Oleanane-type triterpenoids from Sabia limoniacea and their anti-inflammatory activities.

Eighteen new oleanane-type triterpenoids were isolated from the stems of Sabia limoniacea, including sabialimon A (1), a triterpenoid with an unprecedented 6/6/6/7/7 pentacyclic skeleton and seventeen undescribed triterpenoids, sabialimons B-R (2 - 18), along with six previously described analogs (19 - 24). Their structures were fully elucidated via extensive spectroscopic analysis including 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), experimental electronic circular dichroism measurements and X-ray crystallographic studies. Compound 1 is the first triterpenoid that possesses a rare ring system (6/6/6/7/7) with an oxygen-bearing bridge between C-17 and C-18 and a hemiketal form at C-17, which is generated a larger ring by the degradation of C-28 and D/E-ring expansion. Biological evaluation revealed that sabialimon I (9), sabialimon K (11), sabialimon P (16) and 11,13(18)-oleanadien-28-hydroxymethyl 3-one (20) exhibited significantly inhibitory activities against nitric oxide (NO) release with IC50 values of 29.65, 23.41, 18.12 and 26.64 µM, respectively, as compared with the positive control (dexamethasone, IC50 value: 40.35 µM). Furthermore, sabialimon P markedly decreased the secretion of TNF-α, iNOS, IL-6 and NF-κB and inhibited the expression of COX-2 and NF-κB/p65 in LPS-induced RAW264.7 cells in a dose-dependent manner.

Klf4 methylated by Prmt1 restrains the commitment of primitive endoderm.

The second cell fate decision in the early stage of mammalian embryonic development is pivotal; however, the underlying molecular mechanism is largely unexplored. Here, we report that Prmt1 acts as an important regulator in primitive endoderm (PrE) formation. First, Prmt1 depletion promotes PrE gene expression in mouse embryonic stem cells (ESCs). Single-cell RNA sequencing and flow cytometry assays demonstrated that Prmt1 depletion in mESCs contributes to an emerging cluster, where PrE genes are upregulated significantly. Furthermore, the efficiency of extraembryonic endoderm stem cell induction increased in Prmt1-depleted ESCs. Second, the pluripotency factor Klf4 methylated at Arg396 by Prmt1 is required for recruitment of the repressive mSin3a/HDAC complex to silence PrE genes. Most importantly, an embryonic chimeric assay showed that Prmt1 inhibition and mutated Klf4 at Arg 396 induce the integration of mouse ESCs into the PrE lineage. Therefore, we reveal a regulatory mechanism for cell fate decisions centered on Prmt1-mediated Klf4 methylation.

Exploring the Efficacy of Noncovalent SARS-CoV-2 Main Protease Inhibitors: A Computational Simulation Analysis Study.

The SARS-CoV-2 main protease, as a key target for antiviral therapeutics, is instrumental in maintaining virus stability, facilitating translation, and enabling the virus to evade innate immunity. Our research focused on designing non-covalent inhibitors to counteract the action of this protease. Utilizing a 3D-QSAR model and contour map, we successfully engineered eight novel non-covalent inhibitors. Further evaluation and comparison of these novel compounds through methodologies including molecular docking, ADMET analysis, frontier molecular orbital studies, molecular dynamics simulations, and binding free energy revealed that the inhibitors N02 and N03 demonstrated superior research performance (N02 ΔGbind=-206.648 kJ/mol, N03 ΔGbind=-185.602 kJ/mol). These findings offer insightful guidance for the further refinement of molecular structures and the development of more efficacious inhibitors. Consequently, future investigations can draw upon these findings to unearth more potent inhibitors, thereby amplifying their impact in the treatment and prevention of associated diseases.

Comparison of genomic prediction methods for early growth traits of Inner Mongolia cashmere goats based on multi trait models.

Inner Mongolia cashmere goat is an excellent livestock breed formed through long-term natural selection and artificial breeding, and is currently a world-class dual-purpose breed producing cashmere and meat. Multi trait animal model is considered to significantly improve the accuracy of genetic evaluation in livestock and poultry, enabling indirect selection between traits. In this study, the pedigree, genotype, environment, and phenotypic records of early growth traits of Inner Mongolia cashmere goats were used to build multi trait animal model., Then three methods including ABLUP, GBLUP, and ssGBLUP wereused to estimate the genetic parameters and genomic breeding values of early growth traits (birth weight, weaning weight, average daily weight gain before weaning, and yearling weight). The accuracy and reliability of genomic estimated breeding value are further evaluated using the five fold cross validation method. The results showed that the heritability of birth weight estimated by three methods was 0.13-0.15, the heritability of weaning weight was 0.13-0.20, heritability of daily weight gain before weaning was 0.11-0.14, and the heritability of yearling weight was 0.09-0.14, all of which belonged to moderate to low heritability. There is a strong positive genetic correlation between weaning weight and daily weight gain before weaning, daily weight gain before weaning and yearling weight, with correlation coefficients of 0.77-0.79 and 0.56-0.67, respectively. The same pattern was found in phenotype correlation among traits. The accuracy of the estimated breeding values by ABLUP, GBLUP, and ssGBLUP methods for birth weight is 0.5047, 0.6694, and 0.7156, respectively; the weaning weight is 0.6207, 0.6456, and 0.7254, respectively; the daily weight gain before weaning was 0.6110, 0.6855, and 0.7357 respectively; and the yearling weight was 0.6209, 0.7155, and 0.7756, respectively. In summary, the early growth traits of Inner Mongolia cashmere goats belong to moderate to low heritability, and the speed of genetic improvement is relatively slow. The genetic improvement of other growth traits can be achieved through the selection of weaning weight. The ssGBLUP method has the highest accuracy and reliability in estimating genomic breeding value of early growth traits in Inner Mongolia cashmere goats, and is significantly higher than that from ABLUP method, indicating that it is the best method for genomic breeding of early growth weight in Inner Mongolia cashmere goats.

Comparative Investigation on the Phytochemicals and Biological Activities of Jackfruit (Artocarpus heterophyllus Lam.) pulp from Five Cultivars.

Jackfruit is one of the major tropical fruits, but information on the phytochemicals and biological benefits of its pulp is limited. In this study, the phytochemicals and biological activities including antioxidant, antitumor and anti-inflammatory activities of five jackfruit pulp cultivars (M1, M2, M3, M7 and T5) were comparatively investigated. A total of 11 compounds were identified in all cultivars of jackfruit pulp, among which 4-hydroxybenzoic acid, caffeic acid, ferulic acid and tryptophan N-glucoside were reported for the first time in jackfruit. T5 exhibited the highest total phenolic content (7.69 ± 0.73 mg GAE/g DW), antioxidant capacity (109.8, 96.7 and 207 mg VCE/g DW for DPPH, ABTS and FRAP, respectively), antitumor activity (80.31%) and anti-inflammatory activity (78.44%) among five cultivars. These results can provide a reference for growers to choose jackfruit cultivar and offer an insight into the industrial application of jackfruit pulp derived-products.

[Traditional Chinese medicine health care from "long term taking" drugs in Shen Nong's Classic of Materia Medica].

