Application of pectin hydrolyzing bacteria in tobacco to improve flue-cured tobacco quality.

To study the relationship between the diversity of the surface microbial community and tobacco flavor, and to improve tobacco quality using microorganisms. The microbial community composition and diversity of 14 samples of flue-cured tobacco from tobacco-producing areas in Yunnan with varying grades were analyzed by high-throughput sequencing. PICRUSt was used for predicting microbial functions. A strain of Bacillus amyloliquefaciens W6-2 with the ability to degrade pectin was screened from the surface of flued-cured tobacco leaves from Yunnan reroasted tobacco leave. The enzyme preparation was prepared through fermentation and then applied for treating flue-cured tobacco. The improvement effect was evaluated by measuring the content of macromolecule and the changes in volatile components, combined with sensory evaluations. The bacterial communities on the surface of flue-cured tobacco exhibited functional diversity, consisting primarily of Variovorax, Pseudomonas, Sphingomonas, Burkholderia, and Bacillus. These bacterial strains played a role in the aging process of flue-cured tobacco leaves by participating in amino acid metabolism and carbohydrate metabolism. These metabolic activity converted complex macromolecules into smaller molecular compounds, ultimately influence the smoking quality and burning characteristics of flue-cured tobacco. The pectinase preparation produced through fermentation using W6-2 has been found to enhance the aroma and sweetness of flue-cured tobacco, leading to improved aroma, reduced impurities, and enhanced smoothness. Additionally, the levels of pectin, cellulose, and hemicellulose decreased, while the levels of water-soluble sugar and reducing sugar increased, and the contents of esters, ketones, and aldehydes increased, and the contents of benzoic acid decreased. The study revealed the correlation between surface microorganisms and volatile components of Yunnan tobacco leaves, and the enzyme produced by the pectin-degrading bacteria W6-2 effectively improved the quality of flue-cured tobacco.

Metabolomics-based study of chemical compositions in cellulase additives derived from a tobacco-origin Bacillus subtilis and their impact on tobacco sensory attributes.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives.

Complete genome sequence of Bacillus cereus Z4, a biocontrol agent against tobacco black shank, isolated from the Western Pacific Ocean.

Bacillus species have been considered as promising biological control agents due to their excellent antimicrobial ability. Bacillus cereus strain Z4 was isolated from 2000 m deep sea sediments of the Western Pacific Ocean, which possesses significant antifungal activity against Phytophthora nicotianae, the pathogenic fungus of tobacco black shank disease. To reveal the underlying antifungal genetic mechanisms, here, we report the complete genomic sequence of the strain Z4. The genome has one circular chromosome of 5,664,309 bp with a G + C content of 35.31%, 109 tRNAs, and 43 rRNAs. Genomic analysis identified 10 gene clusters related to the biosynthesis of biocontrol active compounds, including bacillibactin, petrobactin, fengycin, and molybdenum cofactor. Meanwhile, 6 gene clusters were responsible for the biosynthesis of metabolites with unknown functions. Strain Z4 also contains a large number of genes encoding carbohydrate-active enzymes and secreted proteins, respectively. The whole genomic analysis of Bacillus cereus Z4 may provide a valuable reference for elucidating its biocontrol mechanism against tobacco black shank.

Application of starch degrading bacteria from tobacco leaves in improving the flavor of flue-cured tobacco.

Starch is an essential factor affecting the quality of flue-cured tobacco, and high starch content can affect the sensory quality and safety. Recently, the degradation of macromolecules in tobacco raw materials by using additional microorganisms to improve their intrinsic quality and safety has become a new research hotspot in the tobacco industry. However, the technical maturity and application scale are limited. Our study analyzed the correlation between microbial community composition and volatile components on the surface of tobacco leaves from 14 different grades in Fujian tobacco-producing areas. The PICRUSt software was utilized to predict the function of the microbial community present in tobacco leaves. Furthermore, dominant strains that produced amylase were screened out, and an enzyme solution was prepared to enhance the flue-cured tobacco flavor. Changes in the content of macromolecules and volatile components were determined, and sensory evaluations were conducted to assess the overall quality of the tobacco leaves. The results showed that the dominant bacterial genera on the surface of Fujian tobacco leaves were Variovorax, Sphingomonas, Bacillus, etc. Bacillus was positively correlated with various volatile components, which contributed to the sweet and aromatic flavors of Fujian flue-cured tobacco. The main genetic functions of Fujian flue-cured tobacco surface bacteria were carbohydrate metabolism and amino acid metabolism. After treating flue-cured tobacco with an enzyme preparation prepared by the fermentation of Paenibacillus amylolyticus A17 #, the content of starch, pectin, and cellulose in flue-cured tobacco decreased significantly compared with the control group. Meanwhile, the content of total soluble sugar and reducing sugar was significantly increased, and the volatile aroma components, such as 3-hydroxy--damascone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-Pyran-4-one, ethyl palmitate, ethyl linolenic acid, etc., were significantly increased. The aroma quality and quantity of flue-cured tobacco were enhanced, while impurities were reduced. The smoke characteristics were improved, with increased fineness, concentration, and moderate strength. The taste characteristics were also improved, with reduced irritation and a better aftertaste. In conclusion, Bacillus, as the dominant genus in the abundance of bacterial communities on tobacco surfaces in Fujian, had an essential impact on the flavor of tobacco leaves by participating in carbohydrate metabolism and finally forming the unique flavor style of flue-cured tobacco in Fujian tobacco-producing areas. Paenibacillus amylolyticus A17 #, a target strain with amylase-producing ability, was screened from the surface of Fujian flue-cured tobacco. The enzyme preparation, produced by the fermentation of Paenibacillus amylolyticus A17 #, was utilized to reduce the content of macromolecules, increase the content of water-soluble total sugar and reducing sugar, and produce a variety of crucial volatile aroma components, which had a significant improvement on the quality of tobacco leaves.

