Stabilizing axin leads to optic nerve hypoplasia in a mouse model of autism.

Autism spectrum disorder (ASD) is a group of neurodevelopment disorders characterized by deficits in social interaction and communication, and repetitive or stereotyped behavior. Autistic children are more likely to have vision problems, and ASD is unusually common among blind people. However, the mechanisms behind the vision disorders in autism are unclear. Stabilizing WNT-targeted scaffold protein Axin2 by XAV939 during embryonic development causes overproduction of cortical neurons and leads to autistic-like behaviors in mice. In this study, we investigated the relationship between vision abnormality and autism using an XAV939-induced mouse model of autism. We found that the mice receiving XAV939 had decreased amplitude of bright light-adaptive ERG. The amplitudes and latency of flash visual evoked potential recorded from XAV939-treated mice were lower and longer, respectively than in the control mice, suggesting that XAV939 inhibits visual signal processing and conductance. Anatomically, the diameters of RGC axons were reduced when Axin2 was stabilized during the development, and the optic fibers had defective myelin sheaths and reduced oligodendrocytes. The results suggest that the WNT signaling pathway is crucial for optic nerve development. This study provides experimental evidence that conditions interfering with brain development may also lead to visual problems, which in turn might exaggerate the autistic features in humans.

Modularization of Immobilized Multienzyme Cascades for Continuous-Flow Enantioselective C-H Amination.

Multienzyme cascades (MECs) have gained much attention in synthetic chemistry but remain far from being a reliable synthetic tool. Here we report a four-enzyme cascade comprising a cofactor-independent and a cofactor self-sustaining bienzymatic module for the enantioselective benzylic C-H amination of arylalkanes, a challenging transformation from bulk chemicals to high value-added chiral amines. The two modules were subsequently optimized by enzyme co-immobilization with microenvironmental tuning, and finally integrated in a gas-liquid segmented flow system, resulting in simultaneous improvements in enzyme performance, mass transfer, system compatibility, and productivity. The flow system enabled continuous C-H amination of arylalkanes (up to 100 mM) utilizing the sole cofactor NADH (0.5 mM) in >90% conversion, achieving a high space-time yield (STY) of 3.6 g·L -1 ·h -1 , which is a 90-fold increase over previously reported values.

Cortex-restricted deletion of Foxp1 impairs barrel formation and induces aberrant tactile responses in a mouse model of autism.

BACKGROUND: Many children and young people with autism spectrum disorder (ASD) display touch defensiveness or avoidance (hypersensitivity), or engage in sensory seeking by touching people or objects (hyposensitivity). Abnormal sensory responses have also been noticed in mice lacking ASD-associated genes. Tactile sensory information is normally processed by the somatosensory system that travels along the thalamus to the primary somatosensory cortex. The neurobiology behind tactile sensory abnormalities, however, is not fully understood. METHODS: We employed cortex-specific Foxp1 knockout (Foxp1-cKO) mice as a model of autism in this study. Tactile sensory deficits were measured by the adhesive removal test. The mice's behavior and neural activity were further evaluated by the whisker nuisance test and c-Fos immunofluorescence, respectively. We also studied the dendritic spines and barrel formation in the primary somatosensory cortex by Golgi staining and immunofluorescence. RESULTS: Foxp1-cKO mice had a deferred response to the tactile environment. However, the mice exhibited avoidance behavior and hyper-reaction following repeated whisker stimulation, similar to a fight-or-flight response. In contrast to the wild-type, c-Fos was activated in the basolateral amygdala but not in layer IV of the primary somatosensory cortex of the cKO mice. Moreover, Foxp1 deficiency in cortical neurons altered the dendrite development, reduced the number of dendritic spines, and disrupted barrel formation in the somatosensory cortex, suggesting impaired somatosensory processing may underlie the aberrant tactile responses. LIMITATIONS: It is still unclear how the defective thalamocortical connection gives rise to the hyper-reactive response. Future experiments with electrophysiological recording are needed to analyze the role of thalamo-cortical-amygdala circuits in the disinhibiting amygdala and enhanced fearful responses in the mouse model of autism. CONCLUSIONS: Foxp1-cKO mice have tactile sensory deficits while exhibit hyper-reactivity, which may represent fearful and emotional responses controlled by the amygdala. This study presents anatomical evidence for reduced thalamocortical connectivity in a genetic mouse model of ASD and demonstrates that the cerebral cortex can be the origin of atypical sensory behaviors.

