MMP-9 facilitates selective proteolysis of the histone H3 tail at genes necessary for proficient osteoclastogenesis.

Although limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, the specific genomic sites targeted for H3NT proteolysis and the functional significance of H3NT cleavage remain largely unknown. Here we report the first method to identify and examine H3NT-cleaved regions in mammals, called chromatin immunoprecipitation (ChIP) of acetylated chromatin (ChIPac). By applying ChIPac combined with deep sequencing (ChIPac-seq) to an established cell model of osteoclast differentiation, we discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most H3NT-cleaved genes displayed significant expression changes during osteoclastogenesis. We also discovered that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9). In contrast to other known H3NT proteases, MMP-9 primarily cleaved H3K18-Q19 in vitro and in cells. Furthermore, our results support CBP/p300-mediated acetylation of H3K18 as a central regulator of MMP-9 H3NT protease activity both in vitro and at H3NT cleavage sites during osteoclastogenesis. Importantly, we found that abrogation of H3NT proteolysis impaired osteoclastogenic gene activation concomitant with defective osteoclast differentiation. Our collective results support the necessity of MMP-9-dependent H3NT proteolysis in regulating gene pathways required for proficient osteoclastogenesis.

A comparative molecular dynamic simulation study on potent ligands targeting mTOR/FRB domain for breast cancer therapy.

Our study aimed to develop and find out the best drug candidate against the mechanistic target of rapamycin (mTOR/FRB) domain having a critical role in the aetiology of breast cancer. The FKBP12-rapamycin-binding (FRB) domain in the essential phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway has been a vital player in the disease progression in breast cancer. By using structure-based drug designing , the best possible targets have been identified and developed. The three-dimensional structure of the target protein was generated using I-TASSER. The ligands were generated against the most suitable target active site using standard tools for active site identification. Furthermore, the seed molecule was drawn using Chemsketch, which was then grown into the pocket using Ligbuilder. The obtained ligands were further validated using online programs for bioavailability and toxicity, followed by molecular dynamic simulations. The study concludes that the equilibrated NVT-NPT complexes indicate LIG2 stability over LIG3. RMSD and RMSF have shown that the complex of LIG2 is more stable than LIG3. LIG2 has the potential antagonistic properties to target the mTOR/FRB domain and has therapeutic implications for breast cancer.

Recent nanotechnological interventions targeting PI3K/Akt/mTOR pathway: A focus on breast cancer.

Breast cancer is the major cause of deaths in women worldwide. Detection and treatment of breast cancer at earlier stages of the disease has shown encouraging results. Modern genomic technologies facilitated several therapeutic options however the diagnosis of the disease at an advanced stage claim more deaths. Therefore more research directed towards genomics and proteomics into this area may lead to novel biomarkers thereby enhancing the survival rates in breast cancer patients. Phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway was shown to be hyperactivated in most of the breast carcinomas resulting in excessive growth, proliferation, and tumor development. Development of nanotechnology has provided many interesting avenues to target the PI3K/Akt/mTOR pathway both at the pre-clinical and clinical stages. Therefore, the current review summarizes the underlying mechanism and the importance of targeting PI3K/Akt/mTOR pathway, novel biomarkers and use of nanotechnological interventions in breast cancer.

Derivation of induced pluripotent stem cells from ferret somatic cells.

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.

BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice.

Signaling pathways are used reiteratively in different developmental processes yet produce distinct cell fates through specific downstream transcription factors. In this study, we used tooth root development as a model with which to investigate how the BMP signaling pathway regulates transcriptional complexes to direct the fate determination of multipotent mesenchymal stem cells (MSCs). We first identified the MSC population supporting mouse molar root growth as Gli1+ cells. Using a Gli1-driven Cre-mediated recombination system, our results provide the first in vivo evidence that BMP signaling activity is required for the odontogenic differentiation of MSCs. Specifically, we identified the transcription factors Pax9, Klf4, Satb2 and Lhx8 as being downstream of BMP signaling and expressed in a spatially restricted pattern that is potentially involved in determining distinct cellular identities within the dental mesenchyme. Finally, we found that overactivation of one key transcription factor, Klf4, which is associated with the odontogenic region, promotes odontogenic differentiation of MSCs. Collectively, our results demonstrate the functional significance of BMP signaling in regulating MSC fate during root development and shed light on how BMP signaling can achieve functional specificity in regulating diverse organ development.

RTD-1 therapeutically normalizes synovial gene signatures in rat autoimmune arthritis and suppresses proinflammatory mediators in RA synovial fibroblasts.

Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1ß-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.

Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells.

SET and MYND domain containing protein 3 (SMYD3) is a histone methyltransferase, which has been implicated in cell growth and cancer pathogenesis. Increasing evidence suggests that SMYD3 can influence distinct oncogenic processes by acting as a gene-specific transcriptional regulator. However, the mechanistic aspects of SMYD3 transactivation and whether SMYD3 acts in concert with other transcription modulators remain unclear. Here, we show that SMYD3 interacts with the human positive coactivator 4 (PC4) and that such interaction potentiates a group of genes whose expression is linked to cell proliferation and invasion. SMYD3 cooperates functionally with PC4, because PC4 depletion results in the loss of SMYD3-mediated H3K4me3 and target gene expression. Individual depletion of SMYD3 and PC4 diminishes the recruitment of both SMYD3 and PC4, indicating that SMYD3 and PC4 localize at target genes in a mutually dependent manner. Artificial tethering of a SMYD3 mutant incapable of binding to its cognate elements and interacting with PC4 to target genes is sufficient for achieving an active transcriptional state in SMYD3-deficient cells. These observations suggest that PC4 contributes to SMYD3-mediated transactivation primarily by stabilizing SMYD3 occupancy at target genes. Together, these studies define expanded roles for SMYD3 and PC4 in gene regulation and provide an unprecedented documentation of their cooperative functions in stimulating oncogenic transcription.

NUMB phosphorylation destabilizes p53 and promotes self-renewal of tumor-initiating cells by a NANOG-dependent mechanism in liver cancer.

UNLABELLED: Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a particular fate whereas the other retains the multipotent characteristics of its parent. The NUMB, a tumor suppressor, in conjunction with another tumor-suppressor protein, p53, preserves this property and acts as a barrier against deregulated expansion of tumor-associated stem cells. In this context, NUMB-p53 interaction plays a crucial role to maintain the proper homeostasis of both stem cells, as well as differentiated cells. Because the molecular mechanism governing the assembly and stability of the NUMB-p53 interaction/complex are poorly understood, we tried to identify the molecule(s) that govern this process. Using cancer cell lines, tumor-initiating cells (TICs) of liver, the mouse model, and clinical samples, we identified that phosphorylations of NUMB destabilize p53 and promote self-renewal of TICs in a pluripotency-associated transcription factor NANOG-dependent manner. NANOG phosphorylates NUMB by atypical protein kinase C zeta (aPKCζ), through the direct induction of Aurora A kinase (AURKA) and the repression of an aPKCζ inhibitor, lethal (2) giant larvae. By radioactivity-based kinase activity assays, we showed that NANOG enhances kinase activities of both AURKA and aPKCζ, an important upstream process for NUMB phosphorylation. Phosphorylation of NUMB by aPKCζ destabilizes the NUMB-p53 interaction and p53 proteolysis and deregulates self-renewal in TICs. CONCLUSION: Post-translational modification of NUMB by the NANOG-AURKA-aPKCζ pathway is an important event in TIC self-renewal and tumorigenesis. Hence, the NANOG-NUMB-p53 signaling axis is an important regulatory pathway for TIC events in TIC self-renewal and liver tumorigenesis, suggesting a therapeutic strategy by targeting NUMB phosphorylation. Further in-depth in vivo and clinical studies are warranted to verify this suggestion.

Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis.

