BackgroundTwo of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent ... more BackgroundTwo of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. ObjectivesTo examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. MethodsA total of 150 specimens representing HIV-1 group M subtypes (A–G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. ResultsIn general, both HR1 (a.a. 540–593) and HR2 (a.a. 628–673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547–549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix intera...
Background. The lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbin... more Background. The lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) augments Ebola virus (EBOV) infection. However, it its unclear whether DC-SIGN promotes only EBOV attachment (attachment factor function, nonessential) or actively facilitates EBOV entry (receptor function, essential). Methods. We investigated whether DC-SIGN on B cell lines and dendritic cells acts as an EBOV attachment factor or receptor. Results. Engineered DC-SIGN expression rendered some B cell lines susceptible to EBOV glycoprotein (EBOV GP)-driven infection, whereas others remained refractory, suggesting that cellular factors other than DC-SIGN are also required for susceptibility to EBOV infection. Augmentation of entry was independent of efficient DC-SIGN internalization and might not involve lectin-mediated endocytic uptake of virions. Therefore, DC-SIGN is unlikely to function as an EBOV receptor on B cell lines; instead, it might concentrate virions onto cells, thereby allowing entry into cell lines expressing low levels of endogenous receptor(s). Indeed, artificial concentration of virions onto cells mirrored DC-SIGN expression, confirming that optimization of viral attachment is sufficient for EBOV GP-driven entry into some B cell lines. Finally, EBOV infection of dendritic cells was only partially dependent on mannose-specific lectins, such as DC-SIGN, suggesting an important contribution of other factors. Conclusions. Our results indicate that DC-SIGN is not an EBOV receptor but, rather, is an attachmentpromoting factor that boosts entry into B cell lines susceptible to low levels of EBOV GP-mediated infection. The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are negative-strand nonsegmented RNA viruses that cause hemorrhagic fever in humans [1]. Fi-Potential conflicts of interest: none reported.
Background: Two of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be poten... more Background: Two of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. Objectives: To examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. Methods: A total of 150 specimens representing HIV-1 group M subtypes (A-G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. Results: In general, both HR1 (a.a. 540-593) and HR2 (a.a. 628-673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547-549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix interaction include residues at positions 628W, 631W, 635I, 638Y, 642I, 645L, 649S, 652Q, 656N, and 659E. Analysis of the 150 specimens revealed that all had identical residues at six of these positions, whereas two positions had minor variations (635 and 649) and two (645 and 659) appeared to have subtype-specific substitutions. Conclusions: This data indicates that there is limited resistance to T-20 in these worldwide populations and that the critical epitopes for effective 5-helix binding are highly conserved across all subtypes. Taken together, these data suggest that T-20 and 5-helix should provide useful additives to current antiretroviral therapy for clinical management of HIV disease.
Alphaviruses are a large class of insect-borne human pathogens and little is known about the host... more Alphaviruses are a large class of insect-borne human pathogens and little is known about the host-factor requirements for infection. To identify such factors, we performed a genome-wide RNAi screen using model Drosophila cells and validated 94 genes that impacted infection of Sindbis virus (SINV), the prototypical alphavirus. We identified a conserved role for SEC61A and valosin-containing protein (VCP) in facilitating SINV entry in insects and mammals. SEC61A and VCP selectively regulate trafficking of the entry receptor NRAMP2, and loss or pharmacological inhibition of these proteins leads to altered NRAMP2 trafficking to lysosomal compartments and proteolytic digestion within lysosomes. NRAMP2 is the major iron transporter in cells, and loss of NRAMP2 attenuates intracellular iron transport. Thus, this study reveals genes and pathways involved in both infection and iron homeostasis that may serve as targets for antiviral therapeutics or for iron-imbalance disorders.
While much research focused on making emphasizes digital and tangible media, few studies have exp... more While much research focused on making emphasizes digital and tangible media, few studies have explored making with biology, or biomaking, where people use cells as fabrication units to grow or "make" desired materials for designing real world applications. This lack is especially glaring considering how biomaking and related industries are often aligned with a growing push toward sustainable production as a way of addressing the pressing environmental issues of the day. In order address how maker frameworks could be used as a productive way of bringing biomaking into K-12 contexts, we report on the design and implementation of a biomaking workshop where teams of high school students both assembled a physical biosensor and imagined applications for this technology to address real world issues. Using classroom observations, analysis of classroom projects, and focus group interviews, we examined student experiences and perceptions of these activities in order to ask: What the...
