TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation.

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1- linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNFRSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

Effects of Plasma-Activated Water Treatment on the Inactivation of Microorganisms Present on Cherry Tomatoes and in Used Wash Solution.

Herein, we investigated the potential of plasma-activated water (PAW) as a wash solution for the microbial decontamination of cherry tomatoes. We analyzed the efficacy of PAW as a bactericidal agent based on reactive species and pH. Immersion for 5 min in PAW15 (generated via plasma activation for 15 min) was determined as optimal for microbial decontamination of fresh produce. The decontamination efficacy of PAW15 exceeded those of mimic solutions with equivalent reactive species concentrations and pH (3.0 vs. 1.7 log reduction), suggesting that the entire range of plasma-derived reactive species participates in decontamination rather than a few reactive species. PAW15-washing treatment achieved reductions of 6.89 ± 0.36, 7.49 ± 0.40, and 5.60 ± 0.05 log10 CFU/g in the counts of Bacillus cereus, Salmonella sp., and Escherichia coli O157:H7, respectively, inoculated on the surface of cherry tomatoes, with none of these strains detected in the wash solution. During 6 days of 25 °C storage post-washing, the counts of aerobic bacteria, yeasts, and molds were below the detection limit. However, PAW15 did not significantly affect the viability of RAW264.7 cells. These results demonstrate that PAW effectively inactivates microbes and foodborne pathogens on the surface of cherry tomatoes and in the wash solution. Thus, PAW could be used as an alternative wash solution in the fresh produce industry without cross-contamination during washing and environmental contamination by foodborne pathogens or potential risks to human health.

Changes in Mechanical Properties of Vesicles by Mucin in Aqueous Solution.

The mechanical properties of vesicles were investigated as they were prepared, according to the ratio of mucin to dipalmitoylphosphatidylcholine (DPPC), using an atomic force microscope (AFM). After the confirmation of the vesicle adsorption on a mica surface, an AFM-tip deflection, caused by the interaction between the tip and the vesicle, was measured. The deflection showed that the tip broke through into the vesicle twice. Each break meant a tip-penetration into the upper and lower portion of the vesicle. Only the first penetration allowed the Hertzian model available to estimate the vesicle mechanical moduli. Two moduli reduced as the ratio of mucin to DPPC increased to 0.5, but the moduli were little changed above the 0.5 ratio. These results seem to be a platform for the effect of the mucin on the plasma-membrane anchoring and cellular signaling.

Selective Binding and Elution of Aptamers for Pesticides Based on Sol-Gel-Coated Nanoporous Anodized Aluminum Oxide Membrane.

Sol-gel-based mesopores allow the entry of target small molecules retained in their cavity and aptamers to bind to target molecules. Herein, sol-gel-based materials are applied to screen-selective aptamers for small molecules, such as pesticides. To enhance the efficiency of aptamer screening using a sol-gel, it is necessary to increase the binding surface. In this study, we applied the sol-gel to an anodized aluminum oxide (AAO) membrane, and the morphological features were observed via electron microscopy after spin coating. The binding and elution processes were conducted and confirmed by fluorescence microscopy and polymerase chain reaction. The sol-gel coating on the AAO membrane formed a hollow nanocolumn structure. A diazinon-binding aptamer was bound to the diazinon-containing sol-gel-coated AAO membrane, and the bound aptamer was effectively retrieved from the sol-gel matrix by thermal elution. As a proof of concept, a sol-gel-coated AAO disc was mounted on the edge of a pipette tip, and the feasibility of the prepared platform for the systematic evolution of ligands by exponential enrichment (SELEX) of the aptamer binding was also confirmed. The proposed approach will be applied to an automated SELEX cycle using an automated dispenser, such as a pipetting robot, in the near future.

Tacrolimus-induced asymptomatic thrombotic microangiopathy diagnosed by laboratory tests in pig-to-rhesus corneal xenotransplantation: A case report.

