The auxin-responsive CsSPL9-CsGH3.4 module finely regulates auxin levels to suppress the development of adventitious roots in tea (Camellia sinensis).

The cutting technique is extensively used in tea breeding, with key emphasis on promoting the growth of adventitious roots (ARs). Despite its importance in tea cultivation, the mechanisms underlying AR development in tea remain unclear. In this study, we demonstrated the essential role of auxins in the initiation and progression of AR and established that the application of exogenous 1-naphthaleneacetic acid-enhanced AR formation in tissue-cultured seedlings and cuttings. Then, we found that the auxin-responsive transcription factor CsSPL9 acted as a negative regulator of AR development by reducing the levels of free indole-3-acetic acid (IAA) in tea plants. Furthermore, we identified CsGH3.4 as a downstream target of CsSPL9, which was activated by direct binding to its promoter. CsGH3.4 also inhibited AR development and maintained low levels of free IAA. Thus, these results revealed the inhibitory effect of the auxin-responsive CsSPL9-CsGH3.4 module on AR development by reducing free IAA levels in tea. These findings have significant theoretical and practical value for enhancing tea breeding practices.

Grain yield improvement by genome editing of TaARF12 that decoupled peduncle and rachis development trajectories via differential regulation of gibberellin signalling in wheat.

Plant breeding is constrained by trade-offs among different agronomic traits by the pleiotropic nature of many genes. Genes that contribute to two or more favourable traits with no penalty on yield are rarely reported, especially in wheat. Here, we describe the editing of a wheat auxin response factor TaARF12 by using CRISPR/Cas9 that rendered shorter plant height with larger spikes. Changes in plant architecture enhanced grain number per spike up to 14.7% with significantly higher thousand-grain weight and up to 11.1% of yield increase under field trials. Weighted Gene Co-Expression Network Analysis (WGCNA) of spatial-temporal transcriptome profiles revealed two hub genes: RhtL1, a DELLA domain-free Rht-1 paralog, which was up-regulated in peduncle, and TaNGR5, an organ size regulator that was up-regulated in rachis, in taarf12 plants. The up-regulation of RhtL1 in peduncle suggested the repression of GA signalling, whereas up-regulation of TaNGR5 in spike may promote GA response, a working model supported by differential expression patterns of GA biogenesis genes in the two tissues. Thus, TaARF12 complemented plant height reduction with larger spikes that gave higher grain yield. Manipulation of TaARF12 may represent a new strategy in trait pyramiding for yield improvement in wheat.

An Essential Role for miRNA167 in Maternal Control of Embryonic and Seed Development.

Maternal cells play a critical role in ensuring the normal development of embryos, endosperms, and seeds. Mutations that disrupt the maternal control of embryogenesis and seed development are difficult to identify. Here, we completely deleted four MICRORNA167 (MIR167) genes in Arabidopsis (Arabidopsis thaliana) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion of MIR167A phenocopied plants overexpressing miRNA167-resistant versions of Auxin Response Factor6 (ARF6) or ARF8, two miRNA167 targets. Both the mir167a mutant and the ARF overexpression lines were defective in anther dehiscence and ovule development. Serendipitously, we found that the mir167a (♀) × wild type (♂) crosses failed to produce normal embryos and endosperms, despite the findings that embryos with either mir167a+/- or mir167a-/- genotypes developed normally when mir167a+/- plants were self-pollinated, revealing a central role of MIR167A in maternal control of seed development. The mir167a phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternal control. Moreover, we found that mir167a mutants flowered significantly later than wild-type plants, a phenotype that was not observed in the ARF overexpression lines. We show that the reproductive defects of mir167a mutants were suppressed by a decrease of activities of ARF6, ARF8, or both. Our results clearly demonstrate that MIR167A is the predominant MIR167 member in regulating Arabidopsis reproduction and that MIR167A acts as a maternal gene that functions largely through ARF6 and ARF8.

Effects of enzymatic hydrolysate of locust bean gum on digestibility, intestinal morphology and microflora of broilers.

