Rapid discrimination of quality grade of black tea based on near-infrared spectroscopy (NIRS), electronic nose (E-nose) and data fusion.

For the manufacturing and sale of tea, rapid discrimination of overall quality grade is of great importance. However, present evaluation methods are time-consuming and labor-intensive. This study investigated the feasibility of combining advantages of near-infrared spectroscopy (NIRS) and electronic nose (E-nose) to assess the tea quality. We found that NIRS and E-nose models effectively identify taste and aroma quality grades, with the highest accuracies of 99.63% and 97.00%, respectively, by comparing different principal component numbers and classification algorithms. Additionally, the quantitative models based on NIRS predicted the contents of key substances. Based on this, NIRS and E-nose data were fused in the feature-level to build the overall quality evaluation model, achieving accuracies of 98.13%, 96.63% and 97.75% by support vector machine, K-nearest neighbors, and artificial neural network, respectively. This study reveals that the integration of NIRS and E-nose presents a novel and effective approach for rapidly identifying tea quality.

Visualizing the nucleoplasmic maturation of human pre-60S ribosomal particles.

Eukaryotic ribosome assembly is a highly orchestrated process that involves over two hundred protein factors. After early assembly events on nascent rRNA in the nucleolus, pre-60S particles undergo continuous maturation steps in the nucleoplasm, and prepare for nuclear export. Here, we report eleven cryo-EM structures of the nuclear pre-60S particles isolated from human cells through epitope-tagged GNL2, at resolutions of 2.8-4.3 Å. These high-resolution snapshots provide fine details for several major structural remodeling events at a virtual temporal resolution. Two new human nuclear factors, L10K and C11orf98, were also identified. Comparative structural analyses reveal that many assembly factors act as successive place holders to control the timing of factor association/dissociation events. They display multi-phasic binding properties for different domains and generate complex binding inter-dependencies as a means to guide the rRNA maturation process towards its mature conformation. Overall, our data reveal that nuclear assembly of human pre-60S particles is generally hierarchical with short branch pathways, and a few factors display specific roles as rRNA chaperones by confining rRNA helices locally to facilitate their folding, such as the C-terminal domain of SDAD1.

Structural dynamics of AAA + ATPase Drg1 and mechanism of benzo-diazaborine inhibition.

The type II AAA + ATPase Drg1 is a ribosome assembly factor, functioning to release Rlp24 from the pre-60S particle just exported from nucleus, and its activity in can be inhibited by a drug molecule diazaborine. However, molecular mechanisms of Drg1-mediated Rlp24 removal and diazaborine-mediated inhibition are not fully understood. Here, we report Drg1 structures in different nucleotide-binding and benzo-diazaborine treated states. Drg1 hexamers transits between two extreme conformations (planar or helical arrangement of protomers). By forming covalent adducts with ATP molecules in both ATPase domain, benzo-diazaborine locks Drg1 hexamers in a symmetric and non-productive conformation to inhibits both inter-protomer and inter-ring communication of Drg1 hexamers. We also obtained a substrate-engaged mutant Drg1 structure, in which conserved pore-loops form a spiral staircase to interact with the polypeptide through a sequence-independent manner. Structure-based mutagenesis data highlight the functional importance of the pore-loop, the D1-D2 linker and the inter-subunit signaling motif of Drg1, which share similar regulatory mechanisms with p97. Our results suggest that Drg1 may function as an unfoldase that threads a substrate protein within the pre-60S particle.

Numerical Analysis on Heat Characteristics of the Ventilation Basket for Fresh Tea Leaves.

Plastic baskets are commonly used as containers for fresh tea leaves during storage and transport after harvest. Nevertheless, there are significant challenges in controlling the core temperature of the basket since fresh tea leaves still maintain a certain degree of respiration after being harvested, with extremely high temperatures being the major factor for the color change of fresh tea leaves. A numerical model was developed to improve the temperature control of the plastic basket, by which the influence of different structural parameters on the core temperature in the plastic baskets with fresh tea leaves was analyzed. The accuracy of the model in predicting airflow and temperature distributions was validated against experimental data. The maximum RMSE was 1.158 °C and the maximum MRE was 5.410% between the simulated and test temperature value. The maximum deviation between the simulated velocity and test velocity was 0.11 m/s, the maximum RE was 29.05% and the maximum SD was 0.024. The results show that a plastic basket with a ventilation duct efficiently decreased the temperature of the fresh tea leaves and significantly affected the heat transfer between the fresh tea leaves and the ambient air compared to the plastic basket without a ventilation duct. Furthermore, the effect on the heat transfer was further expanded by the use of a plastic basket with a ventilation duct when the plastic baskets were stacked. The maximum temperature differences were 0.52 and 0.40 according to the stacked and single-layer products, respectively. The ambient temperature and the bulk density of the fresh tea leaves have a significant influence on the core temperature.

Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.

GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2ß2γ2 hexamers in which the core catalytic α- and ß-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.

Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit.

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.

Structural insights into the membrane microdomain organization by SPFH family proteins.

