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The dormancy of cellular apoptotic machinery has been highlighted as a crucial factor in therapeutic resistance, recurrence, and poor prognosis in patients with malignancy, such as malignant glioma. Increasing evidence indicates that nonsteroidal anti-inflammatory drugs (NSAIDs) confer chemopreventive effects, and indomethacin has been shown to have a novel chemotherapeutic application targeting glioma cells. To extend these findings, herein, we studied the underlying mechanisms of apoptosis activation caused by indomethacin in human H4 and U87 glioma cells. We found that the glioma cell-killing effects of indomethacin involved both death receptor- and mitochondria-mediated apoptotic cascades. Indomethacin-induced glioma cell apoptosis was accompanied by a series of biochemical changes, including reactive oxygen species generation, endoplasmic reticulum (ER) stress, apoptosis signal-regulating kinase-1 (Ask1) activation, p38 hyperphosphorylation, protein phosphatase 2A (PP2A) activation, Akt dephosphorylation, Mcl-1 and FLICE-inhibiting protein (FLIP) downregulation, Bax mitochondrial distribution, and caspases 3/caspase 8/caspase 9 activation. Data on pharmacological inhibition related to oxidative stress, ER stress, free Ca2+, and p38 revealed that the axis of oxidative stress/ER stress/Ask1/p38/PP2A/Akt comprised an apoptotic cascade leading to Mcl-1/FLIP downregulation and glioma apoptosis. Since indomethacin is an emerging choice in chemotherapy and its antineoplastic effects have been demonstrated in glioma tumor-bearing models, the findings further strengthen the argument for turning on the aforementioned axis in order to activate the apoptotic machinery of glioma cells.

In this paper, we address the issues related to the design of fuzzy robust linear regression algorithms. The design of robust linear regression analysis has been studied in the literature of statistics for over two decades. More recently various robust regression models have been proposed for processing noisy data. We proposed a new objective function by using fuzzy complement and derive improved algorithms that can produce good regression analysis from the spoiled data set. Data set from the U.S. Department of Transportation is used to evaluate the performance of the regression algorithms.

Single-stranded DNA-binding protein (SSB) and PriA helicase play important roles in bacterial DNA replication restart process. The mechanism by which PriA helicase is bound and stimulated by SSB in Escherichia coli (Ec) has been established, but information on this process in Gram-positive bacteria are limited. We characterized the properties of SSB from Staphylococcus aureus (SaSsbA, a counterpart of EcSSB) and analyzed its interaction with SaPriA. The gel filtration chromatography analysis of purified SaSsbA showed a stable tetramer in solution. The crystal structure of SaSsbA determined at 1.82 Å resolution (PDB entry 5XGT) reveals that the classic oligonucleotide/oligosaccharide-binding folds are formed in the N-terminal DNA-binding domain, but the entire C-terminal domain is disordered. Unlike EcSSB, which can stimulate EcPriA via a physical interaction between EcPriA and the C-terminus of EcSSB (SSB-Ct), SaSsbA does not affect the activity of SaPriA. We also found that SaPriA can be bound by SaSsbA, but not by SaSsbA-Ct. Although no effect was found with SaSsbA, SaPriA can be significantly stimulated by the Gram-negative Klebsiella pneumoniae SSB (KpSSB). In addition, we found that the conserved SSB-Ct binding site of KpPriA (Trp82, Tyr86, Lys370, Arg697, and Gln701) is not present in SaPriA. Arg697 in KpPriA is known to play a critical role in altering the SSB35/SSB65 distribution, but this corresponding residue in SaPriA is Glu767 instead, which has an opposite charge to Arg. SaPriA E767R mutant was constructed and analyzed; however, it still cannot be stimulated by SaSsbA. Finally, we found that the conserved MDFDDDIPF motif in the Gram-negative bacterial SSB is DISDDDLPF in SaSsbA, i.e., F172 in EcSSB and F168 in KpSSB is S161 in SaSsbA, not F. When acting with SaSsbA S161F mutant, the activity of SaPriA was dramatically enhanced elevenfold. Overall, the conserved binding sites, both in EcPriA and EcSSB, are not present in SaPriA and SaSsbA, thereby no stimulation occurs. Our observations through structure-sequence comparison and mutational analyses indicate that the case of EcPriA-EcSSB is not applicable to SaPriA-SaSsbA because of inherent differences among the species.

The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin β1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-β1 (TGF-β1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-β1 expression. The additions of exogenous fibronectin and TGF-β1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-β1 signaling.

Atypical antipsychotics, such as olanzapine, are commonly prescribed to patients with schizophrenic symptoms and other psychiatric disorders. However, weight gain and metabolic disturbance cause adverse effects, impair patient compliance and limit clinical utility. Thus, a better understanding of treatment-acquired adverse effects and identification of targets for therapeutic intervention are believed to offer more clinical benefits for patients with schizophrenia. Beyond its nutritional effects, studies have indicated that supplementation of chromium brings about beneficial outcomes against numerous metabolic disorders. In this study, we investigated whether olanzapine-induced weight gain and metabolic disturbance involved chromium dynamic mobilization in a female Sprague-Dawley rat model, and whether a dietary supplement of chromium improved olanzapine-acquired adverse effects. Olanzapine medicated rats experienced weight gain and adiposity, as well as the development of hyperglycemia, hyperinsulinemia, insulin resistance, hyperlipidemia, and inflammation. The olanzapine-induced metabolic disturbance was accompanied by a decrease in hepatic Akt and AMP-activated Protein Kinase (AMPK) actions, as well as an increase in serum interleukin-6 (IL-6), along with tissue chromium depletion. A daily intake of chromium supplements increased tissue chromium levels and thermogenic uncoupling protein-1 (UCP-1) expression in white adipose tissues, as well as improved both post-olanzapine weight gain and metabolic disturbance. Our findings suggest that olanzapine medicated rats showed a disturbance of tissue chromium homeostasis by inducing tissue depletion and urinary excretion. This loss may be an alternative mechanism responsible for olanzapine-induced weight gain and metabolic disturbance.

Microglia polarization of classical activation state is crucial to the induction of neuroinflammation, and has been implicated in the pathogenesis of numerous neurodegenerative diseases. Fungal immunomodulatory proteins are emerging health-promoting natural substances with multiple pharmacological activities, including immunomodulation. Herein, we investigated the anti-inflammatory and neuroprotective potential of fungal immunomodulatory protein extracted from Ganoderma microsporum (GMI) in an in vitro rodent model of primary cultures. Using primary neuron/glia cultures consisting of neurons, astrocytes, and microglia, a GMI showed an alleviating effect on lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced inflammatory mediator production and neuronal cell death. The events of neuroprotection caused by GMI were accompanied by the suppression of Nitric Oxide (NO), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Prostaglandin E2 (PGE2) production, along with the inhibition of microglia activation. Mechanistic studies showed that the suppression of microglia pro-inflammatory polarization by GMI was accompanied by the resolution of oxidative stress, the preservation of protein tyrosine phosphatase and serine/threonine phosphatase activity, and the reduction of NF-κB, AP-1, cyclic AMP response element-binding protein (CREB), along with signal transducers and activators of transcription (Stat1) transcriptional activities and associated upstream activators. These findings suggest that GMI may have considerable potential towards the treatment of neuroinflammation-mediated neurodegenerative diseases.

Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.

The neurobehavior of neuropathic pain by chronic constriction injury (CCI) of sciatic nerve is very similar to that in humans, and it is accompanied by a profound local inflammation response. In this study, we assess the potentiality of human amniotic fluid derived mesenchymal stem cells (hAFMSCs) for alleviating the neuropathic pain in a chronic constriction nerve injury model.This neuropathic pain animal model was conducted by four 3-0 chromic gut ligatures loosely ligated around the left sciatic nerve in Sprague-Dawley rats. The intravenous administration of hAFMSCs with 5x105 cells was conducted for three consecutive days.The expression IL-1β, TNF-α and synaptophysin in dorsal root ganglion cell culture was remarkably attenuated when co-cultured with hAFMSCs. The significant decrease of PGP 9.5 in the skin after CCI was restored by administration of hAFMSCs. Remarkably increased expression of CD 68 and TNF-α and decreased S-100 and neurofilament expression in injured nerve were rescued by hAFMSCs administration. Increases in synaptophysin and TNF-α over the dorsal root ganglion were attenuated by hAFMSCs. Significant expression of TNF-α and OX-42 over the dorsal spinal cord was substantially attenuated by hAFMSCs. The increased amplitude of sensory evoked potential as well as expression of synaptophysin and TNF-α expression was alleviated by hAFMSCs. Human AFMSCs significantly improved the threshold of mechanical allodynia and thermal hyperalgesia as well as various parameters of CatWalk XT gait analysis.Human AFMSCs administration could alleviate the neuropathic pain demonstrated in histomorphological alteration and neurobehavior possibly through the modulation of the inflammatory response.

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35-217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35-338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214-338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.