"Taking drugs for a long term" is a qualitative expression of medication method based on the efficacy and safety of Chinese medicine, and the study on it is conducive to the full utilization of the efficacy and rational use of drugs. There are 148 drugs that can be taken for a long time recorded in Shen Nong's Classic of Materia Medica, accounting for 41% of the total drugs. This paper analyzed three-grade classification, natural qualities, four properties and five flavors, and efficacy features of the "long-term taking" drugs(LTTD), thus exploring the herbal source of traditional Chinese medicine health care and the rationality of effect accumulation by long-term taking. It was found that there were more than 110 top-grade LTTD in Shen Nong's Classic of Materia Medica, most of which were herbs, with sweet flavor, flat property, and no toxicity. The efficacies were mainly making body feel light and agile(Qingshen) and prolonging life. Eighty-three LTTD were included in the Chinese Pharmacopoeia(2020 edition). In the modern classification, tonic LTTD accounted for the most, followed by damp-draining diuretic LTTD and exterior-releasing LTTD. Twenty LTTD were included in the "List of Medicinal and Edible Products" and 21 were in the "List of Products Used for Health-care Food", involving in various modern health care effects, such as enhancing immunity, assisting in reducing blood lipids, and anti-oxidation. Shen Nong's Classic of Materia Medica is the classic source of traditional Chinese medicine health care, and its medication thought of taking drugs for a long term to accumulate effects has guiding significance for the regulation of sub-health and chronic diseases nowadays. The efficacy and safety of LTTD have been examined in practice for a long time, and some of the drugs are edible, which is unique in the whole cycle of health-care service, especially in line with the health-care needs in the aging society under the concept of Big Health. However, some records in the book are limited by the understanding of the times, which should be scientifically studied according to the Chinese Pharmacopoeia and the related regulations and technical requirements, under the attitude of eliminating falsifications and preserving the truth and keeping the right essence, so as to achieve further improvement, innovation, and development.

Disinfection effect of hexadecyl pyridinium chloride on SARS-CoV-2 in vitro.

The novel coronavirus (COVID-19 or 2019-nCoV) is a respiratory virus that can exist in the mouth and saliva of patients and spreads through aerosol dispersion. Therefore, stomatological hospitals and departments have become high-infection-risk environments. Accordingly, oral disinfectants that can effectively inactivate the virus have become a highly active area of research. Hexadecyl pyridinium chloride, povidone-iodine, and other common oral disinfectants are the natural primary choices for stomatological hospitals. Therefore, this study investigated the inhibitory effect of hexadecyl pyridinium chloride on SARS-CoV-2 in vitro. Vero cells infected with SARS-CoV-2 were used to determine the disinfection effect; the CCK-8 method was used to determine cytotoxicity, and viral load was determined by real-time PCR. The results showed that hexadecyl pyridinium chloride has no obvious cytotoxic effect on Vero cells in the concentration range 0.0125-0.05 mg/mL. The in vitro experiments showed that hexadecyl pyridinium chloride significantly inhibits the virus at concentrations of 0.1 mg/mL or above at 2 min of action. Thus, the results provide experimental support for the use of hexadecyl pyridinium chloride in stomatological hospitals.

Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish.

The vegetal pole cytoplasm represents a crucial source of maternal dorsal determinants for patterning the dorsoventral axis of the early embryo. Removal of the vegetal yolk in the zebrafish fertilised egg before the completion of the first cleavage results in embryonic ventralisation, but removal of this part at the two-cell stage leads to embryonic dorsalisation. How this is achieved remains unknown. Here, we report a novel mode of maternal regulation of BMP signalling during dorsoventral patterning in zebrafish. We identify Vrtn as a novel vegetally localised maternal factor with dorsalising activity and rapid transport towards the animal pole region after fertilisation. Co-injection of vrtn mRNA with vegetal RNAs from different cleavage stages suggests the presence of putative vegetally localised Vrtn antagonists with slower animal pole transport. Thus, vegetal ablation at the two-cell stage could remove most of the Vrtn antagonists, and allows Vrtn to produce the dorsalising effect. Mechanistically, Vrtn binds a bmp2b regulatory sequence and acts as a repressor to inhibit its zygotic transcription. Analysis of maternal-zygotic vrtn mutants further shows that Vrtn is required to constrain excessive bmp2b expression in the margin. Our work unveils a novel maternal mechanism regulating zygotic BMP gradient in dorsoventral patterning.

Comparison of total endoscopic thyroidectomy with conventional open thyroidectomy for treatment of papillary thyroid cancer: a systematic review and meta-analysis.

