Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC₅₀s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC₅₀ = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC₅₀ = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC₅₀ = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role.     

When Is Rapid On-Site Evaluation Cost-Effective for Fine-Needle Aspiration Biopsy?

Background

Rapid on-site evaluation (ROSE) can improve adequacy rates of fine-needle aspiration biopsy (FNAB) but increases operational costs. The performance of ROSE relative to fixed sampling depends on many factors. It is not clear when ROSE is less costly than sampling with a fixed number of needle passes. The objective of this study was to determine the conditions under which ROSE is less costly than fixed sampling.

Methods

Cost comparison of sampling with and without ROSE using mathematical modeling. Models were based on a societal perspective and used a mechanistic, micro-costing approach. Sampling policies (ROSE, fixed) were compared using the difference in total expected costs per case. Scenarios were based on procedure complexity (palpation-guided or image-guided), adequacy rates (low, high) and sampling protocols (stopping criteria for ROSE and fixed sampling). One-way, probabilistic, and scenario-based sensitivity analysis was performed to determine which variables had the greatest influence on the cost difference.

Results

ROSE is favored relative to fixed sampling under the following conditions: (1) the cytologist is accurate, (2) the total variable cost ($/hr) is low, (3) fixed costs ($/procedure) are high, (4) the setup time is long, (5) the time between needle passes for ROSE is low, (6) when the per-pass adequacy rate is low, and (7) ROSE stops after observing one adequate sample. The model is most sensitive to variation in the fixed cost, the per-pass adequacy rate, and the time per needle pass with ROSE.

Conclusions

Mathematical modeling can be used to predict the difference in cost between sampling with and without ROSE.     

Mechanisms of Resilience in Children of Mothers Who Self-Report with Depressive Symptoms in the First Postnatal Year

Background

Symptoms of maternal postnatal depression are associated with an increased risk of adverse effects on child development. However, some children exposed to postnatal depression have outcomes similar to unexposed children, and can be referred to as resilient. This study aimed to determine the mechanisms of resilience in children exposed to depressive symptoms postnatally.

Method

Data are from a prospective cohort study, the Avon Longitudinal Study of Parents and Children. Self-report questionnaire data were collected during pregnancy and the child’s first 2 years regarding maternal views of parenting and her perception of the child. The Edinburgh Postnatal Depression Scale (EPDS) was completed postnatally at 8 months and the Strengths and Difficulties Questionnaire (SDQ) at age 11 years. Exposed children who scored above the median score of non-exposed children were defined as resilient. Structural equation modeling was used to investigate the development of resilience.

Results

From the core ALSPAC cohort, 1,009 children (6.9%) were exposed to maternal depression at 8 months postnatally. The SDQ total difficulties scores at 11 years of age indicated that 325 (32.2%) were resilient, 684 were non-resilient. Maternal positive feelings about parenting and child non-verbal communication at 15 months increased the likelihood of later resilience.

Conclusions

In this study, resilience was associated with two factors: the child’s nonverbal communication at 15 months and by maternal positive feelings about parenting. Early intervention to support mother-child interaction and foster child development in women identified with postnatal depressive symptoms may benefit later child resilience.     

An Agent-Based Model of Private Woodland Owner Management Behavior Using Social Interactions,Information Flow,and Peer-To-Peer Networks

Privately owned woodlands are an important source of timber and ecosystem services in North America and worldwide. Impacts of management on these ecosystems and timber supply from these woodlands are difficult to estimate because complex behavioral theory informs the owner’s management decisions. The decision-making environment consists of exogenous market factors, internal cognitive processes, and social interactions with fellow landowners, foresters, and other rural community members. This study seeks to understand how social interactions, information flow, and peer-to-peer networks influence timber harvesting behavior using an agent-based model. This theoretical model includes forested polygons in various states of ‘harvest readiness’ and three types of agents: forest landowners, foresters, and peer leaders (individuals trained in conservation who use peer-to-peer networking). Agent rules, interactions, and characteristics were parameterized with values from existing literature and an empirical survey of forest landowner attitudes, intentions, and demographics. The model demonstrates that as trust in foresters and peer leaders increases, the percentage of the forest that is harvested sustainably increases. Furthermore, peer leaders can serve to increase landowner trust in foresters. Model output and equations will inform forest policy and extension/outreach efforts. The model also serves as an important testing ground for new theories of landowner decision making and behavior.     

Tracking Cholera through Surveillance of Oral Rehydration Solution Sales at Pharmacies: Insights from Urban Bangladesh

Background

In Bangladesh, pharmacy-purchased oral rehydration solution (ORS) is often used to treat diarrhea, including cholera. Over-the-counter sales have been used for epidemiologic surveillance in the past, but rarely, if ever, in low-income countries. With few early indicators for cholera outbreaks in endemic areas, diarrhea-related product sales may serve as a useful surveillance tool.

Methodology/Principal Findings

We tracked daily ORS sales at 50 pharmacies and drug-sellers in an urban Bangladesh community of 129,000 for 6-months while simultaneously conducting surveillance for diarrhea hospitalizations among residents. We developed a mobile phone based system to track the sales of ORS and deployed it in parallel with a paper-based system. Our objectives were to determine if the mobile phone system was practical and acceptable to pharmacists and drug sellers, whether data were reported accurately compared to a paper-based system, and whether ORS sales were associated with future incidence of cholera hospitalizations within the community. We recorded 47,215 customers purchasing ORS, and 315 hospitalized diarrhea cases, 22% of which had culture-confirmed cholera. ORS sales and diarrhea incidence were independently associated with the mean daily temperature; therefore both unadjusted and adjusted models were explored. Through unadjusted cross-correlation statistics and generalized linear models, we found increases in ORS sales were significantly associated with increases in hospitalized diarrhea cases up to 9-days later and hospitalized cholera cases up to one day later. After adjusting for mean daily temperature, ORS was significantly associated with hospitalized diarrhea two days later and hospitalized cholera one day later.

Conclusions/Significance

Pharmacy sales data may serve as a feasible and useful surveillance tool. Given the relatively short lagged correlation between ORS sales and diarrhea, rapid and accurate sales data are key. More work is needed in creating actionable algorithms that make use of this data and in understanding the generalizability of our findings to other settings.     

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

Purification,characterization and crystallization of the F-ATPase from Paracoccus denitrificans

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.