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.

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.

Lyso-globotriaosylsphingosine induces endothelial dysfunction via autophagy-dependent regulation of necroptosis.

Fabry disease is a lysosomal storage disorder characterized by the lysosomal accumulations of glycosphingolipids in a variety of cytotypes, which include endothelial cells. The disease is inherited and originates from an error in glycosphingolipid catabolism caused by insufficient α-galactosidase A activity, which causes uncontrolled progressive storage of intracellular globotriaosylceramide (Gb3) in the vasculature and extracellular accumulation of lyso-Gb3 (a deacetylated soluble form of Gb3). Necrosis can lead to inflammation, which exacerbates necrosis and creates a positive feedback loop that triggers necroinflammation. However, the role played by necroptosis, a form of programmed necrotic cell death, in the cell-to-cell inflammatory reaction between epithelial and endothelial cells is unclear. Thus, the present study was undertaken to determine whether lyso-Gb3 induces necroptosis and whether necroptosis inhibition protects endothelial dysfunction against lyso-Gb3 inflamed retinal pigment epithelial cells. We found lyso-Gb3 induced necroptosis of a retinal pigment epithelial cell line (ARPE-19) in an autophagy-dependent manner and that conditioned media (CM) from ARPE-19 cells treated with lyso-Gb3 induced the necroptosis, inflammation, and senescence of human umbilical vein endothelial cells. In addition, a pharmacological study showed CM from lyso-Gb3 treated ARPE-19 cells induced endothelial necroptosis, inflammation, and senescence were significantly inhibited by an autophagy inhibitor (3-MA) and by two necroptosis inhibitors (necrostatin and GSK-872), respectively. These results demonstrate lyso-Gb3 induces necroptosis via autophagy and suggest that lyso-Gb3 inflamed retinal pigment epithelial cells trigger endothelial dysfunction via the autophagy-dependent necroptosis pathway. This study suggests the involvement of a novel autophagy-dependent necroptosis pathway in the regulation of endothelial dysfunction in Fabry disease.

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.

Fluvoxamine Treatment of Patients with Symptomatic COVID-19 in a Community Treatment Center: A Preliminary Result of Randomized Controlled Trial.

BACKGROUND: This study aimed to evaluate whether fluvoxamine reduces clinical deterioration in adult patients with mild to moderate coronavirus disease 2019 (COVID-19), and to identify risk factors for clinical deterioration in patients admitted to a community treatment center (CTC). MATERIALS AND METHODS: A randomized, placebo-controlled trial was conducted in a CTC, in Seoul, Korea from January 15, 2021, to February 19, 2021. Symptomatic adult patients with positive results of severe acute respiratory syndrome coronavirus 2 real time-polymerase chain reaction within 3 days of randomization were assigned at random to receive 100 mg of fluvoxamine or placebo twice daily for 10 days. The primary outcome was clinical deterioration defined by any of the following criteria: oxygen requirement to keep oxygen saturation over 94.0%, aggravation of pneumonia with dyspnea, or World Health Organization clinical progression scale 4 or greater. RESULTS: Of 52 randomized participants [median (interquartile range) age, 53.5 (43.3 - 60.0) years; 31 (60.0%) men], 44 (85.0%) completed the trial. Clinical deterioration occurred in 2 of 26 patients in each group (P >0.99). There were no serious adverse events in either group. Clinical deterioration occurred in 15 (6.0%) of 271 patients admitted to the CTC, and all of them were transferred to a hospital. In multivariate analysis, age between 55 and 64, fever and pneumonia at admission were independent risk factors for clinical deterioration. CONCLUSION: In this study of adult patients with symptomatic COVID-19 who were admitted to the CTC, there was no significant differences in clinical deterioration between patients treated with fluvoxamine and placebo (ClinicalTrials.gov Identifier: NCT04711863).

Higher Branched-Chain Amino Acid Intake Is Associated with Handgrip Strength among Korean Older Adults.

Sarcopenia is a disease of old age characterized by decreased muscle mass and strength. Branched-chain amino acids (BCAAs) promote muscle mass synthesis and increase muscle strength. We aimed to develop a dietary amino acid database and to examine the association between BCAA intake and handgrip strength in Korean older adults. Data from the Korea National Health and Nutrition Examination Survey 2014-2018 were used. Overall, 4852 participants aged ≥65 years were included in the study. Demographic, lifestyle, and health data were obtained through interviews and questionnaires. The 24-h recall method was used to assess dietary intake. An amino acid database was established using the 9th revision of the Korean Standard Food Composition Table. The mean handgrip strength was estimated from triplicate measurements obtained using the dominant hand. Multivariable linear regression analysis was performed to assess the association between BCAA intake and handgrip strength. Grains greatly contributed to amino acid intake; however, a significant negative association was observed between handgrip strength and increased BCAA intake through grains. In the fully adjusted model, leucine intake and handgrip strength showed a positive association. Thus, consuming BCAAs (especially leucine) via a variety of food sources can help maintain muscle health in older people.

