Photoreceptor laminin drives differentiation of human pluripotent stem cells to photoreceptor progenitors that partially restore retina function.

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32 days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2 weeks post transplantation as measured by full-field electroretinogram. At 4 weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20 weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.

Stepwise versus Concerted: Theoretical Insights into the Stereoselectivity in Aryl Imine Formation Assisted by Acid and Water.

The wide applications of the aryl Schiff base require extensive understanding of the mechanism of its formation, which remains unclear. In this work, the detailed formation mechanisms between benzaldehyde and aniline or 4-(9-anthryl) ethynyl aniline were investigated at the CCSD(T)//B3LYP level, and the influence of water molecules and acid catalysis and the stereoselectivity were addressed. The results show that the participation of explicit water molecules greatly accelerates the reactions by alleviating the ring tension of the transition states, and acid catalysis strongly favors the imine formation and provides driving force for the forward reaction. In acidic conditions, both N-protonated carbinolamine formations and imine formations are achieved under mild conditions with the assistance of water molecules, and the proton transfer is more advanced than the C-N and C═N bond formation, which is in good agreement with the experimental observations. In contrast, under neutral conditions, even with the assistance of two water molecules, the reaction is hard to take place at room temperature owing to the high Gibbs free energy barriers with the proton transfer and the C-N or C═N bond formation concerted. The analysis of stereoselectivity shows that the formation of trans imine is both kinetically and thermodynamically more favorable than the cis one under the acidic condition with the assistance of water molecules, and the presence of conjugated substituent 4-(9-anthryl) ethynyl of aniline marginally raises the energy barriers. This work provides a systematic view of the mechanism for the formation of aryl imine and is expected to offer insights for the control of the dynamic covalent chemistry and the synthesis of covalent organic frameworks.

Enhanced Biodegradation of Methyl Orange Through Immobilization of Shewanella oneidensis MR-1 by Polyvinyl Alcohol and Sodium Alginate.

Shewanella oneidensis MR-1 has great potential for use in remediating azo dye pollution. Here, a new high-efficiency biodegradation method was developed utilizing S. oneidensis MR-1 immobilized by polyvinyl alcohol (PVA) and sodium alginate (SA). After determining the optimal immobilization conditions, the effects of various environmental factors on methyl orange (MO) degradation were analyzed. The biodegradation activity of the immobilized pellets was evaluated by analyzing the MO removal efficiency, and characterization was performed using scanning electron microscopy. The MO adsorption kinetics can be described using pseudo-second-order kinetics. Compared with free bacteria, the MO degradation rate of the immobilized S. oneidensis MR-1 increased from 41% to 92.6% after 21 days, suggesting that the immobilized bacteria performed substantially better and had more stable removal rates. These factors indicate the superiority of bacteria entrapment in addition to its easy application. This study demonstrates that the application of immobilized S. oneidensis MR-1 entrapped by PVA-SA can be used to establish a reactor with stable and high MO removal rates.

A fluorescent probe for specifically measuring the overall thioredoxin and glutaredoxin reducing activity in bacterial cells.

Thioredoxins (Trxs) and glutaredoxins (Grxs) are the two major thiol-dependent reductases, participating in many important cellular events such as defense against oxidative stress, DNA synthesis and repair. Both Trxs and Grxs have diverse disulfide-containing substrates in the cells to exert their activities, with overlapping functions. Specific methods for measuring the intracellular overall activities of Trxs and Grxs are still lacking. Here we find that TRFS-green, a disulfide containing fluorescent probe which was used to detect thioredoxin reductase (TrxR) in mammalian cells, is a substrate of bacterial Trxs and Grxs, but not a substrate of bacterial TrxR and GSH. This property made TRFS-green work as a probe to measure the overall activities of Trxs and Grxs in bacterial cells. Using various E. coli Trx or Grx null mutant strains, the contribution of different Trxs and Grxs to cellular redox regulation has been clarified, judged by the reducibility towards TRFS-green. E. coli Grx2 and Grx3 unexpectedly exhibited higher activity in reducing the disulfide probe than the other redoxins. In addition, the bacterial disulfide reductase activity was detected to be affected in the ofloxacin bactericidal process. These results show that TRFS-green may be a useful tool for investigating bacterial redox regulation and demonstrating the critical role of E. coli Grxs in maintaining the bacterial intracellular redox balance.

Oncolytic Vaccinia Virus Expressing Aphrocallistes vastus Lectin as a Cancer Therapeutic Agent.

