Preparation of cellulose-based flexible SERS and its application for rapid and ultra-sensitive detection of thiram on fruits and vegetables.

In response to the prevalent issue of thiram as a common pesticide residue on the surface of fruits and vegetables, our research team employed an acidic hydrated metal salt low co-fusion solvent to dissolve cellulose lysis slurry. Subsequently, a regenerated cellulose membrane (RCM) was successfully prepared via sol-gel method. Uniformly sized Ag nanoparticles (NPs) were deposited on RCM utilizing the continuous ion layer adsorption and reaction (SILAR) technique. The resulting Ag NPs/RCM flexible surface-enhanced Raman spectroscopy (SERS) substrates exhibited a minimum detection limit of 5 × 10-9 M for Rhodamine 6G (R6G), demonstrating good uniformity (RSD = 4.86 %) and reproducibility (RSD = 3.07 %). Moreover, the substrate displayed a remarkable sensitivity of 10-10 M toward thiram standard solution. Given its inherent flexibility, the substrate proves advantageous for the detection of three-dimensional environments such as fruit and vegetable surfaces, and its practicality has been confirmed in the detection of thiram residue on apples, tomatoes, pears, and other fruits and vegetables.

Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Genomic and transcriptomic-based analysis of agronomic traits in sugar beet (Beta vulgaris L.) pure line IMA1.

Sugar beet (Beta vulgaris L.) is an important sugar-producing and energy crop worldwide. The sugar beet pure line IMA1 independently bred by Chinese scientists is a standard diploid parent material that is widely used in hybrid-breeding programs. In this study, a high-quality, chromosome-level genome assembly for IMA1was conducted, and 99.1% of genome sequences were assigned to nine chromosomes. A total of 35,003 protein-coding genes were annotated, with 91.56% functionally annotated by public databases. Compared with previously released sugar beet assemblies, the new genome was larger with at least 1.6 times larger N50 size, thereby substantially improving the completeness and continuity of the sugar beet genome. A Genome-Wide Association Studies analysis identified 10 disease-resistance genes associated with three important beet diseases and five genes associated with sugar yield per hectare, which could be key targets to improve sugar productivity. Nine highly expressed genes associated with pollen fertility of sugar beet were also identified. The results of this study provide valuable information to identify and dissect functional genes affecting sugar beet agronomic traits, which can increase sugar beet production and help screen for excellent sugar beet breeding materials. In addition, information is provided that can precisely incorporate biotechnology tools into breeding efforts.

Perceptions and preparedness toward tobacco cessation counseling amongst clinical medical students in Chongqing, Southwest China: A cross-sectional study.

Background: Medical students play an indispensable role in providing smoking cessation counseling. Despite the rapid increase in tobacco use, there is little data on what Chinese medical students know or are taught about it. This study aims to investigate the relationship between medical students' tobacco education level, clinical experience, and tobacco cessation counseling (TCC) provided by medical students. Methods: This cross-sectional study was carried out among clinical medical students of Chongqing medical university. An anonymous, self-administered questionnaire included items on demographic information, perceptions, and perceived preparedness, clinical medical students' self-reported level of education about alternative tobacco products, and traditional cigarettes. We assessed their perspectives toward TCC using a 5-point Likert scale. Descriptive and binary logistic regression analyses were carried out. Results: A total of 1,263 medical students completed the questionnaire. The majority of students (85%) expressed a willingness to provide TCC to patients in need. However, only half of the students stated unequivocally that they knew some ways and methods of tobacco cessation, while 18% stated that they did not know methods of tobacco cessation. Tobacco education and clinical experience were significantly associated with the ability to provide TCC. Our findings revealed that students with more clinical experience (undergraduates: B = 0.326, P < 0.001; postgraduates: B = 0.518, P < 0.001) were significantly more likely to have a greater self-reported comprehensive ability to provide TCC. Conclusion: Tobacco education and clinical experience can enhance the ability of medical students to provide smoking cessation counseling. There is a need to focus on alternative tobacco products with changing times, and curriculum planners should collaborate to incorporate comprehensive tobacco prevention and cessation training into the medical school curriculum.

Multi-omics Analyses Provide Insight into the Biosynthesis Pathways of Fucoxanthin in Isochrysis galbana.

Isochrysis galbana is considered an ideal bait for functional foods and nutraceuticals of humans because of its high fucoxanthin (Fx) content. However, multi-omics analysis of the regulatory networks for Fx biosynthesis in I. galbana has not been reported. In this study, we report a high-quality genome assembly of I. galbana LG007, which has a genome size of 92.73 Mb, with a contig N50 of 6.99 Mb and 14,900 protein-coding genes. Phylogenetic analysis confirmed the monophyly of Haptophyta, with I. galbana sister to Emiliania huxleyi and Chrysochromulina tobinii. Evolutionary analysis revealed an estimated divergence time between I. galbana and E. huxleyi of âˆ¼ 133 million years ago. Gene family analysis indicated that lipid metabolism-related genes exhibited significant expansion, including IgPLMT, IgOAR1, and IgDEGS1. Metabolome analysis showed that the content of carotenoids in I. galbana cultured under green light for 7 days was higher than that under white light, and ß-carotene was the main carotenoid, accounting for 79.09% of the total carotenoids. Comprehensive multi-omics analysis revealed that the content of ß-carotene, antheraxanthin, zeaxanthin, and Fx was increased by green light induction, which was significantly correlated with the expression of IgMYB98, IgZDS, IgPDS, IgLHCX2, IgZEP, IgLCYb, and IgNSY. These findings contribute to the understanding of Fx biosynthesis and its regulation, providing a valuable reference for food and pharmaceutical applications.

Engineering Metabolic Pathways for Cofactor Self-Sufficiency and Serotonin Production in Escherichia coli.

Serotonin is a neurotransmitter that plays an essential regulatory role in numerous cognitive and behavioral functions. Recent advances in synthetic biology have enabled engineering of non-natural biosynthetic pathways for serotonin production in E. coli. Here, an optimized heterologous serotonin biosynthetic pathway was engineered in E. coli and coupled with the biosynthetic and regeneration modules of the endogenous vital cofactor tetrahydrobiopterin (BH4) for efficient serotonin production using whole-cell catalysis. Further metabolic engineering efforts were performed to ensure an adequate endogenous BH4 supply, including enhancements of GTP biosynthesis and intracellular reducing power availability. Using the optimized fed-batch fermentation, an overall maximum serotonin yield of 40.3% (mol/mol) and a peak titer of 1.68 g/L (production rate of 0.016 g/L/h) were achieved. The strategies employed in this study show the promise of using E. coli for pterin self-sufficiency and high-level serotonin production, and the engineered strains hold the potential for use in industrial applications.

Broadband photodetector based on SnTe nanofilm/n-Ge heterostructure.

Combining novel two-dimensional materials with traditional semiconductors to form heterostructures for photoelectric detection have attracted great attention due to their excellent photoelectric properties. In this study, we reported the formation of a heterostructure comprising of tin telluride (SnTe) and germanium (Ge) by a simple and efficient one-step magnetron sputtering technique. A photodetector was fabricated by sputtering a nanofilm of SnTe on to a pre-masked n-Ge substrate.J-Vmeasurements obtained from the SnTe/n-Ge photodetector demonstrated diode and photovoltaic characteristics in the visible to near-infrared (NIR) band (i.e. 400-2050 nm). Under NIR illumination at 850 nm with an optical power density of 13.81 mW cm-2, the SnTe/n-Ge photodetector exhibited a small open-circuit voltage of 0.05 V. It also attained a high responsivity (R) and detectivity (D*) of 617.34 mA W-1(at bias voltage of -0.5 V) and 2.33 × 1011cmHz1/2W-1(at zero bias), respectively. Therefore, SnTe nanofilm/n-Ge heterostructure is highly suitable for used as low-power broadband photodetector due to its excellent performances and simple device configuration.

Metabonomics analysis of flavonoids in seeds and sprouts of two Chinese soybean cultivars.

A popular food in China, soybean seeds and sprouts contained many biologically active substances which are beneficial to the human body, such as flavonoids. Northeast of China is the main producing area of soybean. The experimental materials came from the main soybean producing areas in Northeast China, this study compared flavonoids of two China cultivars of soybeans, Heinong52(HN52) and Heinong71(HN71). Here, we also considered the effects of germination on the chemical profile of flavonoids. Using a LC-ESI-MS/MS system, 114 differential flavonoid metabolites were identified. A total of 18 metabolites were significantly different between the two soybean varieties before germination, of which 14 were up-regulated and 4 were down-regulated. After germination, 33 significantly different metabolites were found in the two soybean sprouts, of which 19 were up-regulated and 14 were down-regulated. These experimental results revealed significant up-regulation of metabolites in soybean sprouts compared with soybean seeds, thus suggesting that soybean germination may increase content of flavonoid metabolites. There are six main pathways for the synthesis of flavonoids: isoflavonoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, biosynthesis of secondary metabolites, and biosynthesis of phenylpropanoids. Soybean seeds lack flavone and flavanol biosynthesis and develop the capacity for this biosynthetic pathway after germination as sprouts. Isoflavonoid biosynthesis is the most abundantly utilized pathway.

A SiO2 Microcarrier with an Opal-like Structure for Cross-Linked Enzyme Immobilization.

The opal-like SiO2 microcarriers with different pore diameters named opal-SiO2I and opal-SiO2II were synthesized and utilized as microcarriers to immobilize Rhizopus oryzae lipase (ROL) and Aspergillus oryzae α-amylases (AOA). ROL and AOA can be more stably immobilized on the cross-linked SiO2 opals by neopentyl glycol diglycidyl ether (NGDE), which is the first attempt to use it as a cross-linking agent compared with glutaraldehyde. According to the morphology analysis, multiple layers of SiO2 monodisperse microspheres were regularly packed and formed an opal-like structure, and enzymes were well scattered and immobilized throughout the SiO2 opals. The results showed that the performance of enzymes immobilized on opal-SiO2II with a larger specific surface area was much better than that of opal-SiO2I. The enzyme activity of ROL@opal-SiO2II and AOA@opal-SiO2II cross-linked with 1% NGDE increased 5.32 and 9.32 times compared with their free counterpart, respectively. Furthermore, pH and thermal stability and reusability of ROL/AOA@opal-SiO2II were significantly improved and higher than those of ROL/AOA@opal-SiO2I and free enzymes. This study provides an easily obtained microcarrier opal-SiO2II, which shows potential for efficient different enzyme immobilizations and further industrial applications.

Chitosan Oligosaccharide Production Potential of Mitsuaria sp. C4 and Its Whole-Genome Sequencing.

Chitooligosaccharide is a kind of functional food, which is the degradation product of chitosan (COS) catalyzed by the endo-chitosanase (COSE) enzyme. A COSE with a molecular weight of 34 kDa was purified and characterized from a newly isolated Mitsuaria sp. C4 (C4), and a 38.46% recovery rate and 4.79-fold purification were achieved. The purified C4 COSE exhibited optimum activity at 40°C and pH 7.2 and was significantly inhibited in the presence of Cu2+ and Fe3+. The K m and V min of the COSE toward COS were 2.449 g/L and 0.042 g/min/L, respectively. The highest COSE activity reached 8.344 U/ml after optimizing, which represented a 1.34-fold of increase. Additionally, chitooligosaccharide obtained by COSE hydrolysis of COS was verified by using thin-layer chromatography and high-performance liquid chromatography analysis. Whole-genome sequencing demonstrated that the C4 strain contains 211 carbohydrate enzymes, our purified COSE belonging to GHs-46 involved in carbohydrate degradation. Phylogenetic analysis showed that the novel COSE obtained from the C4 strain was clustered into the degree of polymerization = two to three groups, which can perform catalysis in a similar manner to produce (GlcN)2 and (GlcN)3. This work indicates that the C4 strain could be a good resource for enhancing carbohydrate degradation and might represent a useful tool for chitooligosaccharide production in the functional food industry.

Insights into salvianolic acid B biosynthesis from chromosome-scale assembly of the Salvia bowleyana genome.

Salvia bowleyana is a traditional Chinese medicinal plant that is a source of nutritional supplements rich in salvianolic acid B and a potential experimental system for the exploration of salvianolic acid B biosynthesis in the Labiatae. Here, we report a high-quality chromosome-scale genome assembly of S. bowleyana covering 462.44 Mb, with a scaffold N50 value of 57.96 Mb and 44,044 annotated protein-coding genes. Evolutionary analysis revealed an estimated divergence time between S. bowleyana and its close relative S. miltiorrhiza of ~3.94 million years. We also observed evidence of a whole-genome duplication in the S. bowleyana genome. Transcriptome analysis showed that SbPAL1 (PHENYLALANINE AMMONIA-LYASE1) is highly expressed in roots relative to stem and leaves, paralleling the location of salvianolic acid B accumulation. The laccase gene family in S. bowleyana outnumbered their counterparts in both S. miltiorrhiza and Arabidopsis thaliana, suggesting that the gene family has undergone expansion in S. bowleyana. Several laccase genes were also highly expressed in roots, where their encoded proteins may catalyze the oxidative reaction from rosmarinic acid to salvianolic acid B. These findings provide an invaluable genomic resource for understanding salvianolic acid B biosynthesis and its regulation, and will be useful for exploring the evolution of the Labiatae.