To investigate the effects of different levels of enzymatic hydrolysate of dietary locust bean gum on nutrient digestibility, intestinal morphology and microflora of broilers, a total of 768 one-day-old Arbor Acres (AA) broiler chicks were randomly divided into 6 treatments with 8 replicates per treatment and 16 birds per replicate. The treatments were as follows: (1) CON, basal diet; (2) ANT, basal diet +62.5 mg/kg flavomycin; (3) LBG, basal diet +0.1% locust bean gum; (4) ELBG-0.1, basal diet +0.1% enzymatic hydrolysate of LBG; (5) ELBG-0.2, basal diet +0.2% enzymatic hydrolysate of LBG; and (6) ELBG-0.3, basal diet +0.3% enzymatic hydrolysate of LBG. The digestibilities of ether extract, crude protein and dry matter were increased (p < .01) in broilers fed the ELBG-0.3 diet compared with the CON and LBG diets on day 21. Duodenal villus height and the ratio of the villus height to crypt depth were greater (p < .01) in broilers fed the ELBG-0.3 diet than the CON, ANT and LBG diets. Jejunum villus height was higher (p < .05) in broilers fed the ELBG-0.2 and ELBG-0.3 diets than the CON diet. The number of caecal Escherichia coli was reduced (p = .01) in broilers fed the ELBG-0.2 and ELBG-0.3 diets compared with the CON diet. The number of caecal Lactobacilli was greater (p < .05) in broilers fed the ELBG-0.3 diet than the CON and ANT diets. In summary, the addition of 0.3% locust bean enzymatic hydrolysate can increase the surface area of intestinal villi and the number of beneficial bacteria, inhibit the proliferation of harmful bacteria, maintain the balance of intestinal microflora and improve the digestibility of nutrients.

Diagnostic values of shear wave elastography and strain elastography for breast lesions.

BACKGROUND: Strain elastography (SE) and shear wave elastography (SWE) have high diagnostic yield for breast lesions, but the optimal parameters remain elusive. AIM: To evaluate the diagnostic yield of SWE and SE for breast lesions by multivariate logistic regression analysis. MATERIAL AND METHODS: A total of 132 patients with 164 breast tumors were enrolled. Breast lesions were classified with the breast imaging reporting and data system (BI-RADS). Maximum (Emax), mean (Emean) and standard deviation (Esd) of elastic modulus, lesion/fat elasticity ratio and elastographic classification were obtained by SWE. Strain ratio (SR) and elastographic score were obtained by SE. A multivariate logistic regression analysis was performed. The diagnostic efficiencies of BI-RADS classification, SWE, SE and their combination were compared plotting ROC curves. RESULTS: There were 110 benign and 54 malignant lesions which had significantly different SWE and SE parameters. The parameters included in the logistic regression were Esd and elastographic classification obtained by SWE and the elastographic score obtained by SE. When combining SWE with SE, Esd, SR and SWE classification were included in the equation. The areas under ROC curves for BI-RADS classification, SWE, SE and their combination were 0.75, 0.88, 0.79 and 0.89, respectively. CONCLUSIONS: The diagnostic value of SWE in combination with SE for breast lesions exceeded that of SE or SWE alone. Esd showed a good diagnostic yield when SWE was used alone or combined with SE.

Effect of pectin oligosaccharides and zinc chelate on growth performance, zinc status, antioxidant ability, intestinal morphology and short-chain fatty acids in broilers.

A 42-day trial was conducted to investigate the effect of pectin oligosaccharides (POS) and zinc chelate (Zn-POS) on growth performance, antioxidant ability, zinc status, intestinal morphology and short-chain fatty acids in broilers. A total of 324 1-day-old Arbor Acres broilers were randomly assigned to three treatments with six cages of 18 chicks. Treatments were: (a) Control, 80 mg/kg Zn from ZnSO4 ; (b) POS, 80 mg/kg Zn from ZnSO4 + 482 mg/kg POS (the same amount of POS as treatment 3); and (c) Zn-POS, 80 mg/kg zinc from Zn-POS. Compared to the Control, both POS and Zn-POS supplementation increased average daily gain and reduced the mortality during day 22-42, and only Zn-POS supplementation decreased the ratio of feed to gain during day 22-42 and 1-42. Moreover, both POS and Zn-POS supplementation improved Zn status and gut function as evidenced by increased metallothionein concentrations in the pancreas, villus height in the duodenum and isobutyrate concentrations in the caecal digesta. Additionally, Zn-POS supplementation increased gene expressions of metallothionein, Zn transporter 1, Zn transporter 2 in the pancreas, nuclear factor erythroid 2-related factor 2 in the liver, the concentrations of acetate, propionate, butyrate and total SCFA in the caecal digesta and the villus height to crypt depth ratio in the duodenum and jejunum, whereas decreased the crypt depth in these two tissues. Altogether, our results revealed that dietary POS or Zn-POS supplementation benefited growth performance, Zn status, antioxidant ability and gut function of broilers. Supplementing Zn-POS in the form of chelate was more effective than feeding POS or ZnSO4 separately.

