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The distinction between malignant mesothelioma and reactive mesothelial proliferation can be challenging both on histology and cytology. Recently, variants of the BRCA1 - associated protein 1 ( BAP1 ) gene resulting in nuclear protein loss were reported in hereditary and sporadic mesothelioma. Using immunohistochemistry, we evaluated the utility of BAP1 expression in the differential diagnosis between mesothelioma and other mesothelial proliferations on a large series of biopsies that included 212 mesotheliomas, 12 benign mesothelial tumors, and 42 reactive mesothelial proliferations. BAP1 stain was also performed in 70 cytological samples (45 mesotheliomas and 25 reactive mesothelial proliferations). BAP1 was expressed in all benign mesothelial tumors, whereas 139/212 (66%) mesotheliomas were BAP1 negative, especially in epithelioid/biphasic compared with sarcomatoid/desmoplastic subtypes (69% vs 15%). BAP1 loss was homogeneous in neoplastic cells except for two epithelioid mesotheliomas showing tumor heterogeneity. By fluorescence in situ hybridization, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%), whereas 9/10 BAP1-positive mesotheliomas were normal. In biopsies interpreted as reactive mesothelial proliferation BAP1 loss was 100% predictive of malignancy, as all 6 cases subsequently developed BAP1-negative mesothelioma, whereas only 3/36 (8%) BAP1-positive cases progressed to mesothelioma. On cytology/cell blocks, benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; all 6 cases showing BAP1 negativity were associated with histological diagnosis of BAP1-negative mesothelioma. BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively. Our results show that BAP1 protein is frequently lost in mesothelioma, especially of epithelioid/biphasic subtype and is commonly associated with homozygous BAP1 deletion. BAP1 immunostain represents an excellent biomarker with an unprecedented specificity (100%) in the distinction between benign and malignant mesothelial proliferations. Finding BAP1 loss in mesothelial cells should prompt to immediately reevaluate the patient; moreover, it might be useful in mapping tumor extent and planning surgical resection.

Hydrogen sulfide (H 2 S) serves as a gasotransmitter in the regulation of organ development and maintenance of homeostasis in tissues. Its abnormal levels are associated with multiple human diseases, such as neurodegenerative disease, myocardial injury, and ophthalmic diseases. Excessive exposure to H 2 S could lead to cellular toxicity, orchestrate pathological process, and increase the risk of various diseases. Interestingly, under physiological status, H 2 S plays a critical role in maintaining cellular physiology and limiting damages to tissues. In mammalian species, the generation of H 2 S is catalyzed by cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), 3-mercapto-methylthio pyruvate aminotransferase (3MST) and cysteine aminotransferase (CAT). These enzymes are found inside the mammalian eyeballs at different locations. Their aberrant expression and the accumulation of substrates and intermediates can change the level of H 2 S by orders of magnitude, causing abnormal structures or functions in the eyes. Detailed investigations have demonstrated that H 2 S donors’ administration could regulate intraocular pressure, protect retinal cells, inhibit oxidative stress and alleviate inflammation by modulating the function of intra or extracellular proteins in ocular tissues. Thus, several slow-releasing H 2 S donors have been shown to be promising drugs for treating multiple diseases. In this review, we discuss the biological function of H 2 S metabolism and its application in ophthalmic diseases.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1 , but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu -like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu /B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness. Blindness linked to DICER1 activity Kaneko et al . report that geographic atrophy, a form of age-related blindness caused by cell death in the retinal pigmented epithelium, is associated with loss of activity of DICER1, a microRNA-processing enzyme. But cell death is not linked to microRNA processing. Rather, the absence of DICER1 allows pathological Alu repeat sequence RNAs to accumulate. DICER1 binds to and degrades Alu RNA to prevent retinal degeneration. This work suggests a novel therapeutic target for an important cause of blindness. In geographic atrophy, a type of macular degeneration, retinal pigmented epithelium (RPE) cells die. This paper finds that DICER1, which processes miRNA precursors, is reduced in RPE from individuals with geographic atrophy. Cell death is not due to loss of miRNA processing, however; rather, the absence of DICER1 allows accumulation of pathological Alu repeat sequence RNAs. This work reveals a novel function of Dicer in degrading Alu RNAs.

Feeding highly fermentable diets to ruminants is one strategy to increase energy intake. The increase in short-chain fatty acid (SCFA) production and reduced ruminal pH associated with highly fermentable diets imposes a challenge to the metabolism and the regulation of intracellular pH homeostasis of ruminal epithelia. The ruminal epithelia respond to these challenges in a coordinated manner. Whereas the enlargement of absorptive surface area is well documented, emerging evidence at the mRNA and transporter and enzyme activity levels indicate that changes in epithelial cell function may be the initial response. It is not surprising that gene expression analysis has identified pathways involved in fatty acid metabolism, ion transport, and intracellular homeostasis to be the pathways dominantly affected during adaptation and after adaptation to a highly fermentable diet. These findings are important because the intraepithelial metabolism of SCFA, particularly butyrate, helps to maintain the concentration gradient between the cytosol and lumen, thereby facilitating absorption. Butyrate metabolism also controls the intracellular availability of butyrate, which is widely regarded as a signaling molecule. Current data indicate that for butyrate metabolism, 3-hydroxy-3-methylglutaryl-CoA synthase and acetyl-CoA acetyltransferase are potential regulatory points with transient up- and downregulation during diet adaptation. In addition to nutrient transport and utilization, genes involved in the maintenance of cellular tight junction integrity and induction of inflammation have been identified as differentially expressed genes during adaptation to highly fermentable diets. This may have important implications on ruminal epithelial barrier function and the inflammatory response often associated with subacute ruminal acidosis. The objective of this review is to summarize ruminal epithelial adaptation to highly fermentable diets focusing on the changes at the enzyme and transporter activity levels, as well as the underlying molecular changes at the mRNA and protein expression levels.

The role of the endoplasmic reticulum stress-regulated kinase, PERK, in mammary gland function was assessed through generation of a targeted deletion in mammary epithelium. Characterization revealed that PERK is required for functional maturation of milk-secreting mammary epithelial cells. PERK-dependent signaling contributes to lipogenic differentiation in mammary epithelium, and perk deletion inhibits the sustained expression of lipogenic enzymes FAS, ACL, and SCD1. As a result, mammary tissue has reduced lipid content and the milk produced has altered lipid composition, resulting in attenuated pup growth. Consistent with PERK-dependent regulation of the lipogenic pathway, loss of PERK inhibits expression of FAS, ACL, and SCD1 in immortalized murine embryonic fibroblasts when cultured under conditions favoring adipocyte differentiation. These findings implicate PERK as a physiologically relevant regulator of the lipogenic pathway.

Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

This study investigated the protective effect and the molecular mechanism of piceatannol on hydrogen peroxide (H2O2)-induced retinal pigment epithelium cell (ARPE-19) damage. Piceatannol treatment significantly inhibited H2O2-induced RPE cell death and reactive oxygen species (ROS) generation by 64.4% and 75.0%, respectively. Results of flow cytometry showed that H2O2-induced ARPE-19 cells apoptosis was ameliorated by piceatannol supplementation, along with decreased relative protein expressions of Bax/Bcl-2, Cleave-Caspase-3, and Cleave-PARP. Moreover, piceatannol treatment induced NF-E2-related factor 2 (Nrf2) signaling activation, which was evidenced by increased transcription of anti-oxidant genes, glutamate-cysteine ligase catalytic subunit (GCLc), SOD, and HO-1. Knockdown of Nrf2 through targeted siRNA alleviated piceatannol-mediated HO-1 transcription, and significantly abolished piceatannol-mediated cytoprotection. LY294002 (PI3K inhibitor) dramatically blocked piceatannol-mediated increasing of Nrf2 nuclear translocation, HO-1 expression, and cytoprotective activity, indicating the involvement of PI3K/Akt pathway in the cytoprotective effect of piceatannol. The results from this suggest the potential of piceatannol in reducing the risk of age-related macular degeneration.

Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type-specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies.

Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).

The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary‐specific deletion of Pten via WAP‐Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV‐Cre, which targets basal/luminal progenitors. Combined Pten‐p53 mutations accelerated formation of claudin‐low, triple‐negative‐like breast cancer (TNBC) that exhibited hyper‐activated AKT signaling and more mesenchymal features relative to Pten or p53 single‐mutant tumors. Twenty‐four genes that were significantly and differentially expressed between WAP‐Cre:Pten/p53 and MMTV‐Cre:Pten/p53 tumors predicted poor survival for claudin‐low patients. Kinome screens identified eukaryotic elongation factor‐2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53‐deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53‐deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro . Our results identify a prognostic signature for claudin‐low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling. Synopsis The tumor suppressors Pten and p53 are frequently lost in triple‐negative breast cancer (TNBC). In double mouse KO, tumors identity changed to a sarcomatoid/mesenchymal subtype; molecular and bioinformatics analyses revealed eEF2K as a potential therapeutic target. Disruption of Pten and p53 via MMTV‐Cre or WAP‐Cre accelerated formation of claudin‐low‐like TNBC. A 24‐gene set that discriminates Pten/p53‐deficient tumors driven by MMTV‐Cre versus WAP‐Cre transgenes could predict clinical outcome for claudin‐low TNBC patients. Kinome screen identified eEF2K inhibitors as most potent growth suppressors for both mouse and human Pten/p53‐deficient TNBC. eEF2K inhibitors might represent a novel therapy for Pten/p53‐deficient TNBC with high AKT signaling. Graphical Abstract The tumor suppressors Pten and p53 are frequently lost in triple‐negative breast cancer (TNBC). In double mouse KO, tumors identity changed to a sarcomatoid/mesenchymal subtype; molecular and bioinformatics analyses revealed eEF2K as a potential therapeutic target.