A novel method to identify Post-Aire stages of medullary thymic epithelial cell differentiation.

Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+ mTECs differentiate further into Post-Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate-mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi (MHCIIhi CD80hi ) compartment into mTECA/hi (CD24- Sca1- ), mTECB/hi (CD24+ Sca1- ), and mTECC/hi (CD24+ Sca1+ ). While mTECA/hi included mostly Aire-expressing cells, mTECB/hi contained Aire+ and Aire- cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor-product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi , mTECB/hi , and mTECC/hi sequentially mirrored the specific genetic program of Early-, Late- and Post-Aire mTECs. Corroborating their Post-Aire nature, mTECC/hi downregulated the expression of tissue-restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire-deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation.

The pro-proliferative effect of insulin in human breast epithelial DMBA-transformed and non-transformed cell lines is PI3K-, mTOR- and GLUT1-dependent.

Type 2 diabetes mellitus (T2DM) is linked to an increased risk of breast cancer. We aimed to investigate how T2DM-associated characteristics (high levels of glucose, insulin, leptin, inflammatory mediators and oxidative stress) influence breast cancer carcinogenesis, in DMBA-treated (MCF-12ADMBA ) and non-treated breast epithelial (MCF-12A) cell lines. Insulin (50 nM) promotes cell proliferation, 3 H-DG uptake and lactic acid production in both cell lines. The stimulatory effects of insulin upon cell proliferation and 3 H-DG uptake were hampered by rapamycin, LY294001 and BAY-876, in both cell lines. In conclusion, hyperinsulinemia, one important characteristic of T2DM, contributes to the initiation of breast cancer by a PI3K- and mTOR-dependent mechanism involving increased GLUT1-mediated glucose uptake. SIGNIFICANCE: The pro-proliferative effect of insulin in human breast epithelial DMBA-transformed and non-transformed cell lines is PI3K-, mTOR- and GLUT1-dependent.

Biocompatibility of the Biopolymer Cyanoflan for Applications in Skin Wound Healing.

There is a great demand for the development of novel wound dressings to overcome the time and costs of wound care performed by a vast number of clinicians, especially in the current overburdened healthcare systems. In this study, Cyanoflan, a biopolymer secreted by a marine unicellular cyanobacterium, was evaluated as a potential biomaterial for wound healing. Cyanoflan effects on cell viability, apoptosis, and migration were assessed in vitro, while the effect on tissue regeneration and biosafety was evaluated in healthy Wistar rats. The cell viability and apoptosis of fibroblasts and endothelial cells was not influenced by the treatment with different concentrations of Cyanoflan, as observed by flow cytometry. Moreover, the presence of Cyanoflan did not affect cell motility and migratory capacity, nor did it induce reactive oxygen species production, even revealing an antioxidant behavior regarding the endothelial cells. Furthermore, the skin wound healing in vivo assay demonstrated that Cyanoflan perfectly adapted to the wound bed without inducing systemic or local oxidative or inflammatory reaction. Altogether, these results suggest that Cyanoflan is a promising biopolymer for the development of innovative applications to overcome the many challenges that still exist in skin wound healing.

Intrathymic Deletion of IL-7 Reveals a Contribution of the Bone Marrow to Thymic Rebound Induced by Androgen Blockade.

Despite the well-documented effect of castration in thymic regeneration, the singular contribution of the bone marrow (BM) versus the thymus to this process remains unclear. The chief role of IL-7 in pre- and intrathymic stages of T lymphopoiesis led us to investigate the impact of disrupting this cytokine during thymic rebound induced by androgen blockade. We found that castration promoted thymopoiesis in young and aged wild-type mice. In contrast, only young germline IL-7-deficient (Il7-/- ) mice consistently augmented thymopoiesis after castration. The increase in T cell production was accompanied by the expansion of the sparse medullary thymic epithelial cell and the peripheral T cell compartment in young Il7-/- mice. In contrast to young Il7-/- and wild-type mice, the poor thymic response of aged Il7-/- mice after castration was associated with a defect in the expansion of BM hematopoietic progenitors. These findings suggest that BM-derived T cell precursors contribute to thymic rebound driven by androgen blockade. To assess the role of IL-7 within the thymus, we generated mice with conditional deletion of IL-7 (Il7 conditional knockout [cKO]) in thymic epithelial cells. As expected, Il7cKO mice presented a profound defect in T cell development while maintaining an intact BM hematopoietic compartment across life. Unlike Il7-/- mice, castration promoted the expansion of BM precursors and enhanced thymic activity in Il7cKO mice independently of age. Our findings suggest that the mobilization of BM precursors acts as a prime catalyst of castration-driven thymopoiesis.

