Roles of cigar microbes in flavor formation during roasted-rice leachate fermentation.

Roasted-rice leachate fermentation, a distinctive local tobacco fermentation method in Sichuan, imparts a mellow flavor and glossy texture to tobacco leaves, along with a roasted rice aroma. In order to find out the impact of roasted-rice leachate on cigar tobacco leaves, the physicochemical properties, volatile flavor profile, and microbial community were investigated. The content of protein significantly decreased after fermentation. The volatile flavor compounds increased following roasted-rice leachate fermentation, including aldehydes, alcohols, acids, and esters. High-throughput sequencing identified Staphylococcus, Pseudomonas, Pantoea, Oceanobacillus, Delftia, Corynebacterium, Sphingomonas, Aspergillus, Weissella, and Debaryomyces as the primary genera. Network and correlation analysis showed Debaryomyces played a crucial role in roasted-rice leachate fermentation, due to its numerous connections with other microbes and positive relationships with linoelaidic acid, aromandendrene, and benzaldehyde. This study is useful for gaining insight into the relationship between flavor compounds and microorganisms and provides references regarding the effect of extra nutrients on traditional fermentation products. KEY POINTS: • Volatile flavor compounds increased following roasted-rice leachate fermentation • Staphylococcus was the primary genera in fermented cigar • Debaryomyces may improve the quality of tobacco leaves.

Prognostic value of miR-223 for pregnancy outcomes in patients with in vitro fertilisation and intracytoplasmic sperm injection.

BACKGROUND: This study aimed to analyse the expression of microRNA-223 (miR-223) in embryo culture medium and its correlation with pregnancy outcomes. METHODS: Two hundred and two patients undergoing in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) were divided into clinical pregnancy group (n = 101) and non-pregnant group (n = 101). The baseline data, clinical indicators, and the expression level of miR-223 in the embryo medium were compared between the two groups. Logistic regression analysis was used to analyse the relationship between each index and the pregnancy outcome. Receiver operator characteristic curve was carried out to evaluate the differential ability of miR-223 in pregnancy status. Bioinformatics methods were used to identify the target genes of miR-223 and elucidate their functions. RESULTS: Compared with pregnancy group, the non-pregnancy group exhibited a reduction in miR-223 expression (p < 0.001). Multivariate analysis revealed that miR-223 reduction was an independent factor for pregnancy failure (p < 0.05). The ROC curve demonstrated the discriminative capability of miR-223 in distinguishing pregnancy and non-pregnancy. In addition, bioinformatics analysis indicated that the target genes of miR-223 were predominantly located in the endocytic vesicle membrane and were primarily enriched in adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathways. CONCLUSION: In this study, levels of miR-223 in the embryo culture medium predicted pregnancy outcomes in subjects undergoing IVF/ICSI. Low expression of miR-223 was a risk factor for adverse pregnancy outcomes in subjects.

In this study, 202 patients who underwent IVF/ICSI were retrospectively analysed and categorised into pregnant and non-pregnant groups based on their pregnancy status. The examination of embryo culture medium samples from both groups revealed that the non-pregnant group exhibited lower miR-223 expression compared to the pregnant group. Subsequent ROC analysis demonstrated the clinical relevance of miR-223 in effectively distinguishing between pregnant and non-pregnant states. Multi-factor analysis further established that the diminished expression of miR-223 independently influenced the likelihood of successful pregnancy.

Physiological, transcriptome and co-expression network analysis of chlorophyll-deficient mutants in flue-cured tobacco.

BACKGROUND: Photosynthetic pigments in higher plants, including chlorophyll (Chl) and carotenoids, are crucial for photosynthesis and photoprotection. Chl-deficient tobacco seedlings generally have a lower photosynthesis rate and higher nitrate-nitrogen (NO3-N) content, which causes a profound influence on tobacco yield and quality. In this study, a stable albino leaf mutant (Al) and slight-green leaf mutant (SG) obtained from the common flue-cured tobacco (Nicotiana tabacum L.) cultivar 'Zhongyan 100' (ZY100) by mutagenesis with ethyl methanesulfonate (EMS) were used as materials. The differences between the Chl-deficient mutants and the wild-type (WT) were analyzed in terms of biomass, photosynthetic fluorescence parameters, and carbon- and nitrogen-related physiological parameters. RNA sequencing (RNA-seq) and weighted gene co-expression network analysis (WGCNA) were used to explore the key pathways and candidate genes regulating differentiated chlorophyll and nitrate content. RESULTS: The results showed that, when compared to the WT, the Chl content and biomass of mutant plants were considerably lower while the NO3-N content was substantially elevated. The net photosynthetic rate, photosynthetic fluorescence parameters, carbohydrate, soluble protein, and carbon- and nitrogen-related enzyme activities all decreased in leaves of mutants and the development of chloroplasts was abnormal. Applying more nitrogen improved the growth and development of mutants, whereas NO3-N content distinctively increased compared with that of the WT. Through transcriptome sequencing, the downregulated genes in mutants were enriched in plant hormone signal transduction and nitrogen metabolism, which are involved in pigment biosynthesis and the carbon fixation pathway. In addition, two hub genes and seven transcription factors identified from the blue module through WGCNA were likely to be key candidate factors involved in chlorophyll synthesis and nitrate accumulation. CONCLUSION: Our results demonstrated that differences in chlorophyll and nitrate content were caused by the combined effects of chloroplast development, photosynthesis, as well as related biological activity. In addition, transcriptome results provide a bioinformatics resource for further functional identification of key pathways and genes responsible for differences in chlorophyll and nitrate content in tobacco plants.

Single-cell transcriptome analyses reveal cellular and molecular responses to low nitrogen in burley tobacco leaves.

Tobacco (Nicotiana tabacum) is cultivated and consumed worldwide. It requires great amounts of nitrogen (N) to achieve the best yield and quality. With a view to sustainable and environmentally friendly agriculture, developing new genotypes with high productivity under low N conditions is an important approach. It is unclear how genes in tobacco are expressed at the cellular level and the precise mechanisms by which cells respond to environmental stress, especially in the case of low N. Here, we characterized the transcriptomes in tobacco leaves grown in normal and low-N conditions by performing scRNA-seq. We identified 10 cell types with 17 transcriptionally distinct cell clusters with the assistance of marker genes and constructed the first single-cell atlas of tobacco leaves. Distinct gene expression patterns of cell clusters were observed under low-N conditions, and the mesophyll cells were the most important responsive cell type and displayed heterogene responses among its three subtypes. Pseudo-time trajectory analysis revealed low-N stress decelerates the differentiation towards mesophyll cells. In combination with scRNA-seq, WGCNA, and bulk RNA-seq results, we found that genes involved in porphyrin metabolism, nitrogen metabolism, carbon fixation, photosynthesis, and photosynthesis-antenna pathway play an essential role in response to low N. Moreover, we identified COL16, GATA24, MYB73, and GLK1 as key TFs in the regulation of N-responsive genes. Collectively, our findings are the first observation of the cellular and molecular responses of tobacco leaves under low N stress and lay the cornerstone for future tobacco scRNA-seq investigations.

Effects of fermentation chamber temperature on microbes and quality of cigar wrapper tobacco leaves.

The natural fermentation of cigar tobacco leaves usually utilizes natural temperature and humidity for fermentation. Cigars produced in China are often fermented in winter, and the low environmental temperatures can lead to slow heating of the tobacco stack, affecting the cigar tobacco leaves quality. This study aimed to determine the minimum chamber temperature required to initiate the process of fermentation for cigar tobacco leaves and to explore the impact of temperature on the microbial community of tobacco leaves. Here, the cigar variety "Dexue 1" were subjected to stacking fermentation under three temperature parameters (20 ℃, 27 ℃, 34 ℃). With an increase in environmental temperature, the temperature inside the stack of cigar leaves increased significantly, the protein, total sugar, starch, and total alkaloid content in fermented tobacco leaves decreased, and the aroma components and amino acid content increased. Microbial richness and community diversity associated with fermented tobacco were highest at chamber temperatures of above 27 ℃. The relative abundance of Chryseobacterium and Rhodococcus was significantly negatively correlated with protein, alkaloids, total sugar, and starch, and positively correlated with amino acids and aroma components. Chryseobacterium and Rhodococcus may be responsible for the degradation of macromolecular substances and the conversion of favorable aromatic substances, thus improving the tobacco leaves quality. This study demonstrated that increasing the fermentation chamber temperature above 27 ℃ was conductive to raising the inner-stack temperature, increased microbial diversity and aromatic quality, reduced the strength and irritation, and extremely enhanced the overall quality of fermented cigar tobacco leaves. KEY POINTS: • The environmental temperature of the fermentation chamber has a significant impact on the quality of tobacco • Temperature > 27 ℃ can initiate the process of cigar tobacco leaves fermentation and increase inner-stack temperature and microbial diversity and abundance • Chryseobacterium and Rhodococcus may be related to the degradation of macromolecular substances and the transformation of aromatic substances, thereby improving the quality of tobacco leaves.

Identification of Bacteria Associated with Tobacco Mildew and Tobacco-Specific Nitrosamines During Tobacco Fermentation.

Tobacco mildew and tobacco-specific nitrosamines (TSNAs) affect the quality of tobacco products during fermentation. Microbes are thought to play key roles in the development of specific properties of fermented tobacco; however, little is known about the bacteria involved in the fermentation process. This study aims to identify key microbes related to mildew and TSNA formation. Tobacco was fermented at 25 °C, 35 °C, and 45 °C for 2, 4, and 6 weeks, with unfermented samples used as controls. Our preliminary exploration found that TSNAs content elevated with the increase of temperature and period, and mildew was easy to occur at low temperature with short period. Hence, samples were divided into three groups: the temperature gradient group (25 °C, 35 °C, and 45 °C for 6 weeks); the low-temperature group (control, 25 °C for 2, 4, and 6 weeks); and the high-temperature group (control, 45 °C for 2, 4, and 6 weeks). After collecting fermented tobacco leaves, 16S rRNA gene sequencing was used to explore the structure and dynamic changes of bacterial community during fermentation. Methylobacterium and Deinococcus were shared between the temperature gradient and high-temperature groups and showed a linear downward trend; these might play a role in the production of TSNAs. Massilia, Ruminiclostridium, and Cellulosilyticum species increased with prolonged fermentation time in the low-temperature group; this might be associated with tobacco mildew. In summary, the microbial diversity of fermented tobacco was explored under different conditions. These findings might provide data and material support to improve the quality of fermented tobacco products; however, further omics based studies are warranted to analysis the gene and protein expression patter in the identified bacteria.

Reducing the nicotine content of tobacco by grafting with eggplant.

BACKGROUND: Nicotine is a stimulant and potent parasympathomimetic alkaloid that accounts for 96-98% of alkaloid content. A reduction in the amount of nicotine in cigarettes to achieve a non-addictive level is necessary. We investigated whether replacing tobacco root with eggplant by grafting can restrict nicotine biosynthesis and produce tobacco leaves with ultra-low nicotine content, and analyzed the gene expression differences induced by eggplant grafting. RESULTS: The nicotine levels of grafted tobacco leaves decreased dramatically. The contents of nornicotine, anabasine, NNN, NNK, NAT, total TSNAs and the nicotine of mainstream cigarette smoke decreased, and the contents of amino acids and the precursors of alkaloids increased in grafted tobacco. Eggplant grafting resulted in the differential expression of 440 genes. LOC107774053 had higher degrees in two PPI networks, which were regulated by LOC107802531 and LOC107828746 in the TF-target network. CONCLUSIONS: Replacing tobacco root with eggplant by grafting can restrict nicotine biosynthesis and produce tobacco leaves with ultra-low or zero nicotine content. The differential expression of LOC107774053 may be associated with eggplant grafting.

Influence of microbiota and metabolites on the quality of tobacco during fermentation.

BACKGROUND: To explore the optimum fermentation conditions for tobacco leaves and also screen the microbiota and metabolites that are beneficial for fermentation. METHODS: Tobacco leaves were fermented at 25 °C, 35 °C, and 45 °C for 2, 4, and 6 weeks, respectively. For identification of the best fermentation temperature, physicochemical properties and sensory quality of fermented tobacco were investigated. Subsequently, based on the appropriate temperature, 16 s rRNA sequencing and metabolomics analysis of tobacco were performed to monitor the change of microbes and metabolites during fermentation process (from 2 to 6 weeks). RESULTS: Sensory quality analysis indicated that fermentation at 45 °C for 6 weeks represented the optimum condition. Metabolomics analysis showed that a total of 415 metabolites were annotated. The increase of fermentation period led to significant changes of metabolites. Results revealed an increase in concentration of L-phenylalanine and sphingosine as well as decreased concentration of betaine and phytosphingosine with the prolongation of fermentation period (2 to 6 weeks). Distinct changes in the microbiota were also observed with prolongation of the fermentation time. Results revealed that Pseudomonas, Pantoea, and Burkholderia were dominant bacteria in fermentation at 45 °C for 6 weeks. With the extension of the fermentation time, the abundance of Pseudomonas increased, while that of Sphingomonas and Methylobacterium decreased. Furthermore, microbiota profiles were tightly relevant to the altered metabolites, especially compounds involved in the sphingolipid metabolism. CONCLUSION: Suitable fermentation conditions were 45 °C for 6 weeks; phytosphingosine and sphingosine might affect tobacco fermentation via the sphingolipid metabolism pathway. This study provides a theoretical basis for guiding tobacco fermentation and gives insights into reducing harmful substances during tobacco fermentation.

Identification of transcription factors of nitrate reductase gene promoters and NRE2 cis-element through yeast one-hybrid screening in Nicotiana tabacum.

BACKGROUND: This study aimed to identify the transcription factors of nitrate reductase genes (NIA1 and NIA2) promoters and hypothetical cis-element of NRE2. Based on the constructed cDNA library of Nicotiana tabacum K326, a yeast one-hybrid system was established using the Matchmaker® Gold Yeast One-Hybrid Library Screening System from Clontech. The transcription factors of NIA1 andNIA2 promoters and NRE2 cis-elements were screened. RESULTS: After sequencing and bioinformatics analysis, 15 cDNA sequences were identified: 9 for NIA1 (including XP_016503563.1 and NP_001312236.1), 3 for NIA2 (including XP_016510250.1), and 3 for NRE2 (including XM_016576899.1). XP_016503563.1 was annotated in PREDICTED: CRM-domain containing factor CFM3, and NP_001312236.1chloroplastic/mitochondrial-like in Nicotiana tabacum. NP_001312236.1 was annotated in Sulfite oxidase-like of Nicotiana tabacum. XP_016510250.1 was annotated as PREDICTED: uncharacterized protein LOC107827596 in Nicotiana tabacum. XM_016576899.1 was annotated in PREDICTED: Nicotiana tabacum RING-H2 finger protein ATL16-like (LOC107759033). CONCLUSION: A yeast one-hybrid library was successfully constructed. The identified transcription factors may provide a theoretical basis for the study of plant nitrate reductase.

Hemodynamic Solution Computation and Pathological Analysis in the Circle of Willis.

Hemodynamic indexes will change significantly compared to the normal range of many vascular diseases, therefore it is necessary to establish hemodynamic computation model. Blood circulation is periodically forced huge fluid flow network, the heart is generator of the entire fluid network, based on this hemodynamic characteristics, the circle of Willis's structure is simplified from the perspective of network and hemodynamics. According to hemodynamic equations and circuit graph theory, models blood flow network of the periodically forced hemodynamic equation, obtains the approximate solution of the harmonic waves form based on averaging computation. We apply this model in the network of the circle of Willis, which may help explain the development processes of cerebral circulation disease. The simulation results show that computing results consistent with the clinical observation of blood flow changes in cerebral infarction.

Biochemical, Physiological and Transcriptomic Comparison between Burley and Flue-Cured Tobacco Seedlings in Relation to Carbohydrates and Nitrate Content.

Burley tobacco is a genotype of chloroplast-deficient mutant with accumulates high levels of tobacco-specific nitrosamines (TSNAs) which would induce malignant tumors in animals. Nitrate is a principle precursor of tobacco-specific nitrosamines. Nitrate content in burley tobacco was significantly higher than that in flue-cured tobacco. The present study investigated differences between the two tobacco types to explore the mechanisms of nitrate accumulation in burley tobacco. transcripts (3079) related to the nitrogen and carbon metabolism were observed. Expression of genes involved in carbon fixation, glucose and starch biosynthesis, nitrate translocation and assimilation were significantly low in burley tobacco than flue-cured tobacco. Being relative to flue-cured tobacco, burley tobacco was significantly lower at total nitrogen and carbohydrate content, nitrate reductase and glutamine synthetase activities, chlorophyll content and photosynthetic rate (Pn), but higher nitrate content. Burley tobacco required six-fold more nitrogen fertilizers than flue-cured tobacco, but both tobaccos had a similar leaf biomass. Reduced chlorophyll content and photosynthetic rate (Pn) might result in low carbohydrate formation, and low capacity of nitrogen assimilation and translocation might lead to nitrate accumulation in burley tobacco.

Syndrome Differentiation Analysis on Mars500 Data of Traditional Chinese Medicine.

Mars500 study was a psychological and physiological isolation experiment conducted by Russia, the European Space Agency, and China, in preparation for an unspecified future manned spaceflight to the planet Mars. Its intention was to yield valuable psychological and medical data on the effects of the planned long-term deep space mission. In this paper, we present data mining methods to mine medical data collected from the crew consisting of six spaceman volunteers. The synthesis of the four diagnostic methods of TCM, inspection, listening, inquiry, and palpation, is used in our syndrome differentiation. We adopt statistics method to describe the syndrome factor regular pattern of spaceman volunteers. Hybrid optimization based multilabel (HOML) is used as feature selection method and multilabel k-nearest neighbors (ML-KNN) is applied. According to the syndrome factor statistical result, we find that qi deficiency is a base syndrome pattern throughout the entire experiment process and, at the same time, there are different associated syndromes such as liver depression, spleen deficiency, dampness stagnancy, and yin deficiency, due to differences of individual situation. With feature selection, we screen out ten key factors which are essential to syndrome differentiation in TCM. The average precision of multilabel classification model reaches 80%.

Transcriptome analysis reveals the mechanism of NaHCO3 promoting tobacco leaf maturation.

NaHCO3 accelerates the aging of tobacco leaves; however, the underlying molecular mechanisms have not been elucidated. This study aimed to explore the mechanism of NaHCO3 in the promotion of tobacco leaf maturation using transcriptome analysis. Leaves on plants or detached leaves of the tobacco variety, Honghua Dajinyuan, were sprayed with or without 1% NaHCO3. The leaf yellowing was observed, the pigment content and enzyme activities were determined and RNA sequencing (RNA-seq) was performed. Spraying NaHCO3 onto detached leaves was found to promote leaf yellowing. Pigment content, catalase activity, and superoxide dismutase activity significantly decreased, whereas peroxidase activity and malondialdehyde content significantly increased. RNA-seq demonstrated that spraying with NaHCO3 upregulated genes associated with cysteine and methionine metabolism; alpha-linolenic acid metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis and downregulated genes related to photosynthesis and carotenoid biosynthesis. Genes correlated with autophagy-other, valine, leucine, and isoleucine degradation, and the MAPK signaling pathway were upregulated while those correlated with DNA replication, phenylalanine, and tyrosine and tryptophan biosynthesis were downregulated in detached leaves sprayed with NaHCO3 compared with the plant leaves sprayed with NaHCO3. Overall, this study is the first to elucidate the molecular and metabolic mechanisms of NaHCO3 in the promotion of tobacco leaf maturation.

Low nitrogen-induced expression of cyclophilin in Nicotiana tabacum.

Leaf morphology and the leaf protein expression profiles of flue-cured tobacco grown in central Henan province of China under low nitrogen (low-N) and normal nitrogen (normal-N) nutrition were examined. The leaf length and width were measured at 50, 60, and 70 days after transplanting. Leaves grown under low-N conditions were shorter and more narrow than those grown under normal-N conditions. The protein expression profiles of tobacco leaves harvested at 70 days after transplanting were analyzed by 2-dimensional electrophoresis, and five differentially expressed proteins including a putative protein were identified. Except for the MCM protein-like protein, the other three differentially expressed proteins of cyclophilin-like protein, vacuolar invertase INV2, MAR-binding protein and the one putative protein showed increased expression in the low-N nutrition group. Among these proteins, the cyclophilin-like protein, which is a stress-responsive signal protein, may play pivotal roles in regulating leaf development under stress conditions. Real-time quantitative PCR analysis showed that the mRNA expression level of the cyclophilin-like protein at day 50, 60 and 70 under low-N conditions was 0.90, 1.43 and 6.9-fold higher than that under normal-N conditions, indicating that the gene expression of cyclophilin-like protein was strongly induced by low-N conditions.

Topping and grafting affect the alkaloid content and gene expression patterns of tobacco (Nicotiana tabacum L.).

In this study, we aimed to determine molecular mechanisms underlying the effects of topping and grafting in tobacco (Nicotiana tabacum L.) by comparing the alkaloid contents and enrichment pathways of differentially expressed genes (DEGs) among plants subjected to different combinations of topping and grafting treatments. Plants of the tobacco variety "Zhongyan 100" and eggplant (Solanum melongena L.) were grafted in four combinations as scions and rootstocks, respectively. The four treatment groups were tobacco with topping, tobacco without topping, topped tobacco grafted onto an eggplant rootstock, and non-topped tobacco grafted onto an eggplant rootstock. Tobacco leaves were collected on the day of topping, at 7 days after topping, and after flue curing, the alkaloid contents of the collected leaves were determined. Leaves of plants subjected to the different treatments were collected for RNA sequencing and screened for DEGs, which were subsequently subjected to functional enrichment analyses. Analyses revealed reductions in the leaf alkaloid contents of tobacco subjected to combined topping and eggplant grafting. Gene annotation indicated that topping influences biological processes such as starch metabolism and stress response, whereas grafting affected the biosynthesis and metabolic pathways of secondary metabolites. Downregulated DEGs between non-topped tobacco and eggplant-grafted topped tobacco and between topped and non-topped tobacco are mainly involved in inositol phosphate metabolic and biosynthetic processes. Downregulated DEGs between different grafting methods (eggplant-grafted non-topped tobacco vs. non-topped tobacco and eggplant-grafted topped tobacco vs. topped tobacco) are mainly involved in sesquiterpene synthase activity and photosynthesis. The findings of this study provide important insights into the molecular mechanisms underlying the effects of topping and grafting on tobacco plants.

Reducing nitrate and tobacco-specific nitrosamine level in burley tobacco leaves through grafting on flue-cured tobacco rootstock.

Nitrosation of pyridine alkaloids in tobacco generates tobacco-specific nitrosamines (TSNAs), which are notable toxicants in tobacco products and smoke. Burley tobacco, a chloroplast- and nitrogen (N)-deficient phenotype that accumulates high levels of nitrate-nitrogen (NO3-N) in its leaves, is particularly susceptible to TSNAs formation. In this study, reciprocal pot and field grafting experiments were conducted using burley tobacco Eyan No.1 and flue-cured tobacco K326 to investigate whether grafting burley tobacco scions on flue-cured tobacco rootstocks could enhance pigment biosynthesis and photosynthesis, while reducing the NO3-N level in burley tobacco leaves. Grafting burley tobacco scions on flue-cured tobacco rootstocks significantly increased the total pigment content, photosynthetic rate, biomass, nitrate reductase and glutamine synthetase activities, as well as ammonium-nitrogen (NH4-N), total soluble and reducing sugar, and soluble protein levels in burley tobacco leaves compared with burley tobacco self-rooting, while decreasing the NO3-N level and nitrate-N to total N ratio. Transcriptomic analysis revealed that grafting resulted in upregulated expression of genes involved in starch, sucrose, porphyrin, chlorophyll, and N metabolism, as well as carbon fixation and carotenoid biosynthesis. The findings suggest that grafting on high N use efficiency rootstock is an exceptionally promising means of decreasing NO3-N accumulation by improving photosynthesis and N metabolism in the scion, thereby reducing the levels of harmful TSNAs.

Corrective surgery for nipple depression in patients with plasmacytic mastitis - A single-center experience.

Background: Plasma cell mastitis (PCM) is a complex breast disease in the clinic. Currently, there are no unified diagnostic criteria for the disease and no standard treatment methods. The effects of hormone, Conventional Chinese medicine and other treatments are uncertain, with long treatment duration and notable side effects. Surgery is the preferred treatment, but the recurrence rate after conventional surgery is very high, which may be related to depression of the nipple. This study aimed to evaluate the efficacy of a novel corrective procedure in patients with cellular mastitis and depressed nipples. Methods: Patients with PCM who received surgical treatment in the Third Medical Center of PLA General Hospital from January 1996 to January 2018 were retrospectively analyzed. According to the presence or absence of nipple depression before surgery, the patients were divided into the nipple depression group and the non-nipple depression group. In the nipple depression group, patients were subdivided into a novel corrective surgery group ("one" suture or half pocket suture) and a conventional corrective surgery group (oil yarn traction valgus correction of nipple depression). Demographic, clinical, therapeutic, and postoperative relapse data were collected and analyzed. Results: Compared with the patients in the non-nipple depression group, patients in the nipple depression group had a significantly higher recurrence risk after surgery (HR = 2.129 95% CI: 1.110-4.083, p = 0.023). Patients who underwent novel corrective surgery had a significantly lower recurrence risk than those who underwent conventional corrective surgery (HR = 0.363 95% CI: 0.150-0.880, p = 0.025). In addition, the novel corrective surgery significantly reduced the postoperative recurrence risk (HR = 0.088 95% CI: 0.009-0.886, p = 0.037). Conclusion: How to correct nipple depression is a critical factor for postoperative recurrence of PCM, and this novel corrective surgery for nipple depression can effectively reduce the postoperative recurrence rate in patients with nipple depression.

CO2 sequestration and CaCO3 recovery with steel slag by a novel two-step leaching and carbonation method.

The steel smelting process produces extensive CO2 and Ca-containing steel slag (SS). Meanwhile, the low value utilization of steel slag results in the loss of Ca resources. CO2 sequestration utilizing SS can reduce carbon emissions while achieving Ca circulation. However, conventional SS carbon sequestration methods suffer from slow reaction rates, finite Ca usage efficiency, and difficulty separating the CaCO3 product from SS. Herein, an innovative two-step leaching (TSL) and carbonation method was presented based on the variations in leaching efficiency of activated Ca under different conditions, aiming at efficient leaching, carbon sequestration, and high-value reuse of SS. This method employed two NH4Cl solutions in sequence for two leaching operations on SS, allowing the Ca leaching rate to be effectively increased. According to the findings, TSL could increase the activated Ca leaching rate by 26.9 % and achieve 223.15 kg CO2/t SS sequestration compared to the conventional one-step leaching (CSL) method. If part of the CaCO3 is recovered as a slagging agent, about 34.1 % of the exogenous Ca introduction could be saved. In addition, the CO2 sequestration of TSL did not significantly decrease after 8 cycles. This work proposes a strategy that has the potential for recycling SS and reducing carbon emissions.

Physiological and transcriptome analysis reveals the differences in nitrate content between lamina and midrib of flue-cured tobacco.

Nitrate is an important precursor of tobacco-specific nitrosamines (TSNAs) and a remarkable difference in nitrate accumulation between lamina and midrib of flue-cured tobacco has long been observed. However, the physiological and molecular mechanisms underpinning this difference remain poorly understood. In this study, physiological and genetic factors impacting nitrate accumulation were identified in pot experiments using flue-cured tobacco K326 with contrasting nitrate content between lamina and midrib. The results showed that three times higher NO3-N content was observed in midrib than that in the lamina, along with lower pigment, NH4-N content, nitrate reductase activity (NRA), sucrose synthetase activity (SSA), and glutamine synthetase activity (GSA) in midrib. Transcriptome analysis revealed that expression of genes involved in porphyrin and chlorophyll metabolism, carotenoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, starch and sucrose metabolism, nitrogen metabolism, and biosynthesis of amino acids were significantly lower in midrib than in lamina. qRT-PCR results showed that the expression level of nitrate transporter genes LOC107782967, LOC107806749, LOC107775674, LOC107829632, LOC107799198, LOC107768465 decreased by 2.74, 1.81, 49.5, 3.5, 2.64 and 2.96-folds while LOC107789301 increased by 8.23-folds in midrib but not in lamina. Reduced chlorophyll content might result in low carbohydrate formation which is the source of energy and carbon skeleton supply, then the low capacity of nitrogen reduction, assimilation and transportation, and the poor ability of nitrate reallocation but the high capacity of accumulation might lead to nitrate accumulation in midrib. The results laid the foundation for reducing nitrate content and TSNA formation in tobacco midribs and their products.

Phyllosphere microbial community of cigar tobacco and its corresponding metabolites.

Cigar is made of a typical fermented tobacco where the microbiota inhabits within an alkaline environment. Our current understanding on cigar fermentation is far from thorough. This work employed both high-throughput sequencing and chromatography-mass spectrometric technologies to provide new scientific reference for this specific fermented system. Typical cigar samples from different regions (the Caribbeans, South America, East Asia, and Southeast Asia) were investigated. The results show that Firmicutes, Actinobacteria, Proteobacteria, Ascomycota, and Basidiomycota were the predominant phyla in the cigar samples. Rather than the fungal community, it was the bacterial community structures that played vital roles to differentiate the cigar from different regions: Staphylococcus was the dominant genus in the Americas; Bacillus was the dominant genus in Southeast Asia; while in East Asia, there was no dominant genus. Such differences in community structure then affected the microflora metabolism. The correlation between microbiota and metabolites revealed that Aspergillaceae, Cercospora, and Staphylococcus were significantly correlated with sclareolide; Bacillus were positively associated with isophorone. Alcaligenaceae was significantly and positively correlated with L-nicotine and hexadecanoic acid, methyl ester. GRAPHICAL ABSTRACT.