Inverse method based on 3D nonlinear physically constrained minimisation in the framework of traction force microscopy.

Traction force microscopy is a methodology that enables to estimate cellular forces from the measurement of the displacement field of an extracellular matrix (ECM)-mimicking hydrogel that a cell is mechanically interacting with. In this paper, a new inverse and physically-consistent methodology is developed and implemented in the context of 3D nonlinear elasticity. The proposed method searches for a displacement field that approximates the measured one, through the imposition of fulfillment of equilibrium with real and known forces acting in the hydrogel. The overall mathematical formulation leads to a constrained optimisation problem that is treated through a Lagrange operator and that is solved numerically by means of a nonlinear finite element framework. In order to illustrate the potential and enhanced accuracy of the proposed inverse method, it is applied to a total of 5 different real cases of cells cultured in a 3D hydrogel that is considered to behave as a nonlinear elastic material. Different error indicators are defined in order to compare ground truth simulated displacements and tractions to the ones recovered by the new inverse as well as by the forward method. Results indicate that the evaluation of displacement gradients leads to errors, in terms of recovered tractions, that are more than three times lower (on average) for the inverse method compared to the forward method. They highlight the enhanced accuracy of the developed methodology and the importance of appropriate inverse methods that impose physical constraints to traction and stress recovery in the context of traction force microscopy.

Oviductal Proteins Effect in Rooster Spermatic Cryopreservation.

BACKGROUND: Cryopreservation induces spermatic cryo capacitation, which can decrease thawed sperm fertilizing capability. OBJECTIVE: To evaluate the effect of uterus-vaginal union protein factors to inhibit sperm cryo capacitation and maintain viability and fertilizing capability of rooster spermatozoa. MATERIALS AND METHODS: Rooster spermatozoa was cryopreserved using Lake extender supplemented with different hen's uterus-vaginal junction protein concentrations, to determine spermatic viability, sperm physiological condition and fertilizing capability in vivo. RESULTS: It was possible to induce spermatic decapacitation in vitro, inhibiting cryo capacitation and allowing fertility results comparable to those obtained with fresh semen. CONCLUSION: Uterus-vaginal protein extracts induce spermatic decapacitation in vitro.

Changes in the membrane carbohydrates from sperm cryopreserved with dimethylsulfoxide or polyvinylpyrrolidone of red-tailed hawk (Buteo jamaicencis).

BACKGROUND: That cryopreservation can induce alterations in sperm. OBJECTIVE: The goal of this study was to evaluate sperm quality and distribution of N-acetylglucosamine, sialic acid and mannose residues in sperm cryopreserved of red-tailed hawk (Buteo jamaicensis). MATERIALS AND METHODS: We studied twenty samples of ejaculated semen for each cryoprotectant dimethylsulfoxide or polyvinylpyrrolidone. Carbohydrate identification was carried out with lectins Triticum vulgaris agglutinin to N-acetylglucosamine and sialic acid and Concanavalia ensiformis for mannose residues. Sperm viability was not altered but motility decreased significantly with both crioprotectants compared with fresh sperm. RESULTS: Neither the number of WGA positive sperm nor the distribution of N-acetylglucosamine and/or sialic acid residues were affected by the cryopreservation procedure. The sperm proportion with fluorescence associated with the presence of mannose residues was higher in thawed sperm. CONCLUSION: Values obtained with the cryopreservation technique proposed in this study by freezing drops in liquid nitrogen, were within normal parameters established for good quality fresh semen. We can say that it can be used for assisted reproduction of Buteo jamaicensis.

An in silico study on the influence of extracellular matrix mechanics on vasculogenesis.

BACKGROUND AND OBJECTIVES: Blood vessels form a network of capillaries throughout the body that perform essential functions for life. Vasculogenesis, i.e. the formation of new blood vessels, is regulated by many factors, biochemical ones being among the most important. However, others such as the biomechanical influence on shape, organization and structure of vessel networks require further investigation. In this paper, we develop a 3D agent-based mechanobiological model of vasculogenesis with the aim of analyzing how the mechanics of the extracellular matrix (ECM) affects vasculogenesis. METHODS: For this purpose, we consider a growing domain composed of different cells: tip cells, which are the driving cells located at the end of the vessels and stalk cells, which are found in the interior of the vascular network. ECM is considered as particles (agents) that surround the growth of the vascular network. Depending on the cell type, different sets of forces are considered, such as chemotactic, mechanical, random and viscoelastic forces among others. RESULTS: The growth of the network is iteratively analyzed and updated at each time step based on a mechanically-driven proliferation rule. The influence of different biomechanical factors, such as ECM stiffness or viscoelasticity are explored through in silico simulations. A number of indicators are defined along the algorithm, like number of cells, branches, tortuosity and anisotropy, in order to compare topological differences of the vascular network during vasculogenesis under different ECM conditions. The obtained results are qualitatively compared with other related works in the literature. CONCLUSIONS: The present study sheds some light and partially explain, from an in silico perspective, the role of ECM mechanics on vasculogenesis. The main conclusions of this work are: (i) increased stiffness increases proliferation, (ii) the network tends to migrate towards stiffer areas, and (iii) increased viscoelasticity decreases proliferation.

Mercury speciation in mine tailings amended with biochar: Effects on mercury bioavailability, methylation potential and mobility.

Biochar is a low-cost and environmentally friendly amendment with strong ability for adsorption of mercury (Hg) from aqueous solutions, contaminated soils, and sediments. In the present study, six biochars were prepared from the pyrolysis of cocoa pod husk, sugarcane bagasse and banana pseudostem at 400 and 600 °C in order to use them as an organic amendment and to evaluate their capacities to reduce the bioavailability, methylation potential, and mobility of Hg present in mine tailings without environmental treatment. To quantify the effects of each variety of biochar, incubation experiments of soil were established by mixing mine tailings with 5% by weight of biochar for 90 days. Once the incubation time concluded, sequential extraction procedures were carried out to determine the fractionation of the Hg species. Speciation analysis results indicated that the remedial effects of biochar depended on the source of organic matter and pyrolysis temperature. The bioavailable and organic Hg fractions decreased respectively by up to 75 and 79%, indicating a methylation potential reduction. Immobile Hg fraction increased to 76% with respect to the control. Adsorption and stabilization to HgS from the soluble forms of Hg reduce the percentage of bioavailable Hg. The organic Hg fraction reduction was correlated with the decrease of the bioavailable Hg fraction and with direct adsorption processes in the biochar structure. Highly porous biochars developed at high temperature, with large contents of superficial polar functional groups (H/C), and high pH, electrical conductivity, ash percentage and cation exchange capacity values favor the stabilization and adsorption of Hg in mine tailings. In summary, the application of biochar could be an effective method for the remediation of Hg-contaminated mine tailings, transforming the Hg species into less toxic, soluble, reactive, and bioavailable forms.

Advanced in silico validation framework for three-dimensional traction force microscopy and application to an in vitro model of sprouting angiogenesis.

In the last decade, cellular forces in three-dimensional hydrogels that mimic the extracellular matrix have been calculated by means of Traction Force Microscopy (TFM). However, characterizing the accuracy limits of a traction recovery method is critical to avoid obscuring physiological information due to traction recovery errors. So far, 3D TFM algorithms have only been validated using simplified cell geometries, bypassing image processing steps or arbitrarily simulating focal adhesions. Moreover, it is still uncertain which of the two common traction recovery methods, i.e., forward and inverse, is more robust against the inherent challenges of 3D TFM. In this work, we established an advanced in silico validation framework that is applicable to any 3D TFM experimental setup and that can be used to correctly couple the experimental and computational aspects of 3D TFM. Advancements relate to the simultaneous incorporation of complex cell geometries, simulation of microscopy images of varying bead densities and different focal adhesion sizes and distributions. By measuring the traction recovery error with respect to ground truth solutions, we found that while highest traction recovery errors occur for cases with sparse and small focal adhesions, our implementation of the inverse method improves two-fold the accuracy with respect to the forward method (average error of 23% vs. 50%). This advantage was further supported by recovering cellular tractions around angiogenic sprouts in an in vitro model of angiogenesis. The inverse method recovered higher traction peaks and a clearer pulling pattern at the sprout protrusion tips than the forward method. STATEMENT OF SIGNIFICANCE: Biomaterial performance is often studied by quantifying cell-matrix mechanical interactions by means of Traction Force Microscopy (TFM). However, 3D TFM algorithms are often validated in simplified scenarios, which do not allow to fully assess errors that could obscure physiological information. Here, we established an advanced in silico validation framework that mimics real TFM experimental conditions and that characterizes the expected errors of a 3D TFM workflow. We apply this framework to demonstrate the enhanced accuracy of a novel inverse traction recovery method that is illustrated in the context of an in vitro model of sprouting angiogenesis. Together, our study shows the importance of a proper traction recovery method to minimise errors and the need for an advanced framework to assess those errors.

