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Objective: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs., Design: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human β-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR., Results: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum., Conclusions: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Aurora-A kinase is important for cellular proliferation and is implicated in the tumorigenesis of several malignancies, including of the ovary. Information regarding the expression patterns of Aurora-A in normal Müllerian epithelium as well as benign, borderline and malignant epithelial ovarian neoplasms is limited., Methods: We investigated Aurora-A expression by immunohistochemistry in 15 benign, 19 borderline and 17 malignant ovarian serous tumors, and 16 benign, 8 borderline, and 2 malignant ovarian mucinous tumors. Twelve fimbriae from seven patients served as normal Müllerian epithelium controls. We also examined Aurora-A protein expression by western blot in normal fimbriae and tumor specimens., Results: All normal fimbriae (n = 12) showed nuclear but not cytoplasmic Aurora-A immunoreactivity by immunohistochemistry. Benign ovarian tumors also showed strong nuclear Aurora-A immunoreactivity. Forty-eight percent (13/27) of borderline tumors demonstrated nuclear Aurora-A immunoreactivity, while the remainder (52%, 14/27) lacked Aurora-A staining. Nuclear Aurora-A immunoreactivity was absent in all malignant serous tumors, however, 47% (8/17) demonstrated perinuclear cytoplasmic staining. These results were statistically significant when tumor class (benign/borderline/malignant) was compared to immunoreactivity localization or intensity (Fisher Exact Test, p < 0.01). Western blot analysis confirmed the greater nuclear Aurora-A expression in control Müllerian epithelium compared to borderline and malignant tumors., Conclusion: Aurora-A kinase is differentially expressed across normal Müllerian epithelium, benign and borderline serous and mucinous ovarian epithelial neoplasms and malignant serous ovarian tumors., with nuclear expression of unphosphorylated Aurora-A being present in normal and benign neoplastic epithelium, and lost in malignant serous neoplasms. Further studies of the possible biological and clinical implications of the loss of nuclear Aurora-A expression in ovarian tumors, and its role in ovarian carcinogenesis are warranted., (© 2021. The Author(s).)

The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Ohigashi et al.)

Nitric oxide (NO) has been implicated in Na +  homeostatic control in water-breathing fishes. It is, however, uncertain whether air-breathing fish relies on NO to coordinate Na + /K + -ATPase (NKA)-driven Na +  transport during acute hypoxemia. We, thus, examined the action of nitric oxide synthase (NOS) inhibitor, L-NAME on NO availability, inducible NOS (iNOS) protein abundance and the regulatory dynamics of NKA in osmoregulatory epithelia of Anabas testudineus kept at induced hypoxemia. As expected in nonstressed fish, in vivo L-NAME (100 ng g -1 ) challenge for 30 min declined NO production in serum (40%) and osmoregulatory tissues (average 51.6%). Surprisingly, the magnitude of such reduction was less in hypoxemic fish after L-NAME challenge due to the net gain of NO (average 23.7%) in these tissues. Concurrently, higher iNOS protein abundance was found in branchial and intestinal epithelia of these hypoxemic fish. In nonstressed fish, L-NAME treatment inhibited the NKA activity in branchial and intestinal epithelia while stimulating its activity in renal epithelia. Interestingly in hypoxemic fish, L-NAME challenge restored the hypoxemia-inhibited NKA activity in branchial and renal epithelia. Similar recovery response was evident in the NKAα protein abundance in immunoblots and immunofluorescence images of branchial epithelia of these fish. Analysis of Nkaα1 isoform transcript abundance (Nkaα1a, α1b, α1c) also showed spatial and preferential regulation of Nkaα1 isoform switching. Collectively, the data indicate that L-NAME challenge activates iNOS/NO system in the branchial ionocyte epithelia of hypoxemia-stressed Anabas and demands multidimensional regulation of NKA to restore the Na +  transport rate probably to defend against acute hypoxemia., (© 2021 The Authors. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology published by Wiley Periodicals LLC.)

Metamorphosis (Greek for a state of transcending-form or change-in-shape) refers to a dramatic transformation of an animal's body structure that occurs after development of the embryo or larva in many species. The development of a fly (or butterfly) from a crawling larva (or caterpillar) that forms a pupa (or chrysalis) before eclosing as a flying adult is a classic example of metamorphosis that captures the imagination and has been immortalized in children's books. Powerful genetic experiments in the fruit fly Drosophila melanogaster have revealed how genes can instruct the behaviour of individual cells to control patterns of tissue growth, mechanical force, cell-cell adhesion and cell-matrix adhesion drive morphogenetic change in epithelial tissues. Together, the distribution of mass, force and resistance determines cell shape changes, cell-cell rearrangements, and/or the orientation of cell divisions to generate the final form of the tissue. In organising tissue shape, genes harness the power of self-organisation to determine the collective behaviour of molecules and cells, which can often be reproduced in computer simulations of cell polarity and/or tissue mechanics. This review highlights fundamental discoveries in epithelial morphogenesis made by pioneers who were fascinated by metamorphosis, including D'Arcy Thompson, Conrad Waddington, Dianne Fristrom and Antonio Garcia-Bellido., (Copyright © 2020 Elsevier Inc. All rights reserved.)