Dual leucine zipper kinase (DLK) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family mainly involved in neuronal degeneration. However, the role of DLK signaling in the neuropathic pain has not yet been fully determined. Chronic constrictive injury (CCI) was conducted by four 3-0 chromic gut ligatures loosely ligated around the sciatic nerve. Escalated DLK expression over the dorsal root ganglion was observed from one to four rings of CCI. Remarkable expression of DLK was observed in primary dorsal root ganglion cells culture subjected to electrical stimulation and attenuated by DLK short hairpin RNA (shRNA) treatment. Intrathecal injection of DLK shRNA attenuates the expression of DLK over the dorsal root ganglion and hippocampus neurons and increased the threshold of mechanical allodynia and decreased thermal hyperalgesia. In CatWalk gait analysis, significant decreases of print area, maximum contact maximum intensity, stand phase, single stance, and regular index by CCI were alleviated by the DLK shRNA administration. In conclusion, the expression of DLK was up-regulated in chronic constrictive injury and attenuated by the administration of DLK shRNA, which paralleled the improvement of neurobehavior of neuropathic pain. The modulation of DLK expression is a potential clinic treatment option for neuropathic pain.

PURPOSE:The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model. MATERIALS AND METHODS:Seventy two Sprague-Dawley rats weighing 200-250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups. RESULTS:NT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology. CONCLUSION:Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.

Studies indicate that perinatal opioid exposure produces a variety of short- and long-term neurobehavioral consequences. However, the precise modes of action are incompletely understood. Buprenorphine, a mixed agonist/antagonist at the opioid receptors, is currently being used in clinical trials for managing pregnant opioid addicts. This study provides evidence of depression-like consequence following prenatal exposure to supra-therapeutic dose of buprenorphine and sheds light on potential mechanisms of action in a rat model involving administration of intraperitoneal injection to pregnant Sprague-Dawley rats starting from gestation day 7 and lasting for 14 days. Results showed that pups at postnatal day 21 but not the dams had worse parameters of depression-like neurobehaviors using a forced swimming test and tail suspension test, independent of gender. Neurobehavioral changes were accompanied by elevation of oxidative stress, reduction of plasma levels of brain-derived neurotrophic factor (BDNF) and serotonin, and attenuation of tropomyosin-related kinase receptor type B (TrkB) phosphorylation, extracellular signal-regulated kinase (ERK) phosphorylation, protein kinase A activity, cAMP response element-binding protein (CREB) phosphorylation, and CREB DNA-binding activity. Since BDNF/serotonin and CREB signaling could orchestrate a positive feedback loop, our findings suggest that the induction of oxidative stress, reduction of BDNF and serotonin expression, and attenuation of CREB signaling induced by prenatal exposure to supra-therapeutic dose of buprenorphine provide evidence of potential mechanism for the development of depression-like neurobehavior.

Recently it has been proposed a model for fibrils of human insulin in which the fibril growth proceeds via stacking LVEALYL (fragment 11-17 from chain B of insulin) into pairs of tightly interdigitated [Formula: see text]-sheets. The experiments have also shown that LVEALYL has high propensity to self-assembly and binding to insulin. This necessitates study of oligomerization of LVEALYL and its binding affinity to full-length insulin. Using the all-atom simulations with Gromos96 43a1 force field and explicit water it is shown that LVEALYL can aggregate. Theoretical estimation of the binding free energy of LVEALYL to insulin by the molecular mechanic Poisson-Boltzmann surface area method reveals its strong binding affinity to chain B, implying that, in agreement with the experiments, LVEALYL can affect insulin aggregation via binding mechanism. We predict that, similar to LVEALYL, peptide RGFFYT (fragment B22-27) can self-assemble and bind to insulin modulating its fibril growth process. The binding affinity of RGFFYT is shown to be comparable with that of LVEALYL.

Cored sediments from the gas hydrate prospect area offshore southwestern Taiwan exhibit porosities, water contents, and bulk densities comparable to sediments in other gas-hydrate potential areas. Short-distance transport and rapid deposition of the sediments are implied by their poor sorting and detrital and clay mineralogy. These features and the organicrich nature of the sediments present an environment suitable for formation of gas hydrates. High methane concentrations of porewaters and gas-escape structures of the sediments further imply possible occurrence of gas hydrates in the region. Prominent overgrowth microstructures on authigenic pyrite framboids at shallow depths are consistent with sulfate reduction and pyrite precipitation related to migration and oxidation of methane possibly released from gas hydrate zones at later stages of early diagenesis.