Portfolios have recently gained traction within computer science education as a way to assess stu... more Portfolios have recently gained traction within computer science education as a way to assess students' computational thinking and practices. Whereas traditional assessments such as exams tend to capture learning within artificial settings at a single point in time, portfolios provide more authentic opportunities to document a trajectory of students' learning and practices in everyday contexts. Furthermore, because communication itself has been defined as an important computational thinking practice, portfolios give students a place to practice this skill in the classroom. In this study, we report on the implementation of a digital portfolio with a class of 21 high school students used to capture the process of creating of an electronic textile mural project. While students' understanding of computational concepts were only partially captured within the portfolios, their engagements with computational practices-such as debugging and iterationwere better highlighted. Much of this was due to the students' existing communicative strategies themselves, both in terms of how precise they were in describing issues, as well as how they leveraged images and code to explain their process. Recommendations for designing more effective portfolio assessments are discussed, which include greater emphasis on creating shared classroom discourse, and leveraging students' existing experiences with multimedia.
Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients ... more Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (microLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein ...
Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an impro... more Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cellintrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.
We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex... more We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(∇34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(∇34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its late...
Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP... more Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP) epitope, a structure crucial for the virulence of several respiratory pathogens. In this study, ChoP expression analysis comparing organisms from acute and chronic infections revealed that expression of ChoP at 37°C was higher among strains from chronic infections. Coimmunoprecipitation experiments and mass spectrometry analysis demonstrated that ChoP was on the protein elongation factor Tu. The presence of ChoP at the surface was confirmed by immunofluorescence and flow cytometry analysis of intact bacteria. Pretreatment of bronchial epithelial cells or mice with a platelet-activating factor receptor (PAFR) antagonist reduced adhesion and invasion of the ChoP-positive P. aeruginosa isolates. Results of this study suggest that ChoP expression may represent a novel phenotype expressed by the chronic infection isolates that could mediate P. aeruginosa colonization of the epithelial airway by means of the interaction with the PAFR.
We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early... more We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early part of the HIV-1 epidemic among commercial sex workers (CSWs) in Kinshasa, Democratic Republic of Congo (formerly Zaire). Serologic analysis revealed that 27 (28.7%) of the 94 specimens were seropositive by both peptide and whole-virus lysate EIAs and that 24 were positive by molecular screening assays, using generic primers that can detect all known groups of HIV-1. Phylogenetic analyses of the gag(p24), C2V3, and gp41 regions of these 24 specimens showed that all were group M; none of them had any evidence of group O, N, or SIVcpz-like sequences. On the basis of env sequence analysis, the 24 group M specimens were classified as subtypes G (37.5%), A (21%), F1 (12.5%), CRF01_AE (8%), D (4%), and H (4%); 3 (12.5%) were unclassifiable (U). Similar analysis of the gag(p24) region revealed that the majority of infections were subtype A; however, one-third of the specimens were subtype G. Parallel analysis of gag(p24) and env regions revealed discordant subtypes in many specimens that may reflect possible dual and/or recombinant viruses. These data suggest a predominance of subtype G (both pure G and recombinant CRF02_AG) during the early part of the epidemic in Kinshasa. Infections with group N or SIVcpz-like viruses were not present among these CSWs in Kinshasa.
Proceedings of the 2017 Conference on Interaction Design and Children, 2017
We report on the development and implementation of biomakerlab, a wetlab starter kit for syntheti... more We report on the development and implementation of biomakerlab, a wetlab starter kit for synthetic biology activities in K-12. In synthetic biology, participants make their own DNA-gene by gene-and then grow their designs into real applications by inserting them into microorganisms to develop different traits and characteristics provided by the genes. High school students worked with biomakerlab to make logo designs using microorganisms they manipulated to produce differently colored pigments. Our analysis focuses on student engagement with production activities and design challenges in biomaking. In the discussion, we address differences and overlaps between traditional maker activities and biomaker activities for education.
The study of Drosophila, and other genetically tractable insects, has expanded our understanding ... more The study of Drosophila, and other genetically tractable insects, has expanded our understanding of innate immunity and more recently antiviral innate mechanisms. The Drosophila antiviral program includes inflammatory signaling cascades as well as antiviral RNA silencing and autophagy. This review will highlight the recent discoveries in antiviral immunity in insects and will reveal some of the lessons learned.
