Strigolactones alleviate AlCl3 stress by vacuolar compartmentalization and cell wall blocking in apple.

Poor management and excess fertilization of apple (Malus domestica Borkh.) orchards are causing increasingly serious soil acidification, resulting in Al toxicity and direct poisoning of roots. Strigolactones (SLs) are reported to be involved in plant responses to abiotic stress, but their role and mechanism under AlCl3 stress remain unknown. Here, we found that applying 1 µm GR24 (an SL analoge) significantly alleviated AlCl3 stress of M26 apple rootstock, mainly by blocking the movement of Al through cell wall and by vacuolar compartmentalization of Al. RNA-seq analysis identified the core transcription factor gene MdWRKY53, and overexpressing MdWRKY53 enhanced AlCl3 tolerance in transgenic apple plants through the same mechanism as GR24. Subsequently, we identified MdPMEI45 (encoding pectin methylesterase inhibitor) and MdALS3 (encoding an Al transporter) as downstream target genes of MdWRKY53 using chromatin immunoprecipitation followed by sequencing (ChIP-seq). GR24 enhanced the interaction between MdWRKY53 and the transcription factor MdTCP15, further increasing the binding of MdWRKY53 to the MdPMEI45 promoter and inducing MdPMEI45 expression to prevent Al from crossing cell wall. MdWRKY53 also bound to the promoter of MdALS3 and enhanced its transcription to compartmentalize Al in vacuoles under AlCl3 stress. We therefore identified two modules involved in alleviating AlCl3 stress in woody plant apple: the SL-WRKY+TCP-PMEI module required for excluding external Al by blocking the entry of Al3+ into cells and the SL-WRKY-ALS module allowing internal detoxification of Al through vacuolar compartmentalization. These findings lay a foundation for the practical application of SLs in agriculture.

Clinicopathologic Features of Gastrointestinal Tract Langerhans Cell Histiocytosis.

Gastrointestinal (GI) tract involvement by Langerhans cell histiocytosis (LCH) is rare and its clinicopathologic characteristics have only been described in case reports and small series. We reviewed hematoxylin and eosin- and CD1a, S100, and Langerin immunohistochemical-stained slides from 47 patients with well-documented demographic and clinical findings. Our cases included 8 children and 39 adults, with a mean follow-up of 63 months. All pediatric patients had concurrent multisystem LCH, presented with GI symptoms, and showed non-polypoid lesions. Seven (88%) showed multifocal GI disease, including five with multiple GI organ involvement. All sampled lesions from children exhibited infiltrative growth. More than half had died of the disease or manifested persistent LCH at last follow-up. Twenty-five of 39 (64%) adults had LCH involving only the GI tract (single-system), with the remaining 14 (36%) exhibiting multi-system disease. Adult single-system GI LCH was typically encountered incidentally on screening/surveillance endoscopy (72%). Most exhibited isolated colorectal involvement (88%) as a solitary polyp (92%), with a well-demarcated/noninfiltrative growth pattern (70%), and excellent prognosis (100%). In comparison, adult patients with multi-system LCH more frequently presented with GI symptoms (92%, P<0.001), non-colorectal GI site involvement (50%, P=0.02), multifocal GI lesions (43%, P=0.005), non-polypoid lesions (71%, P<0.001), infiltrative histologic growth pattern (78%, P=0.04), and persistent disease (57%, P<0.001). Adult multi-system LCH patients appear to exhibit similar clinicopathologic features to those of pediatric patients. These results demonstrate that adults with single-system LCH involving the GI tract have an excellent prognosis, while multi-system LCH occurring at any age carries an unfavorable prognosis. High-risk features of GI LCH include pediatric age, GI symptomatology, non-colorectal GI involvement, multifocal GI disease, non-polypoid lesions, and infiltrative growth pattern.

Melatonin promotes Al3+ compartmentalization via H+ transport and ion gradients in Malus hupehensis.

Soil acidification in apple (Malus domestica) orchards results in the release of rhizotoxic aluminum ions (Al3+) into soil. Melatonin (MT) participates in plant responses to abiotic stress; however, its role in AlCl3 stress in apple remains unknown. In this study, root application of MT (1 µM) substantially alleviated AlCl3 stress (300 µM) in Pingyi Tiancha (Malus hupehensis), which was reflected by higher fresh and dry weight, increased photosynthetic capacity, and longer and more roots compared with plants that did not receive MT treatment. MT functioned mainly by regulating vacuolar H+/Al3+ exchange and maintaining H+ homeostasis in the cytoplasm under AlCl3 stress. Transcriptome deep sequencing analysis identified the transcription factor gene SENSITIVE TO PROTON RHIZOTOXICITY 1 (MdSTOP1) was induced by both AlCl3 and MT treatments. Overexpressing MdSTOP1 in apple increased AlCl3 tolerance by enhancing vacuolar H+/Al3+ exchange and H+ efflux to the apoplast. We identified 2 transporter genes, ALUMINUM SENSITIVE 3 (MdALS3) and SODIUM HYDROGEN EXCHANGER 2 (MdNHX2), as downstream targets of MdSTOP1. MdSTOP1 interacted with the transcription factor NAM ATAF and CUC 2 (MdNAC2) to induce MdALS3 expression, which reduced Al toxicity by transferring Al3+ from the cytoplasm to the vacuole. Furthermore, MdSTOP1 and MdNAC2 coregulated MdNHX2 expression to increase H+ efflux from the vacuole to the cytoplasm to promote Al3+ compartmentalization and maintain cation balance in the vacuole. Taken together, our findings reveal an MT-STOP1 + NAC2-NHX2/ALS3-vacuolar H+/Al3+ exchange model for the alleviation of AlCl3 stress in apple, laying a foundation for practical applications of MT in agriculture.

Melatonin promotes the recovery of apple plants after waterlogging by shaping the structure and function of the rhizosphere microbiome.

The dynamics of the physiological adaptability of plants and the rhizosphere soil environment after waterlogging remain unclear. Here we investigated the mechanisms regulating plant condition and shaping of the rhizosphere microbiome in a pot experiment. In the experiment, we added melatonin to waterlogged plants, which promoted waterlogging relief. The treatment significantly enhanced photosynthesis and the antioxidant capacity of apple plants, and significantly promoted nitrogen (N) utilization efficiency by upregulating genes related to N transport and metabolism. Multiperiod soil microbiome analysis showed the dynamic effects of melatonin on the diversity of the microbial community during waterlogging recovery. Random forest and linear regression analyses were used to screen for potential beneficial bacteria (e.g., Azoarcus, Pseudomonas and Nocardioides) specifically regulated by melatonin and revealed a positive correlation with soil nutrient levels and plant growth. Furthermore, metagenomic analyses revealed the regulatory effects of melatonin on genes involved in N cycling in soil. Melatonin positively contributed to the accumulation of plant dry weight by upregulating the expression of nifD and nifK (N fixation). In summary, melatonin positively regulates physiological functions in plants and the structure and function of the microbial community; it promoted the recovery of apple plants after waterlogging stress.

A mutation in the promoter of the arabinogalactan protein 7-like gene PcAGP7-1 affects cell morphogenesis and brassinolide content in pear (Pyrus communis L.) stems.

Dwarfing rootstocks and dwarf cultivars are urgently needed for modern pear cultivation. However, germplasm resources for dwarfing pear are limited, and the underlying mechanisms remain unclear. We previously showed that dwarfism in pear is controlled by the single dominant gene PcDw (Dwarf). We report here that the expression of PcAGP7-1 (ARABINOGALACTAN PROTEIN 7-1), a key candidate gene for PcDw, is significantly higher in dwarf-type pear plants because of a mutation in an E-box in the promoter. Electrophoretic mobility shift assays and transient infiltration showed that the transcription factors PcBZR1 and PcBZR2 could directly bind to the E-box of the PcAGP7-1 promoter and repress transcription. Moreover, transgenic pear lines overexpressing PcAGP7-1 exhibited obvious dwarf phenotypes, whereas RNA interference pear lines for PcAGP7-1 were taller than controls. PcAGP7-1 overexpression also enhanced cell wall thickness, affected cell morphogenesis, and reduced brassinolide (BL) content, which inhibited BR signaling via a negative feedback loop, resulting in further dwarfing. Overall, we identified a dwarfing mechanism in perennial woody plants involving the BL-BZR/BES-AGP-BL regulatory module. Our findings provide insight into the molecular mechanism of plant dwarfism and suggest strategies for the molecular breeding of dwarf pear cultivars.

