Is Bipolar Radiofrequency-Assisted Liposuction Equivalent to Open Anterior Platysmaplasty in Facelift Surgery?

BACKGROUND: During facelift surgery, anterior platysmaplasty (AP) has been used for decades, but it limits lateral advancement and can induce contour irregularities. Radiofrequency (RF)-assisted-liposuction in the anterior neck can avoid these disadvantages by tightening skin without open surgery. PURPOSE: The purpose of the study was to compare the esthetic outcomes of facelift surgery with those of AP and RF. STUDY DESIGN, SETTING, SAMPLE: A 5-year retrospective cohort study was performed on facelift patients treated by a single surgeon. Exclusions were single-side surgery, previous facelift, chin/lip augmentation/reduction, and inadequate data. PREDICTOR VARIABLE: The predictor variable was neck management technique (AP vs RF). MAIN OUTCOME VARIABLES: The primary outcome variable was the change in cervicomental angle (CMA) following surgery as measured on facial photographs. Secondary outcomes included distance changes from the central CMA point in vertical and horizontal planes to repeatable reference planes. COVARIATES: Covariates were age, body mass index, American Society of Anesthesiologists classification, smoking, and simultaneous procedures. ANALYSES: The statistical analysis was performed using Wilcoxon rank-sum, Fisher's exact, Kruskal-Wallis tests, Pearson's correlation, and linear regressions. The level of statistical significance was P < .05. RESULTS: There were 132 patients included in the study; 67 received AP and 65 received RF. AP trended toward better performance in CMA change in the unadjusted analysis (-18.7° ± 13.8° vs -22.3° ± 13.7°, respectively, P = .08). AP and RF performed similarly in the adjusted analysis (P = .29). Techniques were similar in horizontal distance change to the CMA (P = .31). RF was associated with less change in the vertical distance to the CMA in the unadjusted analysis (-11.9 mm ± 11.0 mm vs -6.7 mm ± 8.7 mm, respectively, P = .01) and adjusted analysis (ß = 4.3 mm, 95% confidence interval .8 to 7.9 mm, P = .02). CONCLUSION AND RELEVANCE: Utilization of the RF technique for management of the anterior neck in facelift surgery is associated with similar outcomes to the AP technique in horizontal distance to the CMA, but AP performed better in CMA change and vertical distance to the CMA.

Paediatric Gastrointestinal, Hepatic and Pancreatic Inflammatory Myofibroblastic Tumours, A Single Centre Experience.

ABSTRACT: Inflammatory myofibroblastic tumours (IMTs) are rare soft tissue tumours. Reports of gastrointestinal tract, liver and pancreas tumours are limited. The objective of this study is to identify presenting features, contributing prognostic / etiological factors and any variability in outcomes in the context of different historical treatments. We retrospectively reviewed the records of seven children treated at our hospital between 2006 and 2019 and assessed the demographic, presentation, treatment, immunohistochemistry, and outcomes of their tumours. Age range at presentation was 4 months-15 years with a male predominance. Presentations were typically due to local mass effect or incidental discovery. Systemic symptoms were rare. Outcomes were good with six out of seven stable or in remission irrespective of treatment. Surgical resection where possible is the treatment of choice. Medical therapy had good outcomes with chemotherapy acting as first line treatment when required. The only negative prognostic factor identified was local spread at the time of presentation.

Dextran sulfate sodium mouse model of inflammatory bowel disease evaluated for systemic genotoxicity via blood micronucleus and Pig-a gene mutation assays.

Inflammatory bowel disease (IBD) is an important risk factor for gastrointestinal cancers. Inflammation and other carcinogenesis-related effects at distal, tissue-specific sites require further study. In order to better understand if systemic genotoxicity is associated with IBD, we exposed mice to dextran sulfate sodium salt (DSS) and measured the incidence of micronucleated cells (MN) and Pig-a mutant phenotype cells in blood erythrocyte populations. In one study, 8-week-old male CD-1 mice were exposed to 0, 1, 2, 3 or 4% w/v DSS in drinking water. The 4-week in-life period was divided into four 1-week intervals-alternately on then off DSS treatment. Low volume blood samples were collected for MN analysis at the end of each week, and cardiac blood samples were collected at the end of the 4-week period for Pig-a analyses. The two highest doses of DSS were observed to induce significant increases in reticulocyte frequencies. Even so, no statistically significant treatment-related effects on the genotoxicity biomarkers were evident. While one high-dose mouse showed modestly elevated MN frequencies during the DSS treatment cycles, it also exhibited exceptionally high reticulocyte frequencies (e.g. 18.7% at the end of the second DSS cycle). In a second study, mice were treated with 0 or 4% DSS for 9-18 consecutive days. Exposure was continued until rectal bleeding or morbidity was evident, at which point the treatment was terminated and blood was collected for MN analysis. The Pig-a assay was conducted on samples collected 29 days after the start of treatment. The initial blood specimens showed highly elevated reticulocyte frequencies in DSS-exposed mice (mean ± SEM = 1.75 ± 0.10% vs. 13.04 ± 3.66% for 0 vs. 4% mice, respectively). Statistical analyses showed no treatment-related effect on MN or Pig-a mutant frequencies. Even so, the incidence of MN versus reticulocytes in the DSS-exposed mice were positively correlated (linear fit R2 = 0.657, P = 0.0044). Collectively, these results suggest that in the case of the DSS CD-1 mouse model, systemic effects include stress erythropoiesis but not remarkable genotoxicity. To the extent MN may have been slightly elevated in a minority of individual mice, these effects appear to be secondary, likely attributable to stimulated erythropoiesis.

Discovery of an Isothiazolinone-Containing Antitubercular Natural Product Levesquamide.

Antitubercular agent levesquamide is a new polyketide-nonribosomal peptide (PK-NRP) hybrid marine natural product isolated from Streptomyces sp. RKND-216. The structure contains a rare isothiazolinone moiety which has only been reported in collismycin SN. Structure elucidation by NMR spectroscopy was a significant challenge due to a deficiency of protons in this aromatic moiety. Therefore, the genome of Streptomyces sp. RKND-216 was sequenced to identify the levesquamide biosynthetic gene cluster (BGC). Analysis of the BGC provided structural insights and guided stable-isotope labeling experiments, which led to the assignment of the fused pyridine-isothiazolinone moiety. The BGC and the labeling experiments provide further insights into the biosynthetic origin of isothiazolinones. Levesquamide exhibited antimicrobial activity in the microplate alamarBlue assay (MABA) and low oxygen recovery assay (LORA) against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 9.65 and 22.28 µM, respectively. Similar activity was exhibited against rifampicin- and isoniazid-resistant M. tuberculosis strains with MIC values of 9.46 and 9.90 µM, respectively. This result suggests levesquamide has a different mode of action against M. tuberculosis compared to the two first-line antitubercular drugs rifampicin and isoniazid. Furthermore, levesquamide shows no cytotoxicity against the Vero cell line, suggesting it may have a useful therapeutic window.

Ultra-Micro-Scale-Fractionation (UMSF) as a Powerful Tool for Bioactive Molecules Discovery.

Herein is detailed the development and validation of an ultra-micro-scale-fractionation (UMSF) technique for the discovery of plant-based, bioactive molecules, coupling the advantages of ultra-performance liquid chromatography mass spectrometry (UPLC-MS) separations with microtiter plate-based bioassay screens. This novel one-step approach simultaneously uses UPLC to collect chemical profile information, while performing high-resolution fractionation, greatly improving workflow compared to methods relying on high-performance liquid chromatography (HPLC), solid phase extraction or flash systems for chromatographic separations. Using the UMSF technique, researchers are able to utilize smaller quantities of starting materials, reduce solvent consumption during fractionation, reduce laborious solvent dry down times, replace costly single-use solid-phase-extraction cartridges with reusable analytical-sale UPLC columns, reduce fractionation times to less than 10 min, while simultaneously generating chemical profile data of active fractions and enjoying superior chromatographic resolution. Using this technique, individual bioactive components can be readily purified, identified, and bioassayed in one step from crude extracts, thereby eliminating ambiguous synergistic effects often reported in plant-based natural products research. A successful case-study is presented illustrating the versatility of this technique in identifying lupulone as the principal cytotoxic component from hops (Humulus lupulus L.), using a brine shrimp (Artemia franciscana) model. These results confirm and expand upon previous cell-based bioassay studies using a more complex, multicellular organism, and add to our understanding of structure-function activity relationships for secondary metabolites in hops and the Cannabaceae plant family.

