Browsing by Author "Babu, Mohan"

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Open Access
Analysis of Strobilurin Fungicides, Neonicotinoid Insecticides and Degradation Products in Atmospheric Particles
(Faculty of Graduate Studies and Research, University of Regina, 2021-05) Behdarvandan, Asal; Raina-Fulton, Renata; Babu, Mohan; deMontigny, David
In this study, a liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous detection of concentration of 8 neonicotinoid insecticides with 5 degradation products of neonicotinoid insecticides and 7 strobilurin fungicides in the particle phase of atmospheric samples. Detection limits ranged from 0.5 to 3 ng/mL and the recovery percent ranged between 83.5% to 108.3% with relative standard deviation <13% for all analytes. The sampling site was located in Omak, WA within the Okanogan County agricultural region where vineyards and apple orchards are the dominant crops. Matrix effect was determined to evaluate the performance of the developed sample clean-up procedure to remove the matrix. The determined matrix effect revealed that the highest exhibited matrix effects were for metabolites of neonicotinoids, while strobilurin fungicides showed soft to moderate matrix effects in >75% of samples. Neonicotinoids that were detected included imidacloprid, clothianidin, acetamiprid, and thiamethoxam. The highest detection frequency in 2016 was shown for imidacloprid (47%), while in 2018 acetamiprid had the highest detection frequency (67%). This is the first time that one of the neonicotinoid degradation products (desmethyl-thiamethoxam) was detected in the particle phase of air samples worldwide. For strobilurin fungicides, azoxystrobin, kresoxim methyl, trifloxystrobin, and pyraclostrobin were detected in the particle phase. The highest detection frequency was shown for pyraclostrobin (60% in 2016 and 78% in 2018) with the highest concentration (9.82 pg/m3 and 9.87 pg/m3 in 2016 and 2018, respectively).
Open Access
Anti-candida and Anti-virulence Activity of Essential Oils and Their Components
(Faculty of Graduate Studies and Research, University of Regina, 2021-10) Shahina, Zinnat; Dahms, Tanya; Suh, Dae-Yeon; Babu, Mohan; Cameron, Andrew; Lee, Samuel A.
Candida albicans is one of the most common fungi associated with lifethreatening infections. Treatment of the associated infections is often ineffective in the light of resistance, and so there is an urgent need to discover novel antifungals. Rather than killing the fungal cells, which requires quite high specificity and can lead to the emergence of further resistance, inhibiting growth and virulence factors in fungal cells represents a good alternative for the development of new antifungal drugs. Recently, there has been a resurged interest in essential oils and their active components, in relation to their pharmacological properties. The primary objective of this research was to evaluate the antifungal activity of cinnamon bark and rosemary essential oils, along with its major components cinnamadehyde, and 1,8-cineole, α-pinene, respectively, and two other common essential oil components, namely eugenol and citral, against C. albicans. Despite many reports on the antimicrobial activities of essential oils, the results have been diverse and the evaluation methods inconsistent. At higher essential oil concentrations, reactive oxygen species were generated, impacting a wide range of processes including cell membrane depolarization, vacuolar segregation, mitochondrial dysfunction, cell-cycle checkpoint deficiency, and mitotic catastrophe, which resulted in C. albicans death. At lower fractional MICs, essential oils had a ROS-independent response that inhibited mycelial growth and biofilm formation, which may be attributable to defects at the cell membrane. However, microtubule inhibition also plays a role in limiting hyphal growth. I explored the microtubule defects and how they relate to Kar3p, a member of the kinesin-14 family shown to be linked to microtubule stability. Interestingly, both tubulin and Kar3 protein was delocalized with essential oil components exposure at levels for which endogenous ROS levels were normal. I further demonstrate that the level of Kar3 is associated with resistance and susceptibility of C. albicans to essential oil components in both liquid and on solid growth media. All mutant strains grown on hyphae-inducing media in the presence of essential oil components exhibited an enhanced tendency to form pseudohyphal cells, a common phenotype for the homozygous and heterozygous deletion strains. Towards these ends, microtubule defects were linked with theoretical binding between essential oil components and α-tubulin and Kar3p adjacent to cofactor binding sites, consistent with experimentally observed hyphal defects and biofilm inhibition. This study for the first time uncovers a new mode of essential oilmediated microtubule defects, which does not follow the known mechanisms of conventional microtubule inhibition. The essential oil components appear to impact C. albicans eventually giving rise to psuedohyphal formation, microtubule loss, hyphal and biofilm reduction. Since those essential oil components impact multiple Candida targets, they should be less susceptible to resistance. Further, many components showed antivirulence, and may represent an effective approach for inhibiting Candida, especially in the context of physically preventing Candida from entering its host through catheters and prosthetics.
Open Access
Characterizaion and Chemical Rescure of the Phenotype of ors-3, a Knockout mutant line of PpORS in Physcomitrella patens
(Faculty of Graduate Studies and Research, University of Regina, 2019-04) Aslam, Misbah; Suh, Dae-Yeon; Babu, Mohan; Ashton, Neil; Weger, Harold
2ʹ-Oxoalkylresorcinol synthase from Physcomitrella patens (PpORS) is basal to all plant type III polyketide synthases in phylogenetic trees, and may resemble closely their most recent common ancestor. PpORS knockouts were previously generated and partially characterized (Li et al., Planta, 2018, 247: 527–541). This study aimed to investigate further the in planta functions and evolutionary roles of PpORS. Ors-3 (an ors knockout line) was first subjected to dehydration stress. The ability of ors-3 to recover after dehydration is significantly compromised in contrast to the control strain. Ors-3 also loses water faster than the control strain. These results together with the previous data suggested that ors mutants possess a defective cuticle (Li et al., 2018). In ors-3, the expression of two putative paralogs of PpORS was also examined to reveal that their expression levels did not appear to be up-regulated in ors-3 as compared to those in the control. Chemical rescue of ors-3 has been achieved. Thus, the ability of ors-3 to survive dehydration is restored in a dose dependent manner by in vitro PpORS products, purified oxoalkylresorcinol, and also by long chain alkylresorcinol analogs. Exogenous 14Clabelled in vitro PpORS products are incorporated as an insoluble biopolymer, and most of the radoioactivity was recovered after acid hydrolysis. Taken together, these data indicate that PpORS-produced 2ʹ-oxoalkylresorcinols are constituents of the moss cuticular biopolymer that confer resistance to dehydration, and imply that an ancestral ORS in early land plants may have contributed to their successful colonization of the land.
Open Access
Characterization of Neuronal Protein Complexes to Identify Molecular Underpinnings of Autism Spectrum Disorder
(Faculty of Graduate Studies and Research, University of Regina, 2018-07) Wu, Zhuoran; Babu, Mohan; Fitzpatrick, Dennis; Weger, Harold
Autism spectrum disorder (ASD), a complex neurodevelopmental disorder that affects brain development, social and communication skills in children and adults, poses a tremendous burden on caregivers and the healthcare system. Although advances in genetics has enabled the discovery of hundreds of ASD-associated genes, the biological context of how these ASD-linked risk genes contribute to the pathophysiology of the disorder remains unclear. The study of protein-protein interactions (PPI) offers a valuable framework for elucidating this biological context, so far only few human proteomics studies were targeted to ASD. Many cell-context-dependent human ASD interactions involved in neuronal processes are unknown. This study addresses this gap by systematically. Fourty six literature-validated ASD-linked proteins expressed in differentiated SH-SY5Y cells were studied using immunoprecipitation coupled with mass spectrometry (IP/MS) to generate a high-quality PPI network, outlining the organization of individual co-purified proteins in multiprotein complexes. Enrichment analysis confirmed the disease relevance of the PPI network and revealed significant enrichment for genes involved in gene expression, cellular transport and mitochondrial (mt) processes. From the high-confidence PPI network, two previously unreported interacting protein pairs of interest (i.e. FMRP-TWNK and DYRK1A-TRMT61B) linking mitochondria with ASD were selected for further characterization. Co-purifying proteins were validated experimentally by co-immunoprecipitation. Knockouts of the interacting protein pairs of interest were generated, with knockout efficacy confirmed by immunoblotting. The interaction between the ASD-linked fragile X mental retardation protein (FMRP) and the mt twinkle DNA helicase (TWNK) were found to play a role in maintaining mtDNA integrity. As well, a functional role between the dual-specificity tyrosine-phosphorylation regulated kinase (DYRK1A) and the mt tRNA methyltransferase 61B (TRMT61B) in tRNA methylation were established. While additional work is needed to dissect the molecular mechanisms of these interactions, the findings from this study offers support to the validity of the molecular models that were proposed. In conclusion, through this Master’s thesis, I was able to demonstrate that the ASD-linked PPI network generated by IP/MS can offer a powerful means for discovering new ASD links and providing future directions for understanding the role of mt in ASD biology.
