Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays.

As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1ß and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.

Noncoding Variations in the Gene Encoding Ceramide Synthase 6 are Associated with Type 2 Diabetes in a Large Indigenous Australian Pedigree.

Type 2 diabetes (T2D) is a chronic disease that disproportionately affects Indigenous Australians. We have previously reported the localization of a novel T2D locus by linkage analysis to chromosome 2q24 in a large admixed Indigenous Australian pedigree (Busfield et al. (2002). American Journal of Human Genetics, 70, 349-357). Here we describe fine mapping of this region in this pedigree, with the identification of SNPs showing strong association with T2D: rs3845724 (diabetes p = 7 × 10-4), rs4668106 (diabetes p = 9 × 10-4) and rs529002 (plasma glucose p = 3 × 10-4). These associations were successfully replicated in an independent collection of Indigenous Australian T2D cases and controls. These SNPs all lie within the gene encoding ceramide synthase 6 (CERS6) and thus may regulate ceramide synthesis.

Cane Toad Skin Extract-Induced Upregulation and Increased Interaction of Serotonin 2A and D2 Receptors via Gq/11 Signaling Pathway in CLU213 Cells.

Recent evidences show that activation of serotonin 2A receptors (5-HT2A R) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT2A R in certain areas of brain, such as the pre-frontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D2 R) availability, primarily in the striatum of the brain along with dysfunction of 5-HT2A R-D2 R heteromer regulation. We thus hypothesized that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD. In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 µg/mL, P < 0.05) of the Australian cane toad skin significantly increased the levels of 5-HT2A R and D2 R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT2A R and D2 R and formation of expression of 5-HT2A R-D2 R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signaling pathway (Gq/11 -PLCß) along with c-FOS transcription factor preferentially in 5-HT2A -mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT2A R by a mechanism that appears to involve Gq/11 -PLCß signaling pathway and c-FOS transcription factor activation. We indicate this enhanced 5-HT2A R and D2 R expression and their interaction to induce increased 5-HT2A R-D2 R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. J. Cell. Biochem. 118: 979-993, 2017. © 2016 Wiley Periodicals, Inc.

Identifying the science and technology dimensions of emerging public policy issues through horizon scanning.

Public policy requires public support, which in turn implies a need to enable the public not just to understand policy but also to be engaged in its development. Where complex science and technology issues are involved in policy making, this takes time, so it is important to identify emerging issues of this type and prepare engagement plans. In our horizon scanning exercise, we used a modified Delphi technique. A wide group of people with interests in the science and policy interface (drawn from policy makers, policy adviser, practitioners, the private sector and academics) elicited a long list of emergent policy issues in which science and technology would feature strongly and which would also necessitate public engagement as policies are developed. This was then refined to a short list of top priorities for policy makers. Thirty issues were identified within broad areas of business and technology; energy and environment; government, politics and education; health, healthcare, population and aging; information, communication, infrastructure and transport; and public safety and national security.

Potential for layered double hydroxides-based, innovative drug delivery systems.

Layered Double Hydroxides (LDHs)-based drug delivery systems have, for many years, shown great promises for the delivery of chemical therapeutics and bioactive molecules to mammalian cells in vitro and in vivo. This system offers high efficiency and drug loading density, as well as excellent protection of loaded molecules from undesired degradation. Toxicological studies have also found LDHs to be biocompatible compared with other widely used nanoparticles, such as iron oxide, silica, and single-walled carbon nanotubes. A plethora of bio-molecules have been reported to either attach to the surface of or intercalate into LDH materials through co-precipitation or anion-exchange reaction, including amino acid and peptides, ATPs, vitamins, and even polysaccharides. Recently, LDHs have been used for gene delivery of small molecular nucleic acids, such as antisense, oligonucleotides, PCR fragments, siRNA molecules or sheared genomic DNA. These nano-medicines have been applied to target cells or organs in gene therapeutic approaches. This review summarizes current progress of the development of LDHs nanoparticle drug carriers for nucleotides, anti-inflammatory, anti-cancer drugs and recent LDH application in medical research. Ground breaking studies will be highlighted and an outlook of the possible future progress proposed. It is hoped that the layered inorganic material will open up new frontier of research, leading to new nano-drugs in clinical applications.

Apoptosis and microRNA aberrations in cancer.