Identification of Vertebral Fractures by Convolutional Neural Networks to Predict Nonvertebral and Hip Fractures: A Registry-based Cohort Study of Dual X-ray Absorptiometry.

Background Detection of vertebral fractures (VFs) aids in management of osteoporosis and targeting of fracture prevention therapies. Purpose To determine whether convolutional neural networks (CNNs) can be trained to identify VFs at VF assessment (VFA) performed with dual-energy x-ray absorptiometry and if VFs identified by CNNs confer a similar prognosis compared with the expert reader reference standard. Materials and Methods In this retrospective study, 12 742 routine clinical VFA images obtained from February 2010 to December 2017 and reported as VF present or absent were used for CNN training and testing. All reporting physicians were diagnostic imaging specialists with at least 10 years of experience. Randomly selected training and validation sets were used to produce a CNN ensemble that calculates VF probability. A test set (30%; 3822 images) was used to assess CNN agreement with the human expert reader reference standard and CNN prediction of incident non-VFs. Statistical analyses included area under the receiver operating characteristic curve, two-tailed Student t tests, prevalence- and bias-adjusted κ value, Kaplan-Meier curves, and Cox proportional hazard models. Results This study included 12 742 patients (mean age, 76 years ± 7; 12 013 women). The CNN ensemble demonstrated an area under the receiver operating characteristic curve of 0.94 (95% confidence interval [CI]: 0.93, 0.95) for VF detection that corresponded to sensitivity of 87.4% (534 of 611), specificity of 88.4% (2838 of 3211), and prevalence- and bias-adjusted κ value of 0.77. On the basis of incident fracture data available for 2813 patients (mean follow up, 3.7 years), hazard ratios adjusted for baseline fracture probability were 1.7 (95% CI: 1.3, 2.2) for CNN versus 1.8 (95% CI: 1.3, 2.3) for expert reader-detected VFs for incident non-VF and 2.3 (95% CI: 1.5, 3.5) versus 2.4 (95% CI: 1.5, 3.7) for incident hip fracture. Conclusion Convolutional neural networks can identify vertebral fractures on vertebral fracture assessment images with high accuracy, and these convolutional neural network-identified vertebral fractures predict clinical fracture outcomes. © RSNA, 2019 Online supplemental material is available for this article.

NMR characterization of novel pyranoanthocyanins derived from the pulp of Panax quinquefolius L. (North American ginseng).

Three major pigments (one natural and two derived) were determined to be present in the berry pulp of Panax quinquefolius L. (North American ginseng). The first was a simple anthocyanin (pelargonidin 3-O-lathyroside) along with two novel pyranoanthocyanins, structurally similar to those recently discovered in Staghorn sumac. The three anthocyanins were structurally characterized using NMR (1 H, gCOSY, gHSQC, gHMBC, TOCSY, ROESY, and 13 C DEPTq135) and High Resolution MS. All three anthocyanins had the disaccharide lathyrose (2-O-(ß-D-xylopyranosyl)-ß-D-galactopyranoside) attached at the 3-O position. In the tradition of naming novel anthocyanin aglycones based on botanical origin, the new pyranoanthocyanin aglycones have been given the common names Panaxidin A (pelaragonidin-4-vinylcatechol) and Panaxidin B (pelargonidin-4-vinylphenol). Copyright © 2015 Her Majesty the Queen in Right of Canada Magnetic Resonance in Chemistry © 2015 John Wiley & Sons, Ltd.

