Kinetic Effects of Transferrin-Conjugated Gold Nanoparticles on the Antioxidant Glutathione-Thioredoxin Pathway.

Nanoparticle-based therapeutics are being clinically translated for treating cancer. Even when thought to be biocompatible, nanoparticles are being increasingly identified as altering cell regulation and homeostasis. Antioxidant pathways are important for maintaining cell redox homeostasis and play important roles by maintaining ROS levels within tolerable ranges. Here, we sought to understand how a model of a relatively inert nanoparticle without any therapeutic agent itself could antagonize a cancer cell lines' antioxidant mechanism. A label-free protein expression approach was used to assess the glutathione-thioredoxin antioxidative pathway in a prostate cancer cell line (PC-3) after exposure to gold nanoparticles conjugated with a targeting moiety (transferrin). The impact of the nanoparticles was also corroborated through morphological analysis with TEM and classification of pro-apoptotic cells by way of the sub-G0/G1 population via the cell cycle and annexin V apoptosis assay. After a two-hour exposure to nanoparticles, major proteins associated with the glutathione-thioredoxin antioxidant pathway were downregulated. However, this response was acute, and in terms of protein expression, cells quickly recovered within 24 h once nanoparticle exposure ceased. The impact on PRDX-family proteins appears as the most influential factor in how these nanoparticles induced an oxidative stress response in the PC-3 cells. An apparent adaptive response was observed if exposure to nanoparticles continued. Acute exposure was observed to have a detrimental effect on cell viability compared to continuously exposed cells. Nanoparticle effects on cell regulation likely provide a compounding therapeutic advantage under some circumstances, in addition to the action of any cytotoxic agents; however, any therapeutic advantage offered by nanoparticles themselves with regard to vulnerabilities specific to the glutathione-thioredoxin antioxidative pathway is highly temporal.

Cell Size as a Primary Determinant in Targeted Nanoparticle Uptake.

Nanoparticle (NP) internalization by cells is complex, highly heterogeneous, and fundamentally important for nanomedicine. We report powerful probabilistic statistics from single-cell data on quantitative NP uptake of PEG-coated transferrin receptor-targeted gold NPs for cancer-derived and fibroblast cells according to their cell size, receptor expression, and receptor density. The smaller cancer cells had a greater receptor density and more efficient uptake of targeted NPs. However, simply due to fibroblasts being larger with more receptors, they exhibited greater NP uptake. While highly heterogeneous, targeted NP uptake strongly correlated with receptor expression. When uptake was normalized to cell size, no correlation existed. Consequently, skewed population distributions in cell sizes explain the distribution in NP uptake. Furthermore, exposure to the transferrin receptor-targeted NPs alters the fibroblast size and receptor expression, suggesting that the receptor-targeted NPs may interfere with the metabolic flux and nutrient exchange, which could assist in explaining the altered regulation of cells exposed to nanoparticles.

Signal Detection to Measure Allostatic Load.

PURPOSE: To illustrate a means to calculate allostatic load in hospitalized patients using big data from the electronic medical record (EMR). ORGANIZING CONSTRUCT: To describe the development of the Troubled Outcome Risk (TOR) scale using signal detection in big data. METHODS: Using both retrospective and prospective observational studies, I describe a mechanism to determine meaning from retrospective data then use the results to improve nursing surveillance to reduce length of stay (LOS) and nursing sensitive indicators on an inpatient medical surgical unit. FINDINGS: Results from the retrospective study contained over 290,000 individual data points and established an interpretation standard for the TOR score using an algorithm to detect signals. The prospective observational study used the TOR scale and developed an interpretation standard to assist unit charge nurses in assigning staff to patients based on a fully objective measure of patient allostatic load. CONCLUSIONS: The TOR scale in conjunction with existing nurse staffing methodology reduced inpatient LOS by 0.3 days, reduced allostatic load as measured by the TOR scale, and changed staffing patterns from purely geographic to patient-need based. CLINICAL RELEVANCE: The TOR scale demonstrates that careful evidence-based criteria can be easily gathered from the EMR and used to positively impact nursing practice and patient outcomes.

Chemical Mechanisms of Nanoparticle Radiosensitization and Radioprotection: A Review of Structure-Function Relationships Influencing Reactive Oxygen Species.

Metal nanoparticles are of increasing interest with respect to radiosensitization. The physical mechanisms of dose enhancement from X-rays interacting with nanoparticles has been well described theoretically, however have been insufficient in adequately explaining radiobiological response. Further confounding experimental observations is examples of radioprotection. Consequently, other mechanisms have gained increasing attention, especially via enhanced production of reactive oxygen species (ROS) leading to chemical-based mechanisms. Despite the large number of variables differing between published studies, a consensus identifies ROS-related mechanisms as being of significant importance. Understanding the structure-function relationship in enhancing ROS generation will guide optimization of metal nanoparticle radiosensitisers with respect to maximizing oxidative damage to cancer cells. This review highlights the physico-chemical mechanisms involved in enhancing ROS, commonly used assays and experimental considerations, variables involved in enhancing ROS generation and damage to cells and identifies current gaps in the literature that deserve attention. ROS generation and the radiobiological effects are shown to be highly complex with respect to nanoparticle physico-chemical properties and their fate within cells. There are a number of potential biological targets impacted by enhancing, or scavenging, ROS which add significant complexity to directly linking specific nanoparticle properties to a macroscale radiobiological result.

Cross-Correlative Single-Cell Analysis Reveals Biological Mechanisms of Nanoparticle Radiosensitization.

Nanoparticle radiosensitization has been demonstrated well to enhance the effects of radiotherapy, motivate the improvement of therapeutic ratios, and decrease morbidity in cancer treatment. A significant challenge exists in optimizing formulations and translation due to insufficient knowledge of the associated mechanisms, which have historically been limited to physical concepts. Here, we investigated a concept for the role of biological mechanisms. The mere presence of gold nanoparticles led to a down-regulation of thymidylate synthase, important for DNA damage repair in the radioresistant S-phase cells. By developing a cross-correlative methodology to reveal probabilistic gold nanoparticle uptake by cell sub-populations and the associated sensitization as a function of the uptake, a number of revealing observations have been achieved. Surprisingly, for low numbers of nanoparticles, a desensitization action was observed. Sensitization was discovered to preferentially impact S-phase cells, in which impairment of the DNA damage response by the homologous recombination pathway dominates. This small but radioresistant cell population correlates with much greater proliferative ability. Thus, a paradigm is presented whereby enhanced DNA damage is not necessarily due to an increase in the number of DNA double-strand breaks (DSBs) created but can be from a nanoparticle-induced impairment of the damage response by down-regulating repair proteins such as thymidylate synthase.

Assaying the Stability and Inactivation of AAV Serotype 1 Vectors.

Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze-thaw cycles, the resulting transduction efficiency became variable at 60-120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments.

Fathers Matter: Involving and Engaging Fathers in the Child Welfare System Process.

Research suggests that children with involved and engaged fathers tend to have more positive outcomes relative to physical, cognitive, and social emotional health. Of children who become involved in the child welfare system, involving multiple parents in the case (e.g. mother and father) often results in a greater chance of a child returning home, fewer placement episodes, and reduced trauma that may be caused by separation anxiety. With the rise of single parenting homes (which are mostly maternal) in the United States, child welfare agencies are examining the efficacy of engaging multiple caregivers (esp. fathers) in the child welfare process. Research suggests that in order to involve fathers in child welfare processes, practices and policies must be intentional in implementing systems and protocols that encourage involvement of all parents regardless of relationship status of the parents. However, few child welfare agencies are required to inquire about fathers or involve fathers in the child's case. The purpose of this paper is to highlight efforts of the Connecticut Comprehensive Outcome Review (CCOR) process and discuss challenges and lessons learned from interviews and listening forums/focus groups that included social workers and fathers who are involved in the child welfare system in the state of Connecticut. Recommendations and considerations on engaging and involving fathers are discussed.

Targeted over-expression of glutamate transporter 1 (GLT-1) reduces ischemic brain injury in a rat model of stroke.

Following the onset of an ischemic brain injury, the excitatory neurotransmitter glutamate is released. The excitotoxic effects of glutamate are a major contributor to the pathogenesis of a stroke. The aim of this study was to examine if overexpression of a glutamate transporter (GLT-1) reduces ischemic brain injury in a rat model of stroke. We generated an adeno-associated viral (AAV) vector expressing the rat GLT-1 cDNA (AAV-GLT1). Functional expression of AAV-GLT1 was confirmed by increased glutamate clearance rate in non-stroke rat brain as measured by in vivo amperometry. AAV-GLT1 was injected into future cortical region of infarction 3 weeks prior to 60 min middle cerebral artery occlusion (MCAo). Tissue damage was assessed at one and two days after MCAo using TUNEL and TTC staining, respectively. Behavioral testing was performed at 2, 8 and 14 days post-stroke. Animals receiving AAV-GLT1, compared to AAV-GFP, showed significant decreases in the duration and magnitude of extracellular glutamate, measured by microdialysis, during the 60 minute MCAo. A significant reduction in brain infarction and DNA fragmentation was observed in the region of AAV-GLT1 injection. Animals that received AAV-GLT1 showed significant improvement in behavioral recovery following stroke compared to the AAV-GFP group. We demonstrate that focal overexpression of the glutamate transporter, GLT-1, significantly reduces ischemia-induced glutamate overflow, decreases cell death and improves behavioral recovery. These data further support the role of glutamate in the pathogenesis of ischemic damage in brain and demonstrate that targeted gene delivery to decrease the ischemia-induced glutamate overflow reduces the cellular and behavioral deficits caused by stroke.

Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro.

Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity.