Autophagy protects against palmitate-induced apoptosis in hepatocytes

BACKGROUND: Non-alcoholic fatty liver disease, one of the most common liver diseases, has obtained increasing attention. Palmitate (PA)-induced liver injury is considered a risk factor for the development of non-alcoholic fatty liver disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism in response to various stresses. In this study, we investigated whether autophagy plays a protective role in the progression of PA-induced hepatocytes injury. RESULTS: Annexin V-FITC/PI staining by FCM analysis, TUNEL assay and the detection of PARP and cleaved caspase3 expression levels demonstrated that PA treatment prominently induced the apoptosis of hepatocytes. Meanwhile, treatment of PA strongly induced the formation of GFP-LC3 dots, the conversion from LC3I to LC3II, the decrease of p62 protein levels and the increase of autophagosomes. These results indicated that PA also induced autophagy activation. Autophagy inhibition through chloroquine pretreatment or Atg5shRNA infection led to the increase of cell apoptosis after PA treatment. Moreover, induction of autophagy by pretreatment with rapamycin resulted in distinct decrease of PA-induced apoptosis. Therefore, autophagy can prevent hepatocytes from PA-induced apoptosis. In the further study, we explored pathway of autophagy activation in PA-treated hepatocytes. We found that PA activated PKCα in hepatocytes, and had no influence on mammalian target of rapamycin and endoplasmic reticulum stress pathways. CONCLUSIONS: These results demonstrated that autophagy plays a protective role in PA-induced hepatocytes apoptosis. And PA might induce autophagy through activating PKCα pathway in hepatocytes

3D printing of polymeric optical components by two-photon polymerization

Femtosecond laser direct writing achieves high precision into micro-metric in additive manufacturing of customized-geometric structures and was used to fabricate the high quality micro optical lenses in various photoresist, including SU-8, by two-photon polymerization. The fabricates axicons could approximately create the Bessel beam, which has smaller beam diameter and extended depth of focus, so that widely employed in imaging systems. The axicons and any geometric micro-lenses of certain applications were designed and optimized using ZEMAX before the lenses being fabricated by femtosecond laser direct writing, and they serve as core components of imaging systems, microscopy, and other research fields.Published versio

Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale

Abstract Background Methanol, synthesized from CO2, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production. Results In the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine). Conclusions This study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits

Additional file 1 of Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale

Supplementary Material

Self-reconstructing Bessel beam created by two-photon-polymerized micro-axicon for light-sheet fluorescence microscopy

Observing micro-organisms with depth-resolving capability is important in optical microscopy for biomedical sciences and industries. We demonstrate the fabrication and use of a two-photon polymerized micro-axicon lens that generates a self-reconstructing pencil-like Bessel beam for light-sheet fluorescence microscopy (LSFM), providing 3D internal structures of micro-organisms. The fabricated SU-8 micro-axicon of 100 µm diameter transforms the input Gaussian beam from a single-mode fiber into a non-diffractive Bessel-Gauss beam. The focused spot size of the Bessel-Gauss beam is 2.3 ± 0.25 µm with a long propagation distance over 160 µm, which is well-suited for LSFM. The self-reconstruction capability of the generated Bessel-Gauss beam is investigated thoroughly through both simulations and experiments. Since this micro-axicon can be directly 3D-printed on single-mode fibers’ end facets or small mobile substrates, this can replace the bulky objective lens from conventional light-sheet microscopes. This will facilitate the wide-spread use of 3D tomographic imaging of micro-organisms, especially in compact micro-fluidic devices and lab-on-a-chip architectures.National Research Foundation (NRF)Published versionNational Research Foundation of the Republic of Korea (NRF2012R1A3A1050386); Research collaboration agreement by Panasonic Factory Solutions Asia Pacific (PFSAP) and Singapore Centre for 3D Printing (RCA-15/027); Korea Forest Service (Korea Forestry Promotion Institute) through the R&D Program for Forest Science Technology (2020229C10-2022-AC01); Basic Research Program (NK224C) funded by the Korea Institute of Machinery and Materials

Surface third-harmonic generation at a two-photon-polymerized micro-interferometer for real-time on-chip refractive index monitoring

A micro-interferometer based on surface third-harmonic generation (THG) at twophoton- polymerized SU-8 cuboids for real-time monitoring of the refractive index changes of target fluids, which can be easily integrated into microfluidic photonic systems, is demonstrated. The third-harmonic (TH) interferogram is selectively generated only from the target volume by a simple vertical pumping, thereby eliminating the needs for complicated coupling and alignments. The dependence of the generated TH to the input pump polarization state is thoroughly investigated. The THG efficiency by linearly polarized excitation is found to be 2.6 × 10−7, which is the most efficient at the SU-8-air interface and independent of the input polarization direction. The THG efficiency from the SU-8-air interface is 12.17 times higher than that from the glass-air interface and 4.93 times higher than that from the SU-8- glass interface. Real-time monitoring of argon gas pressure is demonstrated using the microinterferometer. The surface TH from two-photon-polymerized 3D structures offers novel design flexibility to the nonlinear optical light sources for microfluidic and microelectronic devices.NRF (Natl Research Foundation, S’pore)Published versio

Injection-seeded high-repetition-rate short-pulse micro-laser based on upconversion nanoparticles

We demonstrate a high repetition-rate upconversion green pulsed micro-laser, which is prepared by the fast thermal quenching of lanthanide-doped upconversion nanoparticles (UCNPs) via femtosecond-laser direct writing. The outer rim of the prepared upconversion hemi-ellipsoidal microstructure works as a whispering-gallery-mode (WGM) optical resonator for the coherent photon build-up of third-harmonic ultra-short seed pulses. When near-infrared (NIR) femtosecond laser pulses of wavelength 1545 nm are focused onto the upconversion WGM resonator, the optical third-harmonic is generated at 515 nm together with the upconversion luminescence. The weak third-harmonic (TH) seed pulses are coherently amplified in the hemi-ellipsoidal upconversion resonator as a result of the resonant interaction between the incident femtosecond laser field, the TH, the upconversion luminescence and the WGM. This upconversion lasing preserves the original repetition rate of the NIR pump laser and the output polarization state is also coherently aligned to the pump laser polarization. Because of the isotropic nature of the upconversion micro-ellipsoids, the upconversion lasing shows maximum intensity with a linearly polarized pump beam and minimum intensity with a circularly polarized pump beam. Our scheme devised for realizing high-repetition-rate lasing at higher photon energies in a compact micro platform will open up new ways for on-chip optical information processing, high-throughput microfluidic sensing, and localized micro light sources for optical memories.Accepted versionThe authors acknowledge funding support received from Panasonic Factory Solutions Asia Pacific (PFSAP) and Singapore Centre for 3D Printing (RCA-15/027), National Research Foundation of the Republic of Korea (NRF-2012R1A3A1050386, NRF-2018R1A4A1025623), Korea Forest Service (Korea Forestry Promotion Institute) through the R&D Program for Forest Science Technology (2020229C10-2022AC01), and Basic Research Program (NK224C) funded by the Korea Institute of Machinery and Materials