Construction and Characterization of a Fine-Tuned Lytic Phage Display System

Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our current understanding of molecular genetics. The lytic nature of this bacterial virus along with the conformation of its wild-type capsid protein (gpD) assembly offer many advantages for the virus for a phage display platform. Protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and persevere, or even increase, solubility. In this study, the exploitation of gpD for the design of a dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. In this system, gpD expression is encoded by mutant infecting phage particles, λDam, that can only produce a wild type length gpD allele within specialized strains of E. coli that can suppress the mutation. However, the functionality of gpD alleles, produced by passage of λDam through various suppressor strains, varies dramatically in their ability to restore functional packaging to the λDam phage, imparting a first dimension of decorative control. As a second dimension of decorative control, a D::eGFP translational fusion on a multicopy plasmid, encoding gpD::eGFP, complements the Dam mutation in trans and is regulated by the temperature-labile CI[Ts]857 repressor, allowing for the conditional expression of D::eGFP by thermoregulation. In combination, the effective exploitation of these two variables has permitted the effective development of a fine-tuned λ lytic phage display system. Of the suppressor-imparted alleles, gpDQ68S, gpDQ68Y, and gpDwt: the allele with the poorest functionality, gpDQ68S (SupD), in combination with submaximal expression of gpD::eGFP conferred the highest incorporation of the fusion into the λDam phage capsid in all combinations. Differences in size, fluorescence, and absolute protein decoration between phage preparations was achieved by varying the temperature of the suppressor host carrying the D::eGFP fusion plasmid. The effective preparation with these two variables provides a simple means to manage fusion decoration on the surface of phage λ for a variety of fusion partners and applications

Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage lambda capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by lambda Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to lambda Dam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the lambda Dam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage lambda.UW Start-up funds; Drug Safety and Effectiveness Cross-Disciplinary Training (DSECT) Scholarship; Canadian Institute of Health Research (CIHR

Immunogenicity and antitumor activity of the superlytic λF7 phage nanoparticles displaying a HER2/neu-derived peptide AE37 in a tumor model of BALB/c mice

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.canlet.2018.03.030 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Phage display technique has been increasingly researched for vaccine design and delivery strategies in recent years. In this study, the AE37 (Ii-Key/HER-2/neu 776–790) peptide derived from HER2 (human epidermal growth factor receptor protein) was used as a fused peptide to the lambda phage (λF7) coat protein gpD, and the phage nanoparticles were used to induce antitumor immunogenicity in a TUBO model of breast cancer in mice. Mice were immunized with the AE37 peptide displaying phage, λF7 (gpD::AE37) every 2-week intervals over 6-weeks, then the generated immune responses were evaluated. An induction of CTL immune response by the λF7 (gpD::AE37) construct compared to the control λF7 and buffer groups was observed in vitro. Moreover, in the in vivo studies, the vaccine candidate showed promising prophylactic and therapeutic effects against the HER2 overexpressing cancer in BALB/c mice.Mashhad University of Medical Sciences, Mashhad, Iran bach (MUMS GN: 922610)NSERC, Canada (NSERC GN: 214684

Metabolic Patterns across core features in Dementia with Lewy Bodies (DLB)

OBJECTIVE: To identify brain regions whose metabolic impairment contributes to DLB clinical core features expression and to assess the influence of severity of global cognitive impairment on the DLB-hypometabolic-pattern. METHODS: Brain FDG-PET and information on core features were available in 171 patients belonging to the imaging repository of the European DLB-consortium. Principal component analysis was applied to identify brain regions relevant to the local data variance. A linear regression model was applied to generate core feature-specific patterns controlling for the main confounding variables (MMSE, Age, Education, Gender, and Center). Regression analysis to the locally-normalized intensities was performed to generate a MMSE score-sensitive map. RESULTS: Parkinsonism negatively covaried with bilateral parietal, precuneus and anterior cingulate metabolism, visual-hallucinations with bilateral dorsolateral-frontal cortex, posterior cingulate and parietal metabolism and RBD with bilateral parieto-occipital cortex, precuneus and ventrolateral-frontal metabolism. VH and RBD shared a positive covariance with metabolism in medial temporal lobe, cerebellum, brainstem, basal ganglia, thalami, orbitofrontal and sensorimotor cortex. Cognitive fluctuations negatively covaried with occipital metabolism and positively with parietal lobes metabolism. MMSE positively covaried with metabolism in left superior frontal gyrus, bilateral-parietal cortex, and left precuneus, and negatively with metabolism in insula, medial frontal gyrus, hippocampus in the left hemisphere and in right cerebellum. INTERPRETATION: Regions of more preserved metabolism are relatively consistent across the variegate DLB spectrum. By contrast, core features were associated to more prominent hypometabolism in specific regions thus suggesting a close clinical-imaging correlation, reflecting the interplay between topography of neurodegeneration and clinical presentation in DLB patients. This article is protected by copyright. All rights reserved