Data-Driven Computational Simulation in Bone Mechanics.

The data-driven approach was formally introduced in the field of computational mechanics just a few years ago, but it has gained increasing interest and application as disruptive technology in many other fields of physics and engineering. Although the fundamental bases of the method have been already settled, there are still many challenges to solve, which are often inherently linked to the problem at hand. In this paper, the data-driven methodology is applied to a particular problem in tissue biomechanics, a context where this approach is particularly suitable due to the difficulty in establishing accurate and general constitutive models, due to the intrinsic intra and inter-individual variability of the microstructure and associated mechanical properties of biological tissues. The problem addressed here corresponds to the characterization and mechanical simulation of a piece of cortical bone tissue. Cortical horse bone tissue was mechanically tested using a biaxial machine. The displacement field was obtained by means of digital image correlation and then transformed into strains by approximating the displacement derivatives in the bone virtual geometric image. These results, together with the approximated stress state, assumed as uniform in the small pieces tested, were used as input in the flowchart of the data-driven methodology to solve several numerical examples, which were compared with the corresponding classical model-based fitted solution. From these results, we conclude that the data-driven methodology is a useful tool to directly simulate problems of biomechanical interest without the imposition (model-free) of complex spatial and individually-varying constitutive laws. The presented data-driven approach recovers the natural spatial variation of the solution, resulting from the complex structure of bone tissue, i.e. heterogeneity, microstructural hierarchy and multifactorial architecture, making it possible to add the intrinsic stochasticity of biological tissues into the data set and into the numerical approach.

Effect of Chronic Consumption of Sweeteners on Microbiota and Immunity in the Small Intestine of Young Mice.

The consumption of sweeteners has increased as a measure to reduce the consumption of calories and thus combat obesity and diabetes. Sweeteners are found in a large number of products, so chronic consumption has been little explored. The objective of the study was to evaluate the effect of chronic sweetener consumption on the microbiota and immunity of the small intestine in young mice. We used 72 CD1 mice of 21 days old, divided into 3 groups: (i) No treatment, (ii) Group A (6 weeks of treatment), and (iii) Group B (12 weeks of treatment). Groups A and B were divided into 4 subgroups: Control (CL), Sucrose (Suc), Splenda® (Spl), and Svetia® (Sv). The following were determined: anthropometric parameters, percentage of lymphocytes of Peyer's patches and lamina propria, IL-6, IL-17, leptin, resistin, C-peptide, and TNF-α. From feces, the microbiota of the small intestine was identified. The BMI was not modified; the mice preferred the consumption of Splenda® and Svetia®. The percentage of CD3+ lymphocytes in Peyer's patches was increased. In the lamina propria, Svetia® increased the percentage of CD3+ lymphocytes, but Splenda® decreases it. The Splenda® and Svetia® subgroups elevate leptin, C-peptide, IL-6, and IL-17, with reduction of resistin. The predominant genus in all groups was Bacillus. The chronic consumption of sweeteners increases the population of lymphocytes in the mucosa of the small intestine. Maybe, Bacillus have the ability to adapt to sweeteners regardless of the origin or nutritional contribution of the same.

Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries.

OBJECTIVES: Sample preparation for high-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences. METHODS: A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination. RESULTS: Among sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components. CONCLUSIONS: We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.

In silico dynamic characterization of the femur: Physiological versus mechanical boundary conditions.

It is established that bone tissue adapts and responds to mechanical loading. Several studies have suggested an existence of positive influence of vibration on the bone mass maintenance. Thus, some bone regeneration therapies are based on vibration of bone tissue under circumstances of disease to stimulate its formation. Frequency of loading should be properly selected and therefore a correct characterization of the dynamic properties of this tissue may be critical for the success of such orthopedic techniques. On the other hand, many studies implement vibration techniques with in silico models. Numerical results are exclusively dependent on properties of bone tissue, i.e. geometry, density distribution and stiffness, as well as boundary conditions. In the present study, the influence of boundary conditions and material properties on the dynamic characteristics of bone tissue was explored in a human femur. Bone shape and density were directly reconstructed from computer tomographies, whereas natural frequencies and modes of vibration were obtained for different boundary conditions including physiological and mechanical ones. Results of this study show the moderate effect of material properties compared to the much substantial effect of boundary conditions. A factor of 2 in the natural frequency was obtained depending on imposed boundary conditions, highlighting the importance in the selection of appropriate conditions in the analysis of the bone organ.

