Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.     

Differential Expression of IGF Family Members in Heat‐Stressed Embryos Produced In Vitro from OPU‐Derived Oocytes of Nelore (Bos indicus) and Holstein (Bos taurus) Cows

The IGF system is related to embryo quality. We aim to determine the effect of the heat stress on the mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4, and PAPPA in in vitro production (IVP) blastocysts from Nelore and Holstein after ovum pick up (OPU) to better understand the differences between these breeds. Oocytes from four Nelore and seven Holstein were collected in six OPU sessions. Following in vitro maturation and fertilization using six Nelore or Holstein sires, embryos were divided into control (cultured at 39°C) and heat stress (HS; exposed to 41°C for 9 h). Blastocysts were submitted to RNA extraction. The IGF1 expression was higher in blastocysts under HS in both breeds, and the expression of IGFBP2 and IGFBP4 was higher in Holstein blastocysts under HS. The high PAPPA expression and the low expression of IGFBP2 and IGFBP4 are associated with a more efficient degradation of IGFBPs, which results in greater IGF bioavailability in Nelore blastocysts and may contribute to the superior HS tolerance in Nelore, when compared to Holstein.     

Ovine rumen papillae biopsy via oral endoscopy; a rapid and repeatable method for serial sampling

AIMS: To explore and validate the utility of rumen endoscopy for collection of rumen papillae for gene expression measurement.

METHODS: Four adult Coopworth ewes were fasted for either 4 or 24 hours. Animals were sedated, placed in a dorsally recumbent position at 45 degrees with the head upright, and an endoscope inserted via a tube inserted into the mouth. Biopsies of rumen papillae were taken from the ventral surface of the rumen atrium under visual guidance. Two biopsies were collected from one of the animals that had been fasted for 4 hours, and three from one of the animals that had been fasted for 24 hours. Video of the rumen atrium and reticulum was also collected. The animals recovered uneventfully. Biopsies were subsequently used for extraction and sequencing of mRNA.

RESULTS: The ventral surface of the rumen atrium was accessible after 4 hours off pasture, but a larger region was accessible after 24 hours of fasting. Sedation allowed access for endoscope use for around 5 to 10 minutes after which increased saliva flow was noted. Rumen papillae biopsies were easily collected, with samples from a variety of sites collected in the ～10 minute time window. High quality RNA was obtained for stranded mRNA sequencing. Of the resulting reads, 69–70% mapped uniquely to version 3.1 of the ovine genome, and 48–49% to a known gene. The rumen mRNA profiles were consistent with a previously reported study.

CONCLUSIONS: This method for obtaining rumenal tissue was found to be rapid and resulted in no apparent short or long term effects on the animal. High quality RNA was successfully extracted and amplified from the rumen papillae biopsies, indicating that this technique could be used for future gene expression studies. The use of rumen endoscopy could be extended to collection of a variety of rumen and reticulum anatomical measurements and deposition and retrieval of small sensors from the rumen. Rumen endoscopy offers an attractive and cost effective approach to repeated rumen biopsies compared with serial slaughter or use of cannulated animals.     

The Unwanted Horse in the United States: An Overview of the Issue

The unwanted horse is one of the most significant welfare issues facing the U.S. horse industry. This paper discussed the causes of the problem, the types of horses affected, options, pending legislation and efforts by the industry to solve it.     

Use of the Anemon Index to evaluate the quality of analgesia during fentanyl and sevoflurane anaesthesia in pigs