Melatonin enhances KCl salinity tolerance by maintaining K+ homeostasis in Malus hupehensis.

Large amounts of potash fertilizer are often applied to apple (Malus domestica) orchards to enhance fruit quality and yields, but this treatment aggravates KCl-based salinity stress. Melatonin (MT) is involved in a variety of abiotic stress responses in plants. However, its role in KCl stress tolerance is still unknown. In the present study, we determined that an appropriate concentration (100 µm) of MT significantly alleviated KCl stress in Malus hupehensis by enhancing K+ efflux out of cells and compartmentalizing K+ in vacuoles. Transcriptome deep-sequencing analysis identified the core transcription factor gene MdWRKY53, whose expression responded to both KCl and MT treatment. Overexpressing MdWRKY53 enhanced KCl tolerance in transgenic apple plants by increasing K+ efflux and K+ compartmentalization. Subsequently, we characterized the transporter genes MdGORK1 and MdNHX2 as downstream targets of MdWRKY53 by ChIP-seq. MdGORK1 localized to the plasma membrane and enhanced K+ efflux to increase KCl tolerance in transgenic apple plants. Moreover, overexpressing MdNHX2 enhanced the KCl tolerance of transgenic apple plants/callus by compartmentalizing K+ into the vacuole. RT-qPCR and LUC activity analyses indicated that MdWRKY53 binds to the promoters of MdGORK1 and MdNHX2 and induces their transcription. Taken together, our findings reveal that the MT-WRKY53-GORK1/NHX2-K+ module regulates K+ homeostasis to enhance KCl stress tolerance in apple. These findings shed light on the molecular mechanism of apple response to KCl-based salinity stress and lay the foundation for the practical application of MT in salt stress.

Brassinolide alleviates Fe deficiency-induced stress by regulating the Fe absorption mechanism in Malus hupehensis Rehd.

KEY MESSAGE: Exogenous brassinolide promotes Fe absorption through mechanism I strategy, thus improving the tolerance of Malus hupehensis seedlings to Fe deficiency stress. Iron (Fe) deficiency is a common nutritional disorder that results in decreased yield and poor fruit quality in apple production. As a highly active synthetic analog of brassinosteroids, brassinolide (BL) plays numerous roles in plant responses to abiotic stresses. However, its role in Fe deficiency stress in apple plants has never been reported. Herein, we found that the exogenous application of 0.2 mg L-1 BL could significantly enhance the tolerance of apple seedlings to Fe deficiency stress and result in a low etiolation rate and a high photosynthetic rate. The functional mechanisms of this effect were also explored. We found that first, exogenous BL could improve Fe absorption through the mechanism I strategy. BL induced the activity of H+-ATPase and the expression of MhAHA family genes, resulting in rhizosphere acidification. Moreover, BL could enhance the activity of Fe chelate reductase and absorb Fe through direct binding with the E-box of the MhIRT1 or MhFRO2 promoter via the transcription factors MhBZR1 and MhBZR2. Second, exogenous BL alleviated osmotic stress by increasing the contents of osmolytes (proline, solution proteins, and solution sugar) and scavenged reactive oxygen species by improving the activities of antioxidant enzymes. Lastly, exogenous BL could cooperate with other endogenous plant hormones, such as indole-3-acetic acid, isopentenyl adenosine, and gibberellic acid 4, that respond to Fe deficiency stress indirectly. This work provided a theoretical basis for the application of exogenous BL to alleviate Fe deficiency stress in apple plants.

Morphologic and molecular analysis of early-onset gastric cancer.