CDC-42 regulation of non-muscle myosin/NMY-2 is required for polarity maintenance in the one-cell embryo of Caenorhabditis elegans CDC-42 and NMY-2 regulate polarity throughout embryogenesis, but their contribution to later events of morphogenesis are less understood. We have shown that epidermal enclosure requires the GTPase CED-10/Rac1 and WAVE/Scar complex, its effector, to promote protrusions that drive enclosure through the branch actin regulator Arp2/3. Our analysis here of RGA-8, a homolog of SH3BP1/Rich1/ARHGAP17/Nadrin, with BAR and RhoGAP motifs, suggests it regulates CDC-42, so that actin and myosin/NMY-2 promote ventral enclosure during embryonic morphogenesis. Genetic and molecular data suggest RGA-8 regulates CDC-42, and phenocopies the CDC-42 pathway regulators WASP-1/WSP-1 and the F-BAR proteins TOCA-1 and TOCA-2. Live imaging shows RGA-8 and WSP-1 enrich myosin and regulate F-actin in migrating epidermal cells during ventral enclosure. Loss of RGA-8 alters membrane recruitment of active CDC-42. We propose TOCA proteins and RGA-8 use BAR domains to localize and regenerate CDC-42 activity, thus regulating F-actin levels, through the branched actin regulator WSP-1, and myosin enrichment. RhoGAP RGA-8 thus polarizes epithelia, to promote cell migrations and cell shape changes of embryonic morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The separation of benign from malignant mesothelial proliferations can be a difficult problem for the surgical pathologist. c-MET is a receptor tyrosine kinase that is overexpressed and detectable by immunohistochemistry in many malignancies, including malignant mesothelioma. Whether c-MET is also expressed in benign mesothelial reactions is unclear from the literature. To determine whether c-MET immunohistochemistry can separate benign from malignant mesothelial processes, we stained 2 tissue microarrays containing 33 reactive epithelioid mesothelial proliferations (E-RMPs), 23 reactive spindle cell mesothelial proliferations, 45 epithelioid malignant mesotheliomas (EMMs), and 26 sarcomatoid/desmoplastic mesotheliomas (SMMs) for c-MET and compared the results with immunohistochemistry for two established markers, BAP1 and methylthioadenosine phosphorylase (MTAP). Membrane staining for c-MET was evaluated using a 12-point H-score classified as negative (score = 0), trace (score = 1-3), moderate (score = 4-6), and strong (score = 8-12). Staining was seen in only 3 of 33 (all trace) E-RMPs compared with 36 of 45 (80%) EMMs (chi-square comparing reactive and malignant = 39.80, p = 1.2 × 10 -8 ). The H-score was >3 (moderate or strong) in 24 of 45 (53%) EMMs. Addition of BAP1 staining to the c-MET-negative/trace EMM increased sensitivity to 75% (32/42), whereas similar addition of MTAP staining increased sensitivity to 77% (33/43). No benign spindle cell proliferations showed staining compared with 10 of 26 (38%) positive SMMs, but only 4 (15%) SMMs were classified as moderate or strong. We conclude that moderate/strong c-MET staining can be used to support a diagnosis of EMM vs an epithelial reactive proliferation. c-MET is too insensitive to use for detecting SMM., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination., Competing Interests: Disclosures: Dr. Graupera reported, "the Graupera laboratory has research agreements with ArQule and Venthera. None of those have a relationship with cancer or LKB1." Dr. Gomis reported shares of Inbiomotion (<$10,000). No other disclosures were reported., (© 2020 Hermanova et al.)

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3K GOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3K GOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-β pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3K GOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-β signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

A series of histochemical techniques have been employed to localize alkaline phosphatase, acid phosphatase, non-specific esterase, catalase and peroxidase; and to visualize and characterize glycoprotein (GPs) moieties in the epithelium of gill arch, gill filaments and secondary lamellae of an angler catfish Chaca chaca. The epithelium of gill arch and gill filament shows strong alkaline phosphatase activity in the deeper layer epithelial cells; strong non-specific esterase activities in the outer layer epithelial cells; and weak acid phosphatase activity throughout the epithelium. The activity of these enzymes in the secondary lamellae is weak. The catalase and peroxidase show strong activities in the blood cells of the secondary lamellae. Various classes of GPs have been identified and characterized in the mucous secretions of the gill epithelium of C. chaca. These include-GPs with oxidizable vicinal diols, GPs with sialic acid residues without O-acyl substitution and GPs with O-sulphate esters. The functional significance of different enzymes in gill epithelium and the GPs in the mucus secreted on the surface has been discussed with the physiology of the gills in relation to the characteristic habit and habitat of the fish., (© 2019 Blackwell Verlag GmbH.)

The airway epithelium is seriously damaged upon pulmonary Pseudomonas aeruginosa infection, especially in cystic fibrosis (CF) sufferers. Therefore, the discovery of novel anti-infective agents accelerating healing of infected injured tissues is crucial. The antipseudomonal peptides esculentin-1a(1-21)NH 2 and its diastereomer Esc(1-21)-1c (Esc peptides) hold promise in this respect. In fact, they stimulate airway epithelial wound repair, but no mechanistic insights are available. Here we demonstrated that this process occurs through promotion of cell migration by an indirect activation of epidermal growth factor receptor mediated by metalloproteinases. Furthermore, we showed an increased expression of metalloproteinase 9, at both gene and protein levels, in peptide-treated bronchial epithelial cells with a functional or mutated form of CF transmembrane conductance regulator. In addition, the two peptides counteracted the inhibitory effect of Pseudomonas lipopolysaccharide (mimicking an infection condition) on the wound healing activity of the airway epithelium, and they enhanced the production of interleukin-8 from both types of cells. Finally, no immunogenicity was discovered for Esc peptides, suggesting their potential safety for clinical usage. Besides representing a step forward in understanding the molecular mechanism underlying the peptide-induced wound healing activity, these studies have contributed to highlight Esc peptides as valuable therapeutics with multiple functions.

Since spermatheca is able to transport spermatozoa and maintain a specific microenvironment for the storage of viable sperm cells for long periods of time, specific morphofunctional features must be involved in this capacity, and an efficient nutritional and oxygen supply must be required. In this study, we investigated the histological features of spermathecae and fat bodies in six species of three genera of epidemiological importance for Chagas' disease. The association of the reproductive system with the fat bodies and tracheal system was also focused in these species. The reproductive system, tracheae and fat bodies were fixed in 4% formaldehyde, and embedded in glycol methacrylate. The sections were stained with H.E., picrosirius red and Periodic-Acid Schiff methods for morphological analyses. Paraffin-embedded spermatheca sections were submitted to immunofluorescence for detection of V-ATPase. In P. lignarius, R. montenegrensis and R. prolixus, the spermatheca contains a slightly dilated tubular distal portion. In P. megistus and T. tibiamaculata, the spermatheca shows a large bulbous distal portion, and in T. infestans, a large oval-shaped distal portion. In all species, this portion was surrounded by a thin muscular layer, and the epithelial height varied according to the shape of this terminal portion. All spermathecal proximal portions showed simple columnar epithelium surrounded by a thick muscular layer. The epithelial cells of spermathecae showed PAS-positive cytoplasm and V-ATPase immunofluorescence in the apical surface. Tracheoles and polysaccharide-rich fat body cells were found next or in close contact to the oviduct or spermathecal tissues. The results indicate that the spermatheca proximal portion is related to contraction and sperm transport, whose oxygen and energy supply is guaranteed by the associated tracheal branches and fat bodies. In the storage portion, fat bodies and tracheae seem to be crucial for the maintenance of an optimal spermathecal microenvironment and storage of viable sperm cells. The participation of V-ATPase in the spermathecae epithelial cells may contribute for the maintenance of an optimal luminal milieu to spermatozoa, by alkalinization and/or acidification of lumen, similarly to the other epithelial cell types in insects. Further studies are necessary to clarify the role of this proton pump in the spermathecal epithelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Intestinal epithelial injury in septic patients predicts subsequent development of multiple organ failure, but its regulation by host factors remains unclear. Sphingosine kinase 1 is an enzyme-regulating inflammatory response., Methods: Cecal ligation and puncture was used to induce sepsis in C57BL/6 mice with and without N,N-dimethylsphingosine, a SphK1 inhibitor. Symptom severity was monitored by murine sepsis severity score. The intestinal barrier function was determined using 4KDa fluorescein-dextran. Bacterial load in the bloodstream was determined by 16S rRNA gene amplification., Results and Conclusions: Our preliminary experimental data showed that expression of sphingosine kinase 1 in ileum was increased by sixfold in septic mice. Pharmacological blockade of sphingosine kinase 1 alleviated septic symptoms. The intestinal permeability and bacterial load in the bloodstream were also reduced in these animals. We hypothesized that inhibition of sphingosine kinase 1 may reduce pro-inflammatory cytokine production, and alleviate intestinal epithelial injury during sepsis. Further mechanistic studies and clinical specimen analyses are warranted.

Oral potentially malignant disorders (OPMD) may develop malignant characteristics and transform into oral squamous cell carcinoma (OSCC) in a range of 1% to 2% of cases. Chronic alcohol consumption is associated with carcinogenesis, but its mechanism has not yet been fully elucidated. ALDH1A1 and 2, isoenzymes responsible for aldehyde oxidation involved in ethanol metabolism may be associated with the development of malignant head and neck neoplasms. The aim of this study was to analyze the expression of ALDH1A1 and ALDH2 in oral leukoplakia with epithelial dysplasia (OLP) and OSCC. A retrospective study was conducted on 27 cases of OLP and 30 cases of OSCC. Clinical data were obtained from medical records, and all cases were classified as mild, moderate, and severe for OLP, and well-differentiated, moderately differentiated, or poorly differentiated for OSCC cases. The ALDH1A1 and ALDH2 expression in OLP and OSCC was evaluated by the immunohistochemical technique. There was predominance of the male sex, in both OLP and OSCC cases. Oral tongue was the most affected site in both groups. OLP showed positive protein expression of ALDH1A1 in all cases, both basal and suprabasal epithelial layers, whereas ALDH2 showed less protein expression. In OSCC, the immunohistochemical reaction for ALDH1A1 expression was negative in 70%, whereas ALDH2 expression was positive in all cases. This study demonstrated the gradual loss of ALDH1A1 expression in OSCC in comparison with OLP, and the increased ALDH2 expression in OSCC.

Heat-stressed broilers usually reduce their feed intake, leading to energy imbalance and disturbing the homeostasis in the small intestine. This study was aimed to explore heat-stress-mediated physiological features that may be ascribed to impairments in the intestinal tract of broilers. The results revealed that heat exposure increased the activities of trypsin and Na + /K + -ATPase, while it decreased the activities of amylase, lipase, and maltase as well as the proliferating cell nuclear antigen cells in the jejunum after 14 days of heat exposure. Meanwhile, heat stress upregulated the mRNA expressions of AMPKα1, LKB1, and HIF-1α and protein expressions of p-AMPKα Thr172 and p-LKB1 Thr189 in the small intestine after 7 or 14 days of heat exposure. In conclusion, chronic heat exposure impeded the development of digestive organs, disordered the activities of intestinal digestive enzymes, and impaired the intestinal epithelial cells by increasing the cell apoptosis and declining cell proliferation, which might be correlated with the adenosine 5'-monophosphate-activated protein kinase signaling pathway. Additionally, heat stress upregulated the gene expression of HIF-1α, which indicated that heat stress may disturb the homeostasis in the intestine.

Eosinophilic esophagitis (EoE) is a chronic, allergen-driven inflammatory disease of the esophagus characterized predominantly by eosinophilic inflammation, leading to esophageal dysfunction. Converging data have placed the esophageal epithelium at the center of disease pathogenesis. In particular, the main EoE disease susceptibility loci at 2p23 and 5p22 encode for gene products that are produced by the esophageal epithelium: the intracellular protease calpain 14 and thymic stromal lymphopoietin, respectively. Furthermore, genetic and functional data establish a primary role for impaired epithelial barrier function in disease susceptibility and pathoetiology. Additionally, the EoE transcriptome, a set of genes dysregulated in the esophagi of patients with EoE, is enriched in genes that encode for proteins involved in esophageal epithelial cell differentiation. This transcriptome has a high proportion of esophagus-specific epithelial genes that are notable for the unexpected enrichment in genes encoding for proteases and protease inhibitors, as well as in IL-1 family genes, demonstrating a previously unappreciated role for innate immunity responses in the esophagus under homeostatic conditions. Among these pathways, basal production of the serine protease inhibitor, Kazal-type 7 (SPINK7) has been demonstrated to be part of the normal differentiation program of esophageal epithelium. Profound lost expression of SPINK7 occurs in patients with EoE and is sufficient for unleashing increased proteolytic activity (including urokinase plasminogen activator), impaired barrier function, and production of large quantities of proinflammatory and proallergic cytokines, including thymic stromal lymphopoietin. Collectively, we put forth a model in which the esophagus is normally equipped as an anti-inflammatory sensing organ and that defects in this pathway, mediated by epithelial protease/protease inhibitor imbalances, unleash inflammatory responses resulting in disorders, such as EoE., (Copyright © 2018. Published by Elsevier Inc.)

The Ras oncogene (Rat Sarcoma oncogene, a small GTPase) is a key driver of human cancer, however alone it is insufficient to produce malignancy, due to the induction of cell cycle arrest or senescence. In a Drosophila melanogaster genetic screen for genes that cooperate with oncogenic Ras (bearing the Ras V12 mutation, or Ras ACT ), we identified the Drosophila Src (Sarcoma virus oncogene) family non-receptor tyrosine protein kinase genes, Src42A and Src64B , as promoting increased hyperplasia in a whole epithelial tissue context in the Drosophila eye. Moreover, overexpression of Src cooperated with Ras ACT in epithelial cell clones to drive neoplastic tumourigenesis. We found that Src overexpression alone activated the Jun N-terminal Kinase (JNK) signalling pathway to promote actin cytoskeletal and cell polarity defects and drive apoptosis, whereas, in cooperation with Ras ACT , JNK led to a loss of differentiation and an invasive phenotype. Src + Ras ACT cooperative tumourigenesis was dependent on JNK as well as Phosphoinositide 3-Kinase (PI3K) signalling, suggesting that targeting these pathways might provide novel therapeutic opportunities in cancers dependent on Src and Ras signalling.

Objective: The aim of this study was to investigate the role of inducible nitric oxide synthase (iNOS) in lethal prostate cancer (PCa) by studying the iNOS immunoreactivity in tumor tissue from men diagnosed with localized PCa., Materials and Methods: This study is nested within a cohort of men diagnosed with incidental PCa undergoing transurethral resection of the prostate (the Swedish Watchful Waiting Cohort). To investigate molecular determinants of lethal PCa, men who died from PCa (n = 132) were selected as cases; controls (n = 168) comprised men with PCa who survived for at least 10 years without dying from PCa during follow-up. The immunoreactivity of iNOS in prostate tumor epithelial cells and in cells of the surrounding stroma was scored as low/negative, moderate or high. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) for lethal PCa according to iNOS category., Results: There was no association between iNOS immunoreactivity in stroma and lethal disease. However, when comparing high versus low/negative iNOS immunoreactivity in epithelial cells, the OR for lethal PCa was 3.80 (95% CI 1.45-9.97)., Conclusion: Patients with localized PCa have variable outcomes, especially those with moderately differentiated tumors. Identifying factors associated with long-term PCa outcomes can elucidate PCa tumor biology and identify new candidate prognostic markers. These findings support the hypothesis that high iNOS in tumor epithelium of the prostate is associated with lethal disease.

Besides androgens, estrogen signaling plays a key role in normal development and pathologies of the prostate. Irreversible synthesis of estrogens from androgens is catalyzed by aromatase. Interestingly, animals lacking aromatase do not develop cancer or prostatitis, whereas those with overexpression of aromatase and, consequently, high estrogen levels develop prostatitis and squamous metaplasia via estrogen receptor 1 (ERα). Even with this evidence, the aromatase expression in the prostate is controversial. Moreover, little is known about the occurrence of age-dependent variation of aromatase and its association with histopathological changes commonly found in advanced age, a knowledge gap that is addressed herein. For this purpose, the immunoexpression of aromatase was evaluated in the prostatic complex of young adult to senile Wistar rats. ERα was also investigated, to extend our understanding of estrogen responsiveness in the prostate. Moderate cytoplasmic immunoreactivity for aromatase was detected in the glandular epithelium. Eventually, some basal cells showed intense staining for aromatase. The expression pattern for aromatase appeared similar in the normal epithelium when young and senile rats were compared; this result was corroborated by Western blotting. Conversely, in senile rats, there was an increase in the frequency of basal cells intensely stained for aromatase, which appeared concentrated in areas of intraepithelial proliferation and prostatitis. These punctual areas also presented increased ERα positivity. Together, these findings suggest a plausible source for hormonal imbalance favoring estrogen production, which, by acting through ERα, may favor the development of prostatic lesions commonly found in advanced age., (Copyright © 2018 Endocrine Society.)

