Objective: To investigate the clinicopathological features, immunophenotype, differential diagnosis, molecular genetic changes and prognosis of salivary gland-type clear cell carcinoma (CCC) of the lung. Methods: Eight cases of salivary gland-type CCC of the lung diagnosed at Fudan University Shanghai Cancer Center and Shanghai Pulmonary Hospital, China from March 2017 to December 2020 were retrieved and analyzed. The pathological sections of these cases were studied using immunohistochemical staining, fluorescence in situ hybridization (FISH), and RNA-seq fusion gene detection based on next generation sequencing technique. The patients were followed up and the relevant literature was reviewed. Results: The 8 patients included 3 males and 5 females, with age ranging from 43 to 64 years (average, 58 years). All patients underwent radical lobectomy and lymph node dissection, while only one had lymph node metastases. The eight patients were followed up for 6 to 45 months, and were all recurrence-free. Histopathologically, the tumor was mainly composed of eosinophilic and clear cells arranged in trabecular, ribbon and nest patterns. Hyalinization was often observed in the stroma around the nest. Immunohistochemical staining showed that 8/8 cases were positive for EMA and CK7; 5/8 cases were positive for p63 and p40; 4/8 cases were positive for SOX10; and the cases were all negative for S-100, SMA and calponin. EWSR1 gene fusion was detected in all cases by FISH. RNA-seq fusion gene was detected in 6 cases based on next generation sequencing. The EWSR1-ATF1 gene fusion was detected in 5 cases, among which one case also had the ATF1-SPTLC2 gene fusion. All 5 cases with EWSR1-ATF1 gene fusion showed that EWSR1 exon 12/13 fused with ATF1 exon 3. And EWSR1-CREM gene fusion was detected in one case. Conclusions: Salivary gland-type CCC of the lung is an extremely rare primary lung tumor arising from the bronchial mucosa. The diagnosis and differential diagnosis of this tumor depend on classic histomorphology, especially the auxiliary detection of EWSR1 fusion gene. The primary treatment choice of this tumor is complete surgical resection. Lymph node metastases may occur, but the overall prognosis is good.

目的： 探讨肺原发涎腺型透明细胞癌的临床病理学特征、免疫表型、鉴别诊断、分子遗传学特征及预后。 方法： 收集2017年3月至2020年12月在复旦大学附属肿瘤医院和同济大学附属上海市肺科医院确诊的8例肺原发涎腺型透明细胞癌的临床病理资料，对标本进行免疫组织化学、荧光原位杂交（FISH）检测和基于二代测序技术的RNA-seq融合基因检测，随访患者并复习相关文献。 结果： 8例患者年龄43~64岁，平均58岁。男女比为3∶5。8例患者均行根治性肺叶切除加淋巴结清扫，1例发生淋巴结转移，术后随访6~45个月均无复发。组织病理学主要由排列呈小梁状、带状、巢状的嗜酸性细胞及透明细胞组成，细胞巢周围间质中常有玻璃样变物包绕。免疫表型：8例肿瘤细胞上皮细胞膜抗原（EMA）、细胞角蛋白（CK）7均呈阳性表达，5例p63、p40呈阳性表达，4例SOX10呈阳性表达，S-100蛋白、平滑肌肌动蛋白（SMA）、Calponin均呈阴性表达。8例FISH均检测出EWSR1融合基因。6例进行了基于二代测序技术的RNA-seq融合基因检测，5例检测到EWSR1-ATF1融合基因（其中1例同时有ATF1-SPTLC2融合基因），5例EWSR1-ATF1基因融合均显示EWSR1外显子12/13与ATF1外显子3融合。1例为EWSR1-CREM基因融合。 结论： 肺原发涎腺型透明细胞癌是一种罕见的起源于支气管黏膜的原发肺肿瘤，该肿瘤诊断和鉴别诊断依赖于特征性形态学表现，特别是EWSR1融合基因检测辅助诊断，治疗以手术完整切除为主，可以发生淋巴结转移，但总体预后较好。.

Gene silencing in S. pombe occurs by heterochromatin formation at the centromere (cen), mating-type (mat) and telomere loci. It is mediated by silencing factors including Swi6, Clr1-4, Rhp6 and Pola. RNAi pathway also plays a role in establishment of silencing at the mat and cen loci. Recently, the stress response factors, Atf1 and Pcr1were shown to play an RNAi-independent role in silencing at the mat3 locus through a cis-acting Atf1-binding site located within the repression element REIII and recruitment of the silencing factors Clr3 and Clr6. Another cis-acting site, named repression element REII abutting the mat2 locus, also establishes heterochromatin structure through Clr5 and histone deacetylases but independently of H3-Lys9-methylation and RNAi. Here, we report the occurrence of binding sites for another oxidative response factor, the pombe AP1- like factor Pap1, at the mating-type, centromere and telomere loci. By genetic studies we show that these sites play a role in silencing at the outer repeats of centromeres as well as mating-type locus and this effect is mediated through Pap1 binding site and interaction with and recruitment of the HP1/Swi6. Importantly, pap1Δ cells display a silencing defect even in absence of the oxidative stress. Such a role of Pap1 in heterochromatin formation may be evolutionarily conserved.

Epigenetic inheritance of heterochromatin requires DNA-sequence-independent propagation mechanisms, coupling to RNAi, or input from DNA sequence, but how DNA contributes to inheritance is not understood. Here, we identify a DNA element (termed "maintainer") that is sufficient for epigenetic inheritance of pre-existing histone H3 lysine 9 methylation (H3K9me) and heterochromatin in Schizosaccharomyces pombe but cannot establish de novo gene silencing in wild-type cells. This maintainer is a composite DNA element with binding sites for the Atf1/Pcr1 and Deb1 transcription factors and the origin recognition complex (ORC), located within a 130-bp region, and can be converted to a silencer in cells with lower rates of H3K9me turnover, suggesting that it participates in recruiting the H3K9 methyltransferase Clr4/Suv39h. These results suggest that, in the absence of RNAi, histone H3K9me is only heritable when it can collaborate with maintainer-associated DNA-binding proteins that help recruit the enzyme responsible for its epigenetic deposition.

Keywords: Atf1; Clr4; Deb1; H3K9me; ORC; Pcr1; Suv39h; epigenetic inheritance; heterochromatin; maintainer.

Background: Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols.

Results: AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain.

Conclusions: This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes.

Keywords: Esterase; Medium-chain-length α,ω-diols; Metabolic versatility; Monooxygenase; Pseudomonas putida; Substrate preference; ω-Oxyfunctionalization.

'Intracranial mesenchymal tumor, FET-CREB fusion-positive' occurs primarily in children and young adults and has previously been termed intracranial angiomatoid fibrous histiocytoma (AFH) or intracranial myxoid mesenchymal tumor (IMMT). Here we performed genome-wide DNA methylation array profiling of 20 primary intracranial mesenchymal tumors with FET-CREB fusion to further study their ontology. These tumors resolved into two distinct epigenetic subgroups that were both divergent from all other analyzed intracranial neoplasms and soft tissue sarcomas, including meningioma, clear cell sarcoma of soft tissue (CCS), and AFH of extracranial soft tissue. The first subgroup (Group A, 16 tumors) clustered nearest to but independent of solitary fibrous tumor and AFH of extracranial soft tissue, whereas the second epigenetic subgroup (Group B, 4 tumors) clustered nearest to but independent of CCS and also lacked expression of melanocytic markers (HMB45, Melan A, or MITF) characteristic of CCS. Group A tumors most often occurred in adolescence or early adulthood, arose throughout the neuroaxis, and contained mostly EWSR1-ATF1 and EWSR1-CREB1 fusions. Group B tumors arose most often in early childhood, were located along the cerebral convexities or spinal cord, and demonstrated an enrichment for tumors with CREM as the fusion partner (either EWSR1-CREM or FUS-CREM). Group A tumors more often demonstrated stellate/spindle cell morphology and hemangioma-like vasculature, whereas Group B tumors more often demonstrated round cell or epithelioid/rhabdoid morphology without hemangioma-like vasculature, although robust comparison of these clinical and histologic features requires future study. Patients with Group B tumors had inferior progression-free survival relative to Group A tumors (median 4.5 vs. 49 months, p = 0.001). Together, these findings confirm that intracranial AFH-like neoplasms and IMMT represent histologic variants of a single tumor type ('intracranial mesenchymal tumor, FET-CREB fusion-positive') that is distinct from meningioma and extracranial sarcomas. Additionally, epigenomic evaluation may provide important prognostic subtyping for this unique tumor entity.

Keywords: ATF1; CREB1; CREM; EWSR1; angiomatoid fibrous histiocytoma (AFH); brain tumor; clear cell sarcoma; intracranial mesenchymal tumor with FET-CREB fusion; intracranial myxoid mesenchymal tumor; molecular neuropathology; sarcoma.

DNA methylation is considered to play an important role in the development of diabetic retinopathy. Here, our goal was to investigate the precise role of methyl-CpG binding domain protein 2 (Mbd2) in the apoptosis of retinal ganglion cells (RGCs) in the early diabetic retina. Mbd2 was significantly upregulated after high glucose (HG) treatment and played a proapoptotic role in RGCs during HG-induced apoptosis. Combining ChIP and gene microarray datasets, the results showed that Mbd2 possessed potential binding sites for miR-345-5p, thereby elevating the expression levels of miR-345-5p via the enhancement of promoter demethylation. Activating transcription factor 1 (Atf1) played an anti-apoptotic role during the process of apoptosis in RGCs and acted as the target gene for miR-345-5p. Furthermore, the number of surviving RGCs in the diabetic retina was increased in Mbd2-knockout mice when compared with wild-type mice and the visual function became better accordingly. Collectively, our data demonstrated that the HG-induced overexpression of Mbd2 in the retina was partly responsible for the apoptosis of retinal neuronal cells through the miR-345-5p/Atf1 axis. Therefore, the targeting of Mbd2 might represent a novel therapeutic strategy for the treatment of neurodegeneration in the early diabetic retina.

Keywords: Atf1; Mbd2; RGCs; apoptosis; diabetes; miR-345-5p.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Tyrosol and hydroxytyrosol derived from virgin olive oil and olives extract, have wide applications both as functional food components and as nutraceuticals. However, they have low bioavailability due to their low absorption and high metabolism in human liver and small intestine. Acetylation of tyrosol and hydroxytyrosol can effectively improve their bioavailability and thus increase their potential use in the food and cosmeceutical industries. There is no report on the bioproductin of tyrosol acetate and hydroxytyrosol acetate so far. Thus, it is of great significance to develop microbial cell factories for achieving tyrosol acetate or hydroxytyrosol acetate biosynthesis. In this study, a de novo biosynthetic pathway for the production of tyrosol acetate and hydroxytyrosol acetate was constructed in Escherichia coli. First, an engineered E. coli that allows production of tyrosol from simple carbon sources was established. Four aldehyde reductases were compared, and it was found that yeaE is the best aldehyde reductase for tyrosol accumulation. Subsequently, the pathway was extended for tyrosol acetate production by further overexpression of alcohol acetyltransferase ATF1 for the conversion of tyrosol to tyrosol acetate. Finally, the pathway was further extended for hydroxytyrosol acetate production by overexpression of 4-hydroxyphenylacetate 3-hydroxylase HpaBC.

Keywords: Escherichia coli; Hydroxytyrosol acetate; Metabolic engineering; Tyrosol acetate.

The low-grade inflammation is pivotal in obesity and its comorbidities; however, the inflammatory proteins are out of target for traditional drug therapy. Omega-3 (ω3) fatty acids can modulate the downstream signaling of Toll-like receptor (TLR) and tumor necrosis factor-α receptor (TNFα) through GPR120, a G-protein-coupled receptor, a mechanism not yet elucidated in humans. This work aims to investigate if the ω3 supplementation, at a feasible level below the previously recommended level in the literature, is enough to disrupt the inflammation and endoplasmic reticulum stress (ER-stress), and also if in acute treatment (3 h) ω3 can activate the GPR120 in peripheral blood mononuclear cells (PBMC) and leukocytes from overweight non-alcoholic fatty liver disease (NAFLD) participants. The R270H variant of the Ffar4 (GPR120 gene) will also be explored about molecular responses and blood lipid profiles. A triple-blind, prospective clinical trial will be conducted in overweight men and women, aged 19-75 years, randomized into placebo or supplemented (2.2 g of ω3 [EPA+DHA]) groups for 28 days. For sample calculation, it was considered the variation of TNFα protein and a 40% dropout rate, obtaining 22 individuals in each group. Volunteers will be recruited among patients with NAFLD diagnosis. Anthropometric parameters, food intake, physical activity, total serum lipids, complete fatty acid blood profile, and glycemia will be evaluated pre- and post-supplementation. In the PBMC and neutrophils, the protein content and gene expression of markers related to inflammation (TNFα, MCP1, IL1β, IL6, IL10, JNK, and TAK1), ER-stress (ATF1, ATF6, IRE1, XBP1, CHOP, eIF2α, eIF4, HSP), and ω3 pathway (GPR120, β-arrestin2, Tab1/2, and TAK1) will be evaluated using Western blot and RT-qPCR. Participants will be genotyped for the R270H (rs116454156) variant using the TaqMan assay. It is hypothesized that attenuation of inflammation and ER-stress signaling pathways in overweight and NAFLD participants will be achieved through ω3 supplementation through binding to the GPR120 receptor. TRIAL REGISTRATION: ClinicalTrials.gov #RBR-7x8tbx. Registered on May 10, 2018, with the Brazilian Registry of Clinical Trials.

Keywords: Endoplasmic reticulum stress; GPR120; Inflammation; NAFLD; Omega-3; Overweight.

