Survey of Pharmaceutical Industry's Best Practices around In Vitro Transporter Assessment and Implications for Drug Development: Considerations from the International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Assessing the Feasibility of Biorefineries for a Sustainable Citrus Waste Management in Korea.

Citrus fruits are one of the most widely used fruits around the world and are used as raw fruits, but are also processed into products such as beverages, and large amounts of by-products and waste are generated in this process. Globally, disposal of citrus waste (CW) through simple landfilling or ocean dumping can result in soil and groundwater contamination, which can negatively impact ecosystem health. The case of Korea is not much different in that these wastes are simply buried or recycled wastes are used as livestock feed additives. However, there are many reports that CW, which is a waste, has high potential to produce a variety of products that can minimize environmental load and increase added value through appropriate waste management. In this study, we aim to explore the latest developments in the evaluation and valorization of the growing CW green technologies in an effort to efficiently and environmentally transform these CW for resource recovery, sustainability, and economic benefits. Recent research strategies on integrated biorefinery approaches have confirmed that CW can be converted into various bioproducts such as enzymes, biofuels and biopolymers, further contributing to energy security. It was found that more efforts are needed to scale up green recovery technologies and achieve diverse product profiling to achieve zero waste levels and industrial viability.

Intelligent Complementary Multi-Modal Fusion for Anomaly Surveillance and Security System.

Recently, security monitoring facilities have mainly adopted artificial intelligence (AI) technology to provide both increased security and improved performance. However, there are technical challenges in the pursuit of elevating system performance, automation, and security efficiency. In this paper, we proposed intelligent anomaly detection and classification based on deep learning (DL) using multi-modal fusion. To verify the method, we combined two DL-based schemes, such as (i) the 3D Convolutional AutoEncoder (3D-AE) for anomaly detection and (ii) the SlowFast neural network for anomaly classification. The 3D-AE can detect occurrence points of abnormal events and generate regions of interest (ROI) by the points. The SlowFast model can classify abnormal events using the ROI. These multi-modal approaches can complement weaknesses and leverage strengths in the existing security system. To enhance anomaly learning effectiveness, we also attempted to create a new dataset using the virtual environment in Grand Theft Auto 5 (GTA5). The dataset consists of 400 abnormal-state data and 78 normal-state data with clip sizes in the 8-20 s range. Virtual data collection can also supplement the original dataset, as replicating abnormal states in the real world is challenging. Consequently, the proposed method can achieve a classification accuracy of 85%, which is higher compared to the 77.5% accuracy achieved when only employing the single classification model. Furthermore, we validated the trained model with the GTA dataset by using a real-world assault class dataset, consisting of 1300 instances that we reproduced. As a result, 1100 data as the assault were classified and achieved 83.5% accuracy. This also shows that the proposed method can provide high performance in real-world environments.

Development of a Real-Time Pixel Array-Type Detector for Ultrahigh Dose-Rate Beams.

Although research into ultrahigh dose-rate (UHDR) radiation therapy is ongoing, there is a significant lack of experimental measurements for two-dimensional (2D) dose-rate distributions. Additionally, conventional pixel-type detectors result in significant beam loss. In this study, we developed a pixel array-type detector with adjustable gaps and a data acquisition system to evaluate its effectiveness in measuring UHDR proton beams in real time. We measured a UHDR beam at the Korea Institute of Radiological and Medical Sciences using an MC-50 cyclotron, which produced a 45-MeV energy beam with a current range of 10-70 nA, to confirm the UHDR beam conditions. To minimize beam loss during measurement, we adjusted the gap and high voltage on the detector and determined the collection efficiency of the developed detector through Monte Carlo simulation and experimental measurements of the 2D dose-rate distribution. We also verified the accuracy of the real-time position measurement using the developed detector with a 226.29-MeV PBS beam at the National Cancer Center of the Republic of Korea. Our results indicate that, for a current of 70 nA with an energy beam of 45 MeV generated using the MC-50 cyclotron, the dose rate exceeded 300 Gy/s at the center of the beam, indicating UHDR conditions. Simulation and experimental measurements show that fixing the gap at 2 mm and the high voltage at 1000 V resulted in a less than 1% loss of collection efficiency when measuring UHDR beams. Furthermore, we achieved real-time measurements of the beam position with an accuracy of within 2% at five reference points. In conclusion, our study developed a beam monitoring system that can measure UHDR proton beams and confirmed the accuracy of the beam position and profile through real-time data transmission.