The lateral segregation of membrane constituents into functional microdomains, conceptually known as lipid raft, is a universal organization principle for cellular membranes in both prokaryotes and eukaryotes. The widespread Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are enriched in functional membrane microdomains at various subcellular locations, and therefore were hypothesized to play a scaffolding role in microdomain formation. In addition, many SPFH proteins are also implicated in highly specific processes occurring on the membrane. However, none of these functions is understood at the molecular level. Here we report the structure of a supramolecular complex that is isolated from bacterial membrane microdomains and contains two SPFH proteins (HflK and HflC) and a membrane-anchored AAA+ protease FtsH. HflK and HflC form a circular 24-mer assembly, featuring a laterally segregated membrane microdomain (20 nm in diameter) bordered by transmembrane domains of HflK/C and a completely sealed periplasmic vault. Four FtsH hexamers are embedded inside this microdomain through interactions with the inner surface of the vault. These observations provide a mechanistic explanation for the role of HflK/C and their mitochondrial homologs prohibitins in regulating membrane-bound AAA+ proteases, and suggest a general model for the organization and functionalization of membrane microdomains by SPFH proteins.

VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography.

Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.

GC-MS analysis combined with sensory analysis revealed the various aroma characteristics of black tea resulted from different grafting rootstocks.

The study was aim to investigate the effects of grafting on volatile compounds and sensory quality of black tea. Seven groups of black tea were prepared from one nongrafted tea tree "Yinghong9 (YJ)" and six grafted tea trees by grafting scion of "YingHong9" on different rootstocks. Sensory analysis indicated marked/slight variations among seven samples, among which, the one grafting on HuangZhiXiangDanCong (HZX) stood out with floral and fruity aroma. The result of chemometrics analysis suggested various effects on compounds caused by different rootstocks. A total of 38 differential compounds were identified, showing mainly quantitative variations, with 36 being identified in all samples. The significant higher contents of volatiles, such as geraniol, phenylethyl alcohol, (E)-nerolidol, decanal, and linalool oxides, in HZX compared with YJ were observed, which explained why floral and fruity aroma stood out among the whole aroma profile of HZX. Both results of sensory and instrumental analysis suggested certain correlation between compound variations and aroma characteristics. Moreover, different rootstocks influenced the aroma quality in different ways. PRACTICAL APPLICATION: In conclusion, the study illuminates the various effects of grafting on the volatile compounds and aroma quality, which enlightens the possibility of changing aroma quality of black tea by grafting scions on different rootstocks. And thus, it can help guide the practical production when cultivating new varieties.

Structural snapshots of human pre-60S ribosomal particles before and after nuclear export.

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.

Structural and functional insights into the tetrameric photosystem I from heterocyst-forming cyanobacteria.

Two large protein-cofactor complexes, photosystem I and photosystem II, are the central components of photosynthesis in the thylakoid membranes. Here, we report the 2.37-Å structure of a tetrameric photosystem I complex from a heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Four photosystem I monomers, organized in a dimer of dimer, form two distinct interfaces that are largely mediated by specifically orientated polar lipids, such as sulfoquinovosyl diacylglycerol. The structure depicts a more closely connected network of chlorophylls across monomer interfaces than those seen in trimeric PSI from thermophilic cyanobacteria, possibly allowing a more efficient energy transfer between monomers. Our physiological data also revealed a functional link of photosystem I oligomerization to cyclic electron flow and thylakoid membrane organization.

Structure of the substrate-engaged SecA-SecY protein translocation machine.

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA's two-helix finger is close to the polypeptide, while SecA's clamp interacts with the polypeptide in a sequence-independent manner by inducing a short ß-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.

Non-targeted Metabolomic Analysis Based on Ultra-High-Performance Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry Reveals the Effects of Grafting on Non-volatile Metabolites in Fresh Tea Leaves ( Camellia sinensis L.).

To investigate the effects of grafting on non-volatile metabolites in tea, non-targeted metabolomic analyses of fresh leaves were performed on the basis of ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF/MS). One non-grafted YingHong No. 9 and four grafted tea [grafting scion YingHong No. 9 on four different rootstocks, BaiMao No. 2 (BM2), BaiYeDanCong (BY), HeiYeShuiXian (HY), and WuLingHong (WLH)] were chosen as materials. In total, 32 differential metabolites were identified, including phenolic acids, flavan-3-ols, dimeric catechins, flavonol and flavonol/flavone glycosides, etc. Partial least squares discrimination analysis and hierarchical cluster analysis showed various effects of different rootstocks on metabolites. Thereinto, rootstocks of WLH and BY showed extremely outstanding performance in up- and downregulating these metabolites, respectively. Differential metabolites were enriched into three crucial pathways, including biosynthesis of phenylpropanoids, flavonoid biosynthesis, and flavone and flavonol biosynthesis, which might influence the quality of tea. This study provides a theoretical basis for grafting-related variations of non-volatile metabolites in fresh tea leaves.

Study on the effects of rapid aging technology on the aroma quality of white tea using GC-MS combined with chemometrics: In comparison with natural aged and fresh white tea.

