High-Resolution Transcriptomic Landscape of the Human Submandibular Gland.

Saliva-secreting and transporting cells are part of the complex cellular milieu of the human salivary gland, where they play important roles in normal glandular physiology and diseased states. However, comprehensive molecular characterization, particularly at single-cell resolution, is still incomplete, in part due to difficulty in procuring normal human tissues. Here, we perform an in-depth analysis of male and female adult human submandibular gland (SMG) samples by bulk RNA sequencing (RNA-seq) and examine the molecular underpinnings of the heterogeneous cell populations by single-cell (sc) RNA-seq. Our results from scRNA-seq highlight the remarkable diversity of clusters of epithelial and nonepithelial cells that reside in the SMG that is also faithfully recapitulated by deconvolution of the bulk-RNA data sets. Our analyses reveal complex transcriptomic heterogeneity within both the ductal and acinar subpopulations and identify atypical SMG cell types, such as mucoacinar cells that are unique to humans and ionocytes that have been recently described in the mouse. We use CellChat to explore ligand-receptor interactome predictions that likely mediate crucial cell-cell communications between the various cell clusters. Finally, we apply a trajectory inference method to investigate specific cellular branching points and topology that offers insights into the dynamic and complex differentiation process of the adult SMG. The data sets and the analyses herein comprise an extensive wealth of high-resolution information and a valuable resource for a deeper mechanistic understanding of human SMG biology and pathophysiology.

Genetic Study of Elf5 and Ehf in the Mouse Salivary Gland.

Salivary gland (SG) development, maturation, and homeostasis require coordinated roles of transcription factors (TFs) that dictate specific cell identities and fate. The ETS family of proteins are important transcriptional drivers of diverse cell lineages, tissue development, and differentiation programs and hence are also likely to play an important role in the SG. Here we have leveraged genomic and epigenomic data of the SG to examine the expression profile of ETS genes and identified 2 closely related paralogs, Elf5 and Ehf, that are highly expressed in distinct epithelial subpopulations. By using a well-defined mouse knockout model of Elf5, we show that Elf5, despite its enriched expression in the acinar cells, is functionally dispensable for maintaining the homeostatic state of the adult SG epithelium. The lack of a discernible phenotype of the Elf5-null SG might be due to possible functional redundancy with Ehf or other ETS factors. To probe this possibility and to examine the specific consequences of Ehf loss in the SG, we used CRISPR-Cas9 to generate mice in which the DNA-binding ETS domain of Ehf is disrupted due to an insertion mutation. We demonstrate that the Ehf mutant (EhfMut) mice exhibit a distinct cellular phenotype with decreased granular convoluted tubules that are accompanied by an increased accumulation of the intercalated Sox9-positive ductal cell population. Interestingly, the ductal phenotype of the EhfMut animals is highly pronounced in males, reaffirming the established sexual dimorphism of the SG that exists in rodents. Our results show that unlike Elf5, Ehf plays a nonredundant role in directing ductal cell differentiation of the SG and highlights the phenotypic subtlety in mutant mice of closely related TFs and the importance of careful consideration of cell type-specific studies.

In vitro evaluation of the cis-[Ru(phen)2(pPDIp)]2+⁎⁎ complex for antimicrobial photodynamic therapy against Sporothrix brasiliensis and Candida albicans.

BACKGROUND: Photodynamic therapy (PDT) activates a photosensitizer by visible light to generate cytotoxic oxygen species that lead to cell death. With proper illumination, PDT is often used in applications on superficial and sub-surface lesions. Sporotrichosis infection occurs by Sporothrix fungi which causes a skin wound, worsened by Candida albicans infections. This study investigated the photosensitizing efficiency of the Ru(phen)2(pPDIp)(PF6)2 complex, RupPDIp, against S. brasiliensis and C. albicans. MATERIAL AND METHODS: RupPDIp efficiency against these fungi was tested using 450 nm (blue light and 36 J/cm2) and 525 nm (green light, 25.2 J/cm2) at 0.05-20 µM concentrations. To ensure PDT effectiveness, control groups were tested in the absence and in the presence of RupPDIp under light irradiation and in the dark. RESULTS: RupPDIp eliminated both fungi at ≤5.0 µM. Green light showed the best results, eliminating S. brasiliensis and C. albicans colonies at RupPDIp 0.5 µM and 0.05 µM, respectively. CONCLUSION: RupPDIp is a promising photosensitizer in aPDT, eliminating 106 CFU/mL of both fungi at 450 nm and 525 nm, with lower light doses and concentrations when treated with the green light compared to the blue light.

A Global Vista of the Epigenomic State of the Mouse Submandibular Gland.

The parotid, submandibular, and sublingual glands represent a trio of oral secretory glands whose primary function is to produce saliva, facilitate digestion of food, provide protection against microbes, and maintain oral health. While recent studies have begun to shed light on the global gene expression patterns and profiles of salivary glands, particularly those of mice, relatively little is known about the location and identity of transcriptional control elements. Here we have established the epigenomic landscape of the mouse submandibular salivary gland (SMG) by performing chromatin immunoprecipitation sequencing experiments for 4 key histone marks. Our analysis of the comprehensive SMG data sets and comparisons with those from other adult organs have identified critical enhancers and super-enhancers of the mouse SMG. By further integrating these findings with complementary RNA-sequencing based gene expression data, we have unearthed a number of molecular regulators such as members of the Fox family of transcription factors that are enriched and likely to be functionally relevant for SMG biology. Overall, our studies provide a powerful atlas of cis-regulatory elements that can be leveraged for better understanding the transcriptional control mechanisms of the mouse SMG, discovery of novel genetic switches, and modulating tissue-specific gene expression in a targeted fashion.

Understanding the electronic properties of BaTiO3 and Er3+ doped BaTiO3 films through confocal scanning microscopy and XPS: the role of oxygen vacancies.

Photonic and electronic properties exist inherently in ferroelectric barium titanate (BaTiO3); severe luminescence quenching also exists due to the insufficient confinement of excitons. In this sense, high optical emission can only be achieved by its chemical and structural modification. Thin BaTiO3 and Er:BaTiO3 films were grown by the spin coating method on a glass substrate at room temperature. Self-trapping of excitons in the thin BaTiO3 film and its structural modification due to the doping with Er3+ ions (Er:BaTiO3) are verified using scanning confocal fluorescence microscopy (SCFM), where self-trapping excitons never occured in its pure state. By thermal treatment and doping (BaTiO3 and Er:BaTiO3) we obtained localization of the excitons, which would further induce lattice strain around the surface defects, to accommodate the self-trapped excitons. With such a self-trapped state, the structure of BaTiO3 generates broadband emission of several overlapping bands between 1.95 and 2.65 eV at room temperature, while the structure Er:BaTiO3 showed defined emission bands at 2.24 and 2.35 eV, with very weak contributions of the emission due to the self-trapping state. The influence of the variation of the excitation wavelength using 1PE and 2PE on the emission bands of BaTiO3 and Er:BaTiO3 is also investigated. The results of enhanced emission bands suggest a clear dependence of the emission intensity on the excitation energy, where a ∼3 fold enhancement in emission has been demonstrated under Er3+ (1.55 eV) excitation, which can be attributed to effective energy transfer between the Er3+ ions. As a result, it is concluded that the developed BaTiO3 and Er:BaTiO3 can pave the way for future photonic devices.

Transcriptomic and Single-Cell Analysis of the Murine Parotid Gland.

The salivary complex of mammals consists of 3 major pairs of glands: the parotid, submandibular, and sublingual glands. While the 3 glands share similar functional properties, such as saliva secretion, their differences are largely based on the types of secretions they produce. While recent studies have begun to shed light on the underlying molecular differences among the glands, few have examined the global transcriptional repertoire over various stages of gland maturation. To better elucidate the molecular nature of the parotid gland, we have performed RNA sequencing to generate comprehensive and global gene expression profiles of this gland at different stages of maturation. Our transcriptomic characterization and hierarchical clustering analysis with adult organ RNA sequencing data sets has identified a number of molecular players and pathways that are relevant for parotid gland biology. Moreover, our detailed analysis has revealed a unique parotid gland-specific gene signature that may represent important players that could impart parotid gland-specific biological properties. To complement our transcriptomic studies, we have performed single-cell RNA sequencing to map the transcriptomes of parotid epithelial cells. Interrogation of the single-cell transcriptomes revealed the degree of molecular and cellular heterogeneity of the various epithelial cell types within the parotid gland. Moreover, we uncovered a mixed-lineage population of cells that may reflect molecular priming of differentiation potentials. Overall our comprehensive studies provide a powerful tool for the discovery of novel molecular players important in parotid gland biology.

TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

ΔNp63 regulates IL-33 and IL-31 signaling in atopic dermatitis.

Atopic dermatitis (AD) is the most common inflammatory skin disease with no well-delineated cause or effective cure. Here we show that the p53 family member p63, specifically the ΔNp63, isoform has a key role in driving keratinocyte activation in AD. We find that overexpression of ΔNp63 in transgenic mouse epidermis results in a severe skin phenotype that shares many of the key clinical, histological and molecular features associated with human AD. This includes pruritus, epidermal hyperplasia, aberrant keratinocyte differentiation, enhanced expression of selected cytokines and chemokines and the infiltration of large numbers of inflammatory cells including type 2 T-helper cells - features that are highly representative of AD dermatopathology. We further demonstrate several of these mediators to be direct transcriptional targets of ΔNp63 in keratinocytes. Of particular significance are two p63 target genes, IL-31 and IL-33, both of which are key players in the signaling pathways implicated in AD. Importantly, we find these observations to be in good agreement with elevated levels of ΔNp63 in skin lesions of human patients with AD. Our studies reveal an important role for ΔNp63 in the pathogenesis of AD and offer new insights into its etiology and possible therapeutic targets.

ΔNp63 regulates select routes of reprogramming via multiple mechanisms.

Somatic cells can be converted into induced pluripotent stem cells (iPSCs) by forced expression of various combinations of transcription factors, but the molecular mechanisms of reprogramming are poorly understood. Specifically, evidence that the reprogramming process can take many distinct routes only begins to emerge. It is definitively established that p53 deficiency greatly enhances reprogramming, revealing p53's barrier function for induced pluripotency, but the role of its homologs p63 and p73 are unknown. Here we report that in stark contrast to p53, p73 has no role in reprogramming. However, p63 is an enabling (rather than a barrier) factor for Oct4, Sox2 and Klf4 (OSK) and Oct4 and Sox2 (OS), but not for Oct4 and Klf4 (OK) reprogramming of mouse embryonic fibroblasts. Specifically, p63 is essential during reprogramming for maximum efficiency, albeit not for the ability to reprogram per se, and is dispensable for maintaining stability and pluripotency of established iPSC colonies. ΔNp63, but not TAp63, is the principal isoform involved. Loss of p63 can affect reprogramming via several mechanisms such as reduced expression of mesenchymal-epithelial transition and pluripotency genes, hypoproliferation and loss of the most reprogrammable cell populations. During OSK and OS reprogramming, different mechanisms seem to be critical, such as regulation of epithelial and pluripotency genes in OSK reprogramming versus regulation of proliferation in OS reprogramming. Finally, our data reveal three different routes of reprogramming by OSK, OS or OK, based on their differential p63 requirements for iPSC efficiency and pluripotency marker expression. This supports the concept that many distinct routes of reprogramming exist.

Interaction between the TP63 and SHH pathways is an important determinant of epidermal homeostasis.

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

Tp63 in oral development, neoplasia, and autoimmunity.

The Tp63 gene encodes for multiple isoforms of the p63 transcription factor, a member of the p53 family of proteins. Much like its more famous sibling, the biological role of p63 is quite complex, with wide-ranging effects on development, differentiation, and cellular lineage choices. The crucial function of p63 is epitomized by the striking phenotype of p63 knockout mice. These animals have a profound block in the development of stratified epithelia and aplasia of multiple ectodermal appendages, as well as orofacial clefting and limb defects. Remarkably, a similar spectrum of phenotypic alterations is observed in human syndromes resulting from Tp63 gene mutations. p63 is an important hub in the transcriptional and signaling networks of epithelial cells; thus, it is not surprising that dysregulation of this transcription factor is associated with squamous cell carcinoma. Finally, as a testament to the growing repertoire of p63-associated diseases, autoantibodies to p63 are associated with chronic ulcerative stomatitis, an oral immunologically mediated disease. Over the past decade, our understanding of the broad biologic and pathophysiological roles of p63 has grown significantly. In this review, we discuss the molecular attributes of Tp63 and the clinical consequences of Tp63 dysregulation, particularly as it pertains to oral tissues.

[Creating an integrated nursing curriculum]. / Construção de um currículo integrado de enfermagem.

During the last two decades, Brazilian society has gone through great changes into political, ideological and economical fields. These changes left their strings into society, specially in population health. The nurse formation based on the Law n(o) 5540/68 and on the Statement n(o) 163/72, no more meets population demands. Since 1992, the Nursing Faculty of UERJ-FEUerj intensifies the reflection movement upon teaching-learning process searching for transforming its own reality. The making of this project presents two complementary and important reasons: FEUerj docents and discents' desire in elaborating a curriculum which searches for nurses' formation that articulates teaching-work-community, theory and practice, based on a Critical Theory of Education, on the line of PROBLEMATIZATION, and the accomplishment of Statement n(o) 314/94 from the CFE and from the Letter of Order MEC n(o) 1171/15/dez/94. From debating, the professional profile has been defined from the social environment where the profession is performed and the alumnate's characteristics; area determination or group of attributions, according to professional praxis adequation, concept hierachization, processes, etc., which in the process of 'classification and syntheses' of knowledge results into a netlike chained and related tree. In the first phase of the curriculum study, it has diagnosed as principal condition, the actual curriculum 'DECONTEXTUALIZATION' and the 'US' to be faced to lead it to an end the Curriculum Reformulation Proposal. The Process of Pedagogical Abilitation for professors, workshops, researches on the desirable and present profile, seminars, performance, abilities and principles systematization, identification of areas which compose the integrated curriculum, subjects localization into areas and articulation between professional subjects and other activities, has been implemented. Based on this work on the problematized pedagogy first step, an instrument 'Research on the Professional Profile for the Nursing Graduation Course', was built, identifying the performances and principles which sustain them on each subject making the present profile clear and desirable to docents and discents.

[The surgical exploration of the spermatic cord and testis with deferento-vesiculography (DVG) in the study of azoospermias (author's transl)]. / Il ruolo dell'esplorazione chirurgica funicolo testicolare con deferento-vesciculografia (dvg) nello studio delle azoospermie.

The Authors refer about their investigation of the azoospermias with surgical exploration of the spermatic cord and testis with deferento-vesiculography (DVG). Thirty-three patients were examined with DVG from a total of 50 observations. The Authors conclude that such investigation, associated to the study of others parameters in the male sterility, reveals itself very useful to provide information about azoospermias. Therefore, while testicular biopsy informs on the histomorfologic aspect of the testis, the DVG provides the best investigation of the morphology of the seminal ducts.