An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing.

Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.

Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy.

Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma (NB). We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR.GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKT cells. These CAR.GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR.GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy.

Medulloblastoma expresses CD1d and can be targeted for immunotherapy with NKT cells.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Current therapies are toxic and not always curative that necessitates development of targeted immunotherapy. However, little is known about immunobiology of this tumor. In this study, we show that MB cells in 9 of 20 primary tumors express CD1d, an antigen-presenting molecule for Natural Killer T cells (NKTs). Quantitative RT-PCR analysis of 61 primary tumors revealed an elevated level of CD1d mRNA expression in a molecular subgroup characterized by an overactivation of Sonic Hedgehog (SHH) oncogene compared with Group 4. CD1d-positive MB cells cross-presented glycolipid antigens to activate NKT-cell cytotoxicity. Intracranial injection of NKTs resulted in regression of orthotopic MB xenografts in NOD/SCID mice. Importantly, the numbers and function of peripheral blood type-I NKTs were preserved in MB patients. Therefore, CD1d is expressed on tumor cells in a subset of MB patients and represents a novel target for immunotherapy.

Effect of different exercise regimens on LVEF and restenosis incidence in patients after PCI: a network meta-analysis and an overview of systematic reviews.

Objective: We aimed to evaluate the effects of different exercise rehabilitation (ER) programs on LVEF and the incidence of restenosis in patients after percutaneous coronary intervention (PCI) through a systematic review and an integrated network meta-analysis (NMA) to provide a reference for the clinical formulation of ER programs for PCI patients. Methods: Meta-analyses of the effects of different types of ER programs on LVEF and the incidence of reinfarction in post-PCI patients were retrieved from 11 domestic and foreign databases. The methodological and reporting quality of the included systematic reviews were evaluated using the AMSTAR 2 and PRISMA statements. The GRADE scoring system was used to evaluate the quality of evidence found in the studies included in the meta-analysis, and studies with high and intermediate-quality evidence were qualitatively analyzed. Stata software (version 16.0) was used to conduct an integrated NMA of the original RCTs with moderate and low risk of bias. Result: Sixteen meta-analyses were included in this evaluation. The reporting quality of the included meta-analyses was relatively complete, and the methodological quality was low. Seventy RCTs were included in the NMA. The results showed that all types of rehabilitative exercises were safe and effectively increased LVEF and reduced the incidence of coronary restenosis in patients. The comprehensive exercise program was the most likely to improve LVEF, and the comprehensive exercise program, early exercise program, and high-intensity interval exercise were better than aerobic exercise. Comprehensive exercise programs, early exercise programs, and aerobic exercise reduced the incidence of restenosis in patients. However, Chinese Qigong did not reduce the incidence of restenosis in patients, and there was a risk of bias and inconsistency in the quantitative analysis of restenosis incidence. Conclusion: Comprehensive exercise programs have the greatest therapeutic significance in improving cardiac output and reducing restenosis rates in post-PCI patients. The early exercise program has great potential but requires kinesiologists to work with physicians to structure the program and strengthen out-of-hospital management. Aerobic exercise has the least therapeutic significance, and Chinese Qigong is suitable for promotion based on its better efficacy than aerobic exercise and may be an alternative to aerobic exercise, but more experimental evidence is needed. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/, PROSPERO CRD42022374590.

