Engineering the synthesis of unsaturated fatty acids by introducing desaturase improved the stress tolerance of yeast.

BACKGROUND: Yeast is often used to build cell factories to produce various chemicals or nutrient substances, which means the yeast has to encounter stressful environments. Previous research reported that unsaturated fatty acids were closely related to yeast stress resistance. Engineering unsaturated fatty acids may be a viable strategy for enhancing the stress resistance of cells. RESULTS: In this study, two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed in S. cerevisiae to determine how unsaturated fatty acids affect cellular stress tolerance of cells. After cloning and plasmid recombination, the recombinant S. cerevisiae cells were constructed. Analysis of membrane fatty acid contents revealed that the recombinant S. cerevisiae with overexpression of OLE1 and FAD2 genes contained higher levels of fatty acids C16:1 (2.77 times), C18:1 (1.51 times) and C18:2 (4.15 times) than the wild-type S. cerevisiae pY15TEF1. In addition, recombinant S. cerevisiae cells were more resistant to multiple stresses, and exhibited improved membrane functionality, including membrane fluidity and integrity. CONCLUSION: These findings demonstrated that strengthening the expression of desaturases was beneficial to stress tolerance. Overall, this study may provide a suitable means to build a cell factory of industrial yeast cells with high tolerance during biological manufacturing. © 2023 Society of Chemical Industry.

Engineering acetyl-CoA metabolism to enhance stress tolerance of yeast by regulating membrane functionality.

Zygosaccharomyces rouxii has excellent fermentation performance and good tolerance to osmotic stress. Acetyl-CoA is a crucial intermediate precursor in the central carbon metabolic pathway of yeast. This study investigated the effect of engineering acetyl-CoA metabolism on the membrane functionality and stress tolerance of yeast. Firstly, exogenous supplementation of acetyl-CoA improved the biomass and the ability of unsaturated fatty acid synthesis of Z. rouxii under salt stress. Q-PCR results suggested that the gene ACSS (coding acetyl-CoA synthetase) was significantly up-expressed. Subsequently, the gene ACSS from Z. rouxii was transformed and heterologously expressed in S. cerevisiae. The recombinant cells exhibited better multiple stress (salt, acid, heat, and cold) tolerance, higher fatty acid contents, membrane integrity, and fluidity. Our findings may provide a suitable means to enhance the stress tolerance and fermentation efficiency of yeast under harsh fermentation environments.

Arginine deiminase pathway of Tetragenococcus halophilus contributes to improve the acid tolerance of lactic acid bacteria.

Arginine deiminase pathway, controlled by arginine deiminase, ornithine carbamoyltransferase and carbamate kinase, could affect and modulate the intracellular pH homeostasis of lactic acid bacteria under acid stress. Herein, strategy based on exogenous addition of arginine had been proposed to improve the robustness of Tetragenococcus halophilus during acid stressed condition. Results indicated cells cultured in the presence of arginine acquired high tolerance to acid stress mainly through maintaining the homeostasis of intracellular microenvironment. Additionally, metabolomic analysis and q-PCR showed the content of intracellular metabolites and expression levels of genes involved in ADI pathway significantly increased when cells encountered acid stress with the presence of exogenous arginine. Furthermore, Lactococcus lactis NZ9000 with heterologous overexpression of arcA and arcC from T. halophilus exhibited high stress tolerance to acidic condition. This study may provide an insight into the systematical understanding about the mechanism underlying acid tolerance and improve the fermentation performance of LAB during harsh condition.

EFHD1, a novel mitochondrial regulator of tumor metastasis in clear cell renal cell carcinoma.

The biological function of many mitochondrial proteins in mechanistic detail has not been well investigated in clear cell renal cell carcinoma (ccRCC). A seven-mitochondrial-gene signature was generated by Lasso regression analysis to improve the prediction of prognosis of patients with ccRCC, using The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium cohort. Among those seven genes, EFHD1 is less studied and its role in the progression of ccRCC remains unknown. The decreased expression of EFHD1 was validated in clinical samples and was correlated with unfavorable outcome. Overexpression of EFHD1 in ccRCC cells resulted in the reduction of mitochondrial Ca2+ , and the inhibition of cell migration and invasion in vitro and tumor metastasis in vivo. Mechanistically, EFHD1 physically bound to the core mitochondrial calcium transporter (mitochondrial calcium uniporter, MCU) through its N-terminal domain. The interaction between EFHD1 and MCU suppressed the uptake of Ca2+ into mitochondria, and deactivated the Hippo/YAP signaling pathway. Further data revealed that the ectopic expression of EFHD1 upregulated STARD13 to enhance the phosphorylation of YAP protein at Ser-127. The knockdown of STARD13 or the overexpression of MCU partly abrogated the EFHD1-mediated induction of phosphorylation of YAP at Ser-127 and suppression of cell migration. Taken together, the newly identified EFHD1-MCU-STARD13 axis participates in the modulation of the Hippo/YAP pathway and serves as a novel regulator in the progression of ccRCC.

Effects of salt concentration on the quality and microbial diversity of spontaneously fermented radish paocai.

Paocai is a traditional Chinese fermented vegetable product popular in Asian countries. As an important additive, salt concentration is closely related to the quality of paocai. The aim of this study was to investigate the effect of salt concentration on the physicochemical characteristics, microbial diversity, and flavor profiles of spontaneously fermented radish, and the cross-correlation between microorganisms and flavor compounds was also revealed. Analysis of the microbial diversity of paocai showed that Firmicutes, Proteobacteria, and Ascomycota were detected as the main phyla with different salt concentrations, Weissella and Lactobacillus were the predominant bacterial genera, and Yarrowia dominated the fungal genera. Based on LEfSe analysis, Lactobacillus, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Microbacterium, Lactococcus, Staphylococcus, and Weissella were regarded as differential genera caused by differences in salinity. Analysis of the flavor compounds showed that 17 free amino acids, 5 isothiocyanates, 3 terpenes, 15 sulphur-containing compounds, 16 esters, 8 organic acids, 9 aldehydes, 8 ketones, 25 alcohols, 7 nitriles, 2 lactones, and 10 hydrocarbons were detected. Then, the correlation between the microbial community and flavor compounds was revealed, and the results indicated that several bacterial genera significantly correlated with flavors, including Lactobacillus, Kosakonia, Weissella, Leuconostoc, and Staphylococcus, while fungi had weak correlations with flavors. In addition, Metacyc pathway analysis was carried out to elucidate the effect of salt content on the metabolic pathways, showing that most flavor-related pathways were up-regulated with the increase in salt content. Results presented in this study may contribute to further understanding the role of salt in the fermentation of paocai and provide effective references for quality control of traditional fermented vegetables.

Food yeasts: occurrence, functions, and stress tolerance in the brewing of fermented foods.

With the rapid development of systems biology technology, there is a deeper understanding of the molecular biological mechanisms and physiological characteristics of microorganisms. Yeasts are widely used in the food industry with their excellent fermentation performances. While due to the complex environments of food production, yeasts have to suffer from various stress factors. Thus, elucidating the stress mechanisms of food yeasts and proposing potential strategies to improve tolerance have been widely concerned. This review summarized the recent signs of progress in the variety, functions, and stress tolerance of food yeasts. Firstly, the main food yeasts occurred in fermented foods, and the taxonomy levels are demonstrated. Then, the main functions of yeasts including aroma enhancer, safety performance enhancer, and fermentation period reducer are discussed. Finally, the stress response mechanisms of yeasts and the strategies to improve the stress tolerance of cells are reviewed. Based on sorting out these related recent researches systematically, we hope that this review can provide help and approaches to further exert the functions of food yeasts and improve food production efficiency.

Metabolomics analysis of salt tolerance of Zygosaccharomyces rouxii and guided exogenous fatty acid addition for improved salt tolerance.

