D-Mannose prevents bone loss under weightlessness.

BACKGROUND: Astronauts undergo significant microgravity-induced bone loss during space missions, which has become one of the three major medical problems hindering human's long-term space flight. A risk-free and antiresorptive drug is urgently needed to prevent bone loss during space missions. D-mannose is a natural C-2 epimer of D-glucose and is abundant in cranberries. This study aimed to investigate the protective effects and potential mechanisms of D-mannose against bone loss under weightlessness. METHODS: The hind legs of tail-suspended (TS) rats were used to mimic weightlessness on Earth. Rats were administered D-mannose intragastrically. The osteoclastogenic and osteogenic capacity of D-mannose in vitro and in vivo was analyzed by micro-computed tomography, biomechanical assessment, bone histology, serum markers of bone metabolism, cell proliferation assay, quantitative polymerase chain reaction, and western blotting. RNA-seq transcriptomic analysis was performed to detect the underlying mechanisms of D-mannose in bone protection. RESULTS: The TS rats showed lower bone mineral density (BMD) and poorer bone morphological indices. D-mannose could improve BMD in TS rats. D-mannose inhibited osteoclast proliferation and fusion in vitro, without apparent effects on osteoblasts. RNA-seq transcriptomic analysis showed that D-mannose administration significantly inhibited the cell fusion molecule dendritic cell-specific transmembrane protein (DC-STAMP) and two indispensable transcription factors for osteoclast fusion (c-Fos and nuclear factor of activated T cells 1 [NFATc1]). Finally, TS rats tended to experience dysuria-related urinary tract infections (UTIs), which were suppressed by treatment with D-mannose. CONCLUSION: D-mannose protected against bone loss and UTIs in rats under weightlessness. The bone protective effects of D-mannose were mediated by inhibiting osteoclast cell fusion. Our findings provide a potential strategy to protect against bone loss and UTIs during space missions.

CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65.

The E3 ubiquitin ligase complex CDC20-activated anaphase-promoting complex/Cyclosome (APC/CCDC20 ) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co-activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well-known cell cycle-related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA-binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11-dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle-independent function of CDC20, establishes APC11CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone-related diseases.

ZIM1 Combined with Hydrogel Inhibits Senescence of Primary PαS Cells during In Vitro Expansion.

Bone marrow stem cells (BMSCs) are a promising source of seed cells in bone tissue engineering, which needs a great quantity of cells. Cell senescence occurs as they are passaged, which could affect the therapeutic effects of cells. Therefore, this study aims to explore the transcriptomic differences among the uncultured and passaged cells, finding a practical target gene for anti-aging. We sorted PαS (PDGFR-α+SCA-1+CD45-TER119-) cells as BMSCs by flow cytometry analysis. The changes in cellular senescence phenotype (Counting Kit-8 (CCK-8) assay, reactive oxygen species (ROS) test, senescence-associated ß-galactosidase (SA-ß-Gal) activity staining, expression of aging-related genes, telomere-related changes and in vivo differentiation potential) and associated transcriptional alterations during three important cell culture processes (in vivo, first adherence in vitro, first passage, and serial passage in vitro) were studied. Overexpression plasmids of potential target genes were made and examed. Gelatin methacryloyl (GelMA) was applied to explore the anti-aging effects combined with the target gene. Aging-related genes and ROS levels increased, telomerase activity and average telomere length decreased, and SA-ß-Gal activities increased as cells were passaged. RNA-seq offered that imprinted zinc-finger gene 1 (Zim1) played a critical role in anti-aging during cell culture. Further, Zim1 combined with GelMA reduced the expression of P16/P53 and ROS levels with doubled telomerase activities. Few SA-ß-Gal positive cells were found in the above state. These effects are achieved at least by the activation of Wnt/ß-catenin signaling through the regulation of Wnt2. The combined application of Zim1 and hydrogel could inhibit the senescence of BMSCs during in vitro expansion, which may benefit clinical application.

DUSP5 promotes osteogenic differentiation through SCP1/2-dependent phosphorylation of SMAD1.