BACKGROUND: Evidence suggests that early-onset gastric cancers are distinct from traditional gastric cancers; however, detailed genomic and morphologic characterization of these cancers has not been performed. METHODS: Genomic analysis was performed for 81 patients with gastric cancer who were 50 years old or younger; pathology slides were available for 53 of these patients, and they were re-reviewed to perform a morphologic-molecular correlation analysis. The results were compared with corresponding cBioPortal data and The Cancer Genome Atlas (TCGA) analysis, which represent traditional gastric cancers. The TP53 molecular signature was established to determine the pattern of somatic mutational damage. Variants of potential germline origin were also identified from next-generation sequencing data. RESULTS: A higher rate of CDH1 mutations (22.2% of early-onset gastric cancers vs 11.4% of traditional gastric cancers; P = .0042) but a similar rate of TP53 mutations (63% of early-onset gastric cancers vs 56.6% of traditional gastric cancers; P = .2674) were seen in early-onset cancers in comparison with traditional gastric cancers. The diffuse/mixed types correlated with the TCGA genomically stable type, and the remaining Lauren types correlated with the TCGA chromosomal instability type. Diffuse and indeterminate histologic types (overall survival, 26.25 months for the intestinal type, 20.5 months for the mixed type, 12.62 months for the diffuse type, and 9 months for the indeterminate type; P = .027) and the presence of a CDH1 gene mutation (overall survival, 9 months for mutant CDH1 and 22 months for wild-type CDH1; P = .013) significantly correlated with worse survival. The TP53 gene frequently showed transition mutations (65.5%) involving the CpG sites (49%). Variants of potential germline origin were seen in high-penetrance genes (CDH1 and APC) and moderate-penetrance genes (ATM, NBN, and MUTYH) in 9.9% of cancers. CONCLUSIONS: Early-onset gastric cancer has distinct genomic alterations, such as CDH1 mutations, but shares with traditional gastric cancers a high frequency of TP53 mutations and the TP53 mutagenic signature. Diffuse and indeterminate histologic types and the presence of a CDH1 mutation are associated with worse overall survival. Endogenous factors leading to cytosine deamination and potential germline alterations in moderate-penetrance cancer susceptibility genes may be implicated in the pathogenesis of these cancers.

Resveratrol improves the iron deficiency adaptation of Malus baccata seedlings by regulating iron absorption.

BACKGROUND: Resveratrol (Res), a phytoalexin, has been widely reported to participate in plant resistance to fungal infections. However, little information is available on its role in abiotic stress, especially in iron deficiency stress. Malus baccata is widely used as apple rootstock in China, but it is sensitive to iron deficiency. RESULTS: In this study, we investigated the role of exogenous Res in M. baccata seedings under iron deficiency stress. Results showed that applying 100 µM exogenous Res could alleviate iron deficiency stress. The seedlings treated with Res had a lower etiolation rate and higher chlorophyll content and photosynthetic rate compared with the apple seedlings without Res treatment. Exogenous Res increased the iron content in the roots and leaves by inducing the expression of MbAHA genes and improving the H+-ATPase activity. As a result, the rhizosphere pH decreased, iron solubility increased, the expression of MbFRO2 and MbIRT1 was induced, and the ferric-chelated reductase activity was enhanced to absorb large amounts of Fe2+ into the root cells under iron deficiency conditions. Moreover, exogenous Res application increased the contents of IAA, ABA, and GA3 and decreased the contents of DHZR and BL for responding to iron deficiency stress indirectly. In addition, Res functioned as an antioxidant that strengthened the activities of antioxidant enzymes and thus eliminated reactive oxygen species production induced by iron deficiency stress. CONCLUSION: Resveratrol improves the iron deficiency adaptation of M. baccata seedlings mainly by regulating iron absorption.

Intratumoral budding and automated CD8-positive T-cell density in pretreatment biopsies can predict response to neoadjuvant therapy in rectal adenocarcinoma.

Tumor budding and CD8-positive (+) T-cells are recognized as prognostic factors in colorectal adenocarcinoma. We assessed CD8+ T-cell density and intratumoral budding in pretreatment rectal cancer biopsies to determine if they are predictive biomarkers for response to neoadjuvant therapy and survival. Pretreatment biopsies of locally advanced rectal adenocarcinoma from 117 patients were evaluated for CD8+ T-cell density using automated quantitative digital image analysis and for intratumoral budding and correlated with clinicopathological variables on postneoadjuvant surgical resection specimens, response to neoadjuvant therapy, and survival. Patients with high CD8+ T-cell density (≥157 per mm2) on biopsy were significantly more likely to exhibit complete/near complete response to neoadjuvant therapy (66% vs. 33%, p = 0.001) and low tumor stage (0 or I) on resection (62% vs. 30%, p = 0.001) compared with patients with low CD8+ T-cell density. High CD8+ T-cell density was an independent predictor of response to neoadjuvant therapy with a 2.63 higher likelihood of complete response (95% CI 1.04-6.65, p = 0.04) and a 3.66 higher likelihood of complete/near complete response (95% CI 1.60-8.38, p = 0.002). The presence of intratumoral budding on biopsy was significantly associated with a reduced likelihood of achieving complete/near complete response to neoadjuvant therapy (odds ratio 0.36, 95% CI 0.13-0.97, p = 0.048). Patients with intratumoral budding on biopsy had a significantly reduced disease-free survival compared with patients without intratumoral budding (5-year survival 39% vs 87%, p < 0.001). In the multivariable model, the presence of intratumoral budding on biopsy was associated with a 3.35-fold increased risk of tumor recurrence (95% CI 1.25-8.99, p = 0.02). In conclusion, CD8+ T-cell density and intratumoral budding in pretreatment biopsies of rectal adenocarcinoma are independent predictive biomarkers of response to neoadjuvant therapy and intratumoral budding associates with patient survival. These biomarkers may be helpful in selecting patients who will respond to neoadjuvant therapy and identifying patients at risk for recurrence.

Gastric neuroendocrine tumours from long-term proton pump inhibitor users are indolent tumours with good prognosis.

AIMS: Proton pump inhibitors (PPIs) are among the most widely used medications in the United States. Most PPI users have persistent hypergastrinaemia during treatment. However, gastric neuroendocrine tumours diagnosed in long-term PPI users are rarely reported. Their clinicopathological features and prognosis are not characterised. It remains unclear whether or not they can be classified as Type III sporadic tumours. METHODS AND RESULTS: We retrospectively characterised 66 gastric neuroendocrine tumours from patients without atrophic gastritis and gastrinoma from two tertiary care medical centres, including 38 tumours in patients who had used PPIs for at least 1 year and 28 tumours from patients without long-term PPI use (control group, Type III tumours). Compared to controls, tumours from long-term PPI users tended to be in the pT1-2 category (98% versus 79%, P = 0.09) and less often invaded the serosa (3% versus 18%, P = 0.08) or lymphovascular spaces (11% versus 32%, P = 0.06). Using Kaplan-Meier analysis, long-term PPI users had significantly longer overall survival than controls (P = 0.035). While three control patients developed distant metastasis and seven died, long-term PPI users were without distant metastasis (P = 0.06) or death (P = 0.002) during follow-up. However, five long-term PPI users developed additional gastric neuroendocrine tumour(s), while none of the controls did (P = 0.07). CONCLUSIONS: Our results show that gastric neuroendocrine tumours of long-term PPI users are probably less aggressive compared to Type III sporadic tumours and have an indolent disease course. Our findings support the classification of gastric neuroendocrine tumours in long-term PPI users as a separate subtype.

Transcriptome profiling of anthocyanin biosynthesis in the peel of 'Granny Smith' apples (Malus domestica) after bag removal.

BACKGROUND: Bagging is commonly used to enhance red pigmentation and thereby improve fruit quality of apples (Malus domestica). The green-skinned apple cultivar 'Granny Smith' develops red pigmentation after bagging removal, but the signal transduction pathways mediating light-induced anthocyanin accumulation in apple peel are yet to be defined. The aim of this study was to identify the mechanisms underpinning red pigmentation in 'Granny Smith' after bag removal based on transcriptome sequencing. RESULTS: The anthocyanin content in apple peel increased considerably after bag removal, while only trace amounts of anthocyanins were present in the peel of unbagged and bagged fruits. RNA sequencing identified 18,152 differentially expressed genes (DEGs) among unbagged, bagged, and bag-removed fruits at 0, 4, and 10 days after bag removal. The DEGs were implicated in light signal perception and transduction, plant hormone signal transduction, and antioxidant systems. Weighted gene co-expression network analysis of DEGs generated a module of 23 genes highly correlated with anthocyanin content. The deletion of - 2026 to - 1870 bp and - 1062 to - 964 bp regions of the MdMYB1 (LOC103444202) promoter induced a significant decrease in glucuronidase activity and anthocyanin accumulation in apple peel. CONCLUSIONS: Bagging treatment can induce red pigmentation in 'Granny Smith' via altering the expression patterns of genes involved in crucial signal transduction and biochemical metabolic pathways. The - 2026 to - 1870 bp and - 1062 to - 964 bp regions of the MdMYB1 promoter are essential for MdMYB1-mediated regulation of anthocyanin accumulation in the 'Granny Smith' apple cultivar. The findings presented here provide insight into the mechanisms of coloration in the peel of 'Granny Smith' and other non-red apple cultivars.

