Diversification of Toll-like receptor 1 in swamp eel (Monopterus albus).

Toll-like receptor 1 (TLR1) is a pattern recognition receptor that plays critical roles in triggering immune activation via detecting bacterial lipoproteins and lipopeptides. In this study, the genetic characteristic of TLR1 was studied for an important aquaculture fish, swamp eel Monopterus albus. The eel has been seriously threatened by infectious diseases. However, a low level of genetic heterogeneity in the fish that has resulted from a demographic bottleneck presents further challenges in breeding for disease resistance. A comparison with the homologue of closely related species M. javanensis revealed that amino acid replacement (nonsynonymous) but not silent (synonymous) differences have accumulated nonrandomly over the coding sequences of the receptors at the early stage of their phylogenetic split. The combined results from comparative analyses of nonsynonymous-to-synonymous polymorphisms showed that the receptor has undergone significant diversification in M. albus driven by adaptive selection likely after the genetic bottleneck. Some of the changes reported here have taken place in the structures mediating heterodimerization with co-receptor TLR2, ligand recognition, and/or formation of active signaling complex with adaptor, which highlighted key structural elements and strategies of TLR1 in arms race against exogenous challenges. The findings of this study will add to the knowledge base of genetic engineering and breeding for disease resistance in the eel.

Cu-Catalyzed Direct Asymmetric Mannich Reaction of 2-Alkylazaarenes and Isatin-Derived Ketimines.

The first direct catalytic asymmetric Mannich reaction of 2-alkylazaarenes and ketimines was realized with a chiral Cu-bis(oxazoline) complex as the catalyst. The asymmetric addition of 2-alkylpyridines to isatin-derived ketimines proceeded smoothly to afford α,ß-functionalized 2-substituted pyridines bearing 3-amino-3,3-disubstituted oxindole motifs with excellent results (≤99% yield, 99:1 dr, and 98% ee). The catalytic system was also extended to 2-alkylbenzothiazoles as nucleophiles for the asymmetric Mannich reaction of ketimines.

Catalytic Asymmetric Synthesis of Vicinally Bis(trifluoromethyl)-Substituted Molecules via Normal [3 + 2] Cycloaddition of N-2,2,2-Trifluoroethyl Benzothiophene Ketimines and ß-Trifluoromethyl Enones.

An organocatalytic asymmetric [3 + 2] cycloaddition of ß-trifluoromethyl enones with 3-(N-2,2,2-trifluoroethyl) benzothiophene ketimines and 2-(N-2,2,2-trifluoroethyl) benzothiophene ketimines was described for the first time. A wide spectrum of vicinally bis(trifluoromethyl)-substituted spiro pyrrolidine-benzothiophenones were obtained with excellent stereocontrol (all cases >20:1 dr and up to 99% ee). The highlight of this work is the extremely high efficiency in the construction of spirocyclic benzothiophenone derivatives possessing a vicinally bis(trifluoromethyl)-substituted pyrrolidine moiety with four contiguous stereocenters.

Palladium-catalyzed decarboxylative α-allylation of thiazolidinones and azlactones with sulfonamido-substituted acyclic allylic carbonates.

A palladium-catalyzed decarboxylative α-allylation of thiazolidinones and azlactones with aza-π-allylpalladium zwitterionic intermediates, in situ generated from sulfonamido-substituted allylic carbonates, is successfully developed. This method allows the formation of a series of structurally diverse 5-alkylated thiazolidinones and 2-piperidones under mild conditions in moderate to high yields (up to 99% yield).

Electrodeposition of High-Quality Ni/SiC Composite Coatings by Using Binary Non-Ionic Surfactants.

In order to increase the hardness, wear resistance and corrosion resistance of nickel-based coatings, pure nickel is often co-electrodeposited with silicon carbide (SiC) particles. However, SiC particles tend to agglomerate and precipitate in the bath, which reduces the amounts of nanoparticles and causes nonuniformity. Herein, we solve these problems by using binary non-ionic surfactants (Span 80 and Tween 60) to effectively disperse SiC particles (binary-SiC) in the bath, which suppresses nanoparticles agglomeration and leads to uniformly distributed SiC particles in the composite coatings. In comparison to the Ni/SiC coatings electrodeposited from the commonly used SDS-modified SiC, the coatings prepared with binary-SiC (Ni/binary-SiC) show finer crystallization and a smoother surface. In addition, the Ni/binary-SiC coatings exhibit higher hardness (556 Hv) and wear resistance (2.95 mg cm-2). Furthermore, higher corrosion resistance is also achieved by the Ni/binary-SiC coatings.

Genetic diversity of Toll-like receptor 9 in swamp eels (Monopterus albus).

