Metabolic design of Escherichia coli for muconic acid production

Adipic acid(AA) is a versatile bulk chemical to be used for raw materials such as nylon 6,6. Currently, AA biosynthesis from bio-resources have received a lot of attention in recent years as environment-friendly and renewable AA production process. Muconic acid(MA), also known as 2,4-hexadienedioic acid, is expected as a biosynthesis precursor of AA. There are Several studies on MA biosynthesis using Escherichia coli introduced foreign genes. In those studies, MA is synthesized from intermediate products of shikimate pathway. However, the production volume is not sufficient and it is a hindrance to industrialization. In this study, we aimed to the high efficiency biosynthesis of MA using metabolic designed Escherichia coli. First, we designed the metabolism to increase the accumulation of phosphoenolpyruvic acid (PEP), which is one of the starting materials of the shikimate pathway. Next, we determined the optimal MA synthetic pathway branched from the shikimate pathway. Specifically, we examined three types of MA production pathway with PEP accumulation strain as parent and selected the pathway with the highest MA production. Finally, we examined efficient production of MA using fusion proteins. Shikimate pathway protein and MA production pathway protein were combined to direct carbon flux into MA production

Trigger digit after corticosteroid injection

Purpose: The aim of this study was to elucidate whether intrasynovial corticosteroid injections for trigger digit reduced the volume of the tendon and pulley on high-resolution ultrasonography. Methods: Twenty-three digits of 20 patients with trigger digit were included. Each affected finger was graded clinically according to the following classification: grade I for pre-triggering, grade II for active triggering, grade III for passive triggering, and grade IV for presence of contracture. Axial ultrasound examinations were performed before an intrasynovial corticosteroid injection and at an average of 31 days after the injection. The transverse diameter, thickness, and cross-sectional area of the tendon and the thickness of the pulley were measured by two independent, blinded researchers. Results: At least 1 grade of improvement was achieved in this study group by the time of the second examination. The transverse diameter and cross-sectional area of the tendon and the thickness of the pulley significantly decreased (P<0.05). Conclusion: The injection of a single dose of betamethasone improved clinical symptoms by reducing the volume of both the tendon and pulley, which may be related to the fact that tendon and pulley ruptures are delayed by corticosteroid injections

Intrapulmonary metastasis of non–small cell lung cancer: A prognostic assessment

AbstractObjective: According to the revised TNM classification in 1997, intrapulmonary metastasis within the same lobe of the primary tumor is designated as T4 and intrapulmonary metastasis in a different lobe is M1. However, their prognostic implications remain unclear. To assess their prognoses, we retrospectively analyzed the postoperative survival of patients with and without intrapulmonary metastasis. Methods: From January 1982 to December 1996, 2340 patients with non–small cell lung cancer underwent surgical resection. The survival of patients having complete resection (n = 1534) was analyzed according to their intrapulmonary metastasis status: patients without intrapulmonary metastasis (n = 1393), those with metastasis in the same lobe (n = 105), and those with metastasis in a different lobe (n = 18). For comparison, patients with T4 disease without intrapulmonary metastasis in the same lobe (n = 54) and those with M1 disease without metastasis in a different lobe (distant M1, n = 18) were also analyzed. Results: The overall 5-year survivals were as follows: no intrapulmonary metastasis, 60%; stage T4 disease with no intrapulmonary metastasis, 34%; pulmonary metastasis in the same lobe, 34%; pulmonary metastasis in a different lobe, 11%; and distant M1, 6%. The differences in survival between patients with no pulmonary metastasis and those with metastasis in the same lobe (P <.001, log-rank test) and between patients with metastasis in the same lobe and those with distant M1 (P <.001) were significant. In contrast, there was no significant difference between patients with metastasis in the same lobe and those with T4 disease and no intrapulmonary metastasis or between patients with metastasis to a different lobe and those with distant M1. Conclusions: Prognostically, intrapulmonary metastasis within the same lobe of the primary tumor was comparable with T4 and that in a different lobe was comparable with M1. In terms of postoperative prognosis, the revised TNM classification for intrapulmonary metastasis seems to be appropriate.J Thorac Cardiovasc Surg 2001;122:24-

Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression

<p>Abstract</p> <p>Background</p> <p>Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid <it>Saccharomyces cerevisiae </it>strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass.</p> <p>Results</p> <p>The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours.</p> <p>Conclusions</p> <p>We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.</p

A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape from a Bio-nanocapsule via an endocytic uptake pathway

BACKGROUND: An affibody-displaying bio-nanocapsule (Z(HER2)-BNC) with a hepatocyte specificity derived from hepatitis B virus (HBV) was converted into an affibody, Z(HER2), that recognizes HER2 receptors. This affibody was previously reported to be the result of the endocytosis-dependent specific uptake of proteins and siRNA into target cancer cells. To assist the endosomal escape of inclusions, a helper lipid with pH-sensitive fusogenic ability (1,2-dioleoyl-sn-glycero-3-phos phoethanolamine; DOPE) was conjugated with a Z(HER2)-BNC. FINDINGS: In this study, we displayed a pH-sensitive fusogenic GALA peptide on the surface of a particle in order to confer the ability of endosomal escape to a Z(HER2)-BNC. A GALA-displaying Z(HER2)-BNC purified from yeast uneventfully formed a particle structure. Furthermore, endosomal escape of the particle was facilitated after endocytic uptake and release of the inclusions to the cytoplasm without the cell toxicity. CONCLUSION: The genetic fusion of a GALA peptide to the virus-like particle confers the ability of endosomal escape

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>T. reesei </it>effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant <it>Saccharomyces cerevisiae</it>, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.</p> <p>Results</p> <p>In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.</p

Paradoxical activation of c-Src as a drug-resistant mechanism

抗がん剤抵抗性の新規メカニズムの解明 --薬剤抵抗性がん細胞は抗がん剤により増殖する--. 京都大学プレスリリース. 2021-03-25.ATP-competitive inhibitors have been developed as promising anti-cancer agents. However, drug-resistance frequently occurs, and the underlying mechanisms are not fully understood. Here, we show that the activation of c-Src and its downstream phosphorylation cascade can be paradoxically induced by Src-targeted and RTK-targeted kinase inhibitors. We reveal that inhibitor binding induces a conformational change in c-Src, leading to the association of the active form c-Src with focal adhesion kinase (FAK). Reduction of the inhibitor concentration results in the dissociation of inhibitors from the c-Src-FAK complex, which allows c-Src to phosphorylate FAK and initiate FAK-Grb2-mediated Erk signaling. Furthermore, a drug-resistant mutation in c-Src, which reduces the affinity of inhibitors for c-Src, converts Src inhibitors into facilitators of cell proliferation by enhancing the phosphorylation of FAK and Erk in c-Src-mutated cells. Our data thus reveal paradoxical enhancement of cell growth evoked by target-based kinase inhibitors, providing potentially important clues for the future development of effective and safe cancer treatment

T2 tumors larger than five centimeters in diameter can be upgraded to T3 in non–small cell lung cancer

AbstractObjective: Among the TNM criteria, tumor size is a well-assessed factor in the prognosis of small tumors. A 3-cm cutoff point separates T1 from T2 tumors, whereas a size larger than 3 cm is not ascribed any prognostic value. Instead, N2 is considered to be the worst prognostic factor for intrathoracic extended disease. Method: The prognosis of 545 patients with non–small cell lung cancer larger than 3 cm in diameter (T2, T3, and T4) was studied. These tumors were completely resected by pneumonectomy (n = 126) or lobectomy (n = 411) or were partially resected (n = 8). Survivals were compared according to the following factors: tumor size (3.1-5 cm, 5.1-7 cm, >7 cm), nodal status, age, sex, histologic type, degree of pleural involvement, operative procedure, stage, and T factor. For the multivariate analysis, the Cox proportional hazard model was used with the same variables. Results: The univariate analysis showed that age, sex, degree of pleural involvement, operative procedure, tumor size, nodal status, and stage were all significant prognostic factors. Further comparison of survival between different tumor sizes (≤5 cm vs >5 cm) in the same nodal category demonstrated a significantly poor prognosis for larger tumors in N0 (P =.00374) and N2+N3 (P =.0157), but not in N1 (P =.3452). T2 tumors (n = 349) were divided, according to size, into T2a (n = 238) and T2b (n = 111), and survival was compared with those in T3 and T4. The 5-year survivals were 51.3%, 35.1%, 47.8%, and 25.3%, respectively. The difference between T2a and T2b was statistically significant (log-rank P =.0170, Breslow P =.0055). Conclusions: A tumor size of more than 5 cm in diameter was indicative of a poor prognosis in non–small cell lung cancer, because patients with T2b tumors had a significantly different survival from that of patients with T2a tumors, and the survival curve was located between those for patients with T3 and T4 tumors. Consequently, T2b might be upgraded to at least T3.J Thorac Cardiovasc Surg 2001;122:907-1

Novel Treatment Criteria for Persistent Ductus Arteriosus in Neonates

BackgroundThe indications for ductus arteriosus ligation in very-low-birth-weight infants (VLBWIs) with persistent ductus arteriosus (PDA) are unclear. Increased left ventricular end-diastolic dimension (LVDd) is commonly found in patients with PDA. Here, the enlargement of LVDd in term and preterm neonates without congenital heart disease was estimated by two-dimensional echocardiography.MethodsThe value of the measured LVDd was divided by the normal LVDd as an index (LVDd ratio) to compare 30 patients who underwent PDA ligation with 30 patients treated with indomethacin and 30 patients who did not undergo radical therapy.ResultsAn LVDd ratio between 122% and 197% (mean, 142%) was considered to be an indication for the ligation procedure. The proportion of patients exceeding 130% in the LVDd ratio was 87% (26/30) in those patients who underwent ligation. Catecholamines and/or vasodilators were required in 83% patients for the treatment of low ejection fraction or hypertension after operations, suggesting that patients had been in preload and/or afterload remodeling failure during the operation. The percentage of patients with less than 115% in the LVDd ratio was 90% in the non-radical-therapy patients. The LVDd ratios of 130% and 115% were regarded as cut-off values for surgical ligation and indomethacin treatment.ConclusionThe LVDd ratio is a useful measure to determine the treatment of VLBWIs with PDA