In silico design of magnesium implants: Macroscopic modeling.

Magnesium-based biomedical implants offer many advantages versus traditional ones although some challenges are still present. In this context, mathematical modeling and computational simulation may be a useful and complementary tool to evaluate in silico the performance of magnesium biomaterials under different conditions. In this paper, a phenomenologically-based model to simulate magnesium corrosion is developed. The model describes the physico-chemical interactions and evolution of species present in this phenomenon. A set of 7 species is considered in the model, which allows to simulate hydrogen release, pH evolution, corrosion products formation as well as degradation of magnesium. The model is developed under the continuum media theory and is implemented in a finite element framework. In the results section, the effect of model parameters on outcomes is firstly explored. Second, model results are qualitative validated versus two examples of application found in the literature. Two main conclusions are derived from this work: (i) the model captures well the experimental trends and allows to analyze the main variables present in magnesium corrosion, (ii) even though further validation is needed the model may be a useful standard in the design of degradable metal implants.

Model of dissolution in the framework of tissue engineering and drug delivery.

Dissolution phenomena are ubiquitously present in biomaterials in many different fields. Despite the advantages of simulation-based design of biomaterials in medical applications, additional efforts are needed to derive reliable models which describe the process of dissolution. A phenomenologically based model, available for simulation of dissolution in biomaterials, is introduced in this paper. The model turns into a set of reaction-diffusion equations implemented in a finite element numerical framework. First, a parametric analysis is conducted in order to explore the role of model parameters on the overall dissolution process. Then, the model is calibrated and validated versus a straightforward but rigorous experimental setup. Results show that the mathematical model macroscopically reproduces the main physicochemical phenomena that take place in the tests, corroborating its usefulness for design of biomaterials in the tissue engineering and drug delivery research areas.

Effect of Supplementation with n-3 Fatty Acids Extracted from Microalgae on Inflammation Biomarkers from Two Different Strains of Mice.

BACKGROUND: Diabetes mellitus is considered a chronic noncommunicable disease in which inflammation plays a main role in the progression of the disease and it is known that n-3 fatty acids have anti-inflammatory properties. One of the most recent approaches is the study of the fatty acids of microalgae as a substitute for fish oil and a source rich in fatty acids EPA and DHA. OBJECTIVE: To analyze the effect of supplementation with n-3 fatty acids extracted from microalgae on the inflammatory markers from two different strains of mice. METHODS: Mice of two strains, db/db and CD1, were supplemented with n-3 fatty acids extracted from microalgae in lyophilized form and added to food; the experiment was carried out from week 8 to 16 of life. Flow cytometry was performed to determine the percentage of TCD4+ cells producing Th1 and Th2 cytokines. RESULTS: Supplementation with microalgae fatty acids decreased the percentage of TCD4+ cells producing IFN-γ and TNF-α and increased the ones producing IL-17A and IL-12 in both strains; on the other hand, supplementation decreased percentage of TCD4+ cells producing IL-4 and increased the ones producing TGF-ß. CONCLUSIONS: Microalgae n-3 fatty acids could be a useful tool in the treatment of diabetes as well as in the prevention of the appearance of health complications caused by inflammatory states.

Periodontitis is associated with preeclampsia in pregnant women.

