Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049, and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions.

RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1⁺ mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1's ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress.IMPORTANCE Control of mRNA localization, stability, turnover, and translation by RNA-binding proteins (RBPs) influences essential processes in all eukaryotes, including signaling by mitogen-activated protein kinase (MAPK) pathways. We describe that in the fission yeast Schizosaccharomyces pombe the RBP Rnc1 negatively regulates cell length at division during unperturbed growth and recovery after acute stress by reducing the activity of the MAPK Sty1, which regulates cell growth and differentiation during environmental cues. This mechanism relies on Rnc1 binding to specific mRNAs encoding both enhancers and negative regulators of Sty1 activity. Remarkably, multiple phosphorylation of Rnc1 by Sty1 favors RBP binding and destabilization of the above mRNAs. Thus, posttranscriptional modulation of MAP kinase signaling by RNA-binding proteins emerges as a major regulatory mechanism that dictates the growth cycle and cellular adaptation in response to the changing environment in eukaryotic organisms.

Like all developmental processes, odontogenesis is highly complex and dynamically regulated, with hundreds of genes co-expressed in reciprocal networks. Tooth agenesis (missing one or more/all teeth) is a common human craniofacial anomaly and may be caused by genetic variations and/or environmental factors. Variants in PAX9, MSX1, AXIN2, EDA, EDAR and WNT10A genes are associated with tooth agenesis. Currently, variants in ATF1, DUSP10, CASC8, IRF6, KDF1, GREM2, LTBP3 and components and regulators of WNT signaling WNT10B, LRP6, DKK, KREMEN1 are at the forefront of interest. Due to the interconnectedness of the signaling pathways of carcinogenesis and odontogenesis, tooth agenesis could be a suitable marker for early detection of cancer predisposition. Variants in genes associated with tooth agenesis could serve as prognostic or therapeutic targets in cancer. This review aims to summarize existing knowledge of development and clinical genetics of teeth. Concurrently, the review proposes possible approaches for future research in this area, with particular attention to roles in monitoring, early diagnosis and therapy of tumors associated with defective tooth development.

Recent studies showed that long non-coding RNAs (lncRNAs) could play critical roles in tumors progression. However, the performance of LINC01354 is still limited in non-small cell lung cancer (NSCLC). In the current study, our results showed that LINC01354 was significantly increased in NSCLC tissues and cell lines. High LINC01354 expression was associated with advanced TNM stage and poor prognosis in NSCLC patients. Loss-of-function assays revealed that knockdown of LINC01354 reduced lung cancer cells proliferation and invasive ability in vitro. Subsequently, mechanism studies showed that LINC01354 positively regulated the ATF1 expression via competitive binding to miR-340-5p. Therefore, our results illustrated that LINC01354 might act as an oncogenic role by modulating the miR-340-5p/ATF1 axis, providing a novel therapeutic therapy for NSCLC treatment.

OBJECTIVE

Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required.

METHODS

A new human CCS cell line (designated RSAR001) was established from the pleural effusion of a 44-year-old man with multiple lung metastases and pleural dissemination. The cell line and its xenograft were characterized including their morphology, immunohistochemistry, cytogenetic analysis, reverse transcription-polymerase chain reaction, direct sequencing analysis, and fluorescence in situ hybridization analysis.

RESULTS

The cell line has been maintained for over 12 months with more than 50 passages. RSAR001 cells exhibited a fascicular or diffuse growth pattern of short spindle- or oval-shaped cells with clear cytoplasm in heterotransplanted tumor, that was similar to the primary tumor. Immunophenotypically, RSAR001 cells in vitro and in vivo exhibited almost the same characteristics as the primary tumor. Cytogenetic analyses revealed a translocation, t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction and direct sequencing analysis detected transcripts of the Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) type 1 fusion gene. Fluorescence in situ hybridization using a break-apart probe for the EWSR1 gene on 22q12 showed a rearrangement.

CONCLUSIONS

These findings indicate that the RSAR001 cell line harbors EWSR1-ATF1 type 1 chimeric fusion gene, which is specific to CCS. RSAR001 cells might be useful for investigating biological behaviors and developing new treatments such as molecular-targeting antitumor drugs or immunological drugs for CCS.

In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Keywords: ATF1; Hanseniaspora uvarum; ethyl acetate; fermentation; genetic modification; transformation.

