Hotspots of Dissolved Arsenic Generated from Buried Silt Layers along Fluctuating Rivers.

Previous studies along the banks of the tidal Meghna River of the Ganges-Brahmaputra-Meghna Delta demonstrated the active sequestration of dissolved arsenic (As) on newly formed iron oxide minerals (Fe(III)-oxides) within riverbank sands. The sand with high solid-phase As (>500 mg/kg) was located within the intertidal zone where robust mixing occurs with oxygen-rich river water. Here we present new evidence that upwelling groundwater through a buried silt layer generates the dissolved products of reductive dissolution of Fe(III)-oxides, including As, while mobilization of DOC by upwelling groundwater prevents their reconstitution in the intertidal zone by lowering the redox state. A three end-member conservative mixing model demonstrated mixing between riverbank groundwater above the silt layer, upwelling groundwater through the silt layer, and river water. An electrochemical mass balance model confirmed that Fe(III)-oxides were the primary electron acceptor driving the oxidation of DOC sourced from sediment organic carbon in the silt. Thus, the presence of an intercalating silt layer in the riverbanks of tidal rivers can represent a biogeochemical hotspot of As release while preventing its retention in the hyporheic zone.

Mass fluxes of dissolved arsenic discharging to the Meghna River are sufficient to account for the mass of arsenic in riverbank sediments.

Shallow (<30 m) reducing groundwater commonly contains abundant dissolved arsenic (As) in Bangladesh. We hypothesize that dissolved As in iron (Fe)-rich groundwater discharging to rivers is trapped onto Fe(III)-oxyhydroxides which precipitate in shallow riverbank sediments under the influence of tidal fluctuations. Therefore, the goal of this study is to compare the calculated mass of sediment-bound As that would be sequestered from dissolved groundwater As that discharges through riverbanks of the Meghna River to the observed mass of As trapped within riverbank sediments. To calculate groundwater discharge, a Boussinesq aquifer analytical groundwater flow model was developed and constrained by cyclical seasonal fluctuations in hydraulic heads and river stages observed at three sites along a 13 km reach in central Bangladesh. At all sites, groundwater discharges to the river year-round but most of it passes through an intertidal zone created by ocean tides propagating upstream from the Bay of Bengal in the dry season. The annualized groundwater discharge per unit width at the three sites ranges from 173 to 891 m2/yr (average 540 m2/yr). Assuming that riverbanks have been stable since the Brahmaputra River avulsed far away from this area 200 years ago and dissolved As is completely trapped within riverbank sediments, the mass of accumulated sediment As can be calculated by multiplying groundwater discharge by ambient aquifer As concentrations measured in 1969 wells. Across all sites, the range of calculated sediment As concentrations in the riverbank is 78-849 mg/kg, which is higher than the observed concentrations (17-599 mg/kg). This discovery supports the hypothesis that the dissolved As in groundwater discharge to the river is sufficient to account for the observed buried deposits of As along riverbanks.

Sustained Aurora Kinase B Expression Confers Resistance to PI3K Inhibition in Head and Neck Squamous Cell Carcinoma.

Tumor suppressor mutations in head and neck squamous cell carcinoma (HNSCC) dominate the genomic landscape, hindering the development of effective targeted therapies. Truncating and missense mutations in NOTCH1 are frequent in HNSCC, and inhibition of PI3K can selectively target NOTCH1 mutant (NOTCH1MUT) HNSCC cells. In this study, we identify several proteins that are differentially regulated in HNSCC cells after PI3K inhibition based on NOTCH1MUT status. Expression of Aurora kinase B (Aurora B), AKT, and PDK1 following PI3K inhibition was significantly lower in NOTCH1MUT cell lines than in wild-type NOTCH1 (NOTCH1WT) cells or NOTCH1MUT cells with acquired resistance to PI3K inhibition. Combined inhibition of PI3K and Aurora B was synergistic, enhancing apoptosis in vitro and leading to durable tumor regression in vivo. Overexpression of Aurora B in NOTCH1MUT HNSCC cells led to resistance to PI3K inhibition, while Aurora B knockdown increased sensitivity of NOTCH1WT cells. In addition, overexpression of Aurora B in NOTCH1MUT HNSCC cells increased total protein levels of AKT and PDK1. AKT depletion in NOTCH1WT cells and overexpression in NOTCH1MUT cells similarly altered sensitivity to PI3K inhibition, and manipulation of AKT levels affected PDK1 but not Aurora B levels. These data define a novel pathway in which Aurora B upregulates AKT that subsequently increases PDK1 selectively in NOTCH1MUT cells to mediate HNSCC survival in response to PI3K inhibition. These findings may lead to an effective therapeutic approach for HNSCC with NOTCH1MUT while sparing normal cells. SIGNIFICANCE: Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Contribution of sedimentary organic matter to arsenic mobilization along a potential natural reactive barrier (NRB) near a river: The Meghna river, Bangladesh.

