Artificial Bloodfeeder Glytube: Evaluating Different Types of Membranes and Blood Sources for Feeding Aedes aegypti and Aedes albopictus

ABSTRACT Mosquito colony maintenance in the laboratory is essential for research but presents logistical and ethical problems with the use of live animals for bloodfeeding. The Glytube is an artificial bloodfeeding system for mosquitoes that uses Parafilm-M® membrane and human blood to feed Aedes aegypti. This study evaluated the efficiency of Glytube with different types of membranes and chicken blood to feed Ae. aegypti and Ae. albopictus. We evaluated 2 artificial (thread seal tape [TST], Parafilm-M) and 2 natural membranes (pork, sheep intestine). The results for Ae. aegypti suggest that TST was the best membrane because it presented a high percentage of fed females (63%), a high average number of eggs per female (54.65), and an egg viability rate significantly similar to control (mouse). For Ae. albopictus, there was no significant difference between the membranes and the control; however, the use of TST is suggested due to the low cost and easy manipulation. The treatments that used chicken blood did not present significant differences in the egg viability when compared with the control. The Glytube functionality can be increased by replacing the Parafilm-M membrane by TST and human to chicken blood.

Abstract 13114: P22 phox Protects the Heart Against Pressure Overload

Introduction: p22 phox forms a complex with NADPH oxidases, major sources of O 2 - and H 2 O 2 . However, the role of p22 phox during stress remains to be elucidated. Purpose: To investigate the role of endogenous p22 phox during pressure overload (PO). Methods and Results: The level of p22 phox protein in isolated cardiomyocytes after 4 weeks of transverse aortic constriction (TAC) was significantly higher than after sham operation (1.7-fold, p<0.05). The cardiac phenotype of cardiac-specific p22 phox knockout ( p22 phox cKO) mice was normal at baseline. However, four weeks after TAC, p22 phox cKO mice exhibited a lower left ventricular ejection fraction (32.0±10.0 vs 53.2±8.4%, p<0.05), a higher lung weight to tibial length ratio (23.0±6.0 vs 13.1±6.6, p<0.05), and more interstitial fibrosis (6.1±1.0 vs 4.4±1.1%, p<0.05) than control mice, indicating that the loss of p22 phox exacerbates TAC-induced cardiac dysfunction. The level of oxidative stress in the heart, evaluated by dityrosine immunoblot, was significantly lower in p22 phox cKO mice than in control mice (0.71±0.04 vs 1.00±0.04, p<0.01). The peak Ca 2+ amplitude in isolated cardiomyocytes was lower in p22 phox cKO mice than in control mice at baseline (2.4±0.1 vs 3.0±0.2, p<0.01). Although mRNA expression of SERCA2a did not differ, there was significantly less SERCA2a protein in p22 phox cKO mice than in control mice (0.62±0.10 vs 1.00±0.23, p<0.01) at baseline. The amount of biotinylated iodoacetamide labeled SERCA2a was significantly smaller in p22 phox cKO hearts than in control mouse hearts (0.4-fold, p<0.01), indicating that cysteine residues in SERCA2a are oxidized to a greater extent in p22 phox cKO hearts than in control mouse hearts. Since cysteine oxidation decreases the stability of SERCA2a, our results suggest that p22 phox stabilizes SERCA2a by preventing cysteine oxidation. Conclusions: Endogenous p22 phox is protective against PO, possibly by maintaining SERCA2a stability.

Maf and Mafb control mouse pallial interneuron fate and maturation through neuropsychiatric disease gene regulation

​Maf (c-Maf) and Mafb transcription factors (TFs) have compensatory roles in repressing somatostatin (SST+) interneuron (IN) production in medial ganglionic eminence (MGE) secondary progenitors in mice. Maf and Mafb conditional deletion (cDKO) decreases the survival of MGE-derived cortical interneurons (CINs) and changes their physiological properties. Herein, we show that (1) Mef2c and Snap25 are positively regulated by Maf and Mafb to drive IN morphological maturation; (2) Maf and Mafb promote Mef2c expression which specifies parvalbumin (PV+) INs; (3) Elmo1, Igfbp4 and Mef2c are candidate markers of immature PV+ hippocampal INs (HIN). Furthermore, Maf/Mafb neonatal cDKOs have decreased CINs and increased HINs, that express Pnoc, an HIN specific marker. Our findings not only elucidate key gene targets of Maf and Mafb that control IN development, but also identify for the first time TFs that differentially regulate CIN vs. HIN production.

Interference and Control of ALS-Resistant Mouse-Ear Cress (Arabidopsis thaliana) in Winter Wheat

In 2015, winter wheat growers in Virginia reported commercial failures of thifensulfuron to control mouse-ear cress. This was the first reported case of field-evolved acetolactate synthase (ALS) resistance in mouse-ear cress, so research was conducted to evaluate alternative herbicide options as well as to document potential yield loss in winter wheat from mouse-ear cress. Efficacy studies were conducted at three site-years in 2015 to 2016 and 2016 to 2017 as well as a POST greenhouse trial. In the PRE study, flumioxazin, pyroxasulfone, saflufenacil, and metribuzin resulted in more than 80% mouse-ear cress control 15 wk after planting across all sites with no observable wheat injury. No differences were observed in wheat yield in two of three sites in the PRE herbicide study; yield differences were attributed to common chickweed and not to mouse-ear cress. In the POST herbicide study, 2,4-D, dicamba, and metribuzin resulted in greater than 75% control in the field and greenhouse. Metribuzin, dicamba, and pyroxsulam resulted in crop injury 3 wk after treatment at some sites, but injury was transient. Yield from all POST treatments was similar to the nontreated plots. No yield loss was observed by mouse-ear cress densities greater than 300 plants m–2, indicating that mouse-ear cress is not very competitive with winter wheat. Growers should make herbicide decisions based on other weeds in the field and can incorporate the aforementioned herbicides for mouse-ear cress control.

Dysfunctional CLEC-2 and GPVI-Mediated Signaling in Syk Y342F Knock-in Mouse Platelets

Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors like GPVI, the protease activated receptors (PARs), and C-type lectin-like receptor 2 (CLEC-2). GPVI is an ITAM-bearing receptor, while Clec-2 is a HemITAM-bearing receptor. Both ITAM and hemITAM-bearing receptors signal through spleen tyrosine kinase (Syk). Syk activity is dependent on phosphorylation of several residues including Y342, Y346, and Y519/520 (Y348, Y352, and Y525/526 in human). However, the importance of each residue on either phosphorylation of other residues or activation of Syk is unknown. Therefore, we produced a Syk Y342F knock-in mouse line via CRISPR/Cas9 to investigate the importance of Y342 on Syk-mediated signaling using a platelet system. Syk Y342F mice are viable, have no gross abnormalities, and their blood cell counts are normal. We were unable to detect any Syk Y342 phosphorylation following stimulation of Y342F platelets with the GPVI agonist collagen-related peptide (CRP) (Figure 1). Further, Platelet reactivity is reduced in response to the GPVI agonists CRP (Figure 2) and collagen, as well as the CLEC-2 agonist rhodocytin. When using CLEC-2 antibody as an agonist no response is observed in Syk Y342F mouse platelets even though WT littermate control mouse platelets responded well (Figure 3). Signaling initiated by either GPVI or CLEC-2 is also inhibited, as Syk Y519/520 phosphorylation is impaired. Furthermore, PLCg2 and SLP-76 phosphorylation are reduced in Syk Y342F platelets compared to platelets from littermate control mice following stimulation of either GPVI or CLEC-2. Although reactivity of Y342F platelets is clearly reduced compared to WT platelets, there was no difference in occlusion time following ferric chloride injury of the carotid artery. Similarly, there was no difference in mortality rate following pulmonary thromboembolism using Syk Y342F mice compared to WT littermate control mice. Nevertheless, these data demonstrate that phosphorylation of Y342 on Syk following stimulation of either ITAM or hemITAM-bearing receptors is important for Syk activation. Disclosures No relevant conflicts of interest to declare.

Immunization with plasmid DNA expressing Heat Shock Protein 40 confers prophylactic protection against chronic Toxoplasma gondii infection in Kunming mice

Toxoplasma gondii causes one of the most common protozoal diseases of humans and animals worldwide. With the aim of designing an effective vaccine against T. gondii infection, we examined the immunogenicity of a DNA vaccine expressing heat shock protein 40 (HSP40) against challenge with T. gondii (type I RH and type II Pru) strains in Kunming mice. The plasmid pVAX1-HSP40 was constructed and used to immunize mice by intramuscular injection for three sequential immunizations with two-week intervals. This immunization regimen significantly reduced parasite cyst burden in pVAX1-HSP40-immunized mice (1871.9 ± 142.3) compared with control mouse groups immunized with pVAX1 (3479.2 ± 204.4), phosphate buffered saline (3024.4 ± 212.8), or left untreated (3275.0 ± 179.8) as healthy controls (p < 0.01). However, immunization failed to protect mice against challenge with the virulent RH strain. There was a significant increase in T lymphocyte subclasses (CD3e+CD4+ T and CD3e+CD8a+ T lymphocytes) in splenic tissues in immunized mice compared with controls (p < 0.05). However, the level of antibodies, lymphocyte proliferation and concentration of cytokines (IFN-γ, IL-2, IL-4, IL-10 and IL-12p70) were not significantly different between immunized and control mouse groups (p < 0.05). These data indicate that pVAX1-HSP40 induced specific immune responses and achieved a significant reduction in the number of brain cysts in Pru-infected mice, and thus can be tested in future immunization studies along with plasmids containing other immunogenic proteins as a cocktail vaccine to fully abolish chronic toxoplasmosis.