Thymic epithelial cells require p53 to support their long-term function in thymopoiesis in mice.

Thymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.

Thymic crosstalk restrains the pool of cortical thymic epithelial cells with progenitor properties.

Cortical (cTEC) and medullary (mTEC) thymic epithelial cells establish key microenvironments for T-cell differentiation and arise from thymic epithelial cell progenitors (TEP). However, the nature of TEPs and the mechanism controlling their stemness in the postnatal thymus remain poorly defined. Using TEC clonogenic assays as a surrogate to survey TEP activity, we found that a fraction of cTECs generates specialized clonal-derived colonies, which contain cells with sustained colony-forming capacity (ClonoTECs). These ClonoTECs are EpCAM+MHCII-Foxn1lo cells that lack traits of mature cTECs or mTECs but co-express stem-cell markers, including CD24 and Sca-1. Supportive of their progenitor identity, ClonoTECs reintegrate within native thymic microenvironments and generate cTECs or mTECs in vivo. Strikingly, the frequency of cTECs with the potential to generate ClonoTECs wanes between the postnatal and young adult immunocompetent thymus, but it is sustained in alymphoid Rag2-/-Il2rg-/- counterparts. Conversely, transplantation of wild-type bone marrow hematopoietic progenitors into Rag2-/-Il2rg-/- mice and consequent restoration of thymocyte-mediated TEC differentiation diminishes the frequency of colony-forming units within cTECs. Our findings provide evidence that the cortical epithelium contains a reservoir of epithelial progenitors whose abundance is dynamically controlled by continual interactions with developing thymocytes across lifespan.

Intermediate expression of CCRL1 reveals novel subpopulations of medullary thymic epithelial cells that emerge in the postnatal thymus.

Cortical and medullary thymic epithelial cells (cTECs and mTECs, respectively) provide inductive microenvironments for T-cell development and selection. The differentiation pathway of cTEC/mTEC lineages downstream of common bipotent progenitors at discrete stages of development remains unresolved. Using IL-7/CCRL1 dual reporter mice that identify specialized TEC subsets, we show that the stepwise acquisition of chemokine (C-C motif) receptor-like 1 (CCRL1) is a late determinant of cTEC differentiation. Although cTECs expressing high CCRL1 levels (CCRL1(hi) ) develop normally in immunocompetent and Rag2(-/-) thymi, their differentiation is partially blocked in Rag2(-/-) Il2rg(-/-) counterparts. These results unravel a novel checkpoint in cTEC maturation that is regulated by the cross-talk between TECs and immature thymocytes. Additionally, we identify new Ulex europaeus agglutinin 1 (UEA)(+) mTEC subtypes expressing intermediate CCRL1 levels (CCRL1(int) ) that conspicuously emerge in the postnatal thymus and differentially express Tnfrsf11a, Ccl21, and Aire. While rare in fetal and in Rag2(-/-) thymi, CCRL1(int) mTECs are restored in Rag2(-/-) Marilyn TCR-Tg mice, indicating that the appearance of postnatal-restricted mTECs is closely linked with T-cell selection. Our findings suggest that alternative temporally restricted routes of new mTEC differentiation contribute to the establishment of the medullary niche in the postnatal thymus.

Thymocyte selection regulates the homeostasis of IL-7-expressing thymic cortical epithelial cells in vivo.

