Isoamyl acetate, produced via fermentation, is a natural flavor chemical with applications in the food industry. Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) can catalyze the esterification of isoamyl alcohol with acetyl coenzyme A. The respective genes were cloned and expressed in an appropriate ack-pta(-) strain of Escherichia coli. The engineered strains produce isoamyl acetate when isoamyl alcohol is added to the culture medium. Aerobic shake flask experiments examined isoamyl acetate production over various growth times, temperatures, and initial optical densities. The strain carrying the pBAD-ATF1 plasmid exhibited a high molar ester yield from glucose (1.13) after 48 h of aerobic growth at 25 degrees C. Low-cost media components, such as fusel oil, sorghum glucose and corn steep liquor, were found to give a high yield of isoamyl acetate. High-cell-density gave an increased isoamyl acetate yield of 0.18 g/g of glucose consumed.

OBJECTIVE

Angiomatoid fibrous histiocytoma (AFH) is a rare neoplastic disease usually occurring in the dermis or subcutis of the extremities of young adults or children. Although sporadic cases in deep soft tissue and visceral organs have been reported, we present here the first description of AFH developing in a large artery.

RESULTS

Paraffin sections of the surgical specimen were stained with haematoxylin and eosin, and immunohistochemistry was performed (CKAE1/AE3, EMA, CD34, p63, CD38, smooth muscle actin, and desmin). In addition, FISH and RT-PCR were applied in order to check for EWRS rearrangement. The histomorphological features, and FISH analysis revealing rearrangement of EWSR, indicated the definitive diagnosis of AFH. RT-PCR confirmed EWSR rearrangement, and detected an EWSR1-ATF1 fusion transcript.

CONCLUSIONS

A thoracic location of AFH has not been reported until very recently, and shares a differential diagnosis with diverse neoplasms, including spindle cell carcinoma and low-grade sarcoma. We describe the first reported case of thoracic AFH arising in a large vessel, and highlight distinctive histological and molecular features.

In human and mouse stem cells and brain, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can occur outside of CG dinucleotides. Using protein binding microarrays (PBMs) containing 60-mer DNA probes, we evaluated the effect of 5mC and 5hmC on one DNA strand on the double-stranded DNA binding of the mouse B-ZIP transcription factors (TFs) CREB1, ATF1, and JUND. 5mC inhibited binding of CREB1 to the canonical CRE half-site |GTCA but enhanced binding to the C/EBP half-site |GCAA. 5hmC inhibited binding of CREB1 to all 8-mers except TGAT|GCAA, where binding is enhanced. We observed similar DNA binding patterns with ATF1, a closely related B-ZIP domain. In contrast, both 5mC and 5hmC inhibited binding of JUND. These results identify new DNA sequences that are well-bound by CREB1 and ATF1 only when they contain 5mC or 5hmC. Analysis of two X-ray structures examines the consequences of 5mC and 5hmC on DNA binding by CREB and FOS|JUN.

Ethyl acetate has attracted much attention as an important chemical raw material and a flavor component of alcoholic beverages. In this study, the biosynthetic pathway for the production of ethyl acetate in Chinese liquor yeast was unblocked. In addition to engineering Saccharomyces cerevisiae to increased intracellular CoA and acetyl-CoA levels, we also increased the combining efficiency of acetyl-CoA to ethanol. The genes encoding phosphopantothenate-cysteine ligase, acetyl-CoA synthetase, and alcohol acetyltransferase were overexpressed by inserting the strong promoter PGK1p and the terminator PGK1t, respectively, and then combine them. Our results finally showed that the ethyl acetate levels of all engineering strains were improved. The final engineering strain CLy12a-ATF1-ACS2-CAB2 had a significant increase in ethyl acetate yield, reaching 610.26 (± 14.28) mg/L, and the yield of higher alcohols was significantly decreased. It is proved that the modification of ethyl acetate metabolic pathway is extremely important for the production of ethyl acetate from Saccharomyces cerevisiae.