BACKGROUND: Zygosaccharomyces rouxii plays an irreplaceable role in the manufacture of traditional fermented foods, which are produced in a high-salt environment. However, there is little research on strategies for improving salt tolerance of Z. rouxii. RESULTS: In this study, metabolomics was used to reveal the changes in intracellular metabolites under salt stress, and the results show that most of the carbohydrate contents decreased, the contents of xanthohumol and glycerol increased (fold change 4.07 and 5.35, respectively), while the contents of galactinol, xylitol and d-threitol decreased (fold change -9.43, -5.83 and -3.59, respectively). In addition, the content of four amino acids and six organic acids decreased, while that of the ten nucleotides increased. Notably, except for stearic acid (C18:0), all fatty acid contents increased. Guided by the metabolomics results, the effect of addition of seven exogenous fatty acids (C12:0, C14:0, C16:0, C18:0, C16:1, C18:1, and C18:2) on the salt tolerance of Z. rouxii was analyzed, and the results suggested that four exogenous fatty acids (C12:0, C16:0, C16:1, and C18:1) can increase the biomass yield and maximum growth rate. Physiological analyses demonstrated that exogenous fatty acids could regulate the distribution of fatty acids in the cell membrane, increase the degree of unsaturation, improve membrane fluidity, and maintain cell integrity, morphology and surface roughness. CONCLUSION: These results are applicable to revealing the metabolic mechanisms of Z. rouxii under salt stress and screening potential protective agents to improve stress resistance by adding exogenous fatty acids. © 2022 Society of Chemical Industry.

Targeting PFKL with penfluridol inhibits glycolysis and suppresses esophageal cancer tumorigenesis in an AMPK/FOXO3a/BIM-dependent manner.

As one of the hallmarks of cancer, metabolic reprogramming leads to cancer progression, and targeting glycolytic enzymes could be useful strategies for cancer therapy. By screening a small molecule library consisting of 1320 FDA-approved drugs, we found that penfluridol, an antipsychotic drug used to treat schizophrenia, could inhibit glycolysis and induce apoptosis in esophageal squamous cell carcinoma (ESCC). Gene profiling and Ingenuity Pathway Analysis suggested the important role of AMPK in action mechanism of penfluridol. By using drug affinity responsive target stability (DARTS) technology and proteomics, we identified phosphofructokinase, liver type (PFKL), a key enzyme in glycolysis, as a direct target of penfluridol. Penfluridol could not exhibit its anticancer property in PFKL-deficient cancer cells, illustrating that PFKL is essential for the bioactivity of penfluridol. High PFKL expression is correlated with advanced stages and poor survival of ESCC patients, and silencing of PFKL significantly suppressed tumor growth. Mechanistically, direct binding of penfluridol and PFKL inhibits glucose consumption, lactate and ATP production, leads to nuclear translocation of FOXO3a and subsequent transcriptional activation of BIM in an AMPK-dependent manner. Taken together, PFKL is a potential prognostic biomarker and therapeutic target in ESCC, and penfluridol may be a new therapeutic option for management of this lethal disease.

Structural characterization and bioactivity of novel exopolysaccharides produced by Tetragenococcus halophilus.

In this study, exopolysaccharides produced by Tetragenococcus halophilus were extracted and purified. Two fractions named EPS-1 and EPS-2 were obtained with average molecular weights of 2613.4 and 93.4 kDa, respectively. Analysis of the structures demonstrated that EPS-1 was mainly composed of galactose, glucose, mannose and glucuronic acid, and EPS-2 mostly consisted of glucose and mannose. 800 MHz nuclear magnetic resonance spectroscopy elucidated different and partial primary structures of EPS-1 and EPS-2. The microtopography was observed by atomic force microscope and scanning electron microscope. In addition, the two fractions exhibited antioxidant activities on DPPH, hydroxyl and ABTS•+ radicals. Furthermore, EPS-1 and EPS-2 could protect Lactococcus lactis against cryogenic and lyophilized stress. These results indicated that exopolysaccharides from T. halophilus in this study would have great potential in the application of food and pharmaceutical industry.

Targeting PP2A with lomitapide suppresses colorectal tumorigenesis through the activation of AMPK/Beclin1-mediated autophagy.

Colorectal cancer (CRC) is one of the most common malignancies worldwide, and effective therapy remains a challenge. In this study, we take advantage of a drug repurposing strategy to screen small molecules with novel anticancer activities in a small-molecule library consisting of 1056 FDA-approved drugs. We show, for the first time, that lomitapide, a lipid-lowering agent, exhibits antitumor properties in vitro and in vivo. Activated autophagy is characterized as a key biological process in lomitapide-induced CRC repression. Mechanistically, lomitapide stimulated mitochondrial dysfunction-mediated AMPK activation, resulting in increased AMPK phosphorylation and enhanced Beclin1/Atg14/Vps34 interactions, provoking autophagy induction. Autophagy inhibition or AMPK silencing significantly abrogated lomitapide-induced cell death, indicating the significance of AMPK-regulated autophagy in the antitumor activities of lomitapide. More importantly, PP2A was identified as a direct target of lomitapide by limited proteolysis-mass spectrometry (LiP-SMap), and the bioactivity of lomitapide was attenuated in PP2A-deficient cells, suggesting that the anticancer effect of lomitapide occurs in a PP2A-dependent manner. Taken together, the results of the study reveal that lomitapide can be repositioned as a potential therapeutic drug for CRC treatment.

Targeted Immunotherapies in Gastrointestinal Cancer: From Molecular Mechanisms to Implications.

Gastrointestinal cancer is a leading cause of cancer-related mortality and remains a major challenge for cancer treatment. Despite the combined administration of modern surgical techniques and chemoradiotherapy (CRT), the overall 5-year survival rate of gastrointestinal cancer patients in advanced stage disease is less than 15%, due to rapid disease progression, metastasis, and CRT resistance. A better understanding of the mechanisms underlying cancer progression and optimized treatment strategies for gastrointestinal cancer are urgently needed. With increasing evidence highlighting the protective role of immune responses in cancer initiation and progression, immunotherapy has become a hot research topic in the integrative management of gastrointestinal cancer. Here, an overview of the molecular understanding of colorectal cancer, esophageal cancer and gastric cancer is provided. Subsequently, recently developed immunotherapy strategies, including immune checkpoint inhibitors, chimeric antigen receptor T cell therapies, tumor vaccines and therapies targeting other immune cells, have been described. Finally, the underlying mechanisms, fundamental research and clinical trials of each agent are discussed. Overall, this review summarizes recent advances and future directions for immunotherapy for patients with gastrointestinal malignancies.

Incorporation of Exogenous Fatty Acids Enhances the Salt Tolerance of Food Yeast Zygosaccharomyces rouxii.

Fatty acids have great effects on the maintenance of the cell membrane structure, cell viability, and cell metabolisms. In this study, we sought to elucidate the effects of exogenous fatty acids on the salt tolerance of food yeast Zygosaccharomyces rouxii. Results showed that Z. rouxii can grow by using exogenous fatty acids (C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, and C18:2) as the sole carbon source. Four fatty acids (C12:0, C16:0, C16:1, and C18:1) can improve the salt tolerance of cells, enhance the formation of the cell biofilm, regulate the chemical compositions, restore growth in the presence of cerulenin, regulate the contents of membrane fatty acids, and control the expression of key genes in the fatty acid metabolism. Our results reveal that Z. rouxii can synthesize membrane fatty acids from exogenous fatty acids and the supplementation of these fatty acids can override the need for de novo fatty acid biosynthesis.

Heat preadaptation improved the ability of Zygosaccharomyces rouxii to salt stress: a combined physiological and transcriptomic analysis.

Zygosaccharomyces rouxii plays important roles in the brewing process of fermented foods such as soy sauce, where salt stress is a frequently encountered condition. In this study, effect of heat preadaptation on salt tolerance of Z. rouxii and the protective mechanisms underlying heat preadaptation were investigated based on physiological and transcriptomic analyses. Results showed that cells subjected to heat preadaptation (37 °C, 90 min) prior to salt stress aroused many physiological responses, including maintaining cell surface smooth and intracellular pH level, increasing Na+/K+-ATPase activity. Cells subjected to heat preadaptation increased the amounts of unsaturated fatty acids (palmitoleic C16:1, oleic C18:1, linoleic C18:2) and decreased the amounts of saturated fatty acids (palmitic C16:0, stearic C18:0) which caused the unsaturation degree (unsaturated/saturated = U/S ratio) increased by 2.4 times when compared with cells without preadaptation under salt stress. Besides, salt stress led to increase in contents of 5 amino acids (valine, proline, threonine, glycine, and tyrosine) and decrease of 2 amino acids (serine and lysine). When comparing the cells pre-exposed to heat preadaptation followed by challenged with salt stress and the cells without preadaptation under salt stress, the serine, threonine, and lysine contents increased significantly. RNA sequencing revealed that the metabolic level of glycolysis by Z. rouxii was weakened, while the metabolic levels of the pentose phosphate pathway and the riboflavin were enhanced in cells during heat preadaptation. Results presented in this study may contribute to understand the bases of adaptive responses in Z. rouxii and rationalize its exploitation in industrial processes.Key points• Heat preadaptation can improve high salinity tolerance of Z. rouxii.• Combined physiological and transcriptomic analyses of heat preadaptation mechanisms.• Provide theoretical support for the application of Z. rouxii.

A Study of Nitrogen Deficiency Inversion in Rice Leaves Based on the Hyperspectral Reflectance Differential.

To achieve rapid, accurate, and non-destructive diagnoses of nitrogen deficiency in cold land japonica rice, hyperspectral data were collected from field experiments to investigate the relationship between the nitrogen (N) content and the difference in the spectral reflectance relationship and to establish the hyperspectral reflectance difference inversion model of differences in the N content of rice. In this study, the hyperspectral reflectance difference was used to invert the nitrogen deficiency of rice and provide a method for the implementation of precision fertilization without reducing the yield of chemical fertilizer. For the purpose of constructing the standard N content and standard spectral reflectance the principle of minimum fertilizer application at maximum yield was used as a reference standard, and the acquired rice leaf nitrogen content and leaf spectral reflectance were differenced from the standard N content and standard spectral reflectance to obtain N content. The difference and spectral reflectance differential were then subjected to discrete wavelet multiscale decomposition, successive projections algorithm, principal component analysis, and iteratively retaining informative variables (IRIVs); the results were treated as partial least squares (PLSR), extreme learning machine (ELM), and genetic algorithm-extreme learning machine (GA-ELM). The results of hyperspectral dimensionality reduction were used as input to establish the inverse model of N content differential in japonica rice. The results showed that the GA-ELM inversion model established by discrete wavelet multi-scale decomposition obtained the optimal results in data set modeling and training. Both the R2 of the training data set and the validation data set were above 0.68, and the root mean square errors (RMSEs) were <0.6 mg/g and were more predictive, stable, and generalizable than the PLSR and ELM predictive models.

A Silicon Optical Bench-Based Forward-View Two-Axis Scanner for Microendoscopy Applications.

Optical microendoscopy enabled by a microelectromechanical system (MEMS) scanning mirror offers great potential for in vivo diagnosis of early cancer inside the human body. However, an additional beam folding mirror is needed for a MEMS mirror to perform forward-view scanning, which drastically increases the diameter of the resultant MEMS endoscopic probe. This paper presents a new monolithic two-axis forward-view optical scanner that is composed of an electrothermally driven MEMS mirror and a beam folding mirror both vertically standing and integrated on a silicon substrate. The mirror plates of the two mirrors are parallel to each other with a small distance of 0.6 mm. The laser beam can be incident first on the MEMS mirror and then on the beam folding mirror, both at 45°. The MEMS scanner has been successfully fabricated. The measured optical scan angles of the MEMS mirror were 10.3° for the x axis and 10.2° for the y axis operated under only 3 V. The measured tip-tilt resonant frequencies of the MEMS mirror were 1590 Hz and 1850 Hz, respectively. With this compact MEMS design, a forward-view scanning endoscopic probe with an outer diameter as small as 2.5 mm can be made, which will enable such imaging probes to enter the subsegmental bronchi of an adult patient.

MEMS Mirrors for LiDAR: A review.

In recent years, Light Detection and Ranging (LiDAR) has been drawing extensive attention both in academia and industry because of the increasing demand for autonomous vehicles. LiDAR is believed to be the crucial sensor for autonomous driving and flying, as it can provide high-density point clouds with accurate three-dimensional information. This review presents an extensive overview of Microelectronechanical Systems (MEMS) scanning mirrors specifically for applications in LiDAR systems. MEMS mirror-based laser scanners have unrivalled advantages in terms of size, speed and cost over other types of laser scanners, making them ideal for LiDAR in a wide range of applications. A figure of merit (FoM) is defined for MEMS mirrors in LiDAR scanners in terms of aperture size, field of view (FoV) and resonant frequency. Various MEMS mirrors based on different actuation mechanisms are compared using the FoM. Finally, a preliminary assessment of off-the-shelf MEMS scanned LiDAR systems is given.