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of BM-derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)-4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven toward osteoblastic differentiation revealed an increase in vascular endothelial growth factor A (Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC-derived VEGFA mRNA and protein and that this was enhanced by BMP-4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA- mediated knock-down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP-4-treated cocultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma-derived intracrine VEGFA in osteoblastogenesis.

Expression profiling of circulating tumor cells in metastatic breast cancer.

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients' blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.

NEMO is essential for Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13-induced gene expression and protection against death receptor-induced cell death, and its N-terminal 251 residues are sufficient for this process.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 was originally believed to protect virally infected cells against death receptor-induced apoptosis by interfering with caspase 8/FLICE activation. Subsequent studies revealed that K13 also activates the NF-κB pathway by binding to the NEMO/inhibitor of NF-κB (IκB) kinase gamma (IKKγ) subunit of an IKK complex and uses this pathway to modulate the expression of genes involved in cellular survival, proliferation, and the inflammatory response. However, it is not clear if K13 can also induce gene expression independently of NEMO/IKKγ. The minimum region of NEMO that is sufficient for supporting K13-induced NF-κB has not been delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis remains to be determined. In this study, we used microarray analysis on K13-expressing wild-type and NEMO-deficient cells to demonstrate that NEMO is required for modulation of K13-induced genes. Reconstitution of NEMO-null cells revealed that the N-terminal 251 amino acid residues of NEMO are sufficient for supporting K13-induced NF-κB but fail to support tumor necrosis factor alpha (TNF-α)-induced NF-κB. K13 failed to protect NEMO-null cells against TNF-α-induced cell death but protected those reconstituted with the NEMO mutant truncated to include only the N-terminal 251 amino acid residues [the NEMO(1-251) mutant]. Taken collectively, our results demonstrate that NEMO is required for modulation of K13-induced genes and the N-terminal 251 amino acids of NEMO are sufficient for supporting K13-induced NF-κB. Finally, the ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptor-induced apoptosis and to activate the NF-κB pathway by binding to adaptor protein NEMO/IKKγ. However, whether K13 can also induce gene expression independently of NEMO and the minimum region of NEMO that is sufficient for supporting K13-induced NF-κB remain to be delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis is not clear. We demonstrate that NEMO is required for modulation of K13-induced genes and its N-terminal 251 amino acids are sufficient for supporting K13-induced NF-κB. The ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. Our results suggest that K13-based gene therapy approaches may have utility for the treatment of patients with NEMO mutations and immunodeficiency.

Pluripotency factor-mediated expression of the leptin receptor (OB-R) links obesity to oncogenesis through tumor-initiating stem cells.

Misregulation of a pluripotency-associated transcription factor network in adult tissues is associated with the expansion of rare, highly malignant tumor-initiating stem cells (TISCs) through poorly understood mechanisms. We demonstrate that robust and selective expression of the receptor for the adipocyte-derived peptide hormone leptin (OB-R) is a characteristic feature of TISCs and of a broad array of embryonic and induced pluripotent stem cells and is mediated directly by the core pluripotency-associated transcription factors OCT4 and SOX2. TISCs exhibit sensitized responses to leptin, including the phosphorylation and activation of the pluripotency-associated oncogene STAT3 and induction of Oct4 and Sox2, thereby establishing a self-reinforcing signaling module. Exposure of cultured mouse embryonic stem cells to leptin sustains pluripotency in the absence of leukemia inhibitory factor. By implanting TISCs into leptin-deficient ob/ob mice or into comparably overweight Lepr(db/db) mice that produce leptin, we provide evidence of a central role for the leptin-TISC-signaling axis in promoting obesity-induced tumor growth. Differential responses to extrinsic, adipocyte-derived cues may promote the expansion of tumor cell subpopulations and contribute to oncogenesis.

Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma.

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.

Profiling of Circulating Tumor Cells for Screening of Selective Inhibitors of Tumor-Initiating Stem-Like Cells.

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

A20 is induced by Kaposi sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 and blocks K13-induced nuclear factor-kappaB in a negative feedback manner.