BACKGROUND: Despite the fact that thyroid surgery has evolved towards minimal incisions and endoscopic approaches, the role of total endoscopic thyroidectomy (TET) in thyroid cancer has been highly disputed. We performed a systematic review and meta-analyses of peer reviewed studies in order to evaluate the safety and effectiveness of TET compared with conventional open thyroidectomy (COT) in papillary thyroid cancer (PTC). METHOD: Medical literature databases such as PubMed, Embase, the Cochrane Library, and Web of science were systematically searched for articles that compared TET and COT in PTC treatment from database inception until March 2019. The quality of the studies included in the review was evaluated using the Downs and Black scale using Review Manager software Stata V.13.0 for the meta-analysis. RESULTS: The systematic review and meta-analysis were based on 5664 cases selected from twenty publications. Criteria used to determine surgical completeness included postoperative thyroglobulin (TG) levels, recurrence of the tumor after long-term follow-up. Adverse event and complication rate scores included transient recurrent laryngeal nerve (RLN) palsy, permanent RLN palsy, transient hypocalcaemia, permanent hypocalcaemia, operative time, number of removed lymph nodes, length of hospital stay and patient cosmetic satisfaction. TET was found to be generally equivalent to COT in terms of surgical completeness and adverse event rate, although TET resulted in lower levels of transient hypocalcemia (OR 1.66; p < 0.05), a smaller number of the retrieved lymph nodes (WMD 0.46; p < 0.05), and better cosmetic satisfaction (WMD 1.73; p < 0.05). COT was associated with a shorter operation time (WMD - 50.28; p < 0.05) and lower rates of transient RLN palsy (OR 0.41; p < 0.05). CONCLUSIONS: The results show that in terms of safety and efficacy, TET was similar to COT for the treatment of thyroid cancer. Indeed, the tumor recurrence rates and the level of surgical completeness in TET are similar to those obtained for COT. TET was associated with significantly lower levels of transient hypocalcemia and better cosmetic satisfaction, and thus is the better option for patients with cosmetic concerns. Overall, randomized clinical trials and studies with larger patient cohorts and long-term follow-up data are required to further demonstrate the value of the TET.

Patient with severe fever with thrombocytopenia syndrome virus infection and central nervous system disturbance in Dongyang, Zhejiang Province, China, 2017.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever that was first described in China in 2011. We report a patient who died of Severe fever with thrombocytopenia syndrome virus (SFTSV) infection, with a rapidly progressive central nervous system (CNS) disturbance, in Dongyang, Zhejiang Province, China, in 2017. CASE PRESENTATION: A 64-year-old man was admitted to hospital after 4 days of fever. SFTSV was detected 1 day after the patient was admitted to hospital. The patient presented with CNS disturbance and died 4 days after admission. Detailed clinical and epidemiological investigations and laboratory tests were conducted. Reduced platelet, white blood cell, lymphocyte, and neutrophil counts, elevated lactate dehydrogenase, creatine kinase, aspartate aminotransferaseand alanine aminotransferase concentrations, and an increased activated partial thromboplastin time were observed. In a phylogenetic analysis, the isolate clustered close to a strain derived from South Korea. CONCLUSIONS: This is the first case of SFTSV infection with CNS disturbance in Dongyang, Zhejiang Province, China. The surveillance of suspected cases of SFTS is important in SFTSV endemic regions.

[Toxic effects of combination of Aconiti Lateralis Radix Praeparata with Trichosanthis Fructus on inflammatory response and myocardial fibrosis in pressure overload rats based on ß2-AR/PKA signal].