Background: The relationship between thymidine phosphorylase (TP) and angiogenesis at the early stage of esophageal squamous cell carcinoma has been unclear., Methods: Using 14 samples of normal squamous epithelium, 11 samples of low-grade intraepithelial neoplasia, and 64 samples of superficial esophageal cancer, microvessel density (MVD) was estimated using immunostaining for CD34 and CD105. TP expression was also evaluated in both cancer cells and stromal monocytic cells (SMCs). We then investigated the correlation between MVD and TP expression in both cancer cells and SMCs., Results: On the basis of the above parameters, MVD was significantly higher in cancerous lesions than in normal squamous epithelium. In terms of CD34 and CD105 expression, MVD showed a gradual increase from normal squamous epithelium, to low-grade intraepithelial neoplasia, and then to M1 and M2 cancer, and M3 or deeper cancer. M1 and M2 cancer showed overexpression of TP in both cancer cells and SMCs. There was no significant correlation between TP expression in cancer cells and MVD estimated from CD34 (rS = 0.16, P = 0.21) or CD105 (rS = 0.05, P = 0.68) expression. Significant correlations were found between TP expression in SMCs and CD34-related (rS = 0.46, P < 0.001) and CD105-related (rS = 0.34, P < 0.01) MVD. In M3 or deeper cancers, there were no significant correlations between TP expression in cancer cells or SMCs and venous invasion, lymphatic invasion, and lymph node metastasis., Conclusion: TP expression is activated in both cancer cells and stromal monocytic cells at the very early stage of ESCC progression. TP expression in SMCs, rather than in cancer cells, is significantly correlated with angiogenesis.

Apoptosis is crucial during the morphogenesis of most organs and tissues, and is utilized for tissues to achieve their proper size, shape and patterning. Many signaling pathways contribute to the precise regulation of apoptosis. Here we show that Jun N-terminal Kinase (JNK) activity contributes to the coordinated removal of interommatidial cells via apoptosis in the Drosophila pupal retina. This is consistent with previous findings that JNK activity promotes apoptosis in other epithelia. However, we found that JNK activity is repressed by Cindr (the CIN85 and CD2AP ortholog) in order to promote cell survival. Reducing the amount of Cindr resulted in ectopic cell death. Increased expression of the Drosophila JNK basket in the setting of reduced cindr expression was found to result in even more severe apoptosis, whilst ectopic death was found to be reduced if retinas were heterozygous for basket. Hence Cindr is required to properly restrict JNK-mediated apoptosis in the pupal eye, resulting in the correct number of interommatidial cells. A lack of precise control over developmental apoptosis can lead to improper tissue morphogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Effects of hypoxia on the osmorespiratory functions of the posterior gills of the shore crab Carcinus maenas acclimated to 12ppt seawater (DSW) were studied. Short-circuit current (Isc) across the hemilamella (one epithelium layer supported by cuticle) was substantially reduced under exposure to 1.6, 2.0, or 2.5mg O 2 /L hypoxic saline (both sides of epithelium) and fully recovered after reoxygenation. Isc was reduced equally in the epithelium exposed to 1.6mg O 2 /L on both sides and when the apical side was oxygenated and the basolateral side solely exposed to hypoxia. Under 1.6mg O 2 /L, at the level of maximum inhibition of Isc, conductance was decreased from 40.0mScm -2 to 34.7mScm -2 and fully recovered after reoxygenation. Isc inhibition under hypoxia and reduced 86 Rb + (K + ) fluxes across apically located K + channels were caused preferentially by reversible inhibition of basolaterally located and ouabain sensitive Na + ,K + -ATPase mediated electrogenic transport. Reversible inhibition of Isc is discussed as decline in active transport energy supply down regulating metabolic processes and saving energy during oxygen deprivation. In response to a 4day exposure of Carcinus to 2.0mg O 2 /L, hemolymph Na + and Cl - concentration decreased, i.e. hyperosmoregulation was weakened. Variations of the oxygen concentration level and exposure time to hypoxia lead to an increase of the surface of mitochondria per epithelium area and might in part compensate for the decrease in oxygen availability under hypoxic conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses., Methods: Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry., Results: Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues., Conclusion: Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.

Introduction and Hypothesis: The objective was to investigate the expression of endothelial nitric oxide synthase (eNOS) and phosphodiesterase (PDE) 5 in vaginal tissue of premenopausal women experiencing stress urinary incontinence (SUI) with and without sexual dysfunction., Methods: Women presenting for treatment of SUI were screened using the Female Sexual Function Index (FSFI) and 10 were selected who met the criteria for female sexual dysfunction (FSD) and 10 asymptomatic controls. Vaginal tissue specimens were obtained from those premenopausal women aged ≥40 years who had had sexual activity ≥2 times every month for the last 6 months and who were scheduled to undergo surgery for SUI. FSD criteria was FSFI scores <18 and arousal domain scores <3. The control group had FSFI scores ≥26 and individual domain scores ≥4. The expressions of eNOS and PDE 5 were compared in the two groups using immunofluorescence staining and western blotting., Results: The mean total FSFI scores were 30.4 ± 2.6 and 15.3 ± 2.3 in the control and FSD groups respectively. In immunofluorescence staining, eNOS and PDE5 were localized in the vaginal epithelium. In western blotting, the expressions of eNOS and PDE5 were significantly lower in the FSD group than in the control group (p = 0.003 and p = 0.038 respectively)., Conclusions: eNOS and PDE5 in the vagina may play important roles in the pathophysiology of FSD.

Reactive oxygen species (ROS) generated after tissue injury play a crucial role during wound healing through initiating acute inflammation, clarifying infection and dead tissue, and mediating various intracellular signal transduction. Prostaglandin E 2 (PGE 2 ) has been identified as one of the major factors responsible for inflammation and tissue repair. In this study, we tested our hypothesis that ROS produced by damaged human keratinocytes induces the synthesis of PGE 2 . In vitro epithelial wounding model was used to observe the production of ROS and secretion of PGE 2 as well as the involved signal pathway. The mechanical injury caused the rapid production of ROS in in vitro cultured keratinocytes, which was significantly blocked by an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. The increased intracellular ROS caused by mechanical injury stimulates PGE 2 production in a time-dependent manner via the activation of cyclooxygenase-2 (COX-2), which was stimulated by phosphorylation of extracellular signal-regulated protein kinase (ERK). These results indicate ROS-induced ERK activation leading to the activation of COX-2 and the synthesis of PGE 2 in human keratinocytes responding to mechanical injury in the acute phase., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

MTOR (mechanistic target of rapamycin [serine/threonine kinase]) plays a crucial role in many major cellular processes including metabolism, proliferation and macroautophagy/autophagy induction, and is also implicated in a growing number of proliferative and metabolic diseases. Both MTOR and autophagy have been suggested to be involved in lung disorders, however, little is known about the role of MTOR and autophagy in pulmonary epithelium in the context of acute lung injury (ALI). In the present study, we observed that lipopolysaccharide (LPS) stimulation induced MTOR phosphorylation and decreased the expression of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β)-II, a hallmark of autophagy, in mouse lung epithelium and in human bronchial epithelial (HBE) cells. The activation of MTOR in HBE cells was mediated by TLR4 (toll-like receptor 4) signaling. Genetic knockdown of MTOR or overexpression of autophagy-related proteins significantly attenuated, whereas inhibition of autophagy further augmented, LPS-induced expression of IL6 (interleukin 6) and IL8, through NFKB signaling in HBE cells. Mice with specific knockdown of Mtor in bronchial or alveolar epithelial cells exhibited significantly attenuated airway inflammation, barrier disruption, and lung edema, and displayed prolonged survival in response to LPS exposure. Taken together, our results demonstrate that activation of MTOR in the epithelium promotes LPS-induced ALI, likely through downregulation of autophagy and the subsequent activation of NFKB. Thus, inhibition of MTOR in pulmonary epithelial cells may represent a novel therapeutic strategy for preventing ALI induced by certain bacteria.

Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate l-arginine (2.0 mmol l -1 ), and addition of exogenous NO via the NO donor SNAP (10 -6 to 10 -4  mol l -1 ), decreased the epithelial short-circuit current (I sc ). Inhibition of endogenous NO production by the NOS inhibitor l-NAME (10 -4  mol l -1 ) increased I sc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10 -5  mol l -1 ) supressed the NO-mediated decrease in I sc , and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on I sc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10 -6  mol l -1 ) and Methylene Blue (10 -4  mol l -1 ), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10 -4  mol l -1 ) led to S-nitrosation of proteins, including Na + /K + -ATPase. Blocking of NOS with l-NAME (10 -6  mol l -1 ) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in I sc Yohimbine (10 -4  mol l -1 ), an inhibitor of α 2 -adrenoceptors, did not block NO effects, suggesting that NO is not involved in the α-adrenergic control of NaCl secretion., (© 2016. Published by The Company of Biologists Ltd.)

Background: Ameloblastoma (AM) is a benign odontogenic neoplasm characterized by local invasiveness and recurrence. We compared the immunohistochemical expression of matrix metalloproteinases in different clinical types of AM as well as in normal odontogenic tissue., Methods: Thirteen cases of solid AMs, five cases of unicystic AM and eight pericoronal follicles (PF) were selected and subjected to immunohistochemical investigation for matrix metalloproteinase-1, matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions., Results: The expressions of MMP-1 and MMP-2 were very high in the cytoplasm of cells throughout the entire epithelium and in fibroblasts from the adjacent connective tissue. MMP-9 expression was observed in the same location although with weaker staining. The Kruskal-Wallis test showed statistically significant differences in the epithelial expressions of MMP-1 and MMP-2; there was lower expression among solid AMs when compared with unicystic AM and PF. Compared to both types of AM, higher stromal expression of MMP-9 was found in PF., Conclusion: MMP-1, MMP-2 and MMP-9 seem to be associated with AM tumour behaviour as well as physiological tissue remodelling within PF., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Esophageal squamous cell carcinoma (ESCC) is the most frequent esophageal tumor in the world. ESCC presents late diagnosis, highly aggressive behavior and poor survival. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1 (GLUT-1), and transcription factor induced by hypoxia (HIF1α), responsible for the transcription of proteins cited. The objective of this study is to evaluate the alterations of these proteins and their association with clinicopathological data in ESCC. We performed immunohistochemistry to determine HIF-1α, GLUT-1, PKM1, PKM2, HK2 and Ki67-expression in ESCC patients and controls. Also, we used RT-qPCR to evaluated mRNA expression of GLUT-1 in esophageal mucosa of individuals without cancer, but are alcohol drinkers and tobacco smokers. Our results showed the exclusively expression of GLUT-1 in tumors cells and dysplastic samples. We also observed a compartmentalization of the expression of PKM1 and PKM2 in relation to tumor cells and stroma associated to tumor areas. All of the proteins evaluated, excepted GLUT-1, were frequently detected in normal mucosa. No correlations between clinicopathological features and protein expressions were observed. GLUT-1 expression appears in initial tumor lesions and is maintained through ESCC evolution. We reported for the first time PKM1 staining in normal esophagus and ESCC, being mostly present in more differentiated cells., (Copyright © 2016. Published by Elsevier Inc.)

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen--a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 μM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity--a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Biological oxidative responses are involved in the toxicity of multiwall carbon nanotubes (MWCNTs), which may cause asbestos-like pathogenicity. Superoxide dismutase 2 (SOD-2) has been proposed as a biomarker of early responses to mesothelioma-inducing fibers. This study was conducted to investigate the alteration of SOD-2 expression in the human mesothelial cell lines Met-5A after exposure to nontoxic doses of MWCNTs and the potential signaling pathway. The parameters measured included the viability, morphological change, superoxide formation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and messenger RNA (mRNA)/protein levels of SOD-2. Our results showed that MWCNTs upregulated SOD-2 expression at both mRNA and protein level. Coincidently, both superoxide formation and ERK1/2 phosphorylation were observed in Met-5A cells exposed to MWCNTs and were diminished by pretreatment with the reactive oxidative species (ROS) scavenger, N-acetyl-l-(+)-cysteine (NAC). To further investigate the role of ROS/ERK1/2 in MWCNTs-induced SOD-2 overexpression, prior to MWCNTs exposure, cells were pretreated with the Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) inhibitor (U0126) or with NAC. Both pretreatments decreased the MWCNTs-induced overexpression of SOD-2. These results suggest that upregulation of SOD-2 in Met-5A cells exposed to MWCNTs is mediated by ROS formation and ERK1/2 activation., (© The Author(s) 2015.)

The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 μM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 μM). Similar changes were observed in response to fiber damage caused by injection of 5 μl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to control ion concentrations in the fiber mass and the Na,K-ATPase response may reflect the critical contribution of the epithelium to lens ion homeostasis., (Published by Elsevier Ltd.)

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.

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.

Intranuclear and cytoplasmic inclusions in the renal proximal tubular epithelium were observed in nontreated male and female Wistar Hannover rats in a 26-week study (32 weeks of age) and a 104-week study (110 weeks of age). The incidence rates were less than 5% in these two studies. In affected animals, the inclusions were observed in more than 60% of proximal tubular epithelium as various sized (approximately 1-8 μm in diameter) round and eosinophilic materials, but not in distal tubules, Henle's loop, or collecting ducts. Ultrastructurally, inclusions appeared finely granular, homogenous with middle-electron density, and without a limiting membrane. These inclusions were determined to be protein histochemically stained by Azan-Mallory and immunoreactive with an antibody against D-amino acid oxidase (DAO). There was no abnormality in in-life observations or in clinical test values suggestive of renal dysfunction. There were no associated degenerative or inflammatory changes in the kidneys, and no similar inclusions were observed in the other organs. These inclusions are very similar to propiverine hydrochloride (propiverine) and norepinephreine/serotonin reuptake inhibitor-induced inclusions. This is the first report of accumulation of DAO and formation of inclusions occurring spontaneously in rat kidneys. The data are important for toxicological studies using Wistar Hannover rats., (© 2014 by The Author(s).)

We previously demonstrated that the neutral sphingomyelinase (nSMase) 2 is the sole sphingomyelinase activated during cigarette smoke (CS)-induced oxidative stress of human airway epithelial cells, leading to ceramide generation and subsequent apoptosis of affected cells. Since then, we reported that nSMase2 is a phosphoprotein, the degree of enzymatic activity and stability of which are dictated by its degree of phosphorylation. Simultaneously, the non-receptor tyrosine kinase and proto-oncogene Src has increasingly become a target of interest in both smoking-related lung injury, such as chronic obstructive pulmonary disease, and lung cancer. Within this context, we tested and now present Src as a regulator of ceramide generation via modulation of nSMase2 phosphorylation and activity during CS-induced oxidative stress. Specifically, we provide evidence that Src activity is necessary for both CS-induced ceramide accumulation in vivo (129/Sv mice) and in vitro (human airway epithelial cells) and for nSMase2 activity during CS-induced oxidative stress. Moreover, because nSMase2 is exclusively phosphorylated on serines, we show that this occurs through Src-dependent activation of the serine/threonine kinase p38 mitogen-activated protein kinase during oxidative stress. Finally, we provide evidence that Src and p38 mitogen-activated protein kinase activities are critical for regulating nSMase2 phosphorylation. This study provides insights into a molecular target involved in smoking-related lung injury, represented here as nSMase2, and its modulation by the oncogene Src.

Background: The aim of this study is to evaluate the expression of human telomerase reverse transcription (hTERT) enzyme in chronic periodontitis (CP) and aggressive periodontitis (AgP) compared with healthy individuals., Methods: A total of 79 individuals consented to participate in the study. The study sample comprised healthy individuals (n = 30), patients with CP (n = 30), and patients with AgP (n = 19). Gingival tissue was collected and evaluated for hTERT expression by Western blot and immunohistochemical methods. Reverse transcription polymerase chain reaction was performed using the gingival crevicular fluid (GCF) samples., Results: The hTERT messenger RNA (mRNA) and protein expression was significantly higher in AgP compared with CP (P <0.001). In GCF, 53.33% of patients with CP and 68.42% of patients with AgP were showing hTERT mRNA expression, but it was not detected in the control group. The AgP tissue showed higher hTERT expression compared with CP (P <0.001). The hTERT mRNA expression did not show a correlation with gingival index (GI), plaque index (PI), probing depth (PD), and clinical attachment loss (AL) in patients with AgP, whereas hTERT protein expression was strongly correlated with GI, PI, PD, and AL in patients with AgP. The protein expression of hTERT shows significant but moderate correlation with GI and AL in patients with CP., Conclusion: High expression of hTERT might be associated with periodontal disease progression, suggesting that hTERT could be a potential prognostic marker.

Glycogen synthase kinase 3β (GSK3β) and phosphorylated GSK3β at Ser9 (pS9GSK3β) are crucial in cellular proliferation and metabolism. GSK3β and pS9GSK3β are deregulated in many diseases including tumors. Data on altered expression of GSK3β and pS9GSK3β are mainly limited to tumor tissues, thus the expression of GSK3β and pS9GSK3β in normal human tissue has been largely unknown. Thus, we examined the immunohistochemical localization of GSK3β and pS9GSK3β in human fetal and adult tissues, and also compared the expression pattern of GSK3β and pS9GSK3β with that of the CK7 and CK20. We found GSK3β expression in neurons of brain, myenteric plexus in gastrointestinal tract, squamous epithelium of skin, and mammary gland. The expression of pS9GSK3β was restricted to the epithelial cells of breast and pancreaticobiliary duct, distal nephron of kidney, gastrointestinal tract, fallopian tube, epididymis, secretory cell of prostatic gland, and umbrella cell of urinary tract. The staining pattern of pS9GSK3β and CK7 was overlapped in most organs except for gastrointestinal tract where CK7 was negative and CK20 was positive. Our results show that the expression of GSK3β may be associated with differentiation of ectodermal derived tissues and pS9GSK3β with that of epithelial cells of endodermal derived tissues in human. In addition, the expression of pS9GSK3β in the selective epithelial cells may indicate its association with secretory or barrier function of specific cells and may serve as another immunohistochemical marker for epithelial cells.

Peritoneal fibrosis resulting from long-term peritoneal dialysis is a major cause of failure of peritoneal ultrafiltration function and main reason of dropout from peritoneal dialysis. Epithelial-mesenchymal transition (EMT) of peritoneal mesochelial cells (HPMCs) is a major contributor of peritoneal fibrosis. Recently, the association between histone acetylation and kinds of fibrosis including liver, lung and kidney fibrosis is well established. Thus, in this study we tried to profile whether histone acetylation is also operates EMT process in HPMCs and what's the regulatory mechanism. We established an EMT model of HPMCs through high glucose treatment. And hyperacetylation of H3 histone was found using western blot in EMT model. After treated with C646, a histone acetyltransferase (HAT) inhibitor, high glucose-induced EMT in HPMCs was counteracted. To further understand the molecular mechanism of C646 rescues high glucose-induced EMT, CHIP-qPCRwas used to examine the modulation of histone H3 acetylation at promoters of series signaling target genes. We found that the H3 acetylation level at TGF-β1 gene promoter was down-regulation by C646 treatment. Moreover, we also found that TGF-β1/Smad3 signaling was blocked. Hence, our results suggest that histone H3 acetylation activated TGF-β1/Smad3 signaling during EMT of HPMCs, and C646 can rescue the mesenchymal phenotype transition. These findings may provide a novel pathogenic mechanism and therapeutic target for peritoneal fibrosis.

Epithelial repair in the Drosophila embryo is achieved through 2 dynamic cytoskeletal machineries: a contractile actomyosin cable and actin-based cellular protrusions. Rho family small GTPases (Rho, Rac, and Cdc42) are cytoskeletal regulators that control both of these wound repair mechanisms. Cdc42 is necessary for cellular protrusions and, when absent, wounds are slow to repair and never completely close. Rac proteins accumulate at specific regions in the wound leading edge cells and Rac-deficient embryos exhibit slower repair kinetics. Mutants for both Rho1 and its effector Rok impair the ability of wounds to close by disrupting the leading-edge actin cable. Our studies highlight the importance of these proteins in wound repair and identify a downstream effector of Rho1 signaling in this process.

Objective: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells., Methods: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs)., Results: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p < 0.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p < 0.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R(2) = 0.875, p < 0.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA., Conclusions: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

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., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in Ras(V12)-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA-VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis., (© 2014. Published by The Company of Biologists Ltd.)

Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15-lipoxygenase-2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15-LOX-2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15-LOX-2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

Apical constriction is a cell shape change that promotes epithelial bending. Activation of nonmuscle myosin II (Myo-II) by kinases such as Rho-associated kinase (Rok) is important to generate contractile force during apical constriction. Cycles of Myo-II assembly and disassembly, or pulses, are associated with apical constriction during Drosophila melanogaster gastrulation. It is not understood whether Myo-II phosphoregulation organizes contractile pulses or whether pulses are important for tissue morphogenesis. Here, we show that Myo-II pulses are associated with pulses of apical Rok. Mutants that mimic Myo-II light chain phosphorylation or depletion of myosin phosphatase inhibit Myo-II contractile pulses, disrupting both actomyosin coalescence into apical foci and cycles of Myo-II assembly/disassembly. Thus, coupling dynamic Myo-II phosphorylation to upstream signals organizes contractile Myo-II pulses in both space and time. Mutants that mimic Myo-II phosphorylation undergo continuous, rather than incremental, apical constriction. These mutants fail to maintain intercellular actomyosin network connections during tissue invagination, suggesting that Myo-II pulses are required for tissue integrity during morphogenesis., (© 2014 Vasquez et al.)

Collagenase-3 (matrix metalloproteinase-13) is a metalloproteinase (MMP) that is associated with bone lesions and exhibits variable expression patterns in odontogenic cysts; it may play a role in regulating focal proliferation and maturation of jaw cyst epithelium. We studied the localization, staining intensity and distribution of collagenase-3 in 13 periapical granulomas with epithelium, 16 periapical granulomas without epithelium and 10 radicular cysts using archived formalin fixed, paraffin embedded tissues. A monoclonal antibody against human collagenase-3 was used to evaluate its expression. Immunohistochemical staining intensities of collagenase-3 in all periapical lesions were (-), 4 (10%); (+), 1 (3%); (++), 22 (56%) and (+++), 12 (31%); differences were not statistically significant. Immunohistochemical distribution of collagenase-3 in epithelial cells was (-), 17 (44%); (+), 17 (44%); (++), 5 (13%); in fibroblasts it was (-), 8 (20%); (+), 23 (59%); (++), 8 (21%); in plasma cells it was (-), 7 (18%); (+), 22 (56%); (++), 10 (26%); in macrophages it was (-), 7 (18%); (+), and 15 (38%); and (++), 17 (44%). Statistically significant differences were found in epithelial cells (p = 0.00) and fibroblasts (p = 0.02), whereas differences were not statistically significant for plasma cells and macrophages. Collagenase-3 may play a role in the conversion of a periapical granuloma with epithelium to radicular cyst. MMP's influence not only epithelial rest cell migration, but also invasion of various stromal cells into granulomatous tissue.

Background: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT., Materials and Methods: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments., Results: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003)., Conclusions: We concluded that these proteins/enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases' roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)