Acute T-cell leukemias remain dependent on Notch signaling despite PTEN and INK4A/ARF loss.

NOTCH1 is activated by mutation in more than 50% of human T-cell acute lymphoblastic leukemias (T-ALLs) and inhibition of Notch signaling causes cell-cycle/growth arrest, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor phosphatase and tensin homolog (PTEN) is also mutated or lost in up to 20% of cases. It was recently observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition, raising concern that patients with PTEN-negative disease may fail Notch inhibitor therapy. As these studies were limited to established cell lines, we addressed this issue using a genetically defined mouse retroviral transduction/bone marrow transplantation model and observed primary murine leukemias to remain dependent on NOTCH1 signaling despite Pten loss, with or without additional deletion of p16(Ink4a)/p19(Arf). We also examined 13 primary human T-ALL samples obtained at diagnosis and found no correlation between PTEN status and resistance to Notch inhibition. Furthermore, we noted in the mouse model that Pten loss accelerated disease onset and produced multiclonal tumors, suggesting NOTCH1 activation and Pten loss may collaborate in leukemia induction. Thus, in contrast to previous findings with established cell lines, these results indicate PTEN loss does not relieve primary T-ALL cells of their "addiction" to Notch signaling.

Millet-based crop planting strategies in the Songhua River Region during the liaojin (907-1234 AD) dynasties: A case of the Luotong Mountain City site.

Introduction: Millet-based dryland agriculture is the traditional mode of agricultural cultivation in northern China and has been of great significance to the emergence and development of Chinese civilization. However, although they are both millet-based agricultural production methods, with various subtypes in different regions of northern China. In the Songhua River Region in northeast China, the ecological environment and abundant natural resources led to the slow development of agriculture, and it was only after the Liaojin Dynasties that a mature farming industry was formed. Material and Method: We used the plant flotation instrument to flotation the soil samples unearthed in the Luotong Mountain City, a Liaojin period site in Songhua River Region, northeast China, and collected the charred plant seeds. Then observing them with the electron microscope, we identified and counted the plant seeds in this site. Result: It was found that this region is still a millet-based crop utilization structure, and a total of 11 types of charred agricultural crop seeds were excavated from flotation at the Luotong Mountain City site. And the barnyard millet crops occupy a prominent advantage, with ubiquity of more than 91%. Discussion: The ancestors of this region were still engaged in a millet-based agricultural strategy during this period, with a certain lag compared to the Central Plains'agricultural strategy where Triticeae crops had become dominant. In addition, the crop structure with the millet-based agriculture of the region is also somewhat different from that of the Central Plains. Through comparative studies of surrounding sites and reference to historical documents, it was found that this difference in crop structure is a phenomenon unique to the Songhua River Region and is related to the dietary habits of the local settled Jurchen nomads, who ate barnyard millet meal.

Multi-omics analyses reveal MdMYB10 hypermethylation being responsible for a bud sport of apple fruit color.

Apple bud sports offer a rich resource for clonal selection of numerous elite cultivars. The accumulation of somatic mutations as plants develop may potentially impact the emergence of bud sports. Previous studies focused on somatic mutation in the essential genes associated with bud sports. However, the rate and function of genome-wide somatic mutations that accumulate when a bud sport arises remain unclear. In this study, we identified a branch from a 10-year-old tree of the apple cultivar 'Oregon Spur II' as a bud sport. The mutant branch showed reduced red coloration on fruit skin. Using this plant material, we assembled a high-quality haplotype reference genome consisting of 649.61 Mb sequences with a contig N50 value of 2.04 Mb. We then estimated the somatic mutation rate of the apple tree to be 4.56 × 10 -8 per base per year, and further identified 253 somatic single-nucleotide polymorphisms (SNPs), including five non-synonymous SNPs, between the original type and mutant samples. Transcriptome analyses showed that 69 differentially expressed genes between the original type and mutant fruit skin were highly correlated with anthocyanin content. DNA methylation in the promoter of five anthocyanin-associated genes was increased in the mutant compared with the original type as determined using DNA methylation profiling. Among the genetic and epigenetic factors that directly and indirectly influence anthocyanin content in the mutant apple fruit skin, the hypermethylated promoter of MdMYB10 is important. This study indicated that numerous somatic mutations accumulated at the emergence of a bud sport from a genome-wide perspective, some of which contribute to the low coloration of the bud sport.

