Modulating the expression and activity of the nuclear import protein, Karyopherin β1, in cancer cells

Cancer is primarily a disease of disordered gene expression; the dysregulation of thousands of different genes has been associated with the progression of many types of cancer. Previous research from our laboratory aimed at identifying genes differentially expressed in cervical cancer compared to normal patient tissue, found Karyopherin β1 (Kpnβ1), the primary nuclear import protein, to be significantly overexpressed in cervical cancer tissue. Further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were only minimally affected. These results suggest that Kpnβ1 has potential as an anti-cancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. In this study, we investigated the biological effects associated with Kpnβ1 overexpression in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype. Our data revealed that Kpnβ1 overexpression, above what was already detected in cancer cells, resulted in reduced proliferation and an associated delay in cell cycle progression. Additionally, overexpression of Kpnβ1 caused changes in the morphology and adhesion properties of cells. Co-expression of Ran, an important nuclear transport factor, binding partner and regulator of Kpnβ1, resulted in a further reduction in proliferation (greater than that of overexpression of either Kpnβ1 or Ran alone), suggesting that cells are particularly unable to handle an imbalance in the levels of Kpnβ1 and Ran. Previous work from our laboratory using a newly identified small molecule, Inhibitor of Nuclear Import-43 (INI-43), showed that INI-43 significantly reduced the proliferation of cancer cells of different tissue origins and interfered with the nuclear import function of Kpnβ1. In order to investigate the specificity of INI-43 for Kpnβ1 in this study, we performed experiments to determine whether overexpression of Kpnβ1 could rescue cancer cells from the negative effects associated with INI-43 treatment. Results revealed that stable overexpression of Kpnβ1 was able to rescue cell viability, as well as the inhibitory effects that INI-43 had on the nuclear import of Kpnβ1 cargoes NFκB p65 and NFAT. Kpnβ1 overexpression was also able to rescue cells from an INI-43 induced G2/M cell cycle block. In addition, treatment of cells with INI-43 enhanced the degradation of Kpnβ1, indicating that INI-43 is likely acting by targeting Kpnβ1. In order to determine the effects of Kpnβ1 dysregulation in HeLa cells, live cell time-lapse videomicroscopy was used to study cells as the progressed through the cell cycle. Inducible expression of Kpnβ1-EGFP, as well as the treatment of cells with the small molecule inhibitor INI-43, were used as mechanisms of dysregulation. Results revealed that modulation of Kpnβ1 by either overexpression or inhibition caused a disruption in mitotic progression, with the appearance of distinct mitotic abnormalities. The treatment of cells expressing Kpnβ1-EGFP with INI-43 resulted in a significant reduction in (or rescue of) the negative effects associated with either condition alone. Taken together, this data suggests that a precise balance of Kpnβ1 expression is required for the correct functioning of cancer cells; when the balance is perturbed in either direction (i.e. with overexpression of Kpnβ1 or INI-43-mediated inhibition of Kpnβ1) negative effects associated with a variety of biological processes are observed. In addition, results from rescue experiments conducted using Kpnβ1 overexpression in combination with INI-43 treatment suggest that that INI-43 is acting, at least in part, by targeting Kpnβ1

Boardman Lake Trail

Today in the United States, a significant portion of energy use is devoted to transportation needs. To address sustainable energy use in transportation, Team Wicked Awesome formed in the class LIB 322: “Wicked Problems in Sustainability” at Grand Valley State University in the winter of 2015. Looking to wicked problem solving methods, we examined Traverse City’s need for alternative options for transportation (other than single occupant vehicles). As an alternative mode of transportation, we looked into a number of ways to promote bicycle ridership and came to discover there was a tentative plan to complete a portion of the Traverse Area Recreation and Transportation Trail (T.A.R.T) that would give an entire side of the Boardman Lake more access to downtown and other neighborhoods via bicycle. We discovered that there was a lack of communication between local cycle groups, other community stakeholders and city officials in addressing the completion of the Boardman Lake Trail. We will hold a summit in order to open a dialogue with the community members and other stakeholders to discuss the state of the trail by integrating stakeholders and experts in a discussion panel. As part of the Wicked Problems model of problem solving we hope to bridge gaps that currently exist between these interested parties. We do not intend to create a new effort, but instead we will increase community involvement in the current effort and encourage city officials to place higher priority on the project. This summit will provide the public with information that will educate them not only on the prospects of the trail\u27s completion, but what obstacles exist, and what the community at large can do to help complete the trail. We hope this event will encourage citizens of the community to engage in collaborative effort and to take ownership and responsibility for the future of their city

Roles of Ras1 Membrane Localization during Candida albicans Hyphal Growth and Farnesol Response