Open Access
Determination of Endocrine Disrupting Chemicals in Water and Wastewater in Saskatchewan
(Faculty of Graduate Studies and Research, University of Regina, 2019-04) Aborkhees, Ghada Abobaker; Raina-Fulton, Renata; Babu, Mohan; Thirunavukkarasu, Ondiveerapan; Henni, Amr
A liquid chromatography-negative ion electrospray ionization-tandem mass spectrometry (LC-ESI--MS/MS) method was developed for the simultaneous analysis of endocrine disruptive chemicals including bisphenol A, 4-octylphenol, 4-nonylphenol, diethylstilbestrol, 17-β-estradiol, estriol, estrone, 17-α-ethinylestradiol, prednisone and prednisolone. Mass spectrometric detection of these target analytes is sensitive to the presence of additives in the mobile phase as well as those used in the sample preparation steps. To avoid issues with reduced mass spectrometry sensitivity, the sample preparation involved elution of the target analytes from the solid-phase sorbent with acetonitrile, drying, and reconstitution with methanol, water, and 2-propanol. Recoveries for all the analytes were from 82.4 to 112%. The detection limits for bisphenol A, octylphenol and nonylphenol using LC-ESI--MS/MS were 1-2 ng/L and for diethylstilbestrol, 17-β-estradiol, estriol, estrone, 17-α-ethinylestradiol, prednisone, and prednisolone were 1-10 ng/L. Mass spectrometric signal suppression was moderate to severe for all analytes except diethylstibestrol. Standard addition calibration was used for quantitation of the target analytes at five wastewater treatment plants in Saskatchewan. Estriol was detected in all the raw wastewater samples with concentrations from 20.4 to 285 ng/L. Estrone and 17-β estradiol were detected in 83% and 67% of the raw wastewater samples at concentrations ranging from 31 to 157 ng/L, and 14 to 100 ng/L, respectively. Concentrations of estrone and 17-β estradiol were higher in raw wastewater than predicted from excretion estimation based on population, indicating non-domestic contribution. In raw wastewater, the concentration of 4-octylphenol was from 127 to 318 ng/L (detected in 33% of raw wastewater samples), and the concentration of 4- nonylphenol was 50 ± 15 ng/L (detected in one location). Bisphenol A was detected in 100% of the raw wastewater samples with concentrations from 133 to 645 ng/L. The concentration of prednisone in raw wastewater was 302 ± 76 ng/L (detected 66%), while prednisolone, which is a metabolite of prednisone, was detected in one raw wastewater sample at 18 ± 1 ng/L. The wastewater treatment process was efficient in the removal of natural estrogens and prednisone with percentage removal of 80-100%, and for BPA generally from 56-100%. The removal efficiency of 4-nonylphenol and prednisolone were very low.
Open Access
Effect of Physioxia on Protein Levels in Adult Mouse (Mus Musculus) Neural Stem and Progenitor Cells
(Faculty of Graduate Studies and Research, University of Regina, 2018-12) Lanigan, Adam Thomas; Manzon, Richard; Chao, Tzu-Chiao; Yost, Christopher; Babu, Mohan
Neural Stem and Progenitor Cells (NSPCs) from the adult subventricular zone hold therapeutic potential for a variety of neurological diseases due to their capabilities of self-renewal and potential for differentiation into the cell types of the brain. The potential for autologous cell replacement using NSPCs in cases of neurological injury or disease alleviates many of the ethical and practical issues that are a concern of other stem cell varieties. However, an understanding of many of the basic mechanisms that influence NSPC behaviour under normal physiological conditions is still required to develop appropriate strategies for NSPC expansion in vitro or for endogenous recruitment. Typically studies of NSPCs are conducted under conditions of atmospheric O2 levels, when in reality NSPCs reside in a niche that is subject to much lower concentrations of O2. As such, most studies concerning NSPCs may be limited in terms of their physiological relevance, and culture at lower levels of O2 may better approximate the conditions NSPCs experience in situ within their neurological niche. With this in mind, adult mouse (Mus musculus) NSPCs were cultured in both physiologically relevant levels of O2 (5%) and atmospheric conditions (21% O2) and then subsequently harvested for quantitative proteomic analysis using a data-independent acquisition (DIA) mass spectrometry (MS)-based approach. While cell count experiments suggested there was a difference in cellular activity between NSPCs in each treatment, there were no statistically significant changes in individual protein levels between O2 treatment groups following MS analysis, likely due to high variation among samples. However, MSanalysis still resulted in the identification of 878 unique proteins across treatment groups with quantitative protein expression values. To the best of our knowledge, this is the first quantitative accounting of the proteome of adult NSPCs from mice using DIA-MS-based techniques, which has resulted in a data set that can be compared to future studies of NSPCs. Additionally, this paper provides a framework for future studies by implementing coarse-grain functional categorization of proteins in order to reduce a large proteomic data set into a partitioning scheme that may be more amenable to deriving biologically meaningful conclusions.
