PEComa with ASPSCR1::TFE3 fusion: expanding the molecular genetic spectrum of TFE3-rearranged PEComa with an emphasis on overlap with alveolar soft part sarcoma.

AIMS: Mesenchymal neoplasms involving TFE3 gene fusions are diverse, mainly include alveolar soft part sarcoma (ASPS) that is characterised by ASPSCR1::TFE3 fusion, and a small subset of perivascular epithelioid cell tumours (PEComas) referred to as TFE3-rearranged PEComa, that most frequently harbours SFPQ::TFE3 fusion. Historically, ASPS and TFE3-rearranged PEComa are considered two distinctive entities despite their known morphological overlap. However, recent studies have suggested a potential histogenetic relationship between them, and several neoplasms that showed morphological features more closely fit PEComa rather than ASPS but harboured ASPSCR1::TFE3 fusion have been documented. In this study, we report three cases of PEComa with ASPSCR1::TFE3 fusion. METHODS AND RESULTS: Clinicopathological features were assessed and partner agnostic targeted next-generation sequencing on clinically validated platforms were performed. The patients are two females and one male with age at presentation ranging from 21 to 51 years. All three tumours were located in the viscera (rectum, kidney and cervix). On a relatively limited follow-up period (range = 9-15 months), all patients are alive without evidence of recurrent or metastatic disease. The neoplasms were composed of tight nested architecture of epithelioid clear cells separated by a delicate vascular network, two of which were associated with sheets of plump spindle cells, and none showed significant discohesive tumour morphology. Immunohistochemically, in addition to TFE3 protein, all three neoplasms demonstrated co-expression of melan-A and smooth muscle actin. RNA-sequencing identified ASPSCR1::TFE3 fusion in all three cases that were confirmed by subsequent fluorescence in-situ hybridisation analyses. CONCLUSIONS: Our study expands the molecular genetic spectrum of TFE3-rearranged PEComa and further indicates its close relationship to ASPS.

Polyphenol oxidase inhibited by 4-hydroxycinnamic acid and naringenin: Multi-spectroscopic analyses and molecular docking simulation at different pH.

It is still unclear how pH affects the inhibitory effects of phenolic acids and flavonoids on polyphenol oxidase (PPO). In this study, 4-hydroxycinnamic acid and naringenin were selected to investigate their interactions with PPO from pH 6.8 to 5.0. Results showed that acidic pH could enhance the inhibitory effect of inhibitors and a greater enhancement effect was observed in 4-hydroxycinnamic acid. Fluorescence emission spectra indicated that 4-hydroxycinnamic acid and naringenin interacted with PPO and quenched its intrinsic fluorescence, which was also enhanced by acidic pH. Circular dichroism suggested that 4-hydroxycinnamic acid and naringenin could reversibly bind to PPO molecules and transform α-helix into ß-sheet. Molecular docking results revealed that 4-hydroxycinnamic acid and naringenin interacted with PPO through hydrogen bond and hydrophobic interaction, and more interactions were observed near the carboxyl group. These results indicated that acidic pH could significantly enhance the inhibitory effect of phenolic acid on PPO.

Comparing the effect of benzoic acid and cinnamic acid hydroxyl derivatives on polyphenol oxidase: activity, action mechanism, and molecular docking.

BACKGROUND: Polyphenol oxidase (PPO) is considered to have a key role in the food industry because it initiates enzymatic browning in the processing and storage of fruit and vegetables. Increasing numbers of benzoic and cinnamic acid derivatives have been found to be efficient inhibitors of polyphenol oxidase, but a comparison study on activity and action mechanism is lacking. In this study, 18 benzoic acid and cinnamic acid hydroxy derivatives were selected and investigated. RESULTS: Three substrates, four activators and 11 inhibitors were identified from benzoic and cinnamic acid derivatives. 2,4-Dihydroxycinnamic acid and benzoic acid showed the strongest inhibitory effect on PPO, with IC50 of 0.092 and1.425 mmol L-1 , respectively. Benzoic acid reversibly inhibited PPO in a competitive manner, while 2,4-dihydroxycinnamic acid showed a mixed-type inhibition. Both of them showed that static-type fluorescence quenching and electrostatic interaction were the main driving force in the bonding process. Compared with benzoic acid, 2,4-dihydroxycinnamic acid more easily formed hydrogen bonds in the active site of PPO, making the interaction more stable. CONCLUSION: Comparative analysis showed that the inhibition effect of cinnamic acid hydroxyl derivatives on PPO was stronger than that of benzoic acid derivatives. Benzoic acid and 2,4-dihydroxycinnamic acid were the strongest inhibitors. PPO inhibitors identified from benzoic and cinnamic acid derivatives are expected to be promising inhibitors for controlling fruit and vegetable browning. © 2021 Society of Chemical Industry.

Effect of Galangal Essential Oil Emulsion on Quality Attributes of Cloudy Pineapple Juice.

Galangal essential oil is obtained from the rhizomes of galangal with proven anti-inflammatory, antioxidant, antiviral, and antimicrobial properties, which are valuable in the food industry. To explore the effect of galangal essential oil on the quality of pineapple juice, 0.05, 0.1, 0.2, and 0.4% galangal essential emulsion were added, and their influence on the physical stability, physicochemical properties, microbial quantity, and aroma profiles of cloudy pineapple juice were evaluated. The essential oil emulsion of galangal is a milky white liquid with a strong aroma of galangal. The pH values of emulsion increased from 4.35 to 5.05 with the increase in essential oil concentration, and there was no significant difference in the particle size of the pineapple juice. The results showed that the galangal essential oil emulsion was stable and the stability of the cloudy pineapple juice was significantly enhanced by the essential oil emulsion determined using LUMiSizer. The cloudy pineapple juice with a 0.2% essential oil emulsion showed the most stability during storage. The lightness of the cloudy pineapple juice increased instantly with the essential oil emulsion addition. In addition, the microbial quantity of the cloudy pineapple juice was decreased by the individual essential oil emulsion or combined with thermal treatment to hold a longer shelf life. The microbial counts in pineapple juice treated by 0.4% essential oil emulsion and thermal treatment only increased from 1.06 to 1.59 log CFU/ml after 4 days of storage at 25°C. Additionally, the pH and total soluble solids showed a slightly increasing trend; however, the value of titratable acidity, free radical scavenging capacity, and ascorbic acid content of the cloudy pineapple juice showed no significant change. Finally, the results of the electronic nose showed that the aroma components of the pineapple juice were changed by the essential oil emulsion and thermal treatment, and the difference was especially evident in the content of the sulfur, sulfur organic, and aromatics compounds. Consequently, the results indicated that galangal essential oil emulsion can be used as juice additives to improve the quality attributes and extend the shelf-life of cloudy pineapple juice.

Anti-browning effect of Rosa roxburghii on apple juice and identification of polyphenol oxidase inhibitors.

Enzymatic browning control of cloudy fruit juice with natural substances has received much attention for improving its nutritional and commercial value. This study explored the anti-browning potential of Rosa roxburghii in apple juice. The anti-browning effects and mechanisms were evaluated by serial measurements of appearance, browning index, polyphenol oxidase (PPO) activity, UPLC-QE-Orbitrap-MS identification, inhibition kinetics and molecular docking. The results showed that Rosa roxburghii juice (0.25%-1.25% w/w) could effectively inhibit browning and PPO activity of apple juice. Ascorbic acid (1.67 g/100 g) as a reducing agent was a main anti-browning factor. Furthermore, seven phenolic compounds in Rosa roxburghii were screened as PPO inhibitors. Representative phenolic inhibitors induced mixed or competitive inhibition of PPO, mainly driven by hydrophobic forces and hydrogen bonds. This work demonstrates that Rosa roxburghii is a promising natural anti-browning ingredient to control the browning of cloudy apple juice due to abundant ascorbic acid and PPO inhibitors.

Necroptotic-susceptible dendritic cells exhibit enhanced antitumor activities in mice.

INTRODUCTION: Priming of tumor-specific T cells is a key to antitumor immune response and inflammation, in turn, is crucial for proper T-cell activation. As antigen-presenting cells can activate T cells, dendritic cells (DCs) loaded with tumor antigens have been used as immunotherapeutics against certain cancer in humans but their efficacy is modest. Necroptosis is a form of programmed cell death that results in the release of inflammatory contents. We previously generated mice with DC deficiency in a negative regulator of necroptosis, Fas-associated death domain (FADD), and found that these mice suffer from systemic inflammation due to necroptotic DCs. We hypothesize that FADD-deficient DCs could serve as a better vaccine than wild-type (WT) DCs against tumors. MATERIALS AND METHODS: FADD-deficient and WT mouse DCs loaded with the relevant tumor peptide were injected onto mice before or after the syngeneic tumor challenge. DC vaccinations were repeated two more times and anti-PD-1 antibodies were coinjected in some experiments. Tumor sizes were measured by caliper, and the percentages of tumor-free mice or mice survived were examined over time. The cytometric analysis was carried out to analyze various immune populations. RESULTS: In two separate tumor models, we find that mice receiving FADD-deficient DCs as vaccine rejected tumors significantly better than those receiving a WT DC vaccine. Tumor growth was severely hampered, and survival extended in these mice. More activated CD8 T cells together with elevated cytokines were observed in mice receiving the FADD-deficient DC vaccine. Furthermore, we observed these effects were potent enough to protect against tumor challenge postinjection and can work in conjunction with anti-PD-1 antibodies to reduce the tumor growth. CONCLUSIONS: Necroptotic-susceptible DCs are better antitumor vaccines than WT DCs in mice. Our findings suggest that necroptosis-driven inflammation by DCs may be a novel avenue to generating a strong adaptive antitumor response in the clinical setting.

The clinicopathological spectrum of pseudomyogenic hemangioendothelioma: report of an additional series with review of the literature.

We present here our experience with 24 cases of pseudomyogenic hemangioendothelioma (PMHE) to further delineate its clinicopathological spectrum. There were 18 males and 6 females with a median age of 28 years (range 10~64 years). Most patients presented with erythematous nodules or papules, with or without pain. The majority (63%) occurred in the lower extremities, whereas a minority involved the trunk (25%), upper extremities (8%), and head and neck (4%). Six cases (25%) had a primary bone origin. With physical and radiological examinations, 16 cases (67%) manifested as multifocal disease, involving multiple tissue planes or different bones within the anatomic region. Six cases (25%) involved skin, soft tissue, and bone simultaneously. Histologically, all cases showed features consistent with a PMHE characterized by loose fascicles or sheets of plump spindled to epithelioid cells harboring brightly eosinophilic cytoplasm and vesicular nuclei. In addition, five cases (21%) contained a prominent myxoid matrix, and one case displayed perineural and intravascular invasion. The follow-up information available in 18 patients revealed local recurrence in 4 patients (22%) and persistent disease in 8 patients (44%), respectively. One patient developed bilateral pulmonary metastases which showed significant remission after systemic chemotherapy. None of the patients died of the disease. As the clinical appearance of PMHE can be deceptive, a radiological examination is essential in identifying an insidious multifocal disease. Although PMHE has a predilection for the distal extremities of young males, this rare tumor type could also occur in unusual sites and affect middle-aged adults of both genders. The striking myoid appearance in association with myxoid stromal change may represent a potential diagnostic pitfall. Biologically, PMHE has an indolent clinical behavior, albeit metastatic disease may occur in rare instance.

Primary pseudomyogenic hemangioendothelioma of the vulva: a rare location for a rare entity.

BACKGROUND: Pseudomyogenic hemangioendothelioma (PMHE) is a recently described vascular neoplasm which typically occurs in the lower extremities of young to middle-aged adults. CASE PRESENTATION: We present here a unique case of PMHE arising primarily in the vulva of a 51-year-old woman who presented with a painful vulvar nodule. Clinically, it was thought as Bartholin gland cyst, vulvar hematoma or papilloma. On surgery, two nodules were found with one located in the superficial dermis and the other in the deep subcutis. Histologically, these two nodules showed similar features, composed of fascicles or sheets of plump spindled to epithelioid cells with eosinophilic cytoplasm. Given the morphological resemblance to a myogenic tumor, the lesion was initially diagnosed as a rhabdomyosarcoma by the referring pathologist. However, a comprehensive reevaluation of the submitted slides made us reconsider a PMHE, which was subsequently confirmed by immunohistochemical study. CONCLUSION: This case demonstrates that PMHE can also develop in the female external genitalia albeit extremely rare. This disease should be included in the differential diagnostic list of vulvar tumors with spindled to epithelioid morphology and cytokeratin-positive immunophenotypes.

Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway.

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3-/- mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

Deletion of FADD in macrophages and granulocytes results in RIP3- and MyD88-dependent systemic inflammation.

Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.

Premalignant PTEN-deficient thymocytes activate microRNAs miR-146a and miR-146b as a cellular defense against malignant transformation.

Cancer develops by a multistep process during which cells acquire characteristics that allow them to evade apoptosis and proliferate unchecked. Sequential acquisition of genetic alterations drives this process but also causes cellular stress, frequently prompting cells to enter a premalignant period during which they mount a defense against transformation. T cell-specific deletion of the tumor suppressor PTEN in mice induces premalignancy in the thymus and development of CD4(+) T-cell lymphomas in the periphery. Here we sought to identify factors mediating the cellular defense against transformation during the premalignant period. We identified several microRNAs upregulated specifically in premalignant thymocytes, including miR-146a, miR-146b, and the miR-183/96/182 cluster. CD4-driven T cell-specific transgenic overexpression of mir-146a and mir-146b significantly delayed PTEN-deficient lymphomagenesis and delayed c-myc oncogene induction, a key driver of transformation in PTEN-deficient T-cell malignancies. We found that miR-146a and miR-146b targeting of Traf6 attenuates TCR signaling in the thymus and inhibits downstream NF-κB-dependent induction of c-myc. Additionally, c-myc repression in mature CD4 T cells by miR-146b impaired TCR-mediated proliferation. Hence, we have identified 2 miRNAs that are upregulated as part of the cellular response against transformation that, when overrepresented, can effectively inhibit progression to malignancy in the context of PTEN deficiency.

H2 control of natural T regulatory cell frequency in the lymph node correlates with susceptibility to day 3 thymectomy-induced autoimmune disease.

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by natural regulatory T cells (nTregs) and development of autoimmune ovarian dysgenesis (AOD) and autoimmune dacryoadenitis (ADA) in A/J and (C57BL/6J × A/J) F(1) hybrids (B6A), but not in C57BL/6J (B6) mice. Previously, using quantitative trait locus (QTL) linkage analysis, we showed that D3Tx-AOD is controlled by five unlinked QTL (Aod1-Aod5) and H2. In this study, using D3Tx B6-Chr(A/J)/NaJ chromosome (Chr) substitution strains, we confirm that QTL on Chr16 (Aod1a/Aod1b), Chr3 (Aod2), Chr1 (Aod3), Chr2 (Aod4), Chr7 (Aod5), and Chr17 (H2) control D3Tx-AOD susceptibility. In addition, we also present data mapping QTL controlling D3Tx-ADA to Chr17 (Ada1/H2), Chr1 (Ada2), and Chr3 (Ada3). Importantly, B6-ChrX(A/J) mice were as resistant to D3Tx-AOD and D3Tx-ADA as B6 mice, thereby excluding Foxp3 as a susceptibility gene in these models. Moreover, we report quantitative differences in the frequency of nTregs in the lymph nodes (LNs), but not spleen or thymus, of AOD/ADA-resistant B6 and AOD/ADA-susceptible A/J, B6A, and B6-Chr17(A/J) mice. Similar results correlating with experimental allergic encephalomyelitis and orchitis susceptibility were seen with B10.S and SJL/J mice. Using H2-congenic mice, we show that the observed difference in frequency of LN nTregs is controlled by Ada1/H2. These data support the existence of an LN-specific, H2-controlled mechanism regulating the prevalence of nTregs in autoimmune disease susceptibility.

Coupling of the cell cycle and apoptotic machineries in developing T cells.

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4(+)CD8(+) thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4(+)CD8(+) thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4(+)CD8(+) thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4(+)CD8(+) thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.