Identification of Novel/Rare EWSR1 Fusion Partners in Undifferentiated Mesenchymal Neoplasms.

Recurrent gene fusions (GFs) in translocated sarcomas are recognized as major oncogenic drivers of the disease, as well as diagnostic markers whose identification is necessary for differential diagnosis. EWSR1 is a 'promiscuous' gene that can fuse with many different partner genes, defining different entities among a broad range of mesenchymal neoplasms. Molecular testing of EWSR1 translocation traditionally relies on FISH assays with break-apart probes, which are unable to identify the fusion partner. Therefore, other ancillary molecular diagnostic modalities are being increasingly adopted for accurate classification of these neoplasms. Herein, we report three cases with rare GFs involving EWSR1 in undifferentiated mesenchymal neoplasms with uncertain differential diagnoses, using targeted RNA-seq and confirming with RT-PCR and Sanger sequencing. Two GFs involved hormone nuclear receptors as 3' partners, NR4A2 and RORB, which have not been previously reported. NR4A2 may functionally replace NR4A3, the usual 3' partner in extraskeletal myxoid chondrosarcoma. The third GF, EWSR1::BEND2, has previously been reported in a subtype of astroblastoma and other rare entities, including a single case of a soft-tissue tumor that we discuss in this work. In conclusion, our findings indicate that the catalogue of mesenchymal neoplasm-bearing EWSR1 fusions continues to grow, underscoring the value of using molecular ancillary techniques with higher diagnostic abilities in the routine clinical setting.

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L-1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

Endoglin and MMP14 Contribute to Ewing Sarcoma Spreading by Modulation of Cell-Matrix Interactions.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.

A Novel NFIX-STAT6 Gene Fusion in Solitary Fibrous Tumor: A Case Report.

Solitary fibrous tumor is a rare subtype of soft-tissue sarcoma with a wide spectrum of histopathological features and clinical behaviors, ranging from mildly to highly aggressive tumors. The defining genetic driver alteration is the gene fusion NAB2-STAT6, resulting from a paracentric inversion within chromosome 12q, and involving several different exons in each gene. STAT6 (signal transducer and activator of transcription 6) nuclear immunostaining and/or the identification of NAB2-STAT6 gene fusion is required for the diagnostic confirmation of solitary fibrous tumor. In the present study, a new gene fusion consisting of Nuclear Factor I X (NFIX), mapping to 19p13.2 and STAT6, mapping to 12q13.3 was identified by targeted RNA-Seq in a 74-year-old female patient diagnosed with a deep-seated solitary fibrous tumor in the pelvis. Histopathologically, the neoplasm did not display nuclear pleomorphism or tumor necrosis and had a low proliferative index. A total of 378 unique reads spanning the NFIXexon8-STAT6exon2 breakpoint with 55 different start sites were detected in the bioinformatic analysis, which represented 59.5% of the reads intersecting the genomic location on either side of the breakpoint. Targeted RNA-Seq results were validated by RT-PCR/ Sanger sequencing. The identification of a new gene fusion partner for STAT6 in solitary fibrous tumor opens intriguing new hypotheses to refine the role of STAT6 in the sarcomatogenesis of this entity.

Biocatalyzed Redox Processes Employing Green Reaction Media.

The application of biocatalysts to perform reductive/oxidative chemical processes has attracted great interest in recent years, due to their environmentally friendly conditions combined with high selectivities. In some circumstances, the aqueous buffer medium normally employed in biocatalytic procedures is not the best option to develop these processes, due to solubility and/or inhibition issues, requiring biocatalyzed redox procedures to circumvent these drawbacks, by developing novel green non-conventional media, including the use of biobased solvents, reactions conducted in neat conditions and the application of neoteric solvents such as deep eutectic solvents.

Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 µM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-ß/ß-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc.

From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of (18) O from the cosubstrate H2 (18) O2 , demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids.

Nanomedical research and development in Spain: improving the treatment of diseases from the nanoscale.

The new and unique possibilities that nanomaterials offer have greatly impacted biomedicine, from the treatment and diagnosis of diseases, to the specific and optimized delivery of therapeutic agents. Technological advances in the synthesis, characterization, standardization, and therapeutic performance of nanoparticles have enabled the approval of several nanomedicines and novel applications. Discoveries continue to rise exponentially in all disease areas, from cancer to neurodegenerative diseases. In Spain, there is a substantial net of researchers involved in the development of nanodiagnostics and nanomedicines. In this review, we summarize the state of the art of nanotechnology, focusing on nanoparticles, for the treatment of diseases in Spain (2017-2022), and give a perspective on the future trends and direction that nanomedicine research is taking.

