication. This finding provides new insights to better understand viroid biology and sheds light on the noncoding RNA-based regulation of splicing. Our discovery also establishes RPL5 as a novel negative factor regulating viroid replication in the nucleus and highlights a potential means for viroid control.

Mixed fermentation of Spirulina platensis with Lactobacillus plantarum and Bacillus subtilis by random-centroid optimization.

Spirulina platensis is a high-nutrient blue-green alga with a long history as a food supplement. In this study, the mixed fermentation of Spirulina with Lactobacillus plantarum and Bacillus subtilis was investigated using random-centroid optimization to improve deodorization of off-flavor and hydrolysis of protein. Fermented Spirulina with the maximum total viable counts of both organisms achieved best sensory characteristics and degree of proteolysis among those with different maximum biomass. The mixed fermentation noticeably reduced the volatile compounds of Spirulina, and yielded the highest relative contents (>85%) of acetoin and other odorants collectively producing a creamy aroma. Approximately one-third of the Spirulina proteins were hydrolyzed, yielding over 16% polypeptides and increasing the ratio of essential amino acids to total free amino acids to 1.5-fold compared with unfermented Spirulina. Fermentation with B. subtilis and L. plantarum effectively improved the odor and protein availability of Spirulina.

Protective effect of Letinous edodes foot peptides against ethanol­induced liver injury in L02 cells.

The aim of the present study was to evaluate the protective effect and mechanism of Letinous edodes foot peptides on ethanol­induced L02 cells. A cell model of ethanol­induced damage was established in vitro to study the effects of the Letinous edodes foot peptides on human L02 hepatocytes. The expression and activity of superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), following treatment were examined to determine the anti­alcoholism and hepatoprotective functions of Letinous edodes foot peptides. Taking Letinous edodes foot peptides prior to ethanol exposure was more beneficial, which significantly increased SOD activity and the mRNA expression of ADH and ALDH suppressed by ethanol. In addition, the intracellular MDA content, and AST and ALT activity decreased in ethanol­induced L02 cells pretreated with the peptides, when compared with the control. Furthermore, Letinous edodes foot peptides inhibited the ethanol­induced activation of the proinflammatory cytokines, interleukin­6 and tumor necrosis factor­α, and promoted the metabolic regulation factors, AMP­activated protein kinase­α2 and peroxisome proliferator­activated receptor­α.

Antioxidant and hypoglycemic effects of Diospyros lotus fruit fermented with Microbacterium flavum and Lactobacillus plantarum.

Diospyros lotus, a member of the Ebenaceae family, has long been used as a traditional sedative in China. In this study, the antioxidant and hypoglycemic effects of non-fermented and microorganism-fermented D. lotus were explored. The total phenolic and vitamin C contents of microorganism-fermented D. lotus for 24-72 h were less than those of non-fermented. High-performance liquid chromatography showed that the tannic, catechinic, and ellagic acid contents increased significantly upon fermentation for 24 h. D. lotus fermented with Microbacterium flavum for 24 h exhibited the highest DPPH radical scavenging activity (IC50 = 4.18 µg mL-1), and the highest ABTS radical scavenging activity was exhibited at 72 h of fermentation (IC50 = 29.18 µg mL-1). The anti-α-glucosidase activity of fermented D. lotus was higher (2.06-4.73-fold) than that of non-fermented one. Thus, fermented D. lotus is a useful source of natural antioxidants, and a valuable food, exhibiting antioxidant and hypoglycemic properties.

Isolation, Purification and Structural Characterization of Two Novel Water-Soluble Polysaccharides from Anredera cordifolia.

Anredera cordifolia, a climber and member of the Basellaceae family, has long been a traditional medicine used for the treatment of hyperglycemia in China. Two water-soluble polysaccharides, ACP1-1 and ACP2-1, were isolated from A. cordifolia seeds by hot water extraction. The two fractions, ACP1-1 and ACP2-1 with molecular weights of 46.78 kDa ± 0.03 and 586.8 kDa ± 0.05, respectively, were purified by chromatography. ACP1-1 contained mannose, glucose, galactose in a molar ratio of 1.08:4.65:1.75, whereas ACP2-1 contained arabinose, ribose, galactose, glucose, mannose in a molar ratio of 0.9:0.4:0.5:1.2:0.9. Based on methylation analysis, ultraviolet and Fourier transform-infrared spectroscopy, and periodate oxidation the main backbone chain of ACP1-1 contained (1→3,6)-galacturonopyranosyl residues interspersed with (1→4)-residues and (1→3)-mannopyranosyl residues. The main backbone chain of ACP2-1 contained (1→3)-galacturonopyranosyl residues interspersed with (1→4)-glucopyranosyl residues.