Tacrolimus-associated thrombotic microangiopathy (TA-TMA) is a rare complication. TA-TMA is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ damage due to thrombus. We report asymptomatic TA-TMA diagnosed by laboratory tests in pig-to-rhesus corneal xenotransplantation. Corneal transplantation had been conducted from a wild-type SNU miniature pig to a rhesus macaque. The veterinary records were retrospectively reviewed in this case. The immunosuppressive regimen consisted of rituximab, basiliximab, and IVIg as inductive therapies, and steroids with tacrolimus (0.1 mg/kg/day) as maintenance therapies. Although there were no clinical symptoms, increased levels of lactate dehydrogenase, total bilirubin, blood urea nitrogen, and creatinine and decreased levels of hemoglobin and platelet were observed in laboratory tests on Day (D) 61. Systemic TA-TMA was tentatively diagnosed. Tacrolimus was discontinued starting on D71. Dalteparin, clopidogrel bisulfate (D77-D99), and IVIg (D72) were administered as a conservative treatment. Abnormal laboratory results were reversed on D99. When low-dose tacrolimus (0.07 mg/kg/day) was re-administered on D131 to prevent rejection of the graft, TMA was detected again by laboratory tests on D161, confirming the initial diagnosis. Discontinuation of tacrolimus on D162 and re-administration of Dalteparin (D161-D196) corrected the laboratory values on D161. This report shows that in pig-to-rhesus corneal xenotransplantation, clinically asymptomatic TMA can be induced by tacrolimus, and the discontinuation of tacrolimus and administration of anticoagulant seems sufficient to correct the laboratory TMA.

Gastrostomy tube placement for long-term oral drug administration in non-human primates.

BACKGROUND: Non-human primates (NHPs) are often used as recipients in preclinical transplantation research that in most cases involves administration of various drugs including immunosuppressants. Long-term oral drug administration, particularly tacrolimus, is challenging in the transplant recipient NHPs. Oral drug administration method using the mixture of drug and fruit juice has been used in NHPs, but this is not always effective in all monkeys. To those monkeys who are poorly compliant, oral drug administration in restraint or administration using gastrostomy tube should be necessary. The aim of this study was to compare the efficacy of between oral drug administration in restraint and administration using gastrostomy tube and to report complications and solutions to overcome the problems related to gastrostomy tube for long-term oral drug dosing in rhesus monkeys. METHODS: Fifteen of 4- to 5-year-old male and female healthy rhesus monkeys weighing 5.0-6.8 kg were used as recipients for porcine pancreatic islet transplantation. Oral drug administration in restraint was used for four monkeys, and gastrostomy tube was placed to other 11 monkeys (8-French Feeding tube, n=6; Tri-Funnel Replacement Gastrostomy tube, n=5). Oral immunosuppressive drugs such as sirolimus and tacrolimus were administered through the tube. The efficacy and the extent of ease for administration and related complications were compared between two groups. RESULTS AND CONCLUSIONS: The complication of gastrostomy included a transient inflammation in the skin and peritonitis caused by a leakage around implantation site (one case), which could be overcome by changing suture method and tube type to interlocking box suture and Tri-Funnel Replacement Gastrostomy tube, respectively. Despite these complications, oral drug administration using gastrostomy tube allowed us to perform accurate dosage of drug administration and to reduce the stress that both the monkey and the researcher may experience. Taken together, this study showed that gastrostomy tube placement is a better alternative to oral drug administration in restraint for long-term oral drug administration in rhesus monkeys who tend to refuse to eat the mixture of drug and fruit juice.

Induction, management, and complications of streptozotocin-induced diabetes mellitus in rhesus monkeys.

BACKGROUND: Diabetes mellitus (DM) model using streptozotocin (STZ) which induces chemical ablation of ß cell in the pancreas has been widely used for various research purposes in non-human primates. However, STZ has been known to have a variety of adverse effects such as nephrotoxicity, hepatotoxicity, and even mortality. The purpose of this study is to report DM induction by STZ, toxicity associated with STZ and procedure and complication of exogenous insulin treatment for DM management in rhesus monkeys (Macaca mulatta) that are expected to be transplanted with porcine islets within 2 months. METHODS: Streptozotocin (immediately dissolved in normal saline, 110 mg/kg) was slowly infused via central catheter for 10 minutes in 22 rhesus monkeys. Clinical signs, complete blood count and blood chemistry were monitored to evaluate toxicity for 1 week after STZ injection. Monkey basal C-peptides were measured and intravenous glucose tolerance test was performed to confirm complete induction of DM. Exogenous insulin was subcutaneously injected to maintain blood glucose in diabetic rhesus monkeys and the complications were recorded while in insulin treatment. RESULTS: Severe salivation and vomiting were observed within 1 hour after STZ injection in 22 rhesus monkeys. One monkey died at 6 hours after STZ injection and the reason for the death was unknown. Pancreatitis was noticed in one monkey after STZ injection, but the monkey recovered after 5 days by medical treatment. Serum total protein and albumin decreased whereas the parameters for the liver function such as aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly increased (P<.05) after STZ injection, but they were resolved within 1 week. Azotemia was not observed. Monkey fasting C-peptide levels after STZ injection were <0.1 ng/mL in 18 rhesus monkeys, but 0.34, 0.22, 0.16 ng/mL in three monkeys, respectively. The value of daily insulin requirement was 0.92±0.26IU/kg/d (range=0.45-1.29) in the monkeys. Diabetic ketoacidosis was observed in one rhesus monkeys, but the monkey recovered after 24 hours by fluid and insulin treatment. CONCLUSIONS: Streptozotocin was effective for inducing DM in rhesus monkeys, but various adverse effects such as pancreatitis, liver toxicity or death were observed. Therefore, careful and suitable medical managements should be implemented to eliminate the risks of mortality and severe adverse effects.

Changes of N/L ratio and cortisol levels associated with experimental training in untrained rhesus macaques.

BACKGROUND: Animal training prior to beginning an experiment is an essential procedure and a very important course because it may affect the results of hematologic and hormonal variables and the functions influenced by those factors. Because this training inevitably causes animal stress, we wondered how much time is needed for untrained monkeys to recover from stresses associated with experimental training. METHODS: We measured the hematological and stress hormonal (e.g., cortisol) changes on weekly basis before and after experimental monkey chair training in newly acquired rhesus monkeys. RESULTS: The neutrophil-to-lymphocyte (N/L) ratio significantly increased during the initial phase of the training and then gradually decreased after 3 weeks. Elevated serum cortisol levels in the initial phase also significantly decreased after 3 weeks of chair training. CONCLUSIONS: We conclude that at least a 3-week period is needed in monkey chair training for recovery from training stress. These results suggest that many researchers using nonhuman primates should provide enough time (>3 weeks) for adaptation to the experimental environment prior to beginning a study.

Heavy metal concentrations in hair of newly imported China-origin rhesus macaques (Macaca mulatta).

Macaque monkeys are good sentinel to humans for environmental pollutions because their similarities in genetic and physiological characteristics. So, their reference values about exposures to heavy metals are required for proper data interpretation. Here, we report several heavy metals concentrations in the hair of rhesus monkeys which are widely used in biomedical research. The hair of 28 imported rhesus monkeys from an animal farm in southwest China were examined for the presence of eight heavy metals (Arsenic, Beryllium, Cadmium, Chromium, Iron, Lead, Mercury, and Selenium). The analyzed data in parts per million (ppm) for hair concentrations of heavy metals in rhesus monkeys were as follow: As (0.654±0.331), Be (0.005±0.003), Cd (0.034±0.022), Cr (11.329±4.259), Fe (87.106±30.114), Pb (0.656±0.613), Hg (0.916±0.619), and Se (3.200±0.735). The concentrations of Be, Cr, and As showed significant higher in females than in males (P<0.05). We present here the reference values of several heavy metals in healthy China-origin rhesus monkeys. These data may provide valuable information for veterinarians and investigators using rhesus monkeys in experimental studies.

Reference values of hematology, chemistry, electrolytes, blood gas, coagulation time, and urinalysis in the Chinese rhesus macaques (Macaca mulatta).

BACKGROUND: Due to increasing preclinical study using the rhesus monkeys, the physiological data of these monkeys are very important for the objective evaluation of experimental results. Here, we report the physiological values of the Chinese rhesus monkeys acclimated under a well-controlled laboratory environment. METHODS: Seventy healthy rhesus monkeys of both genders (29 males and 41 females) were used in this study. Available data on hematology, serum biochemistry, electrolytes, blood gas, coagulation time, urinalysis, water consumption, urine volume, and body weight were examined. RESULTS: The lymphocyte values were 1.5-2.5 times higher than the neutrophil values in both genders. In serum biochemistry, there was no significant difference between the two genders. Interestingly, the values of alkaline phosphatase from the monkeys were very high. According to isoenzyme analysis, the percentage of alkaline phosphatase that originated from the bone was 60%, and this value was significantly higher than that from the liver (39%, P = 0.020). CONCLUSION: Physiological data are very important parameters that can be directly and indirectly related to organ function (e.g. islet, kidney, liver, heart, etc.) in the transplantation model. In this respect, our data constitute valuable references for preclinical study using the Chinese rhesus monkeys.