Molecular Biology and Biochemistry, Department of

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Homeodomain-Interacting Protein Kinase Promotes Tumorigenesis and Metastatic Cell Behavior

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-01-17
Abstract: 

Aberrations in signaling pathways that regulate tissue growth often lead to tumorigenesis. Homeodomain-interacting protein kinase (Hipk) family members are reported to have distinct and contradictory effects on cell proliferation and tissue growth. From these studies, it is clear that much remains to be learned about the roles of Hipk family protein kinases in proliferation and cell behavior. Previous work has shown that Drosophila Hipk is a potent growth regulator, thus we predicted that it could have a role in tumorigenesis. In our study of Hipk-induced phenotypes, we observed the formation of tumor-like structures in multiple cell types in larvae and adults. Furthermore, elevated Hipk in epithelial cells induces cell spreading, invasion and epithelial-tomesenchymal transition (EMT) in the imaginal disc. Further evidence comes from cell culture studies, in which we expressed Drosophila Hipk in human breast cancer cells and showed that it enhances proliferation and migration. Past studies have shown that Hipk can promote the action of conserved pathways implicated in cancer and EMT, such as Wnt/Wingless, Hippo, Notch and JNK. We show that Hipk phenotypes are not likely to arise from activation of a single target, but rather through a cumulative effect on numerous target pathways. Most Drosophila tumor models involve mutations in multiple genes, such as the wellknown RasV12 model, in which EMT and invasiveness occur after the additional loss of the tumor suppressor gene scribble.Our study reveals that elevated levels of Hipk on their own can promote both hyperproliferation and invasive cell behavior, suggesting that Hipk family members could be potent oncogenes and drivers of EMT.

Document type: 
Article
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Hipk Is Required For JAK/STAT Activity during Development and Tumorigenesis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2019-12-31
Abstract: 

Drosophila has been instrumental as a model system in studying signal transduction and revealing molecular functions in development and human diseases. A point mutation in the Drosophila Janus kinase JAK (called hop) causes constitutive activation of the JAK/STAT pathway. We provide robust genetic evidence that the Homeodomain interacting protein kinase (Hipk) is required for endogenous JAK/STAT activity. Overexpression of Hipk can phenocopy the effects of overactive JAK/STAT mutations and lead to melanized tumors, and loss of Hipk can suppress the effects of hyperactive JAK/STAT. Further, the loss of the pathway effector Stat92E can suppress Hipk induced overgrowth. Interaction studies show that Hipk can physically interact with Stat92E and regulate Stat92E subcellular localization. Together our results show that Hipk is a novel factor required for effective JAK/STAT signaling.

Document type: 
Article
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Autopolyploidy Genome Duplication Preserves Other Ancient Genome Duplications in Atlantic Salmon (Salmo Salar)

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2017-02-27
Abstract: 

Salmonids (e.g. Atlantic salmon, Pacific salmon, and trouts) have a long legacy of genome duplication. In addition to three ancient genome duplications that all teleosts are thought to share, salmonids have had one additional genome duplication. We explored a methodology for untangling these duplications from each other to better understand them in Atlantic salmon. In this methodology, homeologous regions (paralogous/duplicated genomic regions originating from a whole genome duplication) from the most recent genome duplication were assumed to have duplicated genes at greater density and have greater sequence similarity. This assumption was used to differentiate duplicated gene pairs in Atlantic salmon that are either from the most recent genome duplication or from earlier duplications. From a comparison with multiple vertebrate species, it is clear that Atlantic salmon have retained more duplicated genes from ancient genome duplications than other vertebrates--often at higher density in the genome and containing fewer synonymous mutations. It may be that polysomic inheritance is the mechanism responsible for maintaining ancient gene duplicates in salmonids. Polysomic inheritance (when multiple chromosomes pair during meiosis) is thought to be relatively common in salmonids compared to other vertebrate species. These findings illuminate how genome duplications may not only increase the number of duplicated genes, but may also be involved in the maintenance of them from previous genome duplications as well.

Document type: 
Article
File(s): 

Meta-Analysis Suggests Evidence of Novel Stress-Related Pathway Components in Orsay Virus - Caenorhabditis Elegans Viral Model

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2019-03-13
Abstract: 

The genetic model organism, Caenorhabditis elegans (Celegans), shares many genes with humans and is the best-annotated of the eukaryotic genome. Therefore, the identification of new genes and pathways is unlikely. Nevertheless, host-pathogen interaction studies from viruses, recently discovered in the environment, has created new opportunity to discover these pathways. For example, the exogenous RNAi response in Celegans by the Orsay virus as seen in plants and other eukaryotes is not systemic and transgenerational, suggesting different RNAi pathways between these organisms. Using a bioinformatics meta-analysis approach, we show that the top 17 genes differentially-expressed during C. elegans infection by Orsay virus are functionally uncharacterized genes. Furthermore, functional annotation using similarity search and comparative modeling, was able to predict folds correctly, but could not assign easily function to the majority. However, we could identify gene expression studies that showed a similar pattern of gene expression related to toxicity, stress and immune response. Those results were strengthened using protein-protein interaction network analysis. This study shows that novel molecular pathway components, of viral innate immune response, can be identified and provides models that can be further used as a framework for experimental studies. Whether these features are reminiscent of an ancient mechanism evolutionarily conserved, or part of a novel pathway, remain to be established. These results reaffirm the tremendous value of this approach to broaden our understanding of viral immunity in Celegans.

Document type: 
Article
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Fabrication of 3D Fingerprint Phantoms via Unconventional Polycarbonate Molding

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-06-25
Abstract: 

Fingerprint biometrics is a valuable and convenient security tool; every fingerprint is highly detailed and unique, we always have them on “hand”. Herein we describe a novel bench-top method of making 3D fingerprint replicas (namely, fingerprint phantoms) by exploring a unique microfabrication approach using conventional polymeric materials, to aid the development of reliable and accurate fingerprint biometrics. By pressing an impression of human fingerprints onto solvent-softened plastic plates (e.g., polycarbonate chips), followed by casting with polydimethylsiloxane (PDMS, a popular elastomer), we can produce a flexible, nanoscale detailed, 3D reproduction of the fingerprint (“phantom”). By testing with standard optical fingerprint scanners, we have shown that all three levels of fingerprint details can be precisely recorded and match well with the original fingerprint. Superior to artificial fingerprint patterns, these phantoms have the exact 3D features of fingerprints and introduce no variability compared to human sampling, which make them perfect targets for standardizing fingerprint scanners and for biometric applications. We envision that the microcontact replication protocol via unconventional PC molding promises a practical, bench-top, instrumentation-free method to mass reproduce many other micro/nanostructures with high fidelity.

Document type: 
Article
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EFHC1, Implicated in Juvenile Myoclonic Epilepsy, Functions at the Cilium and Synapse to Modulate Dopamine Signaling

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2019-02-27
Abstract: 

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy.

Document type: 
Article
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Implications of HIV-1 Nef for “Shock and Kill” Strategies to Eliminate Latent Viral Reservoirs

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-11-30
Abstract: 

Finding a cure for HIV is challenging because the virus is able to integrate itself into the host cell genome and establish a silent state, called latency, allowing it to evade antiviral drugs and the immune system. Various “shock and kill” strategies are being explored in attempts to eliminate latent HIV reservoirs. The goal of these approaches is to reactivate latent viruses (“shock”), thereby exposing them to clearance by viral cytopathic effects or immune-mediated responses (“kill”). To date, there has been limited clinical success using these methods. In this review, we highlight various functions of the HIV accessory protein Nef and discuss their double-edged effects that may contribute to the limited effectiveness of current “shock and kill” methods to eradicate latent HIV reservoirs in treated individuals.

Document type: 
Article
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Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-10-01
Abstract: 

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics. 

Document type: 
Article
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Exploring the Molecular Properties of Collagen Type IV with Atomic Force Microscopy

Author: 
Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-04-13
Abstract: 

Collagen type IV is a network-forming collagen that provides support and anchorage to cells. Its basic structural unit is a 410 nm long and 1.5 nm in diameter triple helix, with natural discontinuities in the triple-helical defining Gly-X-Y sequence. The C-terminal globular domain (NC1) in a collagen IV molecule plays an important role in forming networks, and has recently been reported to be structurally triggered by chloride ions to form hexamers outside the cell. How this hexamer assembles in vitro remains unknown. Here, I aim to use atomic force microscopy (AFM) to investigate the molecular basis of collagen type IV network assembly by studying the effects of different solvent conditions on the stability of the NC1 domain. Studying the dissociation of this hexametric domain can shed light onto how it assembles in solution and under what ionic conditions. The flexibility of the collagen type IV molecule is also investigated by performing statistical analysis of AFM-imaged chains and estimating persistence length, a mechanical property that quantifies the flexibility of a polymer. Here, I investigate the effects of triple helix interruptions on the flexibility of the molecule, by comparing collagen type IV to other fibrillar collagens that are continuously triple-helical. In addition, I determine a position-dependent flexibility profile of the molecule showcasing the effects of over-lapping interruptions, from a α1(IV)]2–α2(IV) mouse collagen type IV, on the persistence length.

Document type: 
Thesis
File(s): 

Implementation of Couples’ Voluntary HIV Counseling and Testing Services in Durban, South Africa

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Background

Couples’ voluntary HIV counseling and testing (CVCT) is an evidence-based intervention that significantly reduces HIV incidence in couples. Despite the high prevalence of HIV and HIV couple serodiscordance in South Africa, there are few CVCT services.

Methods

From February-June 2013, The Rwanda Zambia HIV Research Group provided support, training, and technical assistance for local counselors and promoters to pilot CVCT services in five hospital-based clinics in Durban, South Africa. Client-level data (age, gender, years cohabiting, pregnancy status, previous testing, antiretroviral treatment (ART) status, neighborhood, and test site) collected as a component of routine CVCT service operation is presented stratified by couple serostatus.

Results

Twenty counselors and 28 promoters completed training. Of 907 couples (1,814 individuals) that underwent CVCT, prevalence of HIV was 41.8 % and prevalence of HIV serodiscordance was 29.5 % (19.3 % M-F+, 10.3 % M + F-). Most participants were 25–34 years of age, and this group had the highest prevalence. Previous individual HIV testing was low (50 % for men, 63 % for women). Only 4 % of couples reported previous CVCT. Most (75 %) HIV+ partners were not on ART, and HIV+ individuals in discordant couples were more likely to be on ART than those in concordant positive couples. Pregnancy among HIV+ women was not associated with previous HIV testing or ART use.

Conclusions

Implementation of standard CVCT services was found to be feasible in Durban. The burden of HIV and couple serodiscordance in Durban was extremely high. CVCT would greatly benefit couples in Durban as an HIV prevention strategy.

Document type: 
Article
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