To study the effects of combination of Aconiti Lateralis Radix Praeparata( Fuzi) with Trichosanthis Fructus( Gualou) on cardiac function,electrocardiogram,inflammatory response and myocardial fibrosis in pressure overload( PO) rats,and further explore the mechanism based on ß2-AR/PKA signaling. PO rat model was established by constricting the abdominal aorta. Twelve weeks after the operation,these rats were randomly divided into model goup( PO),low dose Fuzi group( FL,5. 4 g·kg-1·d-1),Gualou group( GL,5. 4 g·kg-1·d-1),Fuzi and Gualou combination group( FG,5. 4 g·kg-1·d-1+5. 4 g·kg-1·d-1) and high dose Fuzi group( FH,10. 8 g·kg-1·d-1). At the same time,sham operation group was set. After intervention for 6 weeks,carotid blood pressure,cardiac function,electrocardiogram and heart mass index were measured. HE staining was used to observe the inflammatory response in the rat heart and kidney. Masson staining was used to determine the myocardial fibrosis. Western blot was used to detect the protein expression of ß2-AR and PKA. As compared with sham operation group,the blood pressure and heart mass index were obviously increased in PO model group,but there was no significant difference in various treatment groups in the above indexes. As compared with PO model group,FH treatment significantly increased the ejection fraction( EF) and GL treatment effectively enhanced the cardiac output( CO),but other treatment groups had no significant effect on these parameters. Moreover,FG treatment can synergistically attenuate QT and QTc internal prolongation,but it also aggravated inflammatory response in the heart and kidney tissues and promoted myocardial fibrosis as compared to FZ or GL alone treatment,with toxic effects equivalent to FH treatment group. Following FG and FH treatment,simultaneously,ß2-AR and PKA protein levels were significantly elevated,indicating that the increasing toxicity of FG could be associated with activation of ß2-AR/PKA signaling. These results suggested that combination of FZ and GL could synergistically enhance toxicity of FZ in special pathological states such as pressure overload,and caution should be taken in clinical application.

[Intelligent and lean manufacturing for Chinese medicine:theory and its applications].

To cope with the " six major scientific problems" and the " five major technical challenges" of intelligent manufacturing and lean production of Chinese medicine( CM),we systematically proposed strategies,methods and the engineering theory of intelligent and lean manufacturing for CM by integrating the holistic view of traditional Chinese medicine and the concepts inspired from international advanced pharmaceutical technology. Moreover,the translational research of the theory and methods was successfully applied to six CMs such as Xuesaitong Injection. Several intelligent production lines were designed and built on the basis of the theory and methods,which greatly accelerated the digitalization,networking,and intelligence manufacture for CM. As a conclusion,the theory and applications provide technical demonstration for technical upgrading and high-quality development of CM industry.

PCAF-mediated acetylation of Lin28B increases let-7 biogenesis in lung adenocarcinoma H1299 cells.

BACKGROUND: Lin28B and its paralog Lin28A are small RNA binding proteins that have similar inhibitory effects, although they target separate steps in the maturation of let-7 miRNAs in mammalian cells. Because Lin28B participates in the promotion and development of tumors mostly by blocking the let-7 tumor suppressor family members, we sought to explore the associated mechanisms to gain insights into how Lin28B might be decreased in human cancer cells to increase let-7 levels and reverse malignancy. RESULTS: We demonstrated that the histone acetyltransferase PCAF, via its cold shock domain, directly interacts with and subsequently acetylates Lin28B in lung adenocarcinoma-derived H1299 cells. RT-qPCR assays showed that both let-7a-1 and let-7g were increased in PCAF-transfected H1299 cells. Lin28B is acetylated by ectopic PCAF and translocates from the nucleus to the cytoplasm in H1299 cells. CONCLUSIONS: The effects of acetylated Lin28B on let-7a-1 and let-7g are similar to that of stable knockdown of Lin28B in H1299 cells. The new role of PCAF in mediating Lin28B acetylation and the specific release of its target microRNAs in H1299 cells may shed light on the potential application of let-7 in the clinical treatment of lung cancer patients.

Cost-effectiveness of afatinib and erlotinib as second-line treatments for advanced squamous cell carcinoma of the lung.

AIM: To investigate the cost-effectiveness of afatinib and erlotinib as second-line therapy for advanced squamous cell carcinoma of the lung. MATERIALS & METHODS: A decision-analytic model was developed for projecting the economic outcomes. Clinical parameters and utilities were from the LUX-Lung 8 trial. Costs were mainly estimated from the Chinese health system. The outcome was the incremental cost-effectiveness ratio. RESULTS: The afatinib strategy generated additional 0.154 quality-adjusted life-years compared with erlotinib, with incremental costs of ¥16,852. Relative to erlotinib, afatinib resulted in an incremental cost-effectiveness ratio of ¥109,429 per quality-adjusted life-year gained. The overall survival time of afatinib had a considerable impact on the model outcomes. CONCLUSION: Afatinib is a cost-effective treatment option compared with erlotinib in patients with squamous cell carcinoma.

