Infected animals will produce reactive oxygen species (ROS) and other inflammatory molecules that help fight pathogens, but can inadvertently damage host tissue. Therefore specific responses, which protect and repair against the collateral damage caused by the immune response, are critical for successfully surviving pathogen attack. We previously demonstrated that ROS are generated during infection in the model host Caenorhabditis elegans by the dual oxidase Ce-Duox1/BLI-3. Herein, an important connection between ROS generation by Ce-Duox1/BLI-3 and upregulation of a protective transcriptional response by SKN-1 is established in the context of infection. SKN-1 is an ortholog of the mammalian Nrf transcription factors and has previously been documented to promote survival, following oxidative stress, by upregulating genes involved in the detoxification of ROS and other reactive compounds. Using qRT-PCR, transcriptional reporter fusions, and a translational fusion, SKN-1 is shown to become highly active in the C. elegans intestine upon exposure to the human bacterial pathogens, Enterococcus faecalis and Pseudomonas aeruginosa. Activation is dependent on the overall pathogenicity of the bacterium, demonstrated by a weakened response observed in attenuated mutants of these pathogens. Previous work demonstrated a role for p38 MAPK signaling both in pathogen resistance and in activating SKN-1 upon exposure to chemically induced oxidative stress. We show that NSY-1, SEK-1 and PMK-1 are also required for SKN-1 activity during infection. Evidence is also presented that the ROS produced by Ce-Duox1/BLI-3 is the source of SKN-1 activation via p38 MAPK signaling during infection. Finally, for the first time, SKN-1 activity is shown to be protective during infection; loss of skn-1 decreases resistance, whereas increasing SKN-1 activity augments resistance to pathogen. Overall, a model is presented in which ROS generation by Ce-Duox1/BLI-3 activates a protective SKN-1 response via p38 MAPK signaling.

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp co-expression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression, and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp co-expression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.

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The Caenorhabditis elegans oxidative stress response transcription factor, SKN-1, is essential for the maintenance of redox homeostasis and is a functional ortholog of the Nrf family of transcription factors. The numerous levels of regulation that govern these transcription factors underscore their importance. Here, we add a thioredoxin, encoded by trx-1, to the expansive list of SKN-1 regulators. We report that loss of trx-1 promotes nuclear localization of intestinal SKN-1 in a redox-independent, cell non-autonomous fashion from the ASJ neurons. Furthermore, this regulation is not general to the thioredoxin family, as two other C. elegans thioredoxins, TRX-2 and TRX-3, do not play a role in this process. Moreover, TRX-1-dependent regulation requires signaling from the p38 MAPK-signaling pathway. However, while TRX-1 regulates SKN-1 nuclear localization, classical SKN-1 transcriptional activity associated with stress response remains largely unaffected. Interestingly, RNA-Seq analysis revealed that loss of trx-1 elicits a general, organism-wide down-regulation of several classes of genes; those encoding for collagens and lipid transport being most prevalent. Together, these results uncover a novel role for a thioredoxin in regulating intestinal SKN-1 nuclear localization in a cell non-autonomous manner, thereby contributing to the understanding of the processes involved in maintaining redox homeostasis throughout an organism.KEYWORDS Caenorhabditis elegans; oxidative stress response; thioredoxin; ASJ neurons; cell non-autonomous signaling T HE ability of an organism to maintain redox homeostasis is critical for its survival. At the cellular level, exposure to oxidative insult can irreversibly damage DNA, proteins, and lipids, all of which can lead to cell apoptosis or necrosis (Ray et al. 2012;Thanan et al. 2014). At the organismal level, unresolved oxidative stress is considered a hallmark of numerous life-threatening diseases, including Alzheimer's, Parkinson's disease, atherosclerosis, and several forms of cancer (Hybertson et al. 2011;Thanan et al. 2014). To counteract oxidative insults, organisms have evolved specific pathways capable of sensing and responding to both endogenous and exogenous oxidative stress, termed "the oxidative stress response" (Lushchak 2011). This response is coordinated by oxidative stress response transcription factors, which activate the expression of detoxification and repair enzymes (McCord and Fridovich 1969;Anderson 1998;Lushchak 2011). In mammals, the major oxidative stress transcription factor is the nuclear factor erythroid 2-related factor, Nrf2, one of three Nrf paralogs (Hybertson et al. 2011). To ensure efficient surveillance of redox homeostasis, several mechanisms regulate Nrf2, including those that regulate its subcellular localization and protein turnover (Marinho et al. 2014).The nematode Caenorhabditis elegans utilizes a functional ortholog of mammalian Nrf proteins, SKN-1, to coordinate its oxidative stress response (Walker et al. 2000; found in the regu...

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OBJECTIVESTo present a canine patient with multiple neoplasms identified within a single adrenal gland and to discuss the diagnostics and management of the condition. METHODSA rare canine patient with multiple neoplasms present within a single adrenal gland is described. RESULTSAn 11-year-old neutered female Labrador retriever was presented with a history of tachypnoea, polyuria and polydipsia. Diagnostics included haematology, biochemistry, a low-dose dexamethasone suppression test, plasma ACTH, urine metanephrine and normetanephrine, serial Doppler blood pressure measurements and computed tomography (CT).CT identified a large bi-lobed abdominal mass in the location of the left adrenal gland. The results of endocrine testing were consistent with adrenal-dependant hyperadrenocorticism, although the presence of intermittent hypertension and borderline high metanephrines were suggestive of a concurrent phaeochromocytoma.As a phaeochromocytoma could not be ruled out, preparatory steps were taken preoperatively including ensuring appropriate drugs were available, pre-emptive blood typing and arterial blood pressure monitoring. A unilateral adrenalectomy was performed. Histopathological examination identified a benign myelolipoma and two distinct malignancies within the adrenal gland; a phaeochromocytoma and an adrenocortical adenocarcinoma. Postoperative recovery was uneventful and resolution of all clinical signs was observed within 4 weeks. No signs of recurrence or metastatic disease were noted 5 months postoperatively. The patient was euthanised 12 months postoperatively due to metastatic disease. STATEMENT (CONCLUSIONS)Multiple unilateral adrenal neoplasms are rare. This case demonstrates the importance of considering the presence of multiple functional tumours in a single patient and performing detailed endocrine testing to optimise pre/perioperative stabilisation and outcome.