Elevated dissolved arsenic (As) concentrations in the shallow aquifers of Bangladesh are primarily caused by microbially-mediated reduction of As-bearing iron (Fe) (oxy)hydroxides in organic matter (OM) rich, reducing environments. Along the Meghna River in Bangladesh, interactions between the river and groundwater within the hyporheic zone cause fluctuating redox conditions responsible for the formation of a Fe-rich natural reactive barrier (NRB) capable of sequestering As. To understand the NRB's impact on As mobility, the geochemistry of riverbank sediment (<3 m depth) and the underlying aquifer sediment (up to 37 m depth) was analyzed. A 24-hr sediment-water extraction experiment was performed to simulate interactions of these sediments with oxic river water. The sediment and the sediment-water extracts were analyzed for inorganic and organic chemical parameters. Results revealed no differences between the elemental composition of riverbank and aquifer sediments, which contained 40 ± 12 g/kg of Fe and 7 ± 2 mg/kg of As, respectively. Yet the amounts of inorganic and organic constituents extracted were substantially different between riverbank and aquifer sediments. The water extracted 6.4 ± 16.1 mg/kg of Fe and 0.03 ± 0.02 mg/kg of As from riverbank sediments, compared to 154.0 ± 98.1 mg/kg of Fe and 0.55 ± 0.40 mg/kg of As from aquifer sediments. The riverbank and aquifer sands contained similar amounts of sedimentary organic matter (SOM) (17,705.2 ± 5157.6 mg/kg). However, the water-extractable fraction of SOM varied substantially, i.e., 67.4 ± 72.3 mg/kg in riverbank sands, and 1330.3 ± 226.6 mg/kg in aquifer sands. Detailed characterization showed that the riverbank SOM was protein-like, fresh, low molecular weight, and labile, whereas SOM in aquifer sands was humic-like, older, high molecular weight, and recalcitrant. During the dry season, oxic conditions in the riverbank may promote aerobic metabolisms, limiting As mobility within the NRB.

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks.

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans, Drosophila, and yeast, indicating that this is a highly conserved response. The examination of histone deacetylase recruitment to chromatin after heat-shock identified SIRT1 as the major deacetylase subsequently enriched at gene-rich regions. Heat-induced SIRT1 recruitment was antagonized by chromatin remodeler SMARCAD1 depletion and, like hyperthermia, the depletion of the SMARCAD1 or combination of the two impaired DNA end resection and increased replication stress. Altered repair protein recruitment was associated with heat-shock-induced γ-H2AX chromatin changes and DSB repair processing. These results support a novel mechanism whereby hyperthermia impacts chromatin organization owing to H4K16ac deacetylation, negatively affecting the HR-dependent DSB repair.

Consequences of access to water from managed aquifer recharge systems for blood pressure and proteinuria in south-west coastal Bangladesh: a stepped-wedge cluster-randomized trial.

BACKGROUND: Drinking-water salinity has been associated with high blood pressure (BP) among communities in south-west coastal Bangladesh. We evaluated whether access to water from managed aquifer recharge (MAR)-a hydrogeological intervention to lower groundwater salinity by infiltrating rainwater into the aquifers-can reduce community BP. METHODS: We conducted a stepped-wedge cluster-randomized trial with five monthly visits between December 2016 and April 2017 in 16 communities. At each visit following baseline, four communities were randomized to access MAR water. Systolic BP was the primary outcome, measured during each visit using Omron® HEM-907 devices. We also measured participants' 24-hour urinary sodium and households' drinking- and cooking-water salinity each visit. We used multilevel regression models to estimate the effects of MAR-water access on participants' BP. The primary analysis was intention-to-treat. RESULTS: In total, 2911 person-visits were conducted in communities randomized to have MAR-water access and 2834 in communities without MAR-water access. Households without MAR-water access predominantly used low-salinity pond water and 42% (range: 26-50% across visits) of households exclusively consumed MAR water when access was provided. Communities randomized to MAR-water access had 10.34 [95% confidence interval (CI): 1.11, 19.58] mmol/day higher mean urinary sodium, 1.96 (95% CI: 0.66, 3.26; p = 0.004) mmHg higher mean systolic BP and 1.44 (95% CI: 0.40, 2.48; p = 0.007) mmHg higher mean diastolic BP than communities without MAR-water access. CONCLUSIONS: Our findings do not support the scale-up of MAR systems as a routine drinking-water source, since communities that shifted to MAR water from the lower-salinity pond-water source had higher urinary sodium and BP.

Evaluation of a field kit for testing arsenic in paddy soil contaminated by irrigation water.

Rice is the primary crop in Bangladesh and rice yield is diminished due to the buildup of arsenic (As) in soil from irrigation with high-As groundwater. Soil testing with an inexpensive kit could help farmers target high-As soil for mitigation or decide to switch to a different crop that is less sensitive to As in soil. A total of 3,240 field kit measurements of As in 0.5 g of fresh soil added to 50 mL of water were compared with total soil As concentrations measured on oven-dried homogenized soil by X-ray fluorescence (XRF). For sets of 12 soil samples collected within a series of rice fields, the average of kit As measurements was a linear function of the average of XRF measurements (r2=0.69). Taking into account that the kit overestimates water As concentrations by about a factor of two, the relationship suggests that about a quarter of the As in paddy soil is released in the kit's reaction vessel. Using the relationship and considering XRF measurements as the reference, the 12-sample average determined correctly whether soil As was above or below a 30 mg/kg threshold in 86% of cases where soil As was above the threshold and in 79% of cases where soil As was below the threshold. We also used a Bayesian approach using 12 kit measurements to estimate the probability that soil As was above a given threshold indicated by XRF measurements. The Bayesian approach is theoretically optimal but was only slightly more accurate than the linear regression. These results show that rice farmers can identify high-As portions of their fields for mitigation using a dozen field kit measurements on fresh soil and base their decisions on this information.

Similar retardation of arsenic in gray Holocene and orange Pleistocene sediments: Evidence from field-based column experiments in Bangladesh.

Groundwater flow has the potential to introduce arsenic (As) in previously uncontaminated aquifers. The extent to which As transport is retarded by adsorption is particularly relevant in Bangladesh where low-As wells offer the best chance of reducing chronic exposure to As of a large rural population dependent on groundwater. In this study, column experiments were conducted with intact cores in the field to measure As retardation. Freshly collected cores of reduced iron (Fe-II) dominated gray sediment of Holocene age as well as oxidized Fe (III)-coated orange sediment of Pleistocene age were eluted at pore-water velocities of 40-230 cm/day with anoxic groundwater pumped directly from a well and containing 320 µg/L As. Up to 100 µg/L As was immediately released from gray sand but the main As breakthrough for both gray and orange sand occurred between 30 and 70 pore volumes, depending on flow rate. The early release of As from gray sand is attributed to the presence of a weakly bound pool of As. The sorption of As was kinetically limited in both gray and orange sand columns. We used a reversible multi-reaction transport model to simulate As breakthrough curves while keeping the model parameters as constant as possible. Contrary to the notion that dissolved As is sorbed more strongly to orange sands, we show that As was similarly retarded in both gray and orange sands in the field.

Groundwater Chemistry and Blood Pressure: A Cross-Sectional Study in Bangladesh.

Background: We assessed the association of groundwater chemicals with systolic blood pressure (SBP) and diastolic blood pressure (DBP). Methods: Blood pressure data for ≥35-year-olds were from the Bangladesh Demographic and Health Survey in 2011. Groundwater chemicals in 3534 well water samples from Bangladesh were measured by the British Geological Survey (BGS) in 1998-1999. Participants who reported groundwater as their primary source of drinking water were assigned chemical measures from the nearest BGS well. Survey-adjusted linear regression methods were used to assess the association of each groundwater chemical with the log-transformed blood pressure of the participants. Models were adjusted for age, sex, body mass index, smoking status, geographical region, household wealth, rural or urban residence, and educational attainment, and further adjusted for all other groundwater chemicals. Results: One standard deviation (SD) increase in groundwater magnesium was associated with a 0.992 (95% confidence interval (CI): 0.986, 0.998) geometric mean ratio (GMR) of SBP and a 0.991 (95% CI: 0.985, 0.996) GMR of DBP when adjusted for covariates except groundwater chemicals. When additionally adjusted for groundwater chemicals, one SD increase in groundwater magnesium was associated with a 0.984 (95% CI: 0.972, 0.997) GMR of SBP and a 0.990 (95% CI: 0.979, 1.000) GMR of DBP. However, associations were attenuated following Bonferroni-correction for multiple chemical comparisons in the full-adjusted model. Groundwater concentrations of calcium, potassium, silicon, sulfate, barium, zinc, manganese, and iron were not associated with SBP or DBP in the full-adjusted models. Conclusions: Groundwater magnesium had a weak association with lower SBP and DBP of the participants.