In phytopathogenic fungi, the Hog1 MAPK pathway has roles in osmoregulation, fungicide sensitivity, and other processes. The ATF1/CREB-activating transcription factor Atf1 is a regulator that functions downstream of the Hog1 MAPK pathway. Here, we identified a gene, designated CsAtf1, that encodes a bZIP transcription factor in Colletotrichum siamense, which is the main pathogen that causes Colletotrichum leaf fall disease in rubber trees in China. CsAtf1 localizes to the nucleus. Its mRNA expression correlates positively with that of CsPbs2 and CsHog1 in the Hog1 MAPK pathway in response to activator (anisomycin), inhibitor (SB203580) and fludioxonil treatments. The CsAtf1 deletion mutant showed slightly retarded mycelial growth, small conidia, slow spore germination, and abnormal appressorium formation. This mutant showed the increased spore germination rate after fludioxonil treatment and more resistance to the fungicide fludioxonil than did the wild-type fungus. However, unlike deletion of Pbs2 or Hog1, which resulted in greater sensitivity to osmotic stress, the CsAtf1 deletion induced slightly increased resistance to osmotic stress and the cell wall stress response. The ΔCsAtf1 strain also exhibited significantly reduced virulence on rubber tree leaves. These data revealed that CsAtf1 plays a key role in the regulation of fludioxonil sensitivity and in pathogenicity regulation in C. siamense.

Keywords: Colletotrichum siamense; Transcription Factor; fludioxonil sensitivity; virulence.

Mesenchymal tumors and tumor-like lesions of the gastrointestinal (GI) tract are uncommon. They vary from reactive tumefactive lesions and benign neoplasms to highly aggressive sarcomas. Among them, GI stromal tumors (GISTs) are most common, followed, with less frequency, by smooth muscle and neurogenic tumors. The major challenge resides in correctly identifying GISTs and providing a comprehensive report (including risk assessment and genotyping) that represents the basis for an optimized surgical-oncological treatment and/or adjuvant therapy. On the other hand, the challenge of benign lesions is to find a good name (well understandable and reproducible diagnostic term) that helps avoid diagnostic ambiguity and prognostic uncertainty so that overprognostication and overtreatment can be prevented. Moreover, several recently described genetically defined benign and malignant entities need be correctly diagnosed due to their special "targeted" therapeutic options and to further characterize their clinicopathological and biological properties in the future. These recent entities include aggressive epithelioid inflammatory myofibroblastic sarcoma (ALK-RANBP2-driven), malignant gastrointestinal neuroectodermal tumor (EWSR1-ATF1/CREB-related), NTRK-rearranged neoplasms, and, most recently, colorectal NUTM1-rearranged sarcomas. This review highlights the major clinicopathological features of gastrointestinal mesenchymal lesions in light of recent developments.

Mesenchymale Tumoren und tumorartige Veränderungen des Gastrointestinaltraktes sind relativ selten. Sie variieren von reaktiven Veränderungen und gutartigen Neoplasien bis hin zu hochaggressiven Sarkomen. Dabei stellen gastrointestinale Stromatumoren (GIST) die häufigsten Entitäten dar, gefolgt mit Abstand von glattmuskulären und neurogenen Tumoren. Die Herausforderung liegt oft darin, GIST korrekt zu identifizieren und einen therapeutisch wegweisenden Befundbericht einschließlich des Risikoprofils und Genotyps zu erstellen, damit eine optimierte chirurgisch-onkologische Therapie geplant und entsprechend umgesetzt werden kann. Auf der anderen Seite liegt die Herausforderung bei gutartigen Veränderungen meist in der Namensfindung, damit jegliche prognostische Unsicherheit und damit Übertherapien vermieden werden können. Ferner wurden in jüngster Zeit diverse genetisch definierte Tumorentitäten beschrieben, deren korrekte Identifizierung für die Therapie und auch für die weitere Charakterisierung dieser neuen Entitäten erforderlich ist. Dazu gehören insbesondere aggressive epitheloide inflammatorische myofibroblastische Sarkome (ALK+), maligne gastrointestinale neuroektodermale Tumoren (EWSR1+), NTRK-positive Tumoren und kolorektale NUTM1-rearrangierte Sarkome. Im Folgenden wird eine Übersicht zu den wichtigsten klinisch-pathologischen Eigenschaften dieser Tumoren vermittelt und ihre diagnostischen Aspekte beleuchtet.

Keywords: Gastrointestinal stromal tumors; Gene fusions; Imatinib; Neuroectodermal tumors; Sarcoma.

Malignant gastrointestinal neuroectodermal tumor (GNET) is a rare malignant primary gastrointestinal mesenchymal tumor which can be diagnosed via fine-needle aspiration (FNA) cytology. In the context of FNA, the diagnosis requires a cell block and the use of significant resources including immunohistochemical stains and molecular testing. The differential diagnosis of GNET includes clear cell sarcoma (CCS), gastrointestinal stromal tumor (GIST), gastric schwannoma, metastatic melanoma, malignant perivascular epithelioid cell tumor (PEComa) and granular cell tumor, among others. Here we describe a case which was initially diagnosed as malignant granular cell tumor by FNA which was later revised to GNET following the finding of an EWSR1-ATF1 fusion gene rearrangement.

Keywords: EWSR1 ATF1 fusion partner; Endoscopic ultrasound; Fine-needle aspiration cytology; GNET; Malignant gastrointestinal neuroectodermal tumor; Stomach mass.

Neuronal damage or degeneration is the main feature of neurological diseases. Regulation of neurogenesis and neuronal differentiation is important in developing therapies to promote neuronal regeneration or synaptic network reconstruction. Neurogenesis is a multistage process in which neurons are generated and integrated into existing neuronal circuits. Neuronal differentiation is extremely complex because it can occur in different cell types and can be caused by a variety of inducers. Recently, natural compounds that induce neurogenesis and neuronal differentiation have attracted extensive attention. In this paper, the potential neural induction effects of medicinal plant-derived natural compounds on neural stem/progenitor cells (NS/PCs), the cultured neuronal cells, and mesenchymal stem cells (MSCs) are reviewed. The natural compounds that are efficacious in inducing neurogenesis and neuronal differentiation include phenolic acids, polyphenols, flavonoids, glucosides, alkaloids, terpenoids, quinones, coumarins, and others. They exert neural induction effects by regulating signal factors and cell-specific genes involved in the process of neurogenesis and neuronal differentiation, including specific proteins (β-tubulin III, MAP-2, tau, nestin, neurofilaments, GFAP, GAP-43, NSE), related genes and proteins (STAT3, Hes1, Mash1, NeuroD1, notch, cyclin D1, SIRT1, reggie-1), transcription factors (CREB, Nkx-2.5, Ngn1), neurotrophins (BDNF, NGF, NT-3) and signaling pathways (JAK/STAT, Wnt/β-catenin, MAPK, PI3K/Akt, GSK-3β/β-catenin, Ca2+/CaMKII/ATF1, Nrf2/HO-1, BMP). The natural compounds with neural induction effects are of great value for neuronal regenerative medicine and provide promising prevention and treatment strategies for neurological diseases.

Keywords: Natural compounds; mesenchymal stem cells.; neural stem/progenitor cells; neurite outgrowth; neurogenesis; neuronal cells; neuronal differentiation.

Aspirin can prevent or inhibit inflammation-related cancers, such as colorectal cancer and hepatocellular carcinoma (HCC). However, the effectiveness of chemotherapy may be compromised by activating oncogenic pathways in cancer cells. Elucidation of such chemoresistance mechanisms is crucial to developing novel strategies to maximize the anti-cancer effects of aspirin. Here, we report that aspirin markedly induces CREB/ATF1 phosphorylation in HCC cells, which compromises aspirin's anti-HCC effect. Inhibition of AMP-activated protein kinase (AMPK) abrogates the induction of CREB/ATF1 phosphorylation by aspirin. Mechanistically, activation of AMPK by aspirin results in decreased expression of the urea cycle enzyme carbamoyl-phosphate synthase 1 (CPS1) in HCC cells and xenografts. Treatment with aspirin or CPS1 knockdown stimulates soluble adenylyl cyclase expression, thereby increasing cyclic AMP (cAMP) synthesis and stimulating PKA-CREB/ATF1 signaling. Importantly, abrogation of aspirin-induced CREB/ATF1 phosphorylation could sensitize HCC to aspirin. The bis-benzylisoquinoline alkaloid berbamine suppresses the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), leading to protein phosphatase 2A-mediated downregulation of CREB/ATF1 phosphorylation. The combination of berbamine and aspirin significantly inhibits HCC in vitro and in vivo. These data demonstrate that the regulation of cAMP-PKA-CREB/ATF1 signaling represents a noncanonical function of CPS1. Targeting the PKA-CREB/ATF1 axis may be a strategy to improve the therapeutic effects of aspirin on HCC.

Keywords: AMPK; CREB; acetylsalicylic acid; aspirin; drug–drug synergism; liver cancer.

Objective: To describe the application of a targeted RNA sequencing assay to detect fusion transcripts in formalin-fixed paraffin-embedded (FFPE), non-decalcified samples of clear cell odontogenic carcinoma (CCOC) and related tumors, and to add to knowledge of the genetic drivers of CCOC.

Study design: Five FFPE tissues, including intraosseous CCOC (n = 3), clear cell carcinoma of the salivary gland (CCC, n = 1), and Ewing sarcoma (ES, n = 1), were analyzed by targeted RNA-seq to detect fusions.

Results: The 3 intraosseous CCOC samples harbored EWSR1 translocations: EWSR1-ATF1 (n = 2) and EWSR1-CREM (n = 1); the CCC sample contained an EWSR1-ATF1 fusion; and the ES sample contained an EWSR1-FLI1 fusion detected by RNA-seq.

Conclusions: These results demonstrate that targeted RNA-seq is a valuable tool to detect fusions in FFPE samples of rare tumors such as CCOC and CCC. The results also confirm the observations that CCOC is driven by fusions between EWSR1 and CREB family transcription factors, including ATF1 and CREM. To our knowledge, this is the second report of CCOC with an EWSR1-CREM translocation.

Malignant gastrointestinal neuroectodermal tumor (GNET) is an extremely rare soft tissue sarcoma and has been designated as a new entity recently. At present, GNET virtually exclusively occurs in the gastrointestinal tract. Here we report a case of extra-GNET that arose in the right heart. A 62-year-old male complained of chest distress and breathing difficulty while lying down at night for over 1 month at admission. The radiological findings revealed an occupying lesion involving the right atrium and the right ventricle without any abdominal abnormalities. The patient then underwent a surgical resection. Microscopically, neoplastic cells proliferated in the pattern of nests and sheets with fibrous separation. Focal areas with cellular dyscohesion imparted a vague pseudopapillary pattern. These tumor cells were small to medium in size with fine chromatin and predominantly pale eosinophilic cytoplasm. The nuclei were typically round to oval with somewhat irregular contours and contained small nucleoli. The mitotic figures were easily found. Immunohistochemically, the neoplastic cells were positive for S100 and SOX-10 but negative for HMB-45, A103, and CD99. EWSR1-AFTF1 rearrangement was detected by fluorescence in situ hybridization and further confirmed by whole-transcriptome sequence analysis. The patient had pulmonary metastasis 8 months later and soon died of the disease. The overall survival of the patient was 20 months. In summary, we reported an extremely rare case of cardiac GNET, indicating that the location of GNET should not be confined to the GI tract as initially defined. Due to the lack of a specific effective treatment and the occurrence of early metastasis, cardiac GNET conferred a poor prognosis. More clinical and experimental studies are warranted to better manage this disease in the future.

Keywords: EWSR1-ATF1; cardiac malignancy; case report; extra-gastrointestinal; malignant gastrointestinal neuroectodermal tumor.

Objective: To determine the functional effects of ATF1, WNT10B and GREM2 gene variants identified in individuals with tooth agenesis (TA).

Settings and sample population: Stem cells from human exfoliated deciduous teeth (SHED) were used as an in vitro model system to test the effect of TA-associated variants.

Materials and methods: Plasmid constructs containing reference and mutant alleles for ATF1 rs11169552, WNT10B rs833843 and GREM2 rs1414655 variants were transfected into SHED for functional characterization of variants. Allele-specific changes in gene transcription activity, protein expression, cell migration and proliferation, and expression of additional tooth development genes (MSX1, PAX9 and AXIN2) were evaluated. Data analyses were performed using Student's t-test. P-values ≤ .05 were considered statistically significant.

Results: Mutant variants resulted in significantly decreased transcriptional activity of respective genes (P < 0.05), although no changes in protein localization were noted. Expression of MSX1 was significantly decreased in ATF1- and GREM2-mutant cells, whereas PAX9 or AXIN2 mRNA expression was not significantly altered. Mutant WNT10B had no significant effect on the expression of additional TA genes. ATF1- and GREM2-mutant cells presented increased cell migration. Cell proliferation was also affected with all three mutant alleles.

Conclusions: Our results demonstrate that ATF1, WNT10B and GREM2 mutant alleles have modulatory effects on gene/protein function that may contribute to TA.

Keywords: functional assays; genetic variation; hypodontia; oligodontia.

Polyfunctional thiols like 3-sulfanylhexan-1-ol (3SH) and its ester 3-sulfanylhexyl acetate (3SHA) are important aroma determinants in wine with exceptionally low odor thresholds. 3SH is largely found in grape must bound to glutathione and cysteine and requires enzymatic action to be perceived sensorially. The wine yeast Saccharomyces cerevisiae is ineffective in releasing volatile thiols from their precursor configuration. For this purpose, a yeast strain was constructed that expresses the carbon-sulfur lyase encoding the tnaA gene from Escherichia coli and overexpresses its native alcohol acetyltransferase encoding genes, ATF1 and ATF2. The resulting yeast strain, which co-expresses tnaA and ATF1, showed elevated 3SH-releasing capabilities and the esterification of 3SH to its acetate ester 3SHA. Levels of over 7000 ng/L of 3SHA in Sauvignon blanc wines were achieved. Enhanced release and esterification of 3SH were also shown in the fermentation of guava and passionfruit pulp and three hop varieties. This study offers prospects for the development of flavor-enhancing yeast strains with optimized thiol-releasing and esterification capabilities in a diverse set of beverage matrices.

Keywords: 3-sulfanylhexan-1-ol; 3-sulfanylhexyl acetate; ATF1; Saccharomyces cerevisiae; aroma; esters; thiols; tnaA; wine; yeast.

This comprehensive literature review represents a summary of all cases of clear cell carcinoma (CCC) of the salivary glands that are documented in the literature. PubMed was used to collect available reports of CCC; 97 reports detailing 254 cases, published between 1983 and 2020, were retrieved. Clinically the tumor manifests most commonly as a painless mass or swelling on the palate, and the duration of symptoms prior to seeking care ranges from 1 week to 6 years. Local tumor recurrence was present in 18.8% of the cases. By histopathology, CCC shows a mixture of growth patterns including solid (25.1%), nested (78.6%), sheet-like (23.5%), cords (46.1%), and trabeculae (42.4%). Immunohistochemical studies are positive for one or more cytokeratins (99.1%), PAS (95.1%), EMA (77.8%), and p63 (96.3%), but negative for S-100 (96.3%), PASD (91.1%), SMA (91.0%), and calponin (95.1%). Molecular features were reported in 113 cases; 96.0% were positive for an EWSR1 rearrangement by EWSR1 break apart FISH testing and 14.8% were positive for the rearrangement EWSR1-ATF1 tested by qPCR or targeted RNA sequencing. Clinical patterns and genetic studies imply that this tumor is the extraosseous counterpart of clear cell odontogenic carcinoma, an intraosseous odontogenic tumor of the jaws.

Keywords: clear cell carcinoma; clear cell sarcoma; hyalinizing; immunohistochemistry; salivary gland.

microRNA-34, a highly conserved microRNA in evolution, is of great interest in recent years. miR-34 regulates multiple targets and performed many functions in cells, such as 1) those mediated by targeting ALDH2, Atf1, Bcl-2 have been implicated in cancer cell apoptosis; 2) it targets CCNE2, CDK4, CDK6 and others to regulate the cell cycle; and 3) it regulates immune homeostasis in Drosophila through Dlg1, Eip75B and others. Meanwhile, miR-34 family is a multifunctional miRNA family identified to be pivotal in organ development, organismal or organ senescence, stress response, spermatogenesis and signal transduction. In this review, we focus on the emerging roles of miR-34 family in mammals, arthropods, and Nematoda by discussing novel functions of old and new targets, mechanisms identified to mediate or interact by miR-34, which provides references for screening of tumor target drugs and creation of green target biopesticide.

Keywords: aging; function; miR-34 family; microRNA; target.

Background: As a rare salivary gland malignancy, clear cell carcinoma (CCC) is easily misdiagnosed. This study identified the features that allow better recognition of the clinicopathological and molecular characteristics and the prognosis of CCC, focusing on high-grade transformation (HGT) in this tumor and cases arising in uncommon sites.

Methods: Clinicopathological and follow-up data for 10 CCC samples were retrieved. Immunohistochemical (IHC) staining was performed, and fluorescence in situ hybridization (FISH) was used to detect EWSR1 gene rearrangements, EWSR1-ATF1 gene fusions, and MAML2 gene rearrangements.

Results: Histologically, typical CCCs comprised bland polygonal or round cells with clear cytoplasm. In contrast with typical CCCs, HGT tumor cells exhibited nuclear pleomorphism, high nuclear-to-cytoplasmic ratios, high mitotic activity, and necrosis. Rare morphologic features such as pseudopapillae, gland-like spaces, and entrapped ducts were also observed. Occasionally, tumors involving the oral cavity might arise from the overlying epithelium of the mucosal surface. Immunohistochemically, all the cases expressed p63, p40, and CK5/6, while myoepithelial-related markers were uniformly negative in all cases. HGT exhibited a wild type p53 expression pattern. FISH demonstrated EWSR1 rearrangement (10/10) and EWSR1-ATF1 fusion (4/5); however, MAML2 remained intact (0/3).

Conclusions: CCCs with HGT or occurring in uncommon sites are extremely rare. Combining morphology based IHC and molecular detection provided reliable evidence that the HGT component represented a transformation of CCC rather than the coexistence of another tumor and helped differentiating CCCs in uncommon sites from their mimics, avoiding potential misdiagnosis and inappropriate therapy. The overall prognosis for CCCs is good, except for the HGT cases, which needed continued treatment.

Keywords: ATF1; Clear cell carcinoma; EWSR1; High-grade transformation; Hyalinizing; MAML2.

Malignant gastrointestinal neuroectodermal tumor (MGNET) is a sarcoma typically involving the gastrointestinal tract with neuroectodermal differentiation and EWSR1-ATF1/CREB1 fusions. Recently, rare MGNET cases were reported in extragastrointestinal sites. We identified 2 cases of MGNET arising in unprecedented laryngeal and intracranial locations, respectively. Both cases showed spindle and epithelioid tumor cells with amphophilic to clear cytoplasm and occasionally prominent nucleoli, arranged in solid, fascicular, and pseudoalveolar patterns. Case 1 exhibited moderate to marked nuclear atypia and focal intraepithelial component. In contrast, case 2 comprised predominantly low-grade epithelioid cells with extensive pseudopapillary structures. Both tumors showed an S100/SOX10-positive and HMB45/melan-A-negative immunoprofile as well as EWSR1-ATF1 fusion. A chief obstacle in diagnosing case 1 was the histologic and immunophenotypic resemblance to melanoma. The striking pseudopapillary architecture and the intracranial location of case 2 rendered differential diagnoses including meningioma and ependymoma. With the peculiar locations and morphology, these cases posed great diagnostic challenge.

Keywords: ATF1; Clear cell sarcoma; EWSR1; Malignant gastrointestinal neuroectodermal tumor; Melanoma.

Binding of platelet-derived growth factor-BB (PDGF-BB) to its cognate receptor (PDGFR) promotes lens epithelial cell (LEC) proliferation and migration. After cataract surgery, these LEC behaviors have been proposed as an influential cause of posterior capsule opacification (PCO). Stimulated PDFGR undergoes dimerization and tyrosine phosphorylation providing docking sites for a SH2-domain-containing noncatalytic region of tyrosine kinase (Nck). Nck is an adaptor protein acting as a linker of the proximal and downstream signaling events. However, the functions of Nck1 protein in LEC have not been investigated so far. We reported here a crucial role of Nck1 protein in regulating PDGFR-mediated LEC activation using LEC with a silenced expression of Nck1 protein. The knockdown of Nck1 suppressed PDGF-BB-stimulated LEC proliferation and migration and disrupted the cell cycle progression especially G1/S transition. LEC lacking Nck1 protein failed to exhibit actin polymerization and membrane protrusions. The downregulation of Nck1 protein in LEC impaired PDGFR-induced phosphorylation of intracellular signaling proteins, including Erk1/2, Akt, CREB and ATF1, which resulted in inhibition of LEC responses. Therefore, these data suggest that the loss of Nck1 expression may disturb LEC activation and Nck1 may potentially be a drug target to prevent PCO and lens-related disease.

While soft tissue sarcomas typically have a spindled or pleomorphic appearance, a subset of malignant soft tissue neoplasms can have a prominent epithelioid morphology. In complex anatomic sites such as the mediastinum, such tumors can often be mistaken for a carcinoma or mesothelioma. Frequent expression of cytokeratin staining can further confound the diagnostic process and familiarity with these entities can help prevent an erroneous diagnosis. Particular entities that have been reported to occur in the mediastinum with such features include dedifferentiated liposarcoma, pleomorphic liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, SMARCA4-deficient thoracic sarcoma, alveolar soft part sarcoma and clear cell sarcoma. Many of these tumors exhibit unique clinical, genetic, molecular or immunohistochemical features which allow for accurate characterization. For example, pleomorphic liposarcoma contains bizarre appearing lipoblasts and dedifferentiated liposarcoma exhibits MDM2 gene amplification that is typically confirmed by fluorescence in-situ hybridization. Malignant peripheral nerve sheath tumor will often arise in association with a nerve or neurofibroma. Synovial sarcoma consistently exhibits rearrangements involving the SS18 gene and SMARCA4-deficient thoracic sarcoma shows loss of SMARCA4 staining in the tumor cells. Alveolar soft part sarcoma demonstrates an ASPL-TFE3 fusion transcript. Clear cell sarcoma often shows an EWSR1-ATF1 fusion transcript. When encountering a sarcoma of the mediastinum with epithelioid features, familiarity with these and other characteristics can help insure a correct diagnosis.

Keywords: Soft tissue sarcoma; epithelioid; epithelioid-like; mediastinum; mesenchymal neoplasm.