The Brominated Flame Retardant Hexabromocyclododecane Causes Systemic Changes in Polyunsaturated Fatty Acid Incorporation in Mouse Lipids.

Brominated flame retardants are used in many household products to reduce flammability, but often leach into the surrounding environment over time. Hexabromocyclododecane (HBCD) is one brominated flame retardant detected in human blood across the world. HBCD exposure can result in neurological problems and altered lipid metabolism, but to date the two remain unlinked. As lipids constitute ∼50% of brain dry weight, lipid metabolism plays a critical role in neuronal function and homeostasis. To determine the effect of HBCD exposure on brain lipid metabolism, young adult male C57BL/6 mice were exposed to 1 mg/kg HBCD every 3 days for 28 days. Major lipid classes were found to change across brain regions, including the membrane glycerolipids phosphatidylcholine and phosphatidylethanolamine, and sphingolipids such as hexosylceramide. In addition, saturated, monounsaturated, and polyunsaturated fatty acids were enriched within brain lipid species. To understand the source of the brain lipidomic alterations, the blood and liver lipidomes and the cecal microbiome were evaluated. The liver and blood demonstrated changes amongst multiple lipid classes, including triacylglycerol suppression, as well as altered esterified fatty acid content. Significant alterations were also detected in the cecal microbiome, with decreases in the Firmicutes to Bacteriodetes ratio, changes in beta diversity, and pathway alterations associated with metabolic pathways and amino acid biosynthesis. These data demonstrate that HBCD can induce lipidomic alterations across brain regions and organs and supports a potential role of the microbiome in these alterations.

Insights into Brominated Flame Retardant Neurotoxicity: Mechanisms of Hippocampal Neural Cell Death and Brain Region-Specific Transcriptomic Shifts in Mice.

Brominated Flame Retardants (BFRs) reduce flammability in a wide range of products including electronics, carpets, and paint, but leach into the environment to result in continuous, population-level exposure. Epidemiology studies have correlated BFR exposure with neurological problems, including alterations in learning and memory. This study investigated the molecular mechanisms mediating BFR-induced cell death in hippocampal cells and clarified the impact of HBCD exposure on gene transcription in the hippocampus, dorsal striatum, and frontal cortex of male mice. Exposure of hippocampus derived HT-22 cells to various flame retardants, including tetrabromobisphenol-A (TBBPA, current use), hexabromocyclododecane (HBCD, phasing out), or 2,2',4,4'-tetrabromodiphenyl ether (BDE-47, phased out) resulted in time, concentration, and chemical-dependent cellular and nuclear morphology alterations, alterations in cell cycle and increases in annexin V staining. All three BFRs increased p53 and p21 expression; however, inhibition of p53 nuclear translocation using pifthrin-α did not decrease cell death. Transcriptomic analysis upon low (10 nM) and cytotoxic (10 µM) BFR exposure indicated that HBCD and BDE-47 altered genes mediating autophagy-related pathways. Further evaluation showed BFR exposure increased LC3-II conversion and autophagosome formation, and co-exposure with the autophagy inhibitor 3-methyladenine (3-MA) attenuated cytotoxicity. Transcriptomic assessment of select brain regions from subchronically HBCD-exposed male mice demonstrated alteration of genes mediating vesicular transport, with greater impact on the frontal cortex and dorsal striatum compared to the dorsal and ventral hippocampus. Immunoblot analysis demonstrated no increases in cell death or autophagy markers, but did demonstrate increases in the SNARE binding complex SNAP29, specifically in the dorsal hippocampus. These data demonstrate that BFRs can induce chemical-dependent autophagy in neural cells in vitro and provide evidence that BFRs induce region-specific transcriptomic and protein expression in the brain suggestive of change in vesicular trafficking.

Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Therapeutic potential of lipin inhibitors for the treatment of cancer.