FvatfA from the maize pathogen Fusarium verticillioides putatively encodes the Aspergillus nidulans AtfA and Schizasaccharomyces pombe Atf1 orthologous bZIP-type transcription factor, FvAtfA. In this study, a ΔFvatfA deletion mutant was constructed and then genetically complemented with the fully functional FvatfA gene. Comparing phenotypic features of the wild-type parental, the deletion mutant and the restored strains shed light on the versatile regulatory functions played by FvAtfA in (i) the maintenance of vegetative growth on Czapek-Dox and Potato Dextrose agars and invasive growth on unwounded tomato fruits, (ii) the preservation of conidiospore yield and size, (iii) the orchestration of oxidative (H₂O₂, menadione sodium bisulphite) and cell wall integrity (Congo Red) stress defences and (iv) the regulation of mycotoxin (fumonisins) and pigment (bikaverin, carotenoid) productions. Expression of selected biosynthetic genes both in the fumonisin (fum1, fum8) and the carotenoid (carRA, carB) pathways were down-regulated in the ΔFvatfA strain resulting in defected fumonisin production and considerably decreased carotenoid yields. The expression of bik1, encoding the polyketide synthase needed in bikaverin biosynthesis, was not up-regulated by the deletion of FvatfA meanwhile the ΔFvatfA strain produced approximately ten times more bikaverin than the wild-type or the genetically complemented strains. The abolishment of fumonisin production of the ΔFvatfA strain may lead to the development of new-type, biology-based mycotoxin control strategies. The novel information gained on the regulation of pigment production by this fungus can be interesting for experts working on new, Fusarium-based biomass and pigment production technologies. Key points • FvatfA regulates vegetative and invasive growths of F. verticillioides. • FvatfA also orchestrates oxidative and cell wall integrity stress defenses. • The ΔFvatfA mutant was deficient in fumonisin production. • FvatfA deletion resulted in decreased carotenoid and increased bikaverin yields.

Keywords: Cell wall integrity stress; Conidiogenesis; Invasive growth; Mycotoxin production; Oxidative stress; Pigment production; Vegetative growth.

Men diagnosed with localized prostate cancer can develop metastases many years after initial treatment, resulting in a poor prognosis. The purpose of this study was to investigate the mechanisms by which signaling through norepinephrine (NE) may incite relapse of quiescent prostate cancer. We used an unbiased bioinformatics pipeline to examine mechanisms for recurrence related to sympathetic signaling in the bone marrow. A transcription factor cell array identified ATF1, RAR, and E2F as key nodes in prostate cancer cells exiting quiescence through adrenergic signaling. Subsequent secretome analysis identified GAS6 as affecting activity of these three factors, leading to cell cycle reentry. GAS6 expression was downregulated in osteoblasts through activation of the cAMP pathway and was targeted in vitro and in vivo using pharmacological agents (propranolol and phentolamine). Propranolol increased expression of GAS6 by osteoblasts, and phentolamine significantly inhibited expression. Propranolol treatment was sufficient to both increase GAS6 expression in marrow osteoblasts as well as eliminate the effects of NE signaling on GAS6 expression. These results demonstrate a strong correlation between adrenergic signaling, GAS6 expression, and recurrence in prostate cancer, suggesting a novel therapeutic direction for patients at high risk of metastasis.

Epigenetic modifications play critical roles in modulating gene expression, yet their roles in regulatory networks in human cell lines remain poorly characterized. We integrated multiomics data to construct directed regulatory networks with nodes and edges labeled with chromatin states in human cell lines. We observed extensive association of diverse chromatin states and network motifs. The gene expression analysis showed that diverse chromatin states of coherent type-1 feedforward loop (C1-FFL) and incoherent type-1 feedforward loops (I1-FFL) contributed to the dynamic expression patterns of targets. Notably, diverse chromatin state compositions could help C1- or I1-FFL to control a large number of distinct biological functions in human cell lines, such as four different types of chromatin state compositions cooperating with K562-associated C1-FFLs controlling "regulation of cytokinesis," "G1/S transition of mitotic cell cycle," "DNA recombination," and "telomere maintenance," respectively. Remarkably, we identified six chromatin state-marked C1-FFL instances (HCFC1-NFYA-ABL1, THAP1-USF1-BRCA2, ZNF263-USF1-UBA52, MYC-ATF1-UBA52, ELK1-EGR1-CCT4, and YY1-EGR1-INO80C) could act as prognostic biomarkers of acute myelogenous leukemia though influencing cancer-related biological functions, such as cell proliferation, telomere maintenance, and DNA recombination. Our results will provide novel insight for better understanding of chromatin state-mediated gene regulation and facilitate the identification of novel diagnostic and therapeutic biomarkers of human cancers.