BACKGROUND: Recent investigations have demonstrated a positive association between periodontitis and pregnancy complications. The purpose of this study was to determine the effect of periodontitis and the subgingival microbial composition on preeclampsia. METHODS: A case-control study was carried out in Cali, Colombia that included 130 preeclamptic and 243 non-preeclamptic women between 26 to 36 weeks of pregnancy. Sociodemographic data, obstetric risk factors, periodontal status, and subgingival microbial composition were determined in both groups. Preeclampsia was defined as blood pressure>or=140/90 mm Hg, and >or=2+ proteinuria, confirmed by 0.3 g proteinuria/24 hours of urine specimens. Controls were healthy pregnant women. Odds ratios (ORs) for periodontitis and subgingival microbiota compositions were calculated. RESULTS: A total of 83 out of 130 preeclamptic women (63.8%) and 89 out of 243 controls (36.6%) had chronic periodontitis (OR: 3.0; 95% confidence interval (CI): 1.91 to 4.87; P<0.001). Clinical attachment loss increased in the case group (4.0+/-0.10 mm) compared to the control group (3.0+/-0.08 mm) (P<0.001). The average newborn birth weight from preeclamptic mothers was 2.453 g, whereas in controls was 2.981 g (P<0.001). Two red complex microorganisms, Porphyromonas gingivalis and Tannerella forsythensis, and the green complex microorganism Eikenella corrodens were more prevalent in the preeclamptic group than in controls (P<0.01). CONCLUSIONS: Chronic periodontal disease and the presence of P. gingivalis, T. forsythensis, and E. corrodens were significantly associated with preeclampsia in pregnant women. Further research is needed to establish pathogenic mechanisms of active periodontal disease and subgingival periodontopathogens related to preeclampsia development.

First Report of Melon necrotic spot virus in Panama.

Melon (Cucumis melo L.) represents an important crop in Panama where 1,449 ha were cultivated in 2005 with 920.4 ha of this crop planted in Los Santos Province (southeast region of Panama). During April 2005 and January 2006, several melon plants in commercial fields in that area showed stem necrosis at the crown level, and less frequently, small necrotic spots on leaves. In some cases, wilting and plant death were observed. Symptoms were similar to those caused by the carmovirus Melon necrotic spot virus (MNSV). Cysts of Olpidium bornovanus also were observed in the roots of all affected melon plants. Roots from eight symptomatic plants collected in seven fields were positive using doubleantibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with an antiserum specific for MNSV (BIO-RAD, Life Sciences, Barcelona, Spain). To confirm these results, total RNA was extracted from symptomatic plants and used in one-step reverse transcription-polymerase chain reaction (RT-PCR) with Platinum Taq (Invitrogen Life Technologies, Barcelona, Spain). MNSV specific primers designed to amplify a region of the coat protein gene were used in the assays. Amplicons of the expected size (651 bp) were generated from symptomatic plant tissue, but were not produced from healthy plants or the water used as negative controls. To establish the authenticity of this virus, RT-PCR products were purified with the High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and directly sequenced. Nucleotide sequences were analyzed by using the basic local alignment search tool (BLAST) (1). The primers produced two amplicons with different but similar sequences. One sequence (GenBank Accession No. DQ443546) showed 92% identity to the coat protein gene of the MNSV Spanish isolate (GenBank Accession No. AY330700) and the MNSV Dutch isolate (GenBank Accession No. M29671) and 88% identity to the Japanese isolate (GenBank Accession No. AB189944). The second sequence (GenBank Accession No. DQ443547) was 93% identical with the Spanish and Dutch MNSV isolates, 88% identical with the Japanese isolate, and 100% identical with sequences from commercial melon seed previously isolated in our laboratory (GenBank Accession No. DQ443545). Infected seed may be a concern with regard to long distance spread of the virus independent of the vector (3) and should be considered in disease management strategies. MNSV has been previously reported in Japan, the Netherlands, the United Kingdom, the United States (2), Guatemala (4), Mexico, Honduras, and Uruguay (C. Jordá, unpublished). To our knowledge, this is the first report of MNSV in Panama. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) A. A. Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Online Publication, 1996. (3) R. N. Campbell et al. Phytopathology 86:1294, 1996. (4) C. Jordá et al. Plant Dis. 89:338, 2005.

Calcium plus linoleic acid therapy for pregnancy-induced hypertension.

OBJECTIVE: To determine the effect of dietary supplementation of calcium plus conjugated linoleic acid (calcium-CLA) in reducing the incidence of vascular endothelial dysfunction in pregnant women at high risk of developing pregnancy-induced hypertension (PIH). PATIENTS AND METHODS: This randomized, double-blind, placebo-controlled trial conducted at 4 outpatient clinics in 2 developing countries recruited 48 healthy primigravidas younger than 19 years or older than 35 years who had a family history of pre-eclampsia and diastolic notch. Twenty-four participants received daily elemental calcium (600 mg) plus CLA (450 mg) and 24 received placebo from week 18 to 22 of pregnancy until delivery. RESULTS: Calcium-CLA supplementation reduced significantly the incidence of PIH (2 cases [8%] in the study group vs. 10 cases [42%] in the placebo group; relative risk, 0.20; 95% confidence interval, 0.05-0.82; P=.01). Endothelial dysfunction was also significantly reduced after calcium-CLA supplementation (in 18 women [75%] vs. 4 women [17%]; P<.001), compared with the placebo group (in 15 [63%] vs. 9 women [38%]; P=.08). CONCLUSION: In pregnant women at high risk for PIH, calcium-CLA supplementation decreases the incidence of PIH and improves endothelial function.

Prevention of preeclampsia by linoleic acid and calcium supplementation: a randomized controlled trial.

OBJECTIVE: To determine the effect of low doses of linoleic acid and calcium on prostaglandin (PG) levels and the efficacy of this treatment in the prevention of preeclampsia. METHODS: In a randomized, double-blind, placebo-controlled study we treated 86 primigravidas with risk factors for preeclampsia (high biopsychosocial risk [above 3 points], positive roll-over test, and high mean blood pressure [above 85 mmHg)] with daily doses of either 450 mg linoleic acid and 600 mg calcium (n=43) or 450 mg starch and 600 mg lactose placebo (n=43) during the third trimester of pregnancy. RESULTS: Four women in the experimental group (9.3%) developed preeclampsia compared with 16 (37.2%) controls (relative risk 0.25, 95% confidence interval 0.09, 0.69, P < .001). The median serum levels of PGE2 after 4 weeks of treatment increased by 106% in the experimental group (P=.03) and decreased by 33% in the control group (P=.02). The median ratio between thromboxane B2 and PGE2 decreased by 40% in the experimental group (P=.02) and increased by 18% in the control group (P=.14). No significant differences were observed in the median ratio between thromboxane B2 and 6-keto PGF1alpha in either group. No serious maternal or neonatal side effects of treatment occurred in either group. CONCLUSION: The administration of low daily doses of linoleic acid and calcium during the third trimester of pregnancy reduced the incidence of preeclampsia significantly in women at high risk, possibly by correcting the PGE2 levels.

Two groups of TH-like immunoreactive neurons in the frog (Rana pipiens) retina.

The morphology and distribution of TH-like immunoreactive (TH-IR) cells in the retina of Rana pipiens were studied in retinal whole mounts and in radial and horizontal sections. A large majority (96%) of the immunoreactive cells were found in the inner nuclear layer while a few cells were found in the ganglion cell layer. All TH-IR cells had round to oval somata with average diameter of 10 microm. The 2-4 primary processes of these cells distributed extensively to sublamina 1 of the inner plexiform layer (IPL) and sparsely to sublamina 5. Two groups of TH-IR cells were distinguished: one, designated thin cells, had only thin (<2 microm diameter) primary processes; the second, designated thick cells, had one or more primary processes with diameter(s) exceeding 2 microm for a distance of 5 microm or more from the soma. The thin cells did not significantly differ from the thick cells in soma diameter, number of primary processes, horizontal spread of processes or vertical lamination of processes. Nearest neighbor analyses of the two types revealed that the population of TH-IR cells (thick and thin together) have an orderly distribution while the thick cells alone are more randomly distributed, indicating that the thick cells do not comprise a functional population. The total number of TH-IR cells varied between retinas; the variability was due principally to variation of thin cell density. It is hypothesized that the thick cells are a subpopulation of the TH-IR cells which are in a particular physiological state at the time of fixation.

Comparison of DNA migrations in two clamped homogeneous electric field chambers of different sizes. Relation between sample thickness and electrophoresis time.

We present here a method to compare the mathematical descriptions of DNA migration per pulse as a function of pulse time. It is based on obtaining robust estimates and variances of DNA reorientation time, migration velocities during and after DNA reorientation; and on the statistical comparisons of these estimates. We demonstrated an equal description for the migration per pulse of each DNA molecule separated under identical conditions in clamped homogeneous electric field (CHEF) and miniCHEF chambers. However, miniCHEF resolved the patterns in shorter times, because it uses thinner samples. The relationship between sample thickness and CHEF run time is also presented.