Synergistic Promotion on Tyrosinase Inhibition by Antioxidants.

When exposed to ultraviolet radiation, the human skin produces profuse reactive oxygen species (ROS), which in turn activate a variety of biological responses. Mounting ROS levels activate tyrosinase by mobilizing α-melanocyte-stimulating hormone in the epidermis and finally stimulates the melanocytes to produce melanin. Meanwhile, the Keap1-Nrf2/ARE pathway, which removes ROS, is activated at increased ROS levels, and antioxidant compounds facilitates the dissociation of Nrf2. In this study, we explored the possible suppressing effects of antioxidant compounds and tyrosine inhibitors on melanin formation and the promotory effects of these compounds on ROS scavenging. The antioxidant activity of glabridin (GLA), resveratrol (RES), oxyresveratrol (OXYR), and phenylethylresorcinol (PR) were investigated via the stable free radical 2,2-diphenyl-1-picrylhydrazyl method. The inhibitory effects of the four compounds and their mixtures on tyrosinase were evaluated. l-Tyrosine or 3-(3,4-dihydroxyphenyl)-l-alanine (l-DOPA) was used as a substrate. The results showed that all mixtures did not exhibit synergistic effects with the l-tyrosine as a substrate, suggesting that l-tyrosine is not suitable as a substrate. However, the mixtures of "GLA:RES," "GLA:OXYR," "OXYR:RES," and "PR:RES" demonstrated synergistic effects (CI < 0.9, p < 0.05), whereas "GLA:RES" and "PR:OXYR" indicated an additive effect (0.9 ditive1, p < 0.05). Furthermore, we used a molecular docking strategy to study the interactions of the four compounds with tyrosinase and l-DOPA. The molecular docking result is consistent with that of the experiment. Finally, we selected RES + OXYR and used PIG1 cells to verify whether OXYR synergistically promotes RES activity on tyrosinase. The two agents had a synergistic inhibitory effect on tyrosinase activity. These results provided a novel synergistic strategy for antioxidants and tyrosinase inhibitors, and this strategy is useful in skin injury treatment.

[Current situation and prospect of resource evaluation and sustainable utilization on Bletilla].

The plants of Bletilla are one of the groups in Orchidaceae with the highest economic value. As the traditional Chinese medicinal material, Bletillae Rhizoma exhibits excellent efficacy in hemostatic, antibiosis, detumescent, anticancer activities and regenerating tissue to heal wound, which has great development potential. However, Bletillae Rhizoma is mainly collected from wild resources. At present, the quantity of wild resources of Bletilla plants has sharply decreased and is far from meeting the needs. Resource appraisal and breeding and cultivation of excellent germplasms of Bletilla plants are important for scientific utilization of the resources of the genus. This paper reviewed the following researches of Chinese Bletilla resources: species and distribution, genetic diversity, active ingredient evaluation, breeding, as well as seeding production and cultivation techniques. Suggestions were also provided in further researches on the resources evaluation, sustainable development and efficient utilization of Chinese Bletilla plants.

C/EBPα negatively regulates SIRT7 expression via recruiting HDAC3 to the upstream-promoter of hepatocellular carcinoma cells.

Mammalian Sirtuin proteins (SIRTs) are homologs of yeast Sir2, and characterized as class III histone deacetylases of NAD(+) dependence. Unlike their lower counterparts that are directly involved in the extending of lifespan, mammalian SIRTs mainly function in metabolism and cellular homeostasis, among them, SIRT7 is the least understood. SIRT7 is localized in the nucleus and rich in nucleoli associated with RNA polymerase I, and correlated with cell proliferation. In contrast, SIRT7 has recently been demonstrated to specifically deacetylate H3K18ac in the chromatin, and in most cases represses proliferation. Although MicroRNA as miR-125b has been reported to down-regulate SIRT7 by binding to its 3'UTR, however, how SIRT7 gene is regulated remains unclear. Here, we identified the transcription initiation site of human SIRT7 gene at the upstream 23rd A nucleotide respective to the translational codon, and the SIRT7 is a TATA-less and initiator-less gene. The sequences in the upstream region between -256 and -129 bp are identical with important functions in the three species detected. A C/EBPα responding element is found that binds both C/EBPα and C/EBPß in vitro. We showed TSA induced SIRT7 gene transcription and only the HDAC3, but not its catalytic domain depleted mutant, interacted with C/EBPα to occupy the C/EBPα element and repressed SIRT7 gene in the hepatocellular carcinoma cells. To our knowledge, this is the first report on the regulation mechanism of SIRT7 gene, in which, HDAC3 collaborated with C/EBPα to occupy its responding element in the upstream region of SIRT7 gene and repressed its expression in human cells.

