Estrogen Receptor Silencing Induces Epithelial to Mesenchymal Transition in Human Breast Cancer Cells

We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in trans-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from a keratin/actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated geometric fold changes ≥3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. These data support our hypothesis that induced loss of estrogen receptor in previously estrogen/antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may offer a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Oral Health-Related Quality-of-Life According to Dental Caries Severity, Body Mass Index and Sociodemographic Indicators in Children with Special Health Care Needs

This study aimed to assess the impact of dental caries’ severity, body mass index (BMI), and sociodemographic factors on oral health-related quality of life (OHRQoL) for special health care needs (SHCN) children and the suitability of their caregivers as proxies to determine OHRQoL. This cross-sectional study recruited 107 pairs of SHCN children and their caregivers and asked them to complete a questionnaire on sociodemographic issues as well as the Arabic version of the early childhood oral health impact scale (A-ECOHIS). This was followed by a dental examination. Dental caries was measured using the dmft/DMFT index, while caries’ severity was also determined. The children’s height and weight were measured, and BMI (kg/m2) was recorded. Data were analyzed statistically using t-test, one-way ANOVA, and Poisson regression models. Our results revealed that the A-ECOHIS score was 10.93, while the OHRQoL was affected in 95.3% of children. The most-reported item was ‘pain in the teeth, mouth, or jaws’ (48.7%). By regression analysis, caries-free children (Odds Ratio (OR): 0.650) or those who had moderate caries (OR: 0.551) were less likely to have a negative impact on their OHRQoL than those with severe caries. Additionally, those whose caregivers had a maximum primary education (OR: 0.656) or whose occupation was in the health sector (OR: 0.721) were less likely to have a negative impact on their OHRQoL. Those who were ≤ 6 years old (OR: 1.188) were more likely to have a negative impact. BMI did not have a significant impact on the OHRQoL of the children. Further, we detected a significant positive correlation between children’s dmft/DMFT scores and the A-ECOHIS scores reported by the mothers. Given these variables, which included dental caries’ severity, but not BMI, and caregivers’ education level and occupation, plus the child’s age group, we found a significant impact on the OHRQoL. However, we found that mothers were better proxies for their children’s OHRQoL

Effect of PI3K -Akt and ERK1/2 inhibitors on EGF and IGF-1 induced pII cell invasion.

<p>The random invasive movement (A + B; as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041847#pone-0041847-g001" target="_blank">Fig 1</a>) of pII cells in response to EGF (10 ng/ml) or IGF-1 (10 ng/ml) was measured in the absence (untreated; UT) or presence of the ERK1/2 inhibitor PD0325901 or the PI3K-Akt inhibitor LY294002 (at concentrations indicated) added directly to the cell medium. Data for drug treated conditions are expressed as a % of the untreated control (set as 100%) and are means ± SEM for 8 independent determinations. Asterisks denote significant difference from untreated control with p≤0.05.</p

Effect of erlotinib on directional invasion and proliferation of pII cells.

<p>Panel A indicates the net directional invasive movement (A – B; as defined in Fig. 1) of pII cells through agarose towards a source of EGF (50 ng/ml) measured in the absence (untreated; UT) or presence of erlotinib added directly into the well containing cells as described in Methods. Data for drug treated conditions are expressed as a % of the untreated control (set as 100%). Each histobar is the mean ± SEM for 5 independent determinations. Asterisks denote significant difference from untreated control with p≤0.05 (*) and p<0.005 (**). Panel B shows the effect of erlotinib on cell proliferation. Approximately 10⁴ pII cells were seeded in microwell plates and allowed to grow over 4 days in the presence of various concentrations of erlotinib as indicated. Cells were harvested and growth was determined by the MTT assay. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from untreated control, with p<0.001.</p

The effect of IGF-1, EGF, TGFβ, PDGFC and RANTES on the random (total) invasive movement of pII cells.

<p>A single chamber formed in 0.5% agarose containing ITS was loaded with 4×10⁴ cells in media containing either growth factor at the indicated concentration or an equivalent volume of PBS. After incubation for 24 h at 37°C cells that had moved into the agarose in either lateral direction were counted as described in Methods. Histobars represent means ± SEM of 4 independent determinations. Number of invading cells from the well given PBS was set at 100% and used as a normalizer. Asterisks indicate significant difference over PBS control with p≤0.05 (*) and p≤0.001 (**).</p

Effect of IGF-1, EGF and inhibitors on MMP activity.

<p>pII (A) or YS1.2 (B) cells were treated with IGF-1 or EGF (10 and 50 ng/ml) for 30 min and metalloproteinase activity was determined using a fluorogenic substrate as described in Methods. The fluorescence signal was monitored every 5 min for a total of 1 h using a microplate reader with excitation and emission at 490 and 525 nm respectively. Panel C shows the effect of pre-treatment of pII cells with 10 µM PI3K-Akt inhibitor LY294002 or ERK1/2 inhibitor PD0325901 on the EGF induced protease activation. Data are expressed as mean ± SEM of 3–6 independent determinations. In panel A asterisks denote significant difference between EGF at 50 ng/ml and no addition (p<0.05). In panel C asterisks denote significant difference between EGF alone and EGF+LY294002. (p≤0.05).</p