Inversion of High-Arsenic Soil for Improved Rice Yield in Bangladesh.

Rice is the primary crop in Bangladesh, and rice yield is diminished due to the buildup of arsenic (As) in soil from irrigation with high-As groundwater. Implementing a soil inversion, where deeper low-As soil is exchanged with the surface high-As soil in contact with rice roots, may mitigate the negative impacts of As on yield. We compared soil As, soil nutrients, and rice yield in control plots with those in adjacent soil inversion plots. We also estimated the quantity of soil As deposited on a yearly basis via irrigation water, to explore the longevity of a soil inversion to reduce surface As. Soil As, organic carbon, nitrogen, and phosphorus concentrations decreased by about 40% in response to the inversion and remained lowered over four seasons of monitoring. Inversion plot yields increased above control plot yields by 15-30% after a one-season lag despite the recovering but still reduced nutrient levels. Farmers have started conducting soil inversions of their own volition, typically close to where irrigation water enters the field. However, the yield gain will be limited to a few decades at most due to deposition of As via well water, unless the field is irrigated with low-As river or pond water.

SMARCAD1 Phosphorylation and Ubiquitination Are Required for Resection during DNA Double-Strand Break Repair.

The chromatin remodeling factor SMARCAD1, an SWI/SNF ATPase family member, has a role in 5' end resection at DNA double-strand breaks (DSBs) to produce single-strand DNA (ssDNA), a critical step for subsequent checkpoint and repair factor loading to remove DNA damage. However, the mechanistic details of SMARCAD1 coupling to the DNA damage response and repair pathways remains unknown. Here we report that SMARCAD1 is recruited to DNA DSBs through an ATM-dependent process. Depletion of SMARCAD1 reduces ionizing radiation (IR)-induced repairosome foci formation and DSB repair by homologous recombination (HR). IR induces SMARCAD1 phosphorylation at a conserved T906 by ATM kinase, a modification essential for SMARCAD1 recruitment to DSBs. Interestingly, T906 phosphorylation is also important for SMARCAD1 ubiquitination by RING1 at K905. Both these post-translational modifications are critical for regulating the role of SMARCAD1 in DNA end resection, HR-mediated repair, and cell survival after DNA damage.

Reservoirs of faecal indicator bacteria in well-head hand pumps in Bangladesh.

The majority of the population of Bangladesh (90%) rely on untreated groundwater for drinking and domestic use. At the point of collection, 40% of these supplies are contaminated with faecal indicator bacteria (FIB). Recent studies have disproved the theory that latrines discharging to shallow aquifers are the major contributor to this contamination. In this study, we tested the hypothesis that hand pumps are a reservoir of FIB. We sampled the handle, spout, piston and seal from 19 wells in Araihazar Upazila, Bangladesh and identified that the spout and seal were reservoirs of FIB. These findings led to our recommendation that well spouts be regularly cleaned, including the removal of precipitated deposits, and that the seals be regularly changed. It is envisaged that one or both of these interventions will reduce the numbers of FIB in drinking water, thereby reducing the burden of diarrhoeal disease in Bangladesh.

Field Study of Rice Yield Diminished by Soil Arsenic in Bangladesh.

Rice was traditionally grown only during the summer (aman) monsoon in Bangladesh but more than half is now grown during the dry winter (boro) season and requires irrigation. A previous field study conducted in a small area irrigated by a single high-arsenic well has shown that the accumulation of arsenic (As) in soil from irrigating with high-As groundwater can reduce rice yield. We investigated the effect of soil As on rice yield under a range of field conditions by exchanging the top 15 cm of soil between 13 high-As and 13 low-As plots managed by 16 different farmers, and we explore the implications for mitigation. Soil As and rice yields were measured for soil replacement plots where the soil was exchanged and adjacent control plots where the soil was not exchanged. Differences in yield (ranging from +2 to -2 t/ha) were negatively correlated to the differences in soil As (ranging from -9 to +19 mg/kg) between adjacent replacement and control plots during two boro seasons. The relationship between soil As and yield suggests a boro rice yield loss over the entire country of 1.4-4.9 million tons annually, or 7-26% of the annual boro harvest, due to the accumulation of As in soil over the past 25 years.