Open Access
An Efficient Lentiviral-based Proteomics Approach Identifies Mitochondrial Complexes with Neurodegenerative Function
(Faculty of Graduate Studies and Research, University of Regina, 2015-04) Kassir, Sandy; Babu, Mohan; Suh, Dae-Yeon; Dahms, Tanya; Buttigieg, Josef
Perturbations in proteins involved in the mitochondrial processes have emerged as a causative factor in a wide range of life-threatening human diseases, such as neurodegenerative disorders. In fact, mitochondrial dysfunctions are argued to be the first step in triggering the onset of other deleterious events that collectively act causally in the pathogenesis of diseases. Accordingly, dysfunctions in mitochondrial proteins influence the proper functioning of basic processes, such as energy metabolism and reactive species production. Indeed, biochemical and genetic evidence has provided molecular insights into the role of mitochondrial proteins and complexes in isolation, yet our understanding of how mitochondrial proteins cause and/or contribute to the diverse array of human diseases, including neurodegeneration remains unclear. Like any other biological system, mitochondria are linked together by extensive networks of physical (protein-protein) interactions; therefore a detailed understanding of the systems properties is required to unravel their role in neurodegenerative diseases. To address this, I have developed and optimized an effective procedure to identify the physically interacting proteins for generating a mitochondrial protein-protein interaction network for those involved in various neurodegenerative diseases.
Open Access
Escherichia coli and Rhizobium leguminosarum response mechanisms to sub-lethal 2,4-dichlorophenoxyacetic acid
(Faculty of Graduate Studies and Research, University of Regina, 2017-02) Bhat, Supriya Venkatesh; Dahms, Tanya; Suh, Dae Yeon; Yost, Christopher; Babu, Mohan; Dillon, Jo-Anne
The chlorophenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is used extensively worldwide despite its known toxicity and our limited understanding of how it affects non-target organisms. To determine the global effects of 2,4-D at sub-lethal levels on Rhizobium leguminosarum bv. viciae 3841 (Rlv) and Escherichia coli BL21, I used a novel combination of methods involving advanced microscopy and metabolomics. Rlv showed an oxidative stress response, but showed adaptive capabilities with changes to specific metabolic pathways and consequent changes to its phenotype, surface ultrastructure, and physical properties during 2,4-D exposure. Interestingly, auxin and 2,4-D, its structural analogue, induced common morphological changes in vitro, which were similar in shape to bacteroids isolated from plant nodules, implying that these changes may be related to bacteroid differentiation required for nitrogen fixation. In E. coli, 2,4-D altered biofilm formation and induced a filamentous phenotype in the lab strain and a selection of genotypically diverse strains isolated from the environment. The phenotype was observed at concentrations 1000 times below field application levels, and was reversible upon supplementation with polyamines, implicating DNA damage. Cells treated with 2,4-D had more compliant envelopes, significantly remodelled surfaces that were rougher and more hydrophobic and altered vital metabolic pathways. Most of the observed effects could be attributed to oxidative stress, consistent with increased reactive oxygen species as a function of 2,4-D exposure. The characteristic filamentous phenotype and metabolic changes with 2,4-D exposure implicated impact on cell division. I developed correlative atomic force microscopy-quantitative imaging and laser scanning confocal microscopy to simultaneously probe cell surface alterations at the pico-nanoscale with details of molecular changes inside live cells in real-time. At the mechanistic level, 2,4-D at >1 mM altered FtsZ, FtsA and SulA localization within seconds accompanied by DNA damage resulting in immediate inhibition of Z-ring formation and arrest of cell division. There were simultaneous changes to cell surface roughness, elasticity and adhesion in a time-dependent manner. I propose that 2,4-D rapidly blocks cell division in E. coli likely by affecting the higher order assembly of the divisome complex with inhibition of Z-ring formation resulting from oxidative stress and DNA damage.
Open Access
Genetic interaction landscape of the Escherichia coli transcriptional factor machinery
(Faculty of Graduate Studies and Research, University of Regina, 2022-07) Hosseinnia, Ali; Babu, Mohan; Raina-Fulton, Renata; Widdifield, Cory; Hansmeier, Nicole; Kumar, Ayush
In Bacteria, transcription factors (TFs) consist of sensory ligand-binding and DNA-binding helix-turn-helix (HTH) domains to respond to the environmental and internal stimuli. Although Escherichia coli is a well-studied model bacterium, yet half of its TFs’ characteristics remain unclear, including 72 genes of unknown function. Using E. coli synthetic genetic array, a quantitative approach to scoring the fitness of single and double mutant genes, I was able to measure pairwise genetic interactions (GIs) among all TFs in rich and minimal media (RM and MM) to create a differential (DF) GI network. DF network analysis demonstrated GIs altered in RM or MM growth conditions. Both static and DF GI networks were also effective in detecting TF pathways, highlighting new roles for uncharacterized TFs (YjdC, YneJ, YdiP) as regulators of cell division, putrescine pathway and efflux pump, and cold shock adaptation, respectively. Pan-bacterial conservation suggests that TF genes with similar GI profiles are co-conserved in bacterial evolution. Moreover, the E. coli TF GI network provided deep insights on conserved genetic association across bacterial phyla that can be valuable to build TF machinery organization, even in pathogens.
Open Access
Identification and Characterization of Developmentally Regulated Components of the Stress Axis in Petromyzon Marinus
(Faculty of Graduate Studies and Research, University of Regina, 2013-01) Endsin, Matthew Joel; Manzon, Richard; Buttigieg, Josef; Babu, Mohan
Genes resembling elements of the Corticotropin releasing hormone (CRH) receptor-ligand system (CRH system) have been identified in invertebrate species and suggest the CRH system has existed, in some form, for approximately a billion years. It is theorized that vertebrates inherited components of the CRH system from an invertebrate ancestor. The association of the CRH system with the stress response, however, is specific to vertebrate species and theorized to have accompanied the development of hypothalamic pituitary (HP) axes, specifically the HP interrenal (HPI) axis in fish. A functional HPI axis has recently been suggested in the lamprey species Petromyzon marinus, a member of the ancient vertebrate superclass agnatha, by identification of pituitary and inter-renal components corticotrophin (ACTH) and 11- deoxycortisol respectively. This study, however, is the first to identify the hypothalamic components, specifically the CRH system, of the HPI. In P. marinus the expression of six CRH system genes, including three hormones, CRH A, CRH B and UCN III-like; two receptors, CRH Rα and CRH Rβ; and a binding protein, CRH BP, are identified by PCR and in silico methodologies. Analysis of the P. marinus CRH system genes appear to support the occurance of the Agnathan superclass prior to a theorized second vertebrate whole genome duplication (WGD) event. This is supported by the P. marinus CRH hormones appearing to represent two of the four vertebrate CRH family paralogues; CRH A and B both being orthologous to vertebrate CRH, and UCN III-like being orthologous to vertebrate UCN III. Additionally, neither CRH Rα nor CRH Rβ, while identified as distinct from one another and related to other vertebrate receptors, were phylogenetically indistinguishable as either ii type 1 or 2. This suggests, the two P. marinus CRH receptor genes identified appear type 1 or 2. This suggests, the two P. marinus CRH receptor genes identified appear to have arisen out of a lamprey specific duplication event they diverged separately from the formation of the type 1 and type 2 receptors. The P. marinus CRH BP deduced amino acid sequence was found to contain regions highly conserved and functionally significant in other vertebrates as well as invertebrate species, and occupies a unique phylogenetic branch. Expression of these genes in brain, gill, liver, kidney as measured by reverse transcription quantitative PCR (RT qPCR) over the life history of P. marinus (including pre-metamorphic larvae, each of the seven stages of metamorphosis, and juvenile parasites) indicated significant variation in gene expression both between tissues and through the life history. Differences in expression were observed for each P. marinus CRH system gene and correlate with significant physiological changes occurring in the developing P. marinus. Some of these include increases in Na+/K+ -ATPase activity in the gill, possibly relating to salt water tolerance, and lipogenic and lipolytic metabolic phases in the kidney and liver. Interestingly, comparatively high expression levels of CRH A, CRH B and CRH Rβ were observed in the JP gonad relative to other JP organs. This suggests these genes may have a paracrine role in this organ, possibly by local regulation of sex steroids, similar to that observed in mice and humans. Interestingly, CRH system mRNA expression did not vary in response to multiple successive acute stressors, including dewatering and salt water exposure, over a 24 hour period as measured by RT qPCR. This suggests that P. marinus CRH system genes may not respond to such stressors at the level of mRNA expression. Collectively, these data indicate that lamprey contain all necessary components of a complete HPI axis, and that the CRH system likely plays an important role in the normal development.
Open Access
Metabolic adaptation of C-terminal protease A-deficient R. leguminosarum in response to loss of nutrient transport
(Frontiers Media, 2018-01-04) Jun, Dong; Minic, Zoran; Bhat, Supriya V.; Vanderlinde, Elizabeth M.; Yost, Chris K.; Babu, Mohan; Dahms, Tanya E.S.
Post-translational modification expands the functionality of the proteome beyond genetic encoding, impacting many cellular processes. Cleavage of the carboxyl terminus is one of the many different ways proteins can be modified for functionality. Gel-electrophoresis and mass spectrometric-based techniques were used to identify proteins impacted by deficiency of a C-terminal protease, CtpA, in Rhizobium leguminosarum bv. viciae 3841. Predicted CtpA substrates from 2D silver stained gels were predominantly outer membrane and transport proteins. Proteins with altered abundance in the wild type and ctpA (RL4692) mutant, separated by 2D difference gel electrophoresis, were selected for analysis by mass spectrometry. Of those identified, 9 were the periplasmic solute-binding components of ABC transporters, 5 were amino acid metabolic enzymes, 2 were proteins involved in sulfur metabolism, and 1 each was related to carbon metabolism, protein folding and signal transduction. Alterations to ABC-binding-cassette transporters, nutrient uptake efficiency and to amino acid metabolism indicated an impact on amino acid metabolism and transport for the ctpA mutant, which was validated by measured amino acid levels.
Open Access
A Multi-omics Study of Human Mitochondrial Proteins During Neurogenesis
(Faculty of Graduate Studies and Research, University of Regina, 2021-04) Zhang, Qingzhou; Babu, Mohan; Suh, Dae-yon; Fitzpatrick, Dennis; Weger, Harold; Hu, Pingzhao
Mitochondria are double membrane organelles in eukaryotic cells, and play vital roles in neurogenesis. Disruptions of mitochondrial functions may lead to neurodegenerative disorders. Yet, the underlying mechanisms remain largely unknown. To elucidate the dynamic changes of transcriptomes during neurogenesis, the single cell RNA sequencing experiments were conducted on both cultured human embryonic carcinoma stem cells and and retinoic acid-induced differentiated neuron-like cells. The systematic analysis framework I have established revealed significant expression alternations between cell states, persistence of heterogeneity in differentiated neuron-like cells, and dynamic modeling during the differentiation, and also identified mitochondrial proteins as novel neuronal markers. To elucidate mitochondrial interactome in neurogenesis, biochemical fractionation coupled with in-depth mass spectrometry profiling experiments were performed in both cell states. The bioinformatics pipeline I have developed resulted in 6,442 high-quality protein-protein interactions among 600 mitochondrial proteins. Computational modeling further predicted that RAB5IF could play a role in the assembly of the respirasome (composed of complexes I, III and IV). Lastly, due to the complexity of BF-MS data and limitation of current software tool kits, I have developed an optimized approach, termed as Statistical Modeling Elution Data (SMED), to infer protein interactions from BF-MS. SMED took advantage of novel statistical modeling and machine learning strategies to achieve better prediction performances compared to previously published studies. Taken together, the multi-omics methods and computational strategies I have established in this study provided not only new insights of mitochondrial biology, but also a systems biology research focus to leverage the deluge of omics data. The findings from this study could accelerate our understanding of how mitochondrial proteins and their interactions play a role in neurobiology.