Semantic segmentation of microscopic neuroanatomical data by combining topological priors with encoder-decoder deep networks.

Understanding of neuronal circuitry at cellular resolution within the brain has relied on neuron tracing methods which involve careful observation and interpretation by experienced neuroscientists. With recent developments in imaging and digitization, this approach is no longer feasible with the large scale (terabyte to petabyte range) images. Machine learning based techniques, using deep networks, provide an efficient alternative to the problem. However, these methods rely on very large volumes of annotated images for training and have error rates that are too high for scientific data analysis, and thus requires a significant volume of human-in-the-loop proofreading. Here we introduce a hybrid architecture combining prior structure in the form of topological data analysis methods, based on discrete Morse theory, with the best-in-class deep-net architectures for the neuronal connectivity analysis. We show significant performance gains using our hybrid architecture on detection of topological structure (e.g. connectivity of neuronal processes and local intensity maxima on axons corresponding to synaptic swellings) with precision/recall close to 90% compared with human observers. We have adapted our architecture to a high performance pipeline capable of semantic segmentation of light microscopic whole-brain image data into a hierarchy of neuronal compartments. We expect that the hybrid architecture incorporating discrete Morse techniques into deep nets will generalize to other data domains.

Zygosaccharomyces rouxii Combats Salt Stress by Maintaining Cell Membrane Structure and Functionality.

Zygosaccharomyces rouxii is an important yeast that is required in the food fermentation process due to its high salt tolerance. In this study, the responses and resistance strategies of Z. rouxii against salt stress were investigated by performing physiological analysis at membrane level. The results showed that under salt stress, cell integrity was destroyed, and the cell wall was ruptured, which was accompanied by intracellular substance spillover. With an increase of salt concentrations, intracellular Na+ content increased slightly, whereas intracellular K+ content decreased significantly, which caused the increase of the intracellular Na+/K+ ratio. In addition, in response to salt stress, the activity of Na+/K+-ATPase increased from 0.54 to 2.14 µmol/mg protein, and the ergosterol content increased to 2.42-fold to maintain membrane stability. Analysis of cell membrane fluidity and fatty acid composition showed that cell membrane fluidity decreased and unsaturated fatty acid proportions increased, leading to a 101.21% rise in the unsaturated/saturated fatty acid ratio. The results presented in this study offer guidance in understanding the salt tolerance mechanism of Z. rouxii, and in developing new strategies to increase the industrial utilization of this species under salt stress.

Miniature fluorescence molecular tomography (FMT) endoscope based on a MEMS scanning mirror and an optical fiberscope.

We present a novel FMT endoscope by using a MEMS scanning mirror and an optical fiberscope. The diameter of this highly miniaturized FMT device is only 5 mm. To our knowledge, this is the smallest FMT device we found so far. Several phantom experiments based on indocyanine green (ICG) were conducted to demonstrate the imaging ability of this device. Two tumor-bearing mice were systematically injected with tumor-targeted NIR fluorescent probes (ATF-PEG-IO-830) and were then imaged to further demonstrate the ability of this FMT endoscope for imaging small animals.

Genetic Single Neuron Anatomy Reveals Fine Granularity of Cortical Axo-Axonic Cells.

Parsing diverse nerve cells into biological types is necessary for understanding neural circuit organization. Morphology is an intuitive criterion for neuronal classification and a proxy of connectivity, but morphological diversity and variability often preclude resolving the granularity of neuron types. Combining genetic labeling with high-resolution, large-volume light microscopy, we established a single neuron anatomy platform that resolves, registers, and quantifies complete neuron morphologies in the mouse brain. We discovered that cortical axo-axonic cells (AACs), a cardinal GABAergic interneuron type that controls pyramidal neuron (PyN) spiking at axon initial segments, consist of multiple subtypes distinguished by highly laminar-specific soma position and dendritic and axonal arborization patterns. Whereas the laminar arrangements of AAC dendrites reflect differential recruitment by input streams, the laminar distribution and local geometry of AAC axons enable differential innervation of PyN ensembles. This platform will facilitate genetically targeted, high-resolution, and scalable single neuron anatomy in the mouse brain.