Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli.

High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.

Causes and countermeasure for blank absorbance increase in the ROS assay.

A reactive oxygen species (ROS) assay is an in chemico photoreactivity test listed in ICH S10 guideline and OECD Test Guideline No. 495. We currently utilize the ROS assay to assess the photosafety of cosmetic ingredients. We have recently confronted a problem that there was an absorbance increase of blank assessing superoxide anion generation after irradiation, whereas this did not occur in the negative control (sulisobenzone), leading to a dissatisfaction of the acceptance criteria. Therefore, we aimed to investigate the causes and find countermeasures. No significant effects of impurities and manufacturer differences of sodium phosphate and DMSO on blank absorbance increases were observed. In contrast, when Cu2+ was added to the buffer, the increase of blank absorbance after irradiation did not occur. We then confirmed the dose-response relationship and found that adding 0.1 µM of Cu2+ (corresponding to 6 ppb of Cu2+) was sufficient in suppressing the blank absorbance increase, suggesting the need of Cu2+ supplementation to the buffer. Finally, we confirmed that the ROS assay using the buffer supplemented with 0.1 µM of Cu2+ obtained stable test results by using 17 proficiency chemicals listed in TG 495. Our results suggest that the modified ROS assay protocol would be useful for obtaining stable test results.

Metabolomics-Driven Identification of the Rate-Limiting Steps in 1-Propanol Production.

The concerted effort for bioproduction of higher alcohols and other commodity chemicals has yielded a consortium of metabolic engineering techniques to identify targets to enhance performance of engineered microbial strains. Here, we demonstrate the use of metabolomics as a tool to systematically identify targets for improved production phenotypes in Escherichia coli. Gas chromatography/mass spectrometry (GC/MS) and ion-pair LC-MS/MS were performed to investigate metabolic perturbations in various 1-propanol producing strains. Two initial strains were compared that differ in the expression of the citramalate and threonine pathways, which hold a synergistic relationship to maximize production yields. While this results in increased productivity, no change in titer was observed when the threonine pathway was overexpressed beyond native levels. Metabolomics revealed accumulation of upstream byproducts, norvaline and 2-aminobutyrate, both of which are derived from 2-ketobutyrate (2KB). Eliminating the competing pathway by gene knockouts or improving flux through overexpression of glycolysis gene effectively increased the intracellular 2KB pool. However, the increase in 2KB intracellular concentration yielded decreased production titers, indicating toxicity caused by 2KB and an insufficient turnover rate of 2KB to 1-propanol. Optimization of alcohol dehydrogenase YqhD activity using an ribosome binding site (RBS) library improved 1-propanol titer (g/L) and yield (g/g of glucose) by 38 and 29% in 72 h compared to the base strain, respectively. This study demonstrates the use of metabolomics as a powerful tool to aid systematic strain improvement for metabolically engineered organisms.

Reactive oxygen species (ROS) assay-based photosafety screening for complex ingredients: Modification of the ROS assay protocol.

A reactive oxygen species (ROS) assay has been widely used for photosafety assessment; however, the phototoxic potential of complex materials, including plant extracts, essential oils, and functional polymers, is unevaluable because of their undefined molecular weights. The present study was undertaken to modify the ROS assay protocol for evaluating phototoxic potentials of those materials with use of their apparent molecular weight (aMw). On preparing sample solutions for the ROS assay, aMw ranging from 150 to 350 was tentatively employed for test substances. The modified ROS assays were applied to 45 phototoxic and 19 non-phototoxic substances, including 44 chemicals and 20 complex materials (plant extracts) for clarification of the predictive performance. Generation of ROS from photo-irradiated samples tended to increase as aMW grew, resulting in the largest number of false-positive predictions at aMW of 350. Some false-negative predictions were also observed when aMW was set at 200 or less. At aMw of 250, all tested phototoxic substances could be correctly identified as photoreactive with no false-negative predictions. Based on these observations, aMw of 250 was found to be suitable for the ROS assay on complex materials, and the sensitivity, specificity, and positive and negative predictivity for the proposed ROS assay were calculated to be 100, 52.6, 83.3, and 100%, respectively. Thus, the proposed approach may be efficacious for predicting phototoxic potentials of complex materials and contribute to the development of new products with a wide photosafety margin.

In chemico sequential testing strategy for assessing the photoallegic potential.

Several non-animal testing methods to assess photoallergic potential have been developed so far, while none of them have yet to be validated and regulatory accepted. Currently, some photoreactivity assays such as UV-VIS spectral analysis and ROS assay are generally used for initial photosafety assessments because of their high sensitivity. However, they have a low specificity, generating a high percentage of false positive results, and the development of a follow-up assessment method is desired. Therefore, this study aimed to develop an in chemico photoallergy testing method, photo-direct peptide reactivity assay (photo-DPRA). Based on photosafety information, 34 photoallergens and 16 non-photoallergens were selected and subjected to UV-VIS spectral analysis, ROS/micellar ROS assays, photo-DPRA, sequential testing strategy (STS) consisting of all three methods, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Combination of the methods addressing the key events of photoallergy exhibited high prediction performance. Our results showed the proposed strategy would be useful to predict the photoallergic potential of chemicals as the follow-up assessment for false positive chemicals by UV/VIS spectral analysis and ROS assay.

Applicability of an Integrated Testing Strategy consisting of in silico, in chemico and in vitro assays for evaluating the skin sensitization potencies of isocyanates.

