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Rotifers, Brachionus plicatilis, are a valuable first exogenous feed for zebrafish because they can provide continuous nutrition for growing zebrafish larvae when used in a rotifer-zebrafish polyculture. Typically cultured at high salinities (>10 ppt), B. plicatilis are temporarily immobilized when moved to lower salinities (5 ppt) used for polycultures, decreasing their accessibility and attractiveness to the larvae. The nutritional value of rotifers varies based on their diet, typically live algae, which has limited nutritional value and may pose biosecurity risks. After confirming that rotifers consume and can reproduce when fed an irradiated, processed larval fish diet (PD), they were reared at 5 or 15 ppt, and fed various combinations of an algae mix and/or PD. Population densities and percentages of egg-bearing rotifers were quantified daily until the population density plateaued, and then their nutritional value was assessed. Results indicated that rotifers thrived at both salinities. Those fed PD were successfully maintained at >500 rotifers per mL and contained a greater ω-6/ω-3 fatty acid ratio. Our findings indicate that enriching rotifers with PD raised at 5 ppt can potentially eliminate rotifer immobilization in polyculture, while providing a nutritious, attractive diet for zebrafish larvae and decreasing biosecurity risks.

Corynebacterium bovis (Cb), the cause of hyperkeratotic dermatitis in various immunocompromised mouse strains, significantly impacts research outcomes if infected mice are used. Although Cb has been isolated from a variety of species, including mice, rats, cows, and humans, little is known about the differences in the infectivity and clinical disease that are associated with specific Cb isolates. The infectious dose that colonized 50% of the exposed population (ID 50 ) and any associated clinical disease was determined in athymic nude mice (Hsd:Athymic Nude-Foxn1 nu ) inoculated with Cb isolates collected from mice ( n = 5), rat ( n = 1), cow ( n = 1), and humans ( n = 2) The same parameters were also determined for 2 of the mouse isolates in 2 furred immunocompromised mouse strains (NSG [NOD. Cg-Prkdc scid Il2rg tm1Wjl /Sz] and NSG-S [NOD. Cg-Prkdc scid Il2rgt m1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ]). To determine the ID 50 , mice ( n = 6/dose; 3 of each sex) were inoculated topically in 10-fold increments ranging from 1 to 10 8 bacteria. Mice were scored daily for 14 days for the severity of clinical signs. On days 7 and 14 after inoculation, buccal and dorsal skin swabs were evaluated by aerobic culture to determine infection status. The mouse isolates yielded lower ID 50 values (58 to 1000 bacteria) than did the bovine (6460 to 7498 bacteria) and rat (10,000 bacteria) isolates. Human isolates did not colonize mice or cause disease. Mouse isolates produced clinical disease of vary- ing severity in nude mice. Despite significant immunodeficiency, furred NSG and NSG-S mice required a 1000- to 3000-fold higher inoculum for colonization than did athymic nude mice. Once colonized, clinically detectable hyperkeratosis did not develop in the haired strains until 18 to 22 d after inoculation, whereas athymic nude mice that developed clinically detect- able disease showed hyperkeratosis between 6 and 14 d after inoculation. In conclusion, there are significant differences in Cb's ID 50 , disease course, and severity of clinical signs between Cb isolates and among immunodeficient mouse strains.

Embryo surface disinfection in either an iodine or sodium hypochlorite (NaOCl) solution is commonly performed on imported zebrafish embryos to decrease pathogen introduction into a facility. The impact of the consecutive use of these disinfectants and the conductivity of the culture media on embryo survival and development post-disinfection have not been evaluated. Iodine (12.5-25 ppm) is effective at eliminating several Mycobacterium species, whereas NaOCl (50-100 ppm) reduces the number of viable Pseudoloma neurophilia spores. Casper and T5D (tropical 5D wild type) embryos reared in media of differing conductivities (0-10, 100-200, 750-950, and 1500-2000 μS) with and without exposure to NaOCl 100 ppm at 6 h post-fertilization were evaluated for survival, hatching success, and morphological defects at 5 days post-fertilization. Additionally, the consecutive use of iodine (12.5 ppm for 2 min) followed by NaOCl (75 or 100 ppm for 10 min), as well as the inverse, was evaluated. Embryo survival was not impacted by embryo rearing media alone; however, survival significantly decreased when embryos were disinfected with 100 ppm NaOCl in media with a conductivity >750-950 μS. Iodine (12 ppm) and NaOCl (75 ppm) used sequentially resulted in >50% survival, whereas the use of 100 ppm NaOCl resulted in high levels of embryo mortality.

