Sample Size and Geometric Morphometrics Methodology Impact the Evaluation of Morphological Variation.

Geometric morphometrics has had a profound impact on our understanding of morphological evolution. However, factors such as sample size and the views and elements selected for two-dimensional geometric morphometric (2DGM) analyses, which are often dictated by specimen availability and time rather than study design, may affect the outcomes of those analyses. Leveraging large intraspecific sample sizes (n > 70) for two bat species, Lasiurus borealis and Nycticeius humeralis, we evaluate the impact of sample size on calculations of mean shape, shape variance, and centroid size. Additionally, we assessed the concordance of multiple skull 2D views with one another and characterized morphological variation in skull shape in L. borealis and N. humeralis, as well as a closely related species, Lasiurus seminolus. Given that L. seminolus is a morphologically cryptic species with L. borealis, we assessed whether differences in skull shape and in 2DGM approach would allow species discrimination. We found that reducing sample size impacted mean shape and increased shape variance, that shape differences were not consistent across views or skull elements, and that trends shown by the views and elements were not all strongly associated with one another. Further, we found that L. borealis and L. seminolus were statistically different in shape using 2DGM in all views and elements. These results underscore the importance of selecting appropriate sample sizes, 2D views, and elements based on the hypothesis being tested. While there is likely not a generalizable sample size or 2D view that can be employed given the wide variety of research questions and systems evaluated using 2DGM, a generalizable solution to issues with 2DGM presented here is to run preliminary analyses using multiple views, elements, and sample sizes, thus ensuring robust conclusions.

A morfometria geométrica teve um impacto profundo na compreensão da evolução morfológica. No entanto, fatores como o tamanho amostral, vista anatômica e os elementos selecionados para as análises de morfometria geométricas bidimensionais (MG2D), que geralmente são determinados pela disponibilidade de espécimes e de tempo ao invés do design de estudo, podem afetar os resultados dessas análises. Utilizando grandes tamanhos de amostra intraespecífica (n > 70) em duas espécies de morcegos, Lasiurus borealis e Nycticeius humeralis, avaliamos o impacto do tamanho amostral nos cálculos da média e da variância da forma, e do tamanho do centroide. Adicionalmente, avaliamos a concordância entre vários planos 2D do crânio e caracterizamos a variação morfológica da forma cranial em L. borealis e N. humeralis, bem como em uma outra espécie proximamente relacionada, Lasiurus seminolus. Dado que L. seminolus é uma espécie morfologicamente críptica em relação a L. borealis, avaliamos se as diferenças no formato do crânio e na abordagem de MG2D utilizada permitiriam discriminar as espécies. Descobrimos que a redução do tamanho da amostra impactou o formato médio e aumentou a variância da forma, que as diferenças na forma não foram consistentes entre as vistas ou elementos cranianos, e que as tendências apresentadas pelos planos anatômicos e pelos elementos não foram fortemente associadas umas às outras. Adicionalmente, constatamos que as formas de L. borealis e L. seminolus, quantificadas por MG2D, foram estatisticamente diferentes em todas as vistas e elementos. Esses resultados enfatizam a importância em selecionar tamanhos amostrais, vistas 2D e elementos anatômicos adequados, com base na hipótese testada. Devido à grande diversidade de perguntas de pesquisa e de sistemas avaliados com MG2D, provavelmente não exista um tamanho amostral ou vista 2D que possam ser aplicados de forma generalizada. No entanto, uma solução geral para os problemas envolvendo MG2D aqui apresentados é conduzir análises preliminares utilizando várias vistas anatômicas, elementos e tamanhos amostrais, garantindo assim conclusões mais robustas.

Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.

Quasi-static and harmonic indentation of osteonal bone.

UNLABELLED: The purpose of the study was to compare Quasi-Static (QS) and harmonic (CSM) methods of indentation testing. Bone sections were obtained from mid-femoral diaphyses of dogs which received a pair of calcein labels. Labeled (n = 35) and unlabeled (n = 112) osteons were identified. Indentation modulus (IM) and hardness (H) for the CSM method were collected during the entire loading cycle to peak depth, while IM and H for QS method were calculated at a peak depth of 500 nm. RESULTS: The mean (SD) of the IM and H for labeled osteons were as follows: QS IM = 15.3 GPa (3.85) versus CSM IM = 14.7 GPa (3.58); P = .52 and QS H = .39 GPa (.171) versus CSM H = .42 GPa (.146); P = .32. The mean (SD) of the IM and H for unlabeled osteons were as follows: QS IM = 21.5 GPa (2.80) versus CSM IM = 20.6 GPa (2.53); P = .054 and QS H = .64 GPa (.117) versus CSM H = .70 GPa (.120); P = .017. There was no difference in IM and H for the two methods, except for H of the unlabeled osteons. In addition, for the CSM method, IM at 100 nm, 200 nm, 300 nm, 400 nm and 500 nm were not statistically significant different (P = .06). Bone is viscoelastic at an organ level. However, this component of its behavior was not detected at the length scale examined.

Comparison of hand-traced and computer-based cephalometric superimpositions.

OBJECTIVE: To determine the ability to produce comparable superimpositions using hand tracing and digital methods (Dolphin v10). In addition, if the two methods were comparable, we wanted to determine if a difference existed between the best-fit cranial base superimposition and S-N superimpositions using the digital method. METHODS AND MATERIALS: Sixty-four initial (T(1)) and final (T(2)) cephalometric film radiographs were obtained. Cranial base and regional superimpositions were completed independently for each pair of radiographs by either hand tracing and digital methods. To quantitatively evaluate the differences between the two methods, the hand and digital superimpositions were digitized to obtain x-y coordinates of routine cephalometric landmarks at T(2). Linear distance between multiple corresponding (hand and digital) T(2) cephalometric landmark locations (e.g., A point) were measured and defined as the T(2) landmark distance (T(2) LD). Additionally, 61 patient records were used to compare the digital method for best-fit cranial base superimpositions versus S-N superimpositions. A Friedman test was applied to examine for differences. RESULTS: The upper 95% confidence limit for the mean of the T(2) LD for hand and digital superimposition methods was <1 mm for all landmarks except maxillary incisor tip and apex. The upper 95% confidence interval for best-fit vs S-N was >1 mm for most landmarks. CONCLUSION: This study validates the use of superimpositions produced by Dolphin Imaging version 10 and is a necessary step forward toward widespread acceptance of digital superimpositions.

Polycyclic aromatic hydrocarbons with bay-like regions inhibited gap junctional intercellular communication and stimulated MAPK activity.

Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of research has been devoted to predicting the genotoxic, tumor-initiating potential of PAHs based on chemical structure. However, information on the correlation of structure with the non-genetoxic, epigenetic events of tumor promotion is sparse. PAHs containing a bay or bay-like region were shown to be potent inhibitors of gap-junctional intercellular communication (GJIC), an epigenetic event involved in the removal of an initiated cell from growth suppression. We tested the epigenetic toxicity of PAHs containing bay-like regions by comparing the effects of methylated vs. chlorinated isomers of anthracene on the temporal activation of mitogen-activated protein kinase (MAPK) and the regulation of GJIC. Specifically, we used anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9,10-dimethylanthracene, 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. We determined the effect of these compounds on GJIC and on the activation of extracellular receptor kinase (ERK 1 and 2), a MAPK, in F344 rat liver epithelial cells. Results showed that bay or bay-like regions, formed by either chlorine or a methyl group, reversibly inhibited GJIC at the same doses, time, and time of recovery, whereas the linear-planar isomers had no effect on GJIC. Similarly, the GJIC-inhibitory isomers also induced the phosphorylation of ERK 1 and ERK 2, while the non-inhibitory isomers had no effect on the activation of these MAPKs. MAPK activation occurred 10-20 min after the inhibition of GJIC, which indicates that MAPK is not involved in the initial regulation of GJIC; instead altered GJIC may be affecting MAPK activation. The present study revealed that there are structural determinants of PAHs, which clearly affect epigenetic events known to be involved in the non-genetoxic steps of tumor promotion. These events are the release of a cell from growth suppression involving the reduction of GJIC, followed by the activation of intracellular mitogenic events.

Bay or baylike regions of polycyclic aromatic hydrocarbons were potent inhibitors of Gap junctional intercellular communication.

Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens, and a considerable amount of research has been devoted to predicting the tumor-initiating potential of PAHs based on chemical structure. However, there has been little research into the effects of PAHs on the epigenetic events of tumor promotion and no structural correlation has been made thereof. Gap junctional intercellular communication (GJIC) activity was used in this study as an epigenetic biomarker to determine the structure-activity relationships of twelve different PAHs. The PAHs used were naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9, 10-dimethylanthracene, phenanthrene, fluorene, 1-methylfluorene, and fluoranthene. Results showed that PAHs containing bay or baylike regions inhibited GJIC more than did the linear PAHs. The nonnaphthalene PAHs were not cytotoxic as determined by a vital dye uptake assay, but the naphthalene compounds were cytotoxic at the higher doses, indicating that the down regulation of GJIC by these naphthalenes could be a consequence of general membrane damage. Inhibition of GJIC by all the inhibitory PAHs was reversed when the cells were refreshed with PAH-free growth medium. Inhibition of GJIC occurred within 0.5-5 min and correlated with the aqueous solubility of the PAHs. The present study revealed that there are structural determinants of epigenetic toxicity as determined by GJIC activity.

The effects of anthracene and methylated anthracenes on gap junctional intercellular communication in rat liver epithelial cells.

Polycyclic aromatic hydrocarbons (PAHs), many of which are known carcinogens, are derived from the pyrolysis of organic materials. A rich source of PAHs is cigarette smoke, which contains methylated anthracenes and phenanthrenes as the predominant PAHs. The tumor-promoting activity of cigarette smoke has been well documented. The down-regulation of gap junction intercellular communication (GJIC) by nongenotoxic chemicals and several oncogenes has been implicated in tumor promotion. Therefore, we determined the effects of the three isomers of methylanthracene on GJIC in WB-F344 rat liver epithelial cells. Anthracene and 2-methylanthracene did not significantly inhibit GJIC, whereas anthracene methylated in the 1 or 9 position reversibly inhibited GJIC with I50 values of 22 and 36 microM, respectively. Inhibition occurred within 15 min. In conclusion, the biological effect of methylanthracene depends on the ring position of the methyl group, and these inhibitory isomers could play a potential role in tumor promotion of methylated PAH-rich mixtures such as cigarette smoke and crude oil products.

The use of market research to design healthcare offerings. A case study of a senior citizen community.

With a growing elderly population, there is little argument that the healthcare system in the United States must understand the needs and wants of its elderly consumers. This is especially important in a rural community where services can be limited and access to these services is difficult for consumers. Marketing research is one way in which rural healthcare facilities can gain market information not only to enhance their product offerings, but also to ensure that proper and sufficient services are provided. This article presents a case study of a long-term healthcare facility using marketing research.