The skin sensitization potential of chemicals has been traditionally assessed using regulatory accepted in vivo methods, such as guinea pig maximization test or mouse local lymph node assays (LLNAs). A huge effort to reduce and replace the use of animals for safety assessments of chemicals because of regulatory requirements and ethical issues is presently underway, and alternative non-animal methods have been greatly developed. So far, a few studies have investigated the sensitization potencies of isocyanates which is a group of highly reactive chemicals that are known to be occupational allergens. The present study evaluated nine commonly used isocyanates using an in vivo LLNA and assessed the applicability of an Integrated Testing Strategy (ITS) consisting of an in silico Derek Nexus prediction, an in chemico direct peptide reactivity assay (DPRA), and an in vitro human Cell Line Activation Test (h-CLAT) to isocyanates. All nine isocyanates were evaluated as positive using the LLNA, Derek Nexus and DPRA, whereas seven chemicals tested positive using the h-CLAT: hexamethylene diisocyanate tested negative, and 1,5-diisocyanatonaphthalene could not be examined because of a solubility issue. When assessed using the ITS, the positive/negative evaluations of skin sensitization hazard were consistent with those assessed using the LLNA for all nine chemicals. However, the potency prediction results of the ITS tended to be underestimated, compared with those of the LLNA. The data presented in this work provide insights into the performance of non-animal testing approaches for evaluating the skin sensitization potencies of isocyanates.

Acute limbic encephalitis: a new entity?

Clinical cases similar to herpes simplex virus (HSV) encephalitis have accumulated in Japan. Detailed examinations have failed to demonstrate HSV infection. Recently, these cases have been named "non-herpetic acute limbic encephalitis". Only a single autopsy case was so far reported in an abstract form, because many cases showed a good prognosis. The case presented here was that following fever, a 59-year-old woman developed disturbance of consciousness and uncontrollable generalized seizures. Brain MRI revealed abnormal signals in the bilateral medial temporal lobe and along the lateral part of the putamen. Autoantibody against the NMDA glutamate receptor (GluR) IgM-epsilon2 was detected in the serum, and the GluR IgG-delta2 antibody was positive in cerebrospinal fluid. She died 12 days after onset. An autopsy examination revealed scattered foci consisting of neuronal loss, neuronophagia and some perivascular lymphocytic infiltration in the hippocampus and amygdala, but no haemorrhagic necrosis in the brain. HSV-1, -2 and human herpes virus-6 were negative immunohistochemically. We believe that our autopsy case may contribute to understanding the neuropathological background of non-herpetic acute limbic encephalitis.

A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia.

BACKGROUND: Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease. To date, 36 dominantly inherited loci have been reported, and 31 causative genes have been identified. RESULTS: In this study, we analyzed a Japanese family with autosomal dominant SCA using linkage analysis and exome sequencing, and identified CACNA1G, which encodes the calcium channel CaV3.1, as a new causative gene. The same mutation was also found in another family with SCA. Although most patients exhibited the pure form of cerebellar ataxia, two patients showed prominent resting tremor in addition to ataxia. CaV3.1 is classified as a low-threshold voltage-dependent calcium channel (T-type) and is expressed abundantly in the central nervous system, including the cerebellum. The mutation p.Arg1715His, identified in this study, was found to be located at S4 of repeat IV, the voltage sensor of the CaV3.1. Electrophysiological analyses revealed that the membrane potential dependency of the mutant CaV3.1 transfected into HEK293T cells shifted toward a positive potential. We established induced pluripotent stem cells (iPSCs) from fibroblasts of the patient, and to our knowledge, this is the first report of successful differentiation from the patient-derived iPSCs into Purkinje cells. There was no significant difference in the differentiation status between control- and patient-derived iPSCs. CONCLUSIONS: To date, several channel genes have been reported as causative genes for SCA. Our findings provide important insights into the pathogenesis of SCA as a channelopathy.

Recurrence of acute disseminated encephalomyelitis after a 12-year symptom-free interval.

We report a patient with recurrent acute meningoencephalitis who had three episodes of headache, fever and unconsciousness; the first episode was at age 6 and the second, at age 7. After a 12-year symptom-free interval, she had a relapse, exhibiting the same symptoms as those in the previous two episodes. Head magnetic resonance imaging also revealed the recurrence of lesions in the basal ganglia and medial portion of the temporal lobe. The occurrences of stereotyped symptoms with meningoencephalitis and the same lesions in the basal ganglia observed in each episode favor the diagnosis of recurrent acute disseminated encephalomyelitis (ADEM) rather than multiple sclerosis or multiphasic disseminated encephalomyelitis. The occurrence of this rare case suggests that ADEM can relapse after a very long symptom-free interval.

A patient with myotonic dystrophy type 1 (DM 1) accompanied by laryngeal and renal cell carcinomas had a small CTG triplet repeat expansion but no somatic instability in normal tissues.

We examined (CTG)n lengths in various tissues from a 70-year-old man with myotonic dystrophy type 1 (DM 1) who had a small 60-70 (CTG), expansion in his leukocytes. He died of renal cell carcinoma 5 years after a total laryngectomy for laryngeal carcinoma. Southern blot and polymerase chain reaction analyses were done on tissues obtained at autopsy. In the various normal tissues, (CTG). lengths were almost all the same size, whereas the renal cell carcinoma and metastatic tissues had longer lengths. When compared with the lengths in leukocytes about 5 years previously, (CTG)n lengths in the normal tissues were the same size. These findings suggest that both somatic instability and age-dependent (CTG). expansion in DM 1 patients with a small expansion may be less dominant than in patients with large expansions.

Ruthenium-catalyzed ring-closing metathesis accelerated by long-range steric effect.

Ruthenium-based metathesis catalysts with a N-heterocyclic carbene ligand bearing 2,3,4,5-tetraphenylphenyl moieties (1-TPPh and 1-TPPh*) are developed. The highly active catalyst system has been realized in THF by the combination of 1-TPPh* and CuCl as a phosphine scavenger.