GPR65 (TDAG8) is a proton-sensing G protein-coupled receptor predominantly expressed in immune cells. Genome-wide association studies (GWAS) have identified GPR65 gene polymorphisms as an emerging risk factor for the development of inflammatory bowel disease (IBD). Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. To study the role of GPR65 in intestinal inflammation and colitis-associated colorectal cancer (CAC), colitis and CAC were induced in GPR65 knockout (KO) and wild-type (WT) mice using dextran sulfate sodium (DSS) and azoxymethane (AOM)/DSS, respectively. Disease severity parameters such as fecal score, colon shortening, histopathology, and mesenteric lymph node enlargement were aggravated in GPR65 KO mice compared to WT mice treated with DSS. Elevated leukocyte infiltration and fibrosis were observed in the inflamed colon of GPR65 KO when compared to WT mice which may represent a cellular mechanism for the observed exacerbation of intestinal inflammation. In line with high expression of GPR65 in infiltrated leukocytes, GPR65 gene expression was increased in inflamed intestinal tissue samples of IBD patients compared to normal intestinal tissues. Moreover, colitis-associated colorectal cancer development was higher in GPR65 KO mice than WT mice when treated with AOM/DSS. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in murine colitis and CAC models, suggesting potentiation of GPR65 with agonists may have an anti-inflammatory therapeutic effect in IBD and reduce the risk of developing colitis-associated colorectal cancer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Colombians in coastal Tumaco have a lower incidence of Helicobacter pylori-associated gastric cancer compared to individuals from Tuquerres in the high Andes. This is despite nearly universal prevalence of H. pylori infection and chronic gastritis., Methods: H. pylori infection was confirmed by Steiner stain and serology using African and European-origin strains. Gastric histology and serum inflammatory biomarkers in dyspeptic Tumaco or Tuquerres patients were evaluated to predict progression of gastric lesions., Results: H. pylori infection was nearly universal by Steiner stain and serology. IgG response to European-origin H. pylori strains were greater than African-origin. High gastric cancer-risk Tuquerres patients, compared to low-risk Tumaco, had significant odds ratios for lesion progression associated with serum IL-5, trefoil factor 3 (TFF3), and low pepsinogen I/II ratio. Sensitivity and specificity for these parameters was 63.8% and 67.9%, respectively, with correctly classifying patients at 66.7%. Most odds ratios for 26 other biomarkers were significant for the town of residency, indicating an environmental impact on Tumaco patients associated with decreased lesion progression., Conclusion: An IL-5 association with progression of gastric lesions is novel and could be evaluated in addition to TFF3 and pepsinogen I/II ratio as a non-invasive prognostic screen. Results suggest Tumaco patients were exposed to infectious diseases beyond H. pylori such as the documented high incidence of helminthiasis and toxoplasmosis., Impact: Results support a prior recommendation to evaluate TFF3 and pepsinogen I/II together to predict aggressive gastric histology. Our data indicate IL-5 should be further evaluated as prognostic parameter., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Clostridioides difficile is an enteric pathogen that can cause significant clinical disease in both humans and animals. However, clinical disease arises most commonly after treatment with broad-spectrum antibiotics. The organism's ability to cause naturally occurring disease in mice is rare, and little is known about its clinical significance in highly immunocompromised mice. We report on 2 outbreaks of diarrhea associated with C. difficile in mice. In outbreak 1, 182 of approximately 2, 400 NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) and related strains of mice became clinically ill after cessation of a 14-d course of 0.12% amoxicillin feed to control an increase in clinical signs associated with Corynebacterium bovis infection. Most mice had been engrafted with human tumors; the remainder were experimentally naïve. Affected animals exhibited 1 of 3 clinical syndromes: 1) peracute death; 2) severe diarrhea leading to euthanasia or death; or 3) mild to moderate diarrhea followed by recovery. A given cage could contain both affected and unaffected mice. Outbreak 2 involved a small breeding colony (approximately 50 mice) of NOD. CB17- Prkdc scid /NCrCrl (NOD- scid ) mice that had not received antibiotics or experimental manipulations. In both outbreaks, C. difficile was isolated, and toxins A and B were detected in intestinal content or feces. Histopathologic lesions highly suggestive of C. difficile enterotoxemia included fibrinonecrotizing and neutrophilic typhlocolitis with characteristic 'volcano' erosions or pseudomembrane formation. Genomic analysis of 4 isolates (3 from outbreak 1 and 1 from outbreak 2) revealed that these isolates were closely related to a pathogenic human isolate, CD 196. To our knowledge, this report is the first to describe naturally occurring outbreaks of C. difficile -associated typhlocolitis with significant morbidity and mortality in highly immunocompromised strains of mice.

Pulmonary granuloma formation is a complex and poorly understood response to inhaled pathogens and particulate matter. To explore the mechanisms of pulmonary granuloma formation and maintenance, our laboratory has developed a multiwall carbon nanotube (MWCNT)-induced murine model of chronic granulomatous inflammation. We have demonstrated that the MWCNT model closely mimics pulmonary sarcoidosis pathophysiology, including the deficiency of alveolar macrophage ATP-binding cassette (ABC) lipid transporters ABCA1 and ABCG1. We hypothesized that deficiency of alveolar macrophage ABCA1 and ABCG1 would promote pulmonary granuloma formation and inflammation. To test this hypothesis, the effects of MWCNT instillation were evaluated in ABCA1, ABCG1, and ABCA1/ABCG1 myeloid-specific knockout (KO) mice. Histological examination revealed significantly larger pulmonary granulomas in ABCG1-KO and ABCA1/ABCG1 double-KO animals when compared with wild-type animals. Evaluation of BAL cells indicated increased expression of CCL2 and osteopontin, genes shown to be involved in the formation and maintenance of pulmonary granulomas. Single deficiency of alveolar macrophage ABCA1 did not affect MWCNT-induced granuloma formation or proinflammatory gene expression. These observations indicate that the deficiency of alveolar macrophage ABCG1 promotes pulmonary granulomatous inflammation and that this is augmented by additional deletion of ABCA1.

Pancreatic cancer is devastating cancer worldwide with few if any truly effective therapies. Pancreatic cancer has an increasing incidence and may become the second leading cause of death from cancer. Novel, more effective therapeutic approaches are needed as pancreatic cancer patients usually survive for less than a year after being diagnosed. Control of blood sugar levels by the prescription drug metformin in diseases such as diabetes mellitus has been examined in association with pancreatic cancer. While the clinical trials remain inconclusive, there is hope that certain diets and medications may affect positively the outcomes of patients with pancreatic and other cancers. Other natural compounds may share some of the effects of metformin. One "medicinal" fruit consumed by millions worldwide is berberine (BBR). Metformin and BBR both activate AMP-activated protein kinase (AMPK) which is a key mediator of glucose metabolism. Glucose metabolism has been shown to be very important in cancer and its significance is increasing. In the following studies, we have examined the effects of metformin, BBR and a panel of modified BBRs (NAX compounds) and chemotherapeutic drugs on the growth of four different human pancreatic adenocarcinoma cell lines (PDAC). Interestingly, the effects of metformin could be enhanced by BBR and certain modified BBRs. Upon restoration of WT-TP53 activity in MIA-PaCa-2 cells, an altered sensitivity to the combination of certain NAX compounds and metformin was observed compared to the parental cells which normally lack WT-TP53. Certain NAX compounds may interact with WT-TP53 and metformin treatment to alter the expression of key molecules involved in cell growth. These results suggest a therapeutic approach by combining certain pharmaceutical drugs and nutraceuticals to suppress the growth of cancer cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mutations at the TP53 gene are readily detected (approximately 50-75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT-TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT-TP53 activity can have in PDAC cells which normally lack WT-TP53, as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Berberine (BBR) is a common nutraceutical consumed by millions worldwide. BBR has many different effects on human health, e.g., diabetes, diarrhea, inflammation and now more recently it has been proposed to have potent anti-cancer effects. BBR has been shown to suppress the growth of cancer cells more than normal cells. BBR has been proposed to exert its growth-inhibitory effects by many different biochemical mechanisms including: suppression of cell cycle progression, induction of reactive oxygen species, induction of apoptosis and autophagy and interactions with DNA potentially leading to DNA damage, and altered gene expression. Pancreatic cancer is a leading cancer worldwide associated with a poor prognosis. As our population ages, pancreatic cancer has an increasing incidence and will likely become the second leading cause of death from cancer. There are few truly-effective therapeutic options for pancreatic cancer. Surgery and certain chemotherapeutic drugs are used to treat pancreatic cancer patients. Novel approaches to treat pancreatic cancer patients are direly needed as they usually survive for less than a year after being diagnosed. In the following manuscript, we discuss the abilities of BBR and certain chemically-modified BBRs (NAX compounds) to suppress growth of pancreatic cancer cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, highly metastatic malignancy and accounts for 85% of pancreatic cancers. PDAC patients have poor prognosis with a five-year survival of only 5-10%. Mutations at the TP53 gene are readily detected in pancreatic tumors isolated from PDAC patients. We have investigated the effects of restoration of wild-type (WT) TP53 activity on the sensitivity of pancreatic cancer cells to: chemotherapy, targeted therapy, as well as, nutraceuticals. Upon introduction of the WT-TP53 gene into the MIA-PaCa-2 pancreatic cancer cell line, the sensitivity to drugs used to treat pancreatic cancer cells such as: gemcitabine, fluorouracil (5FU), cisplatin, irinotecan, oxaliplatin, and paclitaxel increased significantly. Likewise, the sensitivity to drugs used to treat other cancers such as: doxorubicin, mitoxantrone, and 4 hydroxy tamoxifen (4HT) also increased upon introduction of WT-TP53 into MIA-PaCa-2 cells. Furthermore, the sensitivity to certain inhibitors which target: PI3K/mTORC1, PDK1, SRC, GSK-3, and biochemical processes such as proteasomal degradation and the nutraceutical berberine as increased upon introduction of WT-TP53. Furthermore, in some cases, cells with WT-TP53 were more sensitive to the combination of drugs and suboptimal doses of the MDM2 inhibitor nutlin-3a. However, TP53-independent effects of nutlin-3a were observed upon treatment with either a proteasomal or a PI3K/mTOR inhibitor. These studies indicate the sensitizing effects that WT-TP53 can have in PDAC cells which normally lack WT-TP53 to various therapeutic agents and suggest approaches to improve PDAC therapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, highly metastatic malignancy and accounts for 85% of pancreatic cancers. PDAC patients have poor prognosis with a five-year survival of only 5-10% after diagnosis and treatment. Pancreatic cancer has been associated with type II diabetes as the frequency of recently diagnosed diabetics that develop pancreatic cancer within a 10-year period of initial diagnosis of diabetes in increased in comparison to non-diabetic patients. Metformin is a very frequently prescribed drug used to treat type II diabetes. Metformin acts in part by stimulating AMP-kinase (AMPK) and results in the suppression of mTORC1 activity and the induction of autophagy. In the following studies, we have examined the effects of metformin in the presence of various chemotherapeutic drugs, signal transduction inhibitors and natural products on the growth of three different PDAC lines. Metformin, by itself, was not effective at suppressing growth of the pancreatic cancer cell lines at concentration less than 1000 nM, however, in certain PDAC lines, a suboptimal dose of metformin (250 nM) potentiated the effects of various chemotherapeutic drugs used to treat pancreatic cancer (e.g., gemcitabine, cisplatin, 5-fluorouracil) and other cancer types (e.g., doxorubicin, docetaxel). Furthermore, metformin could increase anti-proliferative effects of mTORC1 and PI3K/mTOR inhibitors as well as natural products such as berberine and the anti-malarial drug chloroquine in certain PDAC lines. Thus, metformin can enhance the effects of certain drugs and signal transduction inhibitors which are used to treat pancreatic and various other cancers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Over the past fifty years, society has become aware of the importance of a healthy diet in terms of human fitness and longevity. More recently, the concept of the beneficial effects of certain components of our diet and other compounds, that are consumed often by different cultures in various parts of the world, has become apparent. These "healthy" components of our diet are often referred to as nutraceuticals and they can prevent/suppress: aging, bacterial, fungal and viral infections, diabetes, inflammation, metabolic disorders and cardiovascular diseases and have other health-enhancing effects. Moreover, they are now often being investigated because of their anti-cancer properties/potentials. Understanding the effects of various natural products on cancer cells may enhance their usage as anti-proliferative agents which may be beneficial for many health problems. In this manuscript, we discuss and demonstrate how certain nutraceuticals may enhance other anti-cancer drugs to suppress proliferation of cancer cells., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Extracellular acidosis is a condition found within the tumor microenvironment due to inadequate blood perfusion, hypoxia, and altered tumor cell metabolism. Acidosis has pleiotropic effects on malignant progression; therefore it is essential to understand how acidosis exerts its diverse effects. TDAG8 is a proton-sensing G-protein-coupled receptor that can be activated by extracellular acidosis., Methods: TDAG8 gene expression was analyzed by bioinformatic analyses and quantitative RT-PCR in human hematological malignancies. Retroviral transduction was used to restore TDAG8 expression in U937, Ramos and other blood cancer cells. Multiple in vitro and in vivo tumorigenesis and metastasis assays were employed to evaluate the effects of TDAG8 expression on blood cancer progression. Western blotting, immunohistochemistry and biochemical approaches were applied to elucidate the underlying mechanisms associated with the TDAG8 receptor pathway., Results: TDAG8 expression is significantly reduced in human blood cancers in comparison to normal blood cells. Severe acidosis, pH 6.4, inhibited U937 cancer cell proliferation while mild acidosis, pH 6.9, stimulated its proliferation. However, restoring TDAG8 gene expression modulated the U937 cell response to mild extracellular acidosis and physiological pH by reducing cell proliferation. Tumor xenograft experiments further revealed that restoring TDAG8 expression in U937 and Ramos cancer cells reduced tumor growth. It was also shown U937 cells with restored TDAG8 expression attached less to Matrigel, migrated slower toward a chemoattractant, and metastasized less in severe combined immunodeficient mice. These effects correlated with a reduction in c-myc oncogene expression. The mechanistic investigation indicated that Gα13/Rho signaling arbitrated the TDAG8-mediated c-myc oncogene repression in response to acidosis., Conclusions: This study provides data to support the concept that TDAG8 functions as a contextual tumor suppressor down-regulated in hematological malignancies and potentiation of the TDAG8 receptor pathway may be explored as a potential anti-tumorigenic approach in blood cancers.

Natural products or nutraceuticals promote anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway. This review will focus on the effects of curcumin (CUR), berberine (BBR) and resveratrol (RES), on the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway, with a special focus on GSK-3. These natural products may regulate the pathway by multiple mechanisms including: reactive oxygen species (ROS), cytokine receptors, mirco-RNAs (miRs) and many others. CUR is present the root of turmeric (Curcuma longa). CUR is used in the treatment of many disorders, especially in those involving inflammatory processes which may contribute to abnormal proliferation and promote cancer growth. BBR is also isolated from various plants (Berberis coptis and others) and is used in traditional medicine to treat multiple diseases/conditions including: diabetes, hyperlipidemia, cancer and bacterial infections. RES is present in red grapes, other fruits and berries such as blueberries and raspberries. RES may have some anti-diabetic and anti-cancer effects. Understanding the effects of these natural products on the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway may enhance their usage as anti-proliferative agent which may be beneficial for many health problems., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Various signaling pathways exert critical roles in the epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). The Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, hedgehog (Hh), Notch and TP53 pathways elicit essential regulatory influences on cancer initiation, EMT and progression. A common kinase involved in all these pathways is moon-lighting kinase glycogen synthase kinase-3 (GSK-3). These pathways are also regulated by micro-RNAs (miRs). TP53 and components of these pathways can regulate the expression of miRs. Targeting members of these pathways may improve cancer therapy in those malignancies that display their abnormal regulation. This review will discuss the interactions of the multi-functional GSK-3 enzyme in the Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, Hh, Notch and TP53 pathways. The regulation of these pathways by miRs and their effects on CSC generation, EMT, invasion and metastasis will be discussed.

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for 7days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in the inflamed gut as the absence of GPR4 ameliorates intestinal inflammation in the acute experimental colitis mouse model., (Copyright © 2016 Elsevier B.V. All rights reserved.)