Open Access
Role of Nudix-Hydrolase ASMTL in Modulating Mitochondrial Biogenesis of Cancer Cells
(Faculty of Graduate Studies and Research, University of Regina, 2021-05) Amin, Shahreen Tina; Babu, Mohan; Fitzpatrick, Dennis; Suh, Dae-Yeon; Weger, Harold; Kumar, Ashok
Cancer cells have enhanced DNA biosynthesis, rendering them susceptible to nucleotide modifications that enter the cellular nucleotide pool during repair or DNA degradation. These modified nucleotides can then be incorporated into newly synthesized DNA, resulting in random mutations or, when overwhelming into DNA damage, culminating into apoptosis, which is the desired effect of anti-cancer chemotherapy. Human cells contain nucleotide sanitation enzymes like ASMTL, which prevent incorporating the non-canonical nucleotides into newly synthesized DNA by removing them from the nucleotide pool, thereby relieving cancer cells from proliferative stress-induced mutations and apoptosis, representing an attractive target for anti-cancer chemotherapy. To identify ASMTL as a target for anti-cancer treatment, we investigated the ASMTL requirement for cancer survival in human cancer cell lines and patients, where ASMTL depletion decreased survival. This decrease in cell survival correlated with 14-3-3 interaction-dependent mitochondrial localization of ASMTL. Analyzing mitochondrial function suggests that ASMTL is imperative in the TP53 dependent BAX-BCL2 pathway, which is turned on by inefficient repair of mtDNA damage. Furthermore, after screening 2040 compounds, we identify small molecules TFBQ and TFHQ as ASMTL inhibitors that potently and selectively engage the ASMTL protein after occupying putative ASMTL MAF active site and impeding ASMTL-14-3-3 interaction. Finally, ASMTL is validated as an anti-cancer target in vivo where ASMTL knockout or inhibition triggers apoptosis and decreased metastasis in xenografts. This study collectively exemplifies the non-oncogene addiction concept for cancer treatment and validates ASMTL as phenotypically lethal to carcinomas.
Open Access
Systems Biology of Host-Pathogen Protein-Protein Interactions
(Faculty of Graduate Studies and Research, University of Regina, 2023-06) Rahmatbakhsh, Matineh; Babu, Mohan; Dahms, Tanya; Hansmeier, Nicole; Hu, Pingzhao
Despite undeniable therapeutic developments in infectiology, emerging infectious diseases continue to be a growing threat to public health, as seen by the current COVID- 19 pandemic caused by the novel virus severe acute respiratory syndrome coronavirus (SARS-CoV-2). This virus is classified as an obligate intracellular parasite that co-opts host cellular proteins, often through protein-protein interactions (PPIs), to ensure its replication. Therefore, this thesis aims to integrate high-throughput proteomic approaches with computational modelling to systematically characterize SARS-CoV-2-human networks for a detailed understanding of SARS-CoV-2 pathogenesis. The angiotensin-converting enzyme (ACE2) receptor of SARS-CoV-2 is displayed on many human cells, including the lungs and other organs. However, despite considerable knowledge explaining the SARS-CoV-2 infection mechanism, organ-specific SARS-CoV- 2-host protein interactions remain understudied. In Chapter 2, we carried out an organ/tissue-unbiased proteomic profiling approach of mapping SARS-CoV-2-human protein interactions using high-throughput mass spectrometry (MS)-based proteomic approaches. First, automated machine learning (ML)-based computational workflows with different algorithmic strategies were devised to generate high-quality tissue-specific and tissue-common SARS-CoV-2-human PPIs. Subsequent clustering of highly conserved networks using an optimized complex-based analysis framework uncovered several virally targeted protein complexes (VTCs), reflecting conserved mechanisms of replication. Finally, organ/tissue-specific interaction revealed that NSP3 protein evades host antiviral innate immune signaling by targeting IFIT5 for de-isgylation. Although host interactome is indirectly affected during viral infection, earlier studies have only focused on characterizing the properties of the viral proteins within the host-viral interactions. However, systematically exploring the host-viral interactions from the perspective of the host interactome is essential and should be included in PPI network for a better understanding of viral pathogenesis. In Chapter 3, we combined cofractionation mass spectrometry (CF-MS) with a novel deep learning-based framework, DeepiCE, to map physiologically relevant viral-host and host interactome. First, through comprehensive statistical validations, we demonstrated the remarkable performance of DeepiCE over the state-of-the-art method for network construction. DeepiCE was then applied to co-elution data from salivary samples of individuals infected with SARS-CoV- 2, which led to the generation of high-quality viral-host and host interactome maps highly relevant to SARS-CoV-2 infection. Subsequent clustering of resulting networks using a sophisticated two-stage clustering framework generated high-quality SARS-CoV-2 affected protein complexes, many of which were enriched for diverse cellular processes related to viral pathogenesis and provided new insights into SARS-CoV-2 infection from both the host and pathogen perspective. Despite arduous and time-consuming experimental efforts, PPIs for many pathogenic microbes with their human host are still unknown, limiting our understanding of the intricate interactions during infection and the identification of therapeutic targets. Since computational tools offer a promising alternative, in Chapter 4, we developed a R/Bioconductor package, HPiP software with a series of amino acid sequence property descriptors and an ensemble machine learning classifiers to predict the yet unmapped interactions between pathogen and host proteins. Using SARS-CoV-1 or the novel SARSCoV- 2 coronavirus-human PPI training sets as a case study, we show that HPiP achieves good performance with PPI predictions between SARS-CoV-2 and human proteins, which we confirmed experimentally using several quality control metrics. HPiP also exhibited strong performance in accurately predicting the previously reported PPIs when tested against the sequences of pathogenic bacteria, Mycobacterium tuberculosis and human proteins. Collectively, our fully documented HPiP software will hasten the exploration of PPIs for a systems-level understanding of many understudied pathogens and uncover molecular targets for repurposing existing drugs.
Open Access
Thermal Stress and the Heat Shock Response in Embryonic and Young of the Year Juvenile Lake Whitefish (Coregonus Clupeaformis)
(Faculty of Graduate Studies and Research, University of Regina, 2015-07) Stefanovic, Daniel Ivan; Manzon, Richard; Buttigieg, Josef; Babu, Mohan
I investigated the effects of thermal stress associated with changing environments and industrial thermal pollution by characterizing the kinetics of the heat shock response (HSR) throughout Lake Whitefish (Coregonus clupeaformis) development. Lake Whitefish are a cold water species that spawn in the late fall to early winter. Their embryos develop at 0.5 – 6 °C, usually under the cover of ice. The HSR is a universal response to thermal and other types of stressors that offer protection at the cellular level. This response is characterized by the synthesis of a group of highly conserved proteins called the heat shock proteins (Hsps). Here I isolated five different hsp cDNAs from Lake Whitefish and quantified changes in mRNA transcript levels in response to varying heat stress conditions in embryos and young of the year (YOY) juveniles. Lake Whitefish were subjected to three different heat shock temperatures (3 °C, 6 °C or 9 °C above control) and six different heat shock durations (0.25, 0.50, 1, 2, 3, and 4 hr) followed by a 2 hr recovery period prior to sampling. In addition, the duration of the HSR was examined by varying the post-heat shock recovery time period prior to sampling. In this recovery experiment, Lake Whitefish were permitted to recover for 1, 2, 4, 8, 12, 16, 24, 36, and 48 hr following a 2 hr heat shock. My data suggest that Lake Whitefish embryos may be resilient to short bouts of heat shock. In embryos, hsp70 mRNA levels were elevated following a 2 hr, 9 °C heat shock. However, levels of the hsp70 did not increase in response to any of the 3 °C or 6 °C heat shocks, irrespective of heat shock duration. It is also significant that embryos did not upregulate mRNA levels of the normally inducible hsp90α or hsp47, in response to any of the heat shock exposures. These embryo data are different from those in 60 days post-hatch YOY juveniles. In YOY juveniles, all three inducible hsps were upregulated in response to both a 6 °C and 9 °C heat shock, suggesting that YOY juveniles will more readily initiate a HSR and that this response is more robust in that it involves multiple hsps. In embryos, once triggered, the HSR was relatively long lasting with hsp70 mRNA levels remaining elevated 48 hr post-heat shock. In contrast, in YOY juveniles the HSR was shorter lived with hsp70 levels beginning to decrease as early as 4 - 8 hr post-heat shock. In summary, my data indicates that Lake Whitefish can initiate a HSR during embryogenesis. In comparison to YOY juveniles, embryos are more resistant to heat stress as they only initiated a HSR at relatively high heat shock temperatures and this response was limited to increases in hsp70. However, once initiated the HSR was relatively long lasting. Collectively, these data will help us better understand the potential impact of thermal stress associated with changing environments and industrial thermal pollution on development of this and other coldwater fish species.