Dual-specificity phosphatases (DUSPs) are defined by their capability to dephosphorylate both phosphoserine/phosphothreonine (pSer/pThr) and phosphotyrosine (pTyr). DUSP5, a member of DUSPs superfamily, is located in the nucleus and plays crucially regulatory roles in the signaling pathway transduction. In our present study, we discover that DUSP5 significantly promotes osteogenic differentiation of mesenchymal stromal cells (MSCs) by activating SMAD1 signaling pathway. Mechanistically, DUSP5 physically interacts with the phosphatase domain of small C-terminal phosphatase 1/2 (SCP1/2, SMAD1 phosphatases) by the linker region. In addition, we further confirm that DUSP5 activates SMAD1 signaling through a SCP1/2-dependent manner. Specifically, DUSP5 attenuates the SCP1/2-SMAD1 interaction by competitively binding to SCP1/2, which is responsible for the SMAD1 dephosphorylation, and thus results in the activation of SMAD1 signaling. Importantly, DUSP5 expression in mouse bone marrow MSCs is significantly reduced in ovariectomized (OVX) mice in which osteogenesis is highly passive, and overexpression of Dusp5 via tail vein injection reverses the bone loss of OVX mice efficiently. Collectively, this work demonstrates that the linker region of DUSP5 maybe a novel chemically modifiable target for controlling MSCs fate choices and for osteoporosis treatment.

H2A Histone Family Member Z (H2AFZ) Serves as a Prognostic Biomarker in Lung Adenocarcinoma: Bioinformatic Analysis and Experimental Validation.

BACKGROUND H2A histone family member Z (H2AFZ) is a special subtype in the H2A histone family, which participates in the regulation of gene transcription. Nevertheless, little is known about the role of H2AFZ in the tumor microenvironment and genetic factors associated with lung cancer. MATERIAL AND METHODS The expression of H2AFZ in LUAD was analyzed via Tumor Immune Estimation Resource (TIMER), the Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) databases at the mRNA level. To detect the protein expression level of H2AFZ, immunohistochemistry (IHC) was performed using LUAD tissues and non-tumor lung tissues. Kaplan-Meier survival analysis and Cox regression analysis were conducted to identify the effect of H2AFZ expression on overall survival (OS) based on TCGA-LUAD and the GEO dataset GSE68465 cohorts, and our LUAD patient cohort was used for validation. Identification of signaling pathways associated with the expression of H2AFZ was performed using Gene Set Enrichment Analysis (GSEA). The influences of expression of H2AFZ on tumor immune-infiltrating cell (TIICs) were assessed via TIMER and CIBERSORT. RESULTS The expression of H2AFZ was increased in LUAD tissues at both mRNA and protein levels. In addition, high expression of H2AFZ predicted poor OS and might be an independent prognostic predictor in LUAD patients. Moreover, H2AFZ affected the relative proportion of TIICs and was positively associated with Myeloid-derived suppressor cells (MDSC) infiltration level in LUAD. CONCLUSIONS H2AFZ was upregulated in LUAD and related to poor prognosis of LUAD patients; thus, it could be an underlying prognostic biomarker correlated with immune infiltration in LUAD.

Overexpression and Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA Synthase Affect Central Metabolic Pathways in Tobacco.

Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-coenzyme A. Our previous studies had shown that, while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. Sequential window acquisition of all theoretical mass spectra quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA, which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

Alveolar bone changes in maxillary and mandibular anterior teeth during orthodontic treatment: A systematic review and meta-analysis.

The association between tooth movement and remodelling of surrounding bone is controversial. To analyse the effect of tooth movement on alveolar bone changes in maxillary and mandibular anterior teeth by cone-beam computed tomography (CBCT). The Embase, Cochrane Library and Medline databases were searched without any language restrictions. Longitudinal studies using CBCT to observe alveolar bone changes of maxillary and mandibular anterior teeth during orthodontic treatment were included. Two independent reviewers performed the study selection, data extraction and methodological quality assessment. A total of 26 studies were included in this review, 14 of which were eligible for quantitative synthesis. In extraction cases, meta-analysis showed vertical bone loss on the labial (0.36 mm) and lingual (0.94 mm) sides of maxillary incisors, and lingual bone thickness decreased significantly at the cervical level (0.57 mm). In non-extraction cases, vertical alveolar bone loss was significant on the labial side (0.97 mm) and lingual side (0.86 mm) of mandibular incisors. Subgroup analysis for skeletal class III patients indicated that vertical alveolar bone loss was 1.16 mm on the labial side and 0.83 mm on the lingual side of mandibular incisors. The absence of high-quality studies and the high heterogeneity of the included studies were limitations of this systematic review and meta-analysis. Based on limited evidence, alveolar bone height and thickness, especially at the cervical level, decreased during both labial and lingual movement of anterior teeth.

