A conserved long intergenic non-coding RNA containing snoRNA sequences, lncCOBRA1, affects Arabidopsis germination and development

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein-coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed gemination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and examine the protein-protein interaction network of lncCOBRA1-interacting proteins.

Investigating the function of CLU in Alzheimer's Disease in in vitro and in vivo models

<p>Alzheimer’s disease (AD) is a neurodegenerative disease that is responsible for 50-80% of dementia cases and is characterised by lack of visuospatial perception, impairment of language and memory. One of the main physiological attributions towards this disease is the accumulation of large insoluble deposits of amyloid beta, a toxic peptide, which results in the generation of amyloid plaques found in between neurons in the brain. Currently no therapeutic treatments are available. Clusterin (CLU) is an apolipoprotein that when defective is the second highest genetic risk factor for AD. It has been strongly debated whether CLU counteracts or promotes AD pathology. With the roles of CLU including but not limited to acting as a chaperone for cholesterol transport and aiding autophagy functionality in cancer models, this thesis investigates these two specific functionalities by overexpressing CLU in an in vitro SH-SY5Y and in an in vivo AD model of Drosophila melanogaster (fruit fly). Conclusions from this study reveal that within D. melanogaster, CLU reduced Aβ42 levels and increased cholesterol effect through the blood brain barrier. Additionally, in human cells, CLU ameliorated the defective flux in autophagy. This thesis sheds light into how CLU plays a protective role within an Alzheimer’s disease mammalian system.</p>

Investigating the function of CLU in Alzheimer's Disease in in vitro and in vivo models

<p>Alzheimer’s disease (AD) is a neurodegenerative disease that is responsible for 50-80% of dementia cases and is characterised by lack of visuospatial perception, impairment of language and memory. One of the main physiological attributions towards this disease is the accumulation of large insoluble deposits of amyloid beta, a toxic peptide, which results in the generation of amyloid plaques found in between neurons in the brain. Currently no therapeutic treatments are available. Clusterin (CLU) is an apolipoprotein that when defective is the second highest genetic risk factor for AD. It has been strongly debated whether CLU counteracts or promotes AD pathology. With the roles of CLU including but not limited to acting as a chaperone for cholesterol transport and aiding autophagy functionality in cancer models, this thesis investigates these two specific functionalities by overexpressing CLU in an in vitro SH-SY5Y and in an in vivo AD model of Drosophila melanogaster (fruit fly). Conclusions from this study reveal that within D. melanogaster, CLU reduced Aβ42 levels and increased cholesterol effect through the blood brain barrier. Additionally, in human cells, CLU ameliorated the defective flux in autophagy. This thesis sheds light into how CLU plays a protective role within an Alzheimer’s disease mammalian system.</p>

Mechanistic Model of Replication Fork Progression in Yeast

Replication fork progression complex plays an essential role during DNA replication. It travels along with the DNA with a particular speed called replication fork speed. Faithful duplication of the genome requires strict control over replication fork speed. Both acceleration and pausing mechanisms of the replication fork complex are regulated at the molecular level. Based on the experimental evidence, DNA replicates faster in normal cells than cancer cells, whereas cancer cells duplicate themselves more quickly than normal cells. Then in principle, accelerating the replication fork complex in cancer cells beyond a specific threshold speed limit can cause DNA damage and plausibly kill them. A modular mathematical model is proposed to explain the dynamics of replication fork control during DNA replication using the underlying molecular mechanisms in yeast which can extend to the mammalian system in the future.

Recombinant Protein Expression and Purification of N, S1, and RBD of SARS-CoV-2 from Mammalian Cells and Their Potential Applications

The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.

Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world’s population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA’s action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Mouse aging cell atlas analysis reveals global and cell type-specific aging signatures

Aging is associated with complex molecular and cellular processes that are poorly understood. Here we leveraged the Tabula Muris Senis single-cell RNA-seq data set to systematically characterize gene expression changes during aging across diverse cell types in the mouse. We identified aging-dependent genes in 76 tissue-cell types from 23 tissues and characterized both shared and tissue-cell-specific aging behaviors. We found that the aging-related genes shared by multiple tissue-cell types also change their expression congruently in the same direction during aging in most tissue-cell types, suggesting a coordinated global aging behavior at the organismal level. Scoring cells based on these shared aging genes allowed us to contrast the aging status of different tissues and cell types from a transcriptomic perspective. In addition, we identified genes that exhibit age-related expression changes specific to each functional category of tissue-cell types. Altogether, our analyses provide one of the most comprehensive and systematic characterizations of the molecular signatures of aging across diverse tissue-cell types in a mammalian system.

Role of Hakai in m6A modification pathway in Drosophila

N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. Here we show that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m6A writer components, and Hakai mutants exhibit typical m6A pathway defects in Drosophila, such as lowered m6A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m6A modification is mostly distributed in 5’ UTRs in Drosophila, in contrast to the mammalian system. Interestingly, we demonstrate that m6A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m6A writer. Together, our work not only advances the understanding of mechanism and regulation of the m6A writer complex, but also provides insights into how Sxl cooperate with the m6A pathway to control its own splicing.

Diet-Derived Exogenous miRNAs As Functional Food Components: Facts and New Perspectives

Exogenous miRNAs derived from dietary substances have been shown to be orally transferred to the mammalian system and proven to remain active to regulate host-gene expression. This way they have become an active area of research as functional food components and aspects for dietary supplementation. They are being studied as a new class of metabolically targeted therapeutics that work through diet manipulation and may hold promise for a therapeutic approach in reducing the risk of life-threatening diseases. However, a substantial amount of evidence also defies this dietary miRNA concept in terms of their absorption, bioavailability, cellular uptake and its physiological effects in the mammalian system. But recent advances in the identification of some unique sequence and structural characteristics of dietary miRNAs and a deeper understanding of their stability in host peripheral blood for its cellular uptake have strengthened the whole concept. The review comprehensively summarizes the mechanism for miRNA extracellular transport, absorption through the gastrointestinal tract (GI), stability in peripheral blood, and cellular uptake in mammalian cells. It recapitulates the shreds of evidence, related to the influence of dietary miRNAs on gene expression based on the source of the origin (plant vs animal), and compares their cross-kingdom behaviour in terms of their unique sequence and stem-loop structure properties that help them to get stabilized in the mammalian system. The review also summarizes the parameters required for maintaining the sustainable uptake and bioavailability of the dietary miRNAs with existing examples of successful in-vivo and in-vitro delivery of dietary miRNA for augmented therapy. Lastly, it provides an overview of the available and required databases, webserver, and tools that can be used for the successful identification of potential dietary miRNA candidates.