Inhibition of p90RSK Ameliorates PDGF-BB-Mediated Phenotypic Change of Vascular Smooth Muscle Cell and Subsequent Hyperplasia of Neointima.

Platelet-derived growth factor type BB (PDGF-BB) regulates vascular smooth muscle cell (VSMC) migration and proliferation, which play critical roles in the development of vascular conditions. p90 ribosomal S6 kinase (p90RSK) can regulate various cellular processes through many different target substrates in several cell types, but the regulatory function of p90RSK on PDGF-BB-mediated cell migration and proliferation and subsequent vascular neointima formation has not yet been extensively examined. In this study, we investigated whether p90RSK inhibition protects VSMCs against PDGF-BB-induced cellular phenotypic changes and the molecular mechanisms underlying the effect of p90RSK inhibition on neointimal hyperplasia in vivo. Pretreatment of cultured primary rat VSMCs with FMK or BI-D1870, which are specific inhibitors of p90RSK, suppressed PDGF-BB-induced phenotypic changes, including migration, proliferation, and extracellular matrix accumulation, in VSMCs. Additionally, FMK and BI-D1870 repressed the PDGF-BB-induced upregulation of cyclin D1 and cyclin-dependent kinase-4 expression. Furthermore, p90RSK inhibition hindered the inhibitory effect of PDGF-BB on Cdk inhibitor p27 expression, indicating that p90RSK may induce VSMC proliferation by regulating the G0/G1 phase. Notably, treatment with FMK resulted in attenuation of neointima development in ligated carotid arteries in mice. The findings imply that p90RSK inhibition mitigates the phenotypic switch and neointimal hyperplasia induced by PDGF-BB.

Phytochemistry and Applications of Cinnamomum camphora Essential Oils.

Camphor tree (Cinnamomum camphora) is an ornamental plant that has been cultivated for a long time to obtain wood or camphor. Furthermore, its essential oil can be used as an alternative medicine and is an important source of perfume. Camphor obtained from camphor trees has long been used as a treatment for various symptoms such as inflammation, infection, congestion, muscle pain, and irritation in various regions. The purpose of this literature review is to provide knowledge of the well-established, wide, and extensive applications of camphor both in traditional and modern applications. Despite many studies focused on the essential oil of the camphor tree, there is a lack of systematic studies of its extraction or separation. Besides, various components of camphor are not fully understood, and further research is needed on the medicinal effects of individual components of C. camphor. The genus Cinnamomum has crucial economic value and theoretical significance. However, further systematic reviews and investigative studies based on existing research are needed to promote the modernization process of traditional applications of camphor. For proper use of the essential oil of C. camphora, it is imperative to consider its possible effects on humans and the environment.

Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production.

Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8 mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.

Topical Treatment for Cutaneous Leishmaniasis: Dermato-Pharmacokinetic Lead Optimization of Benzoxaboroles.

Cutaneous leishmaniasis (CL) is caused by several species of the protozoan parasite Leishmania, affecting an estimated 10 million people worldwide. Previously reported strategies for the development of topical CL treatments have focused primarily on drug permeation and formulation optimization as the means to increase treatment efficacy. Our approach aims to identify compounds with antileishmanial activity and properties consistent with topical administration. Of the test compounds, five benzoxaboroles showed potent activity (50% effective concentration [EC50] < 5 µM) against intracellular amastigotes of at least one Leishmania species and acceptable activity (20 µM < EC50 < 30 µM) against two more species. Benzoxaborole compounds were further prioritized on the basis of the in vitro evaluation of progression criteria related to skin permeation, such as the partition coefficient and solubility. An MDCKII-hMDR1 cell assay showed overall good permeability and no significant interaction with the P-glycoprotein transporter for all substrates except LSH002 and LSH031. The benzoxaboroles were degraded, to some extent, by skin enzymes but had stability superior to that of para-hydroxybenzoate compounds, which are known skin esterase substrates. Evaluation of permeation through reconstructed human epidermis showed LSH002 to be the most permeant, followed by LSH003 and LSH001. Skin disposition studies following finite drug formulation application to mouse skin demonstrated the highest permeation for LSH001, followed by LSH003 and LSH002, with a significantly larger amount of LSH001 than the other compounds being retained in skin. Finally, the efficacy of the leads (LSH001, LSH002, and LSH003) against Leishmania major was tested in vivo LSH001 suppressed lesion growth upon topical application, and LSH003 reduced the lesion size following oral administration.

Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology.

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations.

Improvement of isobutanol production in Saccharomyces cerevisiae by increasing mitochondrial import of pyruvate through mitochondrial pyruvate carrier.

Subcellular compartmentalization of the biosynthetic enzymes is one of the limiting factors for isobutanol production in Saccharomyces cerevisiae. Previously, it has been shown that mitochondrial compartmentalization of the biosynthetic pathway through re-locating cytosolic Ehrlich pathway enzymes into the mitochondria can increase isobutanol production. In this study, we improved mitochondrial isobutanol production by increasing mitochondrial pool of pyruvate, a key substrate for isobutanol production. Mitochondrial isobutanol biosynthetic pathway was introduced into bat1Δald6Δlpd1Δ strain, where genes involved in competing pathways were deleted, and MPC1, MPC2, and MPC3 genes encoding the subunits of mitochondrial pyruvate carrier (MPC) hetero-oligomeric complex were overexpressed with different combinations. Overexpression of Mpc1 and Mpc3 forming high-affinity MPCOX was more effective in improving isobutanol production than overexpression of Mpc1 and Mpc2 forming low-affinity MPCFERM. The final engineered strain overexpressing MPCOX produced 330.9 mg/L isobutanol from 20 g/L glucose, exhibiting about 22-fold increase in production compared to wild type.

Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol.

Saccharomyces cerevisiae naturally produces small amounts of isobutanol and 3-methyl-1-butanol via Ehrlich pathway from the catabolism of valine and leucine, respectively. In this study, we engineered CEN.PK2-1C, a leucine auxotrophic strain having a LEU2 gene mutation, for the production of isobutanol and 3-methyl-1-butanol. First, ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. We also increased transcription of endogenous genes in the valine and leucine biosynthetic pathways by expressing Leu3Δ601, a constitutively active form of Leu3 transcriptional activator. For the production of isobutanol, genes involved in isobutanol production (ILV2, ILV3, ILV5, ARO10, and ADH2) were additionally overexpressed in ald6Δbat1Δ strain expressing LEU3Δ601, resulting in 376.9 mg/L isobutanol production from 100 g/L glucose. To increase 3-methyl-1-butanol production, leucine biosynthetic genes were additionally overexpressed in the final isobutanol-production strain. The resulting strain overexpressing LEU2 and LEU4 (D578Y) , a feedback inhibition-insensitive mutant of LEU4, showed a 34-fold increase in 3-methyl-1-butanol synthesis compared with CEN.PK2-1C control strain, producing 765.7 mg/L 3-methyl-1-butanol.

Study of accelerator-based epithermal neutron flux depending on Li and Be targets.

The Li(p,n) and Be(p,n) reactions are widely used as accelerator-based neutron sources for boron neutron capture therapy (BNCT). Because the energy of the neutrons produced by these reactions is high for BNCT, a moderator is required to reduce the energy to the epithermal energy region. The neutron yields and energy spectra derived from the Li(p,n) and Be(p,n) reactions vary with the incident proton energy; a high proton energy results in a high neutron yield. However, a high proton energy can also increase the neutron energy, and in this case, a high neutron energy requires a thick moderator, which decreases the epithermal neutron flux. Notably, these tradeoffs are not completely clear. Furthermore, the upper limit of the epithermal neutron energy used in different BCNT facilities is not the same. Therefore, epithermal neutron flux estimation with applying an appropriate moderator is necessary for comparing epithernal neutron flux depending on the proton energy, target type, and epithermal neutron range definition. In this study, GEANT4 simulations were performed to determine effective moderator materials and thicknesses. After applying a moderator, we examined the epithermal neutron flux by changing the conditions that can influence the epithermal neutron flux, such as the proton energy, target species (Li and Be), and different definitions of the epithermal neutron region. The results showed that the epithermal neutron flux increases with the increasing proton energy until a certain level for both Li and Be targets. For proton energies above 3 MeV for the Li target and above 7 MeV for the Be target, the epithermal neutron flux decreases or its increase rate gradually decreases. And, when the upper energy limit of the epithermal neutron range is changed from 10 keV to 20 keV or 40 keV, the epithermal neutron flux is more than 109 cm-2 s-1 at a proton energy of 3 MeV for Li target and 8 MeV for Be target.

Design of compact accelerator system for high flux accelerator based neutron source.

Accelerator Based Neutron Sources (ABNS) have been studied for their utility in materials research as well as for boron neutron captured therapy. By making significant efforts to study the (p,n) and (d,n) nuclear reactions, the specifications of the accelerator system have been determined. In this paper, we compare the design results for two types of radio frequency quadrupole (RFQ) accelerators to provide proton and deuteron beams, respectively. Both systems consist of an electron cyclotron resonance (ECR) ion source, a low-energy beam transport system, an RFQ accelerator, a medium-energy beam transport system, a Be target, and a moderator system. In order to achieve a compact accelerator system at a reasonable cost, different requirements must be applied to the design of RFQ accelerators. The proton RFQ has been designed with an operation frequency of 352 MHz, up to 4 MeV acceleration, 10 mA beam intensity, and a continuous-wave (CW) operation mode to achieve 0.84 × 109 n/(s/cm2) of neutron production. However, the deuteron RFQ has been designed with an operation frequency of 200 MHz, up to 2.5 MeV acceleration, 15 mA of beam intensity, and a CW operation mode to achieve 1.02 × 109 n/(s/cm2) of neutron production. In this paper, we describe the merit of the deuteron based neutron source by comparing two types of the RFQ accelerators for proton and deuteron beams including the common system of the ECR ion source and Be target in detail.

The complete mitochondrial genome of Patagonian moray cod, Muraenolepis orangiensis Vaillant, 1888 (Gadiformes, Muraenolepididae).

The full-length mitochondrial genome of Muraenolepis orangiensis (Vaillant, 1888) was studied using PacBio platform and it is first report in a Muraenolepididae family. The circular form of mitochondria genome is 16,833 bp including 13 protein-coding genes, two rRNA, and 22 tRNA. Start codon of 13 protein-coding genes was only ATG but three types of stop codons (TAA, T(AA), and TAG) were detected. To evaluate evolutionary position of M. orangiensis, the phylogenetic tree with other 13 Antarctic fishes belonged to five families were showed that M. orangiensis is unique cluster as a Muraenolepididae family and this study would provide fundamental data to understand the evolutionary relationship of fishes founded in Antarctic area.

Cancer cell­specific anticancer effects of Coptis chinensis on gefitinib­resistant lung cancer cells are mediated through the suppression of Mcl­1 and Bcl­2.

The epidermal growth factor receptor (EGFR)­tyrosine kinase inhibitor (TKI), gefitinib, is an effective therapeutic drug used in the treatment of non­small cell lung cancers (NSCLCs) harboring EGFR mutations. However, acquired resistance significantly limits the efficacy of EGFR­TKIs and consequently, the current chemotherapeutic strategies for NSCLCs. It is, therefore, necessary to overcome this resistance. In the present study, the anticancer potential of natural extracts of Coptis chinensis (ECC) against gefitinib­resistant (GR) NSCLC cells were investigated in vitro and in vivo. ECC inhibited the viability, migration and invasion, and effectively induced the apoptosis of GR cells. These effects were associated with the suppression of EGFR/AKT signaling and the expression of anti­apoptotic proteins, Mcl­1 and Bcl­2, which were overexpressed in GR NSCLC cells. Combination treatment with ECC and gefitinib enhanced the sensitivity of GR cells to gefitinib in vitro, but not in vivo. However, ECC increased the survival of individual zebrafish without affecting the anticancer effect to cancer cells in vivo, which indicated a specific cytotoxic effect of ECC on cancer cells, but not on normal cells; this is an important property for the development of novel anticancer drugs. On the whole, the findings of the present study indicate the potential of ECC for use in the treatment of NSCLC, particularly in combination with EGFR­TKI therapy, in EGFR­TKI­resistant cancers.

Development of an efficient cytosolic isobutanol production pathway in Saccharomyces cerevisiae by optimizing copy numbers and expression of the pathway genes based on the toxic effect of α-acetolactate.

Isobutanol production in Saccharomyces cerevisiae is limited by subcellular compartmentalization of the pathway enzymes. In this study, we improved isobutanol production in S. cerevisiae by constructing an artificial cytosolic isobutanol biosynthetic pathway consisting of AlsS, α-acetolactate synthase from Bacillus subtilis, and two endogenous mitochondrial enzymes, ketol-acid reductoisomerase (Ilv5) and dihydroxy-acid dehydratase (Ilv3), targeted to the cytosol. B. subtilis AlsS was more active than Ilv2ΔN54, an endogenous α-acetolactate synthase targeted to the cytosol. However, overexpression of alsS led to a growth inhibition, which was alleviated by overexpressing ILV5ΔN48 and ILV3ΔN19, encoding the downstream enzymes targeted to the cytosol. Therefore, accumulation of the intermediate α-acetolactate might be toxic to the cells. Based on these findings, we improved isobutanol production by expressing alsS under the control of a copper-inducible CUP1 promoter, and by increasing translational efficiency of the ILV5ΔN48 and ILV3ΔN19 genes by adding Kozak sequence. Furthermore, strains with multi-copy integration of alsS into the delta-sequences were screened based on growth inhibition upon copper-dependent induction of alsS. Next, the ILV5ΔN48 and ILV3ΔN19 genes were integrated into the rDNA sites of the alsS-integrated strain, and the strains with multi-copy integration were screened based on the growth recovery. After optimizing the induction conditions of alsS, the final engineered strain JHY43D24 produced 263.2 mg/L isobutanol, exhibiting about 3.3-fold increase in production compared to a control strain constitutively expressing ILV2ΔN54, ILV5ΔN48, and ILV3ΔN19 on plasmids.

Publisher Correction: Development of an efficient cytosolic isobutanol production pathway in Saccharomyces cerevisiae by optimizing copy numbers and expression of the pathway genes based on the toxic effect of α-acetolactate.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic Engineering of Saccharomyces cerevisiae for Production of Shinorine, a Sunscreen Material, from Xylose.

Shinorine, a mycosporine-like amino acid (MAA), is a small molecule sunscreen produced in some bacteria. In this study, by introducing shinorine biosynthetic genes from cyanobacteria Nostoc punctiform into Saccharomyces cerevisiae, we successfully constructed yeast strains capable of producing shinorine. Sedoheptulose 7-phosphate (S7P), an intermediate of the pentose phosphate pathway, is a key substrate for shinorine biosynthesis. To increase the S7P pool, xylose, which is assimilated via the pentose phosphate pathway, was used as a carbon source after introducing xylose assimilation genes from Scheffersomyces stipitis into the shinorine-producing strain. The resulting xylose-fermenting strain produced a trace amount of shinorine when cells were grown in glucose, but shinorine production was dramatically increased by adding xylose in the medium. Shinorine production was further improved by modulating the pentose phosphate pathway through deleting TAL1 and overexpressing STB5 and TKL1. The final engineered strain JHYS17-4 produced 31.0 mg/L (9.62 mg/g DCW) of shinorine in the optimized medium containing 8 g/L of xylose and 12 g/L of glucose, demonstrating that S. cerevisiae is a promising host to produce this natural sunscreen material.

THz-pump and X-ray-probe sources based on an electron linac.

We describe a compact THz-pump and X-ray-probe beamline, based on an electron linac, for ultrafast time-resolved diffraction applications. Two high-energy electron (γ > 50) bunches, 5 ns apart, impinge upon a single-foil or multifoil radiator and generate THz radiation and X-rays simultaneously. The THz pulse from the first bunch is synchronized to the X-ray beam of the second bunch by using an adjustable optical delay of a THz pulse. The peak power of THz radiation from the multifoil radiator is estimated to be 0.14 GW for a 200 pC well-optimized electron bunch. GEANT4 simulations show that a carbon foil with a thickness of 0.5-1.0 mm has the highest yield of 10-20 keV hard X-rays for a 25 MeV beam, which is approximately 103 photons/(keV pC-electrons) within a few degrees of the polar angle. A carbon multifoil radiator with 35 foils (25 µm thick each) can generate close to 103 hard X-rays/(keV pC-electrons) within a 2° acceptance angle. With 200 pC charge and a 100 Hz repetition rate, we can generate 107 X-rays per 1 keV energy bin per second or 105 X-rays per 1 keV energy bin per pulse. The longitudinal time profile of an X-ray pulse ranges from 400 to 600 fs depending on the acceptance angle. The broadening of the time duration of an X-ray pulse is observed owing to its diverging effect. A double-crystal monochromator will be used to select and transport the desired X-rays to the sample. The heating of the radiators by an electron beam is negligible because of the low beam current.