A graph neural network model to estimate cell-wise metabolic flux using single-cell RNA-seq data.

The metabolic heterogeneity and metabolic interplay between cells are known as significant contributors to disease treatment resistance. However, with the lack of a mature high-throughput single-cell metabolomics technology, we are yet to establish systematic understanding of the intra-tissue metabolic heterogeneity and cooperative mechanisms. To mitigate this knowledge gap, we developed a novel computational method, namely, single-cell flux estimation analysis (scFEA), to infer the cell-wise fluxome from single-cell RNA-sequencing (scRNA-seq) data. scFEA is empowered by a systematically reconstructed human metabolic map as a factor graph, a novel probabilistic model to leverage the flux balance constraints on scRNA-seq data, and a novel graph neural network-based optimization solver. The intricate information cascade from transcriptome to metabolome was captured using multilayer neural networks to capitulate the nonlinear dependency between enzymatic gene expressions and reaction rates. We experimentally validated scFEA by generating an scRNA-seq data set with matched metabolomics data on cells of perturbed oxygen and genetic conditions. Application of scFEA on this data set showed the consistency between predicted flux and the observed variation of metabolite abundance in the matched metabolomics data. We also applied scFEA on five publicly available scRNA-seq and spatial transcriptomics data sets and identified context- and cell group-specific metabolic variations. The cell-wise fluxome predicted by scFEA empowers a series of downstream analyses including identification of metabolic modules or cell groups that share common metabolic variations, sensitivity evaluation of enzymes with regards to their impact on the whole metabolic flux, and inference of cell-tissue and cell-cell metabolic communications.

SSMD: a semi-supervised approach for a robust cell type identification and deconvolution of mouse transcriptomics data.

Deconvolution of mouse transcriptomic data is challenged by the fact that mouse models carry various genetic and physiological perturbations, making it questionable to assume fixed cell types and cell type marker genes for different data set scenarios. We developed a Semi-Supervised Mouse data Deconvolution (SSMD) method to study the mouse tissue microenvironment. SSMD is featured by (i) a novel nonparametric method to discover data set-specific cell type signature genes; (ii) a community detection approach for fixing cell types and their marker genes; (iii) a constrained matrix decomposition method to solve cell type relative proportions that is robust to diverse experimental platforms. In summary, SSMD addressed several key challenges in the deconvolution of mouse tissue data, including: (i) varied cell types and marker genes caused by highly divergent genotypic and phenotypic conditions of mouse experiment; (ii) diverse experimental platforms of mouse transcriptomics data; (iii) small sample size and limited training data source and (iv) capable to estimate the proportion of 35 cell types in blood, inflammatory, central nervous or hematopoietic systems. In silico and experimental validation of SSMD demonstrated its high sensitivity and accuracy in identifying (sub) cell types and predicting cell proportions comparing with state-of-the-arts methods. A user-friendly R package and a web server of SSMD are released via https://github.com/xiaoyulu95/SSMD.

MicroRNA-519d-3p antagonizes osteosarcoma resistance against cisplatin by targeting PD-L1.

Accumulating evidence indicates that a ligand of programmed cell death receptor-1 (PD-L1) participates in the progression and recurrence of multiple malignancies, including osteosarcoma. Nevertheless, the role of PD-L1 in chemoresistance development is not fully understood. In the current study, we aim to clarify the interaction of miR-519d-3p and PD-L1 in the development of cisplatin resistance. Immunohistochemistry, quantitative reverse-transcription polymerase reaction, and Western blot were used to evaluate PD-L1 expression. MTT and transwell migration assays were used to measure cell growth and motility, respectively. ENCORI, miRCode, and miRDB databases were recruited to predict candidate miRNAs targeting PD-L1. The binding sequences of miR-519d-3p and PD-L1 3' untranslated region were identified by dual-luciferase reporter and RNA immunoprecipitation assays. Flow cytometric analysis was conducted to measure the cycle distribution and cell apoptosis. Metastatic mouse models were generated with cisplatin-resistant sublines by intravenous injection. We found that PD-L1 expression was positively correlated to cisplatin resistance and metastasis, whereas miR-519d-3p expression was reduced in cisplatin-resistant specimens and was negatively correlated to cisplatin resistance and metastasis of osteosarcoma. We demonstrated that miR-519d-3p overexpression reversed cisplatin resistance, induced G1/S phase arrest and apoptosis. In addition, we proved that miR-519d-3p inhibited lung metastasis by establishing cisplatin-resistant MG63 metastatic xenograft models. The present findings suggest that miR-519d-3p/PD-L1 axis is a novel signaling pathway contributing to cisplatin resistance. Our study provides new clues for curing refractory osteosarcoma beyond immune checkpoint inhibitors.

Control of myogenesis by rodent SINE-containing lncRNAs.

Staufen1-mediated mRNA decay (SMD) degrades mRNAs that harbor a Staufen1-binding site (SBS) in their 3' untranslated regions (UTRs). Human SBSs can form by intermolecular base-pairing between a 3' UTR Alu element and an Alu element within a long noncoding RNA (lncRNA) called a ½-sbsRNA. Since Alu elements are confined to primates, it was unclear how SMD occurs in rodents. Here we identify mouse mRNA 3' UTRs and lncRNAs that contain a B1, B2, B4, or identifier (ID) element. We show that SMD occurs in mouse cells via mRNA-lncRNA base-pairing of partially complementary elements and that mouse ½-sbsRNA (m½-sbsRNA)-triggered SMD regulates C2C12 cell myogenesis. Our findings define new roles for lncRNAs as well as B and ID short interspersed elements (SINEs) in mice that undoubtedly influence many developmental and homeostatic pathways.

4-Hydroxy-2-pyridones: Discovery and evaluation of a novel class of antibacterial agents targeting DNA synthesis.

The continued emergence of bacteria resistant to current standard of care antibiotics presents a rapidly growing threat to public health. New chemical entities (NCEs) to treat these serious infections are desperately needed. Herein we report the discovery, synthesis, SAR and in vivo efficacy of a novel series of 4-hydroxy-2-pyridones exhibiting activity against Gram-negative pathogens. Compound 1c, derived from the N-debenzylation of 1b, preferentially inhibits bacterial DNA synthesis as determined by standard macromolecular synthesis assays. The structural features of the 4-hydroxy-2-pyridone scaffold required for antibacterial activity were explored and compound 6q, identified through further optimization of the series, had an MIC90 value of 8 µg/mL against a panel of highly resistant strains of E. coli. In a murine septicemia model, compound 6q exhibited a PD50 of 8 mg/kg in mice infected with a lethal dose of E. coli. This novel series of 4-hydroxy-2-pyridones serves as an excellent starting point for the identification of NCEs treating Gram-negative infections.

miR-23a suppresses proliferation of osteosarcoma cells by targeting SATB1.

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Recent studies have shown that miR-23a functions as an oncogene in various human cancer types, but its role in osteosarcoma remains poorly understood. Here, we demonstrated that miR-23a is frequently downregulated in osteosarcoma specimens and cell lines compared with adjacent noncancerous tissues and cell line. Bioinformatics analysis further revealed SATB1 as a potential target of miR-23a. Data from luciferase reporter assays showed that miR-23a directly binds to the 3'UTR of SATB1 messenger RNA (mRNA). Furthermore, we found that expression patterns of miR-23a were inversely correlated with those of SATB1 in osteosarcoma tissues and cell lines, and overexpression of miR-23a suppressed SATB1 expression at both transcriptional and translational levels in osteosarcoma cell lines. In functional assays, miR-23a inhibited osteosarcoma cell proliferation, which could be reversed by overexpression of SATB1. Furthermore, knockdown of SATB1 reduced osteosarcoma cell proliferation, which resembled the inhibitory effects of miR-23a overexpression. Taken together, our data provide compelling evidence that miR-23a functions as a tumor suppressor in osteosarcoma, and its inhibitory effect on tumor are mediated chiefly through downregulation of SATB1.

Comparison of hamstring tendon autograft and tibialis anterior allograft in arthroscopic transtibial single-bundle posterior cruciate ligament reconstruction.

PURPOSE: To compare the outcomes between hamstring tendon autograft and tibialis anterior allograft in arthroscopic transtibial single-bundle posterior cruciate ligament (PCL) reconstruction. METHODS: Thirty-seven patients undergoing isolated single-bundle PCL reconstruction were enrolled in this study, and their data were retrospectively analyzed. They were divided into group A [4-strand hamstring tendon autograft (4SHG), n = 18] and group B [2-strand tibialis anterior allograft (2STAG), n = 19] and followed up for 2 years at least. Several parameters including the International Knee Documentation Committee score, Lysholm knee score, Tegner activity rating and knee laxity arthrometer were evaluated, and physical examination was performed preoperatively and postoperatively, and postoperative complications were also observed in all patients. Meanwhile, the postoperative posterior instability was compared between the affected knee and the contra-lateral knee. RESULTS: Compared with preoperative knee laxity and function, both groups had significant improvement postoperatively (P < 0.01). However, there were no significant differences in knee laxity and function between both groups (n.s.). Compared with contra-lateral knee, the posterior stability was worse in the affected knee (P < 0.01). CONCLUSIONS: The outcomes were similar between 4SHG or 2STAG in PCL reconstruction. Compared with contra-lateral knees, the affected knees have slight residual knee laxity in both groups. LEVEL OF EVIDENCE: Retrospective comparative study, Level III.

Self-adhesive, conductive, and multifunctional hybrid hydrogel for flexible/wearable electronics based on triboelectric and piezoresistive sensor.

Flexible electronics are highly developed nowadays in human-machine interfaces (HMI). However, challenges such as lack of flexibility, conductivity, and versatility always greatly hindered flexible electronics applications. In this work, a multifunctional hybrid hydrogel (H-hydrogel) was prepared by combining two kinds of 1D polymer chains (polyacrylamide and polydopamine) and two kinds of 2D nanosheets (Ti3C2Tx MXene and graphene oxide nanosheets) as quadruple crosslinkers. The introduced Ti3C2Tx MXene and graphene oxide nanosheets are bonded with the PAM and PDA polymer chains by hydrogen bonds. This unique crosslinking and stable structure endow the H-hydrogel with advantages such as good flexibility, electrical conductivity, self-adhesion, and mechanical robustness. The two kinds of nanosheets not only improved the mechanical strength and conductivity of the H-hydrogel, but also helped to form the double electric layers (DELs) between the nanosheets and the bulk-free water phase inside the H-hydrogel. When utilized as the electrode of a triboelectric nanogenerator (TENG), high electrical output performances were realized due to the dynamic balance of the DELs between the nanosheets and the H-hydrogel's inside water molecules. Moreover, flexible sensors, including triboelectric, and strain/pressure sensors, were achieved for human motion detection at low frequencies. This hydrogel is promising for HMI and e-skin.

[Retracted] miR­486 suppresses the development of osteosarcoma by regulating PKC­Î´ pathway.

Following the publication of the above article, a concerned reader drew to the Editor's attention that, for the Transwell invasion and migration assay experiments shown in Figs. 5 and 6, there were multiple instances of apparently overlapping data panels, such that the data would have been derived from the same original sources where they were intended to show the results from differently performed experiments; moreover, certain of the data shown in Fig. 5B were strikingly similar to data that had appeared in Fig. 2 in a previously published paper written by different authors at different research institutes [Tian F, Ding D and Li D: Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells. Int J Oncol 46: 2355­2363, 2015]. In view of the fact that certain of the data in the above article had already appeared in a previously published paper, and given the large number of apparently overlapping data panels identified in the two referenced figures, the Editor of International Journal of Oncology has decided that this paper should be retracted from the publication. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 50: 1590­1600, 2017; DOI: 10.3892/ijo.2017.3928].

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

Genome-wide analysis of gene expression profiles during ear development of maize.

In order to unravel the molecular mechanism of maize ear development, a microarray containing approximately 56,000 probes was used to monitor the gene expression profiles of ears at four developmental stages. The results showed that 2,794 genes, accounting for 5.0% of the total probes, changed significantly during ear development. Among the 2,794 genes, 1,844 genes differentially expressed during the spikelet differentiation phase, 836 genes during the floret primordium differentiation phase and 645 genes during the floret organ differentiation phase. Hierarchical clustering revealed that the differentially expressed genes had 9 major expression patterns. Based on Mips Functional Catalogue, 684 differentially expressed genes were grouped into at least one functional category, including metabolism (30.4%), protein related function (29.2%), biogenesis of cellular components (15.4%) and transcription (13.7%). The analysis revealed that the auxin signaling pathway play an important role in ear development. Moreover, regulation of some transcription factors may play a key role during ear development. RT-PCR and in situ hybridization for some selected genes validated our microarray data and supplied additional information on ear developmental processes.

Toolbox approach to the search for effective ligands for catalytic asymmetric Cr-mediated coupling reactions.

Chromium catalysts derived from chiral sulfonamides represented by A effect the couplings of aldehydes with vinyl, allyl, or alkyl halides. With three distinct sites for structural modification, A affords access to a structurally diverse pool of chiral sulfonamides. The Cr catalysts derived from these sulfonamides exhibit a broad range of catalyst-substrate matching profiles. A strategy is presented to search for a satisfactory chiral sulfonamide for a given substrate. In order to demonstrate the generality and effectiveness of this approach, five diverse C-C bond-forming cases have been selected from the halichondrin synthesis. For each of the cases, two ligands have been deliberately searched for, to induce the formation of (R)- and (S)-alcohols, respectively, at the arbitrarily chosen efficiency level of ">or=80% yield with >or=20:1 stereoselectivity in the presence of

miR-627-3p inhibits osteosarcoma cell proliferation and metastasis by targeting PTN.

Dysregulation of microRNA (miRNA) has been observed in several types of tumors, including osteosarcoma. Biochip analysis was used to identify miRNAs differentially expressed in osteosarcoma tissues. The targeting sites of miR-627-3p were analyzed using miRDB software and fluorescein reporter gene. MTT and Transwell assays were used to analyze the effects of miR-627-3p on the growth and migration of osteosarcoma cells. Western blotting and real-time PCR were used to detect the effects of miR-627-3p on related proteins. In vivo experiments were conducted to verify the effect of miR-627-3p on osteosarcoma. We focused on miR-627-3p because it was the most significantly downregulated miRNA in our screening study. Through luciferase reporter assays, western blotting and real-time PCR we found that miR-627-3p directly targets PTN, and that expression levels of miR-627-3p and PTN are negatively correlated in osteosarcoma cells. Downregulation of miR-627-3p promoted osteosarcoma cell proliferation and metastasis, while its overexpression had the opposite effect. By targeting PTN, miR-627-3p also suppressed expression of Cyclin D1 and MMP2. MiR-627-3p inhibited osteosarcoma metastasis in vivo. Thus, miR-627-3p may be a useful therapeutic target for the treatment osteosarcoma or prevention of metastasis.

Facile synthesis of chiral (right-handed) calcium carbonate with exceptional enantioseparation performance of dibenzoyltartaric acid.

The chiral morphology of calcium carbonate (mainly vaterite) was successfully synthesized by using CaCl2 under l-aspartic acid guiding by diffusion of in-site generation of CO2 and NH3 from (NH4)2CO3 and NH3·H2O. Its structure was characterized by XRD, SEM/TEM and FT-IR, it indicated that the helical-shaped chiral calcium carbonate was uniform in a diameter of 10-20 µm and height of 1 µm, and tortuous nanosheets with a thickness of 50 nm were evolved more abundant from center to edge in counterclockwise. The enantioseparation performance for racemic dibenzoyltartaric acid was evaluated, the chiral calcium carbonate showed certain recognition ability for l-dibenzoyltartaric acid due to its better matching with l-dibenzoyltartaric acid molecules in stereochemical structure. Chiral separation ability for dibenzoyltartaric acid could be significantly improved by dibutyl maleate modification of chiral calcium carbonate; the maximal ee values for dibenzoyltartaric acid increased from 20.62% to 62.15%. The synergism of chiral helical-shaped suprastructure, large proportion of vaterite and modification of surface by functional group resulted in the excellent enantiomer separation.

Organic Gallium Treatment Improves Osteoporotic Fracture Healing Through Affecting the OPG/RANKL Ratio and Expression of Serum Inflammatory Cytokines in Ovariectomized Rats.

This study aimed to investigate the impact of organic gallium (OG) on osteoporotic fracture healing in ovariectomized female Sprague-Dawley rats, as well as study the mechanisms of OG on osteoporotic fracture healing. Forty-five female Sprague-Dawley rats were divided into three groups: sham operation group (Sxas control group), ovariectomized group (Ovx), and Ovx treated with OG group (Ovx + OG). Rat femoral fractures were studied using a standardized fracture-healing model utilizing bone fixation with an intramedullary pin. Six weeks later, analyses of micro-CT, histomorphometric, RNA extraction, RT-qPCR, and serum were performed following sacrifice of all mice. In comparison with Ovx group, OG can significantly increase bone volume (BV), tissue volume (TV), BV/TV radio, bone strength, callus bony area, and as similar to BMP-2 expression. OG treatment elevated OPG messenger RNA (mRNA) and inhibited RANKL mRNA, and showed an effect on OPG/RANKL ratio. OG treatment can inhibit the expression of TNF-α and IL-6. In conclusion, current study results indicate that organic OG can positively affect the OPG/RANKL ratio and inhibit the expression of serum inflammatory cytokines; thus, it can improve osteoporotic fracture healing.

miR-141-3p is a key negative regulator of the EGFR pathway in osteosarcoma.

BACKGROUND: Many studies have used miRNA to modulate osteosarcoma development by regulating protein expression, and these studies showed that the expression of EGFR is increased in osteosarcoma. METHODS: Western blot, real-time PCR and immunohistochemical were used to detect the expression of EGFR and miR-141 in osteosarcoma tissues and cells. The correlation between miR-141 and the grading of osteosarcoma and the correlation with the survival time of the patients were analyzed. After predicting the target effect of miR-141 on EGFR by miRDB, correlation analysis was used to analyze the correlation between miR-141 and EGFR. Luciferase reporter gene, western blot and real-time PCR were used to detect the targeting effect of miR-141 on EGFR. Then we detected the effect of miR-141 on proliferation by MTT and PI staining. The effect of miR-141 on cell apoptosis was detected by Hochest33258 and AV-PI staining, and the effect of miR-141 on cell migration was detected by Transwell. The regulatory effects of miR-141 on related proteins were detected by western blot and real-time PCR. Finally, we transfected EGFR and EGFR DEL (mutation with miR-141 binding site) in osteosarcoma cells, and detected the effects of miR-141 on cell proliferation, apoptosis, migration and related proteins. RESULTS: The expression of miR-141-3p was negatively correlated with the expression of EGFR in osteosarcoma. The overexpression of miR-141-3p was not only closely related to the classification and size of the osteosarcoma but also had a negative effect on the growth and migration of the osteosarcoma through negative regulation of the expression of EGFR. MiR-141 can inhibit the growth and metastasis of osteosarcoma cells by targeting EGFR and affecting its downstream pathway proteins. CONCLUSION: Our study provides miR-141-3p may be a new theoretical basis for the treatment of osteosarcoma.

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria.

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5-1 µg/mL) and Acinetobacter baumannii (8-16 µg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Oxymatrine suppresses the growth and invasion of MG63 cells by up-regulating PTEN and promoting its nuclear translocation.

Studies demonstrated that reduced PTEN levels are associated with poor prognoses of osteosarcoma. The nuclear localization of PTEN is important for its tumor suppressive function. Equally importantly, PTEN is the most significant negative regulator of PI3K/Akt signaling cascade, the constitutively activated pathway in osteosarcoma. In our study MG63 cells and U2OS cells were treated with the indicated concentrations of oxymatrine, in order to find the inhibition of oxymatrine to cells. We found the functions of oxymatrine on proliferation, apoptosis and invasion in cells. Oxymatrine could increase the expression of PTEN and promote its nuclear translocation in MG63 cells. In addition, oxymatrine could induce cell cycle arrest in G1 phase and apoptosis of MG63 cells. The migration and invasion potential of MG63 cells were also markedly inhibited by oxymatrine. Oxymatrine could suppress the growth and invasion of MG63 human osteosarcoma cells by up-regulating PTEN and promoting its nuclear translocation and inhibiting PI3K/Akt signaling pathway.

miR-486 suppresses the development of osteosarcoma by regulating PKC-δ pathway.

Osteosarcoma is one of the most highly malignant types of cancer in adolescents and young adults with a high mortality rate. Despite advances in surgery, radiation therapy and chemotherapy, the prognosis for patients with osteosarcoma has not significantly improved over the past several decades. It is necessary to find new indicators of prognosis and therapeutic targets of osteosarcoma. Through the analysis of 40 osteosarcoma tissues, we found that the expression of miR­486 was low and the expression of PKC­Î´ was high in osteosarcoma. Median survival of patients with low expression of miR-486 (30 months) was shorter than the patients with higher expression of miR­486 (40 months). We further found that miR-486 can inhibit the targeting of PKC­Î´ signaling pathways, and this inhibition can inhibit the growth and invasion of osteosarcoma cells. After transfection of miR­486 for 24 h, the proliferation of osteosarcoma cells was inhibited by ~20%, and the migration was inhibited by ~15%. In the present investigation, we demonstrated that miR­486 is negatively associated with the expression of PKC-δ and could regulate the development of osteosarcoma. miR-486 may be a potential target for the treatment of osteosarcoma.

A concise total synthesis of (-)-cylindricine C through a stereoselective intramolecular aza-[3 + 3] annulation strategy.

[reaction: see text] An enantioselective total synthesis of (-)-cylindricine C is described, featuring a diastereoselective intramolecular aza-[3 + 3] annulation strategy and an interesting halohydrin formation of the C4-5 olefin for construction of C4-ketone. This work provides a unique approach to this family of natural products.