Prebiotic effects of diet supplemented with the cultivated red seaweed Chondrus crispus or with fructo-oligo-saccharide on host immunity, colonic microbiota and gut microbial metabolites.

BACKGROUND: Gastrointestinal microbial communities are diverse and are composed of both beneficial and pathogenic groups. Prebiotics, such as digestion-resistant fibers, influence the composition of gut microbiota, and can contribute to the improvement of host health. The red seaweed Chondrus crispus is rich in dietary fiber and oligosaccharides, however its prebiotic potential has not been studied to date. METHODS: Prebiotic effects were investigated with weaning rats fed a cultivated C. crispus-supplemented diet. Comparison standards included a fructo-oligo-saccharide (FOS) diet and a basal diet. The colonic microbiome was profiled with a 16S rRNA sequencing-based Phylochip array. Concentrations of short chain fatty acids (SCFAs) in the feacal samples were determined by gas chromatography with a flame ionization detector (GC-FID) analysis. Immunoglobulin levels in the blood plasma were analyzed with an enzyme-linked immunosorbent assay (ELISA). Histo-morphological parameters of the proximal colon tissue were characterized by hematoxylin and eosin (H&E) staining. RESULTS: Phylochip array analysis indicated differing microbiome composition among the diet-supplemented and the control groups, with the C. crispus group (2.5% supplementation) showing larger separation from the control than other treatment groups. In the 2.5% C. crispus group, the population of beneficial bacteria such as Bifidobacterium breve increased (4.9-fold, p=0.001), and the abundance of pathogenic species such as Clostridium septicum and Streptococcus pneumonia decreased. Higher concentrations of short chain fatty acids (i.e., gut microbial metabolites), including acetic, propionic and butyric acids, were found in faecal samples of the C. crispus-fed rats. Furthermore, both C. crispus and FOS supplemented rats showed significant improvements in proximal colon histo-morphology. Higher faecal moisture was noted in the 2.5% C. crispus group, and elevated plasma immunoglobulin (IgA and IgG) levels were observed in the 0.5% C. crispus group, as compared to the basal feed group. CONCLUSIONS: The results suggest multiple prebiotic effects, such as influencing the composition of gut microbial communities, improvement of gut health and immune modulation in rats supplemented with cultivated C. crispus.

Invariant natural killer T (iNKT) cell exhaustion in sarcoidosis.

Invariant natural killer T (iNKT) cells are integral components of immune responses during many chronic diseases, yet their surface phenotypes, subset distribution, and polyfunctional capacity in this environment are largely unknown. Therefore, using flow cytometry, we determined iNKT cell phenotypic and functional characteristics in subjects with chronic inflammatory disease sarcoidosis and matched controls. We found that sarcoidosis subjects displayed lower iNKT-cell frequencies, which correlated with lung fibrosis, C-reactive protein levels, and other measures of clinical disease. The CD4(-) CD8(-) (double negative, DN) iNKT-cell population was selectively lower in diseased individuals and the remaining DN iNKT cells exhibited higher frequencies of the activation markers CD69 and CD56. Functionally, both total IFN-γ(+) and the dual-functional IFN-γ(+) TNF-α(+) iNKT cells were decreased in sarcoidosis subjects and these functional defects correlated with total iNKT-cell circulating frequencies. As the loss of polyfunctionality can reflect functional exhaustion, we measured the surface antigens programmed death-1 receptor and CD57 and found that levels inversely correlated with dual-functional iNKT-cell percentages. These findings reveal that, similar to traditional T cells, iNKT cells may also undergo functional exhaustion, and that circulating iNKT-cell frequencies reflect these defects. Programmed death-1 receptor antagonists may therefore be attractive therapeutic candidates for sarcoidosis and other iNKT-cell-mediated chronic diseases.

Distinguishing Vaccinium species by chemical fingerprinting based on NMR spectra, validated with spectra collected in different laboratories.

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R(2) = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.

Fetoscopic endoluminal tracheal occlusion (FETO) for congenital diaphragmatic hernia in Australia and New Zealand: are we willing, able, both or neither?