TP53 is a critical tumor suppressor gene that regulates cell cycle progression, apoptosis, cellular senescence and many other properties critical for control of normal cellular growth and death. Due to the pleiotropic effects that TP53 has on gene expression and cellular physiology, mutations at this tumor suppressor gene result in diverse physiological effects. T53 mutations are frequently detected in numerous cancers. The expression of TP53 can be induced by various agents used to treat cancer patients such as chemotherapeutic drugs and ionizing radiation. Radiation will induce Ataxia telangiectasia mutated (ATM) and other kinases that results in the phosphorylation and activation of TP53. TP53 is also negatively regulated by other mechanisms, such as ubiquitination by ligases such as MDM2. While TP53 has been documented to control the expression of many "classical" genes (e.g., p21 Cip-1 , PUMA, Bax) by transcriptional mechanisms for quite some time, more recently TP53 has been shown to regulate microRNA (miR) gene expression. Different miRs can promote oncogenesis (oncomiR) whereas others act to inhibit tumor progression (tumor suppressor miRs). Targeted therapies to stabilize TP53 have been developed by various approaches, MDM2/MDM4 inhibitors have been developed to stabilize TP53 in TP53-wild type (WT) tumors. In addition, small molecules have been isolated that will reactivate certain mutant TP53s. Both of these types of inhibitors are in clinical trials. Understanding the actions of TP53 may yield novel approaches to suppress cancer, aging and other health problems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that participates in an array of critical cellular processes. GSK-3 was first characterized as an enzyme that phosphorylated and inactivated glycogen synthase. However, subsequent studies have revealed that this moon-lighting protein is involved in numerous signaling pathways that regulate not only metabolism but also have roles in: apoptosis, cell cycle progression, cell renewal, differentiation, embryogenesis, migration, regulation of gene transcription, stem cell biology and survival. In this review, we will discuss the roles that GSK-3 plays in various diseases as well as how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, Wnt/beta-catenin, hedgehog, Notch and TP53. Mutations that occur in these and other pathways can alter the effects that natural GSK-3 activity has on regulating these signaling circuits that can lead to cancer as well as other diseases. The novel roles that microRNAs play in regulation of the effects of GSK-3 will also be evaluated. Targeting GSK-3 and these other pathways may improve therapy and overcome therapeutic resistance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Various, diverse molecules contribute to the tumor microenvironment and influence invasion and metastasis. In this review, the roles of neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) in the tumor microenvironment and sensitivity to therapy will be discussed. The lipocalin family of proteins has many important functions. For example when NGAL forms a complex with MMP-9 it increases its stability which is important in cancer metastasis. Small hydrophobic molecules are bound by NGAL which can alter their entry into and efflux from cells. Iron transport and storage are also influenced by NGAL activity. Regulation of iron levels is important for survival in the tumor microenvironment as well as metastasis. Innate immunity is also regulated by NGAL as it can have bacteriostatic properties. NGAL and MMP-9 expression may also affect the sensitivity of cancer cells to chemotherapy as well as targeted therapy. Thus NGAL and MMP-9 play important roles in key processes involved in metastasis as well as response to therapy. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The roles of the epidermal growth factor receptor (EGFR) signaling pathway in various cancers including breast, bladder, brain, colorectal, esophageal, gastric, head and neck, hepatocellular, lung, neuroblastoma, ovarian, pancreatic, prostate, renal and other cancers have been keenly investigated since the 1980's. While the receptors and many downstream signaling molecules have been identified and characterized, there is still much to learn about this pathway and how its deregulation can lead to cancer and how it may be differentially regulated in various cell types. Multiple inhibitors to EGFR family members have been developed and many are in clinical use. Current research often focuses on their roles and other associated pathways in cancer stem cells (CSCs), identifying sites where therapeutic resistance may develop and the mechanisms by which microRNAs (miRs) and other RNAs regulate this pathway. This review will focus on recent advances in these fields with a specific focus on breast cancer and breast CSCs. Relatively novel areas of investigation, such as treatments for other diseases (e.g., diabetes, metabolism, and intestinal parasites), have provided new information about therapeutic resistance and CSCs.

Antivenom is the definitive treatment for venomous snakebites. Alternative treatments warrant investigation because antivenom is sometimes unavailable, expensive, and can have deleterious side effects. This study assesses the efficacy of trypsin to treat coral snake envenomation in an in vivo porcine model. A randomized, blinded study was conducted. Subjects were 13 pigs injected subcutaneously with 1 mL of eastern coral snake venom (10 mg/mL) in the right distal hind limb. After 1 min, subjects were randomized to have the envenomation site injected with either 1 mL of saline or 1 mL of trypsin (100 mg/mL) by a blinded investigator. Clinical endpoint was survival for 72 h or respiratory depression defined as respiratory rate <15 breaths per minute, falling pulse oximetry, or agonal respirations. Fisher's exact t test was used for between group comparisons. Average time to toxicity for the saline control was 263 min (191-305 min). The development of respiratory depression occurred more frequently in control pigs than treated pigs (p = 0.009). Four of the six pigs that received trypsin survived to the end of the 3-day study. No control pigs survived. Two of the trypsin treatment pigs died with times to toxicity of 718 and 971 min. Survival to 12 and 24 h was significantly greater in the trypsin treatment group (p = 0.002, p = 0.009, respectively). Local injection of trypsin, a proteolytic enzyme, at the site of envenomation decreased the toxicity of eastern coral snake venom and increased survival significantly. Further investigation is required before these results can be extended to human snakebites.

A zebra finch (Taeniopygia guttata) housed in a neuroscience laboratory was observed to have numerous feather mites. Subsequently, similar mites were found on other birds in the animal facility and research space. The most abundant mite was a novel, undescribed species in the genus Neocheyletiella. Whereas known Neocheyletiella mites have previously been characterized as skin parasites of various birds worldwide, the species on the zebra finches is unique because it lives and builds nests in the feathers. Infrequent specimens of a 'true' feather mite, a new species of Megninialges, were present also. Although multiple treatments using a pyrethrin spray were effective in eradicating the mites, topical ivermectin later was found to be more efficacious, better tolerated by the birds, and less labor intensive. This case highlights the general dearth of information regarding ectoparasites in zebra finches, even though these are the most frequently used songbirds in biomedical research. The mite epizootic also underscores the diverse pathogens possible in zebra finches that arrive from outside sources and why ongoing health monitoring of finch colonies is warranted.

Farnesoid X receptor (FXR) is a nuclear receptor that regulates bile acid metabolism and transport. Mice lacking expression of FXR (FXR KO) have a high incidence of foci of cellular alterations (FCA) and liver tumors. Here, we report that Helicobacter hepaticus infection is necessary for the development of increased hepatitis scores and FCA in previously Helicobacter-free FXR KO mice. FXR KO and wild-type (WT) mice were sham-treated or orally inoculated with H. hepaticus. At 12 months post-infection, mice were euthanized and liver pathology, gene expression, and the cecal microbiome were analyzed. H. hepaticus induced significant increases hepatitis scores and FCA numbers in FXR KO mice (P<0.01 and P<0.05, respectively). H. hepaticus altered the beta diversity of cecal microbiome in both WT and FXR KO mice compared to uninfected mice (P<0.05). Significant upregulation of β-catenin, Rela, Slc10a1, Tlr2, Nos2, Vdr, and Cyp3a11 was observed in all FXR KO mice compared to controls (P<0.05). Importantly, H. hepaticus and FXR deficiency were necessary to significantly upregulate Cyp2b10 (P<0.01). FXR deficiency was also a potent modulator of the cecal microbiota, as observed by a strong decrease in alpha diversity. A significant decrease in Firmicutes, particularly members of the order Clostridiales, was observed in FXR KO mice (P<0.05 and FDR<5%, ANOVA). While FXR deficiency strongly affects expression of genes related to immunity and bile acid metabolism, as well as the composition of the microbiome; however, its deficiency was not able to produce significant histopathological changes in the absence of H. hepaticus infection.

Brown recluse spider bites result in necrotic skin lesions for which there is no known antidote. Since venom toxins are proteins, a proteolytic enzyme like trypsin might be effective in reducing toxicity. The aim of this study was to conduct a randomized controlled trial of trypsin to treat brown recluse spider bites in guinea pigs. Subjects were 18 female guinea pigs. Anesthesia for injections was inhaled isoflurane. Analgesia was 0.05 mg/kg of buprenorphine twice a day as needed. Intervention was intradermal injection of 30 μg of brown recluse venom (Spider Pharm, Yarnell, AZ). Immediately after envenomation, subjects were randomized to two groups of nine: trypsin 10 μg in 1 mL normal saline and 1 mL of normal saline. The primary outcome was lesion area over a 10-day time period. Statistical analysis was performed with repeated measures ANOVA. Mean lesion area was smaller but not statistically different in the placebo group. Maximum lesion size occurred at day 4 in both groups, when lesion area was 76.1 ± 108.2 mm(2) in the placebo group and 149.7 ± 127.3 mm(2) in the treatment group. P value was 0.15 for placebo vs. treatment. This study did not establish a role for trypsin as a treatment for brown recluse spider bites in a guinea pig model.

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.

Considerable progress has been made in understanding the roles of Helicobacter pylori in inflammation and gastric cancer; however, far less is known about the roles of enterohepatic Helicobacter species (EHS) in carcinogenesis and their zoonotic or pathogenic potential. We determined the prevalence of EHS infection in a cohort of geriatric rhesus monkeys in which intestinal adenocarcinoma (IAC) is common and investigated the association between EHS infection and IAC. The cohort consisted of 36 animals, 14 of which (age 26-35 years) had IAC. Of the 36 rhesus, 35 (97%) were positive for EHS using PCR or bacterial isolation from faeces, colonic or tumour tissues. Only a single rhesus, which had IAC, was negative for EHS by all detection methods. The EHS identified by 16S rRNA sequencing in this study were from three Helicobacter taxa: Helicobacter macacae (previously rhesus monkey taxon 1), Helicobacter sp. rhesus monkey taxon 2, previously described from strain MIT 99-5507, and Helicobacter sp. rhesus monkey taxon 4, related to Helicobacter fennelliae. Thirteen of 14 monkeys with IAC were positive for either H. macacae (7/13, 54%), EHS rhesus monkey taxon 4 (4/13, 31%) or a mixture of the two EHS (2/13, 15%). These results indicate that EHS are prevalent among aged rhesus macaques with IAC. Using Helicobacter genus-specific florescent in situ hybridization, EHS were detected on the surface of colonic epithelia of infected monkeys. All Helicobacter isolates, including H. macacae, effectively adhered to, invaded, and significantly induced proinflammatory genes, including IL-8, IL-6, TNF-α and iNOS, while downregulating genes involved in the function of inflammasomes, particularly IL-1β, CASPASE-1, NRLP3, NLRP6 and NLRC4 in the human colonic T84 cell line (P<0.0001). These results suggest that EHS may represent an aetiological agent mediating diarrhoea, chronic inflammation, and possibly intestinal cancer in non-human primates, and may play a role in similar disease syndromes in humans. Downregulation of inflammasome function may represent an EHS strategy for long-term persistence in the host and play a role in inducing pathological changes in the host's lower bowel., (© 2014 The Authors.)

Objectives: Gastric colonisation with intestinal flora (IF) has been shown to promote Helicobacter pylori (Hp)-associated gastric cancer. However, it is unknown if the mechanism involves colonisation with specific or diverse microbiota secondary to gastric atrophy., Design: Gastric colonisation with Altered Schaedler's flora (ASF) and Hp were correlated with pathology, immune responses and mRNA expression for proinflammatory and cancer-related genes in germ-free (GF), Hp monoassociated (mHp), restricted ASF (rASF; 3 species), and specific pathogen-free (complex IF), hypergastrinemic INS-GAS mice 7 months postinfection., Results: Male mice cocolonised with rASFHp or IFHp developed the most severe pathology. IFHp males had the highest inflammatory responses, and 40% developed invasive gastrointestinal intraepithelial neoplasia (GIN). Notably, rASFHp colonisation was highest in males and 23% developed invasive GIN with elevated expression of inflammatory biomarkers. Lesions were less severe in females and none developed GIN. Gastritis in male rASFHp mice was accompanied by decreased Clostridum species ASF356 and Bacteroides species ASF519 colonisation and an overgrowth of Lactobacillus murinus ASF361, supporting that inflammation-driven atrophy alters the gastric niche for GI commensals. Hp colonisation also elevated expression of IL-11 and cancer-related genes, Ptger4 and Tgf-β, further supporting that Hp infection accelerates gastric cancer development in INS-GAS mice., Conclusions: rASFHp colonisation was sufficient for GIN development in males, and lower GIN incidence in females was associated with lower inflammatory responses and gastric commensal and Hp colonisation. Colonisation efficiency of commensals appears more important than microbial diversity and lessens the probability that specific gastrointestinal pathogens are contributing to cancer risk.

Helicobacter pylori (H. pylori) and hepatitis C virus (HCV) infect millions of people and can induce cancer. We investigated if H. pylori infection promoted HCV-associated liver cancer. Helicobacter-free C3B6F1 wild-type (WT) and C3B6F1-Tg(Alb1-HCVN)35Sml (HT) male and female mice were orally inoculated with H. pylori SS1 or sterile media. Mice were euthanized at ~12 mo postinoculation and samples were collected for analyses. There were no significant differences in hepatocellular tumor promotion between WT and HT mice; however, HT female mice developed significantly larger livers with more hepatic steatosis than WT female mice. H. pylori did not colonize the liver nor promote hepatocellular tumors in WT or HT mice. In the stomach, H. pylori induced more corpus lesions in WT and HT female mice than in WT and HT male mice, respectively. The increased corpus pathology in WT and HT female mice was associated with decreased gastric H. pylori colonization, increased gastric and hepatic interferon gamma expression, and increased serum Th1 immune responses against H. pylori. HT male mice appeared to be protected from H. pylori-induced corpus lesions. Furthermore, during gastric H. pylori infection, HT male mice were protected from gastric antral lesions and hepatic steatosis relative to WT male mice and these effects were associated with increased serum TNF-α. Our findings indicate that H. pylori is a gastric pathogen that does not promote hepatocellular cancer and suggest that the HCV transgene is associated with amelioration of specific liver and gastric lesions observed during concurrent H. pylori infection in mice.