Modulation of PD-L1 by Astragalus polysaccharide attenuates the induction of melanoma stem cell properties and overcomes immune evasion.

BACKGROUND: Melanoma is a highly aggressive form of skin cancer. The existence of cancer stem cells (CSCs) and tumor immune evasion are two major causes of melanoma progression, but no effective treatment has been found at present. Astragalus polysaccharide (APS) is a principal active component derived from Astragalus membranaceus, showing anti-tumor effects in various tumors including melanoma. However, the underlying mechanism is still unclear. METHODS: The regulation of APS on self-renewal ability and CSC markers expression in melanoma stem cells (MSCs) was measured by tumor sphere formation and tumorigenicity assays, RT-qPCR, and western blot. Flow cytometry was conducted to evaluate the activation of immune system by APS in melanoma mice. Further, the mechanism was explored based on PD-L1 overexpression and knock-down B16 cells. RESULTS: APS attenuated the tumor sphere formation of MSCs in vitro as well as the tumorigenicity in vivo. It also decreased the expression of CD133, BMI1 and CD47. Based on the PD-L1 overexpression and knock-down B16 cells, it was confirmed that APS inhibited the induction of MSCs by down-regulating PD-L1 expression. Meanwhile, APS increased the infiltration of CD4+ and CD8+T cells in tumor tissues because of its inhibitory effect on PD-L1. CONCLUSIONS: APS inhibited MSC induction and overcame tumor immune evasion through reducing PD-L1 expression. This study provided compelling evidence that APS could be a prospective therapeutic agent for treating melanoma.

Prognostic Value of CXCR6 and the Correlation with Immune Infiltration in the Microenvironment of Skin Cutaneous Melanoma.

Increasing evidence has demonstrated that CXCRs are associated with the tumor infiltration of immune cells and regulate the tumor immune response. However, the prognostic value of CXCRs expression in patients with skin cutaneous melanoma (SKCM) remains unclear. In this study, we aimed to investigate the expression characteristics of CXCRs in SKCM tissues, analyze their prognostic value and the correlation with immune cell infiltration. Multiple public databases were used for exploration, including GEPIA2, GSCA, ULCAN, Metascape, STRING, TIMER2.0, HPA, and Cistrome DB database. And a confirmation experiment was conducted on melanoma mice with flow cytometry. Compared with normal tissues, lower expression of CXCR2/7/8 and higher expression of CXCR3/4 were found in SKCM tissues. CXCR3/4/6 were abnormally expressed in each pathological stage. Moreover, CXCRs were closely related to immune-related biological functions, and mainly interacted with CXCLs. The high expression of CXCR3/5/6 indicated better overall survival of patients. Among them, CXCR6 had the most significant prognostic value, and was most related to tumor infiltration of CD8+T cells, which was verified in melanoma mice. Finally, ETS1 and STAT5B were predicted as the transcription factor of CXCR6. Our findings play an important role in the study of prognostic markers in patients with SKCM.

CCR -NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica).

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CCR -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.

Rhodium(II)-Catalyzed C(sp3 )-H Diamination of Arylcyclobutanes.

Activated by multiple consecutive oxidative radical-polar crossover and desaturation processes, the selective diamination of arylcyclobutanes, which is difficult to perform by classical metallonitrene C-H insertion, was achieved in a short time by rhodium(II) catalysis using N-fluorobenzenesulfonimide (NFSI) as the oxidant and nitrogen source.

The ERK1/2-ATG13-FIP200 signaling cascade is required for autophagy induction to protect renal cells from hypoglycemia-induced cell death.

Autophagy, an evolutionarily conserved lysosomal degradation pathway, is known to regulate a variety of physiological and pathological processes. At present, the function and the precise mechanism of autophagy regulation in kidney and renal cells remain elusive. Here, we explored the role of ERK1 and ERK2 (referred as ERK1/2 hereafter) in autophagy regulation in renal cells in response to hypoglycemia. Glucose starvation potently and transiently activated ERK1/2 in renal cells, and this was concomitant with an increase in autophagic flux. Perturbing ERK1/2 activation by treatment with inhibitors of RAF or MEK1/2, via the expression of a dominant-negative mutant form of MEK1/2 or RAS, blocked hypoglycemia-mediated ERK1/2 activation and autophagy induction in renal cells. Glucose starvation also induced the accumulation of reactive oxygen species in renal cells, which was involved in the activation of the ERK1/2 cascade and the induction of autophagy in renal cells. Interestingly, ATG13 and FIP200, the members of the ULK1 complex, contain the ERK consensus phosphorylation sites, and glucose starvation induced an association between ATG13 or FIP200 and ERK1/2. Moreover, the expression of the phospho-defective mutants of ATG13 and FIP200 in renal cells blocked glucose starvation-induced autophagy and rendered cells more susceptible to hypoglycemia-induced cell death. However, the expression of the phospho-mimic mutants of ATG13 and FIP200 induced autophagy and protected renal cells from hypoglycemia-induced cell death. Taken together, our results demonstrate that hypoglycemia activates the ERK1/2 signaling to regulate ATG13 and FIP200, thereby stimulating autophagy to protect the renal cells from hypoglycemia-induced cell death.

Immunosuppressive activity is attenuated by Astragalus polysaccharides through remodeling the gut microenvironment in melanoma mice.

Astragalus polysaccharides (APS), the main effective component of Astragalus membranaceus, can inhibit tumor growth, but the underlying mechanisms remain unclear. Previous studies have suggested that APS can regulate the gut microenvironment, including the gut microbiota and fecal metabolites. In this work, our results showed that APS could control tumor growth in melanoma-bearing mice. It could reduce the number of myeloid-derived suppressor cells (MDSC), as well as the expression of MDSC-related molecule Arg-1 and cytokines IL-10 and TGF-ß, so that CD8+ T cells could kill tumor cells more effectively. However, while APS were administered with an antibiotic cocktail (ABX), MDSC could not be reduced, and the growth rate of tumors was accelerated. Consistent with the changes in MDSC, the serum levels of IL-6 and IL-1ß were lowest in the APS group. Meanwhile, we found that fecal suspension from mice in the APS group could also reduce the number of MDSC in tumor tissues. These results revealed that APS regulated the immune function in tumor-bearing mice through remodeling the gut microbiota. Next, we focused on the results of 16S rRNA, which showed that APS significantly regulated most microorganisms, such as Bifidobacterium pseudolongum, Lactobacillus johnsonii and Lactobacillus. According to the Spearman analysis, the changes in abundance of these microorganisms were related to the increase of metabolites like glutamate and creatine, which could control tumor growth. The present study demonstrates that APS attenuate the immunosuppressive activity of MDSC in melanoma-bearing mice by remodeling the gut microbiota and fecal metabolites. Our findings reveal the therapeutic potential of APS to control tumor growth.

A Single-Nucleotide Polymorphism in the Promoter of a Hairpin RNA Contributes to Alternaria alternata Leaf Spot Resistance in Apple (Malus × domestica).

Apple leaf spot caused by the Alternaria alternata f. sp mali (ALT1) fungus is one of the most devastating diseases of apple (Malus × domestica). We identified a hairpin RNA (hpRNA) named MdhpRNA277 that produces small RNAs and is induced by ALT1 infection in 'Golden Delicious' apple. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five resistance (R) genes that are expressed at high levels in resistant apple variety 'Hanfu' and at low levels in susceptible variety 'Golden Delicious' following ALT1 infection. MdhpRNA277 was strongly induced in 'Golden Delicious' but not 'Hanfu' following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated 'Golden Delicious' versus 'Hanfu'. We identified a single-nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between 'Golden Delicious' (pMdhpRNA277-GD) and 'Hanfu' (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-GD, but not to pMdhpRNA277-HF Transgenic 'GL-3' apple expressing pMdhpRNA277-GD:MdhpRNA277 was more susceptible to ALT1 infection than plants expressing pMdhpRNA277-HF:MdhpRNA277 due to induced mdm-siR277 accumulation and reduced expression of the five target R genes. We confirmed that the SNP in pMdhpRNA277 is associated with A. alternata leaf spot resistance by crossing. This SNP could be used as a marker to distinguish between apple varieties that are resistant or susceptible to A. alternata leaf spot.

Oxidatively Induced Selective Carbon-Carbon Bond Formation From Isolated Rhodium(III) Complexes.

This work focuses on oxidatively induced regioselective intramolecular C-C bond formations based on the RhIII complexes synthesized from dirhodium(II) trifluoroacetate with 2-arylpyridines. With the selection of electron-donating groups on the arene rings of 2-arylpyridines, the unusual meta-ortho C-C bond-forming was favored, which led to the formation of meta-substituted 2-arylpyridine homocoupling dimers. On the contrary, the electron-withdrawing groups have tendency to occur conventional ortho-ortho bond-forming, resulting in the formation of new RhIII complexes possessing the intriguing RhIII (TFA)3 fragment. Preliminary mechanistic experiments suggest that the sequential oxidation of RhIII occurred in the reaction.

Dirhodium(II)-Catalyzed C(sp2 )-H Azidation of Benzaldehydes.

Multiple steps are needed to achieve the C-H functional of aromatic aldehyde, since the C-H functional reaction usually occurs preferentially at the aldehydic C-H bond over the aryl C-H bond. We report an efficient azidation method mediated by dirhodium(II) catalysts to achieve the direct aryl azidation of aromatic aldehydes avoiding the simultaneous use of protected aldehydes and prefunctionalized arenes. The regioselectivity of this method is similar to those of typical aromatic electrophilic substitution reactions. The resulting azidobenzaldehyde products are versatile building blocks or precursors for the synthesis of many biologically active compounds. The mechanism studies indicate that the one-electron oxidative intermediate Rh2 (II,III) N3 is responsible for the azide transfer.

Dirhodium(II)-Catalyzed C(sp2 )-H Azidation of Benzaldehydes.

Invited for the cover of this issue is the group of Yuanhua Wang at Sichuan University. The image depicts the reported dirhodium complexes depicted as Chinese lanterns. Read the full text of the article at 10.1002/chem.201905855.

Pd(NHC)(cinnamyl)Cl-catalyzed Suzuki cross-coupling reaction of aryl sulfonates with arylboronic acids.

A series of N-heterocyclic carbene (NHC)-palladium catalysts have been synthesized and applied to catalyze the Suzuki coupling reaction efficiently between aryl sulfonates and arylboronic acids with the potassium phosphate heptahydrate as a base. The desired yields are obtained even with less reactive aryl tosylates or aryl mesylates as substrates. This method was applied successfully to the synthesis of the (R)-2-(t-butoxycarbonylamino)-3-(biphenyl-4-yl)-propan-1-ol which is the key intermediate of sacubitril, a component of the newly approved antihypertensive drug "Entresto."

Mild and Practical Dirhodium(II)/NHPI-Mediated Allylic and Benzylic Oxidations with Air as the Oxidant.

Aerobic allylic and benzylic oxidations catalyzed by dirhodium(II) complexes with N-hydroxyphthalimide (NHPI) are described. The open flask reaction occurs at mild temperature, using air as the oxidant. Mechanistic studies suggest that dirhodium(II) complexes axially coordinate with NHPI to activate the O-H bond in NHPI and decrease the bond-dissociation energy (BDE).

Mild and Practical Dirhodium(II)/NHPI-Mediated Allylic and Benzylic Oxidations with Air as the Oxidant.

Invited for the cover of this issue is the group of Hong Jin and Yuanhua Wang at Sichuan University. The image depicts a large reaction beaker, in which the oxidation catalyzed by the dirhodium(II) complexes together with N-hydroxyphthalimide is taking place, surrounded by a background that evokes an environmentally friendly process. The idea of the illustration comes directly from the experimental equipment, the picture of which appears in the graphical abstract. Read the full text of the article at 10.1002/chem.201902739.

Dirhodium(II)-catalyzed [3 + 2] cycloaddition of N-arylaminocyclopropane with alkyne derivatives.

Dirhodium(II) complex-catalyzed [3 + 2] reactions between N-arylaminocyclopropanes and alkyne derivatives are described. The cycloaddition products proved to be versatile synthetic intermediates. trans-Cyclic ß-amino acids and derivatives thereof can be conveniently synthesized using this cycloaddition protocol.

Dirhodium(ii)/P(t-Bu)3 catalyzed tandem reaction of α,ß-unsaturated aldehydes with arylboronic acids.

Phosphine ligated dirhodium(ii) acetate is advocated as a catalyst for the synthesis of aryl alkyl ketones by the tandem reaction of α,ß-unsaturated aromatic or aliphatic aldehydes with arylboronic acids. This tandem procedure included arylation followed by the isomerization reaction. This method exhibits good functional group tolerance and has a broad substrate scope. With the conjugated aldehydes, the one-step synthesis of γ,δ-unsaturated ketones was realized through this reaction. It is noteworthy that the length of the Rh-P bond is an important factor affecting catalytic reactions. The comparative analysis of the crystal structures of axially alkylphosphane and arylphosphane ligated dirhodium(ii) acetate revealed that the shorter Rh-P bond length favors the isomerization process as compared to the longer one. In addition, the dirhodium(ii) compound can be recovered after the completion of the reaction.

Recyclable Dirhodium(II) Catalyst Rh2(esp)2 for the Allylic Oxidation of Δ5-Steroids.

The chelating dirhodium(II) catalyst Rh2(esp)2 was shown to efficiently catalyze the allylic oxidation of Δ5-steroids using T-HYDRO (70% tert-butyl hydroperoxide in water) as oxidant. Reaction yields were affected by the coordination ability of the solvent. The noncoordinating solvent n-heptane was determined to be an optimal solvent. At gram scale, the product, Δ5-en-7-one steroid, precipitated from the reaction mixture. The Rh2(esp)2 complex did not undergo catalytic degradation and was recycled using Merrifield-pyridine resin for further allylic oxidation cycles. The results of ultraviolet/visible spectral analysis suggested that the Rh2(II,II) species, rather than the Rh2(II,III) species, was in the catalyst resting state during the reaction, which helps to explain the high durability of the catalyst.

Dirhodium(II)-Catalyzed Carbonylation Peroxidation of α,ß-Unsaturated Esters: Mechanistic Insight into the Role of Aryl Aldehydes.

Peroxidation has received considerable attention as a synthetically useful method used to prepare organic peroxides, which are useful synthetic building blocks in synthetic chemistry. The difunctionalization of alkenes to introduce a peroxide and another functional group has become a useful tool for quickly increasing molecular complexity in synthesis. In this work, a three-component oxidative coupling of aryl aldehydes with α,ß-unsaturated esters and tert-butyl hydroperoxide catalyzed by dirhodium(II) catalyst Rh2(esp)2 (esp = α,α,α',α'-tetramethyl-1,3-benzenedipropanoate) under mild conditions is developed. The synthesized carbonylation peroxidation products (ß-peroxyketones) are stable enough to be isolated by silica gel column and characterized. The ß-peroxyketones used as reactants have been applied to the synthesis of the epoxides, polysubstituted furans, carbazole alkaloids, and biologically important natural products. Interestingly, besides being a reactant, aryl aldehydes also play an important role in avoiding the catalyst deactivation during the reaction as shown by ultraviolet/visible analysis. The excess amount of aldehydes was used to ensure the stability of the Rh2(esp)2 catalyst in the reaction by forming the monoaldehyde ligated dirhodium(II) complex. It is important to note that the aldehydes were also found to reduce the inactive Rh2(esp)2Cl species generated in the reaction.

Characterization of DNA methyltransferase and demethylase genes in Fragaria vesca.

DNA methylation is an epigenetic modification essential for gene regulations in plants, but understanding on how it is involved in fruit development, especially in non-climacteric fleshy fruit, is limited. The diploid woodland strawberry (Fragaria vesca) is an important model for non-climacteric fruit crops. In this study, we identified DNA methyltransferase genes and demethylase genes in Fragaria vesca and other angiosperm species. In accordance with previous studies, our phylogenetic analyses of those DNA methylation modifiers support the clustering of those genes into several classes. Our data indicate that whole-genome duplications and tandem duplications contributed to the expansion of those DNA methylation modifiers in angiosperms. We have further demonstrated that some DNA methylase and demethylase genes reach their highest expression levels in strawberry fleshy fruits when turning from white to red, suggesting that DNA methylation might undergo a dramatic change at the onset of fleshy fruit-ripening process. In addition, we have observed that expression of some DNA demethylase genes increases in response to various abiotic stresses including heat, cold, drought and salinity. Collectively, our study indicates a regulatory role of DNA methylation in the turning stage of non-climacteric fleshy fruit and responses to environment stimuli, and would facilitate functional studies of DNA methylation in the growth and development of non-climacteric fruits.

Dirhodium(II)-Catalyzed Sulfide Oxygenations: Catalyst Removal by Coprecipitation with Sulfoxides.

The dirhodium(II) carboxylate complex Rh2(esp)2 (esp = α,α,α',α'-tetramethyl-1,3-benzenedipropanoate) was shown to catalyze the sulfoxidation of organic sulfides using tert-butyl hydroperoxide as the oxidant. Due to the unique structure of Rh2(esp)2 and its stable Rh2(II,II) catalyst resting state, the rhodium catalyst is able to precipitate as a Rh2(esp)2-sulfoxide complex following the reaction which makes separation of the catalyst from the products very convenient. The precipitated Rh2(esp)2-sulfoxide complexes could be reused to catalyze sulfide oxygenation reactions without considerable loss of activity. Mechanistic studies suggest that the axial ligands fine-tune the redox potential of the dirhodium(II,II) compounds and determine the predominant catalyst species in the oxidation reaction.

The synthesis and synergistic antifungal effects of chalcones against drug resistant Candida albicans.

To identify effective and low toxicity synergistic antifungal compounds, 24 derivatives of chalcone were synthesized to restore the effectiveness of fluconazole against fluconazole-resistant Candida albicans. The minimal inhibitory concentration (MIC80) and the fractional inhibitory concentration index (FICI) of the antifungal synergist fluconazole were measured against fluconazole-resistant Candida albicans. This was done via methods established by the clinical and laboratory standards institute (CLSI). Of the synthesized compounds, 2'-hydroxy-4'-methoxychalcone (8) exhibited the most potent in vitro (FICI=0.007) effects. The structure activity relationship of the compounds are then discussed.