Expression of A20, a negative regulator of the NF-κB pathway, is frequently lost in several subtypes of Hodgkin and non-Hodgkin lymphoma. We report that A20 is expressed in Kaposi sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma cell lines, and its expression correlates closely with the expression of KSHV-encoded viral FLICE inhibitory protein K13. Ectopic expression of K13 induced A20 expression through NF-κB-mediated activation of A20 promoter. In turn, A20 blocked K13-induced NF-κB activity and up-regulation of proinflammatory cytokines CCL20 and IL-8 in a negative feedback fashion. Both the N-terminal deubiquitinating domain and the C-terminal zinc finger domain of A20 were involved in the inhibition of K13-induced NF-κB activity. Overexpression of A20 blocked K13-induced IκBα phosphorylation, NF-κB nuclear translocation, and cellular transformation. Consistent with the above, K13-induced IκBα phosphorylation and NF-κB transcriptional activation were enhanced in A20-deficient cells. Finally, A20 was found to interact physically with K13. Taken collectively, these results demonstrate that K13 is a key determinant of A20 expression in KSHV-infected cells, and A20 is a key negative regulator of K13-induced NF-κB activity. A20 might serve to control the inflammatory response to KSHV infection and protect KSHV-infected cells from apoptosis.

Nanoparticles in the treatment and diagnosis of neurological disorders: untamed dragon with fire power to heal.

The incidence of neurological diseases of unknown etiology is increasing, including well-studied diseases such as Alzhiemer's, Parkinson's, and multiple sclerosis. The blood-brain barrier provides protection for the brain but also hinders the treatment and diagnosis of these neurological diseases, because the drugs must cross the blood-brain barrier to reach the lesions. Thus, attention has turned to developing novel and effective delivery systems that are capable of carrying drug and that provide good bioavailability in the brain. Nanoneurotechnology, particularly application of nanoparticles in drug delivery, has provided promising answers to some of these issues in recent years. Here we review the recent advances in the understanding of several common forms of neurological diseases and particularly the applications of nanoparticles to treat and diagnose them. In addition, we discuss the integration of bioinformatics and modern genomic approaches in the development of nanoparticles. FROM THE CLINICAL EDITOR: In this review paper, applications of nanotechnology-based diagnostic methods and therapeutic modalities are discussed addressing a variety of neurological disorders, with special attention to blood-brain barrier delivery methods. These novel nanomedicine approaches are expected to revolutionize several aspects of clinical neurology.

Regulatory role of miR-146a in corneal epithelial wound healing via its inflammatory targets in human diabetic cornea.

PURPOSE: MiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing. METHODS: Our previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs. RESULTS: Overexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1ß, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a. CONCLUSION: Our study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.

Induction of CCL20 production by Kaposi sarcoma-associated herpesvirus: role of viral FLICE inhibitory protein K13-induced NF-kappaB activation.

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is the etiologic agent of Kaposi sarcoma (KS), an angioproliferative lesion characterized by dramatic angiogenesis and inflammatory infiltration. In this study, we report that expression of chemokine CCL20, a potent chemoattractant of dendritic cells and lymphocytes, is strongly induced in cultured cells either by KSHV infection or on ectopic expression of viral FLICE inhibitory protein K13. This induction is caused by transcriptional activation of CCL20 gene, which is mediated by binding of the p65, p50, and c-Rel subunits of the transcription factor nuclear factor-kappaB (NF-kappaB) to an atypical NF-kappaB-binding site present in the CCL20 gene promoter. The CCL20 gene induction is defective in K13 mutants that lack NF-kappaB activity, and can be blocked by specific genetic and pharmacologic inhibitors of the NF-kappaB pathway. CCR6, the specific receptor for CCL20, is also induced in cultured cells either by KSHV infection or on K13 expression. Finally, expression of CCL20 and CCR6 is increased in clinical samples of KS. These results suggest that KSHV and K13-mediated induction of CCL20 and CCR6 may contribute to the recruitment of dendritic cells and lymphocytes into the KS lesions, and to tumor growth and metastases.

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-α, Hedgehog signaling, adherens junctions, TGF-ß, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.