Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone.

Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.

Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling.

BACKGROUND: Prostate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear. METHODS: To investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines. RESULTS: Low ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor. CONCLUSIONS: ABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.

Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy.

Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Brain Iron Distribution after Multiple Doses of Ultra-small Superparamagnetic Iron Oxide Particles in Rats.

The purpose of this study is to determine the effects of high cumulative doses of ultra-small paramagnetic iron oxide (USPIO) used in neuroimaging studies. We intravenously administered 8 mg/kg of 2 USPIO compounds daily for 4 wk to male Sprague-Dawley rats (Crl:SD). Multiecho gradient-echo MRI, serum iron levels, and histology were performed at the end of dosing and after a 7-d washout period. R2* maps and quantitative susceptibility maps (QSM) were generated from multiecho gradient-echo data. R2* maps and QSM showed iron accumulation in brain ventricles on MR images acquired at the 4- and 5-wk time points. Estimates from QSM data showed ventricular iron concentration was equal to or higher than serum iron concentration. Histologic analysis revealed choroid plexus hemosiderosis and midbrain vacuolation, without iron deposition in brain parenchyma. Serum iron levels increased with administration of both compounds, and a 7-d washout period effectively reduced serum iron levels of one but not both of the compounds. High cumulative doses from multiple, frequent administrations of USPIO can lead to iron deposition in brain ventricles, resulting in persistent signal loss on T2*-weighted images. Techniques such as QSM are helpful in quantifying iron biodistribution in this situation.

Pathology of Aging in NOD scid gamma Female Mice.

In the past decade, NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ (NSG, NOD scid gamma) mice have become a model of choice in several areas of biomedical research; however, comprehensive data on their spontaneous age-related pathology are not currently available in the literature. The prevalence of spontaneous morbidity affecting aged NSG female breeders enrolled in a parasitology study was documented with classification of neoplastic and non-neoplastic (inflammatory, metabolic, degenerative) lesions. Malignant mammary neoplasms were most commonly diagnosed, often accompanied by pulmonary metastases, while a low frequency of lymphoma and histiocytic sarcoma was documented. The major inflammatory conditions were suppurative pleuropneumonia and bronchopneumonia with abscess formation, from which Pasteurella pneumotropica was commonly isolated, followed by otitis media. Both inflammatory and degenerative lesions of the genital tract were identified, along with neoplasms such as endometrial yolk sac carcinomas and granulosa cell tumors. Novel conditions identified included renal tubular degeneration and necrosis associated with 2 concurrent types of intranuclear inclusions, focal or multifocal hyperostosis of the skull, and neuroendocrine tumors of the mesometrium. The majority of degenerative lesions that affected the genital tract, endocrine, and skeletal systems did not represent the actual underlying cause of death but rather were considered incidental findings. This study indicates that both inflammatory and neoplastic conditions contribute to morbidity and mortality in experimentally manipulated aged female NSG mice.

Surface-enhanced resonance Raman scattering nanostars for high-precision cancer imaging.

The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.

Tolerance and efficacy of emamectin benzoate and ivermectin for the treatment of Pseudocapillaria tomentosa in laboratory zebrafish (Danio rerio).

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Deletion of Ptprd and Cdkn2a cooperate to accelerate tumorigenesis.

PTPRD encodes the protein tyrosine phosphatase receptor type D and is frequently inactivated across many human cancers. Despite its frequent inactivation, it is unknown whether loss of PTPRD promotes tumorigenesis in vivo. PTPRD is located on chromosome 9p, as is CDKN2A, and the two loci are frequently deleted together. Here, we show that co-deletion of Ptprd and Cdkn2a cooperate to accelerate tumorigenesis. Interestingly,heterozygous loss of Ptprd was sufficient to promote tumorigenesis in our model, suggesting that Ptprd may be a haploinsufficient tumor suppressor. The loss of Ptprd resulted in changes to the tumor spectrum in mice and increased the frequency of lymphomas. In total, we reveal that Ptprd is a tumor suppressor that can promote tumorigenesis in concert with Cdkn2a loss.

Trichodina xenopodus, a ciliated protozoan, in a laboratory-maintained Xenopus laevis.

A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding.

Effect of linezolid on clinical severity and pulmonary cytokines in a murine model of influenza A and Staphylococcus aureus coinfection.

