Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei,mitochondria, and plasma membranes in mice

Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.     

Epidemiological Analysis of Methicillin-Resistant Staphylococcus aureus Carriage among Veterinary Staff of Companion Animals in Japan

Veterinary staff carrying methicillin-resistant Staphylococcus aureus(MRSA) can be a source of MRSA infection in animals. To identify risk factors of MRSA carriage among veterinary staff, MRSA carriage and epidemiological information (sex, career, contact with MRSA-identified animal patients and others) were analyzed from 96 veterinarians and 70 veterinary technicians working at 71 private veterinary clinics in Japan. Univariate analysis determined sex (percentage of MRSA carriage, male (29.2%) vs. female (10%); P=0.002) and career (veterinarians (22.9%) vs. veterinary technicians (10%); P=0.030) as risk factors. Multivariable analysis revealed that sex was independently associated with MRSA carriage (adjusted odds ratio, 3.717; 95% confidence interval, 1.555–8.889; P=0.003). Therefore, male veterinary staff had a higher risk of MRSA carriage than female staff.     

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD₅₀)/ml, 10¹ HAD₅₀/ml and 10⁶ HAD₅₀/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD₅₀/ml and 10¹ HAD₅₀/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD₅₀/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 10³ HAD₅₀/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.     

Analysis of a pair of END+ and END? viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N^pro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of N^pro determine the difference in characteristics between END-phenomenon-positive (END⁺) and END-phenomenon-negative (END⁻) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END⁺ and END⁻ viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END⁺ and END⁻ viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N^pro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.     

Effects of Intermittent Positive Pressure Ventilation on Cardiopulmonary Function in Horses Anesthetized with Total Intravenous Anesthesia Using Combination of Medetomidine,Lidocaine, Butorphanol and Propofol (MLBP-TIVA)

Effects of intermittent positive pressure ventilation (IPPV) on cardiopulmonary function were evaluated in horses anesthetized with total intravenous anesthesia using constant rate infusions of medetomidine (3.5 µg/kg/hr), lidocaine (3 mg/kg/hr), butorphanol (24 µg/kg/hr) and propofol (0.1 mg/kg/min) (MLBP-TIVA). Five horses were anesthetized twice using MLBP-TIVA with or without IPPV at 4-week interval (crossover study). In each occasion, the horses breathed 100% oxygen with spontaneous ventilation (SB-group, n=5) or with IPPV (CV-group, n=5), and changes in cardiopulmonary parameters were observed for 120 min. In the SB-group, cardiovascular parameters were maintained within acceptable ranges (heart rate: 33–35 beats/min, cardiac output: 27–30 l/min, mean arterial blood pressure [MABP]: 114–123 mmHg, mean pulmonary arterial pressure [MPAP]: 28–29 mmHg and mean right atrial pressure [MRAP]: 19–21 mmHg), but severe hypercapnea and insufficient oxygenation were observed (arterial CO₂ pressure [PaCO₂]: 84–103 mmHg and arterial O₂ pressure [PaO₂]: 155–172 mmHg). In the CV-group, normocapnea (PaCO₂: 42–50 mmHg) and good oxygenation (PaO₂: 395–419 mmHg) were achieved by the IPPV without apparent cardiovascular depression (heart rate: 29–31 beats/min, cardiac output: 17–21 l /min, MABP: 111–123 mmHg, MPAP: 27–30 mmHg and MRAP: 15–16 mmHg). MLBP-TIVA preserved cardiovascular function even in horses artificially ventilated.