Coinfection by Mycobacterium marinum and Mycolicibacterium fortuitum in a captive adult diamondback water snake causing disseminated mycobacteriosis with acute cutaneous ulceration.

An adult male captive diamondback water snake (Nerodia rhombifer) was found dead after a 1-d history of lethargy and cutaneous ulcers. The snake had eaten 2 sunfish (Mola spp.) 5 d before death. Gross examination revealed white-to-tan nodules in the lung and liver and segmental intestinal impactions with digested fish. Histopathology confirmed disseminated granulomas with numerous intrahistiocytic acid-fast bacteria in the skin, skeletal muscle, lung, liver, and intestines. Mycobacterium marinum and Mycolicibacterium fortuitum were identified by culture of the hepatic granuloma, followed by PCR and rpoB gene sequencing. To our knowledge, this is the first description of M. marinum and M. fortuitum coinfection in this species. Although M. fortuitum has been isolated from reptiles, lesions associated with its presence in tissues have not been described previously. Interestingly, the mineralization within granulomas that we observed in our case is not reported in mycobacterial infection in reptiles, whereas this finding is common in mammals.

Neoplastic signatures: Comparative proteomics of canine hepatobiliary neuroendocrine tumors to normal niche tissue.

Hepatobiliary neuroendocrine neoplasms are rare cancers in humans and dogs. To date, no large-scale primary hepatobiliary neoplasm omics analyses exist in any species. This limits the development of diagnostic biomarkers and targeted therapeutics. Neuroendocrine cancers are a heterogenous group of neoplasms categorized by their tissue-of-origin. Because the anatomic niche of neuroendocrine neoplasms shapes tumor phenotype, we sought to compare the proteomes of 3 canine hepatobiliary neoplasms to normal hepatobiliary tissue and adrenal glands with the objective of identifying unique protein signatures. Protein was extracted from formalin-fixed paraffin-embedded samples and submitted for tandem mass spectroscopy. Thirty-two upregulated and 126 downregulated differentially expressed proteins were identified. Remarkably, 6 (19%) of the upregulated proteins are correlated to non-hepatobiliary neuroendocrine neoplasia and 16 (50%) are functionally annotated within the exosome cellular compartment key to neuroendocrine signaling. Twenty-six (21%) downregulated proteins are enriched in metabolic pathways consistent with alterations in cancer. These results suggests that characteristic neoplastic protein signatures can be gleaned from small data sets using a comparative proteomics approach.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

Failure to sense energy depletion may be a novel therapeutic target in chronic kidney disease.

The kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD.

Thioacetamide-induced hepatic fibrosis in the common marmoset.

The common marmoset (Callithrix jacchus) is a nonhuman primate that is used for preclinical research on stem cell transplantation therapies due to its similarity to human beings as well as its small size, enabling researchers to perform experiments without preparing a large number of cells. In this study, we developed a marmoset hepatic fibrosis model for regenerative medicine research. Six female marmosets aged 4-6 years were administered thioacetamide (TAA) at a dose of 2.5-40 mg/kg two or three times a week. Hepatic fibrosis was assessed by liver biopsy when blood chemistry indicated liver damage. Administration of TAA increased total bile acid, aspartate aminotransferase, and total bilirubin and decreased serum albumin levels. Following more than 11 weeks of continuous injection of TAA, histological analyses detected hepatic fibrosis in all animals. Type IV collagen 7S serum levels in animals with hepatic fibrosis were significantly higher than in normal animals as a possible marker of hepatic fibrosis in marmosets. Serial liver biopsies following the last administration of TAA revealed that induced fibrosis remained up to 11 weeks. The results suggest that continuous TAA administration induces persistent hepatic fibrosis in the common marmoset and this nonhuman primate hepatic fibrosis model have the possibility to evaluate the therapeutic effects of test samples to ameliorate hepatic fibrosis.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy.

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

Loop diuretics affect skeletal myoblast differentiation and exercise-induced muscle hypertrophy.

Muscle wasting or sarcopenia contributes to morbidity and mortality in patients with cancer, renal failure, or heart failure, and in elderly individuals. Na+-K+-2Cl- cotransporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle, where it contributes to the generation of membrane ion currents and potential. However, the physiologic function of NKCC1 in myogenesis is unclear. We investigated this issue using the NKCC1 inhibitors bumetanide and furosemide, which are commonly used loop diuretics. NKCC1 protein levels increased during C2C12 murine skeletal myoblast differentiation, similarly to those of the myogenic markers myogenin and myosin heavy chain (MHC). NKCC1 inhibitors markedly suppressed myoblast fusion into myotubes and the expression of myogenin and MHC. Furthermore, phosphorylated and total NKCC1 levels were elevated in mouse skeletal muscles after 6 weeks' voluntary wheel running. Immunofluorescence analyses of myofiber cross-sections revealed more large myofibers after exercise, but this was impaired by daily intraperitoneal bumetanide injections (0.2 or 10 mg/kg/day). NKCC1 plays an essential role in myogenesis and exercise-induced skeletal muscle hypertrophy, and sarcopenia in patients with renal or heart failure may be attributable to treatment with loop diuretics.

An adult case of severe steroid-resistant Henoch-Schönlein purpura nephritis treated with intravenous cyclophosphamide and tonsillectomy.

A previously healthy 37-year-old Canadian man living in Japan visited a hospital in Thailand while traveling because of edematous legs, purpura, arthralgia, bloody stool, and fever after an insect bite. Henoch-Schönlein purpura (HSP) was suspected. His creatinine level was 5.2 mg/dL. He was treated with oral prednisolone (PSL) and oral cyclophosphamide (CPA); after treatment, his creatinine level improved to 2.4 mg/dL. Upon returning to Japan, he was admitted to the National Center for Global Health and Medicine Hospital in Tokyo. A kidney biopsy was performed, and HSP nephritis (HSPN) was diagnosed. Renal dysfunction and proteinuria persisted despite 4 administrations of steroid-pulse therapy and 3 sessions of plasma exchange. Finally, he was treated with intravenous cyclophosphamide (IVCY). His creatinine level and proteinuria markedly improved. His microscopic hematuria disappeared after he underwent tonsillectomy. There have been only a few case reports describing patients with adult-onset HSPN necessitating IVCY. We present here a rare case of steroid-resistant HSPN treated with IVCY and tonsillectomy, with reference to some recent findings.

[Case of serious HIV-associated nephropathy resulting in the introduction of hemodialysis].

A previously healthy 46-year-old black man visited the other hospital because of fever, appetite loss and nausea. Renal dysfunction, liver injury, and a highly markedly elevated LDH level were found. Abdominal CT demonstrated enlarged liver, spleen, kidney and lymph nodes. Human immunodeficiency virus (HIV) was serologically positive. His serum BUN, creatinine and potassium were 74.9 mg/dL, 11.78 mg/dL, and 5.6 mEq/L, respectively. After admission, anuria persisted and the progression of renal failure continued despite various treatment methods, necessitating the introduction of maintenance hemodialysis(HD). A kidney biopsy was performed to confirm classical HIV-associated nephropathy (HIVAN). Antiretroviral therapy (ART) was started. Although urine was transiently excreted, HD could not be discontinued. It has been reported that HIVAN is too difficult to treat and that kidney dysfunction seldom recovers. HIVAN is well-known to occur frequently in black HIV-infected patients. However, in Japan, there have been only a few reports describing patients with serious HIVAN and renal failure necessitating HD. We present here a very rare case with HIVAN, with reference to some recent findings.

Identification of FGF2-response element in the rat bone sialoprotein gene promoter.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Basic fibroblast growth factor (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. In skeletal tissues, FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of bone cells. To determine the molecular mechanism of FGF2 regulation of osteogenesis, we have analyzed the effects of FGF2 on the expression of BSP in the rat osteosarcoma cell line ROS 17/2.8. FGF2 at 10 ng/ml, increased BSP mRNA levels approximately 4-fold; the stimulation was first evident at 3 hr, reached maximal levels at 6 hr. The stability of the BSP mRNA was not significantly affected by FGF2, suggesting that the increased mRNA was due to increased transcription. From transient transfection analyses using various BSP promoter-luciferase constructs, a FGF2 response element (FRE) (nts -92 to -85, "GGTGAGAA") was identified as a target of transcriptional activation by FGF2. Ligation of two copies of the FRE 5' to an SV40 promoter was sufficient to confer FGF responsive transcription. A sequence-specific protein-DNA complex, formed with a double-stranded oligonucleotide encompassing the FRE and nuclear extracts from ROS 17/2.8 cells, but not from fibroblasts, was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed both basal and FGF2-mediated promoter activity. Thus, we have identified a novel FRE within the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription.