De novo root regeneration (DNRR) is the process in which adventitious roots are regenerated from damaged plant tissues or organs. We have developed a simple DNRR system in which adventitious roots are formed from detached leaf explants of Arabidopsis (Arabidopsis thaliana) on B5 medium without external hormones. In this chapter, we introduce the methods used to observe gene expression patterns during rooting from leaf explants. Usually, β-glucuronidase (GUS) staining is used to visualize gene expression patterns, since fluorescent proteins are difficult to observe because of the high autofluorescence in leaf explants. Here, we describe the use of the ClearSee technique with Congo red staining for deep imaging to observe fluorescent proteins. This method diminishes autofluorescence in leaf explants and preserves the stability of fluorescent proteins, thus allowing us to investigate the endogenous molecular actions guiding DNRR.

The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters 11 and 13 ). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.

BACKGROUND: Periodontitis is a chronic inflammation of periodontal supporting tissue caused by local factors. Periodontal surgery can change the gene expression of peripheral blood mononuclear cells. However, little is known about the potential mechanism of surgical treatment for periodontitis.
OBJECTIVE: To explore the potential molecular mechanism of surgical treatment for periodontitis.
METHODS: First, based on the expression profiles of genes related to surgical treatment for periodontitis, a set of expression disorder modules related to surgical treatment for periodontitis were obtained by enrichment analysis. Subsequently, based on crosstalk analysis, we proved that there was a significant crosstalk relationship between module 3 and module 5. Finally, based on predictive analysis of multidimensional regulators, we identified a series of regulatory factors, such as endogenous genes, non-coding RNAs (ncRNAs), and transcription factors, which have potential regulatory effects on periodontitis.
RESULTS: A total of 337 genes related to surgical treatment for periodontitis were obtained, and 3896 genes related to periodontitis were amplified. Eight expression modules of periodontitis were obtained, involving the aggregation of 2672 gene modules. These modules are mainly involved in G-protein coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger, and adenylate cyclase-modulating G-protein coupled receptor signaling pathway. In addition, eight endogenous genes (including EGF, RPS27A, and GNB3) were screened by network connectivity analysis. Finally, based on this set of potential dysfunction modules, 94 transcription factors (including NFKB1, SP1, and STAT3) and 1198 ncRNAs (including MALAT1, CRNDE, and ANCR) were revealed. These core regulators are thought to be involved in the potential molecular mechanism of periodontitis after surgical treatment.
CONCLUSIONS: Based on the results of this study, we can show biologists and pharmacists a new idea to reveal the potential molecular mechanism of surgical treatment for periodontitis, and provide valuable reference for follow-up treatment programs.

BACKGROUND: Metastases from primary breast cancers can involve single or multiple organs at metastatic disease diagnosis. Molecular risk factors for particular patterns of metastastic spread in a clinical population are limited.
METHODS: A case-control design including 1357 primary breast cancers was used to study three distinct clinical patterns of metastasis, which occur within the first six months of metastatic disease: bone and visceral metasynchronous spread, bone-only, and visceral-only metastasis. Whole-genome expression profiles were obtained using whole genome (WG)-DASL assays from formalin-fixed paraffin-embedded (FFPE) samples. A systematic protocol was developed for handling FFPE samples together with stringent data quality controls to identify robust expression profiling data. A panel of published and novel gene sets were tested for association with these specific patterns of metastatic spread and odds ratios (ORs) were calculated.
RESULTS: Metasynchronous metastasis to bone and viscera was found in all intrinsic breast cancer subtypes, while immunohistochemically (IHC)-defined receptor status and specific IntClust subgroups were risk factors for visceral-only or bone-only first metastases. Among gene modules, those related to proliferation increased the risk of metasynchronous metastasis (OR (95% CI) = 2.3 (1.1-4.8)) and visceral-only first metastasis (OR (95% CI) = 2.5 (1.2-5.1)) but not bone-only metastasis (OR (95% CI) = 0.97 (0.56-1.7)). A 21-gene module (BV) was identified in estrogen-receptor-positive breast cancers with metasynchronous metastasis to bone and viscera (area under the curve = 0.77), and its expression increased the risk of bone and visceral metasynchronous spread in this population. BV was further orthogonally validated with NanoString nCounter in primary breast cancers, and was reproducible in their matched lymph nodes metastases and an external cohort.
CONCLUSIONS: This case-control study of WG-DASL global expression profiles from FFPE tumour samples, after careful quality control and RNA selection, revealed that gene modules in the primary tumour have differing risks for clinical patterns of metasynchronous first metastases. Moreover, a novel gene module was identified as a putative risk factor for metasynchronous bone and visceral first metastatic spread, with potential implications for disease monitoring and treatment planning.