Lipins are phosphatidic acid phosphatases (PAP) that catalyze the conversion of phosphatidic acid (PA) to diacylglycerol (DAG). Three lipin isoforms have been identified: lipin-1, -2 and -3. In addition to their PAP activity, lipin-1 and -2 act as transcriptional coactivators and corepressors. Lipins have been intensely studied for their role in regulation of lipid metabolism and adipogenesis; however, lipins are hypothesized to mediate several pathologies, such as those involving metabolic diseases, neuropathy and even cognitive impairment. Recently, an emerging role for lipins have been proposed in cancer. The study of lipins in cancer has been hampered by lack of inhibitors that have selectivity for lipins, that differentiate between lipin family members, or that are suitable for in vivo studies. Such inhibitors have the potential to be extremely useful as both molecular tools and therapeutics. This review describes the expression and function of lipins in various tissues and their roles in several diseases, but with an emphasis on their possible role in cancer. The mechanisms by which lipins mediate cancer cell growth are discussed and the potential usefulness of selective lipin inhibitors is hypothesized. Finally, recent studies reporting the crystallization of lipin-1 are discussed to facilitate rational design of novel lipin inhibitors.

Targeting P21-Activated Kinase-1 for Metastatic Prostate Cancer.

Metastatic prostate cancer (mPCa) has limited therapeutic options and a high mortality rate. The p21-activated kinase (PAK) family of proteins is important in cell survival, proliferation, and motility in physiology, and pathologies such as infectious, inflammatory, vascular, and neurological diseases as well as cancers. Group-I PAKs (PAK1, PAK2, and PAK3) are involved in the regulation of actin dynamics and thus are integral for cell morphology, adhesion to the extracellular matrix, and cell motility. They also play prominent roles in cell survival and proliferation. These properties make group-I PAKs a potentially important target for cancer therapy. In contrast to normal prostate and prostatic epithelial cells, group-I PAKs are highly expressed in mPCA and PCa tissue. Importantly, the expression of group-I PAKs is proportional to the Gleason score of the patients. While several compounds have been identified that target group-I PAKs and these are active in cells and mice, and while some inhibitors have entered human trials, as of yet, none have been FDA-approved. Probable reasons for this lack of translation include issues related to selectivity, specificity, stability, and efficacy resulting in side effects and/or lack of efficacy. In the current review, we describe the pathophysiology and current treatment guidelines of PCa, present group-I PAKs as a potential druggable target to treat mPCa patients, and discuss the various ATP-competitive and allosteric inhibitors of PAKs. We also discuss the development and testing of a nanotechnology-based therapeutic formulation of group-I PAK inhibitors and its significant potential advantages as a novel, selective, stable, and efficacious mPCa therapeutic over other PCa therapeutics in the pipeline.

Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses.

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

Identification and Characterization of Key Differentially Expressed Genes Associated With Metronomic Dosing of Topotecan in Human Prostate Cancer.

Repetitive, low-dose (metronomic; METRO) drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/-), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers.

Transcriptomic Analysis of the Differential Nephrotoxicity of Diverse Brominated Flame Retardants in Rat and Human Renal Cells.

Brominated flame retardants (BFRs) are environmentally persistent, are detected in humans, and some have been banned due to their potential toxicity. BFRs are developmental neurotoxicants and endocrine disruptors; however, few studies have explored their potential nephrotoxicity. We addressed this gap in the literature by determining the toxicity of three different BFRs (tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), and tetrabromodiphenyl ether (BDE-47)) in rat (NRK 52E) and human (HK-2 and RPTEC) tubular epithelial cells. All compounds induced time- and concentration-dependent toxicity based on decreases in MTT staining and changes in cell and nuclear morphology. The toxicity of BFRs was chemical- and cell-dependent, and human cells were more susceptible to all three BFRs based on IC50s after 48 h exposure. BFRs also had chemical- and cell-dependent effects on apoptosis as measured by increases in annexin V and PI staining. The molecular mechanisms mediating this toxicity were investigated using RNA sequencing. Principal components analysis supported the hypothesis that BFRs induce different transcriptional changes in rat and human cells. Furthermore, BFRs only shared nine differentially expressed genes in rat cells and five in human cells. Gene set enrichment analysis demonstrated chemical- and cell-dependent effects; however, some commonalities were also observed. Namely, gene sets associated with extracellular matrix turnover, the coagulation cascade, and the SNS-related adrenal cortex response were enriched across all cell lines and BFR treatments. Taken together, these data support the hypothesis that BFRs induce differential toxicity in rat and human renal cell lines that is mediated by differential changes in gene expression.

Measurement of Cell Death in Mammalian Cells.

Methods for assessing mammalian cell death are presented in this article, which is divided into six sections: (1) a brief overview of cytotoxicity and pathways of cell death; (2) a method to measure cell death using lactate dehydrogenase (LDH) release as a marker of membrane integrity; (3) a flow cytometry method that simultaneously measures two types of cell death, necrosis and apoptosis; (4) use of fluorescence microscopy and nuclear morphology to assess apoptosis and necrosis; (5) the use of multi-well plates and high-content analysis imaging systems to assess nuclear morphology; and (6) a discussion of the use of cytotoxicity assays to determine the mechanisms of cell death. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Measurement of plasma membrane integrity and viability using LDH release Basic Protocol 2: Measurement of necrosis and apoptosis using flow cytometry Basic Protocol 3: Determination of nuclear morphology and membrane integrity Alternate Protocol 1: Assessment of nuclear morphology and membrane integrity using DAPI and PI Alternate Protocol 2: Assessment of nuclear morphology using multi-well plates Basic Protocol 4: Measurement of time-dependent toxicity using cell death markers.

Inhibition of glypican-1 expression induces an activated fibroblast phenotype in a human bone marrow-derived stromal cell-line.

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cell type in the tumor microenvironment. CAFs orchestrate tumor-stromal interactions, and contribute to cancer cell growth, metastasis, extracellular matrix (ECM) remodeling, angiogenesis, immunomodulation, and chemoresistance. However, CAFs have not been successfully targeted for the treatment of cancer. The current study elucidates the significance of glypican-1 (GPC-1), a heparan sulfate proteoglycan, in regulating the activation of human bone marrow-derived stromal cells (BSCs) of fibroblast lineage (HS-5). GPC-1 inhibition changed HS-5 cellular and nuclear morphology, and increased cell migration and contractility. GPC-1 inhibition also increased pro-inflammatory signaling and CAF marker expression. GPC-1 induced an activated fibroblast phenotype when HS-5 cells were exposed to prostate cancer cell conditioned media (CCM). Further, treatment of human bone-derived prostate cancer cells (PC-3) with CCM from HS-5 cells exhibiting GPC-1 loss increased prostate cancer cell aggressiveness. Finally, GPC-1 was expressed in mouse tibia bone cells and present during bone loss induced by mouse prostate cancer cells in a murine prostate cancer bone model. These data demonstrate that GPC-1 partially regulates the intrinsic and extrinsic phenotype of human BSCs and transformation into activated fibroblasts, identify novel functions of GPC-1, and suggest that GPC-1 expression in BSCs exerts inhibitory paracrine effects on the prostate cancer cells. This supports the hypothesis that GPC-1 may be a novel pharmacological target for developing anti-CAF therapeutics to control cancer.

Identification of lipidomic profiles associated with drug-resistant prostate cancer cells.

BACKGROUND: The association of circulating lipids with clinical outcomes of drug-resistant castration-resistant prostate cancer (DR-CRPC) is not fully understood. While it is known that increases in select lipids correlate to decreased survival, neither the mechanisms mediating these alterations nor the correlation of resistance to drug treatments is well characterized. METHODS: This gap-in-knowledge was addressed using in vitro models of non-cancerous, hormone-sensitive, CRPC and drug-resistant cell lines combined with quantitative LC-ESI-Orbitrap-MS (LC-ESI-MS/MS) lipidomic analysis and subsequent analysis such as Metaboanalyst and Lipid Pathway Enrichment Analysis (LIPEA). RESULTS: Several lipid regulatory pathways were identified that are associated with Docetaxel resistance in prostate cancer (PCa). These included those controlling glycerophospholipid metabolism, sphingolipid signaling and ferroptosis. In total, 7460 features were identified as being dysregulated between the cell lines studied, and 21 lipid species were significantly altered in drug-resistant cell lines as compared to nonresistant cell lines. Docetaxel resistance cells (PC3-Rx and DU145-DR) had higher levels of phosphatidylcholine (PC), oxidized lipid species, phosphatidylethanolamine (PE), and sphingomyelin (SM) as compared to parent control cells (PC-3 and DU-145). Alterations were also identified in the levels of phosphatidic acid (PA) and diacylglyceride (DAG), whose levels are regulated by Lipin (LPIN), a phosphatidic acid phosphatase that converts PA to DAG. Data derived from cBioPortal demonstrated a population of PCa patients expressing mutations aligning with amplification of LPIN1, LPIN2 and LPIN3 genes. Lipin amplification in these genes correlated to decreased survival in these patients. Lipin-1 mRNA expression also showed a similar trend in PCa patient data. Lipin-1, but not Lipin-2 or - 3, was detected in several prostate cancer cells, and was increased in 22RV1 and PC-3 cell lines. The increased expression of Lipin-1 in these cells correlated with the level of PA. CONCLUSION: These data identify lipids whose levels may correlate to Docetaxel sensitivity and progression of PCa. The data also suggest a correlation between the expression of Lipin-1 in cells and patients with regards to prostate cancer cell aggressiveness and patient survivability. Ultimately, these data may be useful for identifying markers of lethal and/or metastatic prostate cancer.

Liposomes Targeting P21 Activated Kinase-1 (PAK-1) and Selective for Secretory Phospholipase A2 (sPLA2) Decrease Cell Viability and Induce Apoptosis in Metastatic Triple-Negative Breast Cancer Cells.

P21 activated kinases (or group I PAKs) are serine/threonine kinases whose expression is altered in prostate and breast cancers. PAK-1 activity is inhibited by the small molecule "Inhibitor targeting PAK-1 activation-3" (IPA-3), which has selectivity for PAK-1 but is metabolically unstable. Secretory Group IIA phospholipase A2 (sPLA2) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA2, called Secretory Phospholipase Responsive liposomes or SPRL-IPA-3, and demonstrated their ability to alter prostate cancer growth. The efficacy of SPRL against other types of cancers is not well understood. We addressed this limitation by determining the ability of SPRL to induce cell death in a diverse panel of cells representing different stages of breast cancer, including the invasive but non-metastatic MCF-7 cells, and metastatic triple-negative breast cancer (TNBC) cells such as MDA-MB-231, MDA-MB-468, and MDA-MB-435. We investigated the role of sPLA2 in the disposition of these liposomes by comparing the efficacy of SPRL-IPA-3 to IPA-3 encapsulated in sterically stabilized liposomes (SSL-IPA-3), a formulation shown to be less sensitive to sPLA2. Both SSL-IPA-3 and SPRL-IPA-3 induced time- and dose-dependent decreases in MTT staining in all cell lines tested, but SPRL-IPA-3-induced effects in metastatic TNBC cell lines were superior over SSL-IPA-3. The reduction in MTT staining induced by SPRL-IPA-3 correlated to the expression of Group IIA sPLA2. sPLA2 expression also correlated to increased induction of apoptosis in TNBC cell lines by SPRL-IPA-3. These data suggest that SPRL-IPA-3 is selective for metastatic TNBC cells and that the efficacy of SPRL-IPA-3 is mediated, in part, by the expression of Group IIA sPLA2.

Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Alter the Blood Lipidome and the Hepatic Proteome in a Murine Model of Diet-Induced Obesity.

Perfluoroalkyl substances (PFAS) represent a family of environmental toxicants that have infiltrated the living world. This study explores diet-PFAS interactions and the impact of perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic (PFHxS) on the hepatic proteome and blood lipidomic profiles. Male C57BL/6J mice were fed with either a low-fat diet (10.5% kcal from fat) or a high fat (58% kcal from fat) high carbohydrate (42 g/l) diet with or without PFOS or PFHxS in feed (0.0003% wt/wt) for 29 weeks. Lipidomic, proteomic, and gene expression profiles were determined to explore lipid outcomes and hepatic mechanistic pathways. With administration of a high-fat high-carbohydrate diet, PFOS and PFHxS increased hepatic expression of targets involved in lipid metabolism and oxidative stress. In the blood, PFOS and PFHxS altered serum phosphatidylcholines, phosphatidylethanolamines, plasmogens, sphingomyelins, and triglycerides. Furthermore, oxidized lipid species were enriched in the blood lipidome of PFOS and PFHxS treated mice. These data support the hypothesis that PFOS and PFHxS increase the risk of metabolic and inflammatory disease induced by diet, possibly by inducing dysregulated lipid metabolism and oxidative stress.

Sterically stabilized liposomes targeting P21 (RAC1) activated kinase-1 and secreted phospholipase A2 suppress prostate cancer growth and metastasis.

Metastatic prostate cancer (PCa) has a very high mortality rate in men, in Western countries and lacks reliable treatment. The advanced-stage PCa cells overexpress P21 (RAC1) activated kinase-1 (PAK1) and secreted phospholipase A2 (sPLA2) suggesting the potential utility of pharmacologically targeting these molecules to treat metastatic PCa. The small molecule, inhibitor targeting PAK1 activation-3 (IPA3) is a highly specific allosteric inhibitor of PAK1; however, it is metabolically unstable once in the plasma thus, limiting its utility as a chemotherapeutic agent. In the present study, the efficacy and specificity of IPA3 were combined with the stability and the sPLA2-targeted delivery method of two sterically stabilized liposomes [sterically stabilized long-circulating liposomes (SSL)-IPA3 and sPLA2 responsive liposomes (SPRL)-IPA3, respectively] to inhibit PCa growth and metastasis. It was found that twice-a-week administration of either SSL-IPA3 or SPRL-IPA3 for 3 weeks effectively suppressed the growth of PC-3 cell tumor xenografts implanted in athymic nude mice. Both drug formulations also inhibited the metastasis of intravenously administered murine RM1 PCa cells to the lungs of C57BL/6 mice. Whereas the twice-a-week administration of SSL-IPA3 significantly inhibited the spontaneous PCa metastasis to the lungs in Transgenic Adenocarcinoma of the Mouse Prostate mice, the administration of free IPA3 had no significant therapeutic benefit. The results present two novel IPA3 encapsulated liposomes to treat metastatic PCa.

PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling.

Metastatic prostate cancer (PCa) has high mortality and a poor 5-year survival rate primarily due to the lack of effective treatments. Bone is the primary site of PCa metastasis in humans and the development of reliable therapeutic options for bone metastatic PCa will make a huge impact in reducing the mortality among these patients. Although P21 activated kinases (PAKs) have been studied in the past for their role in cancer, the efficacy of targeting PAKs to treat lung and bone metastatic PCa has not been tested yet. In the current study, we report that targeting PAK1 using IPA-3, an allosteric inhibitor of PAK1 kinase activity, significantly inhibits the murine metastatic PCa (RM1) cell proliferation and motility in vitro, and metastasis to the lungs in vivo. More importantly, we demonstrate for the first time that treatment with IPA-3 can blunt metastatic PCa-induced bone remodeling in vivo as analyzed by the 3-dimensional microcomputer tomography analysis. Our study has identified IPA-3 as a potential drug to treat bone metastatic PCa.

Toxicokinetics of cis- and trans-Permethrin: Influence of Isomer, Maturation, and Sex.

Permethrin exposure of children and adults is widespread in many populations, but knowledge of its relative toxicokinetics (TK) and health risks in immature age groups is lacking. Studies were conducted in rats to determine the influence of immaturity and sex (on plasma and target organ dosimetry of each of the insecticide's 2 isomers, cis- and trans-permethrin [CIS and TRANS]). Postnatal day 15, 21, and 90 (adult), Sprague Dawley rats were orally administered a graduated series of doses of CIS and TRANS in corn oil. Serial sacrifices were conducted over 24 h to obtain plasma, brain, liver, skeletal muscle, and fat profiles of CIS and TRANS. Levels of TRANS decreased relatively rapidly, despite administration of relatively high doses. Concentrations of each isomer in plasma, brain, and other tissues monitored were inversely proportional to the animals' age. The youngest pups exhibited 4-fold higher plasma and brain area under the curves than did adults. Little difference was observed in the TK of CIS or TRANS between adult male and female rats, other than higher initial plasma and liver CIS levels in females. Elevated exposure of the immature brain appears to be instrumental in increased susceptibility to the acute neurotoxicity of high-dose permethrin (Cantalamessa [1993]), but it remains to be established whether age-dependent TK is relevant to long-term, low-level risks.