Association between the level of adherence to dietary guidelines and depression among Korean patients with type 2 diabetes mellitus.

OBJECTIVE: This study aimed to examine the cross-sectional association between adherence to dietary guidelines for type 2 diabetes mellitus (T2DM) and depression. METHODS: A total of 3457 patients with T2DM were included. Dietary information was obtained using 24-h recall, and adherence to dietary guidelines was estimated using the Korean Diabetes Association-Korean Ministry of Health and Welfare index. Depression was evaluated using a questionnaire. Participants who had responded "yes" to any of the three questions related to diagnosis, current condition, and treatment were defined as depression. Multivariable logistic regression analysis was used to examine the association between adherence to dietary guidelines and depression. RESULTS: T2DM patients with a lower score on adherence to dietary guidelines were more likely to report depression than those with a higher score (Odds ratios (OR): 0.51, 95% Confidence Interval (CI): 0.30-0.87). Patients with poor adherence to moderate calorie consumption (OR: 1.65, 95% CI: 1.07-2.52) and regular meal patterns (OR: 1.69, 95% CI: 1.15-2.50) were more likely to report depression. However, patients with poor adherence to low sodium intake were less likely to report depression (OR: 0.60, 95% CI: 0.40-0.88), and no association was observed between moderate carbohydrate consumption, sufficient vegetable/seaweed consumption, and moderate alcohol consumption and depression. CONCLUSION: Good adherence to dietary guidelines was closely associated with a lower prevalence of depression among individuals with type 2 diabetes, but the specific guidelines were inconsistent. Systematic and continuous nutrition education for individuals with T2DM is necessary to improve their mental health outcomes.

Effects of Cudrania tricuspidata on anti-senescence in high glucose-treated endothelial cells via the Akt/p53/p21 pathway.

The roles of Cudrania tricuspidata (CT) in the prevention of senescence and the underlying mechanisms have not been elucidated. In a high glucose (HG)-induced senescent endothelial cell (EC) culture, CT (20 µg/ml) reduced the number of senescence-associated ß-galactosidase-positive cells by 8.3% compared with the control group and increased the expression of p-Sirt1 by more than twofold compared with the control group. Moreover, 20 µg/ml CT treatment doubled the activity of p-Akt, which was inhibited by HG, compared with the control group. In addition, CT treatment decreased the expression of p53, p21, and Rb, which was increased by HG. Overall, CT delays HG-induced senescence via the Akt/p53/p21 pathway, suggesting its potential as a functional agent for the protection of ECs.

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.

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.

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.

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.

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.

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.

Long pentraxin PTX3 mediates acute inflammatory responses against pneumococcal infection.

Streptococcus pneumoniae is an important human pathogen responsible for more than 2 million deaths annually worldwide. The airway epithelium acts as the first-line of defense against pneumococcal infections by regulating acute inflammation against invading pneumococcus. Despite the intact adaptive immunity, failure in early defense due to loss of pattern recognition receptors (PRRs) and/or acute phase proteins (APPs) results in detrimental damage and death. C-reactive protein (CRP), the first found APP, is a member of the pentraxin family of proteins and an important soluble PRR for pneumococcus. CRP and another short pentraxin, serum amyloid P, are critical for acute defense against pneumococcal infection. However, the role of the long pentraxin PTX3 in regulating pneumococcal infections is unknown. In this study, PTX3 expression was upregulated by pneumococcus in epithelial cells and in lungs of mice. In addition, PTX3 potentiated pneumococcal inflammation; overexpression of PTX3 enhanced pneumococcus-induced cytokine expression, whereas knock-down of PTX3 with siPTX3 inhibited the cytokine expression. Furthermore, PTX3 deficiency indeed ameliorated acute inflammation and protected mice against death following pneumococcal infection. Pneumococcal toxin pneumolysin was responsible for PTX3 expression and upregulated PTX3 expression via JNK MAPK signaling. These data implicate PTX3 as a novel therapeutic target for the control of acute inflammation by pneumococcus.

Simulating Intestinal Transporter and Enzyme Activity in a Physiologically Based Pharmacokinetic Model for Tenofovir Disoproxil Fumarate.

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0-24; 3,045 ng · h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0-24 (3,036 ng · h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies.

Preclinical Pharmacokinetics and Pharmacodynamic Target of SCY-078, a First-in-Class Orally Active Antifungal Glucan Synthesis Inhibitor, in Murine Models of Disseminated Candidiasis.

