Characterisation of novel functionality within the Blastocystis tryptophanase gene

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.     

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to β cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for β cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.     

Evaluation of a Prednisolone Acetate-Loaded Subconjunctival Implant for the Treatment of Recurrent Uveitis in a Rabbit Model

Aim

To assess the efficacy of a biodegradable, prednisolone acetate implant in a rabbit uveitis model.

Methods

Randomized, controlled study of biodegradable microfilms preloaded with prednisolone acetate (PA) in a rabbit uveitis model. Experimental uveitis was induced by unilateral intravitreal injection of Mycobacterium tuberculosis H37Ra antigen (50 ug; 1 ug/uL) in preimmunized rabbits. PA-loaded poly[d,l-lactide-co-ε-caprolactone] (PLC) microfilms (n = 10) and blank microfilms (n = 6) were implanted subconjunctivally. An estimate of PA release in vivo was calculated from measured residual PA amounts in microfilms after the rabbits were sacrificed. The eyes were clinically monitored for ocular inflammation for 28 days. Histopathological examination of the enucleated eyes was performed at the end of the study period.

Results

In vitro studies revealed that sandwich PA-loaded microfilm formulations exhibited higher release kinetic compared to homogenous PA-loaded microfilms. The 60–40–60% microfilm released an average of 0.034 mg/day of PA over the period of 60 days in vitro; and we found that approximately 0.12 mg/day PA was released in vivo. Animals implanted with the PA-loaded microfilms exhibited significantly lowered median inflammatory scores when compared against the control group in this model for recurrent uveitis (P<0.001). The implants were clinically well tolerated by all the animals. Histology results showed no significant scarring or inflammation around the PA-loaded microfilms.

Conclusion

Our pilot study demonstrated that a subconjunctival PA-loaded implant is effective in suppressing inflammation in the rabbit model of uveitis, by providing therapeutic levels of PA that attenuated the inflammatory response even after a rechallenge. Longer term studies are now needed to establish the therapeutic potential of such a delivery system for treatment of ocular inflammation.     

Enzymatic Characterization of Insecticide Resistance Mechanisms in Field Populations of Malaysian Culex quinquefasciatus Say (Diptera: Culicidae)

Background

There has been no comprehensive study on biochemical characterization of insecticide resistance mechanisms in field populations of Malaysian Culex quinquefasciatus. To fill this void in the literature, a nationwide investigation was performed to quantify the enzyme activities, thereby attempting to characterize the potential resistance mechanisms in Cx. quinquefasciatus in residential areas in Malaysia.

Methodology/Principal Findings

Culex quinquefasciatus from 14 residential areas across 13 states and one federal territory were subjected to esterases, mixed function oxidases, glutathione-S-transferase and insensitive acetylcholinesterase assays. Enzyme assays revealed that α-esterases and β-esterases were elevated in 13 populations and 12 populations, respectively. Nine populations demonstrated elevated levels of mixed function oxidases and glutathione-S-transferase. Acetylcholinesterase was insensitive to propoxur in all 14 populations. Activity of α-esterases associated with malathion resistance was found in the present study. In addition, an association between the activity of α-esterases and β-esterases was also demonstrated.

Conclusions/Significance

The present study has characterized the potential biochemical mechanisms in contributing towards insecticide resistance in Cx. quinquefasciatus field populations in Malaysia. Identification of mechanisms underlying the insecticide resistance will be beneficial in developing effective mosquito control programs in Malaysia.     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Levels of Cry1Ac1 protein in herbivorous and predatory arthropods in fields of Bacillus thuringiensis cabbage

To investigate the extent of exposure and routes of Cry1Ac1 protein through the food chain, we collected Bt cabbage leaves and arthropods that occurred in the field during two trials. Protein levels in the transgenic leaves were significantly higher during the early stages of plant growth, ranging from 209.1 to 553.6?ng?g^?1 in spring and from 208.2 to 402.8?ng?g^?1 in autumn. Enzyme-linked immunosorbent assays were used to measure protein levels in the arthropods. Among the 16 taxa collected in the field, Cry1Ac1 was detected in the bodies of 10. Concentrations were higher in lepidopteran larvae than in the other taxa. In particular, we found a significant correlation between Cry1Ac1 protein levels in cabbage leaves and in Pieris rapae and Mamestra brassicae. Furthermore, this protein was detected in five out of nine taxa of predators (spiders and coleopterans) and parasitoids. These results will be useful as we identify the arthropods that are directly or indirectly exposed to Bt toxin within the food web and the degree to which they are exposed during the cultivation of Bt cabbage.     

Stimulation of adventitious rooting of Taxus species by thiamine

Summary Results obtained from using root inducing compounds on Taxus species cuttings suggested that rooting could be significantly enhanced by the presence of thiamine. This observation was verified using a root inducing solution containing a set concentration of IBA (0.2%), NAA (0.1%), and supplemented with various concentrations of thiamine. The best rooting response for Taxus cuspidata stem cuttings was found using this solution supplemented with 0.08% thiamine. Rooted cuttings were easily established and developed into vigorous plants. In addition, Taxus brevifolia shoots obtained from tissue cultures via in vitro organogenesis also responded favorably to this 0.08% thiamine supplemented rooting solution.