30921 research outputs found
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Evaluating the genotypic and phenotypic stability of CRISPRi essential gene mutants in Burkholderia cenocepacia K56-2
No full textEssential genes encode the processes required for cell growth and survival within specific
environments. Understanding the products of essential genes is important for antibiotic discovery,
as many essential genes encode the targets of antibiotics. Also, controlling the products of essential
genes is a potential approach to reversibly inhibit growth while exploiting beneficial traits. A
genetic tool to investigate essential gene function by controlling gene expression is CRISPR
interference (CRISPRi). This tool comprises a nuclease-inactive Cas9 (dCas9) and a synthetic
guide RNA (sgRNA), forming a ribonucleoprotein complex that binds to the target gene and
sterically blocks transcription. Previously, we constructed a CRISPRi system in strains of the
Burkholderia cepacia complex, opportunistic bacterial pathogens with biotechnological potential.
Our CRISPRi system uses a chromosomally encoded, rhamnose-inducible dCas9 and a sgRNA
constitutively expressed from a plasmid. We and others have observed that CRISPRi mutants
spontaneously lose their conditional growth phenotype (CGP), hampering their potential use in
controlling bacterial growth. To understand this phenotypic reversion, we performed experimental
evolution experiments in selected 15 CRISPRi mutants under dCas9-inducing and non-inducing
conditions. At time points, we performed targeted gene repression by RT-qPCR and whole genome
sequencing (WGS) in parental and evolved mutants. While analysis across generations revealed
diverse CGPs, all mutants reverted to wild-type growth phenotypes. K-medoids clustering
identified 3 clusters with different relationships between essential gene functions and the CGP.
Under consistent inducing conditions, Cluster 1 exhibited severe gene repression of 78-fold,
dropping to 3-fold. Cluster 2 decreased from 46-fold to 0.5-fold, whereas Cluster 3 demonstrated
wide diversity in gene repression. There was no evidence of single nucleotide polymorphisms
(SNPs) in the dCas9, rhamnose system regions, or the encoded sgRNAs maintained on a plasmid.
However, WGS identified SNPs in a porin and a LysR-type global regulator. Immunoblot analysis
confirmed the absence of dCas9, indicating a lack of expression possibly due to porin malfunction
affecting rhamnose uptake or protein degradation caused by unregulated proteases. Together,
while the CRISPRi machinery is genetically stable, compensatory mutations compromised its
function at longer times. Our work highlights the strengths and limitations of CRISPRi for
extended control of bacterial growth.Mitacs Globalink Graduate Fellowship (2021-2022)
Research Manitoba - Natural Sciences & Engineering (2023-2024)May 202
Extraction of phenolic compounds from wheat by developing a rapid and reliable microwave-assisted method
Get PDFPhenolic compounds (PC) are vital secondary plant metabolites with recognized health benefits, including antioxidant and anti-inflammatory properties. These compounds help plants manage environmental stress and contribute to their resilience, with a higher concentration found in the outer layers of wheat, particularly in the bran. Current extraction methods are time-consuming and often involve the use of acids, alkalis, and organic solvents, which pose environmental concerns. Therefore, efficient, green, and rapid methods for extracting these phenolic compounds are essential, especially in the food industry, which has an increasing demand for sustainable processes.
We hypothesize that Microwave-Assisted Extraction (MAE) will serve as a superior methodology for phenolic compound extraction from wheat, offering improved yield, faster processing, and enhanced environmental sustainability compared to conventional extraction (CE).
This study aims to validate MAE as a green and efficient alternative to CE for quantifying phenolic compounds in Canada Western Red Spring wheat kernels, utilizing whole grains and flour. Additionally, MAE was applied to wheat kernels with varying degrees of pearling to assess its effectiveness across different wheat layers. Two solvent systems were tested: 80% ethanol (v/v) and water, with MAE conditions including temperatures between 140°C and 180°C and extraction times of 5, 10, and 15 minutes. A solid-to-liquid ratio of 1:9 was used for whole kernels, and 1:99 for flour fractions, to prevent starch gelatinization.
Results showed that MAE outperformed CE in terms of TPC yield, with water as a solvent leading the highest TPC of 5.41 ± 0.10 mg/g DW (p < 0.05) at 170°C for 10 minutes, compared to 80% ethanol extracts at 3.52 ± 0.24 mg/g DW (p < 0.05). CE yielded the lowest TPC at 2.78 ± 0.18 mg/g DW (p < 0.05). Among individual phenolic acids, gallic acid was found to be the most prominent in all MAE extracts.
Additionally, phenolic acid extraction was evaluated in six pearled fractions of wheat subjected to different pearling time, ranging from 50 to 450 seconds. Shorter pearling times resulted in higher phenolic acid concentration. Also, TPC values were found to be higher in the outer bran layers, supporting the conclusion that PCs are more concentrated in these parts of the wheat grain.
A significant negative correlation was observed between pearling time and phenolic acid content, emphasizing the importance of optimizing pearling times for enhanced phenolic yields.
It must be stressed that for the first time, a method for extracting phenolic acids is proposed using water as a solvent and extracting the compounds directly from the wheat kernel.
This study highlights the efficiency and sustainability of MAE for phenolic extraction, offering a faster, greener alternative to traditional methods. The findings provide insights into enhancing the phenolic content of wheat-based products, with implications for functional food development and wheat breeding.NSERC Engage Grant (RGPIN-2023-04289) and Sustainable CAPFebruary 202
Bioaccessibility of dietary carotenoid compounds assessed from durum wheat
No full textDurum wheat is a source of both macronutrients and micronutrients, including starch, proteins, fiber, vitamins, and minerals. It is also an important source of carotenoids. Its high carotenoid content makes it ideal for pasta production. Carotenoids are known for their health benefits, which include antioxidative and anti-inflammatory properties. To exert any of these health benefits, potentially absorbed dietary carotenoids must first be bioaccessible. Three Canadian durum wheat varieties (AAC Spitfire, CDC Precision, and Transcend) milled to semolina (RS) and whole flour (WWF) were processed into pasta and cooked to Al dente (Al), fully cooked (FCT), or overcooked (OC) to assess the influence of the food matrix and food processing on carotenoid bioaccessibility and absorption using a static in vitro digestion model. Carotenoid retention after cooking, digestive stability, uptake efficiency, and antioxidant activity were also evaluated using the human Caco-2 cell model. Carotenoid composition of the three wheat varieties was examined using HPLC. The wheat varieties differed in their initial carotenoid contents, with lutein being the predominant carotenoid compound. On average, carotenoid contents decreased by 12-31% after pasta extrusion, and further decreases were observed after cooking. Upon digestion, bioaccessibility of lutein, ranged from 124.5-156.9% in RS and 104.0 -139.1% in WWF pasta. Carotenoids in pasta cooked to Al were significantly (P<0.05) more bioaccessible than FCT and OC, although OC pasta promoted higher extractability. Higher cooking stability of lutein was found in the Transcend variety (75.6%) compared to Spitfire (74.2%) and Precision (69.6%), resulting in its significantly high (P<0.05) lutein bioaccessibility, highlighting the influence of the food matrix. Lutein cellular uptake was low, ranging from 3.4-7.9% for RS pasta and 3.7-5.2% for WWF samples. The cellular antioxidant activity of digested and undigested carotenoids demonstrated their efficiency in attenuating reactive oxygen species-induced oxidative stress, especially at low concentrations. Our results showed that the magnitude of carotenoid bioaccessibility depended on the flour type, the carotenoid physical properties, the cooking duration, and the wheat variety. Knowledge gained from this research provides a foundation for further research into the factors that influence carotenoid bioaccessibility in cereal grain food products.February 202
Urticaria
No full textAbstract Urticaria (hives) is a common disorder that may be associated with angioedema (swelling that occurs beneath the skin). It is generally classified as acute or chronic, and chronic urticaria is further classified as spontaneous or inducible Second-generation, non-sedating histamine type 1 (H1)-receptor antihistamines represent the mainstay of therapy for both acute and chronic urticaria. Second-line treatment for uncontrolled chronic urticaria includes omalizumab (a monoclonal anti-immunoglobulin E [IgE] antibody). In this article, we review the causes, diagnosis and management of urticaria (with or without angioedema).Key take-home messages Urticaria is a common disorder characterized by recurrent, pruritic (itchy) lesions with pale centers (wheals) that usually subside within 48 h; it can be associated with angioedema. Mast cells are the primary effector cells in urticaria. Urticaria is classified as acute (lesions for < 6 weeks) or chronic (lesions for ≥ 6 weeks). Chronic urticaria can be further classified as spontaneous or inducible. The diagnosis of urticaria is based primarily on a thorough clinical history and physical exam; however, diagnostic tests may be helpful in some instances. Second-generation, non-sedating H1-receptor antihistamines are the mainstay of therapy for urticaria. Omalizumab and cyclosporine can be used for more severe, chronic cases. Referral to an allergy specialist or dermatologist should be considered in the following situations: failure of or difficulty tolerating first-line second-generation antihistamine treatments; need for specialized treatment; and the presence of severe symptoms and/or atypical features