Thymic epithelial cells (TECs) help orchestrate thymopoiesis, and TEC differentiation relies on bidirectional interactions with thymocytes. Although the molecular mediators that stimulate medullary thymic epithelial cell (mTEC) maturation are partially elucidated, the signals that regulate cortical thymic epithelial cell (cTEC) homeostasis remain elusive. Using IL-7 reporter mice, we show that TECs coexpressing high levels of IL-7 (Il7(YFP+) TECs) reside within a subset of CD205(+)Ly51(+)CD40(low) cTECs that coexpresses Dll4, Ccl25, Ccrl1, Ctsl, Psmb11, and Prss16 and segregates from CD80(+)CD40(high) mTECs expressing Tnfrsf11a, Ctss, and Aire. As the frequency of Il7(YFP+) TECs gradually declines as mTEC development unfolds, we explored the relationship between Il7(YFP+) TECs and mTECs. In thymic organotypic cultures, the thymocyte-induced reduction in Il7(YFP+) TECs dissociates from the receptor activator of NF-κB-mediated differentiation of CD80(+) mTECs. Still, Il7(YFP+) TECs can generate some CD80(+) mTECs in a stepwise differentiation process via YFP(-)Ly51(low)CD80(low) intermediates. Il7(YFP+) TECs are sustained in Rag2(-/-) mice, even following in vivo anti-CD3ε treatment that mimics the process of pre-TCR ß-selection of thymocytes to the double positive (DP) stage. Using Marilyn-Rag2(-/-) TCR transgenic, we find that positive selection into the CD4 lineage moderately reduces the frequency of Il7(YFP+) TECs, whereas negative selection provokes a striking loss of Il7(YFP+) TECs. These results imply that the strength of MHC/peptide-TCR interactions between TECs and thymocytes during selection constitutes a novel rheostat that controls the maintenance of IL-7-expressing cTECs.

Ecological and Syntaxonomic Analysis of the Communities of Glebionis coronaria and G. discolor (Malvion neglectae) in the European Mediterranean Area.

Nitrophilous communities dominated by Glebionis coronaria and Glebionis discolor in the European Mediterranean area were studied. The nomenclature was corrected according to the current taxonomy, following the International Code of Phytosociological Nomenclature (ICPN). The statistical analysis revealed six new associations and one subassociation, with four in Spain, one in Greece, and one in Italy. Additionally, a subassociation of high relevance due to its endemic character was identified. These grasslands exhibit requirements for organic matter and other edaphic nutrients that are closer to those of Malva neglecta communities than to those of Hordeum murinum subsp. leporinum. We confirmed the published syntaxon with the rank of Resedo albae-Glebionenion coronariae suballiance and its subordination to the Malvion neglectae alliance, and we established the type association for this suballiance. Sisimbrietalia officinalis J. Tüxen in Lohmeyer et al. 1962 em. Rivas-Martínez, Báscones, T. E. Díaz, Fernández-González & Loidi 1991. Stellarietea mediae Tüxen, Lohmeyer & Preising ex von Rochow 1951.

Pseudomonas putida are environmental reservoirs of antimicrobial resistance to ß-lactamic antibiotics.

The adaptive flexibility of bacteria largely contributes to the emergence of antibiotic resistance, eventually leading to the predictable failure of current antimicrobial therapies. It is of utmost importance to improve current approaches and implement new ways to control bacterial growth and proliferation. A promising strategy lies in unraveling the antimicrobial resistance (AMR) dynamics in environmental reservoirs, namely in soil. Environmental microorganisms are antibiotic producers and generally also carriers of AMR mechanisms. Therefore, soil samples were collected from areas distinctly influenced by men: rural farms and urban fluvial shores. Globally, microbial communities collected in farms revealed the highest antibiotic resistance potential. Largely predominant Gram-negative isolates were further screened for their low susceptibility to ß-lactamic agents, and found to belong to Pseudomonaceae family, with predominance of Pseudomonas putida (92 %). Minimal Inhibitory Concentration (MIC) was determined for five ß-lactams and the distributive analysis of cefotaxime MIC performed, allowing the first report of Epidemiological Cut-OFF values for P. putida regarding such antibiotic. Hence, 46 % of the isolates from farms presented acquired resistance to cefotaxime, with fluvial strains presenting an acquisition of AMR in 22 % of the isolates. The response to ß-lactams impact in P. putida is different from Pseudomonas aeruginosa's, the family type strain, showing that data determined for a species should only be extended to other bacteria with caution, even closely related. It becomes crucial to broaden present research, mainly focused on few pathogenic bacteria, to other microorganisms carrying relevant resistance tools or capable of genetic transfer to more virulent strains. Most available data on AMR so far has been obtained from studies performed in restricted clinical or veterinary context, showing the result of a strong selective pressure related to therapy but often disregarding the origin of the AMR mechanisms encountered. The strong impact that environmental microorganisms have (and probably already had in the past) on the evolution and spreading of AMR, is just beginning to be unveiled.

TARGETing Transcriptional Regulation in the Medicinal Plant Catharanthus roseus.

