Dietary Phosphorus Levels Influence Protein-Derived Uremic Toxin Production in Nephrectomized Male Rats.

Gut microbiota-derived uremic toxins (UT) accumulate in patients with chronic kidney disease (CKD). Dietary phosphorus and protein restriction are common in CKD treatment, but the relationship between dietary phosphorus, a key nutrient for the gut microbiota, and protein-derived UT is poorly studied. Thus, we explored the relationship between dietary phosphorus and serum UT in CKD rats. For this exploratory study, we used serum samples from a larger study on the effects of dietary phosphorus on intestinal phosphorus absorption in nephrectomized (Nx, n = 22) or sham-operated (sham, n = 18) male Sprague Dawley rats. Rats were randomized to diet treatment groups of low or high phosphorus (0.1% or 1.2% w/w, respectively) for 1 week, with serum trimethylamine oxide (TMAO), indoxyl sulfate (IS), and p-cresol sulfate (pCS) analyzed by LC-MS. Nx rats had significantly higher levels of serum TMAO, IS, and pCS compared to sham rats (all p < 0.0001). IS showed a significant interaction between diet and CKD status, where serum IS was higher with the high-phosphorus diet in both Nx and sham rats, but to a greater extent in the Nx rats. Serum TMAO (p = 0.24) and pCS (p = 0.34) were not affected by dietary phosphorus levels. High dietary phosphorus intake for 1 week results in higher serum IS in both Nx and sham rats. The results of this exploratory study indicate that reducing dietary phosphorus intake in CKD may have beneficial effects on UT accumulation.

Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors.

Local delivery of immune-activating agents has shown promise in overcoming an immunosuppressive tumor microenvironment (TME) and stimulating antitumor immune responses in tumors. However, systemic therapy is ultimately needed to treat tumors that are not readily locatable or accessible. To enable systemic delivery of immune-activating agents, we employ poly(lactic-co-glycolide) (PLGA) nanoparticles (NPs) with a track record in systemic application. The surface of PLGA NPs is decorated with adenosine triphosphate (ATP), a damage-associated molecular pattern to recruit antigen-presenting cells (APCs). The ATP-conjugated PLGA NPs (NPpD-ATP) are loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death to generate tumor antigens in situ. We show that the NPpD-ATP retains ATP activity in hostile TME and provides a stable "find-me" signal to recruit APCs. Therefore, the PTX-loaded NPpD-ATP helps populate antitumor immune cells in TME and attenuate the growth of CT26 and B16F10 tumors better than a mixture of PTX-loaded NPpD and ATP. Combined with anti-PD-1 antibody, PTX-loaded NPpD-ATP achieves complete regression of CT26 tumors followed by antitumor immune memory. This study demonstrates the feasibility of systemic immunotherapy using a PLGA NP formulation that delivers ICD-inducing chemotherapy and an immunostimulatory signal.

Gut Microbiota and Uremic Retention Solutes in Adults With Moderate CKD: A 6-Day Controlled Feeding Study.

OBJECTIVE: To determine serum and urine concentrations of the uremic retention solutes (URSs), indoxyl sulfate (IS), p-cresol sulfate (PCS), and trimethylamine N-oxide (TMAO), and gut microbiota composition in individuals with moderate chronic kidney disease (CKD) compared with matched adults without CKD in a 6-day controlled feeding study. DESIGN AND METHODS: This study was a secondary analysis in which 8 adults with moderate CKD were matched for age, sex, and race with 8 adults without CKD in a parallel-arm, 6-day controlled feeding study. IS, PCS, and TMAO were quantified using liquid chromatography-mass spectrometry in fecal samples, fasting serum, and fasting spot urine samples collected at the end of the feeding period. RESULTS: Fasting serum URS concentrations were 2.8 to 4.9x higher in CKD compared to controls (all P < .05). No differences were found in the composition of the gut microbiota between patients with and without CKD when analyzing samples for α-diversity, ß-diversity, and only minor abundance differences across taxa were apparent. Estimated glomerular filtration rate (eGFR) was inversely related to each serum URS in the whole cohort (all P < .01). However, within groups the relationships between eGFR and serum URS remained strong for CKD patients for IS and TMAO (both P < .05) but weakened for PCS (P = .10). eGFR was only correlated with urine PCS in the whole cohort (P = .03); within groups, no correlation for eGFR with any urine URS was observed. Only urine TMAO was higher in CKD compared to controls (P < .05). CONCLUSION: Serum URS concentrations are elevated in adults with CKD compared to matched non-CKD adults without differences in gut microbiota composition after consuming the same controlled study diet for 6 days. Future studies are needed to determine if specific dietary components may differentially alter the microbiota and URS.

