Effects of processing methods on the aroma of Poria cocos and its changing regulations during processing.

Poria cocos is a natural source of fungal food raw materials. Processing method is a key effecting the aroma of Poria cocos. In this study, the aroma compounds of Poria cocos products processed using sweating-low-temperature drying (SW-LD), sweating-high-temperature drying (SW-HD), steaming-low-temperature drying (ST-LD), and steaming-high-temperature drying (ST-HD) were compared by headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS), and the changes in aroma compounds of Poria cocos products during processing were analyzed. GC-MS analysis showed SW-HD product had highest content of aroma compounds. Aroma activity value (OAV) analysis indicated that 9 aroma compounds contributed to the overall aroma of Poria cocos. Among 9 compounds of Poria cocos, 1-octen-3-ol, hexanal, nonanal, octanal, trans-2-octenal, and heptanal contributed to mushroom, refreshing, sweet and fatty characters. In addition, the aroma compound changes during the processing were analyzed, revealing that steaming and sweating were the key processes affecting the aroma of Poria cocos products. The findings of this study provide valuable theoretical guidance for the development of Poria cocos processing technology.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster.

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

Effect of shaking manners on floral aroma quality and identification of key floral-aroma-active compounds in Hunan black tea.

Shaking is a key process effecting the floral aroma of Hunan black tea (HBT). In this study, the aroma composition of HBTs shaken in the early withering stage (ES1, ES1 + LS1, and ES2), shaken in the late withering stage (LS1), and not shaken (NS), and the identification of main floral aroma compounds were analyzed using sensory evaluation combined with gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactometry (GC-O), and aroma recombination experiments. Sensory evaluation results showed that the floral aroma of HBT shaken in the early withering stage was with high intensity, whereas HBT shaken in the late withering stage had low-intensity floral aroma. GC-MS identified a total number of 81 differential volatile compounds in HBT, including 30 esters, 18 aldehydes, 15 alcohols, 12 terpenes, 4 ketones, and 2 nitrogen-containing compounds. Further screening of important floral aroma differential compounds was performed using sensory-guided, odor activity value (OAV), and GC-O analysis, which identified three critical floral aroma differential compounds. Eventually, absolute quantification analysis and aroma recombination experiments confirmed that indole and methyl jasmonate were the most critical compounds of HBT determining floral aroma intensity. The findings of this study provide valuable guidance for the production of HBT with rich floral aroma attributes.

Effect of Leaf Grade on Taste and Aroma of Shaken Hunan Black Tea.

Shaken Hunan black tea is an innovative Hunan black tea processed by adding shaking to the traditional Hunan black tea. The quality of shaken black tea is influenced by leaf grades of different maturity. In this study, the taste and aroma quality of shaken Hunan black tea processed with different grades were analyzed by sensory evaluation (SP, HPLC, and HS-SPME/GC-MS). The results showed that shaken Hunan black tea processed with one bud and two leaves has the best quality, which has a sweet, mellow, and slightly floral taste, as well as a floral, honey, and sweet aroma. Moreover, caffeine and EGCG were identified as the most important bitter and astringent substances in shaken Hunan black. Combined with the analysis of GC-MS and OAV analysis, geraniol, jasmone, ß-myrcene, citral, and trans-ß-ocimene might be the most important components that affect the sweet aroma, while methyl jasmonate, indole, and nerolidol were the key components that affect the floral aroma of shaken Hunan black tea. This study lays a foundation for this study of the taste and aroma characteristics of shaken Hunan black tea and guides enterprises to improve shaken black tea processing technology.

Molecular Characterization of LKB1 of Triploid Crucian Carp and Its Regulation on Muscle Growth and Quality.

Liver Kinase B1 (LKB1) is a serine/threonine kinase that can regulate energy metabolism and skeletal muscle growth. In the present study, LKB1 cDNA of triploid crucian carp (Carassius auratus) was cloned. The cDNA contains a complete open reading frame (ORF), with a length of 1326 bp, encoding 442 amino acids. Phylogenetic tree analysis showed that the LKB1 amino acid sequence of the triploid crucian carp had a high sequence similarity and identity with carp (Cyprinus carpio). Tissue expression analysis revealed that LKB1 was widely expressed in various tissues. LKB1 expressions in the brain were highest, followed by kidney and muscle. In the short-term LKB1 activator and inhibitor injection experiment, when LKB1 was activated for 72 h, expressions of myogenic differentiation (MyoD), muscle regulatory factor (MRF4), myogenic factor (MyoG) and myostatin 1 (MSTN1) were markedly elevated and the content of inosine monophosphate (IMP) in muscle was significantly increased. When LKB1 was inhibited for 72 h, expressions of MyoD, MyoG, MRF4 and MSTN1 were markedly decreased. The long-term injection experiment of the LKB1 activator revealed that, when LKB1 was activated for 15 days, its muscle fibers were significantly larger and tighter than the control group. In texture profile analysis, it showed smaller hardness and adhesion, greater elasticity and chewiness. Contrastingly, when LKB1 was inhibited for 9 days, its muscle fibers were significantly smaller, while the gap between muscle fibers was significantly larger. Texture profile analysis showed that adhesion was significantly higher than the control group. A feeding trial on triploid crucian carp showed that with dietary lysine-glutamate dipeptide concentration increasing, the expression of the LKB1 gene gradually increased and was highest when dipeptide concentration was 1.6%. These findings may provide new insights into the effects of LKB1 on fish skeletal muscle growth and muscle quality, and will provide a potential application value in improvement of aquaculture feed formula.

The role of spatiotemporal organization and dynamics of clock complexes in circadian regulation.

Circadian clocks are cell autonomous timekeepers that regulate ∼24-h oscillations in the expression of many genes and control rhythms in nearly all our behavior and physiology. Almost every cell in the human body has a molecular clock and networks of cells containing clock proteins orchestrate daily rhythms in many physiological processes, from sleep-wake cycles to metabolism to immunity. All eukaryotic circadian clocks are based on transcription-translation delayed negative feedback loops in which activation of core clock genes is negatively regulated by their cognate protein products. Our current understanding of circadian clocks has been accumulated from decades of genetic and biochemical experiments, however, what remains poorly understood is how clock proteins, genes, and mRNAs are spatiotemporally organized within live clock cells and how such subcellular organization affects circadian rhythms at the single cell level. Here, we review recent progress in understanding how clock proteins and genes are spatially organized within clock cells over the circadian cycle and the role of such organization in generating circadian rhythms and highlight open questions for future studies.

Dietary Supplementation With Hydroxyproline Enhances Growth Performance, Collagen Synthesis and Muscle Quality of Carassius auratus Triploid.

An eight-week experiment was undertaken to examine the effect of dietary hydroxyproline (Hyp) supplementation on growth performance, collagen synthesis, muscle quality of an improved triploid crucian carp (Carassius auratus Triploid) (ITCC). Six isonitrogenous (340 g/kg diet), isolipidic (60 g/kg diet) and isocaloric (17.80 MJ/kg diet) diets were formulated containing a certain amount of Hyp: 0.09% (the control group), 0.39, 0.76, 1.14, 1.53 and 1.90%. Each diet was randomly assigned to three tanks and each group was fed two times daily until apparent satiation. The results showed that growth performance and feed utilization of ITCC were significantly improved with the dietary Hyp level was increased from 0.09 to 0.76%. Crude protein, threonine and arginine content in the dorsal muscle in 0.76% hydroxyproline group were significantly higher than those in basic diet group (p < 0.05). The muscle textural characteristics increased remarkably with the amount of Hyp in the diet rising from 0.09 to 1.53% (p < 0.05). Meanwhile, the contents of type I collagen (Col I) and Pyridinium crosslink (PYD) in the muscle of fish were significantly increased by dietary Hyp (p < 0.05). The muscle fiber diameter and density of the fish were significantly increased when fed with 0.76% Hyp (p < 0.05). Furthermore, dietary supplementation with an appropriate concentration of Hyp substantially increased the expression of genes involved in collagen synthesis (col1a1, col1a2, p4hα1, p4hß, smad4, smad5, smad9, and tgf-ß) and muscle growth (igf-1, tor, myod, myf5, and myhc) (p < 0.05). In conclusion, dietary supplementation of Hyp can enhance fish growth performance, collagen production, muscle textural characteristics and muscle growth of ITCC. According to the SGR broken-line analysis, the recommended supplementation level of Hyp was 0.74% in the diet for ITCC, corresponding to 2.2% of dietary protein.

Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms.

Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. While the genetic and molecular mechanisms of circadian rhythms are well characterized, what remains poorly understood are the intracellular dynamics of circadian clock components and how they affect circadian rhythms. Here, we elucidate how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in Drosophila clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, we demonstrate that Drosophila clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, we show that core clock genes, period and timeless, are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, we show that loss of Lamin B receptor, a nuclear envelope protein, leads to disruption of PER foci and per gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. Our results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing.

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.