Syntheses of functionalized benzocoumarins by photoredox catalysis.

Direct functionalization of inert C(sp3)-H bonds is an attractive synthetic technology for the preparation of pharmaceutically significant compounds in modern synthetic organic chemistry. In this work, we report a new method for the synthesis of functionalized benzocoumarins through the strategy of activation of multiple C-H bonds on 2-aryl toluenes under visible-light-enabled photoredox conditions. This method has the advantages of high functional group compatibility, mild reaction conditions, and effectively avoiding the use of strong oxidants and precious metal catalysts. Detailed mechanistic investigations, including spectroscopic and electrochemical studies, support the reaction's mechanistic course.

Metagenomic insight into the microbial degradation of organic compounds in fermented plant leaves.

Microbial degradation of organic compounds is an environmentally benign and energy efficient part in product processing. Fermentation of plant leaves involves enzymatic actions of many microorganisms. However, microbes and enzymes discovered from natural degradation communities were still limited by cultural methods. In this study, we used a metagenomics sequence-guided strategy to identify the microbes and enzymes involved in compound degradation and explore the potential synergy among community members in fermented tobacco leaves. The results showed that contents of protein, starch, pectin, lignin, and cellulose varied in fermented leaves from different growing sites. The different compound contents were closely related to taxonomic composition and functional profiles of foliar microbial communities. Microbial communities showed significant correlations with protein, lignin, and cellulose. Vital species for degradations of protein (Bacillus cereus and Terribacillus aidingensis), lignin (Klebsiella pneumoniae and Pantoea ananatis) and cellulose (Pseudomonas putida and Sphingomonas sp. Leaf20) were identified and relating hydrolytic enzymes were annotated. Further, twenty-two metagenome-assembled genomes (MAGs) were assembled from metagenomes and six potential cellulolytic genomes were used to reconstruct the cellulose-degrading process, revealing the potential metabolic cooperation related to cellulose degradation. Our work should deepen the understanding of microbial roles in plant fermentation and provide a new viewpoint for applying microbial consortia to convert plant organic components to small molecules.

Prognostic Role of Soluble Programmed Death Ligand 1 in Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis.

OBJECTIVE: The objective of this study was to explore whether soluble programmed death ligand 1 (sPD-L1) is a potential prognostic biomarker in patients with non-small cell lung cancer (NSCLC). METHODS: A comprehensive search of electronic databases was carried out. Original studies with inclusion of sPD-L1, progression-free survival, and overall survival in NSCLC were eligible. The primary endpoints were overall survival and progression-free survival. Hazard ratios (HRs) and 95% confidence intervals (CIs) were applied for data analysis. RESULTS: Eight studies involving 710 patients with NSCLC were included in the analysis. A pooled data analysis revealed that high levels of sPD-L1 were correlated with poorer overall survival (HR = 2.34; 95% CI = 1.82-3.00; P < 0.001) and progression-free survival (HR = 2.35; 95% CI = 1.62-3.40, P < 0.001). A subgroup analysis revealed that high levels of sPD-L1 were correlated with poor overall survival in patients treated with immunotherapy (HR = 2.40; 95% CI = 1.79-3.22; P < 0.001). CONCLUSION: This pooled analysis of published data suggests that sPD-L1 may serve as a readily available biomarker for survival in NSCLC patients treated with ICI based treatment. Prospective studies with well-designed standard assessment methods should be conducted to validate the prognostic role of sPD-L1 in NSCLC. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021283177.

Bioremediation of Pyropia-processing wastewater coupled with lipid production using Chlorella sp.

Pyropia-processing wastewater (PPW) contains diverse organic nutrients and causes environmental pollution. To explore the nutrient removal efficiency and growth performance of Chlorella sp. on PPW, the cultures were conducted in different culture substrates. Results showed that, after 7 days of incubation, the removal rates of total nitrogen (TN), total phosphorus (TP) and phycobiliprotein (PP) all reached more than 90% by cultivating Chlorella sp. C2 and C. sorokiniana F-275 in PPW. The chemical oxygen demand (COD) removal efficiencies could be over 50%. Meanwhile, the increments of biomass in two tested Chlorella strains were 1.39 and 4.89 times higher than those of BG11 and BBM substrates and the increases in lipid productivity were 1.34 and 10.18- fold, respectively. The C18:3 fatty acid proportions were markedly reduced by 27.89% and 29.10%. These results suggest that Chlorella sp. could efficiently reduce various nutrients in PPW and simultaneously accumulate higher biomass with higher biodiesel characteristics.

Visible-Light-Enabled Oxidative Coupling of Alkenes with Dialkylformamides To Access Unsaturated Amides.

A practical and direct method for oxidative cross-coupling of alkenes with dialkylformamides is established employing visible-light-enabled photoredox catalysis. This strategy allows efficient access to diverse unsaturated amides under mild reaction conditions. The application of an appropriate diaryliodonium salt was demonstrated to be critical to the success of this process. This catalyst system is well tolerant of a variety of useful functional groups.

A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs.

Infection of apple by apple stem grooving virus leads to extensive alterations in gene expression patterns but no disease symptoms.

To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question of whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoot cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Thus, global host gene expression changes do not necessarily lead to virus disease symptoms. Our data suggest that the general approaches to correlate host gene expression changes under viral infection conditions to specific disease symptom, based on the interpretation of transcription profiling data and altered individual gene functions, may have limitations depending on particular experimental systems.

Genetic variation analysis of apple chlorotic leaf spot virus coat protein reveals a new phylogenetic type and two recombinants in China.

In this study, we generated sequences of the apple chlorotic leaf spot virus (ACLSV) coat protein (CP) gene. Genetic variation and phylogenetic analyses were carried out on these sequences along with others reported previously. ACLSV populations clustered into four types: in three of the four types, combinations of three amino acids at positions of 40, 75 and 79 were conserved. The fourth phylogenetic type, newly identified here, was characterized by co-variation of Ser(40)-Tyr(75)-Ser(79). Statistically significant genetic differentiation and infrequent gene flow were detected among the four types. Two natural recombinants were detected for the first time among ACLSV isolates/genotypes from China.

VEGF-specific siRNAs modified with 2'-deoxy effectively suppress VEGF expression and inhibit growth of nasopharyngeal carcinoma xenograft in a mouse model.

Vascular endothelial growth factor (VEGF) is up-regulated in the vast majority of human tumors. The up-regulation of VEGF not only plays important roles in tumor angiogenesis, but also provides a target for tumor treatment with small interfering RNA (siRNA) that targets VEGF; however, it is unclear whether a quite high up-regulation of VEGF will affect the efficiency of RNA interference strategies targeting VEGF. A high level expression of VEGF was found in CNE cells from a nasopharyngeal carcinoma cell line. In this study, we investigate whether VEGF-specific siRNAs can effectively suppress VEGF expression in CNE cells, and study the methods for the use of VEGF-specific siRNAs as potential therapeutic agents. CNE cells with high VEGF expression induced by hypoxia were transfected with VEGF-specific siRNAs. The expression of VEGF was effectively suppressed by VEGF-specific siRNAs, measured by ELISA, Western blot analysis and RT-PCR. Furthermore, experiments in nude mice bearing nasopharyngeal carcinoma xenograft were initiated 5 d after injection of CNE cells. VEGF-specific siRNAs were modified with 2'-deoxy, then injected into the tumors, and a liposome-mediated siRNA transfection system and ultrasound exposure were used to help delivery of the siRNAs. Tumor growth was reduced significantly after 3 weeks' treatment. These studies suggest that VEGF-specific siRNAs still can effectively suppress VEGF expression even in tumor cell lines with a relatively high level of VEGF expression, such as CNE, and VEGF-specific siRNAs modified with 2'-deoxy can be used as potential agents for tumor therapy.

Fenretinide-induced neuronal differentiation of ARPE-19 human retinal pigment epithelial cells is associated with the differential expression of Hsp70, 14-3-3, pax-6, tubulin beta-III, NSE, and bag-1 proteins.

