Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 µM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.

Rate normalization for sweat metabolomics biomarker discovery.

As the demand for real-time exercise performance feedback increases, excreted sweat has become a biosource of interest for continuous human performance assessment. For sweat to truly fulfill this requirement, analyte concentrations must be normalized to adequately assess day-to-day differences within and among individuals. In this manuscript, data are presented highlighting the use of accurate localized sweat rate as a means for ion and global metabolomic data normalization. The results illustrate large sweat rate variability among individuals over the course of two distinct exercises protocols. Furthermore, the data show sweat rate is not symmetrical at similar locations among right and left forearms of individuals (p = 0.0007). Sweat ion conductivity analysis suggest overall sweat rate normalization reduces variability collectively among ion values and participants with principal component analysis showing 77.8% of variation in the data set attributable to sweat rate normalization. Global metabolomic analysis of sweat illustrated overall rate normalization increases the variability among test subjects with 72.7% of the variation explained by sweat rate normalization. Finally, overall rate normalized metabolomic features of sweat significantly correlated (ρ ≥ 0.7, ρ ≤ -0.7) with measured performance metrics of the individual, establishing the potential for sweat to be used as a biosource for performance monitoring. Collectively, these data illustrate the importance of accurate localized sweat rate determination, for analyte data normalization, in support for the use of sweat in biomarker discovery efforts to predict human performance.

Lipid, water, and protein composition to facilitate kinetic modeling of the auditory pathway.

Environments combining JP-8 jet fuel exposure with heightened ambient noise may accelerate hearing loss induced by noise. To reduce animal use and facilitate kinetic modeling of this military aviation fuel, tissue-specific parameters are required, including water, protein, and lipid content. However, tissues involved in hearing, including cochlea, brainstem, frontal, and temporal lobe, have not been characterized before. Therefore, water content was determined by lyophilization of rat auditory tissues and the protein of the freeze dried remainder was quantified using a bicinchoninic acid assay. Lipids were extracted from fresh-frozen rat auditory tissues and separated into neutral lipids, free fatty acids, neutral phospholipids, and acidic phospholipids using solid phase extraction. Phospholipid fractions were confirmed by 31 P nuclear magnetic resonance analysis showing distinct phospholipid profiles. Lipid content in reference tissues, such as kidney and adipose, confirmed literature values. For the first time, lipid content in the rat auditory pathway was determined showing that total lipid content was lowest in cochlea and highest in brainstem compared with frontal and temporal lobes. Auditory tissues displayed distinct lipid fraction profiles. The information on water, protein, and lipid composition is necessary to validate algorithms used in mathematical models and predict partitioning of chemicals of future interest into these tissues. This research may reduce the use of animals to measure partition coefficients for prospective physiological models.

Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages.

The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

Drug Target Prioritization for Alzheimer's Disease Using Protein Interaction Network Analysis.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that accounts for numerous deaths worldwide. AD is the most common cause of dementia, characterized by accumulation of fibrous amyloid beta protein in the brain with clinical symptoms, such as loss of intellectual and social skills, gradually leading to the death of brain cells. The genetic complexity of AD during disease progression requires a systems-level understanding to design viable therapeutics. We present an integrative computational analysis to prioritize AD-associated genes outlined through a protein-protein interaction network. Multiple topological parameters of the network were considered to target proteins which are accountable for disease susceptibility. Furthermore, in silico protein structure modeling and molecular dynamics simulation approaches were implemented to characterize presenilin 2 (PSEN2) protein as one of the leading targets in the network. The findings are constructive to aid future drug discovery endeavors in the treatment of AD.

Individual differences in biophysiological toughness: sustaining working memory during physical exhaustion.

Recent evidence suggests that increased dehydroepiandrosterone sulfate (DHEAS), in combination with decreased cortisol levels have been correlated with enhanced performance outcomes in stressful military environments. This study was implemented to replicate these findings in a group of active duty Air Force members to provide information on the usefulness of these biomarkers indices in the training and operational environment. Seventeen active duty males participated in the 4 sessions of this study. Sessions 1 and 2 were training days for the cognitive testing. Session 3 was a baseline measure of physical fitness, utilizing the VO2 Maximal Treadmill test. Session 4 was centered around a modified-Astrand treadmill protocol designed to induce physical exhaustion. Blood draws for biomarker analysis, cognitive testing (NovaScan), psychomotor vigilance, and physiological measures were collected before, during, and following the treadmill protocol. Results showed that prolonged increases in cortisol negatively correlate with working memory performance. DHEAS release from baseline to poststress was negatively related to the changes in cortisol for 20 minutes following stress. These results indicate that the ratio of DHEAS to cortisol buffers, the effect of cortisol increases that are related to poor working memory performance because of physical exhaustion.

A 13-week nose-only inhalation toxicity study for perfluoro-n-butyl iodide (PFBI) in rats with recommended occupational exposure levels.