Reversible adsorption and flushing of arsenic in a shallow, Holocene aquifer of Bangladesh.

The spatial heterogeneity of dissolved arsenic (As) concentrations in shallow groundwater of the Bengal Basin has been attributed to transport of As (and reactive carbon) from external sources or to the release of As from within grey sand formations. We explore the latter scenario in this detailed hydrological and geochemical study along a 300 m transect of a shallow aquifer extending from a groundwater recharge area within a sandy channel bar to its discharge into a nearby stream. Within the 10-20 m depth range, groundwater ages along the transect determined by the 3H-3He method increase from <10 yr in the recharge area to a maximum of 40 yr towards the stream. Concentrations of groundwater As within the same grey sands increase from 10 to 100 to ∼500 µg/L along this transect. Evidence of reversible adsorption of As between the groundwater and sediment was obtained from a series of push-pull experiments, traditional batch adsorption experiments, and the accidental flooding of a shallow monitoring well. Assuming reversible adsorption and a distribution coefficient, Kd, of 0.15-1.5 L/kg inferred from these observations, a simple flushing model shows that the increase in As concentrations with depth and groundwater age at this site, and at other sites in the Bengal and Red River Basins, can be attributed to the evolution of the aquifer over 100-1000 years as aquifer sands are gradually flushed of their initial As content. A wide range of As concentrations can thus be maintained in groundwater with increases with depth governed by the history of flushing and local recharge rates, without external inputs of reactive carbon or As from other sources.

Megacity pumping and preferential flow threaten groundwater quality.

Many of the world's megacities depend on groundwater from geologically complex aquifers that are over-exploited and threatened by contamination. Here, using the example of Dhaka, Bangladesh, we illustrate how interactions between aquifer heterogeneity and groundwater exploitation jeopardize groundwater resources regionally. Groundwater pumping in Dhaka has caused large-scale drawdown that extends into outlying areas where arsenic-contaminated shallow groundwater is pervasive and has potential to migrate downward. We evaluate the vulnerability of deep, low-arsenic groundwater with groundwater models that incorporate geostatistical simulations of aquifer heterogeneity. Simulations show that preferential flow through stratigraphy typical of fluvio-deltaic aquifers could contaminate deep (>150 m) groundwater within a decade, nearly a century faster than predicted through homogeneous models calibrated to the same data. The most critical fast flowpaths cannot be predicted by simplified models or identified by standard measurements. Such complex vulnerability beyond city limits could become a limiting factor for megacity groundwater supplies in aquifers worldwide.

Redox Zonation and Oscillation in the Hyporheic Zone of the Ganges-Brahmaputra-Meghna Delta: Implications for the Fate of Groundwater Arsenic during Discharge.

Riverbank sediment cores and pore waters, shallow well waters, seepage waters and river waters were collected along the Meghna Riverbank in Gazaria Upazila, Bangladesh in Jan. 2006 and Oct.-Nov. 2007 to investigate hydrogeochemical processes controlling the fate of groundwater As during discharge. Redox transition zones from suboxic (0-2 m depth) to reducing (2-5 m depth) then suboxic conditions (5-7 m depth) exist at sites with sandy surficial deposits, as evidenced by depth profiles of pore water (n=7) and sediment (n=11; diffuse reflectance, Fe(III)/Fe ratios and Fe(III) concentrations). The sediment As enrichment zone (up to ~700 mg kg-1) is associated with the suboxic zones mostly between 0-2 m depth and less frequently between 5-7 m depth. The As enriched zones consist of several 5 to 10 cm-thick dispersed layers and span a length of ~5-15 m horizontally from the river shore. Depth profiles of riverbank pore water deployed along a 32 m transect perpendicular to the river shore show elevated levels of dissolved Fe (11.6±11.7 mg L-1) and As (118±91 µg L-1, mostly as arsenite) between 2-5 m depth, but lower concentrations between 0-2 m depth (0.13±0.19 mg L-1 Fe, 1±1 µg L-1 As) and between 5-6 m depth (1.14±0.45 mg L-1 Fe, 28±17 µg L-1 As). Because it would take more than a few hundred years of steady groundwater discharge (~10 m yr-1) to accumulate hundreds of mg kg-1 of As in the riverbank sediment, it is concluded that groundwater As must have been naturally elevated prior to anthropogenic pumping of the aquifer since the 1970s. Not only does this lend unequivocal support to the argument that As occurrence in the Ganges-Brahmaputra-Meghna Delta groundwater is of geogenic origin, it also calls attention to the fate of this As enriched sediment as it may recycle As into the aquifer.

Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression.

A substantial amount of mitochondrial energy is required for cell-cycle progression. The mechanisms underlying the coordination of the mitochondrial respiration with cell-cycle progression, especially the G2/M transition, remain to be elucidated. Here, we show that a fraction of cyclin B1/Cdk1 proteins localizes to the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins, including the complex I (CI) subunits in the respiratory chain. Cyclin B1/Cdk1-mediated CI phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted cyclin B1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cell-cycle time. Thus, cyclin B1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to increased energy demand for G2/M transition and, subsequently, to upregulate mitochondrial respiration for successful cell-cycle progression.

ß1-Integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

INTRODUCTION: Ductal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS and identified possible molecular mechanisms underlying invasive progression in surviving cells. METHODS: We measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt) which, in three-dimensional laminin-rich extracellular matrix (lrECM) and in vivo, form organotypic DCIS-like lesions with lumina expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in three-dimensional lrECM, a malignant phenotype emerged creating a model for invasive recurrence. RESULTS: P-Akt was up-regulated in clinical DCIS specimens and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in three-dimensional lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in three-dimensional lrECM, a malignant phenotype emerged, characterized by invasive activity, up-regulation of fibronectin, α5ß1-integrin, matrix metalloproteinase-9 (MMP-9) and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of nuclear factor-kappa B (NF-κB) to the ß1-integrin promoter region, associated with up-regulation of α5ß1-integrins. Inhibition of NF-κB or ß1-integrin signaling abrogated emergence of the invasive activity. CONCLUSIONS: P-Akt is up-regulated in some human DCIS lesions and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in three-dimensional lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in three-dimensional lrECM mediated by ß1-integrin and NF-κB signaling.

Rapid and automated multidimensional fluorescence microscopy profiling of 3D human breast cultures.

Three-dimensional (3D) tissue culture provides a physiologically relevant microenvironment for distinguishing malignant from non-malignant breast cell phenotypes. 3D culture assays can also be used to test novel cancer therapies and predict a differential response to radiation between normal and malignant cells in vivo. However, biological measurements in such complex models are difficult to quantify and current approaches do not allow for in-depth multifaceted assessment of individual colonies or unique sub-populations within the entire culture. This is in part due to the limitations of imaging at a range of depths in 3D culture resulting from optical aberrations and intensity attenuation. Here, we address these limitations by combining sample smearing techniques with high-throughput 2D imaging algorithms to accurately and rapidly quantify imaging features acquired from 3D cultures. Multiple high resolution imaging features especially designed to characterize 3D cultures show that non-malignant human breast cells surviving large doses of ionizing radiation acquire a "swelled acinar" phenotype with fewer and larger nuclei, loss of cell connectivity and diffused basement membrane. When integrating these imaging features into hierarchical clustering classification, we could also identify subpopulations of phenotypes from individual human tumor colonies treated with ionizing radiation or/and integrin inhibitors. Such tools have therefore the potential to further characterize cell culture populations after cancer treatment and identify novel phenotypes of resistance.

Unsealed tubewells lead to increased fecal contamination of drinking water.

Bangladesh is underlain by shallow aquifers in which millions of drinking water wells are emplaced without annular seals. Fecal contamination has been widely detected in private tubewells. To evaluate the impact of well construction on microbial water quality 35 private tubewells (11 with intact cement platforms, 19 without) and 17 monitoring wells (11 with the annulus sealed with cement, six unsealed) were monitored for culturable Escherichia coli over 18 months. Additionally, two 'snapshot' sampling events were performed on a subset of wells during late-dry and early-wet seasons, wherein the fecal indicator bacteria (FIB) E. coli, Bacteroidales and the pathogenicity genes eltA (enterotoxigenic E. coli; ETEC), ipaH (Shigella) and 40/41 hexon (adenovirus) were detected using quantitative polymerase chain reaction (qPCR). No difference in E. coli detection frequency was found between tubewells with and without platforms. Unsealed private wells, however, contained culturable E. coli more frequently and higher concentrations of FIB than sealed monitoring wells (p < 0.05), suggestive of rapid downward flow along unsealed annuli. As a group the pathogens ETEC, Shigella and adenovirus were detected more frequently (10/22) during the wet season than the dry season (2/20). This suggests proper sealing of private tubewell annuli may lead to substantial improvements in microbial drinking water quality.