Carcinogenesis arises from the malfunction of genes that control cell growth and division. Therefore, the most effective method of hindering tumourigenesis is to induce the death of immortalized cancer cells. Apoptosis or programmed cell death has shown the most promises in impairing cancer growth. A variety of proteins is involved in the regulation of apoptosis and the malfunction of any these regulators may cause cell proliferation. The microRNAs have been shown to play a central role in the regulation of the cell cycle, including apoptosis. The microRNAs are involved in post-transcriptional gene suppression and have been implicated in the regulation of cell differentiation and development. Aberrations in the microRNA regulation of apoptosis lead to tumourigenesis. The present review assesses the current knowledge of apoptotic regulation in cancer and the effect of microRNA aberrations in tumourigenesis.

Blockage of transdifferentiation from fibroblast to myofibroblast in experimental ovarian cancer models.

BACKGROUND: Tumour stromal myofibroblasts can promote tumour invasion. As these cells are genetically more stable than cancer cells, there has been enormous interest in developing targeted molecular therapies against them. Chloride intracellular channel 4 (CLIC4) and reactive oxygen species (ROS) have been linked with promoting stromal cell transdifferentiation in various cancers, but little is known of their roles in ovarian cancer. In this study, we examined the functional roles that both CLIC4 and ROS play in the process of ovarian cancer cell-stimulated or TGF-beta1 induced fibroblast-to-myofibroblast transdifferentiation. We also examine whether it is possible to reverse such a process, with the aim of developing novel therapies against ovarian cancer by targeting activated transdifferentiated myofibroblasts. RESULTS: We demonstrate that TGF-beta1 induced or CM(SKOV3) activate transdifferentiated myofibroblasts (fibroblasts). These fibroblasts mimic "reactive" stromal myofibroblasts and demonstrate significant up-regulation of CLIC4 expression and increased level of ROS production. Blocking the production of ROS with an antioxidant consequently reduces the expression of CLIC4, and is accompanied by disappearance of alpha-smooth-muscle actin (alpha-SMA), a myofibroblast marker, suggesting ROS acts as a signalling molecule that promotes and enhances CLIC4 activities in the myofibroblast transdifferentiaton process. Down-regulation of CLIC4 with a generic agent or specific siRNA both significantly reduces the expression of factors related to the phenotypes and functions of myofibroblasts, such as alpha-SMA, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), thus reversing the myofibroblast phenotype back to fibroblasts. These results convincingly show that ROS and CLIC4 are responsible for TGF-beta1 induced fibroblast-to-myofibroblast transdifferentiaton and down-regulation of both is sufficient to block transdifferentiated myofibroblasts. CONCLUSION: Molecular targeting of ROS and CLIC4 has the potential to develop novel therapies for ovarian cancer.

Neuroprotective effects of a standardized flavonoid extract from Diospyros kaki leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Flavonoids, extracted from the leaves of Diospyros kaki, are the main therapeutic components of NaoXingQing (NXQ), a potent and patented Chinese herbal remedy widely used in China for the treatment of apoplexy syndrome. AIM OF THE STUDY: To investigate the neuroprotective effects of FLDK-P70, a standardized flavonoid extract, using in vivo rat models of both focal ischemia/reperfusion (I/R) injury induced by middle cerebral artery occlusion (MCAO) and on transient global brain ischemia induced by four-vessel occlusion (4-VO). We also aim to examine the effects of FLDK-P70 on glutamate-induced cell injury of hippocampal neurons as well as on hypoxia-induced injury of cortical neurons in primary cell culture. MATERIALS AND METHODS AND RESULTS: Administration of FLDK-P70 for 12 days (40, 80 mg/kg body weight, p.o., 5 days before and 7 days after 4-VO) increased the survival of hippocampal CA1 pyramidal neurons after transient global brain ischemia. Similarly, administration of FLDK-P70 for 7 days (40, 80 mg/kg body weight, p.o., 3 days before and 4 days after MCAO) significantly reduced the lesion of the insulted brain hemisphere and improved the neurological behavior of rats. In primary rat hippocampal neuronal cultures, pretreatment with FLDK-P70 (5, 10 microg/ml) protected neurons from glutamate-induced excitotoxic neuronal death in a dose-dependent manner. In primary rat cerebral cortical neuronal culture, pretreatment with FLDK-P70 (25, 100 microg/ml) also reduced hypoxia-reoxygen induced neuronal death and apoptosis in a dose-dependent manner. CONCLUSIONS: These in vivo and in vitro results suggest that FLDK-P70 significantly protects rats from MCAO and 4-VO ischemic injury in vivo and protects hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia-induced injury in vitro. The mechanisms of these effects may be due to the antioxidative activity of the flavonoids. These results convincingly demonstrate that FLDK-P70 may be useful for the prevention and treatment of ischemia/reperfusion injury and other related neurodegenerative diseases.

Identification of SQSTM1 mutations in familial Paget's disease in Australian pedigrees.

We have conducted a genome-wide scan on a pedigree containing 372 adult members, of whom 49 have PDB. In the present study, we report linkage of a large pedigree to the PDB3 region on chromosome 5q35-qter with a peak multipoint LOD score of 6.77. Sequestosome 1 (SQSTM/p62) has been identified as the causative PDB gene in this region. Six sequestosome 1 mutations have been described to date. Four mutations have been identified in exon 8, 1210delT and 1215delC both resulting in premature stop codon at amino acid 394, 1215C to T (P392L), 1224insT (E396X), one mutation in exon 7, 1200C to T (P387L) and a G to A splice junction mutation at IVS7+1. These mutations cluster in the C terminus of the protein and are predicted to disrupt the ubiquitin binding properties of sequestosome 1. Sequence analysis of the gene encoding sequestosome 1 revealed a single base pair deletion (1215delC) segregating with the majority of affected members in the pedigree. This deletion introduces a stop codon at position 394, resulting in premature termination of the protein (L394X) and loss of the ubiquitin-associated binding domain. Screening of affected members from 10 further PDB families identified the previously reported P392L mutation in one family. No SQSTM1/p62 coding mutations were found in the remaining 9 families or in 113 age-matched controls.

Linkage of Paget disease of bone to a novel region on human chromosome 18q23.

Paget disease of bone (PDB) is characterized by increased osteoclast activity and localized abnormal bone remodeling. PDB has a significant genetic component, with evidence of linkage to chromosomes 6p21.3 (PDB1) and 18q21-22 (PDB2) in some pedigrees. There is evidence of genetic heterogeneity, with other pedigrees showing negative linkage to these regions. TNFRSF11A, a gene that is essential for osteoclast formation and that encodes receptor activator of nuclear factor-kappa B (RANK), has been mapped to the PDB2 region. TNFRSF11A mutations that segregate in pedigrees with either familial expansile osteolysis or familial PDB have been identified; however, linkage studies and mutation screening have excluded the involvement of RANK in the majority of patients with PDB. We have excluded linkage, both to PDB1 and to PDB2, in a large multigenerational pedigree with multiple family members affected by PDB. We have conducted a genomewide scan of this pedigree, followed by fine mapping and multipoint analysis in regions of interest. The peak two-point LOD scores from the genomewide scan were 2.75, at D7S507, and 1.76, at D18S70. Multipoint and haplotype analysis of markers flanking D7S507 did not support linkage to this region. Haplotype analysis of markers flanking D18S70 demonstrated a haplotype segregating with PDB in a large subpedigree. This subpedigree had a significantly lower age at diagnosis than the rest of the pedigree (51.2+/-8.5 vs. 64.2+/-9.7 years; P=.0012). Linkage analysis of this subpedigree demonstrated a peak two-point LOD score of 4.23, at marker D18S1390 (straight theta=0), and a peak multipoint LOD score of 4.71, at marker D18S70. Our data are consistent with genetic heterogeneity within the pedigree and indicate that 18q23 harbors a novel susceptibility gene for PDB.

HYBRIDIZATION AND CRYPTIC SPECIES IN DICAMPTODON (CAUDATA: DICAMPTODONTIDAE).

The salamander genus Dicamptodon consists of at least four genetically divergent groups of populations with 10-19 fixed allelic differences (out of 31 loci scored) between populations in alternative groups. One of these groups corresponds to D. copei; the other three until now usually have been considered to belong to the single morphologically homogeneous species D. ensatus. Two instances of geographic contact between genetic units occur in the genus, one between D. copei and "D. ensatus" in northern Oregon and the other between two highly divergent types of "D. ensatus" in coastal northern Calfornia. In the former case no hybridization occurs, while in the latter a narrow hybrid zone has been observed. There appears to be selection against hybrids within this hybrid zone, and introgression beyond the zone is apparently nonexistent. The population groups therefore appear to be genetically independent units, and Dicamptodon is best considered to consist of four species: D. copei, D. ensatus, D. tenebrosus, and D. aterrimus.