Anemon I is a new monitoring system that can be used to evaluate autonomic nervous system reactivity in real time by showing a simple, easily interpretated quantitative index (0–200), the Anemon Index (AI) ( Junke et al. 2000 ). This study used the AI to evaluate the quality of analgesia during sevoflurane and fentanyl anaesthesia in pigs. Six healthy pigs, weighing 24.76 ± 3.40 kg, were induced to anaesthesia with 5% sevoflurane (SEVO) in 5 L minute^?1 oxygen. After endotracheal intubation SEVO was given at 1 MAC (2.66%) in 3 L minute^?1 oxygen. Fentanyl was infused IV at 50 µg kg^?1 hour^?1 for the first 30 minutes of anaesthesia, discontinued for 30 minutes, and then infused at 100 µg kg^?1 hour^?1 for another 30 minutes. Three mechanical noxious stimuli (needle prick, pin‐prick and pressure on the abdomen) were applied for 15 seconds at 30, 60 and 90 minutes. The AI, ECG, invasive mean arterial blood pressure (MAP), heart rate (HR), SpO₂ by pulse oximetry, tidal volume, Fe′sevo , Fe ′CO₂ and respiratory rate were recorded before induction (baseline), after induction, after intubation and extubation, and before and during noxious stimulation at 30, 60 and 90 minutes. Recovery times were recorded. Statistically significant differences were determined by anova . Spearman rank‐correlation was used to evaluate the relationship between AI and hemodynamic variables. A p‐value of < 0.05 was considered significant. A significant (p < 0.01) decrease in AI was recorded after anaesthetic induction, from 82.3 ± 21.1 to 52.7 ± 20.3. After intubation, AI increased slightly, but not significantly, to 71.7 ± 27.1. A significant (p < 0.05) increase of AI occurred after extubation. Nociceptive stimuli did not have any measurable effect either on AI or on recorded cardiovascular variables. There was no movement, respiratory changes, or any other visible response to noxious stimulation. The AI did not change significantly with the different doses of fentanyl. Respiratory depression and apnoea were seen in all animals during the fentanyl infusion; therefore, pigs received intermittent positive pressure ventilation. Anaesthesia with sevoflurane and fentanyl resulted in a significant (p < 0.001) decrease in MAP. Heart rate did not change significantly. There was no correlation between AI and cardiovascular variables (HR and MAP). Endotracheal intubation caused an increase and extubation a greater significant increase in the AI. This suggests that intubation and extubation may represent stressful events during general anaesthesia, although further studies are needed to validate the use of the AI in pigs. Sevoflurane anesthetic induction may not prevent the sympathetic stimulus caused by endotracheal intubation in pigs, as indicated by the increased AI values.     

Renal allograft histopathology in dog leukocyte antigen mismatched dogs after renal transplantation

OBJECTIVE: To evaluate allograft histopathology in dog leukocyte antigen (DLA)-mismatched dogs undergoing renal transplantation, with transient immunosuppression. STUDY DESIGN: Prospective study. ANIMALS: Ten healthy adult mongrel dogs. METHODS: Reciprocal renal transplantation and bilateral nephrectomy were performed. Immune conditioning consisted of nonmyeloablative (200 cGy), total body irradiation (TBI), bone marrow transplantation (BMT; 7 dogs), cyclosporine (CSA; 15 mg/kg every 12 hours), mycophenolate mofetil (MMF; 10 mg/kg every 12 hours) and intermittent prednisone (1 mg/kg every 12-24 hours). Biopsies were collected at transplantation, during full immunosuppression (44-90 days), and once medications were reduced or discontinued (228-580 days). Biopsies were evaluated for interstitial, tubular, vascular, and glomerular lesions. Blood urea nitrogen, creatinine, serum CSA concentrations, and clinical score were determined at each biopsy. RESULTS: Seven dogs survived >200 days (mean, 380 days). Transient CSA toxicity was suspected in 6 dogs. Lymphocytic, plasmacytic interstitial inflammation, and tubulitis progressed when immunosuppressive medications were decreased. All 7 dogs had histologic lesions consistent with some degree of allograft rejection at study end. CONCLUSION: Nonmyeloablative TBI, BMT, and short-term immunosuppression with CSA, MMF, and prednisone allowed renal allograft function and dog survival for >200 days. It appears unlikely that total drug withdrawal will be possible in unrelated DLA-mismatched dogs using this protocol. CLINICAL RELEVANCE: Transient immunosuppression with MMF, CSA, and prednisone along with BMT and nonmyeloablative TBI may make kidney transplantation a clinical reality for treatment of kidney failure in dogs. Initiating both MMF and CSA at lower dosages may potentially eliminate early renal allograft injury.