Lectins display a variety of biological functions including insecticidal, antimicrobial, as well as antitumor activities. In this report, a gene encoding Aphrocallistes vastus lectin (AVL), a C-type lectin, was inserted into an oncolytic vaccinia virus vector (oncoVV) to form a recombinant virus oncoVV-AVL, which showed significant in vitro antiproliferative activity in a variety of cancer cell lines. Further investigations revealed that oncoVV-AVL replicated faster than oncoVV significantly in cancer cells. Intracellular signaling elements including NF-κB2, NIK, as well as ERK were determined to be altered by oncoVV-AVL. Virus replication upregulated by AVL was completely dependent on ERK activity. Furthermore, in vivo studies showed that oncoVV-AVL elicited significant antitumor effect in colorectal cancer and liver cancer mouse models. Our study might provide insights into a novel way of the utilization of marine lectin AVL in oncolytic viral therapies.

Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier.

Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos(-/-) and wild-type mice. Inos(-/-) mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos(-/-) mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.

Experimental demonstration of ultracompact air hole photonic crystal ring resonator fabricated on silicon-on-insulator wafer.

A photonic crystal ring resonator (PCRR) of air hole arrays is fabricated on a silicon-on-insulator wafer by using electron-beam lithography and inductively coupled plasma etching. The designed PCRR is modeled and its performance is simulated by the two-dimensional finite difference time domain method. The simulation results show that the PCRR has two resonant wavelengths, 1598 and 1606 nm, and their corresponding quality factors are 3994 and 4015, respectively. A sample of the PCRR structure is fabricated and tested by the established experimental setup. Compared with the simulation results, the experimental resonant wavelengths drift to some extent and the quality factors are reduced by about one order of magnitude. The fabrication error and irregularity are the main reasons for the above results, which can be further reduced by improving the process technology. In addition, one more resonant wavelength emerged for the PCRR sample, which can be attributed to the change of the coupling strength.

Sensitivity improvement of micro-grating accelerometer based on differential detection method.

A differential detection method (DDM) with a utility type and ease of realization for a micro-grating accelerometer is reported so as to reduce the common-mode noise and improve the sensitivity of the micro-grating accelerometer. The theoretical model is established, based on scalar diffraction theory for differential detection. According to the simulation and analysis of the DDM, the theoretical result shows that the sensitivity of the micro-grating accelerometer can be improved by at least a factor of 2. Based on the analysis, the detection circuit is designed with proper parameters and devices for the handheld experimental prototype, which is realized with our micro-grating acceleration sensor fabricated by inductively coupled plasma, lift-off, and anodic bonding of glass/silicon, etc. The prototype experiment is conducted with the turntable. Compared with the single-order detection method whose sensitivities are 6.797 V/g (zeroth order, 1 g=9.8 m/s²) and 7.767 V/g (first-order), the result of the DDM shows that the sensitivity of the micro-grating accelerometer is 18.61 V/g with an improvement of over two times. The overall signal-to-noise ratio improvement is 6.47 dB with the input of 0.86 g.

Current modulation technique used in resonator micro-optic gyro.

Resonator micro-optic gyro (RMOG) is a promising candidate for the next generation inertial rotation sensor based on the Sagnac effect. A current modulation technique used in an external cavity laser diode is proposed to construct the gyroscope system for the first time. The resonance curves before and after eliminating accompanying amplitude modulation are theoretically analyzed, calculated, and simulated; the demodulation curves with different modulation currents are formulated theoretically; and the optimum modulation current corresponding to the maximum sensitivity is obtained. The experiment results from the established RMOG experimental setup demonstrate that a bias stability of 2.7 deg/s (10 s integrated time) over 600 s, and dynamic range of ±500 deg/s are demonstrated in an RMOG with a silica optical waveguide ring resonator having a ring length of 12.8 cm.

Disruption of Bacterial Thiol-Dependent Redox Homeostasis by Magnolol and Honokiol as an Antibacterial Strategy.

Some traditional Chinese medicines (TCMs) possess various redox-regulation properties, but whether the redox regulation contributes to antibacterial mechanisms is not known. Here, ginger juice processed Magnoliae officinalis cortex (GMOC) was found to show strong antibacterial activities against some Gram-positive bacteria, but not Gram-negative bacteria including E. coli, while the redox-related transcription factor oxyR deficient E. coli mutant was sensitive to GMOC. In addition, GMOC and its main ingredients, magnolol and honokiol, exhibited inhibitory effects on the bacterial thioredoxin (Trx) system, a major thiol-dependent disulfide reductase system in bacteria. The effects of magnolol and honokiol on cellular redox homeostasis were further verified by elevation of the intracellular ROS levels. The therapeutic efficacies of GMOC, magnolol and honokiol were further verified in S. aureus-caused mild and acute peritonitis mouse models. Treatments with GMOC, magnolol and honokiol significantly reduced the bacterial load, and effectively protected the mice from S. aureus-caused peritonitis infections. Meanwhile, magnolol and honokiol produced synergistic effects when used in combination with several classic antibiotics. These results strongly suggest that some TCMs may exert their therapeutic effects via targeting the bacterial thiol-dependent redox system.

Development of Crosslinker-Free Polysaccharide-Lysozyme Microspheres for Treatment Enteric Infection.

Antibiotic abuse in the conventional treatment of microbial infections, such as inflammatory bowel disease, induces cumulative toxicity and antimicrobial resistance which requires the development of new antibiotics or novel strategies for infection control. Crosslinker-free polysaccharide-lysozyme microspheres were constructed via an electrostatic layer-by-layer self-assembly technique by adjusting the assembly behaviors of carboxymethyl starch (CMS) on lysozyme and subsequently outer cationic chitosan (CS) deposition. The relative enzymatic activity and in vitro release profile of lysozyme under simulated gastric and intestinal fluids were investigated. The highest loading efficiency of the optimized CS/CMS-lysozyme micro-gels reached 84.9% by tailoring CMS/CS content. The mild particle preparation procedure retained relative activity of 107.4% compared with free lysozyme, and successfully enhanced the antibacterial activity against E. coli due to the superposition effect of CS and lysozyme. Additionally, the particle system showed no toxicity to human cells. In vitro digestibility testified that almost 70% was recorded in the simulated intestinal fluid within 6 h. Results demonstrated that the cross-linker-free CS/CMS-lysozyme microspheres could be a promising antibacterial additive for enteric infection treatment due to its highest effective dose (573.08 µg/mL) and fast release at the intestinal tract.

Analysis of a hollow core photonic bandgap fiber ring resonator based on micro-optical structure.

The fiber ring resonator (FRR) is the key component of resonator fiber optic gyros (R-FOGs). The configuration of a novel hollow core photonic bandgap fiber (HC-PBF) ring resonator is proposed based on the usage of micro-optical structure. The normalized transfer function of such kind of FRR is derived, and the effects of different FRR parameters' on the resonant depth, resonant finesse and sensitivity limited by the shot noise of the detector are simulated. The laboratory sample of integrated HC-PBF ring resonator is fabricated and tested experimentally and the optimal scheme is proposed based on the acquired test data. The experimental setup and results verify the feasibility of the micro-optical coupling structure used in the HC-PBF ring resonator and also support the integration and miniaturization of R-FOGs composed of HC-PBFs.

Novel mechanism of ethaselen in poorly differentiated colorectal RKO cell growth inhibition: Simultaneous regulation of TrxR transcription, expression and enzyme activity.

Thioredoxin reductase (TrxR) is a ubiquitous intracellular redox enzyme that regulates tumor growth and proliferation in various cancer cells. Ethaselen (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane), a novel anticancer agent, is designed to target mammalian TrxR1 with the aims of cancer growth inhibition and TrxR inactivation. In this study, we demonstrated that ethaselen significantly inhibits cell growth in the poorly differentiated colorectal RKO cell line, and simultaneously downregulates mammalian TrxR1 mRNA transcript levels, protein expression and enzyme activity, which differs from its actions in moderately differentiated colorectal LoVo cells. Ethaselen's significant abatement of the Wnt/beta-catenin cell differentiation-related signaling pathway was also observed in RKO cells; this apparently leads to its strong inhibitory effect on cell growth and TrxR1 activity in this cell line. These results suggest that ethaselen has a novel mechanism affecting cell growth in the poorly differentiated RKO colorectal cell line.

Modulating antioxidant systems as a therapeutic approach to retinal degeneration.