Exploration micromechanism of VP35 IID interaction and recognition dsRNA: A molecular dynamics simulation.

Multifunctional viral protein (VP35) encoded by the highly pathogenic Ebola viruses (EBOVs) can antagonize host double-stranded RNA (dsRNA) sensors and immune response because of the simultaneous recognition of dsRNA backbone and blunt ends. Mutation of select hydrophobic conserved basic residues within the VP35 inhibitory domain (IID) abrogates its dsRNA-binding activity, and impairs VP35-mediated interferon (IFN) antagonism. Herein the detailed binding mechanism between dsRNA and WT, single mutant, and double mutant were investigated by all-atom molecular dynamics (MD) simulation and binding energy calculation. R312A/R322A double mutations results in a completely different binding site and orientation upon the structure analyses. The calculated binding free energy results reveal that R312A, R322A, and K339A single mutations decrease the binding free energies by 17.82, 13.18, and 13.68 kcal mol-1 , respectively. The binding energy decomposition indicates that the strong binding affinity of the key residues is mainly due to the contributions of electrostatic interactions in the gas phase, where come from the positively charged side chain and the negatively charged dsRNA backbone. R312A, R322A, and K339A single mutations have no significant effect on VP35 IID conformation, but the mutations influence the contributions of electrostatic interactions in the gas phase. The calculated results reveal that end-cap residues which mainly contribute VDW interactions can recognize and capture dsRNA blunt ends, and the central basic residues (R312, R322, and K339) which mainly contribute favorable electrostatic interactions with dsRNA backbone can fix dsRNA binding site and orientation. Proteins 2017; 85:1008-1023. © 2017 Wiley Periodicals, Inc.

Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

Effects of a NTG-based chemical mutagenesis on the propamocarb-tolerance of the nematophagous fungus Lecanicillium attenuatum.

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide for control of plant-pathogenic nematodes. However, relatively low fungicide-tolerance limits its application in the field. To improve the propamocarb-tolerance of L. attenuatum, a NTG-based mutagenesis system was established. Among different combinations of NTG concentration and treatment time in the first-round NTG treatment, the treatment of 1.0mg/ml NTG for 60min gave a proper conidial lethality rate of 84.6% and the highest positive mutation rate of 7.7%, and then produced the highest propamocarb-tolerant mutant LA-C-R1-T4-M whose EC50 value reached to 1050.0µg/ml. The positive mutation range was 105.1% in the first-round NTG treatment. Multiple-round NTG treatment was further employed to enhance the propamocarb tolerance of L. attenuatum. The positive mutation range was significantly accumulated to 179.3% on the third-round NTG treatment, and then appeared to level-off and remained constant. These results indicated that multiple-round NTG treatment had a significant accumulative effect on fungal tolerance to propamocarb. Among all chemical-mutants, the LA-C-R3-M was the highest tolerant to propamocarb, whose EC50 value was increased 2.79-fold compared to the wild-type strain, and it was mitotic stable after 20 passages on PDA medium. Colony growth, conidia yield and conidial germination on plates, and parasitism of nematode eggs of M. incognita and H. glycines were not significantly changed by the NTG-based mutagenesis compared to the wild-type strain in either single- or multiple-round NTG treatment. In conclusion, we succeeded in improving the propamocarb tolerance of L. attenuatum via the optimized NTG-based mutagenesis system. The improved strain LA-C-R3-M could be potentially applied with propamocarb in the field.