The effects of rapid aging technology on aroma compounds and characteristic of white tea were investigated in comparison with natural aged white tea (NAWT) and fresh white tea (FWT). Volatile compounds of white teas were extracted using headspace-solid phase microextraction (HS-SPME), and analyzed with gas-chromatography-mass spectrometry (GC-MS) coupled with chemometrics. Aroma metabolites analysis showed that 40 compounds were identified to distinguish rapid aged white tea (RAWT) from others. Principal component analysis (PCA), hierarchical cluster analysis (HCA), heatmap analysis and Venn diagram demonstrated that alcohols, aldehydes, ketones, esters, heterocyclics and alkanes exhibited significant variations. Quantitative descriptive analysis of white tea aroma showed that in RAWT, sweet and herbal aroma were significantly improved; whereas grassy green and delicate aroma declined sharply. This study provides a comprehensive investigation on aroma quality of RAWT, offering a potentially rapid way to produce aged white tea.

Study of the aroma formation and transformation during the manufacturing process of oolong tea by solid-phase micro-extraction and gas chromatography-mass spectrometry combined with chemometrics.

Oolong tea is a typical semi-fermented tea and is famous for its unique aroma. The aim of this study was to compare the volatile compounds during manufacturing process to reveal the formation of aroma. In this paper, a method was developed based on head-space solid phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) combined with chemometrics to assess volatile profiles during manufacturing process (fresh leaves, sun-withered leaves, rocked leaves and leaves after de-enzyming). A total of 24 aroma compounds showing significant differences during manufacturing process were identified. Subsequently, according to these aroma compounds, principal component analysis and hierarchical cluster analysis showed that the four samples were clearly distinguished from each other, which suggested that the 24 identified volatile compounds can represent the changes of volatile compounds during the four steps. Additionally, sun-withering, rocking and de-enzyming can influence the variations of volatile compounds in different degree, and we found the changes of volatile compounds in withering step were less than other two manufacturing process, indicating that the characteristic volatile compounds of oolong tea might be mainly formed in rocking stage by biological reactions and de-enzyming stage through thermal chemical transformations rather than withering stage. This study suggested that HS-SPME/GC-MS combined with chemometrics methods is accurate, sensitive, fast and ideal for rapid routine analysis of the aroma compounds changes in oolong teas during manufacturing processing.

Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1.

A key step in ribosome biogenesis is the nuclear export of pre-ribosomal particles. Nmd3, a highly conserved protein in eukaryotes, is a specific adaptor required for the export of pre-60S particles. Here we used cryo-electron microscopy (cryo-EM) to characterize Saccharomyces cerevisiae pre-60S particles purified with epitope-tagged Nmd3. Our structural analysis indicates that these particles belong to a specific late stage of cytoplasmic pre-60S maturation in which ribosomal proteins uL16, uL10, uL11, eL40 and eL41 are deficient, but ribosome assembly factors Nmd3, Lsg1, Tif6 and Reh1 are present. Nmd3 and Lsg1 are located near the peptidyl-transferase center (PTC). In particular, Nmd3 recognizes the PTC in its near-mature conformation. In contrast, Reh1 is anchored to the exit of the polypeptide tunnel, with its C terminus inserted into the tunnel. These findings pinpoint a structural checkpoint role for Nmd3 in PTC assembly, and provide information about functional and mechanistic roles of these assembly factors in the maturation of the 60S ribosomal subunit.

Mechanistic insights into the alternative translation termination by ArfA and RF2.

During cellular translation of messenger RNAs by ribosomes, the translation apparatus sometimes pauses or stalls at the elongation and termination steps. With the exception of programmed stalling, which is usually used by cells for regulatory purposes, ribosomes stalled on mRNAs need to be terminated and recycled to maintain adequate translation capacity. Much ribosome stalling originates in aberrant mRNAs that lack a stop codon. Transcriptional errors, misprocessing of primary transcripts, and undesired mRNA cleavage all contribute to the formation of non-stop mRNAs. Ribosomes stalled at the 3' end of non-stop mRNAs do not undergo normal termination owing to the lack of specific stop-codon recognition by canonical peptide release factors at the A-site decoding centre. In bacteria, the transfer-messenger RNA (tmRNA)-SmpB-mediated trans-translation rescue system reroutes stalled ribosomes to the normal elongation cycle and translation termination. Two additional rescue systems, ArfA-RF2 (refs 13, 14, 15, 16) and ArfB (formerly known as YaeJ), are also present in many bacterial species, but their mechanisms are not fully understood. Here, using cryo-electron microscopy, we characterize the structure of the Escherichia coli 70S ribosome bound with ArfA, the release factor RF2, a short non-stop mRNA and a cognate P-site tRNA. The C-terminal loop of ArfA occupies the mRNA entry channel on the 30S subunit, whereas its N terminus is sandwiched between the decoding centre and the switch loop of RF2, leading to marked conformational changes in both the decoding centre and RF2. Despite the distinct conformation of RF2, its conserved catalytic GGQ motif is precisely positioned next to the CCA-end of the P-site tRNA. These data illustrate a stop-codon surrogate mechanism for ArfA in facilitating the termination of non-stop ribosomal complexes by RF2.