Network meta-analysis of the treatment efficacy of different lasers for peri-implantitis.

The aim of this study was comparing different lasers with conventional non-surgical treatment (CNT) for the management of peri-implantitis, regarding probing depth (PD), plaque index (PLI), clinical attachment level (CAL), and sulcus bleeding index (SBI). Randomized controlled trials (RCTs) on different lasers and CNT for peri-implantitis were searched. Pairwise and network meta-analyses were performed to analyze the PD, PLI, CAL, and SBI outcomes. The risk of bias, evidence quality, statistical heterogeneity, and ranking probability were also evaluated. Eleven studies were included in this study, involving three types of lasers. Diode + CNT had significantly superior efficacy to CNT alone, regarding PD reduction, while Er:YAG + CNT had significantly superior efficacy than CNT in terms of the PLI, CAL, and SBI. The highest probability of being most effective for PD was diode + CNT (49%), while Er:YAG + CNT had the highest probability of improving the PLI, CAL, and SBI (66%, 53%, and 79%, respectively). Diode + CNT was significantly superior for PD management in peri-implantitis compared with CNT alone, while Er:YAG + CNT significantly improved the PLI, CAL, and SBI. Therefore, Er:YAG + CNT might be recommended methods considered for management of peri-implantitis.

Network meta-analysis of the desensitizing effects of lasers in patients with dentine hypersensitivity.

OBJECTIVES: This network meta-analysis compares different lasers, placebo, and no treatment in terms of their effects on dentine hypersensitivity (DH) immediately after treatment and over the long term (1 month). METHODS: A systematic electronic literature search of four databases and a manual search were performed to identify randomized controlled trials (RCTs) examining different laser treatments for the treatment of DH. Pairwise and network meta-analyses were performed to analyze the desensitization effect immediately after treatment and over the long term. The risk of bias was assessed based on the Cochrane guidelines and funnel plots. The quality of the evidence, statistical heterogeneity, inconsistencies, and ranking probability were also evaluated. RESULTS: A total of 11 RCTs were included in the network meta-analysis; 11 and 9 of these studies analyzed immediate and long-term effects, respectively. All four types of laser had a better desensitizing effect than controls immediately after treatment and over the long term, but there were no significant differences among the four different lasers. There was a significant placebo effect immediately after treatment. The laser with the highest probability of being the most effective treatment for DH was Er,Cr:YSGG immediately after treatment and over the long term (73% and 47%, respectively). CONCLUSIONS: All four types of laser had significantly better effects than no treatment on DH immediately after treatment and in the long term, but there were no significant differences among the four lasers. In addition, there was a significant placebo effect, supporting the importance of including a placebo group in future studies. Furthermore, Er,Cr:YSGG may be the most effective laser for the treatment of DH immediately and over the long term. CLINICAL RELEVANCE: This study used network meta-analyses to compare different lasers, placebo, and no treatment over different time periods, which is to provide guidance for selecting an appropriate laser treatment in patients with DH.

Comparison of technical, biological, and esthetic parameters of ceramic and metal-ceramic implant-supported fixed dental prostheses: A systematic review and meta-analysis.