UNLABELLED: Excessive inflammation contributes to the severity of post influenza pneumonia caused by methicillin resistant S.aureus (MRSA). Linezolid, vancomycin, and clindamycin are antibiotics used for MRSA infections. Linezolid has immunomodulatory properties. We report on the effects of the three antibiotics on microbial clearance, pulmonary cytokines and clinical course in a murine model of influenza and MRSA coinfection. METHODS: B6 mice were infected with influenza A virus and 3 days later with MRSA, both intranasally. Treatment with placebo, linezolid, vancomycin or clindamycin started immediately after MRSA infection and continued for 72 hours. Bacterial and viral titers as well as cytokine concentrations in the lungs were assessed 4 and 24 hours after MRSA coinfection. Mice were weighted daily for 13 days. RESULTS: Coinfected mice had increased pulmonary IL-1ß, TNF-α and mKC at 4 and 24 hours, IL-6, IL-10 and IL-12 at 4 hours and IFN-γ at 24 hours after MRSA coinfection (all P<0.05). Compared to placebo, coinfected mice treated with linezolid, vancomycin or clindamycin had decreased pulmonary IL-6 and mKC at 4 hours and IFN-γ at 24 hours after MRSA coinfection (all P<0.05). IL-1ß, TNF-α and IL-12 were similar in antibiotic-treated and placebo groups. All antibiotics similarly reduced MRSA without effect on influenza titers. Linezolid-treated mice had less weight loss on days 4-6 after influenza infection compared to placebo (all P<0.05). On all other days weight change was similar among all groups. CONCLUSIONS: This is the first report comparing the effects of antibiotics on cytokines and clinical outcome in a murine model of influenza and MRSA coinfection. Compared to placebo, antibiotic treatment reduced maximum concentration of IL-6, mKC and IFN-γ in the lungs without any difference among antibiotics. During treatment, only linezolid delayed weight loss compared to placebo.

Anatomic, hematologic, and biochemical features of C57BL/6NCrl mice maintained on chronic oral corticosterone.

Metabolic syndrome is a condition that typically includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension. Disruption of the hypothalamic-pituitary-adrenal axis, a regulator of corticosterone secretion, occurs in some cases of metabolic syndrome and obesity, and Cushing hypercortisolemia is associated with obesity and metabolic disorders. We therefore assessed anatomic and clinical pathology in C57BL/6NCrl mice to evaluate the effects of chronic corticosterone in the drinking water at doses of 25, 50, and 100 µg/mL for 25 d. Treated mice developed obesity, glucose intolerance, electrolyte aberrations, and dyslipidemia that were dose-dependent and most severe in the 100-mu;g/mL treatment group. To evaluate return to normal function, additional C57BL/6NCrl mice received corticosterone-free water for 2 wk after the 25-d treatment period. According to results of gross examination, mice appeared to recover within days of exogenous corticosterone withdrawal; however, adrenal gland vacuolation and protein, lipid, and electrolyte abnormalities persisted. Together, these findings support chronic corticosterone exposure through the drinking water as a potentially useful, noninvasive method to induce some features of metabolic syndrome.

Strategies to prevent, treat, and provoke Corynebacterium-associated hyperkeratosis in athymic nude mice.

Athymic nude mice infected with Corynebacterium bovis typically exhibit transient hyperkeratotic dermatitis. Our vivarium experienced an increased incidence of disease characterized by persistent skin lesions and increased mortality, leading to this study. For detection of infection, skin and buccal swab methods showed comparable sensitivities in nude mice. Various prevention, treatment, and eradication strategies were evaluated through clinical assessment, microbiology, and histopathology. In experimentally naïve athymic nude mice, a 2-wk course of prophylactic amoxicillin-containing diet (1200 ppm amoxicillin; effective dose, 200 mg/kg) was ineffective at preventing infection or disease. There was also no significant difference in disease duration or severity in athymic nude mice that received amoxicillin diet or penicillin-streptomycin topical spray (penicillin, 2500 U/mL; streptomycin, 2500 µg/mL). Prolonged treatment with 4 or 8 wk of amoxicillin diet cleared only a small number of athymic nude mice that had subclinical C. bovis infections. Antibiotic sensitivity of C. bovis isolates demonstrated a small colony isolate with less susceptibility to all antibiotics compared with a large colony isolate. Resistance did not appear to develop after prolonged treatment with amoxicillin. Provocation testing by administration of cyclophosphamide (50 mg/kg i.p. every 48 to 72 h for 90 d) to subclinically infected athymic nude mice resulted in prolonged clinical disease that waxed and waned without progression to severe disease. Our findings suggest that antibiotic prophylaxis and treatment of clinical disease in experimentally naïve mice is unrewarding, eradication of bacterial infection is difficult, and severe disease associated with C. bovis is likely multifactorial.