Cloning and functional characterization of a cation-chloride cotransporter gene OsCCC1.

Potassium (K+) and chloride (Cl-) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K+ and Cl-, it remains unclear if they actively use K+-coupled Cl- cotransporters (KCC), as used in animals, to transport K+ and Cl-. We have cloned an Oryza sativa cDNA encoding for a member of the cation-Cl- cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K--Cl- cotransporters than to other cation-Cl- cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na+, K+, and Cl- contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K+ and Cl- than the latter but that they did not differ in terms of Na+ contents, suggesting OsCCC1 may be involved in K+ and Cl- transport. Results from different tests indicated that the OsCCC1 plays a significant role in K+ and Cl- homeostasis and rice plant development.

New insights into gibberellin signaling in regulating plant growth-metabolic coordination.

The Green Revolution of the 1960s boosted cereal crop yields in part through widespread adoption of semi-dwarf plant varieties, many of which were later found to have mutations in either gibberellins (GAs) homeostasis or DELLA proteins. GA is essential for plant growth and developmental regulation and plays an important role in improving crop plant architecture for enhanced grain yield under high nitrogen conditions. A complex regulatory network governs the spatially and temporally controlled genes expression through integrative GA signaling in response to multiple endogenous and environmental cues. In this review, we summarize current advances in understanding the molecular mechanisms of DELLA-dependent and DELLA-independent GA signaling pathways and their contributions to plant developmental and metabolic adaptations to changes in nitrogen availability. The progress in molecular understanding of the plant growth-metabolic coordination will facilitate breeding strategies for future sustainable agriculture and a new Green Revolution.

Optimization of an artificial cellulase cocktail for high-solids enzymatic hydrolysis of cellulosic materials with different pretreatment methods.

Optimization of the composition of cellulase mixtures is an effective strategy to improve their hydrolytic efficiency and reduce protein demand during enzymatic degradation of lignocelluloses. In this study, the mixture design method was used to optimize the ratios of endoglucanase II (EG II), cellobiohydrolase I (CBH I) and ß-glucosidase I (BG I) from Penicillium oxalicum in an artificial cellulase mixture for the hydrolysis of six different cellulosic materials. The optimal composition of enzyme mixture was distinctly different among not only cellulosic materials with different pretreatment methods but hydrolyses at different solids concentrations. CBH I was most critical for the hydrolysis of two acid-pretreated materials, probably due to its strong adsorption on lignin. A higher proportion of EG II was needed for the hydrolysis of ammonium sulfite pretreated wheat straw. The requirements of specific cellulase components were more pronounced at high solids concentrations, highlighting the importance of considering solids loading when optimizing cellulase cocktails.

Development of a nude mouse model for the study of antlerogenesis--mechanism of tissue interactions and ossification pathway.

In a previous study (Li et al., 2001. J Exp Zool 290:18-30) a nude mouse model was established to investigate deer antler development. In that study we found nude mice could support the singularly implanted antlerogenic periosteum (AP) to form pedicle-like, but not antler-like, bony protuberances. To further develop the model and at the same time to use the updated model for the investigation of antler formation, three experiments were carried out in this study. The results showed that (1) antler-like protuberances were successfully induced on the nude mouse heads via subcutaneous co-transplantation of AP and deer skin, and subsequent exposure through wounding of the deer xenografts; (2) deer skin epidermis and its attached half thickness of dermis were sufficient to interact with the AP, and the interactions were capable of transforming adult scalp skin into velvet; (3) the putative initial inductive molecules were primarily derived from the AP cellular layer, rather than fibrous layer; (4) initiation of the ossification center in the avascular cartilage of each mouse "antler" took place via metaplasia, rather than classical endochondral ossification. Further research is required to identify means for effective stimulation of calcification of the "mouse antlers" in order to create the opportunity to investigate antler regeneration using the nude mouse model. Overall, the nude mouse model, once further developed, has the potential to become a powerful tool to study underlying mechanism of antlerogenesis and organogenesis/regeneration in general.

Non-pharmacological treatments for irritable bowel syndrome: study protocol of an umbrella review of systematic review and meta-analyses.

INTRODUCTION: Non-pharmacological treatments are used in the management of irritable bowel syndrome, and their effectiveness has been evaluated in multiple meta-analyses. The robustness of the results in the meta-analyses was not evaluated. We aimed to assess whether there is evidence of diverse biases in the meta-analyses and to identify the treatments without evidence of risk of bias. METHODS AND ANALYSIS: We will search MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, Web of Science and CINAHL Plus for meta-analyses that evaluate the effectiveness of non-pharmacological treatments. The time of publication will be limited from inception to December 2018. The credibility of the meta-analyses will be evaluated by assessing between-study heterogeneity, small-study effect and excess significance bias. The between-study heterogeneity will be assessed using the Cochrane's Q test, and the extent of the heterogeneity will be classified using the I2 statistics. The existence of a small-study effect in a meta-analysis will be evaluated using the funnel plot method and confirmed by Egger's test. Excess significance bias will be evaluated by comparing the expected number of clinical studies with positive findings with the observed number. ETHICS AND DISSEMINATION: No formal ethical approval is required since we will use publicly available data. We will disseminate the findings of the umbrella review through publication in a peer-reviewed journal and conference presentations. PROSPERO REGISTRATION NUMBER: CRD42018111516.

Dynamic Cytosine DNA Methylation Patterns Associated with mRNA and siRNA Expression Profiles in Alternate Bearing Apple Trees.

Cytosine DNA methylation plays an important role in plants: it can mediate gene expression to affect plant growth and development. However, little is known about the potential involvement of cytosine DNA methylation in apple trees as well as in response to alternate bearing. Here, we performed whole-genome bisulfate sequencing to investigate genomic CG, CHG, and CHH methylation patterns, together with their global mRNA accumulation and small RNA expression in "Fuji" apple trees. Results showed that "Fuji" apple trees have a higher CHH methylation than Arabidopsis. Moreover, genomic methylation analysis revealed that CG and CHG methylation were robustly maintained at the early stage of flower induction. Additionally, differentially methylated regions (DMRs), including hypermethylated and hypomethylated DMRs, were also characterized in alternate bearing (AB) apple trees. Intriguingly, the DMRs were enriched in hormones, redox state, and starch and sucrose metabolism, which affected flowering. Further global gene expression evaluation based on methylome analysis revealed a negative correlation between gene body methylation and gene expression. Subsequent small RNA analyses showed that 24-nucleotide small interfering RNAs were activated and maintained in non-CG methylated apple trees. Our whole-genome DNA methylation analysis and RNA and small RNA expression profile construction provide valuable information for future studies.

Role of heterotypic tissue interactions in deer pedicle and first antler formation-revealed via a membrane insertion approach.

Heterotypic tissue interactions play an indispensable role in organ generation and regeneration. In contrast to the classic examples of tissue interactions prevailing in the formation of tetrapod limbs or pectoral fins that can only take place when the interactive tissues are in intimate contacts, the interactions in deer antler formation are novel in that the inducer and the responder are separated by a distance of 1-2 mm. This feature offers a unique opportunity to explore the mechanism underlying tissue interactions by permitting membrane insertion between the two interactive tissues. Four experiments were conducted in this study. The results showed that the impermeable membranes inhibited antler formation. In contrast, the permeable membrane (0.45 microm in pore size) substantially slowed pedicle growth and antler initiation but did not stop them. Interestingly, the impermeable membrane/sheath only slightly retarded antler elongation. Overall, our results demonstrate that interactions between the two interactive tissues, antlerogenic tissue and the overlying skin, are indispensable for first antler initiation and are achieved through diffusible molecules rather than direct physical contact. As the heterotypic tissue interactions are only required during antler initiation but not elongation, they must be transient in nature, and thus differ from those operating in limb/fin formation that can only be sustained by continuous interactions. A system in which organ development is achieved only through transient tissue interactions must be novel, if not completely unique. Understanding this system will undoubtedly enrich the knowledge in the field of tissue interactions and organogenesis.