The Ecl1 family genes extend the lifespan of fission yeast when overexpressed. They also cause resistance against H(2)O(2) stress. In this study, we found that the bZip transcription factor Atf1 is a direct activator of the induction of extender of chronological lifespan (ecl1 (+)) by H(2)O(2) stress. Based on ChIP analysis, we identified that Atf1 binds to the upstream DNA region of ecl1(+). Previously, we reported that overexpression of ecl1(+) increased the expression of the catalase-encoding ctt1(+). This ecl1(+)-dependent increase of ctt1(+) expression occurred in ∆atf1 mutant. On the other hand, the activation of ctt1 (+) caused by the ∆pyp1 mutation, which enhances Sty1-Atf1 activity, could occur in ∆ecl1 mutant. Based on these results, we propose that Atf1 can regulate ctt1(+) in both an Ecl1-dependent and an Ecl1-independent manner.

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas.

Despite the economic importance of creating cold resilient cattle breeds, our knowledge of the genetic basis of adaptation to cold environments in cattle is still scarce compared to information on other economically important traits. Herein, using whole-genome resequencing of animals showing contrasting phenotypes on temperature maintenance under acute cold stress combined with the existing SNP (single nucleotide polymorphism) functional annotations, we report chromosomal regions and candidate SNPs controlling body temperature in the Siberian cattle populations. The SNP ranking procedure based on regional F_ST calculations, functional annotations, and the allele frequency difference between cold-tolerant and cold-sensitive groups of animals pointed to multiple candidate genes. Among these, GRIA4, COX17, MAATS1, UPK1B, IFNGR1, DDX23, PPT1, THBS1, CCL5, ATF1, PLA1A, PRKAG1, and NR1I2 were previously related to thermal adaptations in cattle. Other genes, for example KMT2D and SNRPA1, are known to be related to thermogenesis in mice and cold adaptation in common carp, respectively. This work could be useful for cattle breeding strategies in countries with harsh climates, including the Russian Federation.

Keywords: body temperature maintenance; cattle; cold adaptation; whole-genome resequencing.

Sin1 is a substrate-binding subunit of Target Of Rapamycin Complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 was also identified as a protein that interacts with Spc1/Sty1 stress-activated protein kinase (SAPK) and therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the Atf1 transcription factor, and the Gpd1 glycelrol-3-phosphate dehydrogenase, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems to be important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR) or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzymes-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with the parental strain ΔPoxKu70 when grown on WR under SSF. Importantly, the mutant ΔPoxAtf1 (P. oxalicum Atf1; also called POX03016) displayed 46.1-183.2% more cellulase and xylanase production than ΔPoxKu70 after two days growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environment-friendly economy encourages studies involving the high value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF were limited. Here, we employed RNA-sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on media containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering/improvement of filamentous fungi to further increase enzyme production.

Pediatric mesotheliomas are rare and their pathogenesis remains undefined. In this study, we report 5 cases of malignant mesothelioma in children, characterized by fusions involving the anaplastic lymphoma kinase (ALK) gene. Four cases occurred in females involving the abdominal cavity and were characterized by a pure epithelioid morphology. The fifth arose in the tunica vaginalis of a 15-year-old male and displayed a biphasic epithelioid-sarcomatoid phenotype. All cases demonstrated the classic morphologic and immunohistochemical features of malignant mesothelioma, including tubulopapillary architecture and cuboidal epithelioid cells with eosinophilic cytoplasm and uniform nuclei with vesicular chromatin. Immunohistochemically, all cases showed labeling for ALK, cytokeratins, WT1, and calretinin, while lacking expression of adenocarcinoma immunomarkers. Four cases demonstrated weak-moderate labeling for PAX8 protein, which resulted in diagnostic challenges with primary peritoneal serous carcinoma. The ALK genetic abnormalities were investigated by a combination of molecular methods. Archer FusionPlex was performed in 2 cases, showing fusions between ALK with either STRN or TPM1 genes, resulting in a transcript that retained the ALK kinase domain. One case was further studied by DNA targeted sequencing, but no additional genetic alterations were observed. In 1 case, cytogenetic analysis showed the presence of a t(2;15)(p23;q22) and fluorescence in situ hybridization confirmed the ALK gene break-apart. In the remaining 2 cases, ALK gene rearrangements were demonstrated by fluorescence in situ hybridization. Unlike adult mesotheliomas, which are tightly linked to asbestos exposure, often show loss of BAP1 expression and have complex karyotypes, ALK-rearranged mesothelioma appears to be similar to other fusion-positive mesotheliomas, such as those harboring EWSR1/FUS-ATF1 fusions, sharing significant morphologic overlap, occurring in young patients and displaying a simple, translocation-driven genetic profile.

Clear cell odontogenic carcinoma (CCOC) is a rare malignant odontogenic tumor (MOT) characterized by sheets and lobules of vacuolated and clear cells. To understand the biology of CCOC, we established a new cell line, CCOC-T, with EWSR1-ATF1 fusion gene from a mandible tumor with distant metastasis and characterized this cell line.

To detect the EWSR1-ATF1 fusion gene, we used three CCOC cases, including the present case, by RT-PCR and FISH analysis. We characterized established CCOC-T cells by checking cell growth, invasion and the expression of odontogenic factors and bone-related factors. Moreover, the gene expression profile of CCOC-T cells was examined by microarray analysis.

Histologically, the primary tumor was comprised of cords and nests containing clear and squamoid cells separated by fibrous septa. In addition, ameloblastomatous islands with palisaded peripheral cells were observed, indicating probable odontogenic origin. This tumor expressed the fusion gene EWSR1-ATF1, which underlies the etiology of hyalinizing clear cell carcinoma (HCCC) and potentially that of CCOC. We found a breakpoint in the EWSR1-ATF1 fusion to be the same as that reported in HCCC. Established CCOC-T cells grew extremely slowly, but the cells showed highly invasive activity. Moreover, CCOC-T cells expressed bone-related molecules, odontogenic factors, and epithelial mesenchymal transition (EMT)-related molecules.

To the best of our knowledge, this is the first report on the establishment of a CCOC cell line. CCOC-T cells serve as a useful in vitro model for understanding the pathogenesis and nature of MOT.

n-Butyl acetate is an important food additive commonly produced via concentrated sulfuric acid catalysis or immobilized lipase catalysis of butanol and acetic acid. Compared with chemical methods, an enzymatic approach is more environmentally friendly; however, it incurs a higher cost due to lipase production. In vivo biosynthesis via metabolic engineering offers an alternative to produce n-butyl acetate. This alternative combines substrate production (butanol and acetyl-coenzyme A (acetyl-CoA)), alcohol acyltransferase expression, and esterification reaction in one reactor. The alcohol acyltransferase gene ATF1 from Saccharomyces cerevisiae was introduced into Clostridium beijerinckii NCIMB 8052, enabling it to directly produce n-butyl acetate from glucose without lipase addition. Extractants were compared and adapted to realize glucose fermentation with in situ n-butyl acetate extraction. Finally, 5.57 g/L of butyl acetate was produced from 38.2 g/L of glucose within 48 h, which is 665-fold higher than that reported previously. This demonstrated the potential of such a metabolic approach to produce n-butyl acetate from biomass.

Keywords: Clostridium; alcohol acyltransferase; biosynthesis; butyl acetate; in situ product extraction.

Gene fusions of EWSR1 with members of the CREB family of transcription factors (CREB1, ATF1, and CREM) have recently been described in exceptional intracranial myxoid mesenchymal tumors. Although this is a known gene fusion found in various mesenchymal tumors, EWSR1 fusion with CREM has only been observed in 3 intracranial myxoid tumors. In this paper, we present 1 such tumor with in-depth histopathological description and long-term follow-up. There is controversy regarding whether these tumors represent a novel entity or simply an intracranial localization of the myxoid variant of angiomatoid fibrous histiocytoma, a rare soft tissue tumor of the extremities. Out of 11 cases mentioned in the literature, the 3 isolated case reports by Dunham et al, Ochalski et al, and Alshareef et al are designated as angiomatoid fibrous histiocytoma, whereas the others are defined as a novel tumoral entity called intracranial myxoid mesenchymal tumor with EWSR1-CREB fusion. We believe the vast morphological and immunohistochemical spectrum of angiomatoid fibrous histiocytoma makes it difficult to dismiss this diagnosis.

Hyalinizing clear cell carcinoma (HCCC) is a rare low-grade tumor of the salivary glands made up of clear cells that form cords and nests in hyalinized stroma. To date, primary HCCCs of the paranasal sinus have not been described. This article presents 2 cases of HCCC of the maxillary sinus with unusual glandular formation and lymphoplasmacytic stroma in case 1 and a characteristic solid nest pattern and fibrocellular and hyalinized stroma in case 2. Immunohistochemical studies excluded myoepithelial origin and sinonasal renal cell-like adenocarcinomas. Negativity for p63 and p40 in case 1 ruled out a squamous cell origin. Both cases showed a rearranged EWSR1 gene. Reverse-transcription polymerase chain reaction detected EWSR1-ATF1 fusion gene transcripts, and Sanger sequencing confirmed an EWSR1 exon 11 fused in-frame to ATF exon 3.

Hyalinizing clear cell carcinoma (HCCC) is a low-grade salivary gland carcinoma characterized by clear cells and hyalinized stroma. Recently, the EWSR1-ATF1 fusion gene was found in HCCCs. We herein describe three cases of HCCC identified in one male and two females, ranging in age from 27 to 67 years. The tumors were located in the root of tongue, nasopharynx, and soft palate. They were composed of nested or cord-like proliferations of epithelial cells with clear to pale eosinophilic cytoplasm, embedded in hyalinized and focally fibroedematous stroma. Tumor-associated lymphoid proliferation and pseudoepitheliomatous hyperplasia were also observed in each one case. MAML2 fusions specific to mucoepidermoid carcinoma were not detected in any of the three cases. We found EWSR1-ATF1 in two of three HCCCs using reverse transcription polymerase chain reaction (RT-PCR) with our original primer sets designed to detect the fusion gene transcripts in formalin-fixed paraffin-embedded (FFPE) tissues. EWSR1 rearrangement was also confirmed by fluorescence in situ hybridization (FISH) on FFPE sections in two cases. There was a good concordance between the two methods (two positive cases and one negative case by both RT-PCR and FISH). Therefore, RT-PCR and FISH using FFPE tissue may be ancillary tools to confirm the diagnosis of HCCC.

Hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland tumor with a specific EWSR1-ATF1 fusion gene and can have mucin production. Mucoepidermoid carcinoma (MEC) with a clear cell component is its morphologic mimic. Using MAML2 fluorescence in situ hybridization (FISH), a total of 49 MEC cases were separated into MAML2 fusion-positive (32 cases) and MAML2 fusion-negative groups (17 cases). This study used EWSR1 FISH to investigate MAML2 fusion-negative cases to identify previously unrecognized HCCC. Among 17 MAML2 fusion-negative cases, 3 had rearrangement of the EWSR1 gene and were reclassified as HCCC. Including 5 previously diagnosed HCCC cases, these 8 HCCC cases had a male-to-female ratio of 1:7, and most (7/8) tumors arose from oral minor salivary glands in the oral cavity (tongue base and palate). EWSR1-ATF1 fusion was confirmed by FISH in all 8 HCCC cases. The histologic features between genetically confirmed HCCC and MEC were compared. HCCC was significantly associated with minor salivary gland involvement, a discrepancy between low-grade cytology and intermediate- to high-grade histology using the MEC grading system, and absence of both epidermoid cells with abundant cytoplasm and goblet cells lining cysts or forming clusters. Clear cells and a hyalinized stroma were not specific for HCCC. HCCC may be erroneously classified as MEC because clear cells may be a minor histologic component and mucin production is not uncommon. Previously diagnosed MEC cases should be reevaluated, especially those arising from minor salivary glands or without MAML2 fusion. Careful histologic evaluation with supporting molecular testing can facilitate pathologic diagnoses.

Malignant gastrointestinal neuroectodermal tumor (M-GNET) and clear cell sarcoma (CCS) of soft tissue represent closely related, extremely rare, malignant mesenchymal neoplasm of uncertain differentiation. Both entities are characterized genetically by the same molecular alterations represented by the presence of EWSR1-ATF1 and, more rarely, EWSR1-CREB1 fusion genes. The latter translocation seems to be more represented in M-GNET that, despite significant morphologic overlap with CCS, tends to lack overt features of melanocytic differentiation. Most M-GNET occur in the lower gastrointestinal tract, whereas occurrence in the upper tract has been reported only exceptionally. The differential diagnosis represents a major challenge, and accurate diagnosis impact significantly on therapeutic planning. We herein report the clinicopathologic features of a molecularly confirmed M-GNET that arose at the base of the tongue and review the pertinent literature.

Keywords: Clear cell sarcoma; Malignant gastrointestinal neuroectodermal tumor; Next-generation sequencing; Oral cavity.

Malignant gastrointestinal neuroectodermal tumour (GNET) is a recently characterised rare and aggressive tumour that typically arises in association with the small intestine of adults. We present a novel case of this entity and expand the spectrum of its reported morphological features. The patient was a 5-year-old female, the youngest reported patient affected by the condition, and presented with extra-abdominal disease. The histopathological features included the presence of a junctional component of the palatal tumour, which mimicked mucosal melanoma, a feature that has not been previously reported in GNET. Whole genome and RNA sequencing was performed that demonstrated the EWSR1-ATF1 translocation characteristic of GNET. Knowledge of this entity and its features, together with careful morphological assessment supplemented by judicious immunohistochemical and molecular studies should enable the correct diagnosis to be established.

We describe the clinical, pathological, and molecular features of a primary adrenal angiomatoid fibrous histiocytoma (AFH) in an 11-year-old girl presenting with pyrexia of unknown origin. We performed next-generation sequencing-based anchored multiplex polymerase chain reaction (Archer® FusionPlex® sarcoma assay), which revealed an EWSR1-ATF1 gene fusion with novel breakpoints in exon 11 of EWSR1 and exon 3 of ATF1. The pyrexia resolved fully after surgical resection, and the patient was disease-free on follow-up at 1 year and 6 months. This case exemplifies the value of molecular testing of pediatric neoplasms presenting at unusual sites for diagnosis and identification of novel gene fusion breakpoints.

The mechanism underlying stringently controlled sequence of events in the eukaryotic cell cycle involves periodic transcription of a number of genes encoding important regulators of cell cycle, growth, proliferation and apoptosis. Deregulated activities of transcription factors that contribute to this programmed gene expression, are associated with many diseases including cancer. A detailed mechanistic understanding of the transcriptional control associated with cell division is, therefore, important. We have reported earlier that the transcription factor Atf1 in Schizosaccharomyces pombe can regulate G2-M transition by directly controlling the expression of the mitotic cyclin Cdc13 (1).To gain a better understanding of the role of Atf1 in cell cycle, we performed a microarray based identification of cell cycle related targets of Atf1. The microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series (accession number GSE71820). Here we report the annotation of the genes whose expression get altered by Atf1 overexpression and also provide details related to sample processing and statistical analysis of our microarray data.