A 13-week study was conducted to develop occupational exposure limits (OELs) for the solvent perfluoro-n-butyl iodide (PFBI). Fischer 344 rats (15 males & 10 females per group) were exposed for 6 h/day to 0 (air control), 500, 1500, or 5000 ppm PFBI vapor for 5 days/week for 13 consecutive weeks (at least 65 exposures) followed by a 4-week recovery period. Clinical observations, body weights, clinical pathology, organ weights, and histopathology as well as detailed evaluations of neurotoxicity and thyroid function parameters were conducted at the end of the treatment period for up to 10 animals/sex/group with 5 males/group held for a 4-week recovery period. Findings in the thyroid target tissue consisted of a minimal thyroid follicular cell hypertrophy occasionally accompanied by hyperplasia, but without an increase in thyroid weight in the 500, 1500, and 5000 ppm males. At > or = 500 ppm, there was also increased thyroid stimulating hormone in females and increased T(3) and T(4) in animals of both sexes. These effects resolved following a 4-week recovery period in the males evaluated. Minor clinical pathology variations in all PFBI exposure groups were not considered biologically significant. A 9.4% reduction in absolute body weight in the 5000 ppm males was observed. Dosimetric adjustments for daily exposure time and uncertainty factors were selected to provide a basis for the proposed OELs. For acute (single event) exposures, a ceiling OEL of 3900 ppm, and for repeated exposures, an 8-h time-weighted average of 40 ppm PFBI were proposed.

FRET-based optical assay for monitoring riboswitch activation.

Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6.

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

Evidence for competitive inhibition of iodide uptake by perchlorate and translocation of perchlorate into the thyroid.

Various published data sets that investigate the potential effect of exogenous perchlorate (ClO4-) on the uptake of iodide in the thyroid and subsequent changes in thyroid hormone levels are available. In order to best use the data towards the prediction of human health effects resulting from ClO4- exposure, the available literature data must be integrated into a self-consistent, coherent, and parsimonious quantitative model based on the most likely mode of action of perchlorate effect on thyroid function. We submit that the simplest mode of action for ClO4- in the thyroid that remains consistent with all available data involves competitive inhibition of iodide transport into the thyroid follicle, transport of perchlorate into the thyroid follicle against a concentration gradient, further transport into the thyroid lumen (where it may again interfere with iodide transport), and, finally, passive diffusion back into the blood. We believe this description of perchlorate's kinetic behavior should serve as the foundation for predictive physiologically based pharmacokinetic (PBPK) models and as a working hypothesis for further experimental exploration.

Changes in cross-fostered Sprague-Dawley rat litters exposed to perchlorate.

Ammonium perchlorate is used as an oxidizer in rocket fuel. It has become a groundwater contaminant, dissociating to ammonium cation and perchlorate anion. The perchlorate ion competes with iodide for uptake into the thyroid, reducing thyroid hormone production. Pregnant Sprague-Dawley rats were given either untreated or perchlorate (1 mg/kg-day) treated drinking water beginning on gestation day 2. One set of control and exposed dams was sacrificed on gestation day 20. The litters from the second set of control and exposed dams were crossed immediately after parturition and were sacrificed at postnatal day 10. Dam serum and thyroid, pooled fetal sera, and male and female pup sera were collected and analyzed for perchlorate, thyroid-stimulating hormone (TSH), triiodothyronine (T(3)), and thyroxine (T(4)). Control pups receiving perchlorate through lactation had serum levels at postnatal day 10 of 0.54 microg/ml and 0.56 microg/ml for male and female pups, respectively, whereas exposed fetuses had serum perchlorate levels of 0.38 +/- 0.04 microg/ml. Female pups receiving perchlorate lactationally had significantly lower levels of serum T(4) than control pups and prenatally exposed pups. Serum T(4) levels in male pups were not affected by perchlorate. Serum thyroid hormone levels from gestational perchlorate exposure were restored to control values by postnatal day 10. In utero perchlorate-exposure decreased serum T(4) levels in the fetus. Gestational studies in conjunction with a cross-fostering study design helped discern thyroid hormonal changes caused by perchlorate exposure during the perinatal period.

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells.

The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)(29) dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

The pharmacokinetics of perchlorate and its effect on the hypothalamus-pituitary-thyroid axis in the male rat.

Perchlorate, an environmental contaminant, is known to disturb the hypothalamus-pituitary-thyroid (HPT) axis by blocking iodide accumulation in the thyroid. Iodide deficiency can lead to hypothyroidism and goiter in rats. The objective of the study was to characterize the pharmacokinetics of perchlorate in male Sprague-Dawley rats relative to inhibition of thyroidal radiolabeled iodide uptake and onset of up-regulation of the HPT axis. Radiolabeled perchlorate (3.3 mg/kg (36)ClO(-)(4)) was excreted in urine (99.5% over a 48-h period). (36)ClO(-)(4) is rapidly distributed into tissues with preferential sequestration into skin, gastrointestinal tract (GT), and thyroid. Calculated half-lives of (36)ClO(-)(4) from the skin, thyroid, plasma, GT, and GT contents were 32.0, 7.6, 7.3, 10.0, and 8.6 h, respectively. Perchlorate was very effective at inhibiting thyroidal uptake of radiolabeled iodide ((125)I(-)). In animals iv dosed with perchlorate followed by an iv challenge of (125)I(-), thyroidal (125)I(-) uptake was diminished by 11, 29, 55, and 82% at 11 h postdosing in the 0.01, 0.1, 1.0, and 3.0 mg/kg perchlorate dose groups, respectively. In perchlorate drinking water studies, dose-dependent inhibition in thyroidal uptake of (125)I(-) initially occurred with corresponding increases in serum thyroid-stimulating hormone (TSH) levels and decreases in thyroid hormone levels. TSH stimulated recovery from the initial perchlorate blocking effects was evident during 14 days of treatment in the 1.0 and 3.0 mg/kg per day treatment groups. However, recovery of serum thyroid hormones at these doses was much slower despite evidence for iodide sufficiency in the thyroid. These results suggest that the typical homeostatic mechanisms of the thyroid may respond differently at high doses of perchlorate used in this rat study (above 1 mg/kg per day) or perchlorate may be acting on the HPT axis by mechanisms other than thyroidal (125)I(-) uptake inhibition.