Metabolic Engineering of Escherichia coli for para-Amino-Phenylethanol and para-Amino-Phenylacetic Acid Biosynthesis

Aromatic amines are an important class of chemicals which are used as building blocks for the synthesis of polymers and pharmaceuticals. In this study we establish a de novo pathway for the biosynthesis of the aromatic amines para-amino-phenylethanol (PAPE) and para-amino-phenylacetic acid (4-APA) in Escherichia coli. We combined a synthetic para-amino-l-phenylalanine pathway with the fungal Ehrlich pathway. Therefore, we overexpressed the heterologous genes encoding 4-amino-4-deoxychorismate synthase (pabAB from Corynebacterium glutamicum), 4-amino-4-deoxychorismate mutase and 4-amino-4-deoxyprephenate dehydrogenase (papB and papC from Streptomyces venezuelae) and ThDP-dependent keto-acid decarboxylase (aro10 from Saccharomyces cerevisiae) in E. coli. The resulting para-amino-phenylacetaldehyde either was reduced to PAPE or oxidized to 4-APA. The wild type strain E. coli LJ110 with a plasmid carrying these four genes produced (in shake flask cultures) 11 ± 1.5 mg l−1 of PAPE from glucose (4.5 g l−1). By the additional cloning and expression of feaB (phenylacetaldehyde dehydrogenase from E. coli) 36 ± 5 mg l−1 of 4-APA were obtained from 4.5 g l−1 glucose. Competing reactions, such as the genes for aminotransferases (aspC and tyrB) or for biosynthesis of L-phenylalanine and L-tyrosine (pheA, tyrA) and for the regulator TyrR were removed. Additionally, the E. coli genes aroFBL were cloned and expressed from a second plasmid. The best producer strains of E. coli showed improved formation of PAPE and 4-APA, respectively. Plasmid-borne expression of an aldehyde reductase (yahK from E. coli) gave best values for PAPE production, whereas feaB-overexpression led to best values for 4-APA. In fed-batch cultivation, the best producer strains achieved 2.5 ± 0.15 g l−1 of PAPE from glucose (11% C mol mol-1 glucose) and 3.4 ± 0.3 g l−1 of 4-APA (17% C mol mol−1 glucose), respectively which are the highest values for recombinant strains reported so far

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

Kato O, Youn J-W, Stansen KC, Matsui D, Oikawa T, Wendisch VF. Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate. BMC Microbiology. 2010;10(1): 321.Background: Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Results: Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer. Conclusions: Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer

Malignancies in Korean Patients with Inflammatory Myopathy

The aim of this study was to assess the prevalence and the common type of malignancies in Korean patients with polymyositis (PM) and dermatomyositis (DM) and to evaluate the differences of clinical and laboratory findings between patients with malignancy and those without malignancy. Forty-one Korean patients, who were diagnosed as PM or DM, were enrolled in this study. They fulfilled the Bohan and Peter's criteria for a definite diagnosis of PM and DM. Patients with PM were 25 and those with DM were 16. Eleven out of 41 patients (26.8%) had malignancies. The malignancy was diagnosed simultaneously or later in 81.8% of patients with inflammatory myopathy (IM). The breast cancer was the most common malignancy. In this study, forty three years old as a screening age for malignancy had 88.9% sensitivity and 50.2% specificity. The serum levels of creatine kinase (CK) were significantly lower in patients with malignancy than those without malignancy

Development of a classification model for Cynanchum wilfordii and Cynanchum auriculatum using convolutional neural network and local interpretable model-agnostic explanation technology

Cynanchum wilfordii is a perennial tuberous root in the Asclepiadaceae family that has long been used medicinally. Although C. wilfordii is distinct in origin and content from Cynancum auriculatum, a genus of the same species, it is difficult for the public to recognize because the ripe fruit and root are remarkably similar. In this study, images were collected to categorize C. wilfordii and C. auriculatum, which were then processed and input into a deep-learning classification model to corroborate the results. By obtaining 200 photographs of each of the two cross sections of each medicinal material, approximately 800 images were employed, and approximately 3200 images were used to construct a deep-learning classification model via image augmentation. For the classification, the structures of Inception-ResNet and VGGnet-19 among convolutional neural network (CNN) models were used, with Inception-ResNet outperforming VGGnet-19 in terms of performance and learning speed. The validation set confirmed a strong classification performance of approximately 0.862. Furthermore, explanatory properties were added to the deep-learning model using local interpretable model-agnostic explanation (LIME), and the suitability of the LIME domain was assessed using cross-validation in both situations. Thus, artificial intelligence may be used as an auxiliary metric in the sensory evaluation of medicinal materials in future, owing to its explanatory ability

A New Method for Investigation of the Hair Shaft: Hard X-Ray Microscopy with a 90-nm Spatial Resolution