Formate production is dispensable for Haemophilus ducreyi virulence in human volunteers.

Haemophilus ducreyi is a causative agent of cutaneous ulcers in children who live in the tropics and of the genital ulcer disease chancroid in sexually active persons. In the anaerobic environment of abscesses and ulcers, anaerobic respiration and mixed acid fermentation (MAF) can be used to provide cellular energy. In Escherichia coli, MAF produces formate, acetate, lactate, succinate, and ethanol; however, MAF has not been studied in H. ducreyi. In human challenge experiments with H. ducreyi 35000HP, transcripts of the formate transporter FocA and pyruvate formate lyase (PflB) were upregulated in pustules compared to the inocula. We made single and double mutants of focA and pflB in 35000HP. Growth of 35000HPΔfocA was similar to 35000HP, but 35000HPΔpflB and 35000HPΔfocA-pflB had growth defects during both aerobic and anaerobic growth. Mutants lacking pflB did not secrete formate into the media. However, formate was secreted into the media by 35000HPΔfocA, indicating that H. ducreyi has alternative formate transporters. The pH of the media during anaerobic growth decreased for 35000HP and 35000HPΔfocA, but not for 35000HPΔpflB or 35000HPΔfocA-pflB, indicating that pflB is the main contributor to media acidification during anaerobic growth. We tested whether formate production and transport were required for virulence in seven human volunteers in a mutant versus parent trial between 35000HPΔfocA-pflB and 35000HP. The pustule formation rate was similar for 35000HP (42.9%)- and 35000HPΔfocA-pflB (62%)-inoculated sites. Although formate production occurs during in vitro growth and focA-pflB transcripts are upregulated during human infection, focA and pflB are not required for virulence in humans.

Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE.

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. The reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana, overexpression of FERULATE 5-HYDROXYLASE (F5H) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d (cadc cadd), cinnamoyl-CoA reductase 1, and reduced epidermal fluorescence 3, plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H, which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

Variability in phenylalanine side chain conformations facilitates broad substrate tolerance of fatty acid binding in cockroach milk proteins.

Diploptera punctata, also known as the Pacific beetle cockroach, is a viviparous cockroach that gives birth to live offspring and secretes a highly concentrated mixture of glycosylated proteins as a source of nourishment for developing embryos. These proteins are lipocalins that bind to lipids and crystallize in the gut of the embryo. A structure of milk crystals harvested from the embryos showed that the milk-derived crystals were heterogeneous and made of three proteins (called Lili-Mips). We hypothesized that the isoforms of Lili-Mip would display different affinities for fatty acids due to the ability of the pocket to bind multiple acyl chain lengths. We previously reported the structures of Lili-Mip from crystals grown in vivo and recombinantly expressed Lili-Mip2. These structures are similar, and both bind to several fatty acids. This study explores the specificity and affinity of fatty acid binding to recombinantly expressed Lili-Mip 1, 2 & 3. We show that all isoforms can bind to different fatty acids with similar affinities. We also report the thermostability of Lili-Mip is pH dependent, where stability is highest at acidic pH and declines as the pH increases to physiological levels near 7.0. We show that thermostability is an inherent property of the protein, and glycosylation and ligand binding do not change it significantly. Measuring the pH in the embryo's gut lumen and gut cells suggests that the pH in the gut is acidic and the pH inside the gut cells is closer to neutral pH. In various crystal structures (reported here and previously by us), Phe-98 and Phe-100 occupy multiple conformations in the binding pocket. In our earlier work, we had shown that the loops at the entrance could adapt various conformations to change the size of the binding pocket. Here we show Phe-98 and Phe-100 can reorient to stabilize interactions at the bottom of the cavity-and change the volume of the cavity from 510 Å3 to 337 Å3. Together they facilitate the binding of fatty acids of different acyl chain lengths.

Proteome-wide quantitative analysis of redox cysteine availability in the Drosophila melanogaster eye reveals oxidation of phototransduction machinery during blue light exposure and age.

The retina is one of the highest oxygen-consuming tissues because visual transduction and light signaling processes require large amounts of ATP. Thus, because of the high energy demand, oxygen-rich environment, and tissue transparency, the eye is susceptible to excess production of reactive oxygen species (ROS) resulting in oxidative stress. Oxidative stress in the eye is associated with the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy. ROS can modify and damage cellular proteins, but can also be involved in redox signaling. In particular, the thiol groups of cysteines can undergo reversible or irreversible oxidative post-translational modifications (PTMs). Identifying the redox-sensitive cysteines on a proteome-wide scale provides insight into those proteins that act as redox sensors or become irreversibly damaged upon exposure to oxidative stress. In this study, we profiled the redox proteome of the Drosophila eye under prolonged, high intensity blue light exposure and age using iodoacetamide isobaric label sixplex reagents (iodo-TMT) to identify changes in cysteine availability. Although redox metabolite analysis of the major antioxidant, glutathione, revealed similar ratios of its oxidized and reduced form in aged or light-stressed eyes, we observed different changes in the redox proteome under these conditions. Both conditions resulted in significant oxidation of proteins involved in phototransduction and photoreceptor maintenance but affected distinct targets and cysteine residues. Moreover, redox changes induced by blue light exposure were accompanied by a large reduction in light sensitivity that did not arise from a reduction in the photopigment level, suggesting that the redox-sensitive cysteines we identified in the phototransduction machinery might contribute to light adaptation. Our data provide a comprehensive description of the redox proteome of Drosophila eye tissue under light stress and aging and suggest how redox signaling might contribute to light adaptation in response to acute light stress.

Biocompatibility and Safety Assessment of Combined Topical Ozone and Antibiotics for Treatment of Infected Wounds.

Wound infections with antibiotic-resistant bacteria, particularly the Gram-negative strains, pose a substantial health risk for patients with limited treatment options. Recently topical administration of gaseous ozone and its combination with antibiotics through portable systems has been demonstrated to be a promising approach to eradicate commonly found Gram-negative strains of bacteria in wound infections. However, despite the significant impact of ozone in treating the growing number of antibiotic-resistant infections, uncontrolled and high concentrations of ozone can cause damage to the surrounding tissue. Hence, before such treatments could advance into clinical usage, it is paramount to identify appropriate levels of topical ozone that are effective in treating bacterial infections and safe for use in topical administration. To address this concern, we have conducted a series of in vivo studies to evaluate the efficacy and safety of a portable and wearable adjunct ozone and antibiotic wound therapy system. The concurrent ozone and antibiotics are applied through a wound interfaced gas permeable dressing coated with water-soluble nanofibers containing vancomycin and linezolid (traditionally used to treat Gram-positive infections) and connected to a portable ozone delivery system. The bactericidal properties of the combination therapy were evaluated on an ex vivo wound model infected with Pseudomonas aeruginosa, a common Gram-negative strain of bacteria found in many skin infections with high resistance to a wide range of currently available antibiotics. The results indicated that the optimized combination delivery of ozone (4 mg h-1) and topical antibiotic (200 µg cm-2) provided complete bacteria eradication after 6 h of treatment while having minimum cytotoxicity to human fibroblast cells. Furthermore, in vivo local and systemic toxicity studies (e.g., skin monitoring, skin histopathology, and blood analysis) on pig models showed no signs of adverse effects of ozone and antibiotic combination therapy even after 5 days of continuous administration. The confirmed efficacy and biosafety profile of the adjunct ozone and antibiotic therapy places it as a strong candidate for treating wound infection with antimicrobial-resistant bacteria and further pursuing human clinical trials.

Biobehavioral and psychosocial stress changes during three 8-12 month spaceflight analog missions with Mars-like conditions of isolation and confinement.

Prior theories about individual and team adaptation to living and working in an isolated and confined environment (ICE) have been derived from the experiences of individuals who winter-over in Antarctica or deploy for long durations in submarines. These theories are typically described as a 3- to 4-stage process with phases of excitement and elevated alertness, then followed by difficult phases, including depression and volatility. To further evaluate the applicability of these theories to long-duration human spaceflight missions, longitudinal stress responses to prolonged isolation and confinement of three 6-person crews during 8-12 months simulated Mars missions were characterized through metabolite profiling (biomarkers in hair and urine samples), wearables monitoring (sleep and activity levels), and self-reported ratings of stress, mood, social participation, and perceived health. These data were normalized, aggregated, and clustered to analyze longitudinal trends in biobehavioral and psychosocial stress measures. As a result, this analysis presents a theoretical model that triangulates aspects of prior theories with new evidence to describe ICE stress at HI-SEAS as 1) eustress of initial adaptation (high stress hormone levels at mission start), 2) deprivation due to prolonged isolation and confinement (decreasing dopamine and serotonin levels), 3) disruption of individual and team dynamics (changes in activity levels, mood, perceived stress, and social participation) and 4) asynchronous coping (changes in sleep-wake cycles, outlook, and team cohesion). These findings support several aspects of prior theories in combination, such as the elevated alertness at mission start and that adverse conditions are most likely to develop after the halfway point of a mission (e.g. for HI-SEAS 8-12 months missions, after approximately 6 months) followed by a period of volatility until the end (e.g. as stated in Rohrer's theory, ups and downs until the end, not a renewed outlook at the end as described in 3rd quarter phenomenon theory).

Effects of porcine reproductive and respiratory syndrome virus (PRRSV) on thyroid hormone metabolism in the late gestation fetus.

Porcine reproductive and respiratory syndrome virus (PRRSV) in late gestation causes a profound suppression of circulating maternal and fetal thyroid hormone during a critical window of development. To understand this relationship, we evaluated thyroid hormone metabolism at the maternal-fetal interface and within fetal tissues, along with hormone metabolite levels in serum. Fetuses were classified using an established model based on viral load in serum and thymus, and preservation status, including uninfected (UNIF), high-viral load viable (HV-VIA), and high-viral load meconium-stained (HV-MEC), with additional controls from sham-inoculated gilts (CON). Expression of three iodothyronine deiodinases, five sulfotransferases, sulfatase, and two solute carriers known to transport thyroid hormone were evaluated in maternal endometrium and fetal placenta, liver, and kidney. Serum thyroxin (T4), reverse triiodothyronine (rT3), and diiodothyronine (T2) were evaluated via liquid chromatography tandem mass spectrometry. Significant changes in gene expression were observed in all four tissues, with the liver being the most severely impacted. We observed local and fetal specific regulation of maternal tissues through significant upregulation of DIO2 and DIO3 expression in the endometrium corresponding to infected but viable fetuses relative to uninfected and control fetuses. Expression levels of DIO2 and DIO3 were significantly higher in the resilient (HV-VIA) fetuses relative to the susceptible (HV-MEC) fetuses. A substantial decrease in serum T4 was confirmed, with no corresponding increase in rT3 or T2. Collectively, these results show that thyroid hormone metabolism is altered at the maternal-fetal interface and within the PRRSV infected fetus and is associated with fetal viability.

Mammary Development in Gilts at One Week Postnatal Is Related to Plasma Lysine Concentration at 24 h after Birth, but Not Colostrum Dose.

Perinatal nutrition affects future milk production. The number of mammary epithelial cells affect milk production capacity. Therefore, it was hypothesized that the level of colostrum intake affects the proliferation rate and the total number of mammary epithelial cells in the gland. The ratio of newly synthesized protein to newly synthesized DNA reflects the relative amount of cellular differentiation to cell division. The study objective was to determine the relationship between the level of colostrum intake and 24 h-level of circulating amino acid, glucose and insulin with mammary parenchyma histological features, cell division and protein synthesis over the first week postnatal. One of two standardized doses of a homogenate colostrum sample, 10% (n = 8) and 20% (n = 8) of birth bodyweight, was fed to gilts over the first 24 h postnatal. Gilts were administered deuterium oxide immediately after birth and daily to label newly synthesized DNA and proteins. Gilts were euthanized on postnatal day seven, and DNA and protein were isolated from mammary parenchyma. DNA and protein fractional synthesis (f) and fractional synthetic rate (FSR) were calculated using mass isotopomer distribution analysis. The ratio of protein f and FSR to DNA f and FSR were calculated and used to indicate the relative amounts of differentiation to cell division. Mammary morphological development was also analyzed by measuring the parenchymal epithelial area and the stromal and epithelial proliferation index on postnatal day seven. Colostrum dose was not related to any of the variables used to evaluate mammary development. However, plasma lysine levels at 24 h postnatal were positively related to average daily gain (ADG; r = 0.54, p = 0.05), DNA f (r = 0.57; p = 0.03) and DNA FSR (r = 0.57; p = 0.03) in mammary parenchyma. Plasma lysine was inversely related to the ratio of protein to DNA f and FSR (r = -0.56; p = 0.04). ADG was related to the parenchymal epithelial area and DNA and protein f and FSR (p < 0.05). These relationships support the idea that the nutritional environment affects early mammary development and that higher lysine levels in the perinatal period favored a greater degree of cell division versus differentiation in mammary of neonatal pigs and thus, warrant further investigations.

Disruption of pulmonary resolution mediators contribute to exacerbated silver nanoparticle-induced acute inflammation in a metabolic syndrome mouse model.

Pre-existing conditions modulate sensitivity to numerous xenobiotic exposures such as air pollution. Specifically, individuals suffering from metabolic syndrome (MetS) demonstrate enhanced acute inflammatory responses following particulate matter inhalation. The mechanisms associated with these exacerbated inflammatory responses are unknown, impairing interventional strategies and our understanding of susceptible populations. We hypothesize MetS-associated lipid dysregulation influences mediators of inflammatory resolution signaling contributing to increased acute pulmonary toxicity. To evaluate this hypothesis, healthy and MetS mouse models were treated with either 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), or saline (control) via intraperitoneal injection prior to oropharyngeal aspiration of silver nanoparticles (AgNP). In mice receiving saline treatment, AgNP exposure resulted in an acute pulmonary inflammatory response that was exacerbated in MetS mice. A targeted lipid assessment demonstrated 18-HEPE, 14-HDHA, and 17-HDHA treatments altered lung levels of specialized pro-resolving lipid mediators (SPMs). 14-HDHA and 17-HDHA treatments more efficiently reduced the exacerbated acute inflammatory response in AgNP exposed MetS mice as compared to 18-HEPE. This included decreased neutrophilic influx, diminished induction of inflammatory cytokines/chemokines, and reduced alterations in SPMs. Examination of SPM receptors determined baseline reductions in MetS mice compared to healthy as well as decreases due to AgNP exposure. Overall, these results demonstrate AgNP exposure disrupts inflammatory resolution, specifically 14-HDHA and 17-HDHA derived SPMs, in MetS contributing to exacerbated acute inflammatory responses. Our findings identify a potential mechanism responsible for enhanced susceptibility in MetS that can be targeted for interventional therapeutic approaches.

Pharmacokinetics of thiamine (vitamin B1) in adult horses after administration of three single intravenous doses.

Thiamine is a vital co-factor for several anti-inflammatory and antioxidant processes that are critical for mitigation of sepsis-associated inflammation, but pharmacokinetic (PK) analysis has not been reported in horses. We hypothesized that IV thiamine hydrochloride (TH) at increasing dosages would result in corresponding increases in plasma thiamine concentrations without causing adverse effects. A randomized cross-over study was performed in 9 healthy horses that each received TH at 5, 10, and 20 mg/kg IV. Blood was collected immediately prior to drug administration and at several time points thereafter. High-performance liquid chromatography with mass spectrometry was used to quantify thiamine concentrations at each time point. Non-compartmental PK methods showed that IV TH resulted in supraphysiologic plasma concentrations with a short half-life (0.77-1.12 h) and no adverse clinical signs were observed. The terminal rate constant decreased as the dosage increased (p < .0001) and clearance significantly decreased at the 20 mg/kg dosage (p = .0011). The area under the curve (AUC) increased in a non-linear fashion. These findings suggest that thiamine follows non-linear elimination kinetics in horses, which is likely due to saturation of renal elimination. Future studies are needed to identify therapeutic plasma concentrations and develop thiamine dosing recommendations for horses.

Developmental atrazine exposure in zebrafish produces the same major metabolites as mammals along with altered behavioral outcomes.

Atrazine (ATZ) is the second most commonly applied agricultural herbicide in the United States. Due to contamination concerns, the U.S. EPA has set the maximum contaminant level in potable water sources at 3 parts per billion (ppb; µg/l). Depending on the time of year and sampling location, water sources often exceed this limit. ATZ is an endocrine disrupting chemical in multiple species observed to target the neuroendocrine system. In this study the zebrafish vertebrate model was used to test the hypothesis that a developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in developing larvae. Adult AB zebrafish were bred, embryos were collected, and exposed to 0, 0.3, 3, or 30 ppb ATZ from 1 to 120 h post fertilization (hpf). Targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals: desethyl atrazine (DEA), deisopropyl atrazine (DIA), and diaminochloroatrazine (DACT). The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group (p < 0.05). Further analysis into behavior during the dark and light phases showed zebrafish larvae exposed to 0.3 ppb ATZ had an increase in total distance moved in the first light phase and time spent moving in the first dark and light phases (p < 0.05). Alternatively, a decrease in total distance moved was observed in the second and third dark phases in zebrafish exposed to 30 ppb ATZ (p < 0.05). No significant differences were observed for any of the morphological measurements following ATZ exposure from 1 to 120 hpf (p > 0.05). These findings suggest that a ATZ exposure during early development generates metabolite profiles similar to mammals and leads to behavioral alterations supporting ATZ as a neurodevelopmental toxicant.

The aqueous extract of Fridericia chica grown in northern Colombia ameliorates toxicity induced by Tergitol on Caenorhabditis elegans.

The aqueous extract of fallen leaves from Fridericia chica (Bonpl.) L.G. Lohmann is utilized as a remedy in communities at northern Colombia. Traditional uses include wound healing, gastrointestinal inflammation, leukemia and psoriasis, among others. The aims of this research were to evaluate the potential of the aqueous extract of fallen leaves of F. chica (AEFchica) to inhibit ethoxylated nonylphenol (Tergitol)-induced toxicity in Caenorhabditis elegans; and to identify its main components. The pharmacological properties of AEFchica was evaluated using a Tergitol-induced toxicity model in Caenorhabditis elegans. Lethality, locomotion, reproduction, and DAF-16 nuclear translocation were quantified. The chemical composition of AEFchica was carried out using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. AEFchica induced very little lethality on C. elegans (5.6%) even at high concentrations (10,000 µg/mL). The extract had no effect on locomotion impairing induced by ethoxylated nonylphenol. However, AEFchica (1000 µg/mL) abrogated Tergitol-induced mortality, recovering up to 53.3% of the nematodes from lethality induced by 10 mM Tergitol. Similarly, it also blocked Tergitol-dependent reproduction inhibition (82.1% recovery), as well as DAF-16 nuclear translocation (>95%), suggesting a prominent role on oxidative stress control. The chemical analysis indicated the presence of a great variety of molecules with known antioxidant, metabolic and immune modulator properties, such as hydroxylated methoxy flavones, N-methyl-1-deoxynojirimycin, and rehmaionoside A. In short, the aqueous extract of F. chica protects C. elegans from the deleterious effects of Tergitol on lethality, reproduction and oxidative stress involving DAF-16-mediated pathway. This extract is a promising source of bioactive phytochemicals for multi-target pharmacological purposes.

Development of a Pediatric Mini-Tablet Formulation for Expedited Preclinical Studies.

Multiple considerations are essential to address the main challenges of dose flexibility and patient adherence in pediatric drug development, particularly for oncology. Mini-tablets, 2 mm in diameter, were manufactured using a rotary tablet press at a set weight and compression force level. The physical characteristics were consistent for mini-tablets throughout multiple batches. Polymeric amorphous solid dispersion (ASD) was used as a solubility enhancing technique to increase solubility and exposure of lapatinib. The effects of the polymeric excipient and disintegrant on drug release properties were investigated. While having a lower apparent solubility and shorter storage stability, hydroxypropyl methylcellulose E3 (HPMCE3) formulation provided a higher percentage of drug release compared to hydroxypropyl methylcellulose phthalate (HPMCP). The intermolecular interaction within the ASD system plays a role in the level of apparent solubility, physical stability, and concentration of free drug available in an aqueous environment. Juvenile porcine models at two different weight groups (10 and 20 kg) were used to obtain the pharmacokinetic parameters of lapatinib. While the dose-normalized exposure of drug was found to be lower in the pig study, the dose flexibility of mini-tablets enabled a constant dose level to be administered to achieve equivalent plasma concentration-time profiles between the two groups. This linear scaling in the amount of drug in pediatric and adult population has also been observed in human clinical studies.

Publisher Correction: Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.

A novel method to measure steroid hormone concentrations in walrus bone from archaeological, historical, and modern time periods using liquid chromatography/tandem mass spectrometry.

RATIONALE: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was validated and utilized to measure and analyze four steroid hormones related to stress and reproduction in individual samples from a novel tissue, Pacific walrus (Odobenus rosmarus divergens, herein walrus) bone. This method determines steroid hormone concentrations in the remote walrus population over millennia from archaeological (>200 bp), historical (200-20 bp), and modern (2014-2016) time periods. METHODS: Lipids were extracted from walrus bone collected from these periods using methanol before LC/MS/MS analysis. Isotopically labeled internal standards for each target hormone were added to every sample. Analytical and physiological validations were performed. Additionally, a tissue comparison was done among paired walrus bone, serum, and blubber samples. A rapid resolution liquid chromatography system coupled to a QqQ mass spectrometer was used to analyze all samples after derivatization for progesterone, testosterone, cortisol, and estradiol concentrations. Multiple reaction monitoring was used for MS analysis and data were acquired in positive electrospray ionization mode. RESULTS: Progesterone, testosterone, cortisol, and estradiol were linear along their respective standard calibration curves based on their R2 values (all > 0.99). Accuracy ranged from 93-111% for all hormones. The recovery of extraction, recovery of hormones without matrix effect, was 92-101%. The overall process efficiency of our method for measuring hormones in walrus bone was 93-112%. Progesterone and testosterone concentrations were not affected by reproductive status among adult females and males, respectively. However, estradiol was different among pregnant and non-pregnant adult females. Overall, steroid hormones reflect a long-term reservoir in cortical bone. This method was also successfully applied to walrus bone as old as 3585 bp. CONCLUSIONS: LC/MS/MS analysis of bone tissue (0.2-0.3 g) provides stress and reproductive data from elusive walruses that were alive thousands of years ago. Based on physiological validations, tissue comparison, and published literature, steroid hormone concentrations measured in walrus cortical bone could represent an accumulated average around a 10-20-year time span. By investigating how stress and reproductive physiology may have changed over the past ~3000 years based on bone steroid hormone concentrations, this method will help answer how physiologically resilient walruses are to climate change in the Arctic.

Fingerprint Stimulated Raman Scattering Imaging Reveals Retinoid Coupling Lipid Metabolism and Survival.

Retinoids play critical roles in development, immunity, and lipid metabolism, and their deficiency leads to various human disorders. Yet, tools for sensing retinoids in vivo are lacking, which limits the understanding of retinoid distribution, dynamics and functions in living organisms. Here, using hyperspectral stimulated Raman scattering microscopy, we discover a previously unknown cytoplasmic store of retinoids in Caenorahbditis elegans. Following the temporal dynamics of retinoids, we reveal that their levels are positively correlated with fat storage, and their supplementation slows down fat loss during starvation. We also discover that retinoids promote fat unsaturation in response to high-glucose stress, and improve organism survival. Together, our studies report a new method for tracking the spatiotemporal dynamics of retinoids in living organisms, and suggest the crucial roles of retinoids in maintaining metabolic homeostasis and enhancing organism fitness upon developmental and dietary stresses.