Transcriptional regulation is a central piece of the highly valuable monoterpenoid indole alkaloid pathway of C. roseus , and the ultimate tool for its understanding and manipulation. Here, we describe the adaptation of the TARGET methodology to identify specific and genome-wide leaf targets of C. roseus candidate transcription factors (TFs).

The Impact of Climate Change on Forest Development: A Sustainable Approach to Management Models Applied to Mediterranean-Type Climate Regions.

Forest ecosystems are divided into three major groups: boreal, temperate, and tropical. These can be subdivided according to the particularities of each type due to its relative location (littoral, mountain, etc.), climatic conditions, or even geological substrate. Climate change affects each type of forest ecosystem differently. However, it seems to affect temperate forests in Mediterranean-type climate regions more intensely. These regions are located over several continents, with major impacts of increased temperature during summer and decreased precipitation during winter. This situation affects Mediterranean forest ecosystems by increasing the risk of fires, which arise more frequently and are more severe. In addition, the emergence of pests and the spread of invasive species are well-known problems affecting these ecosystems. All of these conditions contribute to losses of productivity and biodiversity. To avoid the destruction of forest resources, and since Mediterranean-type climate regions are considered climate change hot spots with increased vulnerability to disturbances, the implementation of adaptive forest management models could contribute to increasing the resilience of such forests, which could also contribute to mitigating climate change.

Intra-articular epithelioid sarcoma of the knee: a diagnostic challenge.

Epithelioid sarcoma is a rare mesenchymal neoplasm characterized by aggregates of epithelioid cells. Intra-articular occurrence is exceedingly rare with only few reports described in the literature. A 22 year-old man presented a progressive mechanical knee pain. Initially, the investigation revealed a non-infectious unspecific synovitis. The patient gradually presented increasing knee enlargement and functional impairment. Intra-articular nodular proliferation with bone invasion was later observed on magnetic resonance imaging reevaluation. Pigmented villonodular synovitis hypothesis was considered. The biopsy ultimately revealed nodules of epithelioid cells with an immunoprofile compatible with epithelioid sarcoma diagnosis. The patient underwent neoadjuvant chemotherapy and radiotherapy and an above-knee amputation was performed.

Benign Metastasizing Leiomyoma of the Uterus: Rare Manifestation of a Frequent Pathology.

Benign Metastasizing Leiomyoma (BML) is a rare condition with few cases reported in the literature. It is usually incidentally diagnosed several years after a primary gynecological surgery for uterine leiomyoma. Differential diagnosis of BML is complex requiring an extensive work-up and exclusion of malignancy. Here, we report two cases of BML based on similarity of histopathological, immunohistochemical, and genetic patterns between lung nodules and uterine leiomyoma previously resected, evidencing the variability of clinical and radiological features of BML. We highlight the importance of 19q and 22q deletions as highly suggestive of BML. These findings are particularly relevant when there is no uterine sample for review.

Correction: Community structure informs species geographic distributions.

[This corrects the article DOI: 10.1371/journal.pone.0197877.].

Community structure informs species geographic distributions.

Understanding what determines species' geographic distributions is crucial for assessing global change threats to biodiversity. Measuring limits on distributions is usually, and necessarily, done with data at large geographic extents and coarse spatial resolution. However, survival of individuals is determined by processes that happen at small spatial scales. The relative abundance of coexisting species (i.e. 'community structure') reflects assembly processes occurring at small scales, and are often available for relatively extensive areas, so could be useful for explaining species distributions. We demonstrate that Bayesian Network Inference (BNI) can overcome several challenges to including community structure into studies of species distributions, despite having been little used to date. We hypothesized that the relative abundance of coexisting species can improve predictions of species distributions. In 1570 assemblages of 68 Mediterranean woody plant species we used BNI to incorporate community structure into Species Distribution Models (SDMs), alongside environmental information. Information on species associations improved SDM predictions of community structure and species distributions moderately, though for some habitat specialists the deviance explained increased by up to 15%. We demonstrate that most species associations (95%) were positive and occurred between species with ecologically similar traits. This suggests that SDM improvement could be because species co-occurrences are a proxy for local ecological processes. Our study shows that Bayesian Networks, when interpreted carefully, can be used to include local conditions into measurements of species' large-scale distributions, and this information can improve the predictions of species distributions.

Mucosal Prolapse Polyp Mimicking Rectal Malignancy: A Case Report.

Mucosal prolapse polyps (MPPs) are rare inflammatory lesions that are part of the mucosal prolapse syndrome. We present the case of a 40-year-old male with history of constipation referred to our institution with suspected rectal malignancy due to hematochezia and a palpable rectal mass. Colonoscopy revealed a 25 mm wide lesion suggestive of subepithelial origin but with marked erythema and erosion in the mucosa. Crypt dilatation and distortion, mixed inflammatory infiltrate and fibrosis were apparent on histological evaluation after bite-on-bite biopsies. Due to the initial suspicion of malignancy, resection was decided after discussion with the patient. However, due to non-elevation partial resection was performed allowing the diagnosis of MPP. Hematochezia ceased after obstipation treatment and endoscopic follow-up showed maintenance of the lesion with the same characteristics except for reduced dimension. MPP may mimic neoplastic lesions and should be considered in the differential diagnosis of rectal masses. History, endoscopy and histological characteristics are all necessary and important in the diagnosis of MPP.

Os pólipos de prolapso mucoso (MPPs) são lesões inflamatórias raras enquadradas na síndrome de prolapso mucoso. Apresentamos o caso de um homem, 40 anos, com antecedentes de obstipação, referenciado à nossa instituição por suspeita de neoplasia do reto devido a hematoquésias e lesão palpável ao toque retal. A colonoscopia mostrou uma lesão com 25 mm, de aspeto subepitelial, com mucosa marcadamente eritematosa e erosionada. As biopsias bite-on-bite revelaram dilatação e distorção das criptas, infiltrado inflamatório misto e fibrose. Devido à suspeita inicial de neoplasia foi decidida resseção após discussão com o doente, que não foi possível devido à elevação inadequada da lesão. Efetuada resseção parcial, permitindo o diagnóstico seguro de MPP. As hematoquésias cessaram após tratamento da obstipação. Os MPPs podem mimetizar lesões neoplásicas anorrectais devendo ser incluídos no diagnóstico diferencial. A conjugação da história clínica com os aspetos endoscópicos e histológicos é fundamental para o diagnóstico correto e orientação adequada.

Pathologic Findings in Prophylactic and Nonprophylactic Hysterectomy Specimens of Patients With Lynch Syndrome.

Women with Lynch syndrome (LS) have a high risk of developing endometrial carcinoma (EC) and, less frequently, ovarian carcinoma. As EC not uncommonly is the first malignancy, prophylactic hysterectomy (PH) has been increasingly implemented. In this study, we report the clinicopathologic features of a series of 70 LS patients who underwent either PH (n=39) or nonprophylactic hysterectomy (NPH) (n=31) at 3 tertiary referral centers. Among the 39 patients with PH, 2 had endometrial tumors seen grossly, whereas 37 showed no macroscopic lesions. Total inclusion of the endometrium was performed in 24/39 (61.5%). Abnormal histologic findings were identified in 9/39 (23.1%) PHs: 3 endometrial endometrioid carcinomas (EECs), including the 2 macroscopic and 1 microscopic (0.6 cm), and 4 atypical and 6 nonatypical hyperplasias. NPH included those performed for endometrial and ovarian cancer treatment. Tumor sampling followed standard protocols. ECs comprised 26 EECs and 1 clear cell carcinoma, with a median size of 3.7 cm. Hyperplasia was observed in 10 (33.3%) as background in EC, in 4 showing atypia. Eight (29.6%) tumors were centered in the lower uterine segment (all EECs). EECs were predominantly well differentiated (53.8%) and FIGO stage I (77.8%). A papillary architecture was common (51.9%) and associated with microcystic elongated and fragmented foci in 4. Mucinous differentiation was observed in 25.9% of endometrial tumors, typically representing <10%. Most endometrial tumors (81.5%) showed tumor-infiltrating lymphocyte counts ≥42/10 high-power fields. Four tumors showed extensive necrosis. Eight patients had ovarian tumors (4 synchronous), including 2 endometrioid carcinomas, 2 clear cell carcinomas, 1 borderline clear cell adenofibroma, 1 Müllerian carcinoma of mixed cell types, 1 primitive neuroectodermal tumor, and 1 metastatic melanoma. Total inclusion of the endometrium should be done in all LS patients' surgical specimens without macroscopic lesions as some of these patients harbor preneoplastic or neoplastic conditions treatable at an early stage. The phenotype of LS-associated endometrial and ovarian tumors is variable and frequently includes features not commonly observed in sporadic cancers, but in our experience carcinomas were in general low grade and low stage.