Various methods have been used to investigate the hair shaft. In the ultrastructural hair field, scanning and transmission electron microscopies are widely used investigative methods, but they have some technical limitations. Recently, X-ray microscopes with sub-micron spatial resolution have emerged as useful instruments because they offer a unique opportunity to observe the interior of an undamaged sample in greater detail. In this report, we examined damaged hair shaft tips using hard X-ray microscopy with a 90 nm spatial resolution. The results of this study suggest that hard X-ray microscopy is an alternative investigative method for hair morphology studies

Effects of Berberine and Hwangryunhaedok-Tang on Oral Bioavailability and Pharmacokinetics of Ciprofloxacin in Rats

Hwangryunhaedok-Tang (HR) and berberine-containing single herbs are used to treat bacterial infection and inflammatory diseases in eastern Asia. The combination of berberine-containing herbal medicines and ciprofloxacin can be an excellent antibacterial chemotherapy against multidrug resistance bacteria. To evaluate the pretreatment effect of berberine and HR, vehicle, berberine (25 and 50 mg/kg/day), and HR (1.4 g/kg/day) were daily administered to rats for five consecutive days. On day 6, ciprofloxacin was administered (10 mg/kg, i.v. and 20 mg/kg, p.o.) to rats. To assess cotreatment effect of berberine and ciprofloxacin, berberine (50 mg/kg) and ciprofloxacin (20 mg/kg) were coadministered by single oral gavage. Pharmacokinetic data were estimated by noncompartmental model. Compared with ciprofloxacin alone (control group), coadministration of berberine (50 mg/kg) and ciprofloxacin significantly decreased Cmax of ciprofloxacin (P<0.05). In addition, the pretreatment of berberine (50 mg/kg/day) and HR (1.4 g/kg/day) significantly decreased Cmax and AUC0→∞, compared with control group (P<0.05). The oral bioavailability of ciprofloxacin was reduced by cotreatment of berberine and pretreatment of berberine and HR. Our results suggest that the expression of P-glycoprotein and organic anion and/or organic cation transporters (OAT/OCT) could take a role in reduced oral bioavailability of ciprofloxacin by berberine and HR

Potential redox-sensitive Akt activation by dopamine activates Bad and promotes cell death in melanocytes

Dopamine (DA) is a well known oxidative neurotoxin. In addition, Akt has been reported to deliver a survival signal that inhibits apoptosis. However, it has also been reported that chronic Akt activation leads to apoptosis in response to oxidative stress. The objective of the present study was to investigate the possible role of the Akt pathway in vitiligo and its possible relationship with DA-induced cell death using Mel-Ab cells. Cultured Mel-Ab cells were treated with DA with and without N-Acetyl-L-cysteine (NAC), which is known to have antioxidative properties. Cell viability was then assessed by a crystal violet assay and Annexin staining was performed. The changes in the expression of Akt were analyzed by western blot analysis. The cell viability was reduced by approximately 60% in response to treatment with 500 µM DA, and NAC effectively prevented this cytotoxic effect. Likewise, treatment with DA produced numerous Annexin positive cells, while treatment with NAC prevented this apoptotic cell death. Akt was slowly phosphorylated after treatment with DA, while NAC clearly inhibited the DA-induced Akt activation. Western blot analysis also showed that treatment with DA induced the activation of Bad. Finally, LY294002 exerted a protective effect against DA-induced apoptotic cell death. DA may induce redox-sensitive Akt activation and increase the level of Bad, which can promote cell death by heterodimerization with survival proteins. Moreover, NAC effectively protects against DA-induced melanocyte death via inhibition of DA-induced Akt activation

Risk of Parkinson disease in stroke patients: A nationwide cohort study in South Korea

BACKGROUND AND PURPOSE Previous studies have examined the risk of stroke in patients with Parkinson disease (PD), but the incidence of PD onset among stroke patients and its risk according to severity of poststroke disabilities have scarcely been investigated. This study aims to determine whether the risk of PD is increased among stroke patients using a retrospective cohort with a large population-based database. METHODS We used data collected by the Korean National Health Insurance Service from 2010 to 2018 and examined 307,361 stroke patients and 380,917 sex- and age-matched individuals without stroke to uncover the incidence of PD. Cox proportional hazards regression was used to calculate the hazard ratio (HR) and 95% confidence interval (CI), and the risk of PD was compared according to presence and severity of disability. RESULTS During 4.31 years of follow-up, stroke patients had a 1.67 times higher risk of PD compared to individuals without stroke (adjusted HR = 1.67, 95% CI = 1.57-1.78). The risk of PD was greater among stroke patients with disabilities than among those without disabilities, even after adjustment for multiple covariates (adjusted HR = 1.72, 95% CI = 1.55-1.91; and adjusted HR = 1.66, 95% CI = 1.56-1.77, respectively). CONCLUSIONS Our study demonstrated an increased risk of PD among stroke patients. Health professionals need to pay careful attention to detecting movement disorders as clues for diagnosing PD