Long noncoding RNAs (lncRNAs) are abnormally expressed in many malignant tumors and involved in regulating the malignant phenotypes of cancer cells. However, the role of LINC00665 in colorectal cancer (CRC) and its regulatory mechanism remain unclear. In this study, real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of LINC00665, miR-9-5p and activating transcription factor 1 (ATF1) mRNA in CRC tissues. The expression of ATF1 in CRC tissues was also detected by immunohistochemistry and Western blot. CCK-8 and colony formation assays were employed to detect cell proliferation. Cell cycle and apoptosis were detected by flow cytometry analysis. Scratch healing assay and Transwell test were exploited to detect cell migration and invasion. The targeting relationships between LINC00665 and miR-9-5p, and miR-9-5p and ATF1 were validated by dual luciferase reporter assay. We found that LINC00665 was significantly overexpressed in CRC tissues, and it was also negatively correlated with the expression of miR-9-5p and positively associated with the expression of ATF1. Besides, LINC00665 promoted the proliferation, migration and invasion of CRC cells, and inhibited cell apoptosis by sponging miR-9-5p. ATF1 was proved to be the downstream target of miR-9-5p and was indirectly regulated by LINC00665. Collectively, it is concluded that LINC00665 contributes to the progression of CRC by regulating miR-9-5p/ATF1 axis.

Keywords: ATF1; CRC; LINC00665; miR-9-5p.

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.

Keywords: MGL2; YJU3; histone acetylation; substituted ester metabolism; wine fermentation.

Enhanced growth and proliferation of cancer cells are accompanied by profound changes in cellular metabolism. These metabolic changes are also common under physiological conditions, and include increased glucose fermentation accompanied by elevated cytosolic pH (pHc)^1,2. However, how these changes contribute to enhanced cell growth and proliferation is unclear. Here, we show that elevated pHc specifically orchestrates an E2F-dependent transcriptional programme to drive cell proliferation by promoting cyclin D1 expression. pHc-dependent transcription of cyclin D1 requires the transcription factors CREB1, ATF1 and ETS1, and the histone acetyltransferases p300 and CBP. Biochemical characterization revealed that the CREB1-p300/CBP interaction acts as a pH sensor and coincidence detector, integrating different mitotic signals to regulate cyclin D1 transcription. We also show that elevated pHc contributes to increased cyclin D1 expression in malignant pleural mesotheliomas (MPMs), and renders these cells hypersensitive to pharmacological reduction of pHc. Taken together, these data demonstrate that elevated pHc is a critical cellular signal regulating G1 progression, and provide a mechanism linking elevated pHc to oncogenic activation of cyclin D1 in MPMs, and possibly other cyclin D1~dependent tumours. Thus, an increase of pHc may represent a functionally important, early event in the aetiology of cancer that is amenable to therapeutic intervention.

Clear cell sarcoma (CCS) affects the deep soft tissues in young adults and is known to have high rates of metastasis, including lymphatic metastasis. In our previous study an xenoplant model of CCS was established, which exhibited local tumor growth, lymphatic metastasis, and distant metastasis in SCID-Beige mice. In the current study, the role of NK cells during metastasis in the same xenoplant murine model was investigated. Injection of murine or human NK cells significantly suppressed the metastasis of HS-MM CCS cells in SCID-Beige mice. Notably, reverse transcription-quantitative PCR analysis demonstrated that injection of NK cells did not alter the mRNA expression levels of ERSR1-ATF1, which is specifically transcribed in CCS, in the buffy coat of circulating blood cells of HS-MM-xenoplanted SCID-Beige mice. BALB/c nude mice xenoplanted with HS-MM cells exhibited local growth without evident metastasis, whereas inoculation with the anti-asialo-GM1 antibody, which has previously been found to abolish NK-cell activity, resulted in metastasis of HS-MM cells in BALB/c nude mice. The injection of the anti-CD96 antibody, which increases the cytotoxicity of NK cells, significantly suppressed the metastasis of HS-MM cells in SCID-Beige mice. These results indicated that NK cells impaired the metastatic tumor microenvironments in the present mice xenoplant model.

Keywords: ERSR1-ATF1; NK cells; clear cell sarcoma; metastatic model; metastatic tumor microenvironment.