SCY-078 (MK-3118) is a novel, semisynthetic derivative of enfumafungin and represents the first compound of the triterpene class of antifungals. SCY-078 exhibits potent inhibition of ß-(1,3)-d-glucan synthesis, an essential cell wall component of many pathogenic fungi, including Candida spp. and Aspergillus spp. SCY-078 is currently in phase 2 clinical development for the treatment of invasive fungal diseases. In vitro disposition studies to assess solubility, intestinal permeability, and metabolic stability were predictive of good oral bioavailability. Preclinical pharmacokinetic studies were consistent with once-daily administration to humans. After intravenous delivery, plasma clearance in rodents and dogs was low, representing <15% and <25% of hepatic blood flow, respectively. The terminal elimination-phase half-life was 5.5 to 8.7 h in rodents, and it was ∼9.3 h in dogs. The volume of distribution at steady-state was high (4.7 to 5.3 liters/kg), a finding suggestive of extensive tissue distribution. Exposure of SCY-078 in kidney tissue, a target organ for invasive fungal disease such as candidiasis, exceeded plasma by 20- to 25-fold for the area under the concentration-time curve from 0 h to infinity (AUC0-∞) and Cmax SCY-078 achieved efficacy endpoints following oral delivery across multiple murine models of disseminated candidiasis. The pharmacokinetic/pharmacodynamic indices Cmax/MIC and AUC/MIC correlated with outcome. Target therapeutic exposure, expressed as the plasma AUC0-24, was comparable across models, with an upper value of 11.2 µg·h/ml (15.4 µM·h); the corresponding mean value for free drug AUC/MIC was ∼0.75. Overall, these results demonstrate that SCY-078 has the oral and intravenous (i.v.) pharmacokinetic properties and potency in murine infection models of disseminated candidiasis to support further investigation as a novel i.v. and oral treatment for invasive fungal diseases.

Development of a Novel Formulation That Improves Preclinical Bioavailability of Tenofovir Disoproxil Fumarate.

Tenofovir disoproxil fumarate (TDF), the bisphosphonate ester prodrug of tenofovir (TFV), has poor bioavailability due to intestinal degradation and efflux transport. Reformulation using U.S. Food and Drug Administration-approved esterase and efflux inhibitors to increase oral bioavailability could provide lower dose alternatives and reduce costs for patients with HIV in resource-limited settings. Inhibition of mucosal and intracellular esterases was studied in human and rat intestinal extracts (S9), where TDF was protected by the carboxylesterase inhibitor bis-para-nitrophenylphosphate, the ester mix EM1, and the generally recognized-as-safe (GRAS) excipient propylparaben. Permeability studies using Madin-Darby canine kidney and Caco-2 cell monolayers demonstrated that TDF was a substrate for the permeability glycoprotein with permeability glycoprotein inhibitors reducing basolateral to apical transport of TDF. These studies also showed that transport was increased by esterase inhibitors. TDF, TFV, and tenofovir monophosphonate ester transport across Caco-2 monolayers with esterase and efflux inhibitors revealed a maximum 38.7-fold increase in apical to basolateral TDF transport with the potent non-GRAS combination of EM1 and GF120918. Transport was increased 22.8-fold by the GRAS excipients, propylparaben, and d-a-tocopheryl polyethylene glycol 1000 succinate (a vitamin E derivative). TFV pharmacokinetics in rats following oral administration of TDF and GRAS esterase and efflux inhibitors confirmed enhanced bioavailability. Area under the curve increased 1.5- to 2.1-fold with various combinations of parabens and d-a-tocopheryl polyethylene glycol 1000 succinate. This significant inhibition of TDF hydrolysis and efflux in vivo exhibits the potential to safely increase TDF bioavailability in humans.

The TRPCs, Orais and STIMs in ER/PM Junctions.

The Ca(2+) second messenger is initiated at ER/PM junctions and propagates into the cell interior to convey the receptor information. The signal is maintained by Ca(2+) influx across the plasma membrane through the Orai and TRPC channels. These Ca(2+) influx channels form complexes at ER/PM junctions with the ER Ca(2+) sensor STIM1, which activates the channels. The function of STIM1 is modulated by other STIM isoforms like STIM1L, STIM2 and STIM2.1/STIM2ß and by SARAF, which mediates the Ca(2+)-dependent inhibition of Orai channels. The ER/PM junctions are formed at membrane contact sites by tethering proteins that generate several types of ER/PM junctions, such as PI(4,5)P2-poor and PI(4,5)P2-rich domains. This chapter discusses several properties of the TRPC channels, the Orai channels and the STIMs, their key interacting proteins and how interaction of the STIMs with the channels gates their activity. The chapter closes by highlighting open questions and potential future directions in this field.

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.