The human retina is facing a big challenge of reactive oxygen species (ROS) from endogenous and exogenous sources. Excessive ROS can cause damage to DNA, lipids, and proteins, triggering abnormal redox signaling, and ultimately lead to cell death. Thus, oxidative stress has been observed in inherited retinal diseases as a common hallmark. To counteract the detrimental effect of ROS, cells are equipped with various antioxidant defenses. In this review, we will focus on the antioxidant systems in the retina and how they can protect retina from oxidative stress. Both small antioxidants and antioxidant enzymes play a role in ROS removal. Particularly, the thioredoxin and glutaredoxin systems, as the major antioxidant systems in mammalian cells, exert functions in redox signaling regulation via modifying cysteines in proteins. In addition, the thioredoxin-like rod-derived cone viability factor (RdCVFL) and thioredoxin interacting protein (TXNIP) can modulate metabolism in photoreceptors and promote their survival. In conclusion, elevating the antioxidant capacity in retina is a promising therapy to curb the progress of inherited retinal degeneration.

Mitochondrial Glrx2 Knockout Augments Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) is one of the most widely used drugs with antipyretic and analgesic effects, and thus hepatotoxicity from the overdose of APAP becomes one of the most common forms of drug-induced liver injury. The reaction towards thiol molecules, such as GSH by APAP metabolite, N-acetyl-p-benzo-quinonimine (NAPQI), is the main cause of APAP-induced hepatotoxicity. However, the role of many other thiol-related regulators in toxicity caused by APAP is still unclear. Here we have found that knockout of the Glrx2 gene, which encodes mitochondrial glutaredoxin2 (Grx2), sensitized mice to APAP-caused hepatotoxicity. Glrx2 deletion hindered Nrf2-mediated compensatory recovery of thiol-dependent redox systems after acetaminophen challenge, resulting in a more oxidized cellular state with a further decrease in GSH level, thioredoxin reductase activity, and GSH/GSSG ratio. The weakened feedback regulation capacity of the liver led to higher levels of protein glutathionylation and thioredoxin (both Trx1 and Trx2) oxidation in Glrx2-/- mice. Following the cellular environment oxidation, nuclear translocation of apoptosis-inducing factor (AIF) was elevated in the liver of Glrx2-/- mice. Taken together, these results demonstrated that mitochondrial Grx2 deficiency deteriorated APAP-induced hepatotoxicity by interrupting thiol-redox compensatory response, enhancing the AIF pathway-mediated oxidative damage.

Thioredoxin reductase inhibitor ethaselen increases the drug sensitivity of the colon cancer cell line LoVo towards cisplatin via regulation of G1 phase and reversal of G2/M phase arrest.

We evaluated the combination treatment of ethaselen (BBSKE) as a thioredoxin reductase (TrxR) inhibitor plus cisplatin (CDDP) on the human colon adenocarcinoma cell line LoVo. Therapeutic effects ranging from nearly additive to clearly synergistic demonstrated an effective combination, i.e., the cytostatic dose of CDDP could be reduced without a loss in efficacy. To further investigate the cellular response mechanisms of these favorable outcomes, we analyzed the cell-cycle profiles, mRNA expression patterns, and protein levels of several key genes after incubation with BBSKE or CDDP separately and in combination. In appropriate conditions, CDDP induced arrest at the G2/M phase accompanied by the enhanced inhibitory phosphorylation of Cdk1 and the elevated protein expression of cyclin B1. BBSKE downregulated expression of cyclin D1 by increasing mRNA and protein levels of p21, and thus induced G1 phase arrest. BBSKE returned Cdk1 to an activated state, and reduced the protein level of cyclin B1 after incubation in combination with CDDP, which was consistent with the reduction in the percentage of cells in G2/M identified by flow cytometry. By regulating the G1 phase and reversing CDDP-induced G2/M phase arrest, BBSKE increases drug sensitivity of LoVo cells toward CDDP, and probably provides a meaningful anticancer strategy for further clinical studies.

Selenium-containing thioredoxin reductase inhibitor ethaselen sensitizes non-small cell lung cancer to radiotherapy.

It has been proposed that thioredoxin reductase (TR) is a mediator that allows non-small cell lung cancer (NSCLC) to develop resistance to irradiation; however, little is known regarding the detailed mechanisms of action. Thus, ethaselen {1, 2-[bis (1,2-benzisoselenazolone-3 (2H)-ketone)] ethane, BBSKE}, a novel organoselenium TR inhibitor, is currently being investigated in a phase I clinical trial in China. However, its radiosensitizing effect remains unexplored. In this study, we found that the activity of TR increased dramatically in both A549 and H1299 cells after radiation, and moreover, could be inhibited by pretreatment with BBSKE (5 µmol/l). As a TR inhibitor, BBSKE enhanced the efficacy of radiation therapy both in vivo and in vitro without observable toxicity. BBSKE was found to suppress irradiation-induced NF-κB activation dramatically when using A549 cells stably transfected with NF-κB luciferase reporter. These results show the critical role of TR in the radioresistance of NSCLC and suggest that BBSKE is a potentially promising agent for the treatment of patients with NSCLC clinically.

Selenium Status in Diet Affects Acetaminophen-Induced Hepatotoxicity via Interruption of Redox Environment.

Aims: Drug-induced liver injury, especially acetaminophen (APAP)-induced liver injury, is a leading cause of liver failure worldwide. Mouse models were used to evaluate the effect of microelement selenium levels on the cellular redox environment and consequent hepatotoxicity of APAP. Results: APAP treatment affected mouse liver selenoprotein thioredoxin reductase (TrxR) activity and glutathione (GSH) level in a dose- and time-dependent manner. Decrease of mouse liver TrxR activity and glutathione level was an early event, and occurred concurrently with liver damage. The decreases in the GSH/glutathione disulfide form (GSSG) ratio and TrxR activity, and the increase of protein S-glutathionylation were correlated with the APAP-induced hepatotoxicity. Moreover, in APAP-treated mice both mild deprivation and excess supplementation with selenium increased the severity of liver injury compared with those observed in mice with normal dietary selenium levels. An increase in the oxidation state of the TrxR-mediated system, including cytosolic thioredoxin1 (Trx1) and peroxiredoxin1/2 (Prx1/2), and mitochondrial Trx2 and Prx3, was found in the livers from mice reared on selenium-deficient and excess selenium-supplemented diets upon APAP treatment. Innovation: This work demonstrates that both Trx and GSH systems are susceptible to APAP toxicity in vivo, and that the thiol-dependent redox environment is a key factor in determining the extent of APAP-induced hepatotoxicity. Dietary selenium and selenoproteins play critical roles in protecting mice against APAP overdose. Conclusion: APAP treatment in mice interrupts the function of the Trx and GSH systems, which are the main enzymatic antioxidant systems, in both the cytosol and mitochondria. Dietary selenium deficiency and excess supplementation both increase the risk of APAP-induced hepatotoxicity.

Targeting Bacterial Antioxidant Systems for Antibiotics Development.

The emergence of multidrug-resistant bacteria has become an urgent issue in modern medicine which requires novel strategies to develop antibiotics. Recent studies have supported the hypothesis that antibiotic-induced bacterial cell death is mediated by Reactive Oxygen Species (ROS). The hypothesis also highlighted the importance of antioxidant systems, the defense mechanism which contributes to antibiotic resistance. Thioredoxin and glutathione systems are the two major thiol-dependent systems which not only provide antioxidant capacity but also participate in various biological events in bacteria, such as DNA synthesis and protein folding. The biological importance makes them promising targets for novel antibiotics development. Based on the idea, ebselen and auranofin, two bacterial thioredoxin reductase inhibitors, have been found to inhibit the growth of bacteria lacking the GSH efficiently. A recent study combining ebselen and silver exhibited a strong synergistic effect against Multidrug-Resistant (MDR) Gram-negative bacteria which possess both thioredoxin and glutathione systems. These drug-repurposing studies are promising for quick clinical usage due to their well-known profile.

Inhibition of thioredoxin reductase 1 correlates with platinum-based chemotherapeutic induced tissue injury.

Platinum-containing drugs (PtDs; e.g. cisplatin, carboplatin, and oxaliplatin) have been widely used as anticancer reagents against various cancers. However, treatment with these drugs results in undesirable adverse effects with unknown mechanisms. Herein, we found a strong correlation between the inhibitory effects of PtDs on cytosolic thioredoxin reductase (TXNRD1) and tissue injury. Of the PtDs tested, cisplatin was found to be the most effective inhibitory PtD against TXRND1, causing the severest kidney injury. The initial inhibition of TXNRD1 in the kidney resulted from cisplatin-induced transcriptional activation of Nrf2-regulated genes including Txnrd1. However, the antioxidant responses in the kidney did not reverse the cisplatin-induced oxidation process. Nephrotoxicity was accompanied with an increase of protein glutathionylation and a cellular thiol redox environment oxidation. These results suggest that the changes of the cellular thiol-dependent redox environment regulated by TXNRD1 is a major event in the adverse effects of cisplatin in kidney.