The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, c... more The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins.
A major neutralizing epitope (here referred to as the T332 epitope) located on the lateral surfac... more A major neutralizing epitope (here referred to as the T332 epitope) located on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope protein has been identified based on the analysis of murine monoclonal antibodies. However, little is known about the humoral immune response against WNV in a natural host or whether DIII in general or the T332 epitope in particular are important targets of neutralizing antibodies in vivo. To characterize the types of antibodies produced during infection with WNV, we studied a group of naturally infected horses. Using immune adsorption assays coupled with the use of virus particles bearing mutations in the T332 epitope, we found that in some animals neutralizing activity against DIII and the T332 epitope was below the limit of detection. In contrast, some animals generated a significant fraction of neutralizing activity to DIII and the T332 epitope. Thus, while antibodies to the T332 epitope did not represent a significant fraction of the total antibody response in the infected animals studied, in some horses, they comprised a significant fraction of neutralizing activity, making this an important but far from dominant neutralizing epitope. Rather, the neutralizing response to WNV generated in infected horses is both variable and polyclonal in nature, with epitopes within and outside of DIII playing important roles.
West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While th... more West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein.
We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein... more We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.
Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through t... more Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through the blood brain barrier (BBB) endothelium to cause aseptic meningitis. To identify cellular components required for CVB and PV infection of human brain microvascular endothelial cells, an in vitro BBB model, we performed comparative RNAi screens and identified 117 genes that influenced infection. Whereas a large proportion of genes whose depletion enhanced infection (17 of 22) were broadly antienteroviral, only 46 of the 95 genes whose depletion inhibited infection were required by both CVB and PV and included components of cell signaling pathways such as adenylate cyclases. Downregulation of genes including Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases displayed specificity in their requirement for either CVB or PV infection. These findings highlight the pathways hijacked by enteroviruses for entry and replication in the BBB endothelium, a specialized and clinically relevant cell type for these viruses.► RNAi screens reveal 117 host molecules involved in enterovirus infection ► Components of immune signaling including MAPKs, Akts, and TLR8 restrict infection ► Adenylate cyclases are required for both PV and CVB infection ► Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases control infection
BackgroundTwo of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent ... more BackgroundTwo of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. ObjectivesTo examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. MethodsA total of 150 specimens representing HIV-1 group M subtypes (A–G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. ResultsIn general, both HR1 (a.a. 540–593) and HR2 (a.a. 628–673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547–549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix intera...
Background. The lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbin... more Background. The lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) augments Ebola virus (EBOV) infection. However, it its unclear whether DC-SIGN promotes only EBOV attachment (attachment factor function, nonessential) or actively facilitates EBOV entry (receptor function, essential). Methods. We investigated whether DC-SIGN on B cell lines and dendritic cells acts as an EBOV attachment factor or receptor. Results. Engineered DC-SIGN expression rendered some B cell lines susceptible to EBOV glycoprotein (EBOV GP)-driven infection, whereas others remained refractory, suggesting that cellular factors other than DC-SIGN are also required for susceptibility to EBOV infection. Augmentation of entry was independent of efficient DC-SIGN internalization and might not involve lectin-mediated endocytic uptake of virions. Therefore, DC-SIGN is unlikely to function as an EBOV receptor on B cell lines; instead, it might concentrate virions onto cells, thereby allowing entry into cell lines expressing low levels of endogenous receptor(s). Indeed, artificial concentration of virions onto cells mirrored DC-SIGN expression, confirming that optimization of viral attachment is sufficient for EBOV GP-driven entry into some B cell lines. Finally, EBOV infection of dendritic cells was only partially dependent on mannose-specific lectins, such as DC-SIGN, suggesting an important contribution of other factors. Conclusions. Our results indicate that DC-SIGN is not an EBOV receptor but, rather, is an attachmentpromoting factor that boosts entry into B cell lines susceptible to low levels of EBOV GP-mediated infection. The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are negative-strand nonsegmented RNA viruses that cause hemorrhagic fever in humans [1]. Fi-Potential conflicts of interest: none reported.