Advancing Inclusion and Quality of Life for Canadian Seniors
No full textThe Coalition for Healthy Aging in Manitoba (CHAM) would like to congratulate the Standing Committee on Human Resources, Skills and Social Development and the Status of Persons with Disabilities (HUMA) on undertaking the study “Advancing Inclusion and Quality of Life for Canadian Seniors”. The three themes of the report (housing, income security and quality of life), well reflect areas of work that need to be undertaken in Canada and around the world
Evaluating the role and therapeutic potential of neuregulin-1 for remyelination in chronic multiple sclerosis
No full textIn in chronic lesions of progressive multiple sclerosis (MS), failure in myelin regeneration and prolonged demyelination lead to permanent neurodegeneration and increased disability. Currently, minimal regenerative treatments are available to promote myelin repair (remyelination) for progressive MS. In acute demyelinating lesions, microglia have remarkable capacity to facilitate remyelination by clearance of cholesterol-rich myelin debris and supporting new oligodendrocytes to remyelinate white matter lesions. However, in chronic demyelinated lesions, microglial cholesterol metabolism and recycling process is dysregulated, resulting in a dysfunctional myelin-derived lipid-accumulating phenotype that eventually leads to microglial cell death. Currently, the underlying molecular mechanisms that drive microglia dysfunction in progressive MS are not fully understood. We previously reported downregulation of neuregulin-1 (Nrg-1), a key myelination factor, in human secondary progressive MS lesions and lesions of the MS mouse model, experimental auto-immune encephalomyelitis (EAE). Restoring Nrg-1 levels in EAE mice showed positive immunomodulatory effects and promoted a pro-regenerative phenotype in microglia in acute demyelinating lesions.
In this study, we demonstrate that persistent dysregulation of Nrg-1 levels contribute to impaired remyelination in chronically demyelinated brain lesions using a cuprizone mouse model of chronic demyelination that models progressive MS and in Nrg-1 conditional knockout mice. Using parallel in vitro and in vivo experiments, we show that therapeutic restoration of Nrg-1 promotes remyelination in chronic demyelinated lesions by enhancing oligodendrogenesis and oligodendrocyte maturation in a microglia-dependent manner. Mechanistically, Nrg-1 treatment revitalizes the innate capacity of microglia for phagocytosis of myelin debris, cholesterol recycling and efflux through activation of LXR and PPAR signaling and upregulation of cholesterol transporters. These changes in microglial function improve their survival and provide paracrine support for oligodendrocyte remyelination in chronic demyelinated lesions. Altogether, this study, for the first time, links dysregulation of Nrg-1 to remyelination failure in chronic demyelinated lesions, and demonstrates the therapeutic potential of Nrg-1 to augment the ability of microglia for remyelination in progressive MS.University of Manitoba Graduate Fellowship (UMGF)
Rady Faculty of Health Sciences (RFHS) Graduate StudentshipFebruary 202
Characterizing the effects of chronic conventional heavy crude oil exposure on the growth, development, and behaviour of larval wood frog (Lithobates sylvaticus)
No full textIn recent years, the need for data on the effects of oil spills on freshwater environments has become increasingly important in Canada. This thesis will address some of these gaps by focusing on the ecological impact of oil spills on amphibians, specifically wood frog (Lithobates sylvaticus) tadpoles, within the context of two major research studies: the Freshwater Oil Spill Remediation Study (FOReSt) and the Floating Wetland Treatments to Enhance Remediation (FloWTER) study which took place at the International Institute for Sustainable Development Experimental Lakes Area (IISD-ELA) in Northwestern Ontario. The FOReSt study aimed to assess the effectiveness of non-invasive oil spill remediation techniques in shoreline habitats, while the FloWTER study investigated the potential use of symbiotic plant-microbe systems via engineered floating wetlands for the degradation of oil-derived hydrocarbons. FOReSt simulated an oil spill event and industry-standard remediation response (shoreline rinsing), characterizing the fate and bioaccumulation of oil components in tadpoles exposed to weathered conventional heavy crude oil (CHV) in shoreline enclosures. Results indicated significant bioaccumulation of total polycyclic aromatic compounds (tPACs) in tadpole tissue, though no measurable effects on growth, development, or survival were observed. Qualitative histological analysis of hepatic tissues found no significant differences between treatment and control groups, suggesting that tadpoles did not experience adverse health impacts under the conditions of the study. The amphibian component within the mesocosm tanks explored the chronic exposure of wood frog tadpoles to a water-accommodated fraction of CHV in combination with floating wetland treatments. Behavioural assays and histological evaluations were conducted to determine the impact of these oil spill response techniques on tadpole health and behaviour. The results showed no significant differences in bioaccumulation, growth, development, or behavioural metrics, such as total distance travelled, or time spent near conspecifics. These findings suggest that the amphibian early life stages studied were resilient to the oil spill scenarios tested under the conditions and remediation methods applied. Further research is needed to fully understand the long-term impacts of oil spills on amphibian populations and freshwater ecosystems.February 202
Survey findings summary report
No full textIn the spring and summer of 2023, University of Manitoba (UM) Senior Scholars and Professors Emeriti were invited to complete an online survey regarding their perspectives on these roles at the UM. In total, 78 individuals consented to the research survey, although not all 78 individuals answered each question
Metagenomics-guided microbiome analysis of the wastewater treatment plants and antibiotic-exposed lab-scale bioreactors: insights into phage-host interactions and dynamics of antibiotic resistance
No full textMicrobes are crucial to the wastewater treatment plants (WWTPs). WWTPs receive large influxes of microbes and are designed to benefit from microbes to treat wastewater. Recent COVID-19 pandemic emphasized their roles in surveilling and predicting infection dynamics in populations. In an era of outbreaks and adverse environmental problems, understanding wastewater microbiome can contribute to the effective surveillance methods and improved wastewater treatment systems. The research presented here focuses on the wastewater surveillance of SARS-CoV-2, phage and bacterial communities and the spread of antibiotic resistance genes (ARGs). The skimmed milk flocculation (SMF) was found to be effective in concentrating viral particles. This method was successfully applied to surveil SARS-CoV-2 in wastewater and establish relationships between SARS-CoV-2 concentration and infection dynamics in populations. Moreover, the methods developed here formed a basis to study the dynamics and interactions of phage and bacterial communities and phages’ potential contribution to the antibiotic resistance in WWTPs and antibiotic-exposed bench-scale wastewater treatment systems. A combined metagenomics and qPCR approach was employed to study phage and bacterial communities, their potential interactions, and dynamics and mechanisms of antibiotic resistance in wastewater treatment systems. In full-scale WWTPs, the profiles of bacteriomes, phageomes, potential hosts of phages and resistomes were distinct and their compositional diversities followed very similar trends, indicating a habitat- and community-driven population dynamics. While host-phage dynamics in WWTPs was partly in support of Kill the Winner (KtW) hypothesis, dynamics of phage and host communities were highly connected in bench-scale reactors, supporting KtW hypothesis. ARGs were rarely associated with phages, highlighting the limited contribution of phages to the spread of ARGs. On the contrary, phages were found out to encode auxiliary metabolic genes (AMGs) and infect ARG-carrying bacteria and functional wastewater bacteria, indicating phages potential roles in shaping microbial communities and metabolism. Overall, the findings of this study provide insights into the phage-host interactions and dynamics in wastewater treatment systems and highlight the limited contribution of phages to the spread of antibiotic resistance. Phages’ potential applications in microbiome engineering and treatment of bacterial infections seems quite promising considering their interactions with prokaryotic communities.May 202
Understanding the marketed plant-based beverages: From ingredients technological function to their nutritional value
Get PDFDespite the market expansion of plant-based beverages (PBB) there is limited information about what is driven the market and the nutritional status of the existing beverages. The objective was to identify the existing gaps in the PBB market with particular emphasis on their composition and nutritional value. PBB are mainly based on individual flour/powder and blends and sunflower oil is frequently present, besides gellan gum to stabilize the emulsion. In general, PBB are low calorie drinks (10–84 Kcal/ 100 mL), with low amount of saturated fat (0.1–1.90 g/ 100 mL) and fibers, and large variation in proteins (0.1–12 g/ 100 mL). The calcium fortification of PBB is comparable to the calcium levels of whole cow's milk, although the vitamin fortification is low. Analysis reveals that salt and oil reduction, as well as fibers enrichment might drive future innovations