Lasting Gammaproteobacteria profile changes characterized hematological cancer patients who developed oral mucositis following conditioning therapy.

Background: Oral mucositis (OM) is a common side effect of conditioning therapy implemented before hematopoietic stem cell transplantation (HSCT). The role of oral microbiome in OM is not fully elucidated. Objective: To determine oral microbiome profile changes post-conditioning in HSCT patients who developed moderate OM, or mild to no OM. Design: Patient groups were: Muc0-1 with OM-score = 0-1 (43 paired samples) and Muc2 with WHO OM-score = 2 (36 paired samples). Bacterial DNA was isolated from oral samples (saliva, swabs of buccal mucosa, tongue, and supragingival plaque) at pre-conditioning (T 0 ), post-conditioning mucositis onset (T Muc ), and one-year post-conditioning (T Year ). 16S-rRNA gene next-generation sequencing was used to determine the relative abundance (RA) of >700 oral species. Alpha-diversity, beta-diversity and linear discriminant analyses (LDA) were performed Muc2 versus Muc0-1. Results: Muc2 oral microbiome alpha- and beta-diversity differed between T 0 and T Muc . Muc2 alpha-diversity and Muc0-1 beta-diversity did not differ between T 0 and T Year . T 0 to T Muc LDA scores were significant in Muc2 for Gammaproteobacteria. For Muc2 patients, the average RA decreased for Haemophilus parainfluenza, a species known as mucosal surfaces protector, but increased for Escherichia-Shigella genera. Conclusions: Post-conditioning OM might contribute to long-term oral microbiome changes affecting Gammaproteobacteria, in HSCT patients.

Oral Microbiome and Cancer Therapy-Induced Oral Mucositis.

Characterization of the role of oral microbiome in cancer therapy-induced oral mucositis (CTOM) is critical in preventing the clinically deleterious effects on patients' health that are associated with CTOM. Funding initiatives related to the National Institutes of Health human microbiome project have resulted in groundbreaking advancements in biology and medicine during the last decade. These advancements have shown that a human being is in fact a superorganism made of human cells and associated symbiotic or commensal microbiota. In this review, we describe the state of science as it relates to fundamental knowledge on oral microbiome and its role in CTOM. We also discuss how state-of-the-art technologies and systems biology tools may be used to help tackle the difficult challenges ahead to develop effective treatments or preventive therapies for oral mucositis. We make a clear distinction between disease processes pertaining to the oral microbiome, which includes opportunistic pathogens that may be defined as pathobionts, and those infectious disease processes initiated by exogenous pathogens. We also explored the extent to which knowledge from the gastrointestinal tract in disease and intestinal mucositis could help us better understand CTOM pathobiology. Finally, we propose a model in which the oral microbiome participates in the current five-step CTOM pathobiology model. With the advent of more sophisticated metagenomics technologies and methods of analysis, much hope lies ahead to implement an effective holistic approach to treat cancer patients affected by CTOM.

Caries-associated oral microbiome in head and neck cancer radiation patients: a longitudinal study.

Head and neck cancer (HNC) therapy often leads to caries development. Our goal was to characterize the oral microbiome of HNC patients who underwent radiation therapy (RT) at baseline (T0), and 6 (T6) and 18 (T18) months post-RT, and to determine if there was a relationship with increased caries. HOMINGS was used to determine the relative abundance (RA) of >600 bacterial species in oral samples of 31 HNC patients. The DMFS score was used to define patient groups with tooth decay increase (DMFS[+]) or no increase (DMFS[-]).A change in microbiome beta-diversity was observed at T6 and T18. The Streptococcus mutans RA increased at T6 in both DMFS[+] and DMFS[-] groups. The RA of Prevotella melaninogenica, the species often associated with caries in young children, decreased at T6 in the DMFS[-] group. The RA of the health-associated species, Abiotrophia defective, decreased in the DMFS[+] group. The oral microbiome underwent significant changes in radiation-treated HNC patients, whether they developed caries or not. Caries rates were not associated with a difference in salivary flow reduction between DMFS[+] andDMFS[-] groups. Patients who develop caries might be more susceptible to certain species associated with oral disease or have fewer potentially protective oral species.

Text mining-based in silico drug discovery in oral mucositis caused by high-dose cancer therapy.

INTRODUCTION: Oral mucositis (OM) is a major dose-limiting side effect of chemotherapy and radiation used in cancer treatment. Due to the complex nature of OM, currently available drug-based treatments are of limited efficacy. OBJECTIVES: Our objectives were (i) to determine genes and molecular pathways associated with OM and wound healing using computational tools and publicly available data and (ii) to identify drugs formulated for topical use targeting the relevant OM molecular pathways. METHODS: OM and wound healing-associated genes were determined by text mining, and the intersection of the two gene sets was selected for gene ontology analysis using the GeneCodis program. Protein interaction network analysis was performed using STRING-db. Enriched gene sets belonging to the identified pathways were queried against the Drug-Gene Interaction database to find drug candidates for topical use in OM. RESULTS: Our analysis identified 447 genes common to both the "OM" and "wound healing" text mining concepts. Gene enrichment analysis yielded 20 genes representing six pathways and targetable by a total of 32 drugs which could possibly be formulated for topical application. A manual search on ClinicalTrials.gov confirmed no relevant pathway/drug candidate had been overlooked. Twenty-five of the 32 drugs can directly affect the PTGS2 (COX-2) pathway, the pathway that has been targeted in previous clinical trials with limited success. CONCLUSIONS: Drug discovery using in silico text mining and pathway analysis tools can facilitate the identification of existing drugs that have the potential of topical administration to improve OM treatment.

Biosemantics guided gene expression profiling of Sjögren's syndrome: a comparative analysis with systemic lupus erythematosus and rheumatoid arthritis.

BACKGROUND: Sjögren's syndrome (SS) shares many clinical and pathological similarities with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). These autoimmune diseases mostly affect women. In this study, concept profile analysis (CPA) and gene expression meta-analysis were used to identify genes potentially involved in SS pathogenesis. METHODS: Human genes associated with SS, SLE, and RA were identified using the CPA tool, Anni 2.1. The differential mRNA expression of genes common to SS and SLE (SS-SLE) was determined in female peripheral blood mononuclear cells (PBMCs) using NCBI-GEO2R. Differentially expressed (DE) SS-SLE PBMC genes in common with the SS-SLE CPA-identified genes were analyzed for differential expression in salivary glands or synovial biopsies, and for genes common to SS and RA and SLE and RA, analyzing differential expression in salivary glands in SS, synovial fibroblasts in RA, and synovial fluid in SLE. Among common genes, DE genes found in salivary gland mRNA expression in patients with SS were used for gene enrichment and SS molecular network construction. Secondary analysis was performed to identify DE genes unique to the disease site tissues, by excluding PBMC and CPA common DE genes to complement the SS network. RESULTS: We identified 22 DE genes in salivary gland datasets in SS that have not previously been clearly associated with SS pathogenesis. Among these, higher levels of checkpoint kinase 1 (CHEK1), V-Ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1), and lymphoid enhancer binding factor 1 (LEF1) were significantly correlated with higher matrix metalloproteinase 9 (MMP9) levels. Higher MMP9 levels have been implicated in degradation of salivary gland structural integrity, leading to hypo-salivation in patients with SS. Salivary gland mRNA expression of MMP9 and the expression of cytokine CXCL10 were higher in patients with SS. CXCL10 has been shown to increase MMP9 expression and therefore may also play an important role in SS pathogenesis. CONCLUSION: Using CPA and gene expression analysis, we identified factors targeting MMP9 expression and/or function, namely CHEK1, CXCL10, ETS1, LEF1, and tissue inhibitor of metalloproteinase 1; altered mRNA expression of these could increase expression/activity of MMP9 in a concerted manner, thereby potentially impacting SS pathogenesis.

Concordance of HOMIM and HOMINGS technologies in the microbiome analysis of clinical samples.

BACKGROUND: Over 700 bacterial species reside in human oral cavity, many of which are associated with local or distant site infections. Extensive characterization of the oral microbiome depends on the technologies used to determine the presence and proportions of specific bacterial species in various oral sites. OBJECTIVE: The objective of this study was to compare the microbial composition of dental plaque at baseline using Human Oral Microbe Identification Microarray (HOMIM) and Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) technologies, which are based on 16S rRNA. METHODS: Dental plaque samples were collected from 96 patients at baseline prior to a dental procedure involving manipulation of gingival tissues. The samples were surveyed for 293 and 597 oral bacterial species via HOMIM and HOMINGS, respectively, based on 16S rRNA gene sequences. We determined the concordance between the two technologies for common species. Genus level analysis was performed using HOMINGS-specific genus identification capabilities. RESULTS: HOMINGS detected twice the number of species in the same dental plaque samples compared to HOMIM. For the species detected by both HOMIM and HOMINGS, there was no difference in relative proportions of overall bacterial composition at the species, genus or phylum levels. Additionally, there was no difference in relative proportion for total species per patient between the two technologies. CONCLUSION: HOMINGS significantly expanded oral bacterial species identification compared to HOMIM. The genus and species probes, combined in HOMINGS, provided a more comprehensive representation of oral bacterial community, critical for future characterization of oral microbes in distant site infections.

Abnormal retinal development in Cloche mutant zebrafish.

BACKGROUND: Functions for the early embryonic vasculature in regulating development of central nervous system tissues, such as the retina, have been suggested by in vitro studies and by in vivo manipulations that caused additional ocular vessels to develop. Here, we use an avascular zebrafish embryo, cloche-/- (clo-/-), to begin to identify necessary developmental functions of the ocular vasculature in regulating development and patterning of the neural retina, in vivo. These studies are possible in zebrafish embryos, which do not yet rely upon the vasculature for tissue oxygenation. RESULTS: clo-/- embryos lacked early ocular vasculature and were microphthalmic, with reduced retinal cell proliferation and cell survival. Retinas of clo mutants were disorganized, with irregular synaptic layers, mispatterned expression domains of retinal transcription factors, morphologically abnormal Müller glia, reduced differentiation of specific retinal cell types, and sporadically distributed cone photoreceptors. Blockade of p53-mediated cell death did not completely rescue this phenotype and revealed ectopic cones in the inner nuclear layer. clo-/- embryos did not upregulate a molecular marker for hypoxia. CONCLUSIONS: The disorganized retinal phenotype of clo-/- embryos is consistent with a neural and glial developmental patterning role for the early ocular vasculature that is independent of its eventual function in gas exchange.

Retinoic Acid Signaling Regulates Differential Expression of the Tandemly-Duplicated Long Wavelength-Sensitive Cone Opsin Genes in Zebrafish.

The signaling molecule retinoic acid (RA) regulates rod and cone photoreceptor fate, differentiation, and survival. Here we elucidate the role of RA in differential regulation of the tandemly-duplicated long wavelength-sensitive (LWS) cone opsin genes. Zebrafish embryos were treated with RA from 48 hours post-fertilization (hpf) to 75 hpf, and RNA was isolated from eyes for microarray analysis. ~170 genes showed significantly altered expression, including several transcription factors and components of cellular signaling pathways. Of interest, the LWS1 opsin gene was strongly upregulated by RA. LWS1 is the upstream member of the tandemly duplicated LWS opsin array and is normally not expressed embryonically. Embryos treated with RA 48 hpf to 100 hpf or beyond showed significant reductions in LWS2-expressing cones in favor of LWS1-expressing cones. The LWS reporter line, LWS-PAC(H) provided evidence that individual LWS cones switched from LWS2 to LWS1 expression in response to RA. The RA signaling reporter line, RARE:YFP indicated that increased RA signaling in cones was associated with this opsin switch, and experimental reduction of RA signaling in larvae at the normal time of onset of LWS1 expression significantly inhibited LWS1 expression. A role for endogenous RA signaling in regulating differential expression of the LWS genes in postmitotic cones was further supported by the presence of an RA signaling domain in ventral retina of juvenile zebrafish that coincided with a ventral zone of LWS1 expression. This is the first evidence that an extracellular signal may regulate differential expression of opsin genes in a tandemly duplicated array.

Synthesis of tetrahydroxybiphenyls and tetrahydroxyterphenyls and their evaluation as amyloid-ß aggregation inhibitors.

3,3',4,4'-Tetrahydroxybiphenyl and three isomeric 3,3″,4,4″-tetrahydroxyterphenyls with varying geometries around the central phenyl ring have been synthesized and evaluated for their in vitro activity against aggregation of Alzheimer's amyloid-ß peptide (Aß). Results from Congo red spectral-shift assays reveal that all four compounds successfully inhibit association of Aß monomers. For the tetrahydroxyterphenyls, efficacy varies with linker geometry: the ortho-arrangement affords the most successful inhibition and the para-geometry the least, perhaps due to differing abilities of these compounds to bind Aß. Of the four small molecules studied, 3,3',4,4'-tetrahydroxybiphenyl is the most effective inhibitor, reducing Aß aggregation by 50% when present in stoichiometric concentrations.

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure.

BACKGROUND: Retinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined. RESULTS: Treatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA. CONCLUSIONS: These data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor mediates the effects of endogenous, as well as exogenous RA, on rod development.

Developmental expression of sema3G, a novel zebrafish semaphorin.

The semaphorins are a large, evolutionarily conserved family of signaling molecules with broad functions during development. The class 3 semaphorins are a subclass of secreted semaphorins found in vertebrates. There have been six class 3 semaphorins identified to date (sema3A to sema3F) and some have been shown to function in axon guidance and cardiovascular development. However, the functions of many class 3 semaphorins and their potential interactions in vivo are still not well understood. As a step toward understanding the actions of all class 3 semaphorins in vivo, we have cloned and analyzed the developmental expression pattern of a novel zebrafish class 3 semaphorin, sema3H [corrected] sema3H [corrected] is expressed in a dynamic pattern throughout the first 3 days of development. It is expressed in the adaxial cells of the somite during somitogenesis. In the brain, sema3H [corrected] is expressed in cell clusters in the midbrain and diencephalon, and is expressed in the telencephalon in close proximity to the olfactory epithelium. sema3H [corrected] also is expressed in the pharyngeal arches, the pectoral fin bud, and the developing pronephros. These results provide a basis for studying how expression of multiple semaphorins could be essential for aspects of early development.

Forced activation of Wnt signaling alters morphogenesis and sensory organ identity in the chicken inner ear.

Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, we used retroviral gene transfer to force the expression of an activated form of beta-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. We hypothesize that Wnt/beta-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct.