Toxicological Implications of Platinum Nanoparticle Exposure: Stimulation of Intracellular Stress, Inflammatory Response, and Akt Signaling In Vitro.

Due to their distinctive physicochemical properties, platinum nanoparticles (PtNPs) have emerged as a material of interest for a number of biomedical therapeutics. However, in some instances NP exposure has been correlated to health and safety concerns, including cytotoxicity, activation of cellular stress, and modification to normal cell functionality. As PtNPs have induced differential cellular responses in vitro, the goal of this study was to further characterize the behavior and toxicological potential of PtNPs within a HepG2 liver model. This study identified that a high PtNP dosage induced HepG2 cytotoxicity. However, lower, subtoxic PtNP concentrations were able to elicit multiple stress responses, secretion of proinflammatory cytokines, and modulation of insulin-like growth factor-1 dependent signal transduction. Taken together, this work suggests that PtNPs would not be overtly toxic for acute exposures, but sustained cellular interactions might produce long term health consequences.

An insight into the microbiome of the Amblyomma maculatum (Acari: Ixodidae).

The aim of this study was to survey the bacterial diversity of Amblyomma maculatum Koch, 1844, and characterize its infection with Rickettsia parkeri. Pyrosequencing of the bacterial 16S rRNA was used to determine the total bacterial population in A. maculatum. Pyrosequencing analysis identified Rickettsia in A. maculatum midguts, salivary glands, and saliva, which indicates successful trafficking in the arthropod vector. The identity of Rickettsia spp. was determined based on sequencing the rickettsial outer membrane protein A (rompA) gene. The sequence homology search revealed the presence of R. parkeri, Rickettsia amblyommii, and Rickettsia endosymbiont ofA. maculatum in midgut tissues, whereas the only rickettsia detected in salivary glands was R. parkeri, suggesting it is unique in its ability to migrate from midgut to salivary glands, and colonize this tissue before dissemination to the host. Owing to its importance as an emerging infectious disease, the R. parkeri pathogen burden was quantified by a rompB-based quantitative polymerase chain reaction (qPCR) assay and the diagnostic effectiveness of using R. parkeri polyclonal antibodies in tick tissues was tested. Together, these data indicate that field-collected A. maculatum had a R. parkeri infection rate of 12-32%. This study provides an insight into the A. maculatum microbiome and confirms the presence of R. parkeri, which will serve as the basis for future tick and microbiome interaction studies.

Knockdown of selenocysteine-specific elongation factor in Amblyomma maculatum alters the pathogen burden of Rickettsia parkeri with epigenetic control by the Sin3 histone deacetylase corepressor complex.

Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex.

Molecular characterization of tick salivary gland glutaminyl cyclase.

Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the bloodmeal of adult female ticks prior to fast-feeding phases in both I. scapularis and A. maculatum suggesting a functional link with bloodmeal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control.

Uncovering latent deficits due to mild traumatic brain injury by using normobaric hypoxia stress.

Memory deficits and other cognitive symptoms frequently associated with mTBI are commonly thought to resolve within 7-10 days. This generalization is based principally on observations made in individuals who are in the unstressed environmental conditions typical of a clinic and so does not consider the impact of physiologic, environmental, or psychological stress. Normobaric hypoxic stress can be generated with normal mean sea level (MSL) air, which is about 21% oxygen (O2) and 78% nitrogen (N), by reducing the percentage of O2 and increasing the percentage of N so that the resultant mixed-gas has a partial pressure of O2 approximating that of specified altitudes. This technique was used to generate normobaric hypoxic equivalents of 8,000, 12,000, and 14,000 feet above MSL in a group of 36 volunteers with a mTBI history and an equal number of controls matched on the basis of age, gender, tobacco smoking consumption, weight, height, and body mass index. Short-term visual memory was tested using the Matching to Sample (M2S) subtest of the BrainCheckers analog of the Automated Neuropsychological Assessment Metrics. Although there were no significant differences in M2S performance between the two groups of subjects at MSL, with increased altitude, the mTBI group performance was significantly worse than that of the control group. When the subjects were returned to MSL, the difference disappeared. This finding suggests that the "hypoxic challenge" paradigm developed here has potential clinical utility for assessing the effects of mTBI in individuals who appear asymptomatic under normal conditions.

Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands.

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and Amblyomma americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases.

Evaluation of sumatriptan-naproxen in the treatment of acute migraine: a placebo-controlled, double-blind, cross-over study assessing cognitive function.

OBJECTIVE: To assess the cognitive effects of acute migraine and the subsequent impact of acute treatment in a controlled setting. BACKGROUND: Cognitive dysfunction may be an associated symptom in patients with migraine with or without aura. The loss of cognitive efficiency in migraine may be disabling and is often under recognized. METHODS: Thirty migraine patients were prospectively studied for cognitive function before and then at the beginning of a migraine using a computerized cognitive battery (Mental Efficacy Workload Test). Each patient then was treated for 2 headaches in a cross-over manner with sumatriptan-naproxen (Treximet®) or placebo in a double-blind, placebo-controlled fashion with cognitive testing repeated at 1 and 2 hours post-dose. RESULTS: Twenty-five of the 30 screened migraine subjects completed study-specific procedures and were included in the data analyses. There were no significant side effects from Treximet or placebo and no serious adverse events. At the onset of headache, there was a statistically significant decline in overall cognitive efficiency compared with the baseline cognitive testing (migraine-free) for all subjects (P = .001 paired samples t-test). For subjects taking Treximet compared with taking placebo, there was a statistically significant return to cognitive efficiency by measures of immediate and sustained attention, visual-spatial awareness, mental flexibility, and reaction time between 1 hour and 2 hours (P = .05). There was no statistical significance between patients taking Treximet or placebo in measures of complex reasoning or fine motor coordination. Subanalysis showed a correlation between headache severity and Performance Index in the Treximet group but not in the placebo group (∼Fig. ). CONCLUSIONS: There is a significant decline in global cognitive efficiency at the onset of an attack of migraine. The use of Treximet allows a significantly faster recovery time in some measures of cognitive efficiency compared with placebo. Decline of cognitive efficiency may be independent of headache severity.

Choice of a stable set of reference genes for qRT-PCR analysis in Amblyomma maculatum (Acari: Ixodidae).

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a widely used laboratory tool to quantify mRNA levels of target genes involved in various biological processes. The most commonly used method for analyzing qRT-PCR data are the normalizing technique where a housekeeping gene is used to determine the transcriptional regulation of the target gene. The choice of a reliable internal standard is pivotal for relative gene expression analysis to obtain reproducible results, especially when measuring small differences in transcriptional expression. In this study, we used geNorm, NormFinder, and BestKeeper programs to analyze the gene expression results using qRT-PCR. Five candidate reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, elongation factor 1-alpha, and glutathione s-transferase, were used to evaluate the expression stability during prolonged blood-feeding on the vertebrate host. These five genes were evaluated in all life stages of Amblyomma maculatum (Koch) as well as in the salivary gland and midgut tissues of adult females to determine which are the most stably expressed gene for use in qRT-PCR studies. Beta-actin is the most stably expressed gene in salivary glands and midguts ofA. maculatum, and throughout all developmental stages both Actin and GAPDH were found to have the most stable expression with the lowest degree of variance. We recommend the use of beta-actin and/ or GAPDH as reference genes for qRT-PCR analysis of gene expression in A. maculatum.

Laboratory-infected Ehrlichia chaffeensis female adult Amblyomma americanum salivary glands reveal differential gene expression.

Ticks are efficient ectoparasites that are able to steal blood, a rich source of nutrients, from their vertebrate hosts. The nymphal developmental stage of ticks plays an important role for pathogen transmission to human and other animal hosts. In this article, we describe a bloodmeal-based sex differentiation tool to generate adult female ticks infected with Ehrlichia chaffeensis to investigate vector-pathogen interactions (functional genomics and gene expression studies). We demonstrate that there is a correlation between the uptake of blood during nymph attachment and the molting into male or female adult ticks. The data obtained from the bloodmeal experiments suggest that nymphs that molt into females presumably imbibe more blood than those that become male during the nymphal stage. The natural low E. chaffeensis infection rate in female adult Amblyomma americanum (L.) is a major limiting factor to investigate Ehrlichia-Amblyomma interactions. To generate Ehrlichia-infected female adult ticks, we inoculated obligate E. chaffeensis (Arkansas strain) infected DH82 cells into heavier engorged nymphs (> 12 mg) and allowed them to molt. Freshly molted adults were used to test the E. chaffeensis infection rate. E. chaffeensis genomic DNA was extracted from individual unfed and partially blood fed tick midgut and salivary gland tissues. The tissue samples were tested for the presence of E. chaffeensis using the nested polymerase chain reaction process. Polymerase chain reaction-amplified fragments were detected in unfed and partially fed tissues, demonstrating successful E. chaffeensis infection of tick tissues. This method was used to successfully show differential expression of selected tick genes in E. chaffeensis-infected midguts and salivary glands.

Two center, randomized pilot study of migraine prophylaxis comparing paradigms using pre-emptive frovatriptan or daily topiramate: research and clinical implications.

