Imaging findings of vertebral osteomyelitis caused by nontuberculous mycobacterial organisms: Three case reports and literature review.

RATIONALE: Prompt diagnosis of nontuberculous Mycobacterial (NTM) vertebral osteomyelitis is challenging, yet necessary to prevent serious morbidity and mortality. Here, we report 3 cases of vertebral osteomyelitis caused by NTM with imaging findings. PATIENT CONCERNS: Case 1, a 58-year-old male patient, was admitted to our hospital because of the presence of a pulmonary mass for 6 months with cough and chest pain.Case 2, a 50-year-old male patient, had fever and cough for 3 years and was diagnosed with tuberculosis. Antituberculosis treatment was ineffective, accompanied by lymph node enlargement and osteosclerotic changes involving vertebral bodies.Case 3, a 66-year-old female patient, was admitted to our hospital with a mass on the top of her head for 1 month, which ruptured in the last 2 weeks. DIAGNOSES: Case 1: Sputum culture revealed Mycobacterium (M.) avium.Case 2: The final culture results of the lymph node biopsy samples were M. intracellulare.Case 3: Culture results of the sputum and pus from the abscess were M. gordon.We found sclerosing lesions in the spine in all 3 NTM patients, which were easily misdiagnosed as metastatic tumors. In 2 cases, there was bone destruction in the ilium with limbic sclerosis, and there were abscesses near the ilium and in front of the sacrum in 1 case. INTERVENTIONS: Case 1 was transferred to other specialist hospital.Case 3 received surgical treatment for cranial lesions and abscess drainage.Case 2 and case 3 received targeted treatment for nontuberculous mycobacteria in our hospital. OUTCOME: The condition of case 1 was unknown.Recovery of case 2 was uneventful because of prolonged illness; however, inflammation gradually improved overall.Case 3 had no recurrence following surgical treatment. LESSONS: In our 3 cases of NTM vertebral osteomyelitis, bone lesions were often misdiagnosed as bony metastases because of the presence of multiple sclerotic lesions. Diagnoses were challenging and delayed. It is important to consider osteomyelitis by NTM when disseminated osteosclerosis with or without osteolytic bone lesions is present in conjunction with continuous inflammatory symptoms and signs. Moreover, an open biopsy of the lesion should be performed for a definitive diagnosis.

Physiological and transcription analyses reveal regulatory pathways of 6-benzylaminopurine delaying leaf senescence and maintaining quality in postharvest Chinese flowering cabbage.

The regulatory role of cytokinins (CTKs) in leaf senescence has been documented in different species, including Chinese flowering cabbage. However, its physiological and molecular basis relatively remains unknown. In this study, exogenous application of a CTK analogue 6-benzylaminopurine (6-BA) significantly retarded leaf senescence of Chinese flowering cabbage, with less chlorophyll degradation and lower accumulation of reactive oxygen species (ROS) and malondialdehyde compared with the control group. Meanwhile, higher levels of soluble sugars and proteins, flavonoids, cellulose, amino acids, total phenols, procanthocyanins, and vitamin C were retained in 6-BA-treated leaves. 6-BA treatment also prevented the decline in endogenous CTK content and the increase in ethylene, abscisic acid, and jasmonic acid contents. Moreover, the comparative transcriptome analysis revealed that a total of 21,895 differently expressed genes (DEGs) were identified from four comparisons of 6-BA treatment versus the control during senescence. Further analysis showed that most of the identified DEGs were enriched in ROS, respiratory metabolism, and phytohormone pathways, and a total of 50 classes of transcription factors that were possibly involved in modulating these DEGs were obtained. The transcriptional levels of 18 DEGs were verified by Quantitative real-time PCR (qRT-PCR), which confirmed the accuracy of the transcriptomic data. Overall, these findings and data provide a comprehensive view of physiological and molecular events concerning with the CTK-mediated leaf senescence and -maintained quality in economical leafy vegetables.

Methyl jasmonate pretreatment improves aroma quality of cold-stored 'Nanguo' pears by promoting ester biosynthesis.

Cold storage is widely used for delaying ripening and senescence; however, fruit aroma diminishes noticeably after long-term cold storage. The esters synthesized by the lipoxygenase (LOX) pathway are responsible for 'Nanguo' pear aroma. As methyl jasmonate (MeJA) is known to act on various fruit qualities, we investigated whether it acts via the LOX pathway in cold-stored 'Nanguo' pears. MeJA treatment increased the content of volatile esters and unsaturated fatty acids and the activities of alcohol acyltransferase, alcohol dehydrogenase, and LOX. It also up-regulated the expression of key genes (PuAAT, PuADH3, PuADH5, PuADH9, PuLOX1, and PuLOX3) in the LOX pathway and that of transcription factors (PuMYB21-like, PuMYB108-like, PuWRKY61, PuWRKY72, and PuWRKY31), whose genes were differentially expressed in preliminary transcriptome analysis. Therefore, considering its effects on LOX pathway-related genes and transcription factors, MeJA may be useful in preventing cold-storage-induced decline in ester biosynthesis, aroma, and consequently the quality of cold-stored 'Nanguo' pears.

PuMYB21/PuMYB54 coordinate to activate PuPLDß1 transcription during peel browning of cold-stored "Nanguo" pears.

Refrigeration is commonly used to extend the storage life of "Nanguo" pears, but fruit in long-term refrigeration is prone to peel browning, which is related to membrane lipid degradation. To determine the mechanism of membrane lipid degradation, we identified two R2R3-MYB transcription factors (TFs), PuMYB21 and PuMYB54, from "Nanguo" pears, which were notably expressed in response to cold stress and during the peel-browning process. The results from yeast one-hybrid, electrophoretic mobility shift, and transient expression assays indicated that both PuMYB21 and PuMYB54 directly bind to the promoter of PuPLDß1 (a key enzyme catalyzing the hydrolysis of membrane phospholipids) and activate its expression, which probably enhances the degradation of membrane phospholipids and eventually results in peel browning. Moreover, the overexpression of PuMYB21 and PuMYB54 can greatly activate the transcription of endogenous PuPLDß1 in both "Nanguo" pear fruits and calli, and their silencing can inhibit its transcription. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays verified that PuMYB21 interacts with PuMYB54 to enhance the expression of PuPLDß1. In summary, we demonstrate that PuMYB21 and PuMYB54 may have roles in membrane lipid metabolism by directly binding to the downstream structural gene PuPLDß1 during the low temperature-induced peel browning of "Nanguo" pears.

Glycine betaine treatment alleviates loss of aroma-related esters in cold-stored 'Nanguo' pears by regulating the lipoxygenase pathway.

Glycine betaine (GB) is known to alleviate chilling injury in many fruit species. Therefore, we studied how GB affects the biosynthesis of esters in 'Nanguo' pears. Based on the kinds of esters, total esters, and the quantity of the main esters, it was evident that aroma losses were alleviated by GB treatment. In addition, unsaturated fatty acids contents (linoleic and linolenic acid) and the activities of lipoxygenase (LOX) and alcohol acyltransferase (AAT) enzymes were also increased. Meanwhile, comparing with the control fruit, the genes directly involved in ester synthesis were up-regulated in the GB-treated fruit. In addition, an increase in the activities and gene expression of antioxidant enzymes was observed in the treated samples. Thus, GB treatment promotes the synthesis of esters by regulating the LOX pathway and increasing antioxidant capacity, thereby effectively improving the quality of esters in cold-stored fruit.

Transcription factor CaNAC1 regulates low-temperature-induced phospholipid degradation in green bell pepper.

Phospholipids constitute the main component of biomembranes. During low-temperature storage and transportation of harvested bell peppers (Capsicum annuum), chilling injury participates in their decay. A primary cause of this chilling injury is phospholipid degradation. In this study, three genes encoding phospholipase D (PLD) were identified from bell peppers and their activities were examined under cold stress. Low temperature (4 °C) induced strong accumulation of the CaPLDα4 transcript, suggesting that it is associated with the phenomenon of phospholipid degradation and destruction of cell membranes. Low temperature also significantly induced increased amounts of NAM-ATAF1/2-CUC2 (NAC) domain transcription factors. CaNAC1 was found to interact with the promoter of CaPLD4 in a yeast one-hybrid screen. Electrophoretic mobility shift and ß-glucuronidase reporter assays demonstrated that CaNAC1 binds to the CTGCAG motif in the CaPLDα4 promoter, thereby activating its transcription and controlling phospholipid degradation. The ubiquitination sites of the CaNAC1 protein were characterized by liquid chromatography-tandem mass spectrometry. We conclude that CaNAC1 is a transcriptional activator of CaPLDα4 and suggested that it participates in the degradation of membrane lipids in bell peppers when they are stored at low temperature.

Proteomic analysis of the potential mechanism of fading of aroma-related esters in "Nanguo" pears after long-term refrigeration.

Low-temperature storage is a key method for delaying the ripening of "Nanguo" pears. However, the aroma of "Nanguo" pears fades after long-term refrigeration. We investigated the potential mechanism of fading of aroma in "Nanguo" pears by analyzing differentially expressed proteins in pears stored at room temperature, which had higher level of aromatic esters and those stored at low temperature, having lower esters. We observed that 293 kinds of proteins were down-regulated and 377 were up-regulated. Gene ontology analysis showed that proteins in the "catalytic activity," "metabolic process," "organelle," and "membrane" proteins were affected by low temperature. KEGG analysis showed that the differentially expressed proteins were involved in oxidative phosphorylation, carbon and fatty acid metabolism. Real-time PCR showed that transcription levels of nine selected genes correlated with differentially expressed proteins. The results revealed that the expression of potentially aroma-related proteins, which are important in further research on improving aroma quality of "Nanguo" pears. PRACTICAL APPLICATIONS: "Nanguo" pears aroma is an important character for attracting consumers and many proteins are involved in the synthesis of aroma. However, their aroma is lost after cold storage and the quality of fruits is affected by low temperature. It is, therefore, of great significance to study the potential proteins that regulate the aroma of refrigerated "Nanguo" pears. In addition, the study results could provide basic and scientific data for the study of improving the aroma quality and genetic improvement of "Nanguo" pears.

Transcriptome profiling reveals the roles of pigment mechanisms in postharvest broccoli yellowing.

Postharvest broccoli is prone to yellowing during storage, which is the key factor leading to a reduction in value. To explore appropriate control methods, it is important to understand the mechanisms of yellowing. We analyzed the genes related to the metabolism of chlorophyll, carotenoids, and flavonoids and the transcription factors (TFs) involved in broccoli yellowing using transcriptome sequencing profiling. Broccoli stored at 10 °C showed slight yellowing on postharvest day 5 and serious symptoms on day 12. There were significant changes in chlorophyll fluorescence kinetics, mainly manifesting as a decrease in the Fv/Fm value and an increase in nonphotochemical quenching, during the yellowing process. Transcriptome sequencing profiles from samples of fresh broccoli and broccoli with slight and severe yellowing revealed 6, 5, and 4 differentially expressed genes involved in chlorophyll metabolism, carotenoid biosynthesis, and flavonoid biosynthesis, respectively. The transcription factor gene ontology categories showed that the MYB, bHLH, and bZip gene families were involved in chlorophyll metabolism. In addition, the transcription factor families included NACs and ethylene response factors (ERFs) that regulated carotenoid biosynthesis. Reverse transcription polymerase chain reaction further confirmed that bHLH66, PIF4, LOB13, NAC92, and APL were vital transcription factors that potentially regulated the CAO and HYD genes and were involved in chlorophyll metabolism and the carotenoid biosynthetic process. The flavonoid biosynthetic pathway was mainly regulated by MYBs, NACs, WRKYs, MADSs, and bZips. The results of the differentially expressed gene (DEG) and pigment content analyses indicated that the transcriptome data were accurately and positively associated with broccoli yellowing.

Low-temperature stress-induced aroma loss by regulating fatty acid metabolism pathway in 'Nanguo' pear.

Low temperatures retard postharvest ripening and prolong the supply period of 'Nanguo' pears. However, quality deterioration, or more specifically, a faded aroma occurs in long-term cold-stored fruits. To understand the implicit mechanism, we analyzed the change in content of the main aroma esters and fatty acids by chromatography-mass spectrometry (GC-MS), and the expression patterns of the main genes in fatty acid metabolism pathways. The results showed that hexyl hexanoate disappeared completely and the content of five other aromas declined significantly in cold-stored fruit during the optimal taste period. The content of saturated and unsaturated fatty acids significantly increased and decreased, respectively in cold-stored fruit. fadD, fadE, fadJ, atoB, fabF, SCD, FAD2, LOX2S, LOX1_5 and HPL genes down-regulated in fatty acid metabolism during shelf life in the fruit after cold storage. In conclusion, the faded aroma of cold-stored fruit was mainly regulated by fatty acid metabolism pathway.

Chlorophyll degradation and carotenoid biosynthetic pathways: Gene expression and pigment content in broccoli during yellowing.

