Biotransformation of 2-keto-4-hydroxybutyrate via aldol condensation using an efficient and thermostable carboligase from Deinococcus radiodurans.

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min-1 mg-1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L-1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L-1 h-1 and a specific productivity of 357.6 mg mg-enzyme-1 h-1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L-1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L-1 h-1 and 583.4 mg mg-enzyme-1 h-1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde.

One-Pot Biosynthesis of 2-Keto-4-hydroxybutyrate from Cheap C1 Compounds Using Rationally Designed Pyruvate Aldolase and Methanol Dehydrogenase.

One-carbon chemicals (C 1s) are potential building blocks as they are cheap, sustainable, and abiotic components. Methanol-derived formaldehyde can be another versatile building block for the production of 2-keto-4-hydroxyacid derivatives that can be used for amino acids, hydroxy carboxylic acids, and chiral aldehydes. To produce 2-keto-4-hydroxybutyrate from C 1s in an environment-friendly way, we characterized an aldolase from Pseudomonas aeruginosa PAO1 (PaADL), which showed much higher catalytic activity in condensing formaldehyde and pyruvate than the reported aldolases. By applying a structure-based rational approach, we found a variant (PaADLV121A/L241A) that exhibited better catalytic activities than the wild-type enzyme. Next, we constructed a one-pot cascade biocatalyst system by combining PaADL and a methanol dehydrogenase (MDH) and, for the first time, effectively produced 2-keto-4-hydroxybutyrate as the main product from pyruvate and methanol via an enzymatic reaction. This simple process applied here will help design a green process for the production of 2-keto-4-hydroxyacid derivatives.

Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting.

Immortalized cell lines can be used for diverse in vitro experiments, providing invaluable data before conducting in vivo studies Callithrix jacchus, the common marmoset, is a non-human primate model utilized for studying various human diseases. However, only a few immortalized marmoset cell lines are currently available. In the present study, we reveal that CRISPR-Cas9-mediated targeting of the p53 gene or CDKN2A locus is an effective means for immortalizing primary marmoset skin fibroblasts. In addition to frameshift mutations that result in premature stop codons, in-frame mutations potentially destroying the DNA-binding motif of p53 are frequently detected in immortalized cells. Like Cdkn2a-deficient mouse cells, CDKN2A-deficient marmoset cells express wild-type p53 proteins normally respond to genotoxic stresses, including adriamycin and etoposide. Taken together, these findings indicate that Cas9- mediated gene targeting of the p53 gene or CDKN2A locus is an effective tool for establishing immortalized marmoset cell lines with defined genetic alterations.

Carfilzomib enhances cisplatin-induced apoptosis in SK-N-BE(2)-M17 human neuroblastoma cells.

Neuroblastoma is a solid malignant tumor of the sympathetic nervous system, which accounts for 8-10% of childhood cancers. Considering the overall high risk and poor prognosis associated with neuroblastoma, effective therapeutics should be developed to improve patient survival and quality of life. A recent study showed that a proteasome inhibitor, carfilzomib (CFZ), reduced cell viability of SK-N-BE(2)-M17 neuroblastoma cells. Therefore, we investigated the molecular mechanisms by which CFZ lower the cell viability of neuroblastoma cells. CFZ reduced cell viability via cell cycle arrest at G2/M and apoptosis, which involved caspase activation (caspases-8, 9, 4, and 3), endoplasmic reticulum stress, reactive oxygen species production, mitochondrial membrane potential loss, and autophagy in a dose- and time-dependent manner. The effect of CFZ was additive to that of cisplatin (Cis), a well-known chemotherapeutic drug, in terms of cell viability reduction, cell cycle arrest, and apoptosis. Importantly, the additive effect of CFZ was maintained in Cis-resistant neuroblastoma cells. These results suggest that CFZ can be used in combination therapy for patients with neuroblastoma to overcome the resistance and adverse side effects of Cis.

Alpha-naphthoflavone induces apoptosis through endoplasmic reticulum stress via c-Src-, ROS-, MAPKs-, and arylhydrocarbon receptor-dependent pathways in HT22 hippocampal neuronal cells.

α-Naphthoflavone (αNF) is a prototype flavone, also known as a modulator of aryl hydrocarbon receptor (AhR). In the present study, we investigated the molecular mechanisms of αNF-induced cytotoxic effects in HT22 mouse hippocampal neuronal cells. αNF induced apoptotic cell death via activation of caspase-12 and -3 and increased expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitor, salubrinal, or by CHOP siRNA transfection reduced αNF-induced cell death. αNF activated mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, and inhibition of MAPKs reduced αNF-induced CHOP expression and cell death. αNF also induced accumulation of reactive oxygen species (ROS) and an antioxidant, N-acetylcysteine, reduced αNF-induced MAPK phosphorylation, CHOP expression, and cell death. Furthermore, αNF activated c-Src kinase, and inhibition of c-Src by a kinase inhibitor, SU6656, or siRNA transfection reduced αNF-induced ROS accumulation, MAPK activation, CHOP expression, and cell death. Inhibition of AhR by an AhR antagonist, CH223191, and siRNA transfection of AhR and AhR nuclear translocator reduced αNF-induced AhR-responsive luciferase activity, CHOP expression, and cell death. Finally, we found that inhibition of c-Src and MAPKs reduced αNF-induced transcriptional activity of AhR. Taken together, these findings suggest that αNF induces apoptosis through ER stress via c-Src-, ROS-, MAPKs-, and AhR-dependent pathways in HT22 cells.

Correction to: Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca2+-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells.

The original version of this article contained mistakes in figures. The western blot data for pro-caspase-3 and cleaved caspase-3 (Fig. 1d), ß-actin (Fig. 1d), PLCγ1 (Fig. 5d), and eIF2α (Fig. 7d) are incorrect. The corrected Figs. 1d, 5d, and 7d are shown below. The corrections do not influence either the validity of the published data or the conclusion described in the article.

Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells.

Fluoxetine (FLX) is an antidepressant drug that belongs to the class of selective serotonin reuptake inhibitors. FLX is known to induce apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of FLX were investigated in SK-N-BE(2)-M17 human neuroblastoma cells. FLX induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid or CHOP siRNA transfection reduced FLX-induced cell death. FLX induced phosphorylation of mitogen-activated protein kinases (MAPKs) family, p38, JNK, and ERK, and an upstream kinase apoptosis signal kinase 1 (ASK1). Inhibition of MAPKs and ASK1 reduced FLX-induced cell death and CHOP expression. We then showed that FLX reduced mitochondrial membrane potential (MMP) and ER stress inhibitors as well as MAPK inhibitors ameliorated FLX-induced loss of MMP. Interestingly, FLX induced hyperacetylation of histone H3 and H4, upregulation of p300 histone acetyltransferase (HAT), and downregulation of histone deacetylases (HDACs). Treatment with a HAT inhibitor anacardic acid or p300 HAT siRNA transfection blocked FLX-induced apoptosis in SK-N-BE(2)-M17 cells. However, FLX did not induce histone acetylation and anacardic acid had no protective effect on FLX-induced cell death and CHOP expression in MYCN non-amplified SH-SY5Y human neuroblastoma and MYCN knockdowned SK-N-BE(2)-M17 cells. These findings suggest that FLX induces apoptosis in neuroblastoma through ER stress and mitochondrial dysfunction via the ASK1 and MAPK pathways and through histone hyperacetylation in a MYCN-dependent manner.

Production of 5,8-dihydroxy-9(Z)-octadecenoic acid from oleic acid by whole recombinant cells of Aspergillus nidulans expressing diol synthase.

The optimal conditions for the production of 5,8-dihydroxy-9(Z)-octadecenoic acid (5,8-diHOME) from oleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans were 40 °C, pH 7.5, 10 % (v/v) dimethyl sulfoxide, 35 g cells l(-1), and 12 g oleic acid l(-1) at 250 rpm in a 250 ml baffled flask. Under these conditions, whole recombinant cells produced 5.2 g 5,8-diHOME l(-1) together with 1 g l(-1) of the intermediate 8-hydroperoxy-9(Z)-octadecenoic acid (8-HPOME) after 60 min. This corresponded to a conversion yield of 43 % (w/w), a volumetric productivity of 5.2 g l(-1 )h(-1), and a specific productivity of 148 mg g cells(-1 )h(-1). This is the first report of the biotechnological production of 5,8-diHOME from oleic acid.

Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca(2+)-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells.

Licochalcone A (LicA), an estrogenic flavonoid, induces apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of LicA were investigated in HepG2 human hepatocellular carcinoma cells. LicA induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by CHOP knockdown or treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid, reduced LicA-induced cell death. LicA also induced reactive oxygen species (ROS) accumulation and the anti-oxidant N-acetylcysteine reduced LicA-induced cell death and CHOP expression. In addition, LicA increased the levels of cytosolic Ca(2+), which was blocked by 2-aminoethoxydiphenyl borate (an antagonist of inositol 1,4,5-trisphosphate receptor) and BAPTA-AM (an intracellular Ca(2+) chelator). 2-Aminoethoxydiphenyl borate and BAPTA-AM inhibited LicA-induced cell death. Interestingly, LicA induced phosphorylation of phospholipase Cγ1 (PLCγ1) and inhibition of PLCγ1 reduced cell death and ER stress. Moreover, the multi-targeted receptor tyrosine kinase inhibitors, sorafenib and sunitinib, reduced LicA-induced cell death, ER stress, and cytosolic Ca(2+) and ROS accumulation. Finally, LicA induced phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and c-Met receptor and inhibition of both receptors by co-transfection with VEGFR2 and c-Met siRNAs reversed LicA-induced cell death, Ca(2+) increase, and CHOP expression. Taken together, these findings suggest that induction of ER stress via a PLCγ1-, Ca(2+)-, and ROS-dependent pathway may be an important mechanism by which LicA induces apoptosis in HepG2 hepatocellular carcinoma cells.

Image quality and radiation dose of 128-slice dual-source CT venography using low kilovoltage combined with high-pitch scanning and automatic tube current modulation.

To compare vascular enhancement, image quality, and radiation dose of 128-slice dual-source CT venography (CTV) between an imaging setting of 120 kVp with low pitch, and a setting of 100 kVp combined with high pitch and automatic tube current modulation. A total of 100 patients with suspected deep vein thrombosis and varicose veins were divided into two groups: Group 1 [50 patients, 120 kVp, low pitch (0.6), and fixed 120 mA) and Group 2 (50 patients, 100 kVp, high pitch (3.0), and automatic tube current modulation]. Two radiologists, who were blinded to the image protocol, assessed vascular enhancement and image noise in the inferior vena cava (IVC), femoral vein, and popliteal vein. They also assigned an image quality score independently using a 5-point visual scale. Effective dose was estimated using the dose-length product (DLP). Group demographics, radiation dose, vascular enhancement, image noise, and image quality in the two groups were analyzed. Mean vascular enhancement of the IVC, femoral vein, and popliteal vein was significantly higher in group 2 than in group 1, and images in group 2 had significantly higher image noise. However, there were no significant differences in subjective image quality score of the IVC, femoral vein, and popliteal vein. The mean DLP in group 2 (402.10 ± 94.29 mGy cm) was significantly lower than that in group 1 (973.36 ± 63.20 mGy cm) (P < 0.001). Lower extremity CTV using 100 kVp, high pitch (3.0), and automatic tube current modulation improved vascular enhancement with acceptable image quality and low radiation dose.

An overview of thoracic actinomycosis: CT features.

BACKGROUND: Thoracic actinomycosis is an uncommon, chronic suppurative bacterial infection caused by actinomyces species, especially Actinomyces israelii. METHODS: It is usually seen in immunocompetent patients with respiratory disorders, poor oral hygiene, alcoholism and chronic debilitating diseases. RESULTS: We illustrate the radiological manifestations of thoracic actinomycoses in various involved areas in the thorax. CONCLUSION: Thoracic actinomycosis can be radiologically divided into the parenchymal type, the airway type including bronchiectasis, the endobronchial form, and the mediastinum or chest wall involvement type. TEACHING POINTS: • Important risk factors for thoracic actinomycosis are underlying respiratory disorders such as emphysema and chronic bronchitis. • Different CT patterns can be distinguished in thoracic actinomycosis: parenchymal, bronchiectatic, endobronchial and extrapulmonary. • Typical CT findings in the parenchymal pattern are a central low density within the parenchymal consolidation and adjacent pleural thickening.

Pulmonary nodular lymphoid hyperplasia associated with Sjögren's syndrome.

Pulmonary nodular lymphoid hyperplasia (NLH) is a term first suggested by Kradin and Mark to describe one or more pulmonary nodules or localized lung infiltrates consisting of reactive lymphoid proliferation. To date, there have been only a few cases of pulmonary NLH reported associated with autoimmune disorders. There is no case of NLH associated with Sjögren's syndrome from Korea in the medical literature. A 56-year-old woman was referred to our hospital with cough productive of sputum and chest tightness. The Computed tomography scans of the chest revealed multiple and well-defined peribronchiolar nodular opacities. A video assisted thoracoscopic surgery (VATS) biopsy was performed and the nodular opacity in the lung parenchyma was pathologically confirmed as NLH. Through meticulous review of patient's record, we found that she had been suffering from dry eye and dry mouth. The symptoms suggested Sjögren's syndrome, which was confirmed by specific laboratory tests including the Schirmer test, anti-nuclear antibody and anti-Ro/La antibody. The patient is followed regularly and has no further progression of symptoms.