Primary pulmonary myxoid sarcoma (PPMS) is a recently reported, exceedingly rare low-grade lung neoplasm characterized by reticular/lace-like growth of spindle to epithelioid cells embedded in an abundant myxoid matrix. Morphologically, it overlaps with a myxoid variant of angiomatoid fibrous histiocytoma (AFH) of the soft tissue. Genetically, they were both reported to harbor EWSR1-CREB1 fusion, while EWSR1-ATF1 has only been reported in AFH thus far. We report a case of primary pulmonary low-grade myxoid spindle cell tumor with morphologic and immunohistochemical features of PPMS but with an EWSR1-ATF1 fusion gene. In addition, we also encountered a case of endobronchial AFH with EWSR1-CREB1 translocation but also focal morphologic features of PPMS. These findings provide new evidence supporting the concept that PPMS and a myxoid variant of AFH represent a continuum with overlapping histologic, immunohistochemical, and genetic features.

Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In Saccharomyces cerevisiae cells, the syntheses of esters and higher alcohols are considerably influenced by intracellular CoA levels catalyzed by pantothenate kinase. In this work, we examined the effects of cofactor CoA and acetyl-CoA synthesis on the metabolism of esters and higher alcohols. Strains 12α-BAP2 and 12α+ATF1 where generated by deleting and overexpressing BAP2 (encoded branched-chain amino acid permease) and ATF1 (encoded alcohol acetyl transferases), respectively, in the parent 12α strains. Then, 12α-BAP2+CAB1 and 12α-BAP2+CAB3 strains were obtained by overexpressing CAB1 (encoded pantothenate kinase Cab1) and CAB3 (encoded pantothenate kinase Cab3) in the 12α-BAP2 strain, and 12α-BAP2+CAB1+ATF1 and 12α-BAP2+CAB3+ATF1 were generated by overexpressing ATF1 in the pantothenate kinase overexpression strains. The acetate ester level in 12α-BAP2 was slightly changed relative to that in the control strain 12α, whereas the acetate ester levels in 12α-BAP2+CAB1, 12α-BAP2+CAB3, 12α-BAP2+CAB1+ATF1, and 12α-BAP2+CAB3+ATF1 were distinctly increased (44-118% for ethyl acetate and 18-57% for isoamyl acetate). The levels of n-propanol, methyl-1-butanol, isopentanol, isobutanol, and phenethylol levels were changed and varied among the six engineered strains. The levels of acetate esters and higher alcohols can be modulated by changing the CoA and acetyl-CoA levels. The method proposed in this work supplies a practical means of breeding yeast strains by modulating acetate ester and higher alcohol production.

The cAMP response element binding protein (CREB) family activating transcription factor 1 (ATF1) and cAMP response element binding protein 1 (CREB1) have been reported in a diverse group of tumors, however, the mechanistic basis for this remains unclear. Here we found that CREB1 and ATF1 unexpectedly regulate glutathione (GSH) biosynthesis by suppressing the expression of glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthase (GSS), two key enzymes of GSH biosynthesis pathway. Mechanistic studies reveal that GCLM and GSS are direct transcriptional targets of CREB1 and ATF1. Through repressing the expression of these two enzymes, CREB1 and ATF1 reduce the GSH biosynthesis and the capability of cells to detoxicate reactive oxygen species (ROS), thereby increasing cellular susceptibility to oxidative stress. Therefore, our findings link CREB1 family to cellular metabolism, and uncover a potential therapeutic approach by targeting GCLM or oxidative stress for the treatment of tumors with relatively high expression of CREB1 family proteins.

Keywords: ATF1; CREB1; GSH; ROS; proliferation; survival.

Mitogen-activated protein kinase (MAPK) cascades are broadly conserved and play essential roles in multiple cellular processes, including fungal development, pathogenicity, and secondary metabolism. Their function, however, also exhibits species and strain specificity. Penicillium oxalicum secretes plant-biomass-degrading enzymes (PBDEs) that contribute to the carbon cycle in the natural environment and to utilization of lignocellulose in industrial processes. However, knowledge of the MAPK pathway in P. oxalicum has been relatively limited. In this study, comparative transcriptomic analysis of P. oxalicum, cultured on different carbon sources, found ten putative kinase genes with significantly modified transcriptional levels. Six of these putative kinase genes were knocked out in the parental strain ∆PoxKu70, and deletion of the gene, Fus3/Kss1-like PoxMK1 (POX00158), resulted in the largest reduction (91.1%) in filter paper cellulase production. Further tests revealed that the mutant ∆PoxMK1 lost 37.1 to 92.2% of PBDE production, under both submerged- and solid-state fermentation conditions, compared with ∆PoxKu70. In addition, the mutant ∆PoxMK1 had reduced vegetative growth and increased pigment biosynthesis. Comparative transcriptomic analysis showed that PoxMK1 deletion from P. oxalicum downregulated the expression of major PBDE genes and known regulatory genes such as PoxClrB and PoxCxrB, whereas the transcription of pigment biosynthesis-related genes was upregulated. Comparative phosphoproteomic analysis revealed that PoxMK1 deletion considerably modified phosphorylation of key transcription- and signal transduction-associated proteins, including transcription factors Mcm1 and Atf1, RNA polymerase II subunits Rpb1 and Rpb9, MAPK-associated Hog1 and Ste7, and cyclin-dependent kinase Kin28. These findings provide novel insights into understanding signal transduction and regulation of PBDE gene expression in fungi.Key points• PoxMK1 is involved in expression of PBDE- and pigment synthesis-related genes.• PoxMK1 is required for vegetative growth of P. oxalicum.• PoxMK1 is involved in phosphorylation of key TFs, kinases, and RNA polymerase II.

Keywords: MAP kinase PoxMK1; Penicillium oxalicum; Plant-biomass-degrading enzymes; Regulation of PBDE gene expression.

Patients with pathogenic mutations in NGLY1 cannot make tears and have global developmental delay and liver dysfunction. Traditionally, NGLY1 cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins before proteasomal degradation. We demonstrate that Ngly1-null mouse embryonic fibroblasts, NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient mouse embryonic fibroblasts swell slower and have reduced aquaporin1 mRNA and protein expression. Ngly1 knockdown and overexpression confirms that Ngly1 regulates aquaporin1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 knockout cells show reduced aquaporin11 mRNA, supporting NGLY1 as regulating expression of multiple aquaporins across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restores normal hypotonic lysis and aquaporin1 protein. We show that transcription factors Atf1/Creb1 regulate aquaporin1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient mouse embryonic fibroblasts. These results identify a non-enzymatic, regulatory function of NGLY1 in aquaporin transcription, possibly related to alacrima and neurological symptoms.

Our previous work reported activating transcription factor 1 (ATF1) is a promotive factor of nasopharyngeal carcinoma (NPC) tumorigenesis. This study is to further explore the association between the human ATF1 rs11169571 polymorphism and the risk of NPC occurrence. The association between ATF1 rs11169571 and risk of NPC occurrence was investigated in clinical samples of 560 patients and 661 controls obtained from southern China with high incidence of NPC. The genotypes were detected by PCR-RFLP. The differential expression activity of alleles -T and -C was analyzed with CNE-2 and C666-1 cells by luciferase reporter assay. Our data suggested that the allelic frequency and genotypes were significantly different between patients and controls. Compared to the TT homozygote, the TC and CC genotypes have been shown to be significantly decreased in NPC patients (OR = 0.494, 95% CI = 0.387-0.629, P < 0.001 and OR = 0.556, 95% CI = 0.364-0.851, P = 0.007, respectively). Compared to the -T allele, the -C allele is a factor of decreased risk in NPC (OR = 0.642, 95% CI = 0.537-0.767, P < 0.001). Luciferase reporter activity revealed that the -T allele confers a higher expression activity than the -C allele in CNE2 cells and C666-1 cells. In conclusion, ATF1 rs11169571 which could affect the expression of ATF1 is associated with NPC risk.

Background: Chondroclasts and osteoclasts have been previously identified as the cells capable of resorbing mineralized cartilage and bone matrices, respectively. While both cell types appear morphologically similar, contain comparable ultrastructural features, and express tartrate-resistant acid phosphatase (TRAP), however, no information is available about the genomic similarities and differences between osteoclasts and chondroclasts.

Methods: To address this question, we laser captured homogeneous populations of TRAP-positive cells that interact with bone (osteoclasts) and TRAP-positive cells that interact with mineralized cartilage (chondroclasts) on the same plane from murine femoral fracture callus sections. We then performed a global transcriptome profiling of chondroclasts and osteoclasts by utilizing a mouse genome Agilent GE 4X44K V2 microarray platform. Multiple computational approaches and interaction networks were used to analyze the transcriptomic landscape of osteoclasts and chondroclasts.