The swamp eel, Monopterus albus, is an important aquaculture species in Asia (mainly China) whose production has seriously suffered from infectious diseases. In spite of the critical requirement for aquaculture practices, to date there is scant information on its immune defence. Here, the genetic characteristics of Toll-like receptor 9 (TLR9), which plays crucial roles in the initiation of host defence against microbial invasion, were analysed. It exhibits a striking lack of genetic variation resulting from a recent demographic bottleneck. A comparison with the homologue of M. javanensis revealed that replacement but not silent differences have nonrandomly accumulated in the coding sequences at the early stage following their split from a common ancestor. Furthermore, the replacements relevant to the type II functional divergence have mainly occurred in structural motifs mediating ligand recognition and receptor homodimerization. These results provide hints to understand the diversity-based strategy of TLR9 in the arms race against pathogens. Furthermore, the findings reported here give credence to the importance of basic immunology knowledge, especially for the key elements, in genetic engineering and breeding for disease resistance in the eel and other fishes.

Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion.

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.

Higher-order dispersion and the spectral behavior in a doubly resonant optical parametric oscillator.

In doubly resonant optical parametric oscillators (DROPOs), it is possible to generate, enhance, and phase lock two frequencies at once. Following intracavity phase conditions, a complex tuning behavior of the signal and idler spectra takes place in DROPOs, cumulating into degeneracy with phase self-locking and coherent wavelength doubling. In this work, we identify group delay matching as the important parameter determining the global tuning behavior and demonstrate the key role of higher-order dispersion in the spectral dependencies. Applicationwise, we suggest a simple way to control the phase self-locking region by varying the intracavity third-order dispersion.

Single frequency MOPA based on Nd:YAG single crystal fiber and rods.

We demonstrate a single frequency 1064 nm master oscillator power amplifier (MOPA) system operating in macro-micro pulse scheme. The repetition rate for the macro pulses was 300 Hz with pulse duration of 300 µs. Micro pulses operated at 25 kHz. The master laser was a single-longitudinal-mode electro-optically Q-switched Nd:YAG laser with an output power of 250 mW and pulse duration of 33 ns. Three stages of power amplifiers based on Nd:YAG single crystal fiber and rods were designed. The final output power reached 31.3 W with pulse duration of 30 ns and linewidth of less than 130 MHz. Micro pulse energy of 13.9 mJ was obtained with a peak power of up to 464 kW. The beam quality factors (M2) were measured to be 1.56 and 1.76 in horizontal and vertical directions, respectively.

High power broadband LiF:F(2)(-) color center laser.

A high power LiF:F(2)(-) color center laser is demonstrated with broadband emission. The excitation source is a quasi-continuous wave diode side-pumped acousto-optically Q-switched Nd:YAG laser. Under an incident 1064-nm laser power of 25.4 W, the highest output power of up to 4.7 W is obtained with a macro pulse repetition rate of 400 Hz and a micro pulse repetition rate of 50 kHz. The broadband emission is centered at 1142 nm with a bandwidth of 13 nm.

Long-term Improvements in Lifespan and Pathology in CNS and PNS After BMT Plus One Intravenous Injection of AAVrh10-GALC in Twitcher Mice.

Krabbe disease is an autosomal recessive disorder resulting from defects in the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency leads to severe neurological features. The only treatment for presymptomatic infantile patients and later-onset patients is hematopoietic stem cell transplantation (HSCT). This treatment is less than ideal with most patients eventually developing problems with gait and expressive language. Several naturally occurring animal models are available, including twitcher (twi) mice, which have been used for many treatment trials. Previous studies demonstrated that multiple injections of AAVrh10-GALC into the central nervous system (CNS) of neonatal twi mice resulted in significant improvements. Recently we showed that one i.v. injection of AAVrh10-GALC on PND10 resulted in normal GALC activity in the CNS and high activity in the peripheral nervous system (PNS). In the present study, a single i.v. injection of AAVrh10-GALC was given 1 day after bone marrow transplantation (BMT) on PND10. The mice show greatly extended lifespan and normal behavior with improved CNS and PNS findings. Since HSCT is the standard of care in human patients, adding this single i.v. injection of viral vector may greatly improve the treatment outcome.

RTP Q-switched single-longitudinal-mode Nd:YAG laser with a twisted-mode cavity.

This paper describes a high pulse repetition frequency Nd:YAG twisted-mode laser that uses an RTP crystal as the electro-optic Q-switch. Stable single-longitudinal-mode laser beams at 1, 5, and 10 kHz were obtained with a linewidth less than 0.1 GHz. Under an incident pump power of 7.5 W and a PRF of 10 kHz, the maximum output power of the single-longitudinal-mode laser was 1.19 W. The corresponding conversion efficiency, single pulse energy and pulse peak power were 15.8%, 119 µJ, and 2.5 kW, respectively.

Intravenous injection of AAVrh10-GALC after the neonatal period in twitcher mice results in significant expression in the central and peripheral nervous systems and improvement of clinical features.

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessive disorder resulting from the defective lysosomal enzyme galactocerebrosidase (GALC). The lack of GALC enzyme leads to severe neurological symptoms. While most human patients are infants who do not survive beyond 2 years of age, older patients are also diagnosed. In addition to human patients, several naturally occurring animal models, including dog, mouse, and monkey, have also been identified. The mouse model of Krabbe disease, twitcher (twi) mouse has been used for many treatment trials including gene therapy. Using the combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of the adeno-associated virus serotype rh10 (AAVrh10) expressing mouse GALC in neonate twi mice we previously have demonstrated a significantly extended normal life and exhibition of normal behavior in treated mice. In spite of the prolonged healthy life of these treated mice and improved myelination, it is unlikely that using multiple injection sites for viral administration will be approved for treatment of human patients. In this study, we have explored the outcome of the single iv injection of viral vector at post-natal day 10 (PND10). This has resulted in increased GALC activity in the central nervous system (CNS) and high GALC activity in the peripheral nervous system (PNS). As we have shown previously, an iv injection of AAVrh10 at PND2 results in a small extension of life beyond the typical lifespan of the untreated twi mice (~40 days). In this study, we report that mice receiving a single iv injection at PND10 had no tremor and continued to gain weight until a few weeks before they died. On average, they lived 20-25 days longer than untreated mice. We anticipate that this strategy in combination with other therapeutic options may be beneficial and applicable to treatment of human patients.

Sixteen novel mutations in the arylsulfatase A gene causing metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused mainly by mutations in the arylsulfatase A (ARSA) gene. In this manuscript we report sixteen novel mutations identified in the ARSA gene of fifteen unrelated patients affected with MLD. Of these 16 mutations nine were missense mutations (p.L11Q, p.S44P, p.L81P, p.R84L, p.V177D, p.P284S, p.R288S, p.G301R, p.P425S), three were nonsense mutations (p.Q51X, p.Y149X, p.C156X), three were frame shift mutations (c.28delG, c.105C>A+106_124dup, c.189delC) and one was a splice-site mutation (c.1102-2A>G). In addition, three previously reported mutations were identified on an allelic background different from the one in the original reports. Two mutations, p.G309S and p.E312D, were identified on the background of the so-called pseudodeficiency (Pd) allele while previously they were reported alone. On the other hand, mutation p.R311X was identified in two unrelated patients not in cis with the Pd mutations, as previously reported.

Extended normal life after AAVrh10-mediated gene therapy in the mouse model of Krabbe disease.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency results in accumulation of certain galactolipids including psychosine which is cytotoxic for myelin-producing cells. Treatment of human patients at this time is limited to hematopoietic stem cell transplantation (HSCT) that appears to slow the progression of the disease when performed in presymptomatic patients. In this study, adeno-associated virus (AAV) serotype rh10-(AAVrh10) expressing mouse GALC was used in treating twitcher (twi) mice, the mouse model of GLD. The combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of viral particles in neonate twi mice resulted in high GALC activity in brain and cerebellum and moderate to high GALC activity in spinal cord, sciatic nerve, and some peripheral organs. Successfully treated mice maintained their weight with no or very little twitching, living up to 8 months. The physical activities of the long-lived treated mice were comparable to wild type for most of their lives. Treated mice showed normal abilities to mate, to deliver pups, to nurse and to care for the newborns. This strategy alone or in combination with other therapeutic options may be applicable to treatment of human patients.

Theoretical investigations on removal reactions of ethenol by H atom.

Ethenol is a recently identified combustion intermediate. However, its chemistry remains unclear. In present work, the removal reactions of ethenol by H atom are investigated. The geometries of all species involved in the reaction are optimized at B3LYP/6-311++G(d,p), and their single point energies are extrapolated to the infinite-basis-set limit at the level CCSD(T). Energies are also calculated at G3B3, CBS-APNO, and CCSD(T)/6-311++G(3df, 2p) for comparison. A total of six elementary reactions, including four abstractions and two additions, with explicit transition states are investigated. The results show that the reactions are selective: for abstractions, the hydrogen atom, linked to the oxygen atom, is the most reactive; while for additions, the preferred carbon site is the head "CH(2)═". The rate constants are estimated in the temperature range 300-3000 K according to the conventional transition state theory with the Eckart tunneling model. The dominant channels are the two additions in the whole temperature range. The abstractions can be competitive at high temperature but still do not dominate. The calculated rate constants for the reverse reaction of (R6), syn-CH(2)═CHOH + H ↔ CH(3)·CHOH, are consistent with the available literature values. Finally, the Fukui functions are calculated to analyze the site reactivity.

Accurate prediction of enthalpies of formation for a large set of organic compounds.

This article describes a multiparameter calibration model, which improves the accuracy of density functional theory (DFT) for the prediction of standard enthalpies of formation for a large set of organic compounds. The model applies atom based, bond based, electronic, and radical environmental correction terms to calibrate the calculated enthalpies of formation at B3LYP/6-31G(d,p) level by a least-square method. A diverse data set of 771 closed-shell compounds and radicals is used to train the model. The leave-one-out cross validation squared correlation coefficient q(2) of 0.84 and squared correlation coefficient r(2) of 0.86 for the final model are obtained. The mean absolute error in enthalpies of formation for the dataset is reduced from 4.9 kcal/mol before calibration to 2.1 kcal/mol after calibration. Five-fold cross validation is also used to estimate the performance of the calibration model and similar results are obtained.

Biochemical and pathological evaluation of long-lived mice with globoid cell leukodystrophy after bone marrow transplantation.

Globoid cell leukodystrophy (GLD) is a disorder of the central and peripheral nervous systems caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). The pathological changes associated with the disease include accumulation of globoid cells and loss of myelin due to production of psychosine, a toxic metabolite responsible for the apoptosis of oligodendrocytes. While most patients present with symptoms before 6 months of age, older patients are also diagnosed. Treatment at this time is limited to hematopoietic stem cell transplantation in asymptomatic and late-onset patients. GLD occurs naturally in several animal species including mice, dogs, and monkeys. In addition, a transgenic (trs) mouse model of GLD was generated in our laboratory. Trs mice develop symptoms slower than twitcher mice and survive an average of 10 days longer. In this study, we evaluated the therapeutic effects of bone marrow transplantation (BMT) using trs mice. BMT prolonged the life of some treated animals to over one year. After BMT, GALC activity reached 15-20% of normal in brain and near normal values in liver and sciatic nerve. In long-lived transplanted animals psychosine levels were normalized in the brain and greatly reduced in the sciatic nerve. Staining of brain sections showed more abundant and better quality myelin and near absence of globoid cells. Electron micrographs of sciatic nerves showed reduced endoneurial edema, increased axon density, and abundant onion bulb structures associated with remyelinating axons. Therefore, BMT can ameliorate many of the biochemical and pathological features of GLD. However, additional therapies may be required to completely correct the features of this disease.

Insulin-like growth factor-1 provides protection against psychosine-induced apoptosis in cultured mouse oligodendrocyte progenitor cells using primarily the PI3K/Akt pathway.

Psychosine (galactosylsphingosine) is a toxic metabolite that accumulates in globoid cell leukodystrophy (GLD) due to the deficiency of galactocerebrosidase (GALC) activity. This results in subsequent programmed cell death of oligodendrocytes and demyelination in human patients and animal models. We investigated the potential role of insulin-like growth factor-1 (IGF-1) in modifying the apoptotic effect of psychosine in cultured mouse oligodendrocyte progenitor cells (OLP-II). We show that psychosine inhibits the phosphorylation of Akt and Erk1/Erk2 (Erk1/2), which are the main anti-apoptotic pathways of the IGF-1 receptor (IGF-1R). Although IGF-1 sustained phosphorylation of both of these pathways, it provided maximum protection to OLP-II cells from psychosine-induced cell death in a PI3K/Akt-dependent manner. The effects of IGF-1 were dose-dependent and resulted in increased IGF-1R autophosphorylation levels. Although relatively high concentrations of IGF-1 also resulted in the activation of the insulin receptor (IR), its effect was more significant on the IGF-1R.

AAV-mediated expression of galactocerebrosidase in brain results in attenuated symptoms and extended life span in murine models of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by a deficiency of galactocerebrosidase (GALC) activity. GALC is required for the lysosomal degradation of galactosylceramide, psychosine, and possibly other galactolipids. This process is extremely important during active myelination. In the absence of functional GALC, psychosine accumulates, resulting in the apoptotic death of myelin-producing cells. While most patients are infants who do not survive beyond 2 years of age, some older patients are also diagnosed. Hematopoietic stem cell transplantation has proven to have a positive effect on the course of some patients with late-onset Krabbe disease. Murine models of this disease provide an excellent opportunity to evaluate therapeutic alternatives including gene therapy. In this study we used serotype 1 AAV to express mouse GALC under the control of the human cytomegalovirus promoter. Direct administration of these viral particles into the brains of neonatal mice with GLD resulted in sustained expression of GALC activity, improved myelination, attenuated symptoms, and prolonged life span. While this treatment also resulted in significant pathological improvements, the treated mice died with symptoms similar to those of the untreated mice. Additional initiatives may be required to prevent the onset of disease and reverse the course of the disease in animal models and human patients.