Many Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. In Candida albicans, a fungal pathogen of humans, Ras1 links environmental cues to morphogenesis. Here, we report the localization and membrane dynamics of Ras1, and we characterize the roles of conserved C-terminal cysteine residues, C287 and C288, which are predicted sites of palmitoylation and farnesylation, respectively. GFP-Ras1 is localized uniformly to plasma membranes in both yeast and hyphae, yet Ras1 plasma membrane mobility was reduced in hyphae compared to that in yeast. Ras1-C288S was mislocalized to the cytoplasm and could not support hyphal development. Ras1-C287S was present primarily on endomembranes, and strains expressing ras1-C287S were delayed or defective in hyphal induction depending on the medium used. Cells bearing constitutively activated Ras1-C287S or Ras1-C288S, due to a G13V substitution, showed increased filamentation, suggesting that lipid modifications are differentially important for Ras1 activation and effector interactions. The C. albicans autoregulatory molecule, farnesol, inhibits Ras1 signaling through adenylate cyclase and bears structural similarities to the farnesyl molecule that modifies Ras1. At lower concentrations of farnesol, hyphal growth was inhibited but Ras1 plasma membrane association was not altered; higher concentrations of farnesol led to mislocalization of Ras1 and another G protein, Rac1. Furthermore, farnesol inhibited hyphal growth mediated by cytosolic Ras1-C288SG13V, suggesting that farnesol does not act through mechanisms that depend on Ras1 farnesylation. Our findings imply that Ras1 is farnesylated and palmitoylated, and that the Ras1 stimulation of adenylate cyclase-dependent phenotypes can occur in the absence of these lipid modifications

Orientación para el cambio : uso del Mapeo de Alcances para guiar el cambio institucional en un Banco Regional de Desarrollo

Versión en inglés disponible en la Biblioteca Digital del IDRC: Positioning for change : using Outcome Mapping to guide institutional change within a Regional Development BankVersión francés en la biblioteca: Positionnement pour le changement : utilisation de la cartographie des incidences pour orienter le changement institutionnel au sein d’une banque régionale de développemen

Positioning for change : using Outcome Mapping to guide institutional change within a Regional Development Bank

French version available in IDRC Digital Library: Positionnement pour le changement : utilisation de la cartographie des incidences pour orienter le changement institutionnel au sein d’une banque régionale de développementSpanish version available in IDRC Digital Library: Orientación para el cambio : uso del Mapeo de Alcances para guiar el cambio institucional en un Banco Regional de DesarrolloUsing the recommendations drawn from an operations audit and identifying the transition of the organization towards new behaviours necessary for sustaining strategic objectives, Outcome Mapping was able to articulate a human dimension of change. The process of using Outcome Mapping to construct the change management framework (CMF) involved mobilization of planning and assessment teams (Project Steering Committee and Change Management Taskforce). The process was guided and facilitated by an external consultant. The study reports on this process

Positionnement pour le changement : utilisation de la cartographie des incidences pour orienter le changement institutionnel au sein d’une banque régionale de développement

Version anglaise disponible dans la Bibliothèque numérique du CRDI: Positioning for change : using Outcome Mapping to guide institutional change within a Regional Development BankVersion espagnole disponible dans la Bibliothèque numérique du CRDI: Orientación para el cambio : uso del Mapeo de Alcances para guiar el cambio institucional en un Banco Regional de Desarroll

A tight balance of Karyopherin β1 expression is required in cervical cancer cells

Background Karyopherin β1 (Kpnβ1) is the main nuclear import protein involved in the transport of cargoes from the cytoplasm into the cell nucleus. Previous research has found Kpnβ1 to be significantly overexpressed in cervical cancer and other cancer tissues, and further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were minimally affected. These results suggest that Kpnβ1 has potential as an anticancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. Here, the biological effects associated with Kpnβ1 overexpression were investigated in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype. Methods To evaluate the effect of Kpnβ1 overexpression on cell biology, cell proliferation, cell cycle, cell morphology and cell adhesion assays were performed. To determine whether Kpnβ1 overexpression influences cell sensitivity to chemotherapeutic agents like Cisplatin, cell viability assays were performed. Expression levels of key proteins were analysed by Western blot analysis. Results Our data revealed that Kpnβ1 overexpression, above that which was already detected in cancer cells, resulted in reduced proliferation of cervical cancer cells. Likewise, normal epithelial cells showed reduced proliferation after Kpnβ1 overxpression. Reduced cancer cell proliferation was associated with a delay in cell cycle progression, as well as changes in the morphology and adhesion properties of cells. Additionally, Kpnβ1 overexpressing HeLa cells exhibited increased sensitivity to cisplatin, as shown by decreased cell viability and increased apoptosis, where p53 and p21 inhibition reduced and enhanced cell sensitivity to Cisplatin, respectively. Conclusions Overall, our results suggest that a tight balance of Kpnβ1 expression is required for cellular function, and that perturbation of this balance results in negative effects associated with a variety of biological processes

Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study

Background: The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society