A global collaboRAtive study of CIC-rearranged, BCOR::CCNB3-rearranged and other ultra-rare unclassified undifferentiated small round cell sarcomas (GRACefUl).

BACKGROUND: Undifferentiated small round cell sarcomas (URCSs) represent a diagnostic challenge, and their optimal treatment is unknown. We aimed to define the clinical characteristics, treatment, and outcome of URCS patients. METHODS: URCS patients treated from 1983 to 2019 at 21 worldwide sarcoma reference centres were retrospectively identified. Based on molecular assessment, cases were classified as follows: (1) CIC-rearranged round cell sarcomas, (2) BCOR::CCNB3-rearranged round cell sarcomas, (3) unclassified URCSs. Treatment, prognostic factors and outcome were reviewed. RESULTS: In total, 148 patients were identified [88/148 (60%) CIC-rearranged sarcoma (median age 32 years, range 7-78), 33/148 (22%) BCOR::CCNB3-rearranged (median age 17 years, range 5-91), and 27/148 (18%) unclassified URCSs (median age 37 years, range 4-70)]. One hundred-one (68.2%) cases presented with localised disease; 47 (31.8%) had metastases at diagnosis. Male prevalence, younger age, bone primary site, and a low rate of synchronous metastases were observed in BCOR::CCNB3-rearranged cases. Local treatment was surgery in 67/148 (45%) patients, and surgery + radiotherapy in 52/148 (35%). Chemotherapy was given to 122/148 (82%) patients. At a 42.7-month median follow-up, the 3-year overall survival (OS) was 92.2% (95% CI 71.5-98.0) in BCOR::CCNB3 patients, 39.6% (95% CI 27.7-51.3) in CIC-rearranged sarcomas, and 78.7% in unclassified URCSs (95% CI 56.1-90.6; p < 0.0001). CONCLUSIONS: This study is the largest conducted in URCS and confirms major differences in outcomes between URCS subtypes. A full molecular assessment should be undertaken when a diagnosis of URCS is suspected. Prospective studies are needed to better define the optimal treatment strategy in each URCS subtype.

Engineering Collariella virescens Peroxygenase for Epoxides Production from Vegetable Oil.

Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO (by substituting one, two, or up to six residues of its access channel by alanines) improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α−linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate.

Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases.

Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, flavoring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosubstrate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosubstrate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, notable differences were observed between them, with rCciUPO being responsible for the highest substrate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies.

The Role of Nitric Oxide in Stem Cell Biology.

Nitric oxide (NO) is a gaseous biomolecule endogenously synthesized with an essential role in embryonic development and several physiological functions, such as regulating mitochondrial respiration and modulation of the immune response. The dual role of NO in embryonic stem cells (ESCs) has been previously reported, preserving pluripotency and cell survival or inducing differentiation with a dose-dependent pattern. In this line, high doses of NO have been used in vitro cultures to induce focused differentiation toward different cell lineages being a key molecule in the regenerative medicine field. Moreover, optimal conditions to promote pluripotency in vitro are essential for their use in advanced therapies. In this sense, the molecular mechanisms underlying stemness regulation by NO have been studied intensively over the current years. Recently, we have reported the role of low NO as a hypoxia-like inducer in pluripotent stem cells (PSCs), which supports using this molecule to maintain pluripotency under normoxic conditions. In this review, we stress the role of NO levels on stem cells (SCs) fate as a new approach for potential cell therapy strategies. Furthermore, we highlight the recent uses of NO in regenerative medicine due to their properties regulating SCs biology.

Genetic Alterations and Deregulation of Hippo Pathway as a Pathogenetic Mechanism in Bone and Soft Tissue Sarcoma.

The Hippo pathway is an evolutionarily conserved modulator of developmental biology with a key role in tissue and organ size regulation under homeostatic conditions. Like other signaling pathways with a significant role in embryonic development, the deregulation of Hippo signaling contributes to oncogenesis. Central to the Hippo pathway is a conserved cascade of adaptor proteins and inhibitory kinases that converge and regulate the activity of the oncoproteins YAP and TAZ, the final transducers of the pathway. Elevated levels and aberrant activation of YAP and TAZ have been described in many cancers. Though most of the studies describe their pervasive activation in epithelial neoplasms, there is increasing evidence pointing out its relevance in mesenchymal malignancies as well. Interestingly, somatic or germline mutations in genes of the Hippo pathway are scarce compared to other signaling pathways that are frequently disrupted in cancer. However, in the case of sarcomas, several examples of genetic alteration of Hippo members, including gene fusions, have been described during the last few years. Here, we review the current knowledge of Hippo pathway implication in sarcoma, describing mechanistic hints recently reported in specific histological entities and how these alterations represent an opportunity for targeted therapy in this heterogeneous group of neoplasm.