Asymmetric Hydrogenation of C = C Bonds in a SpinChem Reactor by Immobilized Old Yellow Enzyme and Glucose Dehydrogenase.

The application of immobilized enzymes in pharmaceutical and bulk chemical production has been shown to be economically viable. We demonstrate the exceptional performance of a method that immobilizes the old yellow enzyme YqjM and glucose dehydrogenase (GDH) on resin for the asymmetric hydrogenation (AH) of C = C bonds in a SpinChem reactor. When immobilized YqjM and GDH are reused 10 times, the conversion of 2-methylcyclopentenone could reach 78%. Which is because the rotor of the SpinChem reactor effectively reduces catalyst damage caused by shear force in the reaction system. When the substrate concentration is 175 mM, an 87% conversion of 2-methylcyclopentenone is obtained. The method is also observed to perform well for the AH of C = C bonds in other unsaturated carbonyl compounds with the SpinChem reactor. Thus, this method has great potential for application in the enzymatic production of chiral compounds.

Direct Asymmetric Reductive Amination of Alkyl (Hetero)Aryl Ketones by an Engineered Amine Dehydrogenase.

The direct asymmetric reductive amination of heteroaryl ketones has been a long-standing synthetic challenge. Here we report the engineering of an amine dehydrogenase (AmDH) from Jeotgalicoccus aerolatus for the asymmetric synthesis of chiral α-(hetero)aryl primary amines in excellent conversions (up to 99 %) and enantioselectivities (up to 99 % ee). The best AmDH variant (Ja-AmDH-M33 ) exhibited high activity and specificity toward alkyl (hetero)aryl ketones, even for those bearing a bulky alkyl chain. An efficient directed evolution approach based on molecular docking was implemented to enlarge the active pocket with a more hydrophobic entrance, which is responsible for the high activity. The Ja-AmDH-M33 was also used for preparative-scale synthesis of pharmaceutically relevant amines and a key intermediate of chiral pincer ligands, which highlighted its practical application in synthetic chemistry.

One pot purification and co-immobilization of His-tagged old yellow enzyme and glucose dehydrogenase for asymmetric hydrogenation.

In this study, a novel kind of Ni-NTA modified monodispersed SiO2 nanoflowers (Ni-NTA@SiO2 nanoflowers) were successfully synthesized. The obtained Ni-NTA@SiO2 nanoflowers were used to specifically adsorb and purify His-tagged old yellow enzyme (OYE1) and glucose dehydrogenase (GDH), which allows access to optically pure (3 S)- 3-methyl-cyclohexanone through asymmetric hydrogenation reaction, and forms a cofactor regeneration system. The protein loading amount on Ni-NTA@SiO2 nanoflowers was 40.17 mg/g support and the activity recoveries of OYE1 and GDH were 81.53% and 79.68%, respectively. The effects of pH and temperature on the activity of free and co-immobilized enzymes were investigated, and the stability as well as reusability were also measured. Compared to free enzymes, the co-immobilized enzymes showed higher thermal and storage stability. The co-immobilized enzymes were applied to asymmetric reduction of CC bonds for the synthesis of a chiral center with excellent enantioselectivity (ee > 99%), and the conversion was 46.02% after 7 cycles. This work introduced a one-pot multi-enzyme purification and co-immobilization strategy to construct efficient cofactor regeneration system with high activity and stability.

Enzymatic Synthesis of Glycerol Carbonate Using a Lipase Immobilized on Magnetic Organosilica Nanoflowers as a Catalyst.

For synthesizing glycerol carbonate (GC) by a reaction between glycerol (GL) and dimethyl carbonate (DMC), a lipase immobilized on magnetic organosilica nanoflowers was prepared and utilized as a biocatalyst. Candida antarctica lipase B (CALB) was chosen as a model enzyme for preparing an immobilized biocatalyst (CALB@nanoflowers). The obtained CALB@nanoflowers was characterized using scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy. Effects of GL/DMC molar ratio, biocatalyst amount, temperature, surfactant and molecular sieve addition, and reaction time on the conversion of GL and the selectivity of CALB@nanoflowers were investigated. The optimal catalytic performance (yield of GC: 88.66% and conversion of GL: 94.24%) was achieved under the condition of 1:20 molar ratio of GL to DMC with 0.2 g of molecular sieves added at 50 °C for 24 h. After recycling seven times, the CALB@nanoflowers maintained over 79% of its initial activity and the yield of GC was 70.31%.

The Mouse Fetal Ovary Has Greater Sensitivity Than the Fetal Testis to Benzo[a]pyrene-Induced Germ Cell Death.

The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary.

Comparison of 2,2-bis(bromomethyl)-1,3-propanediol induced genotoxicity in UROtsa cells and primary rat hepatocytes: relevance of metabolism and oxidative stress.

2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in urethane foams and polyester resins. In a two year dietary study, BMP caused neoplastic lesions at multiple sites including the urinary bladder of both rats and mice. However, liver was not a target tissue. We previously reported that BMP elicited oxidative DNA damage in a human uroepithelial cell line (UROtsa). The present in vitro study investigated the susceptibility of target (UROtsa cells) and non-target cells (primary rat hepatocytes) to BMP-induced genotoxicity. In contrast to hepatocytes, BMP exhibited greater genotoxic potential in UROtsa cells as evidenced by the concentration dependent increase in DNA strand breaks and DNA binding. Total content of intracellular GSH quantified in UROtsa cells (2.7±1.0nmol/mg protein) was 4 fold lower than that in hepatocytes (10.7±0.3nmol/mg protein). HPLC analysis indicated BMP was not metabolized and/or consumed in UROtsa cells at any of the concentrations tested (10-250µM) but was extensively converted to a mono-glucuronide in hepatocytes. These results demonstrate that a target cell line such as UROtsa cells are more susceptible to BMP-induced DNA damage when compared to non-target cells. This increased susceptibility may relate to the deficiency of antioxidant and/or metabolic capabilities in UROtsa cells.

Induction of DNA damage in human urothelial cells by the brominated flame retardant 2,2-bis(bromomethyl)-1,3-propanediol: role of oxidative stress.

2,2-bis(bromomethyl)-1,3-propanediol (BMP) is an extensively used brominated flame retardant found in urethane foams and polyester resins. In a 2-year dietary study conducted by the National Toxicology Program, BMP caused neoplastic lesions at multiple sites including the urinary bladder in both rats and mice. The mechanism of its carcinogenic effect is unknown. In the present study, using SV-40 immortalized human urothelial cells (UROtsa), endpoints associated with BMP induced DNA damage and oxidative stress were investigated. The effects of time (1-24h) and concentration (5-100 µM) on BMP induced DNA strand breaks were assessed via the alkaline comet assay. The results revealed evidence of DNA strand breaks at 1 and 3h following incubation of cells with non-cytotoxic concentrations of BMP. Strand breaks were not present after 6h of incubation. Evidences for BMP associated oxidative stress include: an elevation of intracellular ROS formation as well as induction of Nrf2 and HSP70 protein levels. In addition, DNA strand breaks were attenuated when cells were pre-treated with N-acetyl-l-cysteine (NAC) and oxidative base modifications were revealed when a lesion specific endonuclease, human 8-hydroxyguanine DNA glycosylase 1 (hOGG1) was introduced into the comet assay. In conclusion, these results demonstrate that BMP induces DNA strand breaks and oxidative base damage in UROtsa cells. Oxidative stress is a significant, determinant factor in mediating these DNA lesions. These early genotoxic events may, in part, contribute to BMP-induced carcinogenesis observed in rodents.

Anti-tussive activity of benproperine enantiomers on citric-acid-induced cough in conscious guinea-pigs.

The anti-tussive effect of the R-(+)- and S-(-)-enantiomers of benproperine was evaluated and compared with that of the racemate on cough induced by 7.5% citric acid in conscious guinea-pigs. All the three compounds, intraperitoneally administered 1.5 h before the test, significantly inhibited citric-acid-induced cough. The ID50 values (effective doses for 50% inhibition) (with 95% confidence intervals) were 16.1 (9.1-28.4), 23.3 (11.2-48.6), 25.4 (11.7-55.1) mg kg(-1) for the number of coughs in the 3 min of challenge, and 11.9 (5.3-26.6), 13.5 (5.6-32.4), 19.2 (12.8-28.9) mg kg(-1) for the number of coughs in the 5 min immediately after the challenge, for (+/-)-benproperine, R-(+)-benproperine and S-(-)-benproperine, respectively. These findings suggest that the use of either enantiomer does not show any advantage over the racemate with regard to their anti-tussive effect.