SATB2 and CDX2 are prognostic biomarkers in DNA mismatch repair protein deficient colon cancer.

DNA mismatch repair protein deficient colon cancer frequently displays reduced CDX2 expression, and recent literature has suggested that negative CDX2 expression is a poor prognostic biomarker in colon cancer. We have recently demonstrated that SATB2 is an immunohistochemical marker that is complementary to CDX2. Using a tissue microarray approach, we evaluated SATB2 and CDX2 immunohistochemical expression in 514 patients with colonic adenocarcinoma including 146 with mismatch repair protein deficient tumors and correlated expression with histopathologic variables, molecular alterations, and survival. Overall, SATB2-negative and/or CDX2-negative expression was identified in 33% of mismatch repair protein deficient tumors compared with only 15% of mismatch repair protein proficient tumors (p < 0.001) and in 36% of BRAF V600E mutated compared with only 13% of BRAF wild-type tumors (p < 0.001). Both SATB2-negative and CDX2-negative colonic adenocarcinomas more often displayed lymphatic invasion, venous invasion, and perineural invasion (all with p < 0.05). SATB2-negative expression was also more frequently identified in tumors with mucinous or signet ring cell differentiation (p < 0.01 for both). In a multivariable analysis of survival in patients with mismatch repair protein deficient tumors (n = 131), only tumor stage (p = 0.01) and SATB2-negative and/or CDX2-negative expression (p = 0.009) independently predicted disease-specific survival. Of the 99 patients with stage II or III mismatch repair protein deficient tumors, death from disease only occurred in patients with either SATB2-negative or CDX2-negative tumors, and no patients with SATB2-positive/CDX2-positive tumors developed recurrence or died of disease. SATB2 and CDX2 expression had no effect on patient survival in mismatch repair protein proficient, BRAF-mutated, or KRAS-mutated tumors. In summary, our results suggest that SATB2 and CDX2 are prognostic biomarkers in patients with mismatch repair protein deficient colon cancer and that inclusion of SATB2 and CDX2 immunohistochemistry may be helpful as part of a comprehensive pathologic risk assessment in mismatch repair protein deficient colon cancer.

The effect of promoter methylation on MdMYB1 expression determines the level of anthocyanin accumulation in skins of two non-red apple cultivars.

BACKGROUND: Fruit color in apple (Malus domestica Borkh.) is ascribed mainly to the accumulation of anthocyanin pigments, and is an important trait for determining fruit market acceptance. Bagging is a commonly used treatment to enhance the red pigmentation in apple skin. The MdMYB1 transcription factor gene plays an important role in the biosynthesis of anthocyanin in apple after bag removal, but little is known about how MdMYB1 transcription is regulated. RESULTS: In this study, we investigated pigmentation in the non-red skinned cultivars 'Granny Smith' and 'Golden Delicious' after bag removal. The fruit skins of the two cultivars showed red/pink pigmentation after bag treatment. Transcript levels of MdMYB1, the master regulator of anthocyanin biosynthesis in apple, increased, and showed a correlation with anthocyanin content in both cultivars after bag removal. The MdMYB1 genomic sequences were compared in the two cultivars, which showed that the green-fruited cultivar 'Granny Smith' harbors the MdMYB1-1 and MdMYB1-2 alleles, while the yellow-fruited cultivar 'Golden Delicious' harbors only MdMYB1-2. A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in 'Granny Smith'. Moreover, 'Granny Smith' fruits responded to treatment with 5-aza-2'-deoxycytidine, an inducer of DNA demethylation. An investigation of the MdMYB1 promoter in 'Granny Smith' showed reduced methylation in the regions - 2026 to - 1870 bp, - 1898 to - 1633 bp, and - 541 to - 435 bp after bag removal and 5-aza-2'-deoxycytidine treatments. CONCLUSIONS: Differences in anthocyanin levels between 'Granny Smith' and 'Golden Delicious' can be explained by differential accumulation of MdMYB1-specific mRNA. Different levels of MdMYB1 transcripts in the two cultivars are associated with methylation levels in the promoter region. Hypomethylation of the MdMYB1 promoter is correlated with the formation of red pigmentation in 'Granny Smith' fruit skins. As a result, red pigmentation in Granny Smith' was more intense than in 'Golden Delicious' fruits after bag removal.

Transcriptome Profiling Reveals Transcriptional Regulation by DNA Methyltransferase Inhibitor 5-Aza-2'-Deoxycytidine Enhancing Red Pigmentation in Bagged "Granny Smith" Apples (Malus domestica).

The red color of apples (Malus domestica) is an attractive trait for consumers. The green skinned "Granny Smith" cultivar develops red pigmentation after bagging treatment. DNA methylation plays an important role in various developmental processes in plants. To explore the possible functions of DNA methylation in the pigmentation of bagged "Granny Smith" apples, we first analyzed the anthocyanin content of fruit skin following treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The results revealed an increase in anthocyanin content in bagged fruits following 5-aza-dC treatment, while no anthocyanins were detected in unbagged fruits. In addition, 8482 differentially expressed genes between 5-aza-dC-treated and control groups were identified in bagged fruits by RNA sequencing, including genes encoding transcription factors, enzymes related to anthocyanin accumulation, and methylases. Changes in the expression of these genes may be responsible for 5-aza-dC-induced red pigmentation in bagged fruits of "Granny Smith". The findings provide novel evidence for the involvement of DNA methylation in the red pigmentation of non-red-skinned apples.

High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx.

Isoflurane is a commonly used inhalational anesthetic that can induce neurotoxicity via elevating cytosolic calcium (Ca2+). High glucose regulates the expression of a family of non-selective cation channels termed transient receptor potential canonical (TRPC) channels that may contribute to Ca2+ influx. In the present study, we investigated whether high glucose enhances isoflurane-induced neurotoxicity by regulating TRPC-dependent Ca2+ influx. First, we evaluated toxic damage in mice primary cultured hippocampal neurons and human neuroblastoma cells (SH-SY5Y cells) after hyperglycemia and isoflurane exposure. Next, we investigated cytosolic Ca2+ concentrations, TRPC mRNA expression levels and tested the effect of the TRPC channel blocker SKF96365 on cytosolic Ca2+ levels in cells treated with high glucose or/and isoflurane. Finally, we employed knocked down TRPC6 to demonstrate the role of TRPC in high glucose-mediated enhancement of isoflurane-induced neurotoxicity. The results showed that high glucose could enhance isoflurane-induecd toxic damage in primary hippocampal neurons and SH-SY5Y cells. High glucose enhanced the isoflurane-induced increase of cytosolic Ca2+ in SH-SY5Y cells. High glucose elevated TRPC mRNA expression, especially that of TRPC6. SKF96365 and knock down of TRPC6 were able to inhibit the high glucose-induced increase of cytosolic Ca2+ and decrease isoflurane-induced neurotoxicity in SH-SY5Y cells cultured with high glucose. Our findings indicate that high glucose could elevate TRPC expression, thus increasing Ca2+ influx and enhancing isoflurane-induced neurotoxicity.

Terminal hepatic venule injury in liver biopsies of allogeneic haematopoietic stem cell recipients-a study of 63 cases.

AIMS: Subtle lesions of terminal hepatic venules (THVs) may be overlooked in liver biopsies from haematopoietic stem cell transplant (HSCT) receipients when graft-versus-host disease is the clinical concern. The aim of this study was to evaluate the frequency of THV injury resembling sinusoidal obstruction syndrome (SOS). METHODS AND RESULTS: Sixty-three consecutive biopsies from allogeneic HSCT recipients were scored for injured THVs. Forty-nine (78%) biopsies had injured THVs, and 10 (16%) were diagnosed with SOS (mean ± standard deviation of injured THVs/biopsy: 90 ± 9%). Biopsies diagnosed with other diseases also had injured THVs (36 ± 33%). Biopsies from patients with cyclophosphamide plus fractionated total body irradiation conditioning and biopsies taken within 100 days post-HSCT had significantly more occluded THVs (respectively: 40 ± 38%, P = 0.0188; and 35 ± 35%, P = 0.0076) than those with other conditioning regimens or in biopsies taken >100 days post-HSCT. All biopsies taken at any time in the 6-year post-HSCT period had similar amounts of THV phlebosclerosis (23 ± 25%). CONCLUSIONS: Our results demonstrate a high incidence of THV injuries resembling SOS in post-HSCT liver biopsies. THV injuries were detectable for several years post-HSCT, and were concurrent with other diagnoses. Our results also suggest that SOS may be underdiagnosed.

Predictive value of immunohistochemistry in pre-malignant lesions of the gastrointestinal tract.

Immunohistochemistry can be an important adjunct to histopathology for the diagnosis of pre-malignant lesions of the gastrointestinal tract and in patient risk stratification. The purpose of this review is to provide information and guidance on the usefulness of immunohistochemical markers that facilitate the diagnosis of dysplasia and help to predict risk for the development of carcinoma in pre-malignant lesions of the gastrointestinal tract. Particular emphasis is given to the role of immunohistochemistry in the assessment of epithelial dysplasia in the setting of Barrett's esophagus and inflammatory bowel disease; supplementary immunohistochemistry for subtyping adenomas of the stomach and ampulla and serrated polyps of the colon and rectum; and ancillary markers of squamous neoplasia of the anal canal.

Melatonin alleviates cadmium toxicity by regulating root endophytic bacteria community structure and metabolite composition in apple.

The level of cadmium (Cd) accumulation in orchard soils is increasing, and excess Cd will cause serious damage to plants. Melatonin is a potent natural antioxidant and has a potential role in alleviating Cd stress. This study aimed to investigate the effects of exogenous melatonin on a root endophyte bacteria community and metabolite composition under Cd stress. The results showed that melatonin significantly scavenged the reactive oxygen species and restored the photosynthetic system (manifested by the improved photosynthetic parameters, total chlorophyll content and the chlorophyll fluorescence parameters (Fv/Fm)), increased the activity of antioxidant enzymes (the activities of catalase, superoxide dismutase, peroxidase and ascorbate oxidase) and reduced the concentration of Cd in the roots and leaves of apple plants. High-throughput sequencing showed that melatonin increased the endophytic bacterial community richness significantly and changed the community structure under Cd stress. The abundance of some potentially beneficial endophytic bacteria (Ohtaekwangia, Streptomyces, Tabrizicola and Azovibrio) increased significantly, indicating that the plants may absorb potentially beneficial microorganisms to resist Cd stress. The metabolomics results showed that melatonin significantly changed the composition of root metabolites, and the relative abundance of some metabolites decreased, suggesting that melatonin may resist Cd stress by depleting root metabolites. In addition, co-occurrence network analysis indicated that some potentially beneficial endophytes may be influenced by specific metabolites. These results provide a theoretical basis for studying the effects of melatonin on the endophytic bacterial community and metabolic composition in apple plants.

Noninvasive Markers of Inflammation and Protein Loss Augment Diagnosis of Pediatric Celiac Disease.

INTRODUCTION: Circulating tissue transglutaminase immunoglobulin A concentration is a sensitive and specific indicator of celiac disease, but discrepancies between serologic and histologic findings occur. We hypothesized that fecal markers of inflammation and protein loss would be greater in patients with untreated celiac disease than in healthy controls. Our study aims to evaluate multiple fecal and plasma markers in celiac disease and correlate these findings with serologic and histologic findings as noninvasive means of evaluating disease activity. METHODS: Participants with positive celiac serologies and controls with negative celiac serologies were prospectively enrolled before upper endoscopy. Blood, stool, and duodenal biopsies were collected. Concentrations of fecal lipocalin-2, calprotectin, and alpha-1-antitrypsin and plasma lipocalin-2 were determined. Biopsies underwent modified Marsh scoring. Significance was tested between cases and controls, modified Marsh score and tissue transglutaminase immunoglobulin A concentration. RESULTS: Lipocalin-2 was significantly elevated in the stool ( P = 0.006) but not the plasma of participants with positive celiac serologies. There was no significant difference in fecal calprotectin or alpha-1 antitrypsin between participants with positive celiac serologies and controls. Fecal alpha-1 antitrypsin >100 mg/dL was specific, but not sensitive for biopsy-proven celiac disease. DISCUSSION: Lipocalin-2 is elevated in the stool but not the plasma of patients with celiac disease suggesting a role of local inflammatory response. Calprotectin was not a useful marker in the diagnosis of celiac disease. While random fecal alpha-1 antitrypsin was not significantly elevated in cases compared with controls, an elevation of greater than 100 mg/dL was 90% specific for biopsy-proven celiac disease.