Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

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.

Astragalus polysaccharides promote neural stem cells-derived oligodendrogenesis through attenuating CD8+T cell infiltration in experimental autoimmune encephalomyelitis.

Endogenous neural stem cells (NSCs) have the potential to generate remyelinating oligodendrocytes, which play an important role in multiple sclerosis (MS). However, the differentiation of NSCs into oligodendrocytes is insufficient, which is considered a major cause of remyelination failure. Our previous work reported that Astragalus polysaccharides (APS) had a neuroprotective effect on experimental autoimmune encephalomyelitis (EAE) mice. However, it remains unclear whether APS regulate NSCs differentiation in EAE mice. In this study, our data illustrated that APS administration could promote NSCs in the subventricular zone (SVZ) to differentiate into oligodendrocytes. Furthermore, we found that APS significantly improved neuroinflammation and inhibited CD8+T cell infiltration into SVZ of EAE mice. We also found that MOG35-55-specific CD8+T cells suppressed NSCs differentiation into oligodendrocytes by secreting IFN-γ, and APS facilitated the differentiation of NSCs into oligodendrocytes which was related to decreased IFN-γ secretion. In addition, APS treatment did not show a better effect on the NSCs-derived oligodendrogenesis after CD8+T cell depletion. This present study demonstrated that APS alleviated neuroinflammation and CD8+T cell infiltration into SVZ to induce oligodendroglial differentiation, and thus exerted neuroprotective effect. Our findings revealed that reducing the infiltration of CD8+T cells might contribute to enhancing NSCs-derived neurogenesis. And APS might be a promising drug candidate to treat MS.

Fusarium Wilt Invasion Results in a Strong Impact on Strawberry Microbiomes.

Plant-endophytic microbes affect plant growth, development, nutrition, and resistance to pathogens. However, how endophytic microbial communities change in different strawberry plant compartments after Fusarium pathogen infection has remained elusive. In this study, 16S and internal transcribed spacer rRNA amplicon sequencing were used to systematically investigate changes in the bacterial and fungal diversity and composition in the endophytic compartments (roots, stems, and leaves) of healthy strawberries and strawberries with Fusarium wilt, respectively. The analysis of the diversity, structure, and composition of the bacterial and fungal communities revealed a strong effect of pathogen invasion on the endophytic communities. The bacterial and fungal community diversity was lower in the Fusarium-infected endophytic compartments than in the healthy samples. The relative abundance of certain bacterial and fungal genera also changed after Fusarium wilt infection. The relative abundance of the beneficial bacterial genera Bacillus, Bradyrhizobium, Methylophilus, Sphingobium, Lactobacillus, and Streptomyces, as well as fungal genera Acremonium, Penicillium, Talaromyces, and Trichoderma, were higher in the healthy samples than in the Fusarium wilt samples. The relative abundance of Fusarium in the infected samples was significantly higher than that in the healthy samples, consistent with the field observations and culture isolation results for strawberry wilt. Our findings provide a theoretical basis for the isolation, identification, and control of strawberry wilt disease.

Comparison of miRNA transcriptome of exosomes in three categories of somatic cells with derived iPSCs.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) through epigenetic manipulation. While the essential role of miRNA in reprogramming and maintaining pluripotency is well studied, little is known about the functions of miRNA from exosomes in this context. To fill this research gap,we comprehensively obtained the 17 sets of cellular mRNA transcriptomic data with 3.93 × 1010 bp raw reads and 18 sets of exosomal miRNA transcriptomic data with 2.83 × 107 bp raw reads from three categories of human somatic cells: peripheral blood mononuclear cells (PBMCs), skin fibroblasts(SFs) and urine cells (UCs), along with their derived iPSCs. Additionally, differentially expressed molecules of each category were identified and used to perform gene set enrichment analysis. Our study provides sets of comparative transcriptomic data of cellular mRNA and exosomal miRNA from three categories of human tissue with three individual biological controls in studies of iPSCs generation, which will contribute to a better understanding of donor cell variation in functional epigenetic regulation and differentiation bias in iPSCs.

FvWRKY50 is an important gene that regulates both vegetative growth and reproductive growth in strawberry.

The WRKY transcription factors play important roles in plant growth and resistance, but only a few members have been identified in strawberry. Here we identified a WRKY transcription factor, FvWRKY50, in diploid strawberry which played essential roles in strawberry vegetative growth, and reproductive growth. Knocking out FvWRKY50 by genome editing accelerated flowering time and leaf senescence but delayed anthocyanin accumulation in fruit. Further analysis showed that FvWRKY50 acted as a transcriptional repressor to negatively regulate the expression of flowering- and leaf senescence-related genes, including FvFT2, FvCO, FvFT3, and FvSAUR36. Notably, FvWRKY50 directly upregulated the expression of FvCHI and FvDFR by binding their promoter under normal conditions, but at low temperature FvWRKY50 was phosphorylated by FvMAPK3 and then induced protein degradation by ubiquitination, delaying anthocyanin accumulation. In addition, the homozygous mutant of FvWRKY50 was smaller while the biallelic mutant showed normal size. These new findings provide important clues for us to further reveal the regulatory mechanisms of strawberry growth and fruit ripening.

Rhodium(II)-Catalyzed Desaturative [3+2] Tandem Cyclization of Arylcycloalkanes with ß-Dicarbonyls.

Synthetically important scaffolds, fused tricyclic frameworks containing a 2,3-cyclo[b]dihydrofuran unit, play a crucial role in drug discovery. In this study, we demonstrate that rhodium(II)/N-fluorobenzenesulfonimide can catalyze the in situ generation of highly reactive alkene intermediates from commonly accessible alkanes, which undergo intermolecular [3+2] tandem cyclization with the simultaneously generated ß-dicarbonyl radical to synthesize a series of fused tricyclic frameworks containing a 2,3-cyclo[b]dihydrofuran unit with a quaternary carbon center.

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.

Visible light driven photocatalytic removal of uranium(VI) in strongly acidic solution.

Photocatalytic reduction and removal of toxic uranium(VI) from aqueous solution is a highly economic, non-pollutant and efficient strategy. However, most uranium containing waste waters are highly acidic, but current photocatalysts are still restricted in slightly acidic or neutral media (pH ≥ 4). Herein, a conjugated microporous polymer (CMP), pTTT-Ben, was used for visible light driven photocatalytic reduction of U(VI) in highly acidic condition (pH = 1). A high uranium removal capacity (4710 mg/g) was achieved. The structural information of reduced uranium was investigated by X-ray photoelectron spectroscopy (XPS) and extended X-ray absorption fine structure (EXAFS), revealing the amorphous U(IV) hydrate complex, with an additional interaction between U(IV) and nitrogen atoms on pTTT-Ben. In addition, pTTT-Ben also showed excellent photocatalytic U(VI) reduction performance under natural sunlight irradiation.

Suppression of stemness and enhancement of chemosensibility in the resistant melanoma were induced by Astragalus polysaccharide through PD-L1 downregulation.

Chemotherapy is commonly used in the clinical treatment of melanoma, but it is prone to resistance leading to the poor effectiveness. The mechanisms of resistance are complicated including the cancer stemness. Astragalus polysaccharide (APS) is one of the active components of traditional Chinese herbal medicine Astragalus Membranaceus. Our previous work was reported that APS had an inhibitory effect on the stemness of melanoma. In this study we established chemo-resistant melanoma cells and found that expression of stemness genes were upregulated in the resistant melanoma cells. And APS could downregulate expression of stemness genes. Furthermore, APS combined with cisplatin (DDP) could significantly slow down the tumor growth in the mouse model induced by DDP-resistant cells. In addition, we found that programmed death-ligand 1 (PD-L1) expression could be downregulated and the PI3K/AKT signaling could be affected by APS. These results suggested that APS could be a potential candidate in combination with chemotherapeutic agents, which might play a role in reducing the occurrence of resistance and improving the prognosis of melanoma patients.

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.

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.

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.

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.

Synergistic antifungal effects of curcumin derivatives as fungal biofilm inhibitors with fluconazole.

Lack of novel antifungal agents and severe drug resistance has led to high incidence and associated mortality of invasive fungal infections. To tackle the challenges, novel antifungal agents with anti-resistant potency are highly desirable. Thus, derivatives of curcumin were synthesized to restore the effectiveness of fluconazole (FLC) against FLC-resistant Candida spp. and structure-activity relationships were then discussed. Some novel derivatives showed promising features as novel antifungal lead compounds. Of them, compound 4 showed good alone or synergistic antifungal activity against FLC-resistant Candida spp. Moreover, compound 4 was proven as a potent inhibitor of Candida albicans biofilm formation and yeast-to-hypha morphological transition whether used alone or in combination with FLC, which was further confirmed by the inhibitory effect on cellular surface hydrophobicity of C. albicans. Compound 4 also inhibits intracellular ATP production of C. albicans and disrupts membrane permeability of C. albicans when used in combination with FLC. The results highlighted the potential of curcumin derivatives to overcome fluconazole-related and biofilm-related drug resistance.

Microglia Polarization from M1 toward M2 Phenotype Is Promoted by Astragalus Polysaccharides Mediated through Inhibition of miR-155 in Experimental Autoimmune Encephalomyelitis.

Activated microglia is considered to be major mediators of the neuroinflammatory environment in demyelinating diseases of the central nervous system (CNS). Activated microglia are mainly polarized into M1 type, which plays a role in promoting inflammation and demyelinating. However, the proportion of microglia polarized into M2 type is relatively low, which cannot fully play the role of anti-inflammatory and resistance to demyelinating. Our previous study found that Astragalus polysaccharides (APS) has an immunomodulatory effect and can inhibit neuroinflammation and demyelination in experimental autoimmune encephalomyelitis (EAE), which is a classic animal model of CNS demyelinating disease. In this study, we found that APS was effective in treating EAE mice. It restored microglia balance by inhibiting the polarization of microglia to M1-like phenotype and promoting the polarization of microglia to M2-like phenotype in vivo and in vitro. miR-155 is a key factor in regulating microglia polarization. We found that APS could inhibit the expression level of miR-155 in vivo and in vitro. Furthermore, we performed transfection overexpression and blocking experiments. The results showed that miR-155 mediated the polarization of microglia M1/M2 phenotype, while the selective inhibitor of miR-155 attenuated the inhibition of APS on microglia M1 phenotype and eliminated the promotion of APS on microglia M2 phenotype. Microglia can secrete IL-1α, TNF-α, and C1q after polarizing into M1 type and induce the activation of A1 neurotoxic astrocytes, further aggravating neuroinflammation and demyelination. APS reduced the secretion of IL-1α, TNF-α, and C1q by activated microglia, thus inhibited the formation of A1 neurotoxic astrocytes. In summary, our study suggests that APS regulates the polarization of microglia from M1 to M2 phenotype by inhibiting the miR-155, reduces the secretion of inflammatory factors, and inhibits the activation of neurotoxic astrocytes, thus effectively treating EAE.

Artificial classification of cervical squamous lesions in ThinPrep cytologic tests using a deep convolutional neural network.

The diagnosis of squamous cell carcinoma requires the accurate classification of cervical squamous lesions in the ThinPrep cytologic test (TCT). It primarily relies on a pathologist's interpretation under a microscope. Deep convolutional neural networks (DCNN) have played an increasingly important role in digital pathology. However, they have not been applied to diverse datasets and externally validated. In the present study, a DCNN model based on VGG16 and an ensemble training strategy (ETS) based on 5-fold cross-validation was employed to automatically classify normal and abnormal cervical squamous cells from a multi-center dataset. First, we collected a dataset comprising 82 TCT samples from four hospitals and fine-tuned our model twice on the dataset with and without the ETS. Then, we compared the classifications obtained from the models with those provided by two skilled pathologists to discriminate the performance of the models in terms of classification accuracy and efficiency. Finally, paired sample t-tests were used to validate the consistency between the classification provided by the proposed methods and that of the pathologists. The results showed that ETS slightly, though not significantly, improved the classification accuracy compared with that of the pathologists: P0=0.387>0.05 (DCNN without ETS vs. DCNN with ETS), P1=0.771>0.05 (DCNN with ETS vs. pathologist 1), P2=0.489>0.05 (DCNN with ETS vs. pathologist 2). The DCNN model was almost 6-fold faster than that of the pathologists. The accuracy of our automated scheme was similar to that of the pathologists, but a higher efficiency in the accurate identification of cervical squamous lesions was provided by the scheme. This result allows for wider and more efficient screening and may provide a replacement for pathologists in the future. Future research should address the viability of the practical implementation of such DCNN models in the laboratory setting.

Genome-Wide Identification and Expression Analysis of the Strawberry FvbZIP Gene Family and the Role of Key Gene FabZIP46 in Fruit Resistance to Gray Mold.

A total of 54 FvbZIP genes were identified from the strawberry genome. These genes were found to be unevenly distributed on seven different chromosomes, and two of the genes had no matching chromosomal localization. FvbZIP genes were divided into 10 subfamilies according to protein sequence, and the structures of these genes were found to be highly conserved. Based on the bioinformatics analysis of FvbZIP genes, the expression of FabZIP genes changed during different stages of its growth and of its infection with gray mold disease. FabZIP46 was substantially upregulated, and its expression remained relatively high. FabZIP46 was cloned from cultivated strawberries by homologous cloning. The results of a transient transgenic assay revealed that the damage to the fruit tissue was markedly alleviated in strawberries overexpressing FabZIP46, with the incidence rate being substantially lower than that in the control group. By contrast, a brief silencing of FabZIP46 had the opposite effect. The results revealed that FabZIP46 played a positive role in the resistance of strawberries to Botrytis cinerea. The study findings provide valuable insights into the role of bZIP transcription factors as well as a theoretical reference for the regulation of resistance to gray mold disease in strawberry fruit.

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.

Facial synthesis of key intermediate of obeticholic acid via Pd-catalyzed Kumada-Tamao-Corriu cross-coupling reaction.

Obeticholic acid (OCA) is used to treatment for Primary Biliary Cholangitis and other Famesoid X Receptor related diseases. Through the palladium catalyzed Kumada-Tamao-Corriu cross-coupling reaction, a novel and efficient method for synthesis of OCA with satisfied yield was successfully developed. The absolute configuration of the key intermediate was confirmed by Single-crystal X-ray Diffraction. It affords good strategy for large-scale synthesis of OCA.