Overexpression of PtVDL1 in Phaeodactylum tricornutum Increases Fucoxanthin Content under Red Light.

Phaeodactylum tricornutum is a model diatom with significant biotechnological applications, including enhancing biomass, biofuel, and carotenoid production. Specifically, owing to the capacity of this organism to serve as a valuable source of essential raw materials for pharmaceuticals and nutraceuticals, ongoing research is actively focused on enhancing its productivity. One of the genes involved in various stages of fucoxanthin (Fx) biosynthesis, violaxanthin de-epoxidase like 1 (VDL1), has recently been identified. To validate the intracellular function of this gene and boost Fx production through overexpression, we established and examined three transgenic P. tricornutum lines characterized by elevated P. tricortunum VDL1 ( PtVDL1) expression and evaluate their cell growth and Fx productivity. These transgenic lines exhibited substantially increased PtVDL1 mRNA and protein levels compared to the wild type (WT). Notably, the enzyme substrate violaxanthin was entirely depleted and could not be detected in the transformants, whereas it remained at constant levels in the WT. Interestingly, under standard white light conditions, Fx productivity in the transformants remained unchanged; however, but after 48 h of exposure to red light, it increased by up to 15%. These results indicate that PtVDL1-overexpressing P. tricornutum has industrial potential, particularly for enhancing Fx production under red light conditions.

FRET-based hACE2 receptor mimic peptide conjugated nanoprobe for simple detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a pandemic of acute respiratory disease, namely coronavirus disease (COVID-19). This disease threatens human health and public safety. Early diagnosis, isolation, and prevention are important to suppress the outbreak of COVID 19 given the lack of specific antiviral drugs to treat this disease and the emergence of various variants of the virus that cause breakthrough infections even after vaccine administration. Simple and prompt testing is paramount to preventing further spread of the virus. However, current testing methods, namely RT-PCR, is time-consuming. Binding of the SARS-CoV-2 spike (S) glycoprotein to human angiotensin-converting enzyme 2 (hACE2) receptor plays a pivotal role in host cell entry. In the present study, we developed a hACE2 mimic peptide beacon (COVID19-PEB) for simple detection of SARS-CoV-2 using a fluorescence resonance energy transfer system. COVID19-PEB exhibits minimal fluorescence in its ''closed'' hairpin structure; however, in the presence of SARS-CoV-2, the specific recognition of the S protein receptor-binding domain by COVID19-PEB causes the beacon to assume an ''open'' structure that emits strong fluorescence. COVID19-PEB can detect SARS-CoV-2 within 3 h or even 50 min and exhibits strong fluorescence even at low viral concentrations, with a detection limit of 4 × 103 plaque-forming unit/test. Furthermore, in SARS-CoV-2-infected patient samples confirmed using polymerase chain reaction, COVID19-PEB accurately detected the virus. COVID19-PEB could be developed as a rapid and accurate diagnostic tool for COVID-19.

Electrospun Nanofibrous Membrane-Based Colorimetric Device for Rapid and Simple Screening of Amphetamine-Type Stimulants in Drinks.

With the growth of drug-facilitated crimes, prevention has become increasingly important. Although various drug detection technologies exist, most focus on postconsumption detection. However, the prevention of drug-facilitated crimes requires technology for the quick and easy detection of amphetamine-type stimulants (ATSs) before ingestion. Herein, drug screening kits (DSKs) were developed for the simple detection of ATSs in drinks. The DSKs consisted of polydiacetylene nanofiber-based paper sensors fabricated by electrospinning with 10,12-pentacosadiynoic acid (PCDA) and PCDA-dopamine as sensing materials that can bind ATSs via hydrogen bonding and π-π interactions. Dropping a drink on the DSK provided an immediate visual indication of the presence of ATSs. When ATSs were present in the drink, the color of the DSK clearly changed from blue to red, with the increase in red intensity being more than twofold greater than that observed when water alone was tested. Notably, the result could be confirmed by the naked eye without any analytical instrumentation. A color change indicating the presence of ATSs was successfully observed in various alcoholic and nonalcoholic drinks. These results indicate the potential of DSKs for preventing drug-facilitated crimes caused by unwanted drug intake.

Computational and Histological Analyses for Investigating Mechanical Interaction of Thermally Drawn Fiber Implants with Brain Tissue.

The development of a compliant neural probe is necessary to achieve chronic implantation with minimal signal loss. Although fiber-based neural probes fabricated by the thermal drawing process have been proposed as a solution, their long-term effect on the brain has not been thoroughly investigated. Here, we examined the mechanical interaction of thermally drawn fiber implants with neural tissue through computational and histological analyses. Specifically, finite element analysis and immunohistochemistry were conducted to evaluate the biocompatibility of various fiber implants made with different base materials (steel, silica, polycarbonate, and hydrogel). Moreover, the effects of the coefficient of friction and geometric factors including aspect ratio and the shape of the cross-section on the strain were investigated with the finite element model. As a result, we observed that the fiber implants fabricated with extremely softer material such as hydrogel exhibited significantly lower strain distribution and elicited a reduced immune response. In addition, the implants with higher coefficient of friction (COF) and/or circular cross-sections showed a lower strain distribution and smaller critical volume. This work suggests the materials and design factors that need to be carefully considered to develop future fiber-based neural probes to minimize mechanical invasiveness.

Enhanced pyruvate metabolism in plastids by overexpression of putative plastidial pyruvate transporter in Phaeodactylum tricornutum.

BACKGROUND: The development of microalgal strains for enhanced biomass and biofuel production has received increased attention. Moreover, strain development via metabolic engineering for commercial production is being considered as the most efficient strategy. Pyruvate is an essential metabolite in the cells and plays an essential role in amino acid biosynthesis and de novo fatty acid biosynthesis in plastids. Although pyruvate can be a valuable target for metabolic engineering, its transporters have rarely been studied in microalgae. In this study, we aimed to identify the plastidial pyruvate transporter of Phaeodactylum tricornutum and utilize it for strain development. RESULTS: We identified putative pyruvate transporter localized in the plastid membrane of Phaeodactylum tricornutum. Transformants overexpressing the pyruvate transporter were generated to increase the influx of pyruvate into plastids. Overexpression of a plastidial pyruvate transporter in P. tricornutum resulted in enhanced biomass (13.6% to 21.9%), lipid contents (11% to 30%), and growth (3.3% to 8.0%) compared to those of wild type during one-stage cultivation. CONCLUSION: To regulate the pyruvate influx and its metabolism in plastids, we generated transformants overexpressing the putative plastidial pyruvate transporter in P. tricornutum. They showed that its overexpression for compartmentalizing pyruvate in plastids could be an attractive strategy for the effective production of biomass and lipids with better growth, via enhanced pyruvate metabolism in plastids.

Using Phytochemicals to Investigate the Activation of Nicotine Detoxification via Upregulation of CYP2A6 in Animal Models Exposed Tobacco Smoke Condensate by Intratracheal Instillation.

This study examined the efficacy of standardized Smilax china L. root extract (SSCR) containing chlorogenic acid on detoxifying nicotine from tobacco smoke condensate (TSC) in vitro and in vivo. Chlorogenic acid is an identified bioactive component in SSCR by ultraperformance liquid chromatography/photodiode array/electrospray ionization/mass spectroscopy (UPLC/PDA/ESI/MS). HepG2 liver cells and A549 lung cells were carried for measuring ROS and antioxidant enzymes. Sprague-Dawley rats were treated with nicotine by intratracheal instillation (ITI). Cell viabilities by pretreatments of 5, 12.5, and 25, 50 µg SSCR/mL ranged from 41 to 76% in HepG2 and 65 to 95% in A549. Pretreatments of SSCR inhibited TSC-mediated production of reactive oxygen species (ROS) by 8 and 10% in HepG2 and A549 cells, respectively. However, the expression of CAT, SOD1, and AOX1 was downregulated by SSCR in the both cells. The highest conversion of cotinine was observed at 50 µg/mL of SSCR after 120 min of incubation. SSCR upregulated CYP2A6 3-fold in A549 cells regardless of TSC cotreatment. When Sprague-Dawley rats were treated with nicotine by ITI or subjected to SSCR administration for 14 days, the levels of cotinine in urine increased in SSCR treatment only. The cellular level of antioxidant capacity at 10 or 100 mg/kg body weight/day of SSCR treatment was 1.89 and 1.86 times higher than those of nicotine-control. Results suggest that the intake of SSCR can detoxify nicotine by elevating nicotine conversion to cotinine and antioxidant capacity.

The effect of tobacco smoke exposure on the generation of reactive oxygen species and cellular membrane damage using co-culture model of blood brain barrier with astrocytes.

Brain tissue is known to be vulnerable to the exposure by tobacco smoke. Tobacco smoke can induce generation of reactive oxygen species (ROS), causing inflammatory activity and blood-brain barrier (BBB) impairment. The aim of the present study was to investigate the effect of tobacco smoke on cell cytotoxicity, generation of ROS, and cellular membrane damage in astrocytes and BBB using a co-culture system. Cell viability of U373MG cells was reduced in a dose-dependent manner, ranging from 96.7% to 40.3% by tobacco smoke condensate (TSC). Cell viability of U373MG co-cultured with human brain microvascular endothelial cells (HBMECs) was 104.9% at the IC50 value of TSC. Trans-epithelial electric resistance values drastically decreased 80% following 12-h incubation. The value was maintained until 48 h and then increased at 72-h incubation (85%). It then decreased to 75% at 120 h. Generation of ROS increased in a dose-dependent manner, ranging from 102.7% to 107.9%, when various concentrations of TSC (4-16 mg/mL) were administered to the U373MG monoculture. When TSC was added into U373MG co-cultured with HBMECs, production of ROS ranged from 101.7% to 102.6%, slightly increasing over 12 h. Maximum exposure-generated ROS of 104.8% was reached at 24 h. Cell cytotoxicity and oxidative stress levels in the U373MG co-culture model system with HBMECs were lower than U373MG monoculture. HBMECs effectively acted as a barrier to protect the astrocytes (U373MG) from toxicity of TSC.

Improvement of the phosphoenolpyruvate carboxylase activity of Phaeodactylum tricornutum PEPCase 1 through protein engineering.

In order to mitigate CO2 accumulation and decrease the rate of global warming and climate change, we previously presented a strategy for the development of an efficient CO2 capture and utilization system. The system employs two recombinant enzymes, carbonic anhydrase and phosphoenolpyruvate carboxylase, which were originated from microalgae. Although utilization of this integrated system would require a large quantity of high quality PEPCase protein, such quantities could be produced by increasing the solubility of the Phaeodactylum tricornutum PEPCase 1 (PtPEPCase 1) protein in the Escherichia coli heterologous expression system. We first expressed the putative mitochondria targeting peptide- and chloroplast transit peptide-truncated proteins of PtPEPCase 1, mPtPEPCase 1 and cPtPEPCase 1, respectively, in E. coli. After affinity chromatography, the amount of purified PEPCase protein from 500mL of E. coli culture was greatest for cPtPEPCase 1 (1.99mg), followed by mPtPEPCase 1 (0.82mg) and PtPEPCase 1 (0.61mg). Furthermore, the enzymatic activity of mPtPEPCase 1 and cPtPEPCase 1 showed approximately 1.6-fold (32.19 units/mg) and 3-fold (59.48 units/mg) increases, respectively. Therefore, cPtPEPCase 1 purified using the E. coli heterogeneous expression system could be a strong candidate for a platform technology to capture CO2 and produce value-added four-carbon platform chemicals.