Simultaneous Imaging of Multiple miRNAs in Mitochondria Controlled by Fluorescently Encoded Upconversion Optical Switches for Drug Resistance Studies.

Mitochondrial miRNAs (mitomiRs) are essential regulators of biological processes by influencing mitochondrial gene expression and function. To comprehensively understand related pathological processes and treatments, simultaneous imaging of multiple mitomiRs is crucial. In this study, we present a technique that enables simultaneous monitoring of multiple mitomiRs in living cells using a near-infrared (NIR) photoactivated controlled detection probe (PD-mFleU) with a fluorescence-encoded error correction module and a nonsupervised machine learning data-processing algorithm. This method allows controlled sensing imaging of mitomiRs with a DNA reporter probe that can be activated by NIR light after targeted mitochondrial localization. Multilayer upconversion nanoparticles (UCNPs) are used for encoding probes and error correction. Additionally, the density-based spatial clustering of applications with the noise (DBSCAN) algorithm is used to process and analyze the image. Using this technique, we achieved rapid in situ imaging of the abnormal expression of three mitomiRs (miR-149, miR-590, and miR-671) related to mt-ND1 in drug-resistant cells. Furthermore, upregulating the three mitomiRs simultaneously efficiently reverted drug-resistant cells to sensitive cells. Our study provides an analytical strategy for multiplex imaging of mitomiRs in living cells with potential clinical applications.

Dual Functional Full-Color Carbon Dot-Based Organelle Biosensor Array for Visualization of Lipid Droplet Subgroups with Varying Lipid Composition in Living Cells.

In situ visualization of lipid composition diversity in lipid droplets (LDs) is essential for decoding lipid metabolism and function. However, effective probes for simultaneously localizing and reflecting the lipid composition of LDs are currently lacking. Here, we synthesized full-color bifunctional carbon dots (CDs) that can target LDs as well as respond to the nuance in internal lipid compositions with highly sensitive fluorescence signals, due to lipophilicity and surface state luminescence. Combined with microscopic imaging, uniform manifold approximation and projection, and sensor array concept, the capacity of cells to produce and maintain LD subgroups with varying lipid composition was clarified. Moreover, in oxidative stress cells, LDs with characteristic lipid compositions were deployed around mitochondria, and the proportion of LD subgroups changed, which gradually disappeared when treated with oxidative stress therapeutics. The CDs demonstrate great potential for in situ investigation of the LD subgroups and metabolic regulations.

Response of cross-correlations between high PM2.5 and O3 with increasing time scales to the COVID-19: different trends in BTH and PRD.

The air pollution in China currently is characterized by high fine particulate matter (PM2.5) and ozone (O3) concentrations. Compared with single high pollution events, such double high pollution (DHP) events (both PM2.5 and O3 are above the National Ambient Air Quality Standards (NAAQS)) pose a greater threat to public health and environment. In 2020, the outbreak of COVID-19 provided a special time window to further understand the cross-correlation between PM2.5 and O3. Based on this background, a novel detrended cross-correlation analysis (DCCA) based on maximum time series of variable time scales (VM-DCCA) method is established in this paper to compare the cross-correlation between high PM2.5 and O3 in Beijing-Tianjin-Heibei (BTH) and Pearl River Delta (PRD). At first, the results show that PM2.5 decreased while O3 increased in most cities due to the effect of COVID-19, and the increase in O3 is more significant in PRD than in BTH. Secondly, through DCCA, the results show that the PM2.5-O3 DCCA exponents α decrease by an average of 4.40% and 2.35% in BTH and PRD respectively during COVID-19 period compared with non-COVID-19 period. Further, through VM-DCCA, the results show that the PM2.5-O3 VM-DCCA exponents [Formula: see text] in PRD weaken rapidly with the increase of time scales, with decline range of about 23.53% and 22.90% during the non-COVID-19 period and COVID-19 period respectively at 28-h time scale. BTH is completely different. Without significant tendency, its [Formula: see text] is always higher than that in PRD at different time scales. Finally, we explain the above results with the self-organized criticality (SOC) theory. The impact of meteorological conditions and atmospheric oxidation capacity (AOC) variation during the COVID-19 period on SOC state are further discussed. The results show that the characteristics of cross-correlation between high PM2.5 and O3 are the manifestation of the SOC theory of atmospheric system. Relevant conclusions are important for the establishment of regionally targeted PM2.5-O3 DHP coordinated control strategies.

A modified high-yield method for primary culture of rat retinal microglial cells.

Microglial cells are the main immune cells of the retina. The primary culture of the retinal microglia is critically important in investigating the cells' properties and behaviors in neurodegenerative and inflammatory retinal disease. Here, we described a modified protocol of a microglial cell culture from the neonatal rat retina. In our culture protocol, the retina was isolated from the neonatal rat eye from postnatal day 1 to day 3 and trypsinized into a single-cell suspension. The cells were seeded into a T75 flask, which was pre-coated with poly-D-lysine (PDL) and cultured with dulbecco's modified eagle medium-F12 (DMEM/F12) that contained 10% fetal bovine serum (FBS) with different concentrations. Small bright rounded cells were observed on the top of mixed glial cells on the seventh day, and attained the maximum cell number on the 14th day. Then, the isolation was performed by a shaking method and isolated cells were identified with microglia markers ionized calcium-binding adaptor molecule 1 (IBA1), transmembrane protein 119 (TMEM119), cluster of differentiation 11b (CD11b), as well as astrocyte marker glial fibrillary acidic protein (GFAP) by immunofluorescence staining. Additionally, the initial plating ratio of the mixed glial cell, culture period of isolation, procedures of the isolation, as well as the purification procedure, were optimized for our primary microglial cell culture. The morphological changes and phagocytic function were performed after lipopolysaccharide (LPS) stimulation. Moreover, the release of pro-inflammatory cytokines at different time points of LPS activation were measured. In the present study, we found that the concentration of one retina/T75 flask could harvest the largest number of microglial cells. Besides, we continuously cultured the mixed glial cells as long as one month and isolated the mixed glial cells as much as three times. In our study, we used an isolation-shaking rate of 200 rpm for 2h, which guaranteed the steady rate and resulted in high purification of the primary retinal-microglial cells, with no need of an additional purification procedure. In conclusion, we provided a high-producing protocol for the primary culture of purified rat retinal-microglial cells.

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions.

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.

Glucose-6-Phosphate Dehydrogenase, Redox Homeostasis and Embryogenesis.

Normal embryogenesis requires complex regulation and precision, which depends on multiple mechanistic details. Defective embryogenesis can occur by various mechanisms. Maintaining redox homeostasis is of importance during embryogenesis. NADPH, as produced from the action of glucose-6-phosphate dehydrogenase (G6PD), has an important role in redox homeostasis, serving as a cofactor for glutathione reductase in the recycling of glutathione from oxidized glutathione and for NADPH oxidases and nitric oxide synthases in the generation of reactive oxygen (ROS) and nitrogen species (RNS). Oxidative stress differentially influences cell fate and embryogenesis. While low levels of stress (eustress) by ROS and RNS promote cell growth and differentiation, supra-physiological concentrations of ROS and RNS can lead to cell demise and embryonic lethality. G6PD-deficient cells and organisms have been used as models in embryogenesis for determining the role of redox signaling in regulating cell proliferation, differentiation and migration. Embryogenesis is also modulated by anti-oxidant enzymes, transcription factors, microRNAs, growth factors and signaling pathways, which are dependent on redox regulation. Crosstalk among transcription factors, microRNAs and redox signaling is essential for embryogenesis.

Circular RNA MTO1 intercorrelates with microRNA-630, both associate with Enneking stage and/or pathological fracture as well as prognosis in osteosarcoma patients.

OBJECTIVE: Circular RNA-mitochondrial tRNA translation optimization 1 (circ-MTO1) not only involves in bioprocess of various cancers, but also regulates osteosarcoma progression by regulating microRNA-630 (miR-630). However, the clinical role of circ-MTO1 and miR-630 in osteosarcoma is still obscure. This study aimed to assess the correlation of circ-MTO1 and miR-630 with disease features and prognosis and to explore their association with each other in osteosarcoma patients. METHODS: Forty-four osteosarcoma patients who received neoadjuvant chemotherapy to surgical resection were analyzed in this retrospective study. Then, circ-MTO1 and miR-630 expressions were evaluated in tumor and adjacent non-tumor specimens by reverse transcription quantitative polymerase chain reaction. RESULTS: Circ-MTO1 was lower in tumor than in non-tumor tissues (p<0.001); meanwhile, its elevated tumor expression was correlated with less advanced Enneking stage (p=0.049), good neoadjuvant chemotherapy response (p=0.029), and longer disease-free survival (DFS) (p=0.047). However, no association was found between circ-MTO1 and overall survival (OS) (p=0.122). Additionally, miR-630 in tumor was higher than in non-tumor tissues (p<0.001), while its raised tumor expression was associated with pathological fracture occurrence (p=0.003), advanced Enneking stage (p=0.036), poor neoadjuvant chemotherapy response (p=0.035), and shorter DFS (p=0.011). However, no association was found between miR-630 and OS (p=0.066). In addition, tumor circ-MTO1 was negatively associated with miR-630 (r=-0.323, p=0.032). CONCLUSION: Circ-MTO1 and miR-630 expressions are inter-correlated and dysregulated in osteosarcoma patients. Besides, they associate with Enneking stage and/or pathological fracture, as well as neoadjuvant treatment response and accumulating DFS in these patients.

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase.

OBJECTIVE: To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. RESULTS: The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for L-DOPA biosynthesis. CONCLUSIONS: The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for L-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

Chiral Spiro-Axis Induced Blue Thermally Activated Delayed Fluorescence Material for Efficient Circularly Polarized OLEDs with Low Efficiency Roll-Off.

A spiro-axis skeleton not only introduces circularly polarized luminescence (CPL) into thermally activated delayed fluorescence (TADF) molecules but also enhances the intramolecular through space charge transfer (TSCT) process. Spiral distributed phenoxazine and 2-(trifluoromethyl)-9H-thioxanthen-9-one-10,10-dioxide act as donor and acceptor units, respectively. The resulting TADF enantiomers, (rac)-OSFSO, display emission maxima at 470 nm, small singlet-triplet energy gap (ΔEST ) of 0.022 eV and high photoluminescence quantum yield (PLQY) of 81.2 % in co-doped film. The circularly polarized OLEDs (CP-OLEDs) based on (R)-OSFSO and (S)-OSFSO display obvious circularly polarized electroluminescence (CPEL) signals with dissymmetry factor up to 3.0×10-3 and maximum external quantum efficiency (EQEmax ) of 20.0 %. Moreover, the devices show remarkably low efficiency roll-off with an EQE of 19.3 % at 1000 cd m-2 (roll-off ca. 3.5 %), which are among the top results of CP-OLEDs.

Do Parental Feeding Rate and Feeding Synchrony Affect Nest Survival in a Songbird?

Nest predation risk is an important factor that may promote the evolution of adaptive parental reproductive strategies in animals. In altricial birds, where parents feed their offspring at the nest for a period of time, parent birds must balance the benefit from increased nest visits and the cost from increased nest predation resulting from their nest visits being detected by predators. Empirical evidence has shown a relationship between parental nest visits and nest predation risks, and parents have been found to be able to reduce the chance of being detected by predators by synchronizing their feeding visits while maintaining feeding rate unchanged. However, it remains poorly understood whether the relationships are universal to all species. We investigated whether nest survival is correlated with parental feeding rate and feeding synchrony in the silver-throated tit (Aegithalos glaucogularis), a songbird endemic to China. We found that parental feeding rate varied by brood size, nestling age and the time during the day, but feeding synchrony was unrelated to the above factors. Moreover, we showed no effect of parental feeding rate or synchrony on nest survival. The failure to find a relationship between parental feeding synchrony and nest survival seems exceptional because it is contrary to findings of previous studies. While there was likely a publication bias regarding previous studies, it is also possible that the evolution of feeding synchrony in different species has different explanations, or the complexity of the predator community and other factors affecting nest survival need to be considered in the analyses.

Enzymatic synthesis of structured lipids enriched with conjugated linoleic acid and butyric acid: strategy consideration and parameter optimization.

Structured lipids (SLs) rich in conjugated linoleic acid (CLA) and butyric acid with functions of low calorie and weight loss were synthesized in this study. By comparison of different synthetic routes, transesterification of CLA ethyl ester (CLAee) and tributyrin under vacuum was determined as the best method. The reaction conditions for SL synthesis were screened and the best conditions were as follows: Novozym 435 as the catalyst, enzyme load 6 wt%, temperature 60 °C, substrate molar ratio 2:1 (CLAee/tributyrin), water activity 0.68, reaction time 80 min. Under these conditions, the final product contained 97.5% of SLs, in which the contents of dibutyl-conjugated linoleoyl-glycerol and butyl-diconjugated linoleoyl-glycerol were 78.4% and 19.1%, respectively. The reusability evaluation indicated that the lipase could be reused at least 17 times. The obtained SLs with functions of both fatty acids could replace natural oil in food for inhibition of obesity and thus have great potential for commercial applications.

tert-Butyl Hydroperoxide (tBHP)-Induced Lipid Peroxidation and Embryonic Defects Resemble Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in C. elegans.

G6PD is required for embryonic development in animals, as severe G6PD deficiency is lethal to mice, zebrafish and nematode. Lipid peroxidation is linked to membrane-associated embryonic defects in Caenorhabditis elegans (C. elegans). However, the direct link between lipid peroxidation and embryonic lethality has not been established. The aim of this study was to delineate the role of lipid peroxidation in gspd-1-knockdown (ortholog of g6pd) C. elegans during reproduction. tert-butyl hydroperoxide (tBHP) was used as an exogenous inducer. Short-term tBHP administration reduced brood size and enhanced germ cell death in C. elegans. The altered phenotypes caused by tBHP resembled GSPD-1 deficiency in C. elegans. Mechanistically, tBHP-induced malondialdehyde (MDA) production and stimulated calcium-independent phospholipase A2 (iPLA) activity, leading to disturbed oogenesis and embryogenesis. The current study provides strong evidence to support the notion that enhanced lipid peroxidation in G6PD deficiency promotes death of germ cells and impairs embryogenesis in C. elegans.

The role of the RGD motif in CD97/ADGRE5-and EMR2/ADGRE2-modulated tumor angiogenesis.

CD97/ADGRE5, an adhesion G protein-coupled receptor (aGPCR), is highly expressed in several tumor cell types. CD97 has been shown to modulate tumorigenesis in part by promoting HUVEC migration, invasion and angiogenesis through the interaction with integrin α5ß1 via its ectodomain RGD motif. In this study, we show that CD97 could induce angiogenesis via an alternative RGD-independent mechanism. Overexpression of CD97 with the wild-type or mutant RGD motif in HT1080 cells led to up-regulated MMP-9 and induced angiogenesis as revealed by the in vitro endothelial cell tube formation assay and in ovo chick chorioallantoic membrane assay. By contrast, expression of EMR2/ADGRE2, the CD97-homologous aGPCR that contains a corresponding SGD sequence, fails to induce angiogenesis due to lower MMP-9 expression. Interestingly, a single change of the SGD to RGD sequence allowed EMR2 to up-regulate MMP-9 expression, leading to enhanced angiogenesis. MMP-9 was shown to promote the proliferation, migration, and invasion of HUVEC partly by modulating the levels of VEGF, PIGF, and bFGF. Finally, we showed that the MMP-9 expression was in turn modulated by N-cadherin that was up-regulated by CD97 and EMR2/RGD. Our results indicate that two homologous aGPCRs, CD97 and EMR2, modulate angiogenesis and HUVEC proliferation, migration, and invasion through N-cadherin-regulated MMP-9 expression by RGD-independent and -dependent mechanisms, respectively.

Medical empirical research on forest bathing (Shinrin-yoku): a systematic review.

AIMS: This study focused on the newest evidence of the relationship between forest environmental exposure and human health and assessed the health efficacy of forest bathing on the human body as well as the methodological quality of a single study, aiming to provide scientific guidance for interdisciplinary integration of forestry and medicine. METHOD: Through PubMed, Embase, and Cochrane Library, 210 papers from January 1, 2015, to April 1, 2019, were retrieved, and the final 28 papers meeting the inclusion criteria were included in the study. RESULT: The methodological quality of papers included in the study was assessed quantitatively with the Downs and Black checklist. The methodological quality of papers using randomized controlled trials is significantly higher than that of papers using non-randomized controlled trials (p < 0.05). Papers included in the study were analyzed qualitatively. The results demonstrated that forest bathing activities might have the following merits: remarkably improving cardiovascular function, hemodynamic indexes, neuroendocrine indexes, metabolic indexes, immunity and inflammatory indexes, antioxidant indexes, and electrophysiological indexes; significantly enhancing people's emotional state, attitude, and feelings towards things, physical and psychological recovery, and adaptive behaviors; and obvious alleviation of anxiety and depression. CONCLUSION: Forest bathing activities may significantly improve people's physical and psychological health. In the future, medical empirical studies of forest bathing should reinforce basic studies and interdisciplinary exchange to enhance the methodological quality of papers while decreasing the risk of bias, thereby raising the grade of paper evidence.

Dual Lewis Acid/Lewis Base Catalyzed Acylcyanation of Aldehydes: A Mechanistic Study.

A mechanistic investigation, which included a Hammett correlation analysis, evaluation of the effect of variation of catalyst composition, and low-temperature NMR spectroscopy studies, of the Lewis acid-Lewis base catalyzed addition of acetyl cyanide to prochiral aldehydes provides support for a reaction route that involves Lewis base activation of the acyl cyanide with formation of a potent acylating agent and cyanide ion. The cyanide ion adds to the carbonyl group of the Lewis acid activated aldehyde. O-Acylation by the acylated Lewis base to form the final cyanohydrin ester occurs prior to decomplexation from titanium. For less reactive aldehydes, the addition of cyanide is the rate-determining step, whereas, for more reactive, electron-deficient aldehydes, cyanide addition is rapid and reversible and is followed by rate-limiting acylation. The resting state of the catalyst lies outside the catalytic cycle and is believed to be a monomeric titanium complex with two alcoholate ligands, which only slowly converts into the product.

Thinopyrum ponticum chromatin-integrated wheat genome shows salt-tolerance at germination stage.

A wild wheatgrass, Thinopyrum ponticum (2n = 10x = 70), which exhibits substantially higher levels of salt tolerance than cultivated wheat, was employed to transfer its salt tolerance to common wheat by means of wide hybridization. A highly salt-tolerant wheat line S148 (2n = 42) was obtained from the BC3F2 progenies between Triticum aestivum (2n = 42) and Th. ponticum. In the cross of S148 × salt-sensitive wheat variety Chinese Spring, the BC4F2 seeds at germination stage segregated into a ratio of 3 salt tolerant to 1 salt sensitive, indicating that the salt tolerance was conferred by a dominant gene block. Genomic in situ hybridization analysis revealed that S148 had a single pair of Th. ponticum-T. aestivum translocated chromosomes bearing the salt-tolerance. This is an initial step of molecular breeding for salt-tolerant wheat.

Field and long-term demonstration of a wide area quantum key distribution network.

A wide area quantum key distribution (QKD) network deployed on communication infrastructures provided by China Mobile Ltd. is demonstrated. Three cities and two metropolitan area QKD networks were linked up to form the Hefei-Chaohu-Wuhu wide area QKD network with over 150 kilometers coverage area, in which Hefei metropolitan area QKD network was a typical full-mesh core network to offer all-to-all interconnections, and Wuhu metropolitan area QKD network was a representative quantum access network with point-to-multipoint configuration. The whole wide area QKD network ran for more than 5000 hours, from 21 December 2011 to 19 July 2012, and part of the network stopped until last December. To adapt to the complex and volatile field environment, the Faraday-Michelson QKD system with several stability measures was adopted when we designed QKD devices. Through standardized design of QKD devices, resolution of symmetry problem of QKD devices, and seamless switching in dynamic QKD network, we realized the effective integration between point-to-point QKD techniques and networking schemes.

Minor enantiomer recycling: application to enantioselective syntheses of beta blockers.

Continuous recycling of the minor product enantiomer obtained from the acetylcyanation of prochiral aldehydes provided access to highly enantiomerically enriched products. Cyanohydrin derivatives, which under normal conditions are obtained with modest or poor enantiomeric ratios, were formed with high enantiomeric purity by using a reinforcing combination of a chiral Lewis acid catalyst and a biocatalyst. The primarily obtained products were transformed into ß-adrenergic antagonists (S)-propanolol, (R)-dichloroisoproterenol, and (R)-pronethalol by means of a two-step procedure.

Enantioselective acylphosphonylation-dual Lewis acid-Lewis base activation of aldehyde and acylphosphonate.

Acetoxyphosphonates were obtained by a one-step procedure consisting of reaction of diethyl acetylphosphonate with prochiral aldehydes in the presence of a catalytic system comprising a chiral Lewis acid, an achiral Lewis base, and a Brønstedt base. Best results were obtained using a tridentate Schiff base aluminum(III) Lewis acidic complex, 1H-1,2,3-benzotriazole, and a tertiary amine such as DBU. The target compounds were in most cases obtained in high yields, but with moderate enantiomeric ratios (up to 78:22).

[Isolation, identification and biological activity of myxobacteria from soils].

OBJECTIVE: Purpose of this work was to screen myxobacteria from soils and study their biological activities towards pathogenic bacteria, tumor cells and insects. METHODS: Through inactivated E. coli and filter paper inducing methods, we isolated and purified myxobacteria from soil samples. Then we identified these purified strains based on morphological observation, physiological and biochemical characteristics, and the 16S rDNA sequences homologous analysis. By plate diffusion experiments, oral toxicity tests and tetrazolium assays, we investigated the biological activities of the myxobacterial culture supernatant. RESULTS: We isolated 35 myxobacterial strains and classified them as 4 genera: Myxococcus (9), Corallococcus (9), Nannocystis (11) and Sorangium (6). Eight purified myxobacteria were identified and named. Cytotoxicity tests show that strain C. macrospores S22 had potent and broad-spectrum cytotoxic effect on tumor cell lines including B16, 4T1, HeLa and HCT-116, so did the strains M. fulvus S51, C. exiguus S22 and M. Xanthus S55. Additionally, C. macrospores S22 also shows inhibitory activity to pathogenic bacteria Bacillus subtillis and Candida albicans. CONCLUSION: Myxobacteria are widely distributed in natural soils. C. macrosporus has potent toxicity against cancer cells and pathogenic bacteria; and C. exiguus with antitumor activity. The myxobacterial strains are promising resources for discovery and development of new active natural products and drugs.