OBJECTIVE: To compare the efficacy and clinical benefit of 2 paradigms of migraine prevention using pre-emptive frovatriptan and daily topiramate. The study compares the paradigms of pre-emptive use of frovatriptan, a drug approved for acute migraine, and the daily use of topiramate, a Federal Drug Administration-approved and -accepted standard for migraine prophylaxis. BACKGROUND: Traditionally, preventive treatment of migraine required daily medication. However, recent studies suggest that pre-emptive prophylaxis may be beneficial to those migraineurs who can predict an attack of migraine based on premonitory symptoms and treat during that phase. METHODS: A total of 76 adult subjects with a diagnosis of migraine were screened for the study. During a 1-month baseline period, subjects demonstrated through a daily diary that they predicted at least 50% of migraine attacks during the premonitory phase and treated with their usual medication. Of these, 55 were randomized to either Group A (daily topiramate) or Group B (frovatriptan during premonitory symptoms); 44 completed the study. The treatment period lasted 2 months. The subjects answered the Migraine-Specific Quality of Life Questionnaire at randomization, and at Weeks 4 and 8. The revised Patient Perception of Migraine Questionnaire was answered 24 hours after taking frovatriptan (Group A, for break-through headaches; Group B, treatment during premonitory symptoms). RESULTS: The number of migraine attacks and headache days per month decreased significantly from baseline for both Groups A and B. Subjects in Group A had considerably more adverse events leading to study withdrawal than in Group B (18% vs 4%). Though this study was not powered to directly compare the efficacy of the 2 drugs, topiramate showed superiority over frovatriptan at Month 2 in reduction of headache days, which was a secondary end point in the study (P = .036). CONCLUSIONS: This pilot study demonstrated that statistical benefit for reduction of headache days over baseline for both pre-emptive frovatriptan and daily topiramate. Subjects utilizing pre-emptive frovatriptan experienced fewer adverse events leading to study withdrawal. Subjects utilizing daily topiramate had fewer headache days at Month 2.

A double-blind placebo-controlled pilot study of sublingual feverfew and ginger (LipiGesic™ M) in the treatment of migraine.

BACKGROUND: Therapeutic needs of migraineurs vary considerably from patient to patient and even attack to attack. Some attacks require high-end therapy, while other attacks have treatment needs that are less immediate. While triptans are considered the "gold standard" of migraine therapy, they do have limitations and many patients are seeking other therapeutic alternatives. In 2005, an open-label study of feverfew/ginger suggested efficacy for attacks of migraine treated early during the mild headache phase of the attack. METHODS/MATERIALS: In this multi-center pilot study, 60 patients treated 221 attacks of migraine with sublingual feverfew/ginger or placebo. All subjects met International Headache Society criteria for migraine with or without aura, experiencing 2-6 attacks of migraine per month within the previous 3 months. Subjects had <15 headache days per month and were not experiencing medication overuse headache. Inclusion required that subjects were able to identify a period of mild headache in at least 75% of attacks. Subjects were required to be able to distinguish migraine from non-migraine headache. Subjects were randomized 3:1 to receive either sublingual feverfew/ginger or a matching placebo and were instructed but not required to treat with study medication at the earliest recognition of migraine. RESULTS: Sixty subjects treated 208 evaluable attacks of migraine over a 1-month period; 45 subjects treated 163 attacks with sublingual feverfew/ginger and 15 subjects treated 58 attacks with a sublingual placebo preparation. Evaluable diaries were completed for 151 attacks of migraine in the population using feverfew/ginger and 57 attacks for those attacks treated with placebo. At 2 hours, 32% of subjects receiving active medication and 16% of subjects receiving placebo were pain-free (P= .02). At 2 hours, 63% of subjects receiving feverfew/ginger found pain relief (pain-free or mild headache) vs 39% for placebo (P= .002). Pain level differences on a 4-point pain scale for those receiving feverfew/ginger vs placebo were -0.24 vs -0.04 respectively (P= .006). Feverfew/ginger was generally well tolerated with oral numbness and nausea being the most frequently occurring adverse event. CONCLUSION: Sublingual feverfew/ginger appears safe and effective as a first-line abortive treatment for a population of migraineurs who frequently experience mild headache prior to the onset of moderate to severe headache.

Delay activity in avian prefrontal cortex--sample code or reward code?

In the current study, we examined whether delay activity in the avian equivalent of the prefrontal cortex (PFC) represents a neural correlate of a to-be-remembered sample stimulus or an upcoming reward. Birds were trained on a directed forgetting paradigm in which sample stimuli (red and white) were either followed by a cue to remember (high-frequency tone) or a cue to forget (low-frequency tone). The task also incorporated a differential outcomes procedure in which a correct response on the memory test following a red (remember) sample was rewarded with food, but correct responses on the memory test following the white (remember) sample were not. If delay activity represents a sample code, then it should be seen on both red-remember and white-remember trials. On the other hand, if delay activity represents a reward code, then delay activity should be seen only on red-remember trials, but not white-remember trials. Our findings suggest that activity in the avian PFC represents the outcome associated with each sample (reward or no reward) rather than memory for the sample itself.