Broccoli undergoes yellowing in unfavorable conditions, thereby diminishing the sensory quality and commodity value. This study aimed to investigate systematically cellular and/or biomolecular changes involved in broccoli yellowing by analyzing changes in microstructural integrity, pigment content, and gene expression. On day-5 of storage at 20 °C, the buds turned yellow without blooming and showed structural damage; ultrastructural analysis revealed plastid transformation and abnormal chloroplast development. Genes regulating pigment content and chloroplast structure directly were identified. More specifically, BoCAO and BoNYC1 regulated chlorophyll turnover, affecting chlorophyll a and b contents. Changes in the ß-cryptoxanthin content were influenced by the combined action of up- (BoHYD) and downstream (BoZEP) genes. BoZEP and BoVDE were activated after cold-temperature induction. High BoHO1 expression delayed yellowing at low temperature, inducing BoZEP expression. Color intensity correlated significantly with the chlorophyll b, ß-cryptoxanthin, and ß-carotene contents, which were associated with increased yellowing of plant tissues.

Transcriptome analysis of harvested bell peppers (Capsicum annuum L.) in response to cold stress.

Bell peppers are valued for their plentiful vitamin C and nutritional content. Pepper fruits are susceptible to cold storage, which leads to chilling injury (CI); however, the crucial metabolic product and molecular basis response to cold stress have not been elucidated definitely yet. To comprehensively understand the gene regulation network and CI mechanisms in response to cold stress on a molecular level, we performed high-throughput RNA-Seq analysis to investigate genome-wide expression profiles in bell peppers at different storage temperatures (4 °C and 10 °C). A total of 61.55 Gb of clean data were produced; 3863 differentially expressed genes (DEGs) including 1669 up-regulated and 2194 down-regulated were annotated and classified between the CI group and control. Together, a total of 41 cold-induced transcription factor families comprising 250 transcription factors (TFs) were identified. Notably, numerous DEGs involved in biomembrane stability, dehydration and osmoregulation, and plant hormone signal transduction processes were discovered. The transcriptional level of 20 DEGs was verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Our results present transcriptome profiles of bell peppers in response to cold stress; the data obtained may be useful for the identification of key candidate genes and elucidation of the mechanisms underlying membrane damage during chilling injury.

miRNAs play important roles in aroma weakening during the shelf life of 'Nanguo' pear after cold storage.

Cold storage is commonly employed to delay senescence in 'Nanguo' pear after harvest. However, this technique also causes fruit aroma weakening. MicroRNAs are regulators of gene expression at the post-transcriptional level that play important roles in plant development and in eliciting responses to abiotic environmental stressors. In this study, the miRNA transcript profiles of the fruit on the first day (C0, LT0) move in and out of cold storage and the optimum tasting period (COTP, LTOTP) during shelf life at room temperature and after cold storage were analyzed, respectively. 314 known miRNAs were identified in 'Nanguo' pear; 176 and 135 miRNAs were significantly differentially expressed on the C0 vs. LT0 and on the COTP vs. LTOTP, respectively. After prediction the target genes of these differentially expressed miRNAs, 9 s-lipoxygenase (LOX2S), 13 s-lipoxygenase (LOX1_5), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH1) were found differentially expressed, which were the key genes during aroma formation. The expression pattern of these target genes and the related miRNAs were identified by RT-PCR. mdm-miR172a-h, mdm-miR159a/b, mdm-miR160a-e, mdm-miR395a-i, mdm-miR399a/b/c, mdm/ppe-miR535a/b, and mdm-miR7120a/b may negatively regulate the target genes expression. These results indicate that miRNAs may play key roles in aroma weakening in cold storage 'Nanguo' pear and provide valuable information for studying the molecular mechanisms of miRNAs in the aroma weakening of fruit due to low temperature.

Effects of methyl jasmonate and melatonin treatments on the sensory quality and bioactive compounds of harvested broccoli.

Harvested broccoli is prone to decline in quality with regard to its appearance and nutrition. In this study, freshly harvested broccoli was treated with methyl jasmonate (MeJA) and melatonin (MT) and stored at 20 °C and the changes in sensory qualities and bioactive compounds were analyzed. The control samples began yellowing on day 2, whereas MeJA and MT treatments delayed the yellowing by 2 and 4 days, respectively. Upon yellowing, sweetness and bitterness of control samples increased sharply, accompanied by the accumulation of bioactive compounds, except for sulforaphane; however, no significant change in volatile components was detected. When the samples started losing their green color, MeJA alleviated the bitterness while increasing the sweetness and sulforaphane content. The bitterness, astringency, umami level, and the content of sulfurous volatiles improved significantly in the MT-treated samples. Moreover, these samples showed high antioxidant activity; the protective effect on VC and carotenoids was extremely significant.

Membrane lipid metabolism changes and aroma ester loss in low-temperature stored Nanguo pears.

Cold storage is an effective method used to retard the senescence of Nanguo pears after harvest. However, this causes aroma loss in the fruit. To elucidate the role of membrane lipid metabolism in aroma reduction, we investigated the contents of total aroma eaters and major fatty acid components, the membrane permeability, and the activity and gene expression of key enzymes in membrane lipid metabolism and aroma formation. The results showed that the contents of total aroma esters, oleic acid and linoleic acid, and alcohol dehydrogenase activity were at a lower level in cold stored fruit than that in control fruit. However, the palmitic acid content, membrane permeability, and the activities and gene expression of alcohol acyltransferase, lipoxygenase, phospholipase D, and lipase were higher. In conclusion, the loss of aroma esters may be caused by membrane lipid metabolism disruption during cold storage.

Proteomic analysis of peel browning of 'Nanguo' pears after low-temperature storage.

BACKGROUND: Postharvest ripening of the 'Nanguo' pear (Pyrus ussuriensis Maxim.) can be impeded by low-temperature storage. However, pears after long-term refrigeration are prone to peel browning when returned to room temperature conditions. This study investigated the browning mechanism of 'Nanguo' pear stored at a low temperature by analysing the differentially expressed proteins between healthy fruit and fruit with peel browning. RESULTS: The results showed that 181 proteins underwent statistically significant changes. A categorisation of the disparately accumulated proteins was performed using gene ontology annotation. The results showed that the 'metabolic process', 'cellular process', 'catalytic activity', and 'binding' proteins were the most affected after low-temperature storage. Further analysis revealed that the differentially expressed proteins, which are related to peel browning, are primarily involved in the phenylpropanoid pathway, linoleic acid pathways, fatty acid biosynthesis pathway, glutathione metabolism pathway, photosynthesis pathway, oxidative phosphorylation pathway, and glycolysis pathway. CONCLUSION: This study reveals that there are variations in key proteins in 'Nanguo' pear after low-temperature storage, and the identification of these proteins will be valuable in future functional genomics studies, as well as provide protein resources that can be used in the efforts to improve pear quality. © 2016 Society of Chemical Industry.

Dissemination of blaOXA-23 in Acinetobacter spp. in China: main roles of conjugative plasmid pAZJ221 and transposon Tn2009.

Production of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance in Acinetobacter spp. This study was conducted to reveal the genetic basis of blaOXA-23 dissemination in Acinetobacter spp. in China. A total of 63 carbapenem-resistant OXA-23-producing Acinetobacter sp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carrying blaOXA-23 were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23 Acinetobacter baumannii and three Acinetobacter nosocomialis isolates, while a novel ca. 50-kb plasmid was carried by only two other A. baumannii isolates. Three of these isolates had an additional copy of blaOXA-23 on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes except blaOXA-23. The blaOXA-23 gene of the remaining 35 isolates was chromosome borne. The blaOXA-23 genetic environments were correlated with Tn2009 in 57 isolates, Tn2008 in 5 isolates, and Tn2006 in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers of blaOXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009 have effectively contributed to the wide dissemination of blaOXA-23 in Acinetobacter spp. in China and that horizontal gene transfer may play an important role in dissemination of the blaOXA-23 gene.

SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.).

The genic multiple-allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L.) was identified as a spontaneous mutation. Applying this gene to hybrid seed production, several B. rapa cultivars have been successfully bred in China. A BC(1) population (244 plants) was constructed for mapping the Ms gene. Screening 268 simple sequence repeat (SSR) markers which cover the entire genome of Chinese cabbage was performed with bulked segregant analysis (BSA). On the basis of linkage analysis, the Ms gene was located on linkage group R07. In addition, through the amplified fragment length polymorphism (AFLP) and the sequence-characterized amplified region (SCAR) techniques combining BSA, two SCAR markers which were converted from corresponding AFLP markers flanked the Ms gene. Finally, a genetic map of the Ms gene was constructed covering a total interval of 9.0 cM. Two SCAR markers, syau_scr01 and syau_scr04, flanked the Ms gene at distances of 0.8 and 2.5 cM, respectively. All the SSR markers (cnu_m273, cnu_m030, cnu_m295, and syau_m13) were mapped on the same side of the gene as syau_scr04, the nearest one of which, syau_m13, was mapped at a distance of 3.3 cM. These SSR and SCAR markers may be useful in marker-assisted selection and map-based cloning.