Safe Nanoparticles: Are We There Yet?

The field of nanotechnology has grown over the last two decades and made the transition from the benchtop to applied technologies. Nanoscale-sized particles, or nanoparticles, have emerged as promising tools with broad applications in drug delivery, diagnostics, cosmetics and several other biological and non-biological areas. These advances lead to questions about nanoparticle safety. Despite considerable efforts to understand the toxicity and safety of these nanoparticles, many of these questions are not yet fully answered. Nevertheless, these efforts have identified several approaches to minimize and prevent nanoparticle toxicity to promote safer nanotechnology. This review summarizes our current knowledge on nanoparticles, their toxic effects, their interactions with mammalian cells and finally current approaches to minimizing their toxicity.

Lipidomic changes in the rat hippocampus following cocaine conditioning, extinction, and reinstatement of drug-seeking.

INTRODUCTION: Cocaine dependence affects millions of individuals worldwide; however, there are no pharmacotherapeutic and/or diagnostic solutions. Recent evidence suggests a role for lipid signaling in the development and maintenance of addiction, highlighting the need to understand how lipid remodeling mediates neuroadaptation after cocaine exposure. METHODS: This study utilized shotgun lipidomics to assess cocaine-induced lipid remodeling in rats using a novel behavioral regimen that incorporated multiple sessions of extinction training and reinstatement testing. RESULTS: Mass spectrometric imaging demonstrated widespread decreases in phospholipid (PL) abundance throughout the brain, and high-spatial resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry indicated hippocampus-specific PL alterations following cocaine exposure. We analyzed the expression of genes involved in hippocampal lipid metabolism and observed region-specific regulation. In addition, we found that cocaine exposure differentially regulates mitochondrial biogenesis in the brain. CONCLUSIONS: This work presents a comprehensive lipidomic assessment of cocaine-induced lipid remodeling in the rat brain. Further, these findings indicate a potential interplay between CNS energetics and differential lipid regulation and suggest a role for cocaine in the maintenance of energy homeostasis.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Effect of P21-activated kinase 1 (PAK-1) inhibition on cancer cell growth, migration, and invasion.

P21-activated kinase-1 (PAK-1) is a serine/threonine kinase involved in multiple signaling pathways that mediate cellular functions such as cytoskeletal motility, cell proliferation, and survival. PAK-1 expression is altered in various cancers, including prostate and breast. Our recent studies showed that prostate cancer cells expressing higher levels of PAK-1 were resistant to the cytotoxic effects of the PAK-1 inhibitor, inhibitor targeting PAK-1 activation-3 (IPA-3), compared to those with lower expression. This study expanded these findings to other cancers (breast and melanoma) by testing the hypothesis that genetic and pharmacological inhibition of PAK-1 alters cell growth, migration, and invasion in prostate, breast, and skin cancer cell lines. We also tested the specificity of IPA-3 for PAK-1 and the hypothesis that gene silencing of PAK-1 altered the efficacy of sterically stabilized liposomes (SSL) containing IPA-3 (SSL-IPA-3). PAK-1 expression was identified in four different breast cancer cell lines, and in a melanoma cell line. The expression of PAK-1 correlated to the IC50 of IPA-3 as measured by MTT staining. PAK-1 inhibition using shRNA correlated with decreased cell migration and invasion in prostate cancer DU-145 and breast cancer MCF-7 cells. Decreased migration and invasion also correlated to decreased expression of E-cadherin and alterations in C-X-C Chemokine Receptor type 4 and Homing Cell Adhesion Molecule expression. PAK-1 inhibition increased the cytotoxicity of IPA-3, and the cytotoxicity of SSL-IPA-3 to levels comparable to that of free drug. These data demonstrate that both pharmacological and molecular inhibition of PAK-1 decreased growth in prostate, breast, and melanoma cancer cell lines, and increased the toxicity of IPA-3 and its liposomal formulation. These data also show the specificity of IPA-3 for PAK-1, are some of the first data suggesting that IPA-3 is a therapeutic treatment for breast cancer and melanoma, and demonstrate the efficacy of liposome-encapsulated IPA-3 in breast cancer cells.