Cloning,Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium,Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS-PAGE. Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results:Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS- PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion:In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity. Key Words: Escherichia coli, Gene expression, Lactobacillus, Lipase, Phylogeny     

Clinical comparison of two regimens of lidocaine infusion in horses undergoing laparotomy for colic

ObjectiveTo compare, in horses undergoing laparotomy for colic, the effects of administering or not administering a loading intravenous (IV) bolus of lidocaine prior to its constant rate infusion (CRI). Effects investigated during isoflurane anaesthesia were end-tidal isoflurane concentration (Fe’ISO), cardiovascular function, anaesthetic stability and the quality of recovery.Study designProspective, randomized clinical study.AnimalsThirty-six client-owned horses.MethodsHorses were assigned randomly to receive lidocaine as a CRI (50 μg kg⁻¹ minute⁻¹) either preceded (LB) or not preceded (L) by a loading dose (1.5 mg kg⁻¹ IV over 15 minutes). Lidocaine infusion (LInf) was started (T0) within 20 minutes after induction of general anaesthesia and discontinued approximately 30 minutes before the end of surgery. Anaesthetic depth, Fe’ISO, intra-operative physiological parameters and quality of recovery were assessed or measured. Data were analysed using one-way anova, t-test, Fisher test, Wilcoxon and Kruskal–Wallis tests as appropriate (p < 0.05).ResultsMean ± SD Fe’ISO was 1.21 ± 0.08% in group LB and 1.23 ± 0.06% in group L. Heart rate was significantly higher in group L than in group LB at times T5-T15, T25, T35 and T95. No difference was found between groups in other measured physiological values, nor in any measure taken to improve these parameters. Recovery phase was comparable and satisfactory in all but one full term pregnant horse in group L which fractured a femur during recovery.ConclusionPreloading with a lidocaine bolus prior to a CRI of lidocaine did not influence isoflurane requirements, cardiopulmonary effects (other than a reduction in heart rate at some time points) or recovery compared to no preloading bolus.Clinical relevanceA loading dose of lidocaine prior to CRI does not confer any advantage in horses undergoing laparotomy for colic.     

FEASIBILITY FOR USING HYPOFRACTIONATED STEREOTACTIC VOLUMETRIC MODULATED ARC RADIOTHERAPY (VMAT) WITH ADAPTIVE PLANNING FOR TREATMENT OF THYMOMA IN RABBITS: 15 CASES

Thymoma is a relatively common tumor in rabbits. Treatment with surgery, radiation therapy, and chemotherapy alone or in combination has been reported with varying outcomes. Stereotactic volumetric modulated arc radiotherapy delivered in a hypofractionated manner allows high doses of radiation to be delivered to the target volume while allowing sparing of adjacent critical structures. This therapy is ideally suited for thymomas in rabbits given their size, the difficulty of multiple anesthesia episodes and the complexity of the radiotherapy plans required due to the tumor's proximity to the heart, lungs, and mediastinal structures. Fifteen rabbits with thymoma were prospectively recruited for this observational, single institution, single arm clinical study. All rabbits were imaged with both computed tomography (CT) and magnetic resonance imaging (MRI). A total dose of 40 Gy in six fractions was delivered using a single arc over an 11‐day period with repeat CT simulation done every other fraction for adaptive planning. Follow‐up evaluation was done through repeat CT and MRI imaging and revealed complete responses using the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Two rabbits had died at 618 and 718 days, 10 were alive and three were lost to follow‐up. Observed acute and late effects were graded according to the Veterinary Radiation Therapy Oncology Group (VRTOG) criteria and were found to be minimal.     

Factors influencing the abundance of three common nearshore fishes in south‐west Lake Michigan

Variation in the distribution and abundance of nearshore fishes is critical to understand food web processes and fishery management issues in Lake Michigan. This study characterised patterns in abundance of three common nearshore species, yellow perch Perca flavescens (Mitchell), round goby Neogobius melanostomus (Pallas) and alewife Alosa pseudoharengus (Wilson), in relation to spatio‐temporal, abiotic and biotic factors using gillnet sampling conducted across 5 years at multiple locations representing different substrates. Significant variations were observed in alewife and round goby catches between locations. A negative relationship between round goby and age‐0 yellow perch catch was observed, indicating potential competition between the two species. This study demonstrates that variability in nearshore fish communities can be driven by factors including substrate and interspecific interactions. Given the prominent role these species play in Lake Michigan's food web and thus their importance to fishery production, a thorough understanding of these factors is warranted.     

Effects of agricultural management on nematode–mite assemblages: Soil food web indices as predictors of mite community composition

Biological indicators based on abundances of soil organisms are powerful tools for inferring functional and diversity changes in soils affected by agricultural perturbations. Field plots, combining organic and conventional practices with no tillage, conservation tillage and standard tillage maintained different nematode assemblages and soil food webs. Soil food web indices based on nematode assemblages were reliable predictors of the trophic composition of functional characteristics of soil mite assemblages. Bacterial-feeding and predatory nematodes, together with predatory mites, were abundant in the organic-no till treatments and were associated with high values of the Enrichment and the Structure Index based on nematode assemblages. Conventional-Standard tillage treatments had high abundances of fungal- and plant-feeding nematodes and algivorous mites, associated with high values of the Basal and Channel Index. This study validates the hypothesis that nematode-based soil food web indices are useful indicators of other soil organisms such as mites, with similar functional roles and environmental sensitivities.