Direct Imaging of Integrated Circuits in CPU with 60 nm Super-Resolution Optical Microscope.

Far-field super-resolution optical microscopies have achieved incredible success in life science for visualization of vital nanostructures organized in single cells. However, such resolution power has been much less extended to material science for inspection of human-made ultrafine nanostructures, simply because the current super-resolution optical microscopies modalities are rarely applicable to nonfluorescent samples or unlabeled systems. Here, we report an antiphase demodulation pump-probe (DPP) super-resolution microscope for direct optical inspection of integrated circuits (ICs) with a lateral resolution down to 60 nm. Because of the strong pump-probe (PP) signal from copper, we performed label-free super-resolution imaging of multilayered copper interconnects on a small central processing unit (CPU) chip. The label-free super-resolution DPP optical microscopy opens possibilities for easy, fast, and large-scale electronic inspection in the whole pipeline chain for designing and manufacturing ICs.

Genetic and functional characterization of the hyaluronate lyase HylB and the beta-N-acetylglucosaminidase HylZ in Streptococcus zooepidemicus.

Hyaluronic acid (hyaluronan) is a high molecular weight glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine. The molecular weight of hyaluronan is important for its rheological property, biological function, and application. However, genes important for regulation of hyaluronan production or molecular weight remain poorly understood. Here, we address the roles of two predicated hyaluronate lyase-encoding genes, hylB and hylZ in Streptococcus zooepidemicus. Semi-quantitative RT-PCR assays showed that hylZ was constitutively expressed while the expression level of hylB was growth-phase dependent. Using recombinantly expressed 6His-HylB and -HylZ protein, enzyme assays revealed that HylB was a hyaluronate lyase, and its K(m) and V(max) were 0.57 µM and 1.43 mM min(-1), respectively. 6His-HylZ showed no activity against hyaluronan while it hydrolyzed pNp-ß-GlcNAc suggesting that HylZ was a beta-N-acetylglucosaminidase. Under the optimal conditions (pH 6.0 and 50 °C), the K(m) and V(max) for 6His-HylZ to degrade pNp-ß-GlcNA were 1.16 mM and 26.18 µmol min(-1) mg(-1), respectively. Characterization of ΔhylB and ΔhylZ demonstrated that loss of hylB or/and hylZ had no significant effects on cell growth, lactic acid, and hyaluronan yields. Significantly, as compared to the wild type, ΔhylB produced hyaluronan with an 18 % increase in molecular weight. Our results strongly suggest that hylB encodes a hyaluronate lyase while hylZ encodes a ß-N-acetylglucosaminidase. hylB-deficient strain has the potential to produce high molecular weight hyaluronan.

Bioinspired integrated triboelectric electronic tongue.

An electronic tongue (E-tongue) comprises a series of sensors that simulate human perception of taste and embedded artificial intelligence (AI) for data analysis and recognition. Traditional E-tongues based on electrochemical methods suffer from a bulky size and require larger sample volumes and extra power sources, limiting their applications in in vivo medical diagnosis and analytical chemistry. Inspired by the mechanics of the human tongue, triboelectric components have been incorporated into E-tongue platforms to overcome these limitations. In this study, an integrated multichannel triboelectric bioinspired E-tongue (TBIET) device was developed on a single glass slide chip to improve the device's taste classification accuracy by utilizing numerous sensory signals. The detection capability of the TBIET was further validated using various test samples, including representative human body, environmental, and beverage samples. The TBIET achieved a remarkably high classification accuracy. For instance, chemical solutions showed 100% identification accuracy, environmental samples reached 98.3% accuracy, and four typical teas demonstrated 97.0% accuracy. Additionally, the classification accuracy of NaCl solutions with five different concentrations reached 96.9%. The innovative TBIET exhibits a remarkable capacity to detect and analyze droplets with ultrahigh sensitivity to their electrical properties. Moreover, it offers a high degree of reliability in accurately detecting and analyzing various liquid samples within a short timeframe. The development of a self-powered portable triboelectric E-tongue prototype is a notable advancement in the field and is one that can greatly enhance the feasibility of rapid on-site detection of liquid samples in various settings.

AI-Enabled Portable E-Nose Regression Predicting Harmful Molecules in a Gas Mixture.

The biomimetic electronic nose (e-nose) technology is a novel technology used for the identification and monitoring of complex gas molecules, and it is gaining significance in this field. However, due to the complexity and multiplicity of gas mixtures, the accuracy of electronic noses in predicting gas concentrations using traditional regression algorithms is not ideal. This paper presents a solution to the difficulty by introducing a fusion network model that utilizes a transformer-based multikernel feature fusion (TMKFF) module combined with a 1DCNN_LSTM network to enhance the accuracy of regression prediction for gas mixture concentrations using a portable electronic nose. The experimental findings demonstrate that the regression prediction performance of the fusion network is significantly superior to that of single models such as convolutional neural network (CNN) and long short-term memory (LSTM). The present study demonstrates the efficacy of our fusion network model in accurately predicting the concentrations of multiple target gases, such as SO2, NO2, and CO, in a gas mixture. Specifically, our algorithm exhibits substantial benefits in enhancing the prediction performance of low-concentration SO2 gas, which is a noteworthy achievement. The determination coefficient (R2) values of 93, 98, and 99% correspondingly demonstrate that the model is very capable of explaining the variation in the concentration of the target gases. The root-mean-square errors (RMSE) are 0.0760, 0.0711, and 3.3825, respectively, while the mean absolute errors (MAE) are 0.0507, 0.0549, and 2.5874, respectively. These results indicate that the model has relatively small prediction errors. The method we have developed holds significant potential for practical applications in detecting atmospheric pollution detection and other molecular detection areas in complex environments.

Elucidation of sterol biosynthesis pathway and its co-regulation with fatty acid biosynthesis in the oleaginous marine protist Schizochytrium sp.

Sterols constitute vital structural and regulatory components of eukaryotic cells. In the oleaginous microorganism Schizochytrium sp. S31, the sterol biosynthetic pathway primarily produces cholesterol, stigmasterol, lanosterol, and cycloartenol. However, the sterol biosynthesis pathway and its functional roles in Schizochytrium remain unidentified. Through Schizochytrium genomic data mining and a chemical biology approach, we first in silico elucidated the mevalonate and sterol biosynthesis pathways of Schizochytrium. The results showed that owing to the lack of plastids in Schizochytrium, it is likely to use the mevalonate pathway as the terpenoid backbone pathway to supply isopentenyl diphosphate for the synthesis of sterols, similar to that in fungi and animals. In addition, our analysis revealed a chimeric organization of the Schizochytrium sterol biosynthesis pathway, which possesses features of both algae and animal pathways. Temporal tracking of sterol profiles reveals that sterols play important roles in Schizochytrium growth, carotenoid synthesis, and fatty acid synthesis. Furthermore, the dynamics of fatty acid and transcription levels of genes involved in fatty acid upon chemical inhibitor-induced sterol inhibition reveal possible co-regulation of sterol synthesis and fatty acid synthesis, as the inhibition of sterol synthesis could promote the accumulation of fatty acid in Schizochytrium. Sterol and carotenoid metabolisms are also found possibly co-regulated, as the inhibition of sterols led to decreased carotenoid synthesis through down-regulating the gene HMGR and crtIBY in Schizochytrium. Together, elucidation of the Schizochytrium sterol biosynthesis pathway and its co-regulation with fatty acid synthesis lay the essential foundation for engineering Schizochytrium for the sustainable production of lipids and high-value chemicals.

Rewiring the Metabolic Network to Increase Docosahexaenoic Acid Productivity in Crypthecodinium cohnii by Fermentation Supernatant-Based Adaptive Laboratory Evolution.

Docosahexaenoic acid (DHA, 22:6n-3) plays significant roles in enhancing human health and preventing human diseases. The heterotrophic marine dinoflagellate Crypthecodinium cohnii is a good candidate to produce high-quality DHA. To overcome the inhibition caused by the fermentation supernatant in the late fermentation stage of DHA-producing C. cohnii, fermentation supernatant-based adaptive laboratory evolution (FS-ALE) was conducted. The cell growth and DHA productivity of the evolved strain (FS280) obtained after 280 adaptive cycles corresponding to 840 days of evolution were increased by 161.87 and 311.23%, respectively, at 72 h under stress conditions and increased by 19.87 and 51.79% without any stress compared with the starting strain, demonstrating the effectiveness of FS-ALE. In addition, a comparative proteomic analysis identified 11,106 proteins and 910 differentially expressed proteins, including six stress-responsive proteins, as well as the up- and downregulated pathways in FS280 that might contribute to its improved cell growth and DHA accumulation. Our study demonstrated that FS-ALE could be a valuable solution to relieve the inhibition of the fermentation supernatant at the late stage of normal fermentation of heterotrophic microalgae.

Metabolic Analysis of Schizochytrium Mutants With High DHA Content Achieved With ARTP Mutagenesis Combined With Iodoacetic Acid and Dehydroepiandrosterone Screening.

High DHA production cost caused by low DHA titer and productivity of the current Schizochytrium strains is a bottleneck for its application in competition with traditional fish-oil based approach. In this study, atmospheric and room-temperature plasma with iodoacetic acid and dehydroepiandrosterone screening led to three mutants, 6-8, 6-16 and 6-23 all with increased growth and DHA accumulations. A LC/MS metabolomic analysis revealed the increased metabolism in PPP and EMP as well as the decreased TCA cycle might be relevant to the increased growth and DHA biosynthesis in the mutants. Finally, the mutant 6-23, which achieved the highest growth and DHA accumulation among all mutants, was evaluated in a 5 L fermentor. The results showed that the DHA concentration and productivity in mutant 6-23 were 41.4 g/L and 430.7 mg/L/h in fermentation for 96 h, respectively, which is the highest reported so far in literature. The study provides a novel strain improvement strategy for DHA-producing Schizochytrium.

Imaging Chemical Kinetics of Radical Polymerization with an Ultrafast Coherent Raman Microscope.

Numerous mechanisms have been proposed for polymerization to provide qualitative and quantitative prediction of how monomers spatially and temporally arrange into the polymeric chains. However, less is known about this process at the molecular level because the ultrafast chemical reaction is inaccessible for any form of microscope so far. Here, to address this unmet challenge, a stimulated Raman scattering microscope based on collinear multiple beams (COMB-SRS) is demonstrated, which allows label-free molecular imaging of polymer synthesis in action at speed of 2000 frames per second. The field of view of the developed 2 kHz SRS microscope is 30 × 28 µm2 with 50 × 46 pixels and 7 µs dwell time. By catching up the speed of chemical reaction, COMB-SRS is able to quantitatively visualize the ultrafast dynamics of molecular vibrations with submicron spatial resolution and sub-millisecond temporal resolution. The propagating polymer waves driven by reaction rate and persistent UV initiation are observed in situ. This methodology is expected to permit the development of novel functional polymers, controllable photoresists, 3D printing, and other new polymerization technologies.

Omics Analysis for Dinoflagellates Biology Research.

Dinoflagellates are important primary producers for marine ecosystems and are also responsible for certain essential components in human foods. However, they are also notorious for their ability to form harmful algal blooms, and cause shellfish poisoning. Although much work has been devoted to dinoflagellates in recent decades, our understanding of them at a molecular level is still limited owing to some of their challenging biological properties, such as large genome size, permanently condensed liquid-crystalline chromosomes, and the 10-fold lower ratio of protein to DNA than other eukaryotic species. In recent years, omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, have been applied to the study of marine dinoflagellates and have uncovered many new physiological and metabolic characteristics of dinoflagellates. In this article, we review recent application of omics technologies in revealing some of the unusual features of dinoflagellate genomes and molecular mechanisms relevant to their biology, including the mechanism of harmful algal bloom formations, toxin biosynthesis, symbiosis, lipid biosynthesis, as well as species identification and evolution. We also discuss the challenges and provide prospective further study directions and applications of dinoflagellates.

Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31.

BACKGROUND: Docosahexaenoic acid (DHA, C22:6) and odd-chain fatty acids (OCFAs, C15:0 and C17:0) have attracted great interest, since they have been widely used in food and therapeutic industries, as well as chemical industry, such as biodiesel production and improvement. The oil-producing heterotrophic microalgae Schizochytrium sp. 31 is one of main DHA-producing strains. Recently, it was found that Schizochytrium can also synthesize OCFAs; however, contents and titers of DHA and OCFAs in Schizochytrium are still low, which limit its practical application. RESULTS: In this study, we found that acetyl-CoA carboxylase suffered from a feedback inhibition by C16-CoA in Schizochytrium, and relief of the inhibition resulted in improved both lipid content and the ratio of OCFAs in total fatty acids. Based on this finding, a novel strategy for elevating both DHA and OCFAs contents was established. First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply. Second, the inhibition effect on acetyl-CoA carboxylase was relieved by overexpressing a codon-optimized ELO3 gene from Mortierella alpina, which encodes an elongase enzyme responsible for converting C16 into C18 fatty acids. After the above two-step engineering, contents of DHA and OCFAs were increased by 1.39- and 3.30-fold, reaching a level of 26.70 and 25.08% of dry cell weight, respectively, which are the highest contents reported so far for Schizochytrium. The titers of DHA and OCFAs were elevated by 1.08- and 2.57-fold, reaching a level of 3.54 and 3.32 g/L, respectively. Notably, the OCFAs titer achieved was 2.66-fold higher than the highest reported in Escherichia coli (1.25 g/L), implying potential value for industry application. To reveal the potential metabolic mechanism for the enhanced biosynthesis of both DHA and OCFAs, LC-MS metabolomic analysis was employed and the results showed that the pentose phosphate pathway and the glycolysis pathway were strengthened and intracellular propionyl-CoA concentration were also significantly increased in the engineered Schizochytrium, suggesting an increased supply of NADPH, acetyl-CoA, and propionyl-CoA for DHA and OCFAs accumulation. CONCLUSIONS: The discovery provides a new source of OCFAs production, and proposes a new strategy to improve contents and titers of both DHA and OCFAs in Schizochytrium. These will be valuable for improving commercial potential of Schizochytrium and guiding the engineering strategy in other fatty acids producing heterotrophic microalga.

Near-resonance enhanced label-free stimulated Raman scattering microscopy with spatial resolution near 130 nm.

High-resolution optical microscopes that can break 180 nm in spatial resolution set to conventional microscopies are much-needed tools. However, current optical microscopes have to rely on exogenous fluorescent labels to achieve high resolution in biological imaging. Herein, we report near-resonance enhanced label-free stimulated Raman scattering (SRS) microscopy with a lateral resolution near 130 nm, in which the high-resolution image contrast originates directly from a low concentration of endogenous biomolecules, with sensitivity gains of approximately 23 times. Moreover, by using a 0.3-m-long optical fiber, we developed hyperspectral SRS microscopy based on spectral focusing technology. Attributed to enhancements in spatial resolution and sensitivity, we demonstrated high-resolution imaging of three-dimensional structures in single cells and high-resolution mapping of large-scale intact mouse brain tissues in situ. By using enhanced high-resolution hyperspectral SRS, we chemically observed sphingomyelin distributed in the myelin sheath that insulates single axons. Our concept opens the door to biomedical imaging with ~130 nm resolution.