PURPOSE: We reported earlier that fenretinide can induce neuronal differentiation of ARPE-19 human retinal pigment epithelial cells in culture. The purpose of this study was to investigate the potential involvement of key proteins involved in gene transcription, signal transduction, cell cycle check point, differentiation, neuronal cell survival, and stress response in the neuronal differentiation of ARPE-19 cells by fenretinide. METHODS: Cells in culture were treated with 1.0 microM fenretinide. Cells were analyzed using antibodies against pax-6, neuronal specific enolase (NSE), tubulin beta-III, 14-3-3, bag-1, and Hsp-70 proteins using immunocytochemistry, western blot and ELISA methodologies. RESULTS: We found that pax-6 and NSE were both expressed in the control ARPE-19 cells. Fenretinide induced neuronal differentiation of ARPE-19 cells led to a decrease in pax-6 protein and an increase in tubulin beta-III protein expression after 5 days fenretinide treatment. There was a translocation of 14-3-3 from the cytoplasm to the nucleus, and an increase in nuclear expression of bag-1 after treatment. We also found a time-dependent increase in Hsp70 protein expression in ARPE-19 cells treated with fenretinide. D-407, another human retinal pigment epithelial cell line, but not either Y-79 or PC-12 cells, was also able to be induced into neuronal morphologies by fenretinide. CONCLUSIONS: The fenretinide-induced neuronal differentiation of ARPE-19 cells is associated with an increase in expression of the neuronal specific protein tubulin beta-III, and a decrease in expression of the progenitor cell marker pax-6. Neuronal differentiation of ARPE-19 cells is also associated with nuclear translocation of 14-3-3, a protein involved in signal transduction, cell cycle check point and cell growth, and an increase in expression of bag-1, a protein involved in neuronal cell survival and axon elongation. These results suggest that ARPE-19 cells could be a progenitor cell line that can be differentiated into neuronal cells when treated with factors such as fenretinide.

Immunofluorescence analysis of the expression of Norpeg (Rai14) in retinal Müller and ganglion cells.

Novel retinal pigment epithelial cell gene (Norpeg, Rai14), a developmentally regulated mouse gene, encodes a protein containing six ankyrin repeats and a coiled-coil domain. The objective of the present study was to characterize the Norpeg protein and to analyze its expression in mouse retina using an antibody preparation that we developed. The approximately 110kDa Norpeg was immunoprecipitated and characterized by mass spectrometry. Primary cultures of Müller and ganglion cells isolated from the mouse retina were found to express Norpeg when analyzed by immunoblotting and immunofluorescence microscopy. Immunofluorescence analysis of normal mouse retina sections demonstrated that Norpeg is expressed in cells of the ganglion cell layer, inner nuclear layer as well as in the retinal pigment epithelium. Immunoreactivity was also evident in the radial glial (Müller) cell fibers.

Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein.

NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P(270)KKRKAP(276)) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

[Expressions of P16 and nm23-H1 in nasopharyngeal cacinoma and their clinical significance].

OBJECTIVE: To examine the expressions of P16 and nm23-H(1) protein in nasopharyngeal carcinoma (NPC) and explore their association with clinicopathology and the level of argyrophilic protein of the nucleolar organizer regions (AgNORs) of peripheral blood T lymphocyte (T-AgNORs) before radiotherapy. METHODS: SP immunohistochemistry was used to detect P16 and nm23-H(1) expressions in 65 cases of NPC, and the level of T-AgNORs was measured before radiotherapy. RESULTS: The positivity rate of P16 and nm23-H(1) protein in NPC was 24.6% (16/65) and 61.5% (40/65), respectively. The positivity rates of P16 and nm23-H(1) protein in patients in T(1) and T(2) stages were higher than those in patients in T(3) and T(4) stages, but the difference was not statistically significant (P>0.05). No significant association of P16 expression was noted with lymph node metastasis and post-radiotherapy distant metastasis (P>0.05). In patients with regional lymph node and distant metastases, the positivity rates for nm23-H(1) were 51.2% (21/41) and 33.3% (5/15), which were significantly lower than those in patients without local lymph node (79.2%, 9/24;X(2)= 4.99, P<0.05) or distant metastasis (70%, 35/50; X(2)=7.56, P<0.01). The levels of T-AgNORs in P16- and nm23-positive cases were higher than those in the negative cases (t=5.721, P<0.001). CONCLUSIONS: nm23-H(1) expression in NPC tissue is correlated with NPC metastasis and may be useful for clinical prediction of local lymph node and distant metastases after radiotherpay. nm23 and p16 expressions are also correlated with the level of T-AgNORs, which probably indicate the immune functions of the patients.

Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N-(4-hydroxyphenyl)retinamide.

ARPE-19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N-(4-hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE-19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide-induced neuronal differentiation was dose and time dependent, started within 1-2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde-binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two-dimensional gel followed by matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated that heat-shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.