It is well known that healthy gut microbiota is essential to promote host health and well‐being. The intestinal microbiota of endothermic animals as well as fish are classified as autochthonous or indigenous, when they are able to colonize the host's epithelial surface or are associated with the microvilli, or as allochthonous or transient (associated with digesta or are present in the lumen). Furthermore, the gut microbiota of aquatic animals is more fluidic than that of terrestrial vertebrates and is highly sensitive to dietary changes. In fish, it is demonstrated that [a] dietary form (live feeds or pelleted diets), [b] dietary lipid (lipid levels, lipid sources and polyunsaturated fatty acids), [c] protein sources (soybean meal, krill meal and other meal products), [d] functional glycomic ingredients (chitin and cellulose), [e] nutraceuticals (probiotics, prebiotics, synbiotics and immunostimulants), [f] antibiotics, [g] dietary iron and [h] chromic oxide affect the gut microbiota. Furthermore, some information is available on bacterial colonization of the gut enterocyte surface as a result of dietary manipulation which indicates that changes in indigenous microbial populations may have repercussion on secondary host–microbe interactions. The effect of dietary components on the gut microbiota is important to investigate, as the gastrointestinal tract has been suggested as one of the major routes of infection in fish. Possible interactions between dietary components and the protective microbiota colonizing the digestive tract are discussed.

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Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

The outcome of recurrent brain metastasis is dismal. This study aims to assess the clinical outcomes and toxicity of reirradiation as a salvage treatment for progressive brain metastasis in patients with advanced breast cancer. Between July 2005 and September 2014, the medical records of 56 patients with brain metastasis from breast cancer were retrospectively reviewed. Of these patients, 39 received whole-brain radiotherapy (WBRT) followed by stereotactic radiosurgery (SRS) reirradiation (Group 1), and 17 received SRS followed by WBRT reirradiation (Group 2). Overall survival (OS) and brain progression-free survival rates/times were calculated using the Kaplan–Meier method. Prognostic factors were evaluated using the Cox proportional hazards model. Change in neurologic function was also assessed. The median OS was 10.8 months (range, 1.3–56.8 months). In Group 1, the median PFS time (PFS-1) was 6.5 months and the OS time was 11.4 months. Multivariate analysis revealed that longer OS was significantly associated with a high Karnofsky performance score (KPS) (P = 0.004), controlled extracranial metastasis (P = 0.001) and a good response to reirradiation (P = 0.034). In Group 2, the median PFS time (PFS-2) after reirradiation was 8.5 months and the OS time was 10.8 months. Multivariate analysis revealed that longer OS was significantly associated with a high KPS (P = 0.018). The majority of the patients had improved or stable neurological function. Reirradiation is an effective and a safe treatment for patients with brain metastases from breast cancer. It might delay the progression of intracranial disease and improve neurological function. A suitable patient selection for reirradiation was suggested.

The genotype-specific defense activation in the roots of perennial tree crops to soilborne necrotrophic pathogens remains largely unknown. A recent phenotyping study indicated that the apple rootstock genotypes B.9 and G.935 have contrasting resistance responses to infection by Pythium ultimum. In the current study, a comparative transcriptome analysis by Illumina Solexa HiSeq 3000 platform was carried out to identify the global transcriptional regulation networks between the susceptible B.9 and the resistant G.935 to P. ultimum infection. Thirty-six libraries were sequenced to cover three timepoints after pathogen inoculation, with three biological replicates for each sample. The transcriptomes in the roots of the susceptible genotype B.9 were reflected by overrepresented differentially expressed genes (DEGs) with downregulated patterns and systematic suppression of cellular processes at 48 h post inoculation (hpi). In contrast, DEGs with annotated functions, such as kinase receptors, MAPK signaling, JA biosynthesis enzymes, transcription factors, and transporters, were readily induced at 24 hpi and continued up-regulation at 48 hpi in G.935 roots. The earlier and stronger defense activation is likely associated with an effective inhibition of necrosis progression in G.935 roots. Lack of effector-triggered immunity or existence of a susceptibility gene could contribute to the severely disturbed transcriptome and susceptibility in B.9 roots. The identified DEGs constitute a valuable resource for hypothesis-driven studies to elucidate the resistance/tolerance mechanisms in apple roots and validating their potential association with resistance traits.

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Allele-specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar 'Royal Gala' (RG). A high quality chromosome-level genome was assembled using a homozygous tetra-haploid RG plant, derived from anther cultures. Using RNA-sequencing (RNA-seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of 'Golden Delicious' (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele-specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele-specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome-wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.