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.

A New Era of Morphological Investigations: Reviewing Methods for Comparative Anatomical Studies.

The increased use of imaging technology in biological research has drastically altered morphological studies in recent decades and allowed for the preservation of important collection specimens alongside detailed visualization of bony and soft-tissue structures. Despite the benefits associated with these newer imaging techniques, there remains a need for more "traditional" methods of morphological examination in many comparative studies. In this paper, we describe the costs and benefits of the various methods of visualizing, examining, and comparing morphological structures. There are significant differences not only in the costs associated with these different methods (monetary, time, equipment, and software), but also in the degree to which specimens are destroyed. We argue not for any one particular method over another in morphological studies, but instead suggest a combination of methods is useful not only for breadth of visualization, but also for the financial and time constraints often imposed on early-career research scientists.

Pulmonary anatomy and a case of unilateral aplasia in a common snapping turtle (Chelydra serpentina): developmental perspectives on cryptodiran lungs.

The common snapping turtle (Chelydra serpentina) is a well studied and broadly distributed member of Testudines; however, very little is known concerning developmental anomalies and soft tissue pathologies of turtles and other reptiles. Here, we present an unusual case of unilateral pulmonary aplasia, asymmetrical carapacial kyphosis, and mild scoliosis in a live adult C. serpentina. The detailed three-dimensional (3D) anatomy of the respiratory system in both the pathological and normal adult C. serpentina, and a hatchling are visualized using computed tomography (CT), microCT, and 3D digital anatomical models. In the pathological turtle, the right lung consists of an extrapulmonary bronchus that terminates in a blind stump with no lung present. The left lung is hyperinflated relative to the normal adult, occupying the extra coelomic space facilitated by the unusual mid-carapacial kyphotic bulge. The bronchial tree of the left lung retains the overall bauplan of the normal specimens, with some minor downstream variation in the number of secondary airways. The primary difference between the internal pulmonary structure of the pathological individual and that of a normal adult is a marked increase in the surface area and density of the parenchymal tissue originating from the secondary airways, a 14.3% increase in the surface area to volume ratio. Despite this, the aplasia has not had an impact upon the ability of the turtle to survive; however, it did interfere with aquatic locomotion and buoyancy control under water. This turtle represents a striking example of a non-fatal congenital defect and compensatory visceral hypertrophy.

Effects of intravenous and subcutaneous heartworm homogenate from doxycycline-treated and untreated donor dogs on bronchial reactivity and lung in cats.

A controlled, blind research study was conducted to define the innate response of lungs in specific pathogen free (SPF) cats to intravenous (n=10) or subcutaneous (n=4) administration of homogenate of adult Dirofilaria immitis from donor dogs compared with lung response in control cats (n=6). There was no difference in cats that received heartworm homogenate IV for 18 days from donor dogs treated with doxycycline for 1 month compared with cats given heartworm homogenate from untreated donor dogs. Cats did not develop clinical signs, and no radiographic changes were noted. Cats given SC heartworm homogenate at lower concentration than IV groups did not develop histologic changes. Cats that received IV heartworm homogenate for 18 days developed mild interstitial and peribronchial myofibrocyte proliferation and smooth muscle proliferation of the pulmonary arteries. Bronchial ring contractility in vitro was blunted in the IV homogenate cats to the agonists acetylcholine and 5-hydroxytryptamine. Cats in the SC group had increased sensitivity to histamine at high concentrations but normal contractility and relaxation responses to other agonists. No increase in mast cells was noted in lung tissues of cats given homogenate. In the absence of bronchial wall remodeling, cats given IV homogenate had blunted responses to bronchial constriction, but normal relaxation to nitroprusside and substance P and increased sensitivity to histamine. In the absence adult heartworms, the homogenate of adult heartworms in the circulation of SPF cats induced a direct effect on lung parenchyma and altered bronchial ring reactivity.

Effect of pre-cardiac and adult stages of Dirofilaria immitis in pulmonary disease of cats: CBC, bronchial lavage cytology, serology, radiographs, CT images, bronchial reactivity, and histopathology.

A controlled, blind study was conducted to define the initial inflammatory response and lung damage associated with the death of precardiac stages of Dirofilaria immitis in cats as compared to adult heartworm infections and normal cats. Three groups of six cats each were used: UU: uninfected untreated controls; PreS I: infected with 100 D. immitis L3 by subcutaneous injection and treated topically with selamectin 32 and 2 days pre-infection and once monthly for 8 months); IU: infected with 100 D. immitis L3 and left untreated. Peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected from all cats on Days 0, 70, 110, 168, and 240. CT images were acquired on Days 0, 110, and 240. Cats were euthanized, and necropsies were conducted on Day 240 to determine the presence of heartworms. Bronchial rings were collected for in vitro reactivity. Lung, heart, brain, kidney, and liver tissues were collected for histopathology. Results were compared for changes within each group. Pearson and Spearman correlations were performed for association between histologic, radiographic, serologic, hematologic and bronchoalveolar lavage (BAL) results. Infected cats treated with selamectin did not develop radiographically evident changes throughout the study, were heartworm antibody negative, and were free of adult heartworms and worm fragments at necropsy. Histologic lung scores and CT analysis were not significantly different between PreS I cats and UU controls. Subtle alveolar myofibrosis was noted in isolated areas of several PreS I cats and an eosinophilic BAL cytology was noted on Days 75 and 120. Bronchial ring reactivity was blunted in IU cats but was normal in PreS I and UU cats. The IU cats became antibody positive, and five cats developed adult heartworms. All cats with heartworms were antigen positive at one time point; but one cat was antibody positive, antigen negative, with viable adult females at necropsy. The CT revealed early involvement of all pulmonary arteries and a random pattern of parenchymal disease with severe lesions immediately adjacent to normal areas. Analysis of CT 3D reconstruction and Hounsfield units demonstrated lung disease consistent with restrictive pulmonary fibrosis with an interstitial infiltrate, absence of air trapping, and decrease in total lung volume in Group IU as compared to Groups UU and PreS I. The clinical implications of this study are that cats pretreated with selamectin 1 month before D. immitis L3 infection did not become serologically positive and did not develop pulmonary arterial hypertrophy and myofibrosis.

Disparity and convergence in bipedal archosaur locomotion.

This study aims to investigate functional disparity in the locomotor apparatus of bipedal archosaurs. We use reconstructions of hindlimb myology of extant and extinct archosaurs to generate musculoskeletal biomechanical models to test hypothesized convergence between bipedal crocodile-line archosaurs and dinosaurs. Quantitative comparison of muscle leverage supports the inference that bipedal crocodile-line archosaurs and non-avian theropods had highly convergent hindlimb myology, suggesting similar muscular mechanics and neuromuscular control of locomotion. While these groups independently evolved similar musculoskeletal solutions to the challenges of parasagittally erect bipedalism, differences also clearly exist, particularly the distinct hip and crurotarsal ankle morphology characteristic of many pseudosuchian archosaurs. Furthermore, comparative analyses of muscle design in extant archosaurs reveal that muscular parameters such as size and architecture are more highly adapted or optimized for habitual locomotion than moment arms. The importance of these aspects of muscle design, which are not directly retrievable from fossils, warns against over-extrapolating the functional significance of anatomical convergences. Nevertheless, links identified between posture, muscle moments and neural control in archosaur locomotion suggest that functional interpretations of osteological changes in limb anatomy traditionally linked to postural evolution in Late Triassic archosaurs could be constrained through musculoskeletal modelling.

Skin penetration of minerals in psoriatics and guinea-pigs bathing in hypertonic salt solutions.

Penetration of electrolytes through the human skin was measured in healthy volunteers and in psoriatic patients after bathing in the Dead-Sea or in simulated bath-salt solutions. Significant increases in the levels of serum Br, Rb, Ca and Zn were noticed only in the psoriatic patients after daily bathing in the Dead-Sea for a 4-week regimen. Guinea-pigs "bathed" in simulated Dead-Sea bath-salt solutions containing radionuclides of Ca, Mg, K and Br. Traces of each radionuclide were detected in the blood and in some internal organs after 60 minutes of bathing. The radionuclides showed a physiological pattern in their organ distribution. Even though the whole investigation was carried out in hypertonic solutions, there is a definite penetration of salts through healthy (human and guinea-pigs) and damaged (psoriatic) epidermis. This finding suggests that improvement of the psoriatic condition after bathing in the Dead-Sea, may be partly attributed (in addition to ultraviolet irradiation) to the minerals' effect on the psoriatic skin.

Ruthenium-97 hepatobiliary agents for delayed studies of the biliary tract. I. ru-97 PIPIDA: concise communication.

Failure of early diagnosis of biliary atresia results in the development of cirrhosis and death. Commonly used hepatobiliary agents are not ideal for follow-up studies because of their unfavorable physical properties or short half-life. The excellent physical properties of Ru-97 should overcome these limitations. Therefore, Ru-97 PIPIDA (N,alpha-(p-isopropyl acetanilide) iminoacetic acid) is being investigated as a potential hepatobiliary agent that would allow an improved diagnosis of the disease. Ruthenium-97 PIPIDA and Tc-99m PIPIDA showed similar blood clearance rates in dogs. Ru-97 PIPIDA scintigrams in dogs showed early uptake in liver and gallbladder and slow excretion through the gastrointestinal tract. Biodistribution studies were performed in normal rats and rats with biliary obstruction. The findings suggest that Ru-97 PIPIDA should be useful for delayed studies (1-3 days) of the biliary tract.

Ruthenium-97 DTPA: a new radiopharmaceutical for cisternography.

Ruthenium-97 DTPA (diethylenetriamine penta-acetic acid) was evaluated for its possible use as a cerebrospinal fluid imaging agent. Ru-97 has favorable physical properties that are highly suitable for imaging: decay by electron capture; gamma energy = 216 keV, 85%; T 1/2 = 2.9 days. Dogs were injected with 0.4 mCi Ru-97 DTPA or In-111 DTPA into the cisterna magna. The movement of the agents was monitored with a camera interfaced to a computer, or with a dual-probe system placed over the head and urinary bladder. In addition, blood and urine samples were collected at fixed intervals for 6 hr. High-quality images were obtained up to 48 hr after injection. The results show that the kinetics and excretion of Ru-97 DTPA are similar to those of In-111 DTPA. Radiation dose for identical activities is twice as high for In-111, in part because of greater abundance of the low-energy electron emission of In-111.

Liver scanning for amyloid.

Subsequent liver scans with both 99mTc-colloid and 99mTc-diphosphonate in a patient with biopsy proven liver amyloidosis are described. The colloid liver scans demonstrated the variable pattern which can be obtained at different stages of the disease, whereas, the absence of Tc-diphosphonate uptake, by the infiltrated liver, did not confirm previous suggestions about the amyloid seeking properties of this material.

Carcinoma of the bronchus with atypical presentation: report of a case.

A 64-year-old man presented with dysphagia due to obstruction of lower third of the esophagus. Esophagoscopy showed narrowing of the esophagus with normal mucosa. Chest X rays were normal, but on lung scan there was no perfusion of the left lung. That patient died of aspiration and on post mortem a carcinoma of the bronchus with abscess formation and perforation into the esophagus was found.