Metabolic Correlates of Dopaminergic Loss in Dementia with Lewy bodies

BACKGROUND: Striatal dopamine deficiency and metabolic changes are well-known phenomena in dementia with Lewy bodies and can be quantified in vivo by 123 I-Ioflupane brain single-photon emission computed tomography of dopamine transporter and 18 F-fluorodesoxyglucose PET. However, the linkage between both biomarkers is ill-understood. OBJECTIVE: We used the hitherto largest study cohort of combined imaging from the European consortium to elucidate the role of both biomarkers in the pathophysiological course of dementia with Lewy bodies. METHODS: We compared striatal dopamine deficiency and glucose metabolism of 84 dementia with Lewy body patients and comparable healthy controls. After normalization of data, we tested their correlation by region-of-interest-based and voxel-based methods, controlled for study center, age, sex, education, and current cognitive impairment. Metabolic connectivity was analyzed by inter-region coefficients stratified by dopamine deficiency and compared to healthy controls. RESULTS: There was an inverse relationship between striatal dopamine availability and relative glucose hypermetabolism, pronounced in the basal ganglia and in limbic regions. With increasing dopamine deficiency, metabolic connectivity showed strong deteriorations in distinct brain regions implicated in disease symptoms, with greatest disruptions in the basal ganglia and limbic system, coincident with the pattern of relative hypermetabolism. CONCLUSIONS: Relative glucose hypermetabolism and disturbed metabolic connectivity of limbic and basal ganglia circuits are metabolic correlates of dopamine deficiency in dementia with Lewy bodies. Identification of specific metabolic network alterations in patients with early dopamine deficiency may serve as an additional supporting biomarker for timely diagnosis of dementia with Lewy bodies. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.status: accepte

The effects of a multi-ingredient supplement on markers of muscle damage and inflammation following downhill running in females

BACKGROUND: The effects of a multi-ingredient performance supplement (MIPS) on markers of inflammation and muscle damage, perceived soreness and lower limb performance are unknown in endurance-trained female athletes. The purpose of this study was to determine the impact of MIPS (NO-Shotgun®) pre-loaded 4 weeks prior to a single-bout of downhill running (DHR) on hsC-Reactive Protein (hsCRP), interleukin (IL)-6, creatine kinase (CK), muscle soreness, lower limb circumferences and performance. METHOD: Trained female runners (n = 8; 29 ± 5.9 years) (VO(2max): ≥ 50 ml(-1).kg(-1).min(-1), midfollicular phase (7-11 days post-menses) were randomly assigned in a double-blind manner into two groups: MIPS (n = 4) ingested one serving of NO Shotgun daily for 28 days prior to DHR and 30 min prior to all post-testing visits; Control (CON) (n = 4) consumed an isocaloric maltodextrin placebo in an identical manner to MIPS. hsCRP, IL-6, CK, perceived soreness, limb circumferences, and performance measures (flexibility, squat jump peak power) were tested on 5 occasions; immediately before (PRE), immediately post-DHR, 24, 48 and 72 h post-DHR. RESULTS: There were main effects of time for CK (p = 0.05), pain pressure threshold (right tibialis anterior (p = 0.010), right biceps femoris (p = 0.01), and left iliotibial band (ITB) (p = 0.05) across all time points), and maximum squat jump power (p = 0.04). Compared with 24 h post-DHR, maximum squat jump power was significantly lower at 48 h post-DHR (p = 0.05). Lower body perceived soreness was significantly increased at 24 h (p = 0.02) and baseline to 48 h (p = 0.02) post DHR. IL-6 peaked immediately post-DHR (p = 0.03) and hsCRP peaked at 24 h post-DHR (p = 0.06). Calculation of effect sizes indicated a moderate attenuation of hsCRP in MIPS at 72 h post-DHR. CONCLUSIONS: Consumption of MIPS for 4 weeks prior to a single bout of DHR attenuated inflammation three days post, but did not affect perceived soreness and muscle damage markers in endurance trained female runners following a single bout of DHR