STATEMENT OF PROBLEM: Differences between ceramic and metal-ceramic implant-supported fixed dental prostheses are unclear. PURPOSE: The purpose of this systematic review and meta-analysis was to compare the technical, biological, and esthetic complication rates of implant-supported ceramic and metal-ceramic restorations. MATERIAL AND METHODS: Six databases were searched to identify randomized controlled clinical trials (RCTs) and prospective and retrospective cohort studies of implant-supported fixed dental prostheses. The survival rate, marginal adaptation, marginal bone loss, pocket probing depth, crown color match, and mucosal discoloration of ceramic and metal-ceramic single crowns were assessed. For implant-supported fixed partial dental prostheses (FPDPs), only the survival rate was assessed. The risk of bias was assessed for individual studies and across studies by using the Cochrane guidelines, Newcastle-Ottawa scale, and funnel plots. RESULTS: Twenty studies were included in this meta-analysis. Ceramic and metal-ceramic implant-supported single crowns were compared in terms of the survival rate (OR=0.84 [0.32, 2.23], P=.730), marginal adaptation (mean difference [MD]=0.33 [0.19, 0.47], P<.001), marginal bone loss (MD=-0.03 [-0.07, 0.02], P=.260), pocket probing depth (MD=-0.07 [-0.14, 0.00], P=.060), crown color match (MD=-0.15 [-0.29, 0.00], P=.040), and mucosal discoloration (standardized mean difference [SMD]=-0.14 [-0.86, 0.58], P=.710). The survival rate of ceramic and metal-ceramic implant-supported FPDPs was also compared (odds ratio [OR]=1.92 [1.26, 2.94], P=.003). CONCLUSIONS: No significant difference was observed between ceramic and metal-ceramic implant-supported single crowns in terms of the survival rate, marginal bone loss, pocket probing depth, or mucosal discoloration. However, metal-ceramic single crowns had better marginal adaptation and poorer crown color match than did ceramic single crowns. In addition, current evidence indicates that metal-ceramic implant-supported FPDPs might have a higher survival rate than ceramic FPDPs.

Effect of Lasers on Dentine Hypersensitivity: Evidence From a Meta-analysis.

OBJECTIVES: The aim of the study was to evaluate the immediate and long-term desensitizing effect of lasers in reducing dentine hypersensitivity (DH) compared with negative controls. MATERIAL AND METHODS: Six databases were searched to identify relevant articles published up to June 8, 2018. Randomized controlled trials comparing lasers with placebo or no treatment control in adult patients who suffer from DH were included. The risk of bias was assessed according to the Cochrane guidelines, and the quality of the evidence was evaluated using the Grading of Recommendations Assessment, Development and Evaluation tool. Inverse-variance random effects meta-analyses of standardized mean differences and 95% confidence intervals were performed using the RevMan 5.3 software. RESULTS: Twenty-two randomized controlled trials were finally included in the meta-analysis, and 21 studies of these were conducted to analyze the immediate and long-term effects. All types of lasers had better immediate and long-term desensitizing effects on DH than negative controls. The quality of evidence of the included studies showed that lasers compared with negative controls had moderate-quality immediate and long-term effects on DH. The statistical heterogeneity of these comparisons was high, for which the result of I2 ranged from 90% to 98%. CONCLUSIONS: Our results indicate that all types of lasers had a better desensitizing effect on DH than negative controls, both in immediate and long term. Furthermore, more high-quality studies with a large sample size are needed to confirm our results (PROSPERO CRD42018102260).

Structure of Drosophila Oskar reveals a novel RNA binding protein.

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix-fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3'UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3'UTRs, and provide structural insights into a novel protein-RNA interaction motif involving a hydrolase-related domain.

Porous zirconia ceramic as an alternative to dentin for in vitro dentin barriers cytotoxicity test.

OBJECTIVES: This study assessed the potential of porous zirconia ceramic as an alternative to dentin via an in vitro dentin barrier cytotoxicity test. METHODS: The permeability of dentin and porous zirconia ceramic was measured using a hydraulic-conductance system, and their permeability was divided into two groups: high and low. Using an in vitro dentin barrier test, the cytotoxicity of dental materials by dentin and porous zirconia ceramic was compared within the same permeability group. The L-929 cell viability was assessed by MTT assay. RESULTS: The mean (SD) permeability of the high and low group for dentin was 0.334 (0.0873) and 0.147 (0.0377) µl min-1 cm-2 cm H2O-1 and for zirconia porous ceramic was 0.336 (0.0609) and 0.146 (0.0340) µl min-1 cm-2 cm H2O-1. The cell viability of experimental groups which are the low permeability group was higher than that of the high permeability group for both dentin and porous zirconia ceramic as a barrier except for Maxcem Elite™ by porous zirconia ceramic. There was no significant difference between dentin and porous zirconia ceramic in cell viability, within either the high or low permeability group for all materials. The SD for cell viability of the porous zirconia ceramic was less than that of the dentin, across all materials within each permeability group, except for Maxcem Elite™ in the high permeability group. CONCLUSIONS: Porous zirconia ceramic, having similar permeability to dentin at the same thickness, can be used as an alternative to dentin for in vitro dentin barrier cytotoxicity tests. CLINICAL RELEVANCE: In vitro dentin barrier cytotoxicity tests when a standardized porous zirconia ceramic was used as a barrier could be useful for assessing the potential toxicity of new dental materials applied to dentin before applying in clinical and may resolve the issue of procuring human teeth when testing proceeds.

Enhanced expression of genes involved in initial xylose metabolism and the oxidative pentose phosphate pathway in the improved xylose-utilizing Saccharomyces cerevisiae through evolutionary engineering.

Fermentation of xylose in lignocellulosic hydrolysates by Saccharomyces cerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3 % lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP(+)-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.

TGF-ß signaling regulates differentiation of MSCs in bone metabolism: disputes among viewpoints.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into cells of different lineages to form mesenchymal tissues, which are promising in regard to treatment for bone diseases. Their osteogenic differentiation is under the tight regulation of intrinsic and extrinsic factors. Transforming growth factor ß (TGF-ß) is an essential growth factor in bone metabolism, which regulates the differentiation of MSCs. However, published studies differ in their views on whether TGF-ß signaling regulates the osteogenic differentiation of MSCs positively or negatively. The controversial results have not been summarized systematically and the related explanations are required. Therefore, we reviewed the basics of TGF-ß signaling and summarized how each of three isoforms regulates osteogenic differentiation. Three isoforms of TGF-ß (TGF-ß1/ß2/ß3) play distinct roles in regulating osteogenic differentiation of MSCs. Additionally, other possible sources of conflicts are summarized here. Further understanding of TGF-ß signaling regulation in MSCs may lead to new applications to promote bone regeneration and improve therapies for bone diseases.

Synthesis and structure-activity optimization of 7-azaindoles containing aza-ß-amino acids targeting the influenza PB2 subunit.

The PB2 subunit of influenza virus polymerase has been demonstrated as a promising drug target for anti-influenza therapy. In this work, 7-azaindoles containing aza-ß3- or ß2,3 -amino acids were synthesized possessing a good binding affinity of PB2. The aza-ß-amino acid moieties with diverse size, shape, steric hindrance and configuration were investigated. Then a lead HAA-09 was validated, and the attached aza-ß3-amino acid moiety with acyclic tertiary carbon side chain well occupied in the key hydrophobic cavity of PB2_cap binding domain. Importantly, HAA-09 displays potent polymerase inhibition capacity, low cytotoxicity (selectivity index up to 2915) as well as robust anti-viral activity against A/WSN/33 (H1N1) virus and oseltamivir-resistant H275Y variant. Moreover, HAA-09 exhibited druggability with high plasma stability (t1/2 ≥ 12 h) and no obvious hERG inhibition (IC50 > 10 µM). Also, HAA-09 demonstrated a favorable safety profile when orally administrated in healthy mice at a high dose of 40 mg/kg QD for consecutive 3 days. Besides, in vivo therapeutic efficacy (85.7% survival observed at the day 15 post infection) was demonstrated when HAA-09 was administrated orally at 12.5 mg/kg BID starting 48 h post infection for 9 days. These data support that exploring the interactions between side chains on aza-ß3- or ß2,3 -amino acid moieties and hydrophobic pocket of PB2_cap binding domain is a potential medicinal chemistry strategy for developing potent PB2 inhibitors.

BHLHE40 Maintains the Stemness of PαS Cells In Vitro by Targeting Zbp1 through the Wnt/ß-Catenin Signaling Pathway.

Primary bone mesenchymal stem cells (BMSCs) gradually lose stemness during in vitro expansion, which significantly affects the cell therapeutic effects. Here, we chose murine PαS (SCA-1+PDGFRα+CD45-TER119-) cells as representative of BMSCs and aimed to explore the premium culture conditions for PαS cells. Freshly isolated (fresh) PαS cells were obtained from the limbs of C57/6N mice by fluorescence-activated cell sorting (FACS). We investigated the differences in the stemness of PαS cells by proliferation, differentiation, and stemness markers in vitro and by ectopic osteogenesis and chondrogenesis ability in vivo, as well as the changes in the stemness of PαS cells during expansion in vitro. Gain- and loss-of-function experiments were applied to investigate the critical role and underlying mechanism of the basic helix-loop-helix family member E40 (BHLHE40) in maintaining the stemness of PαS cells. The stemness of fresh PαS cells representative in vivo was superior to that of passage 0 (P0) PαS cells in vitro. The stemness of PαS cells in vitro decreased gradually from P0 to passage 4 (P4). Moreover, BHLHE40 plays a critical role in regulating the stemness of PαS cells during in vitro expansion. Mechanically, BHLHE40 regulates the stemness of PαS cells by targeting Zbp1 through the Wnt/ß-catenin signaling pathway. This work confirms that BHLHE40 is a critical factor for regulating the stemness of PαS cells during expansion in vitro and may provide significant indications in the exploration of premium culture conditions for PαS cells.

Deep mutational scanning of influenza A virus neuraminidase facilitates the identification of drug resistance mutations in vivo.

IMPORTANCE: NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA's mutational effect and drug resistance profiles in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.

Cinobufotalin prevents bone loss induced by ovariectomy in mice through the BMPs/SMAD and Wnt/ß-catenin signaling pathways.

BACKGROUND: Osteoporosis is a chronic bone disease characterized by bone loss and decreased bone strength. However, current anti-resorptive drugs carry a risk of various complications. The deep learning-based efficacy prediction system (DLEPS) is a forecasting tool that can effectively compete in drug screening and prediction based on gene expression changes. This study aimed to explore the protective effect and potential mechanisms of cinobufotalin (CB), a traditional Chinese medicine (TCM), on bone loss. METHODS: DLEPS was employed for screening anti-osteoporotic agents according to gene profile changes in primary osteoporosis. Micro-CT, histological and morphological analysis were applied for the bone protective detection of CB, and the osteogenic differentiation/function in human bone marrow mesenchymal stem cells (hBMMSCs) were also investigated. The underlying mechanism was verified using qRT-PCR, Western blot (WB), immunofluorescence (IF), etc. RESULTS: A safe concentration (0.25 mg/kg in vivo, 0.05 µM in vitro) of CB could effectively preserve bone mass in estrogen deficiency-induced bone loss and promote osteogenic differentiation/function of hBMMSCs. Both BMPs/SMAD and Wnt/ß-catenin signaling pathways participated in CB-induced osteogenic differentiation, further regulating the expression of osteogenesis-associated factors, and ultimately promoting osteogenesis. CONCLUSION: Our study demonstrated that CB could significantly reverse estrogen deficiency-induced bone loss, further promoting osteogenic differentiation/function of hBMMSCs, with BMPs/SMAD and Wnt/ß-catenin signaling pathways involved.

Martynoside rescues 5-fluorouracil-impaired ribosome biogenesis by stabilizing RPL27A.

Martynoside (MAR), a bioactive component in several well-known tonic traditional Chinese herbs, exhibits pro-hematopoietic activity during 5-fluorouracil (5-FU) treatment. However, the molecular target and the mechanism of MAR are poorly understood. Here, by adopting the mRNA display with a library of even-distribution (md-LED) method, we systematically examined MAR-protein interactions in vitro and identified the ribosomal protein L27a (RPL27A) as a key cellular target of MAR. Structural and mutational analysis confirmed the specific interaction between MAR and the exon 4,5-encoded region of RPL27A. MAR attenuated 5-FU-induced cytotoxicity in bone marrow nucleated cells, increased RPL27A protein stability, and reduced the ubiquitination of RPL27A at lys92 (K92) and lys94 (K94). Disruption of MAR binding at key residues of RPL27A completely abolished the MAR-induced stabilization. Furthermore, by integrating label-free quantitative ubiquitination proteomics, transcriptomics, and ribosome function assays, we revealed that MAR restored RPL27A protein levels and thus rescued ribosome biogenesis impaired by 5-FU. Specifically, MAR increased mature ribosomal RNA (rRNA) abundance, prevented ribosomal protein degradation, facilitated ribosome assembly, and maintained nucleolar integrity. Collectively, our findings characterize the target of a component of Chinese medicine, reveal the importance of ribosome biogenesis in hematopoiesis, and open up a new direction for improving hematopoiesis by targeting RPL27A.