Results: Our systematic and comprehensive analyses using hierarchical clustering and principal component analysis (PCA) demonstrate that chondroclasts and osteoclasts are transcriptionally distinct cell populations and exhibit discrete transcriptomic signatures as revealed by multivariate analysis involving scatter plot, volcano plot, and heatmap analysis. TaqMan qPCR was used to validate the microarray results. Intriguingly, the functional enrichment and integrated network analyses revealed distinct Gene Ontology terms and molecular pathways specific to chondroclasts and osteoclasts and further suggest that subsets of metabolic genes were specific to chondroclasts. Protein-protein interaction (PPI) network analysis showed an abundance of structured networks of metabolic pathways, ATP synthesis, and proteasome pathways in chondroclasts. The regulatory network analysis using transcription factor-target gene network predicted a pool of genes including ETV6, SIRT1, and ATF1 as chondroclast-specific gene signature.

Conclusions: Our study provides an important genetic resource for further exploration of chondroclast function in vivo. To our knowledge, this is the first demonstration of genetic landscape of osteoclasts from chondroclasts identifying unique molecular signatures, functional clustering, and interaction network.

Keywords: Chondroclasts; Fracture callus; Gene arrays; Osteoclasts; Regulatory network; Transcriptomics.

Hyalinizing clear cell carcinoma (HCCC) is a rare malignancy, characterized by EWSR1-ATF1 gene fusion, whose behavior is poorly understood, as it was for many years considered a diagnosis of exclusion.

All available salivary gland carcinomas (n = 594) from our institution were reviewed. Diagnosis of HCCC was confirmed by fluorescence in situ hybridization (FISH) for EWSR1. Literature review was performed.

We found 15 patients with HCCCs (10 women, 5 men), 13 with EWSR1 rearrangement. Median age at diagnosis was 57 years (range, 31-87 years). Oral cavity (n = 9) and base of tongue (n = 4) were the most frequent primary sites. Combining our cases with those identified in literature review, the 10-year risk of local recurrence and locoregional nodal metastasis were 49% and 15%, respectively.

Molecularly confirmed HCCC accounted for 2.5% of salivary gland malignancies at our institution. HCCCs are indolent tumors with a propensity for locoregional recurrence. © 2016 Wiley Periodicals, Inc. Head Neck 39: 503-511, 2017.

BACKGROUND

Fermentative aromas play a key role in the organoleptic profile of young wines. Their production depends both on yeast strain and fermentation conditions. A present-day trend in the wine industry consists in developing new strains with aromatic properties using adaptive evolution approaches. An evolved strain, Affinity™ ECA5, overproducing esters, was recently obtained. In this study, dynamics of nitrogen consumption and of the fermentative aroma synthesis of the evolved and its ancestral strains were compared and coupled with a transcriptomic analysis approach to better understand the metabolic reshaping of Affinity™ ECA5.

RESULTS

Nitrogen assimilation was different between the two strains, particularly amino acids transported by carriers regulated by nitrogen catabolite repression. We also observed differences in the kinetics of fermentative aroma production, especially in the bioconversion of higher alcohols into acetate esters. Finally, transcriptomic data showed that the enhanced bioconversion into acetate esters by the evolved strain was associated with the repression of genes involved in sterol biosynthesis rather than an enhanced expression of ATF1 and ATF2 (genes coding for the enzymes responsible for the synthesis of acetate esters from higher alcohols).

CONCLUSIONS

An integrated approach to yeast metabolism-combining transcriptomic analyses and online monitoring data-showed differences between the two strains at different levels. Differences in nitrogen source consumption were observed suggesting modifications of NCR in the evolved strain. Moreover, the evolved strain showed a different way of managing the lipid source, which notably affected the production of acetate esters, likely because of a greater availability of acetyl-CoA for the evolved strain.

Glucose limitation is a major stress condition that cells must respond to by altering their metabolism to ensure survival. Rsv1 is a zinc finger protein previously shown to be required for survival during stationary phase. In this study, we present a novel mechanism regulated by Rsv1 in the fission yeast Schizosaccharomyces pombe that is involved in altering glucose metabolic flux. We found that rsv1 gene expression is induced by Rst2 and Atf1, two transcription factors regulated by the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascade, respectively. The downstream target genes of Rsv1 were identified by genome-wide ChIP sequencing of Rsv1-bound DNA sites and RNA sequencing analysis of Rsv1-dependent transcripts that were differentially expressed under glucose starvation. Rsv1 directly regulated the expression of at least 21 genes that mostly encode transporters and proteins related to sugar metabolism. Among these, gcd1, which encodes glucose dehydrogenase in the gluconate shunt for the pentose phosphate pathway, was most remarkably repressed by Rsv1. The defect in survival of Δrsv1 mutant under glucose starvation condition was mitigated by additional deletion of a gcd1, idn1, or a gene for a putative lactonase (SPCC16c4.10), suggesting the critical importance of downregulating the gluconate shunt and pentose phosphate pathway for long-term survival. These results show an intricate response to glucose starvation: increasing the synthesis of a transcription factor via two signal transduction pathways, which sheds light on the importance of remodeling a metabolic circuit to secure glucose for cell survival.

Background: Intracranial myxoid mesenchymal tumors (IMMT) carrying an EWSR1-CREB gene family fusion are extremely rare and have only been identified in ten previous reports. All but one has been found in children or young adults. Although there appear to be similarities a myxoid variant of angiomatoid fibrous histiocytoma (AFH), clear histological differences exist that appear to distinguish IMMTs as a distinct and novel entity. Previous reports have lacked detailed long-term follow-up and recommendations regarding treatment approach.

Case description: This case describes a 48-year-old female who presented with a left intraventricular mass that was identified on histology as an IMMT with a EWSR1-ATF1 gene fusion. Following initial resection, the tumor demonstrated local recurrence. Repeat resection was performed followed by immediate demonstration of local as well as distant tumor recurrence. Histological analysis of the tumor demonstrated a myxoid mesenchymal tumor distinct from AFH. Fractionated stereotactic radiation therapy was administered following the second resection, and tumor control was achieved at 1 year.

Conclusions: Intracranial myxoid mesenchymal tumor is a novel and rare entity that has been previously identified in only ten cases. This case is particularly remarkable as it is only the second IMMT case to occur in a middle-aged adult and shares striking similarities in clinical presentation to the previously reported case. Given the aggressive recurrence seen with the presented case, we recommend the treatment plan to be surgical resection followed by adjuvant radiation therapy.

Keywords: EWSR1 fusion; EWSR1-ATF1; intraventricular tumor; myxoid mesenchymal tumor.

Clear cell carcinoma (CCC) is a low-grade malignancy that commonly arises in minor salivary glands of the oropharynx and other sites. EWSR1-ATF1 gene fusions seem to be specific for this salivary neoplasm. Testing for EWSR1-ATF1 has expanded the histologic spectrum of CCC. As one important example, many CCCs have a predominantly squamous phenotype with few clear cells, a finding that can cause confusion with squamous cell carcinoma (SqCC). P16 immunohistochemical staining to determine human papillomavirus (HPV) status has become standard practice for all oropharyngeal carcinomas showing squamous differentiation. The purpose of this study was to determine whether this practice could contribute to the difficulty in distinguishing CCC from p16-positive SqCC. The authors' surgical pathology archives were searched for cases of CCC. All cases were evaluated with p16 immunohistochemistry, high-risk HPV RNA in situ hybridization (ISH), and EWSR1 gene break-apart fluorescence ISH. Sixteen CCCs were identified. All harbored an EWSR1 rearrangement. Eleven patients were women and 5 were men. They ranged in age from 30 to 85 years (mean, 58 y). The CCCs arose in the oropharynx (tongue base or tonsil) (n=8, 50%), oral cavity (n=4, 25%), and nasopharynx (n=4, 25%). Each case demonstrated clear cells, but the proportion was highly variable (10% to 90%, mean 48%), with 7 of 16 cases having <50% clear cells. Submitted diagnoses included SqCC (n=3) and mucoepidermoid carcinoma (n=2). Of the 3 patients diagnosed with SqCC, 1 was scheduled to undergo chemoradiation, and 1 had already completed chemoradiation. All 16 CCCs demonstrated p16 staining, with the percentage of p16-positive cells ranging from ≥70% (n=2), 50% to 69% (n=3), and 10% to 49% (n=11). Staining was cytoplasmic and nuclear. All cases were negative for high-risk HPV by RNA ISH. CCCs regularly show squamous features, often lack prominent clear cell changes, frequently arise in the oropharynx, and invariably show p16 staining. These features may cause confusion with SqCC, particularly HPV-related oropharyngeal SqCC. P16 staining is not to be taken as unequivocal evidence of an HPV-related SqCC, even for carcinomas showing squamous differentiation and originating in the oropharynx. Failure to recognize this pitfall could result in overly aggressive treatment of a low-grade carcinoma.