Lipid and carotenoid production by the Rhodosporidium toruloides mutant in cane molasses.

Cane molasses is beneficial for lipid and carotenoid production in microalgae. We made a survey for the lipid and carotenoid production profile of R. toruloides M18 (MT) with various concentrations of molasses under nitrogen-deficited conditions. The production of α-linolenate and torularhodin from MT were 1.22- and 14.68-fold higher than those of the wild-type strain. We observed that molasses at concentrations of 35 g/L and 70 g/L represented a cheap and environmentally friendly strategy for producing lipids and carotenoids. Transcriptome and WGCNA analysis demonstrated that the genes relevant to the lipid and carotenoid production, including MYB, bHLH, Δ-4 desaturase, Δ-12 desaturase and FA2H, were significantly highly expressed. The results indicated that molasses could represent an inexpensive means for achieving high lipids and carotenoids production in R. toruloides.

Stretch-induced sarcoplasmic reticulum calcium leak is causatively associated with atrial fibrillation in pressure-overloaded hearts.

AIMS: Despite numerous reports documenting an important role of hypertension in the development of atrial fibrillation (AF), the detailed mechanism underlying the pathological process remains incompletely understood. Here, we aim to test the hypothesis that diastolic sarcoplasmic reticulum (SR) Ca2+ leak in atrial myocytes, induced by mechanical stretch due to elevated pressure in the left atrium (LA), plays an essential role in the AF development in pressure-overloaded hearts. METHODS AND RESULTS: Isolated mouse atrial myocytes subjected to acute axial stretch displayed an immediate elevation of SR Ca2+ leak. Using a mouse model of transverse aortic constriction (TAC), the relation between stretch, SR Ca2+ leak, and AF susceptibility was further tested. At 36 h post-TAC, SR Ca2+ leak in cardiomyocytes from the LA (with haemodynamic stress), but not right atrium (without haemodynamic stress), significantly increased, which was further elevated at 4 weeks post-TAC. Accordingly, AF susceptibility to atrial burst pacing in the 4-week TAC mice were also significantly increased, which was unaffected by inhibition of atrial fibrosis or inflammation via deletion of galectin-3. Western blotting revealed that type 2 ryanodine receptor (RyR2) in left atrial myocytes of TAC mice was oxidized due to activation and up-regulation of Nox2 and Nox4. Direct rescue of dysfunctional RyR2 with dantrolene or rycal S107 reduced diastolic SR Ca2+ leak in left atrial myocytes and prevented atrial burst pacing stimulated AF. CONCLUSION: Our study demonstrated for the first time the increased SR Ca2+ leak mediated by enhanced oxidative stress in left atrial myocytes that is causatively associated with higher AF susceptibility in pressure-overloaded hearts.

The evolutionary origin and domestication history of goldfish (Carassius auratus).

Goldfish have been subjected to over 1,000 y of intensive domestication and selective breeding. In this report, we describe a high-quality goldfish genome (2n = 100), anchoring 95.75% of contigs into 50 pseudochromosomes. Comparative genomics enabled us to disentangle the two subgenomes that resulted from an ancient hybridization event. Resequencing 185 representative goldfish variants and 16 wild crucian carp revealed the origin of goldfish and identified genomic regions that have been shaped by selective sweeps linked to its domestication. Our comprehensive collection of goldfish varieties enabled us to associate genetic variations with a number of well-known anatomical features, including features that distinguish traditional goldfish clades. Additionally, we identified a tyrosine-protein kinase receptor as a candidate causal gene for the first well-known case of Mendelian inheritance in goldfish-the transparent mutant. The goldfish genome and diversity data offer unique resources to make goldfish a promising model for functional genomics, as well as domestication.

Photooligomerization Determines Photosensitivity and Photoreactivity of Plant Cryptochromes.