AIM: An estimated 140 pregnancies are diagnosed with congenital diaphragmatic hernia (CDH) in Australia and New Zealand each year, with these fetuses having a less than even chance of 1-year survival. Fetoscopic endoluminal tracheal occlusion (FETO) is a relatively new technique that offers a prenatal interventional strategy for selective cases of CDH. This is not routinely offered in Australia or New Zealand. The aim of this systematic review is to critically appraise controlled clinical trials investigating the role of FETO in moderate and severe isolated CDH and explore whether this treatment is justified within our region. METHODS: A systematic literature search of multiple electronic databases was undertaken, with restrictions to human subjects and controlled clinical trials. RESULTS: Nine relevant studies were identified. No current evidence was found in favour of FETO for moderate severity CDH. For severe CDH, the most recent evidence demonstrates significantly improved survival following FETO performed using contemporary percutaneous minimally invasive techniques. Optimum timing for balloon insertion, removal and occlusion duration remains conjectural. Substantial variation in survival rates observed among control groups highlights the impact of post-natal care in prenatally diagnosed CDH. CONCLUSION: Until recently, evidence to support a role for FETO in prenatal CDH management was weak. Recently reported and ongoing controlled trials give cause for optimism, with improved FETO safety and increased survival reported for severe CDH cases. Should Australasia embrace FETO for selected CDH cases, a co-ordinated, evidence-informed service should be established under the guidance of experienced international partnerships.

Helicopter pilot scan techniques during low-altitude high-speed flight.

INTRODUCTION: This study examined pilots' visual scan patterns during a simulated high-speed, low-level flight and how their scan rates related to flight performance. As helicopters become faster and more agile, pilots are expected to navigate at low altitudes while traveling at high speeds. A pilot's ability to interpret information from a combination of visual sources determines not only mission success, but also aircraft and crew survival. METHODS: In a fixed-base helicopter simulator modeled after the U.S. Navy's MH-60S, 17 active-duty Navy helicopter pilots with varying total flight times flew and navigated through a simulated southern Californian desert course. Pilots' scan rate and fixation locations were monitored using an eye-tracking system while they flew through the course. Flight parameters, including altitude, were recorded using the simulator's recording system. RESULTS: Experienced pilots with more than 1000 total flight hours better maintained a constant altitude (mean altitude deviation = 48.52 ft, SD = 31.78) than less experienced pilots (mean altitude deviation = 73.03 ft, SD = 10.61) and differed in some aspects of their visual scans. They spent more time looking at the instrument display and less time looking out the window (OTW) than less experienced pilots. Looking OTW was associated with less consistency in maintaining altitude. DISCUSSION: Results may aid training effectiveness specific to helicopter aviation, particularly in high-speed low-level flight conditions.

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Adaptive tail-length evolution in deer mice is associated with differential Hoxd13 expression in early development.

Variation in the size and number of axial segments underlies much of the diversity in animal body plans. Here we investigate the evolutionary, genetic and developmental mechanisms driving tail-length differences between forest and prairie ecotypes of deer mice (Peromyscus maniculatus). We first show that long-tailed forest mice perform better in an arboreal locomotion assay, consistent with tails being important for balance during climbing. We then identify six genomic regions that contribute to differences in tail length, three of which associate with caudal vertebra length and the other three with vertebra number. For all six loci, the forest allele increases tail length, indicative of the cumulative effect of natural selection. Two of the genomic regions associated with variation in vertebra number contain Hox gene clusters. Of those, we find an allele-specific decrease in Hoxd13 expression in the embryonic tail bud of long-tailed forest mice, consistent with its role in axial elongation. Additionally, we find that forest embryos have more presomitic mesoderm than prairie embryos and that this correlates with an increase in the number of neuromesodermal progenitors, which are modulated by Hox13 paralogues. Together, these results suggest a role for Hoxd13 in the development of natural variation in adaptive morphology on a microevolutionary timescale.

Interferon-inducible chemokines reflect severity and progression in sarcoidosis.

BACKGROUND: Identification of serum proteins that track with disease course in sarcoidosis may have clinical and pathologic importance. We previously identified up-regulated transcripts for interferon-inducible chemokines CXCL9, and CXCL10, in blood of sarcoidosis patients compared to controls. The objective of this study was to determine whether proteins encoded by these transcripts were elevated in serum and identified patients with remitting vs. chronic progressive sarcoidosis longitudinally. METHODS: Serum levels of CXCL9, CXCL10, and proteins associated with inflammation and/or disease activity (sIL2R, ACE, ESR and CRP) were measured in a prospective cohort of sarcoidosis subjects and controls. Comparisons were made between groups and clinical course using pulmonary function measures and a severity score developed by Wasfi et al. RESULTS: In a cross-sectional analysis of 36 non-immunosuppressed sarcoidosis subjects, serum CXCL9, CXCL10, and sIL2R were significantly elevated compared to 46 controls (p < 0.0001). CXCL9 and CXCL10 were strongly inter-correlated (p = 0.0009). CXCL10 and CXCL9 were inversely correlated with FVC% predicted and DLCO% predicted, respectively. CXCL10 and CXCL9 significantly correlated with sarcoidosis severity score. sIL2R, ESR, CRP, and ACE serum levels did not correlate with pulmonary function measures or severity score. In the longitudinal analysis of 26 subjects, changes in serum CXCL10 level over time corresponded with progression versus remission of disease. CONCLUSIONS: Interferon-γ-inducible chemokines, CXCL9 and CXCL10, are elevated in sarcoidosis and inter-correlated with each other. Chemokine levels correlated with measures of disease severity. Serial measurements of CXCL10 corresponded to clinical course.

The Tug1 lncRNA locus is essential for male fertility.

BACKGROUND: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. RESULTS: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. CONCLUSIONS: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals.

Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

Metal exchange in metallothioneins: a novel structurally significant Cd(5) species in the alpha domain of human metallothionein 1a.

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn(2+) ions for Cd(2+) ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human alpha domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the alpha domain of native human metallothionein 1a, was studied. Characteristically this fragment coordinates four Cd(2+) ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd(2+) ion to form a novel Cd(5)alpha-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd(5)alpha species following addition of 5.0 molar equivalents of Cd(2+) to a solution of Cd(4)alpha-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd(5)alpha species in contrast to the derivative-shaped spectrum of the Cd(4)alpha-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd(2+) ions in the Cd(4)alpha species. The (113)Cd chemical shift of the fifth Cd(2+) is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd(2+) ions in Cd(4)alpha-MT by both direct and indirect (113)Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect (1)H-(113)Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd(2+) is not tetrahedral to four thiolates, as is the case with the four Cd(2+) ions in the Cd(4)alpha-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.

The chemical cue tetrabromopyrrole induces rapid cellular stress and mortality in phytoplankton.

Eukaryotic phytoplankton contribute to the flow of elements through marine food webs, biogeochemical cycles, and Earth's climate. Therefore, how phytoplankton die is a critical determinate of the flow and fate of nutrients. While heterotroph grazing and viral infection contribute to phytoplankton mortality, recent evidence suggests that bacteria-derived cues also control phytoplankton lysis. Here, we report exposure to nanomolar concentrations of 2,3,4,5-tetrabromopyrrole (TBP), a brominated chemical cue synthesized by marine γ-proteobacteria, resulted in mortality of seven phylogenetically-diverse phytoplankton species. A comparison of nine compounds of marine-origin containing a range of cyclic moieties and halogenation indicated that both a single pyrrole ring and increased bromination were most lethal to the coccolithophore, Emiliania huxleyi. TBP also rapidly induced the production of reactive oxygen species and the release of intracellular calcium stores, both of which can trigger the activation of cellular death pathways. Mining of the Ocean Gene Atlas indicated that TBP biosynthetic machinery is globally distributed throughout the water column in coastal areas. These findings suggest that bacterial cues play multiple functions in regulating phytoplankton communities by inducing biochemical changes associated with cellular death. Chemically-induced lysis by bacterial infochemicals is yet another variable that must be considered when modeling oceanic nutrient dynamics.