Integrating digital pathology with transcriptomic and epigenomic tools for predicting metastatic uterine tumor aggressiveness.

The incidence of new cancer cases is expected to increase significantly in the future, posing a worldwide problem. In this regard, precision oncology and its diagnostic tools are essential for developing personalized cancer treatments. Digital pathology (DP) is a particularly key strategy to study the interactions of tumor cells and the tumor microenvironment (TME), which play a crucial role in tumor initiation, progression and metastasis. The purpose of this study was to integrate data on the digital patterns of reticulin fiber scaffolding and the immune cell infiltrate, transcriptomic and epigenetic profiles in aggressive uterine adenocarcinoma (uADC), uterine leiomyosarcoma (uLMS) and their respective lung metastases, with the aim of obtaining key TME biomarkers that can help improve metastatic prediction and shed light on potential therapeutic targets. Automatized algorithms were used to analyze reticulin fiber architecture and immune infiltration in colocalized regions of interest (ROIs) of 133 invasive tumor front (ITF), 89 tumor niches and 70 target tissues in a total of six paired samples of uADC and nine of uLMS. Microdissected tissue from the ITF was employed for transcriptomic and epigenetic studies in primary and metastatic tumors. Reticulin fiber scaffolding was characterized by a large and loose reticular fiber network in uADC, while dense bundles were found in uLMS. Notably, more similarities between reticulin fibers were observed in paired uLMS then paired uADCs. Transcriptomic and multiplex immunofluorescence-based immune profiling showed a higher abundance of T and B cells in primary tumor and in metastatic uADC than uLMS. Moreover, the epigenetic signature of paired samples in uADCs showed more differences than paired samples in uLMS. Some epigenetic variation was also found between the ITF of metastatic uADC and uLMS. Altogether, our data suggest a correlation between morphological and molecular changes at the ITF and the degree of aggressiveness. The use of DP tools for characterizing reticulin scaffolding and immune cell infiltration at the ITF in paired samples together with information provided by omics analyses in a large cohort will hopefully help validate novel biomarkers of tumor aggressiveness, develop new drugs and improve patient quality of life in a much more efficient way.

Molecular Approaches to Diagnosis in Ewing Sarcoma: Targeted RNA Sequencing.

Molecular testing of pathognomonic gene fusions is mandatory for small round cell tumor diagnosis, including Ewing sarcoma which is indeed defined by a variety of chimeric genes. Reference laboratories are increasingly implementing NGS-based techniques to overcome several limitations of conventional singleplex determinations. We have been early adopters of a targeted-RNA sequencing method based on Anchored multiplex PCR, which allows assessing several fusion transcripts simultaneously with previous knowledge of only one partner gene. Here we describe in detail our protocol and tips for nucleic acid extraction, library preparation, sequencing, and reporting of gene fusions.

Advances in enzymatic oxyfunctionalization of aliphatic compounds.

Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as "dream biocatalysts" of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases.

Evaluation of NAB2-STAT6 Fusion Variants and Other Molecular Alterations as Prognostic Biomarkers in a Case Series of 83 Solitary Fibrous Tumors.

Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location.

Detection of SARS-CoV-2 and its S and N proteins using surface enhanced Raman spectroscopy.

The COVID-19 pandemic demonstrated the critical need for accurate and rapid testing for virus detection. This need has generated a high number of new testing methods aimed at replacing RT-PCR, which is the golden standard for testing. Most of the testing techniques are based on biochemistry methods and require chemicals that are often expensive and the supply might become scarce in a large crisis. In the present paper we suggest the use of methods based on physics that leverage novel nanomaterials. We demonstrate that using Surface Enhanced Raman Spectroscopy (SERS) of virion particles a very distinct spectroscopic signature of the SARS-CoV-2 virus can be obtained. We demonstrate that the spectra are mainly composed by signals from the spike (S) and nucleocapsid (N) proteins. It is believed that a clinical test using SERS can be developed. The test will be fast, inexpensive, and reliable. It is also clear that SERS can be used for analysis of structural changes on the S and N proteins. This will be an example of application of nanotechnology and properties of nanoparticles for health and social related matters.

Characterizing the Invasive Tumor Front of Aggressive Uterine Adenocarcinoma and Leiomyosarcoma.

The invasive tumor front (the tumor-host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors.