To understand the changes of femoral cartilage in response to treadmill running with different intensities in the hope of differentiating "moderate" and "strenuous" running in a rat model.

We previously reported that Chlamydia trachomatis hypothetical protein CT311 was secreted out of chlamydial inclusion and into host cell cytosol. We now found that CT311 further entered host cell nucleus at the late stage of infection and continued to accumulate in the nucleus of C. trachomatis-infected cells. When CT311 was expressed via a transgene in mammalian cells, CT311 protein was exclusively detected in the nucleus, suggesting that CT311 by itself is sufficient for nuclear targeting. However, preexisting nuclear CT311 did not affect subsequent chlamydial infection. Using deletion constructs, we mapped a nuclear localization signal sequence of CT311 to residues 21 to 63 ((21)AVEGKPLSRAAQLRERRKDLHVSGKPSPRYALKKRALEAKKNK(63)). This sequence was sufficient for targeting a heterologous protein into mammalian cell nucleus and it contains two independent clusters of basic residues ((34)RERRK(38) and (53)KKRALEAKKNK(63) respectively). Deletion or alanine substitution of the basic residues in either cluster led to loss of nuclear targeting activity, suggesting that both clusters are critical for the nuclear targeting function. These observations have demonstrated that the hypothetical protein CT311 possesses a novel nuclear localization signal sequence with dual modules of basic residues for targeting host cell nucleus during Chlamydia trachomatis infection.

Foam cell formation and macrophage polarization are involved in the pathologic development of atherosclerosis, one of the most important human diseases affecting large and medium artery walls. This study was designed to assess the effects of rapamycin and FTY720 (fingolimod) on macrophages and foam cells. Mouse peritoneal macrophages were collected and treated with rapamycin and FTY720 to study autophagy, polarization, and lipid accumulation. Next, foam cells were formed by oxidizing low-density lipoprotein to observe changes in lipid accumulation, autophagy, and polarization in rapamycin-treated or FTY720-treated foam cells. Lastly, foam cells that had been treated with rapamycin and FTY720 were evaluated for sphingosine 1-phosphate receptor (S1prs) expression. Autophagy microtubule-associated protein 1 light chain 3- (LC3-) II was increased, and classically activated macrophage phenotype markers interleukin- (IL-) 6, cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) were increased, whereas alternatively activated macrophage phenotype markers transforming growth factor- (TGF-) β, arginase 1 (Arg1), and mannose receptor C-type 1 (Mrc1) were decreased by rapamycin in peritoneal macrophages. LC3-II was also obviously enhanced, though polarization markers were unchanged in rapamycin-treated foam cells. Moreover, lipid accumulation was inhibited in rapamycin-treated macrophage cells but was unchanged in rapamycin-treated foam cells. For FTY720, LC3-II did not change, whereas TGF-β, Arg1 and Mrc1 were augmented, and IL-6 was suppressed in macrophages. However, LC3-II was increased, and TGF-β, ARG1 and MRC1 were strikingly augmented, whereas IL-6, COX2 and iNOS could be suppressed in foam cells. Furthermore, lipid accumulation was alleviated in FTY720-treated foam cells. Additionally, S1pr1 was markedly decreased in foam cells (P < .05); S1pr2, S1pr3, S1pr4 and S1pr5 were unchanged in rapamycin-treated foam cells. In FTY720-treated foam cells, S1pr3 and S1pr4 were decreased, and S1pr1, S1pr2 and S1pr5 were unchanged. Therefore, we deduced that rapamycin stimulated classically activated macrophages and supressed early atherosclerosis. Rapamycin may also stabilize artery plaques by preventing apoptosis and S1PR1 in advanced atherosclerosis. FTY720 allowed transformation of foam cells into alternatively activated macrophages through the autophagy pathway to alleviate advanced atherosclerosis.

To develop and demonstrate the effectiveness of a novel dexamethasone (Dex) nanoformulation for treating uveitis.

Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein-DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.

The cytosolic protein α-catenin is a postulated force transducer at cadherin complexes. The demonstration of force activation, identification of consequent downstream events in live cells, and development of tools to study these dynamic processes in living cells are central to elucidating the role of α-catenin in cellular mechanics and tissue function. Here we demonstrate that α-catenin is a force-activatable mechanotransducer at cell-cell junctions by using an engineered α-catenin conformation sensor based on fluorescence resonance energy transfer (FRET). This sensor reconstitutes α-catenin-dependent functions in α-catenin-depleted cells and recapitulates the behavior of the endogenous protein. Dynamic imaging of cells expressing the sensor demonstrated that α-catenin undergoes immediate, reversible conformation switching in direct response to different mechanical perturbations of cadherin adhesions. Combined magnetic twisting cytometry with dynamic FRET imaging revealed rapid, local conformation switching upon the mechanical stimulation of specific cadherin bonds. At acutely stretched cell-cell junctions, the immediate, reversible conformation change further reveals that α-catenin behaves like an elastic spring in series with cadherin and actin. The force-dependent recruitment of vinculin—a principal α-catenin effector—to junctions requires the vinculin binding site of the α-catenin sensor. In cells, the relative rates of force-dependent α-catenin conformation switching and vinculin recruitment reveal that α-catenin activation and vinculin recruitment occur sequentially, rather than in a concerted process, with vinculin accumulation being significantly slower. This engineered α-catenin sensor revealed that α-catenin is a reversible, stretch-activatable sensor that mechanically links cadherin complexes and actin and is an indispensable player in cadherin-specific mechanotransduction at intercellular junctions.

Canonical transient receptor potential channel 6 (TRPC6) can play an important role in governing how cells perceive the surrounding material environment and regulate Ca(2+) signaling. We have designed a TRPC6 reporter based on fluorescence resonance energy transfer (FRET) to visualize the TRPC6-mediated calcium entry and hence TRPC6 activity in live cells with high spatiotemporal resolutions. In mouse embryonic fibroblasts (MEFs), platelet-derived growth factor BB (PDGF) can activate the TRPC6 reporter, mediated by phospholipase C (PLC). This TRPC6 activation occurred mainly at lipid rafts regions of the plasma membrane because disruption of lipid raft/caveolae by methyl-β-cyclodextrin (MβCD) or the expression of dominant-negative caveolin-1 inhibited the TRPC6 activity. Culturing cells on soft materials or releasing the intracellular tension by ML-7 reduced this PDGF-induced activation of TRPC6 without affecting the PDGF-regulated Src or inositol 1,4,5-trisphosphate (IP3) receptor function, suggesting a specific role of mechanical tension in regulating TRPC6. We further showed that the release of intracellular tension had similar effect on the diffusion coefficients of TRPC6 and a raft marker, confirming a strong coupling between TRPC6 and lipid rafts. Therefore, our results suggest that the TRPC6 activation mainly occurs at lipid rafts, which is regulated by the mechanical cues of surrounding materials.

A combination of antidepressant drugs and psychotherapy exhibits more promising efficacy in treating fear disorders than either treatment alone, but underlying mechanisms of such treatments remain largely unknown. Here we investigated the role of DNA methylation of the brain-derived neurotrophic factor (Bdnf) gene in the therapeutic effects of ketamine in combination with extinction training in a mouse model of post-traumatic stress disorder (PTSD) induced by inescapable electric foot shocks (IFS). Male mice received ketamine for 22 consecutive days starting 1 h after the IFS (long-term ketamine treatment) or 2 h prior to the extinction training on days 15 and 16 after the IFS (short-term ketamine treatment). The Open Field (OF) and Elevated Plus Maze (EPM) tests were conducted on days 18 and 20. The spontaneous recovery and fear renewal tests were performed on day 23. Mice, subjected to IFS, exhibited anxiety-like behavior and fear relapse, accompanied by the increased levels of DNA methyltransferases, hyper-methylation of Bdnf gene, and decreased BDNF mRNA expression in the medial prefrontal cortex (mPFC) and hippocampus (HIP). Long-term treatment with ketamine combined with extinction training alleviated the IFS-induced abnormalities. These results suggest that long-term ketamine treatment in combination with extinction training may ameliorate fear relapse in the murine model of PTSD, at least in part, by normalizing DNA methylation of Bdnf gene.

Acute graft-versus-host disease (aGVHD) remains a clinical challenge and a major source of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dimethyl fumarate (DMF), an activator of Nrf2, has been shown to have anti-inflammatory and immunomodulatory properties without significant immunosuppression. We therefore hypothesized that DMF could be potentially harnessed for the treatment of aGVHD with retention of graft-versus-tumor effect. In this study, we showed that DMF significantly inhibited alloreactive T cell responses in vitro in mixed lymphocyte reaction assay. Administration of DMF significantly alleviated the severity, histological damage, and the overall mortality of aGVHD in an MHC-mismatched aGVHD model. DMF administration reduced the activation and effector function of donor T cells in vitro and in vivo. In addition, DMF treatment upregulated antioxidant enzymes heme oxygenase-1 and glutathione S-transferase-α1 expressions. Furthermore, DMF treatment markedly increased the frequencies of Treg cells. Depletion of CD25+ cells in DMF recipients aggravated aGVHD mortality compared with IgG control recipients. DMF could promote Treg cell differentiation in a dose dependent manner by upregulating TGF-β expression in vitro. Most importantly, DMF administration preserved graft-versus-leukemia effect after bone marrow transplantation. In conclusion, our findings demonstrated DMF as a promising agent for the prevention of aGVHD after allo-HSCT.

The pituitary-derived somatolactotrophe GH3 cells secrete both growth hormone (GH) and prolactin (PRL). We have found that the hnRNP L and L-like (LL) paralogs differentially regulate alternative splicing of genes in these cells. Here, we show that hnRNP L is essential for PRL only, but LL is essential for both PRL and GH production. Transcriptome-wide RNA sequencing (RNA-Seq) analysis indicates that they differentially control groups of hormone or hormone-related genes involved in hormone production/regulation at total transcript and alternative exon levels. Interestingly, hnRNP L also specifically binds and prevents the aberrant usage of a nonconserved CA-rich intron piece of Prl pre-mRNA transcripts, and many others involved in endocrine functions, to prevent mostly cryptic last exons and mRNA truncation. Essential for the full hnRNP L effect on specific exons is a proline-rich region that emerged during evolution in vertebrate hnRNP L only but not LL. Together, our data demonstrate that the hnRNP L and its paralog, LL, differentially control hormone gene expression programs at multiple levels, and hnRNP L in particular is critical for protecting the transcriptome from aberrant usage of intronic sequences. The multilevel differential control by hnRNPs likely tailors the transcriptome to help refine and safeguard the different gene expression programs for different hormones.

Multidrug resistance (MDR) is a serious obstacle encountered in cancer treatment, in which the overexpression of P-glycoprotein (P-gp) plays an important role. Here, a novel α-hederagenin derivative, designated H6, was designed, synthesized and evaluated for its ability to reverse MDR. Our results showed that H6 could sensitize KBV and MCF7/T cells to paclitaxel and vincristine. Meanwhile, H6 could increase both rhodamine 123 and paclitaxel accumulation in MDR cells without affecting the expression of P-gp. Interestingly, siRNA knockdown of MDR1 further sensitized the cytotoxic activity of paclitaxel when co-administrated with H6. In addition, H6 could directly stimulate P-gp ATPase activity in vitro. Importantly, H6 enhanced the efficacy of paclitaxel against KBV cancer cell-derived xenograft tumors in nude mice. Finally, H6 showed high binding affinity with P-gp with a high docking score. Overall, we show H6 is a novel and potent MDR reversal agent, which has the potential to be administered in combination with conventional anticancer drugs.

Apical periodontitis (AP) is an inflammation affecting the periapical region of tooth root. Microbial pathogens activate inflammasomes and promote the production of pro-inflammatory cytokines. Caspase-1-mediated pyroptosis is a possible mechanism involved in the initiation and progression of AP. The purpose of this study was to evaluate the role of caspase-1 and pyroptosis on AP at different stages. Human periapical inflammatory tissue was collected to study chronic AP stage. Human periodontal ligament fibroblasts (hPDLFs) were stimulated with lipopolysaccharide in vitro for 24h to simulate early AP stage. Experimental AP rat model was established to study acute AP stage from 0d to 28d. The results showed that NLRP3, cleaved caspase-1 and Interleukin (IL)-1β were enhanced in all AP stages. Caspase-1 activation was detectable in most cells. However, the level of pyroptosis was in accordance with the degree of AP inflammation. Early and chronic AP showed a comparable hemostasis state, with pyroptosis remaining in a reduced level. On the contrary, extensive pyroptosis accelerated inflammation and induced cell death in acute AP. VX765, a caspase-1 inhibitor, was used in an experimental AP rat model. The results showed that VX765 suppressed bone loss, suggesting a role of pyroptosis on bone resorption in acute AP. VX765 also inhibited the expressions of IL-1β, Monocyte chemoattractant protein-1 (MCP-1), IL-6 and IL-8 in vitro, thus decreased inflammatory responses during AP. In conclusion, caspase-1 and pyroptosis contributed to AP inflammation and lesion and pyroptosis extent was in line with AP progression.

Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-β, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-β, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.

Matrix remodeling associated 7 (MXRA7) was first noted to co-express with a group of matrix remodeling related genes, and its biological functions had remained unclear. In this study, we investigated the presumed function of MXRA7 in a carbon tetrachloride (CCl4)-induced acute liver injury model in mice. Wild-type, MXRA7-/- mice, and mice that were pulsed with hydrodynamic injection of vehicle or MXRA7-harboring plasmids were challenged with a single dose of CCl4 for injury induction. The sera, spleens, and livers were harvested from mice for assay of cytokines/chemokines expression, cellular responses, or histological features. We found that MXRA7 deficiency alleviated, and MXRA7 overexpression aggravated liver damage in CCl4-challenged mice. FACS analysis showed that MXRA7 deficiency reduced the recruitment of neutrophils through downregulation the expression of CXCL1 and CXCL2 in liver, decreased the number of CD8+ T cells in liver and spleen, suppressed the release of IFNγ and TNFα from T cells, and decreased IFNγ in serum and liver. Western blot assay demonstrated that MXRA7 deficiency suppressed the activation of MAPK pathway and AKT/NF-κB pathway, respectively. Lastly, MXRA7 deficiency or overexpression regulated the expression of two matrix remodeling-related genes (fibronectin and TIMP1) in the liver. We concluded that MXRA7 was an active player in CCl4-induced liver injury, hypothetically by mediating the inflammation or immune compartments and matrix remodeling processes. Further exploration of MXRA7 as a possible new therapeutic target for management of inflammation-mediated liver injury was discussed.

The International Classification of Diseases (ICD) relies on clinical features and lags behind the current understanding of the molecular specificity of disease pathobiology, necessitating approaches that incorporate growing biomedical data for classifying diseases to meet the needs of precision medicine. Our analysis revealed that the heterogeneous molecular diversity of disease chapters and the blurred boundary between disease categories in ICD should be further investigated. Here, we propose a new classification of diseases (NCD) by developing an algorithm that predicts the additional categories of a disease by integrating multiple networks consisting of disease phenotypes and their molecular profiles. With statistical validations from phenotype-genotype associations and interactome networks, we demonstrate that NCD improves disease specificity owing to its overlapping categories and polyhierarchical structure. Furthermore, NCD captures the molecular diversity of diseases and defines clearer boundaries in terms of both phenotypic similarity and molecular associations, establishing a rational strategy to reform disease taxonomy.

The recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route of mcr-1 among Enterobacteriaceae species in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis of Escherichia coli isolates collected in a hospital in Hangzhou, China. We found that mcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread of mcr-1 The mcr-1 gene was found on either chromosomes or plasmids, but in most of the E. coli isolates, mcr-1 was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission of mcr-1 and the coexistence of mcr-1 with other genes encoding β-lactamases and fluoroquinolone resistance in the E. coli isolates. These findings indicate that mcr-1 is heterogeneously disseminated in both commensal and pathogenic strains of E. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology of mcr-1 among hospital-associated E. coli strains.IMPORTANCE Colistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergent mcr-1 colistin resistance gene threatens the clinical utility of colistin and has gained global attention. How mcr-1 spreads in hospital settings remains unknown and was investigated by whole-genome sequencing of mcr-1-carrying Escherichia coli in this study. The findings revealed extraordinary flexibility of mcr-1 in its spread among genetically diverse E. coli hosts and plasmids, nosocomial transmission of mcr-1-carrying E. coli, and the continuous emergence of novel Inc types of plasmids carrying mcr-1 and new mcr-1 variants. Additionally, mcr-1 was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology of mcr-1 and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

Ubiquinone (UQ) has been considered as an electron mediator in electron transfer that generates ATP in Rhizobium under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few Rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. MK and UQ were extracted from the wild type strain 7653R and mutant strain HK116. MK-7 was accumulated under microaerobic condition and UQ-10 was accumulated under aerobic condition in M. huakuii 7653R. The predicted function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results revealed that MK-7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids.

Dicer is the ribonuclease III for synthesis of mature functional microRNAs (miRNAs), which play an important role in regulating cell development. In the mouse ovary, the Dicer1 protein was expressed in both oocyte and granulosa cells of the follicle. In the present study, the role of miRNAs in mouse ovarian development was explored by using Dicer1 conditional knockout (cKO) mouse ovarian tissue (Amhr2 Cre/-; Dicer flox/flox), in which Dicer1 is deleted specifically in follicular granulosa cells. The morphology and gene expression profile of cKO and wild type (WT) mouse ovaries at various stages of development (day 4, day 8, 8 weeks and 8 months) were examined. Comparative analysis of the follicle number indicated that conditional inactivation of Dicer1 in the follicular granulosa cells led to an increased primordial follicle pool endowment, accelerated early follicle recruitment and more degenerate follicles in the cKO ovaries. In addition, significant differences were noted in the expression of some follicle development-related genes between cKO and WT mouse ovaries, such as Amh, Inhba, Cyp17a1, Cyp19a1, Zps, Gdf9 and Bmp15, suggesting the function of miRNAs in regulating gene expression is time- and gene-dependent. With the Dicer1 inactivation, mmu-mir-503, a miRNA that is more abundant in mouse ovary than in other tissues, was down-regulated significantly. Meanwhile, the expression of mmu-mir-503 decreased notably with follicle development in the gonadotropin-primed mouse ovary. Up-regulation of mmu-mir-503 in primary cultured granulosa cells resulted in the decreased expression of both the target gene and non-target gene at the transcriptional level, which involve genes related to granulosa cell proliferation and luteinization. In conclusion, Dicer1 plays important roles in follicular cell development through the differential regulation of gene expression.

The zinc finger protein Klf7 and POU homeodomain protein Brn3a are each required for efficient transcription of TrkA in primary sensory neurons. In this study, we examined whether these transcription factors act in concert to regulate TrkA expression. In vitro, Brn3a and Klf7 can synergistically activate the TrkA enhancer. In vivo, precursor cells that are destined to become TrkA(+) neurons are born. However, both Brn3a and Klf7 are dispensable for the initiation of TrkA expression. At E12.5, while TrkA expression is unaffected in Brn3a-/- trigeminal ganglia and only slightly decreased in Klf7-/- trigeminal ganglia, it is severely reduced in the double mutant Brn3a-/-;Klf7-/- trigeminal ganglia. At birth, all Trk(+) neurons are lost in Brn3a-/-;Klf7-/- trigeminal ganglia. We further demonstrate that the TrkA enhancer is inactive in Brn3a-/-;Klf7-/- trigeminal ganglia. Thus, cooperation between these two transcription factors is required for endogenous TrkA gene expression and the survival of nociceptive sensory neurons.

Aurora kinase A (AURKA), which belongs to the serine/threonine protein kinase family, has been identified as a key driver of the genesis and progression of diverse tumors. The aim of this study was to determine the clinical significance of AURKA in patients with hepatoblastoma (HB) and the effect of inhibiting AURKA in the HB cell line HuH-6.