Immunofluorescence mapping, electron microscopy and genetics in the diagnosis and sub-classification of inherited epidermolysis bullosa: a single-centre retrospective comparative study of 87 cases with long-term follow-up.

BACKGROUND: Epidermolysis bullosa (EB) comprises a heterogeneous group of skin fragility disorders, classified in four major types based on skin cleavage level, i.e. EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), Kindler EB, and in more than 30 subtypes defined by the combination of laboratory and clinical data, including disease course. OBJECTIVES: Our aims were to address whether, in the age of genomics, electron microscopy (TEM) has still a role in diagnosing EB, and whether the genotype per se may be sufficient to sub-classify EB. METHODS: A thoroughly characterized single-centre EB case series was retrospectively evaluated to compare the power of TEM with immunofluorescence mapping (IFM) in establishing the EB type, and the ability of TEM, IFM and genetics to predict selected EB subtypes, i.e. severe dominant EBS (DEBS), severe JEB, severe recessive DEB (RDEB) and DEB self-improving, using genetic and final diagnosis, respectively, as gold standard. RESULTS: The series consisted of 87 patients, including 44 newborns, with a median follow-up of 54 months. Ninety-five mutations were identified in EB-associated genes, including 25 novel variants. Both IFM and TEM were diagnostic in about all cases of JEB (21/21 for both) and DEB (43/44 for IFM, 44/44 for TEM). TEM sensitivity was superior to IFM for EBS (19/20 vs. 16/19). As to EB subtyping, IFM performed better than genetics in identifying severe JEB cases due to laminin-332 defect (14/14 vs. 10/14) and severe RDEB (eight/nine vs. seven/nine). Genetics had no role in self-improving DEB diagnosis; it almost equalled TEM in predicting severe DEBS (eight/nine vs. nine/nine) and enabled to discriminate dominant from recessive non-severe DEB phenotypes and to identify special subtypes, e.g. DEBS with KLHL24 mutations. CONCLUSIONS: Transmission electron microscopy remains relevant to the diagnosis of EBS. IFM and genetics are essential and complementary tools in the vast majority of EB cases.

Diversity of EPT (Ephemeroptera, Plecoptera, Trichoptera) Along Streams Fragmented by Waterfalls in the Brazilian Savanna.

In stream ecosystems, waterfalls intercept the fluvial continuum and limit the dispersion of aquatic organisms. However, the barrier effect may be minimized or absent for insects whose life cycle is not restricted to the aquatic environment (i.e., winged adults), such as the orders Ephemeroptera, Plecoptera and Trichoptera (EPT). In this study, we investigated EPT diversity (abundance, richness, and composition) in savanna headwater streams (Cerrado Domain, Brazil) to investigate the hypothesis that large waterfalls have little effect on instream assemblage structure. During 2017 and 2018, we sampled EPTs and measured environmental conditions at reaches adjacent (downstream and upstream) to waterfalls (5 to 70 m high). We collected 2532 immature specimens of 33 genera, including the first record of 14 genera of Ephemeroptera, 11 Trichoptera, and three Plecoptera for the Tocantins State. Hydro-geomorphic and streambed conditions varied among sites, but they were not related to the presence of waterfalls. Assemblage structure was similar between downstream and upstream reaches, with no significant difference in taxa richness, abundance, and composition. This pattern was consistently observed considering all data combined, and each order separately. These results support the hypothesis that physical barriers have weak effects on EPT assemblages at small spatial scales, where strong dispersal and habitat similarity probably homogenizes the structure of local assemblages in stream corridors adjacent to large waterfalls. The study provides important information about factors driving the distribution of biodiversity in savanna streams, which are relevant to support conservation and management plans in the Brazilian Cerrado.

A new species of Camelobaetidius Demoulin, 1966 (Ephemeroptera: Baetidae), from the Colombian Orinoco River basin.

Three species of Camelobaetidius Demoulin, 1966 have been reported from Colombia until now, based on nymphs. We describe a fourth species based on nymphs from the Colombian Orinoco river basin. The new species can be recognized by: 1) labrum narrowly rounded anteriorly; 2) segment II of labial palp with a short rounded distomedial projection; 3) incisors of right mandible with eleven denticles; 4) gills absent from the bases of coxae; 5) tarsal claws with ten denticles; 6) outer margin of forefemur with a row of about 23 long, spine-like setae; 7) posterior margin of tergum IV with truncate spines, and 8) terminal filament almost as long as cerci. [Zoobank URL: urn:lsid:zoobank.org:act:500CF998-8EF3-43E3-BA8B-F062B92768F3].

Corinnella lourii, a rare new species of Baetidae (Ephemeroptera) from central Brazil.

A new species of the rare genus Corinnella is described based on nymphs from the Rio Pindaíba, a river located close to the Cerrado - Amazonian border. The new species is more related to C. eximia than to C. thomasi, but can be distinguished from them by the following combination of characters: 1) distomedial emargination of labrum broad and with very small process; 2) maxillary palp 2.0× length of galea-lacinia; 3) posterior margin of terga I-IX with pointed spaced spines; 4) claws with four denticles.

Malignant ovarian germ cell tumors in pediatric patients: The AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) study.

OBJECTIVE: Malignant ovarian germ cell tumors (MOGCT) carry an excellent prognosis, and the treatment aims to achieve results with the least possible treatment-related morbidity. The aim of this study was to assess the outcomes of pediatric patients with MOGCT. METHODS: Patients were treated according to their stage: surgery and surveillance for stage I; a modified bleomycin-etoposide-cisplatin (BEP) regimen for stages II (three cycles), III, and IV (three cycles) with surgery on residual disease. RESULTS: Seventy-seven patients were enrolled (median age 11.8 years), 26 with dysgerminoma (Dysg), 13 with immature teratoma and elevated serum alpha-fetoprotein levels (IT + AFP), and 38 with nondysgeminoma (Non-Dysg) staged as follows: 27 stage I, 13 stage II, 32 stage III, 5 stage IV. Among evaluable patients in stage I (5-year event-free survival [EFS] 72.1% [95% CI: 56.4-92.1%]; 5-year overall survival [OS] 100%), seven relapsed (three patients with Dysg and four patients with Non-Dysg) and were rescued with chemotherapy (plus surgery in three patients). Among the evaluable patients with stages II-IV, 48 (98%) achieved complete remission after chemotherapy ± surgery, one (IT + AFP, stage IV) had progressive disease. In the whole series (median follow-up 80 months), the 5-year OS and EFS were 98.5% (95% CI: 95.6-100%) and 84.5% (95% CI: 76.5-93.5%). CONCLUSIONS: We confirm the excellent outcome for MOGCT. Robust data are lacking on surgical staging, surveillance for Non-Dysg with stage I, the management of IT + AFP, and the most appropriate BEP regimen. As pediatric oncologists, we support the role of surveillance after proper surgical staging providing cases are managed by experts at specialized pediatric centers.

First record of the genus Lumahyphes Molineri, 2004 (Ephemeroptera: Leptohyphidae) from Brazil with description of a new species.

The genus Lumahyphes Molineri, 2004 (Ephemeroptera: Leptohyphidae) currently is comprised of three species. Lumahyphes cocal sp. nov. is described based on material from the state of Roraima in northern Brazil. The new species differs from the other described species by having the following combination of characteristics: greyish wing membrane; terga base color whitish and terga I-III & VIII-X washed completely with gray; dorsal structure of penes rounded; spines of penes S-shaped in lateral view; and apex of spines directed anteriorly.

The male imago of Paramaka incognita Domínguez, Grillet, Nieto, Molineri and Guerrero, 2014 (Ephemeroptera: Leptophlebiidae).

The genus Paramaka Savage & Domínguez, 1992 (Ephemeroptera: Leptophlebiidae) is restricted to South America, and it is comprised of four species. The male imago of P. incognita is described for the first time based on material from the state of Roraima in northern Brazil. The male adult differs from other described species of the genus by having the following combination of characteristics: Forewing with costal and subcostal areas basally washed with light yellowish brown; hind wing with base of wing washed with yellowish brown; abdominal terga II-V and VI-IX with lateral margins washed with reddish brown; abdominal sterna VI-VIII with lateral blackish stripes; styliger plate median portion with a pointed, posterior projection that is oriented in a posterior direction; and penes with ventral spines length subequal to length of penes, located on the distal third of the penes.

Five new species of Camelobaetidius Demoulin, 1966 (Ephemeroptera: Baetidae), and redescription of Camelobaetidius mexicanus (Traver & Edmunds, 1968).

The genus Camelobaetidius Demoulin, 1966 has 38 species distributed throughout the Neotropical Region, being one of the most studied genera of the family Baetidae. Based on material from Brazil, the aim of the present paper is to describe five new species of Camelobaetidius with the terminal filament as long as cerci, and to redescribe the nymph of C. mexicanus (Traver & Edmunds, 1968) based on a paratype from Mexico.

Galectin-3 is a marker of favorable prognosis and a biologically relevant molecule in neuroblastic tumors.

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a ß-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient's risk stratification in the clinical setting.

A truncating mutation in the laminin-332α chain highlights the role of the LG45 proteolytic domain in regulating keratinocyte adhesion and migration.

BACKGROUND: Altered function of laminin-332 (α3ß3γ2) consequent to mutations in the LAMA3, LAMB3 and LAMC2 genes causes junctional epidermolysis bullosa non-Herlitz (JEB-nH). JEB-nH patients suffer from skin blistering and have an increased risk of developing aggressive skin carcinomas in adulthood. Laminin-332 is proteolytically processed and its extracellular mature form lacks the α3 chain C-terminal globules 4 and 5 (LG45). The LG45 tandem has cell adhesion and protumorigenic properties. However, mutations that affect this domain are very rare and their functional effects in patients have not been explored to date. OBJECTIVE: To characterize molecularly an adult patient with JEB-nH and altered laminin-332 expression presenting multiple skin carcinomas, and to analyse LG45-mediated biological functions using keratinocytes from the patient. METHODS: A mutational search in laminin-332 genes was performed by hetero-duplex analysis. LAMA3 mRNA and laminin-332 protein levels in patient keratinocytes were investigated by real-time reverse transcriptase polymerase chain reaction and radioimmunoprecipitation assay, respectively. Keratinocyte migration was examined by scratch and Boyden chamber assays. RESULTS: We identified a homozygous LAMA3 mutation, p.Leu1648TrpfsX32, which truncates the last 45 amino acids of the carboxyl terminal LG5 subdomain. Gene expression studies revealed that the mutant transcripts were stable and even increased, precursor laminin-332 molecules were retained intracellularly and the amount of mature extracellular heterotrimers was reduced to about 50%. Finally, the patient's keratinocytes migrated faster than normal keratinocytes. CONCLUSIONS: Structural disruption of LG5 highlights the critical functions of the LG45 proteolytic region in precursor laminin-332 secretion and keratinocyte adhesion and migration. Perturbation of LG45 function might explain the non-aggressive behaviour of carcinomas in this patient.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

ADAR2-editing activity inhibits glioblastoma growth through the modulation of the CDC14B/Skp2/p21/p27 axis.

Grade IV astrocytoma or glioblastoma multiforme (GBM) is one of the most aggressive and lethal tumors affecting humans. ADAR2-mediated A-to-I RNA editing, an essential post-transcriptional modification event in brain, is impaired in GBMs and astrocytoma cell lines. However, the role of ADAR2 editing in astrocytomas remains to be defined. Here, we show that ADAR2 editing rescue in astrocytomas prevents tumor growth in vivo and modulates an important cell cycle pathway involving the Skp2/p21/p27 proteins, often altered in glioblastoma. We demonstrate that ADAR2 deaminase activity is essential to inhibit tumor growth. Indeed, we identify the phosphatase CDC14B, which acts upstream of the Skp2/p21/p27 pathway, as a novel and critical ADAR2 target gene involved in glioblastoma growth. Specifically, ADAR2-mediated editing on CDC14B pre-mRNA increases its expression with a consequent reduction of the Skp2 target protein, as shown both in vitro and in vivo. We found that, compared to normal brain, both CDC14B editing and expression are progressively impaired in astrocytomas from grade I to IV, being very low in GBMs. These findings (1) demonstrate that post-transcriptional A-to-I RNA editing might be crucial for glioblastoma pathogenesis, (2) identify ADAR2-editing enzyme as a novel candidate tumor suppressor gene and (3) provide proof of principle that ADAR2 or its substrates may represent a suitable target(s) for possible novel, more effective and less toxic approaches to the treatment of GBMs.

The adults of Camelobaetidiusjanae Dominique & Thomas, 2001 and C. yacutinga Nieto, 2003, with a new synonym for C. leentvaari Demoulin, 1966 (Ephemeroptera: Baetidae).

The genus Camelobaetidius Demoulin, 1966 has 39 species distributed throughout the Nearctic and Neotropical Regions, being one of the most studied genera of the family Baetidae. Based on material from Brazil, the aim of the present paper is to describe adults of C. janae Dominique & Thomas, 2001 and C. yacutinga Nieto, 2003, and to recognize a new synonym for Camelobaetidius leentvaari Demoulin, 1966 (= C. mantis Traver & Edmunds, 1968 nov. syn.) based on the examination of type material.

A new species of Corinnella Thomas & Dominique, 2006 (Ephemeroptera: Baetidae).

The monotypic genus Corinnella Thomas & Dominique, 2006, was described based on nymphs from French Guyana, and since the original description nothing has been added to knowledge of its systematics. The aim of the present paper is to describe a new species of the genus from Northern Brazil, to improve its diagnoses, and to report the genus for the first time from Brazil. The new species can be recognized by: labrum with discernible anteromedial emargination and process; posterior margin of terga IV with rounded spines; and tarsal claw with seven to nine denticles, with the outer and inner denticles distinctly longer than the others.

Leptohyphidae (Insecta: Ephemeroptera) from northeastern Brazil

New records of Leptohyphidae for Northeastern Brazil are provided. Previously, only Tricorythopsis bahiensis Dias, Salles & Ferreira had been recorded in this region, but we now record one species of Amanahyphes Salles & Molineri, one of Leptohyphes Eaton, three of Traverhyphes Molineri, three of Tricorythodes Ulmer, and seven of Tricorythopsis Traver. Two of these species are recorded for the first time in Brazil. As all of these species are reported, the Northeastern Brazil became the second most diverse geographic region in Brazil in Leptohyphidae.

Leptohyphidae (Insecta: Ephemeroptera) from northeastern Brazil.

New records of Leptohyphidae for Northeastern Brazil are provided. Previously, only Tricorythopsis bahiensis Dias, Salles & Ferreira had been recorded in this region, but we now record one species of Amanahyphes Salles & Molineri, one of Leptohyphes Eaton, three of Traverhyphes Molineri, three of Tricorythodes Ulmer, and seven of Tricorythopsis Traver. Two of these species are recorded for the first time in Brazil. As all of these species are reported, the Northeastern Brazil became the second most diverse geographic region in Brazil in Leptohyphidae.

Anaplastic large cell lymphomas: a study of 75 pediatric patients.

In this article, we describe the morphologic and immunophenotypic features of 75 cases of pediatric anaplastic large cell lymphoma (ALCL). According to the World Health Organization classification, 49 cases were common subtype ALCL, and respectively, 3, 6, and 17 cases were small cell, lymphohistiocytic, or mixed histologic variants. Anaplastic lymphoma kinase positivity was detected in 90.7% of the tumors and, using a panel of 9 T-cell surface markers, 88% could be assigned to the T-cell lineage. A molecular analysis for the T-cell receptor gamma (TCR- gamma) and the heavy chain of the immunoglobulin H rearrangements was performed on 6/9 ALCLs with a null immunophenotype, and a TCR clonal pattern was detected in 5/6 cases. In addition, 94.1% were immunoreactive for 1 or more cytotoxic proteins (Tia1, granzyme B, or perforin), and 15% expressed CD56. Clusterin, CD83, and Pax5, respectively, expressed in 91.3%, 1.7%, and 0% of the ALCLs, were useful biomarkers for the differential diagnosis with Hodgkin's lymphomas.