Background: Two of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be poten... more Background: Two of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. Objectives: To examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. Methods: A total of 150 specimens representing HIV-1 group M subtypes (A-G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. Results: In general, both HR1 (a.a. 540-593) and HR2 (a.a. 628-673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547-549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix interaction include residues at positions 628W, 631W, 635I, 638Y, 642I, 645L, 649S, 652Q, 656N, and 659E. Analysis of the 150 specimens revealed that all had identical residues at six of these positions, whereas two positions had minor variations (635 and 649) and two (645 and 659) appeared to have subtype-specific substitutions. Conclusions: This data indicates that there is limited resistance to T-20 in these worldwide populations and that the critical epitopes for effective 5-helix binding are highly conserved across all subtypes. Taken together, these data suggest that T-20 and 5-helix should provide useful additives to current antiretroviral therapy for clinical management of HIV disease.
Alphaviruses are a large class of insect-borne human pathogens and little is known about the host... more Alphaviruses are a large class of insect-borne human pathogens and little is known about the host-factor requirements for infection. To identify such factors, we performed a genome-wide RNAi screen using model Drosophila cells and validated 94 genes that impacted infection of Sindbis virus (SINV), the prototypical alphavirus. We identified a conserved role for SEC61A and valosin-containing protein (VCP) in facilitating SINV entry in insects and mammals. SEC61A and VCP selectively regulate trafficking of the entry receptor NRAMP2, and loss or pharmacological inhibition of these proteins leads to altered NRAMP2 trafficking to lysosomal compartments and proteolytic digestion within lysosomes. NRAMP2 is the major iron transporter in cells, and loss of NRAMP2 attenuates intracellular iron transport. Thus, this study reveals genes and pathways involved in both infection and iron homeostasis that may serve as targets for antiviral therapeutics or for iron-imbalance disorders.
While much research focused on making emphasizes digital and tangible media, few studies have exp... more While much research focused on making emphasizes digital and tangible media, few studies have explored making with biology, or biomaking, where people use cells as fabrication units to grow or "make" desired materials for designing real world applications. This lack is especially glaring considering how biomaking and related industries are often aligned with a growing push toward sustainable production as a way of addressing the pressing environmental issues of the day. In order address how maker frameworks could be used as a productive way of bringing biomaking into K-12 contexts, we report on the design and implementation of a biomaking workshop where teams of high school students both assembled a physical biosensor and imagined applications for this technology to address real world issues. Using classroom observations, analysis of classroom projects, and focus group interviews, we examined student experiences and perceptions of these activities in order to ask: What the...
Portfolios have recently gained traction within computer science education as a way to assess stu... more Portfolios have recently gained traction within computer science education as a way to assess students' computational thinking and practices. Whereas traditional assessments such as exams tend to capture learning within artificial settings at a single point in time, portfolios provide more authentic opportunities to document a trajectory of students' learning and practices in everyday contexts. Furthermore, because communication itself has been defined as an important computational thinking practice, portfolios give students a place to practice this skill in the classroom. In this study, we report on the implementation of a digital portfolio with a class of 21 high school students used to capture the process of creating of an electronic textile mural project. While students' understanding of computational concepts were only partially captured within the portfolios, their engagements with computational practices-such as debugging and iterationwere better highlighted. Much of this was due to the students' existing communicative strategies themselves, both in terms of how precise they were in describing issues, as well as how they leveraged images and code to explain their process. Recommendations for designing more effective portfolio assessments are discussed, which include greater emphasis on creating shared classroom discourse, and leveraging students' existing experiences with multimedia.
Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients ... more Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (microLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein ...
Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an impro... more Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cellintrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.
We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex... more We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(∇34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(∇34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its late...
Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP... more Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP) epitope, a structure crucial for the virulence of several respiratory pathogens. In this study, ChoP expression analysis comparing organisms from acute and chronic infections revealed that expression of ChoP at 37°C was higher among strains from chronic infections. Coimmunoprecipitation experiments and mass spectrometry analysis demonstrated that ChoP was on the protein elongation factor Tu. The presence of ChoP at the surface was confirmed by immunofluorescence and flow cytometry analysis of intact bacteria. Pretreatment of bronchial epithelial cells or mice with a platelet-activating factor receptor (PAFR) antagonist reduced adhesion and invasion of the ChoP-positive P. aeruginosa isolates. Results of this study suggest that ChoP expression may represent a novel phenotype expressed by the chronic infection isolates that could mediate P. aeruginosa colonization of the epithelial airway by means of the interaction with the PAFR.
We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early... more We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early part of the HIV-1 epidemic among commercial sex workers (CSWs) in Kinshasa, Democratic Republic of Congo (formerly Zaire). Serologic analysis revealed that 27 (28.7%) of the 94 specimens were seropositive by both peptide and whole-virus lysate EIAs and that 24 were positive by molecular screening assays, using generic primers that can detect all known groups of HIV-1. Phylogenetic analyses of the gag(p24), C2V3, and gp41 regions of these 24 specimens showed that all were group M; none of them had any evidence of group O, N, or SIVcpz-like sequences. On the basis of env sequence analysis, the 24 group M specimens were classified as subtypes G (37.5%), A (21%), F1 (12.5%), CRF01_AE (8%), D (4%), and H (4%); 3 (12.5%) were unclassifiable (U). Similar analysis of the gag(p24) region revealed that the majority of infections were subtype A; however, one-third of the specimens were subtype G. Parallel analysis of gag(p24) and env regions revealed discordant subtypes in many specimens that may reflect possible dual and/or recombinant viruses. These data suggest a predominance of subtype G (both pure G and recombinant CRF02_AG) during the early part of the epidemic in Kinshasa. Infections with group N or SIVcpz-like viruses were not present among these CSWs in Kinshasa.
Proceedings of the 2017 Conference on Interaction Design and Children, 2017
We report on the development and implementation of biomakerlab, a wetlab starter kit for syntheti... more We report on the development and implementation of biomakerlab, a wetlab starter kit for synthetic biology activities in K-12. In synthetic biology, participants make their own DNA-gene by gene-and then grow their designs into real applications by inserting them into microorganisms to develop different traits and characteristics provided by the genes. High school students worked with biomakerlab to make logo designs using microorganisms they manipulated to produce differently colored pigments. Our analysis focuses on student engagement with production activities and design challenges in biomaking. In the discussion, we address differences and overlaps between traditional maker activities and biomaker activities for education.
The study of Drosophila, and other genetically tractable insects, has expanded our understanding ... more The study of Drosophila, and other genetically tractable insects, has expanded our understanding of innate immunity and more recently antiviral innate mechanisms. The Drosophila antiviral program includes inflammatory signaling cascades as well as antiviral RNA silencing and autophagy. This review will highlight the recent discoveries in antiviral immunity in insects and will reveal some of the lessons learned.
The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, c... more The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins.
A major neutralizing epitope (here referred to as the T332 epitope) located on the lateral surfac... more A major neutralizing epitope (here referred to as the T332 epitope) located on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope protein has been identified based on the analysis of murine monoclonal antibodies. However, little is known about the humoral immune response against WNV in a natural host or whether DIII in general or the T332 epitope in particular are important targets of neutralizing antibodies in vivo. To characterize the types of antibodies produced during infection with WNV, we studied a group of naturally infected horses. Using immune adsorption assays coupled with the use of virus particles bearing mutations in the T332 epitope, we found that in some animals neutralizing activity against DIII and the T332 epitope was below the limit of detection. In contrast, some animals generated a significant fraction of neutralizing activity to DIII and the T332 epitope. Thus, while antibodies to the T332 epitope did not represent a significant fraction of the total antibody response in the infected animals studied, in some horses, they comprised a significant fraction of neutralizing activity, making this an important but far from dominant neutralizing epitope. Rather, the neutralizing response to WNV generated in infected horses is both variable and polyclonal in nature, with epitopes within and outside of DIII playing important roles.
West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While th... more West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein.
We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein... more We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.
Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through t... more Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through the blood brain barrier (BBB) endothelium to cause aseptic meningitis. To identify cellular components required for CVB and PV infection of human brain microvascular endothelial cells, an in vitro BBB model, we performed comparative RNAi screens and identified 117 genes that influenced infection. Whereas a large proportion of genes whose depletion enhanced infection (17 of 22) were broadly antienteroviral, only 46 of the 95 genes whose depletion inhibited infection were required by both CVB and PV and included components of cell signaling pathways such as adenylate cyclases. Downregulation of genes including Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases displayed specificity in their requirement for either CVB or PV infection. These findings highlight the pathways hijacked by enteroviruses for entry and replication in the BBB endothelium, a specialized and clinically relevant cell type for these viruses.► RNAi screens reveal 117 host molecules involved in enterovirus infection ► Components of immune signaling including MAPKs, Akts, and TLR8 restrict infection ► Adenylate cyclases are required for both PV and CVB infection ► Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases control infection
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Papers by Sheri Hanna