Plant and non-plant species possess cryptochrome (CRY) photoreceptors to mediate blue light regulation of development or the circadian clock. The blue light-dependent homooligomerization of Arabidopsis CRY2 is a known early photoreaction necessary for its functions, but the photobiochemistry and function of light-dependent homooligomerization and heterooligomerization of cryptochromes, collectively referred to as CRY photooligomerization, have not been well established. Here, we show that photooligomerization is an evolutionarily conserved photoreaction characteristic of CRY photoreceptors in plants and some non-plant species. Our analyses of the kinetics of the forward and reverse reactions of photooligomerization of Arabidopsis CRY1 and CRY2 provide a previously unrecognized mechanism underlying the different photosensitivity and photoreactivity of these two closely related photoreceptors. We found that photooligomerization is necessary but not sufficient for the functions of CRY2, implying that CRY photooligomerization is presumably accompanied by additional function-empowering conformational changes. We further demonstrated that the CRY2-CRY1 heterooligomerization plays roles in regulating functions of Arabidopsis CRYs in vivo. Taken together, these results suggest that photooligomerization is an evolutionarily conserved mechanism determining the photosensitivity and photoreactivity of plant CRYs.

The Universally Conserved Residues Are Not Universally Required for Stable Protein Expression or Functions of Cryptochromes.

Universally conserved residues (UCRs) are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. Cryptochromes are photoreceptors in plants or light-independent components of the circadian clocks in mammals. We experimentally analyzed 51 UCRs of Arabidopsis cryptochrome 2 (CRY2) that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, we found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one photoresponses analyzed. Our finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.

Overexpression of acetyl-CoA carboxylase increases fatty acid production in the green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a photosynthetic unicellular model algae with multiple biotechnological advantages, and its fatty acids can be used to produce biofuels. Numerous studies suggest that acetyl-coA carboxylase (ACCa) catalyzes the first committed and rate-limiting step of fatty acid biosynthesis, thereby playing a central role in oil accumulation. Here, we cloned and overexpressed ACCa in C. reinhardtii to directly evaluate its effect on fatty acid synthesis. GC-MS analysis found that the unsaturated FAs contents of the CW15-24 and CW15-85 strains were 55.45% and 56.15%, which were significantly enriched compared to the wild type CW15 (48.39%). Under the optimized conditions, the content of lipid by overexpressed the ACCa gene in the mutant CW15-85 (0.46 g/l) was 1.16-fold greater than control through optimization of N and P sources. Altogether, our data clearly demonstrate that ACCa overexpression in C. reinhardtii can directly increase the synthesis of fatty acids.

Characterization of Flowering Time Mutants.

Timing of flowering, which is adapted to the ambient environment, is one of the key traits to ensure the reproductive success of plants. Our current understanding of the complex genetic control network of this trait is mostly derived from the studies in the model plant species Arabidopsis thaliana. Arabidopsis thaliana is an annual facultative long-day plant, whose flowering time is controlled by numerous environmental and endogenous factors. Here we briefly summarize the genetic pathways that promote flowering of Arabidopsis and describe standard protocols to characterize the flowering time phenotype of Arabidopsis mutants under laboratory conditions.

Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae.

Bioethanol, as a form of renewable and clean energy, has become increasingly important to the energy supply. One major obstacle in ethanol production is developing a high-capacity system. Existing approaches for regulating the ethanol production pathway are relatively insufficient, with nonspecific genetic manipulation. Here, we used CRISPR/Cas9 technology to disrupt the alcohol dehydrogenase (ADH) 2 gene via complete deletion of the gene and introduction of a frameshift mutation in the ADH2 locus. Sequencing demonstrated the accurate knockout of the target gene with 91.4% and near 100% targeting efficiency. We also utilized genome resequencing to validate the mutations in the ADH2 mutants targeted by various single-guide RNAs. This extensive analysis indicated the mutations in the CRISPR/Cas9-engineered strains were homozygous. We applied the engineered Saccharomyces cerevisiae strains for bioethanol production. Results showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain. This work illustrates the applicability of this highly efficient and specific genome engineering approach to promote the improvement of bioethanol production in S. cerevisiae via metabolic engineering. Importantly, this study is the first report of the disruption of a target gene, ADH2, in S. cerevisiae using CRISPR/Cas9 technology to improve bioethanol yield.

A CRY-BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis.

Cryptochromes are blue light receptors that regulate various light responses in plants. Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate blue light inhibition of hypocotyl elongation and long-day (LD) promotion of floral initiation. It has been reported recently that two negative regulators of Arabidopsis cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit cryptochrome function by blocking blue light-dependent cryptochrome dimerization. However, it remained unclear how cryptochromes regulate the BIC gene activity. Here we show that cryptochromes mediate light activation of transcription of the BIC genes, by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters. These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other. Surprisingly, phytochromes also mediate light activation of BIC transcription, suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature.