A clinically realistic large animal model of intra-articular fracture that progresses to post-traumatic osteoarthritis.

OBJECTIVE: Translation of promising treatments for post-traumatic osteoarthritis (PTOA) to patients with intra-articular fracture (IAF) has been limited by the lack of a realistic large animal model. To address this issue we developed a large animal model of IAF in the distal tibia of Yucatan minipigs and documented the natural progression of this injury. DESIGN: Twenty-two fractures were treated using open reduction and internal fixation with either an anatomic reduction or an intentional 2-mm step-off. Pre-operatively, and 3 days, 1, 2, 4, 8, and 12 weeks post-operatively, animals were sedated for synovial fluid draws and radiographs. Limb loading was monitored at the same time points using a Tekscan Walkway. Animals were sacrificed at 12 weeks and the limbs were harvested for histological evaluation. RESULTS: All animals achieved bony union by 12 weeks, facilitating nearly complete recovery of the initial 60% decrease in limb loading. TNFα, IL1ß, IL6, and IL8 concentrations in the fractured limbs were elevated (P < 0.05) at specific times during the 2 weeks after fracture. Histological cartilage degeneration was more severe in the step-off group (0.0001 < P < 0.27 compared to normal) than in the anatomic reconstruction group (0.27 < P < 0.99 compared to normal). CONCLUSIONS: This model replicated key features of a human IAF, including surgical stabilization, inflammatory responses, and progression to osteoarthritic cartilage degeneration, thereby providing a potentially useful model for translating promising treatment options to clinical practice.

Life history as a constraint on plasticity: developmental timing is correlated with phenotypic variation in birds.

Understanding why organisms vary in developmental plasticity has implications for predicting population responses to changing environments and the maintenance of intraspecific variation. The epiphenotype hypothesis posits that the timing of development can constrain plasticity-the earlier alternate phenotypes begin to develop, the greater the difference that can result amongst the final traits. This research extends this idea by considering how life history timing shapes the opportunity for the environment to influence trait development. We test the prediction that the earlier an individual begins to actively interact with and explore their environment, the greater the opportunity for plasticity and thus variation in foraging traits. This research focuses on life history variation across four groups of birds using museum specimens and measurements from the literature. We reasoned that greater phenotypic plasticity, through either environmental effects or genotype-by-environment interactions in development, would be manifest in larger trait ranges (bills and tarsi) within species. Among shorebirds and ducks, we found that species with relatively shorter incubation times tended to show greater phenotypic variation. Across warblers and sparrows, we found little support linking timing of flight and trait variation. Overall, our results also suggest a pattern between body size and trait variation, consistent with constraints on egg size that might result in larger species having more environmental influences on development. Taken together, our results provide some support for the hypothesis that variation in life histories affects how the environment shapes development, through either the expression of plasticity or the release of cryptic genetic variation.

A cryogenic torsion balance using a liquid-cryogen free, ultra-low vibration cryostat.

We describe a liquid-cryogen free cryostat with ultra-low vibration levels, which allows for continuous operation of a torsion balance at cryogenic temperatures. The apparatus uses a commercially available two-stage pulse-tube cooler and passive vibration isolation. The torsion balance exhibits torque noise levels lower than room temperature thermal noise by a factor of about four in the frequency range of 3-10 mHz, limited by residual seismic motion and by radiative heating of the pendulum body. In addition to lowering thermal noise below room-temperature limits, the low-temperature environment enables novel torsion balance experiments. Currently, the maximum duration of a continuous measurement run is limited by accumulation of cryogenic surface contamination on the optical elements inside the cryostat.

Optical biopsy and imaging using optical coherence tomography.

Optical coherence tomography is a new imaging technique that can perform high-resolution, micrometre-scale, cross-sectional imaging in biological systems. The technology has been developed, and reduced to, preliminary clinical practice in ophthalmology. The challenging problem that OCT may address is the development of 'optical biopsy' techniques. These techniques can provide diagnostic imaging of tissue morphology without the need for excision of specimens. Many investigations remain to identify optimal areas for clinical application, and additional engineering must be done to integrate vertically the technology and to reduce it to clinical practice. Nevertheless, preliminary studies indicate the feasibility of developing this technology for a wide range of clinical and research diagnostic imaging applications. The ability to non-excisionally evaluate tissue morphology using a catheter or an endoscope could have a significant impact on the diagnosis and management of a wide range of diseases.

The molecular landscape of ASPM mutations in primary microcephaly.

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a model disease to study human neurogenesis. In affected individuals the brain grows at a reduced rate during fetal life resulting in a small but structurally normal brain and mental retardation. The condition is genetically heterogeneous with mutations in ASPM being most commonly reported. METHODS AND RESULTS: We have examined this further by studying three cohorts of microcephalic children to extend both the phenotype and the mutation spectrum. Firstly, in 99 consecutively ascertained consanguineous families with a strict diagnosis of MCPH, 41 (41%) were homozygous at the MCPH5 locus and all but two families had mutations. Thus, 39% of consanguineous MCPH families had homozygous ASPM mutations. Secondly, in 27 non-consanguineous, predominantly Caucasian families with a strict diagnosis of MCPH, 11 (40%) had ASPM mutations. Thirdly, in 45 families with a less restricted phenotype including microcephaly and mental retardation, but regardless of other neurological features, only 3 (7%) had an ASPM mutation. This report contains 27 novel mutations and almost doubles the number of MCPH associated ASPM mutations known to 57. All but one of the mutations lead to the use of a premature termination codon, 23 were nonsense mutations, 28 deletions or insertions, 5 splicing, and 1 was a translocation. Seventeen of the 57 mutations were recurrent. There were no definitive missense mutations found nor was there any mutation/phenotype correlation. ASPM mutations were found in all ethnic groups studied. CONCLUSION: This study confirms that mutations in ASPM are the most common cause of MCPH, that ASPM mutations are restricted to individuals with an MCPH phenotype, and that ASPM testing in primary microcephaly is clinically useful.

Hail suppression and society.

An interdisciplinary assessment of hail suppression in the past, present, and future has shown it to be currently scientifically uncertain but a potentially beneficial future technology. An established suppression technology would be widely adopted in the Great Plains, providing benefits to agriculture and secondarily to the American consumer. Development of a reliable technology will require a sizable longterm federal commitment to atmospheric and social research. Subcritical funding would be a mistake. Orderly future usage of hail suppression, with its scientific complexities and regional character, will necessitate development of governmental regulations, evaluation procedures, interstate arrangements, and means for compensating those who lose from modification.

Optical coherence tomography.

A technique called optical coherence tomography (OCT) has been developed for noninvasive cross-sectional imaging in biological systems. OCT uses low-coherence interferometry to produce a two-dimensional image of optical scattering from internal tissue microstructures in a way that is analogous to ultrasonic pulse-echo imaging. OCT has longitudinal and lateral spatial resolutions of a few micrometers and can detect reflected signals as small as approximately 10(-10) of the incident optical power. Tomographic imaging is demonstrated in vitro in the peripapillary area of the retina and in the coronary artery, two clinically relevant examples that are representative of transparent and turbid media, respectively.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

Transcriptional regulation of a periodically controlled flagellar gene operon in Caulobacter crescentus.

Temporal regulation of flagellar gene expression in Caulobacter crescentus has been examined by a detailed analysis of the flbG-flaJ-flbH-flaK hook operon. The approximate location of the promoter for this 4.4 X 10(3) base-pair transcriptional unit was determined by deletion mapping, and the flaK gene was shown by nucleotide sequencing to code for the hook protein. flaK messenger RNA was quantified by S1 nuclease mapping with an internal restriction fragment of the gene as the 5'-labeled DNA probe. The results of these assays provide the first direct evidence that periodic expression of a flagellar gene in the C. crescentus cell cycle is regulated at the transcriptional level. The effect of altering the time of gene duplication in the cell cycle was examined by subcloning the complete hook operon on a plasmid that replicates throughout the S phase. The normal periodicity of flaK transcription and translation was maintained in this merodiploid strain, which suggests that replication alone is not sufficient to initiate flagellar gene expression. We also show that the three adjacent transcriptional units III, IV and V are required in trans for transcription of the book operon, and we discuss the possible role of these genes in the hierarchical regulation of the flagellar gene expression.

The Responding Site of the Rex Locus of Drosophila melanogaster.

Rex is a dominant, maternal-effect locus in the heterochromatin of the X chromosome Drosophila melanogaster. It causes an early mitotic exchange-like event between heterochromatic elements of an attached- XY in X/attached-XY embryos of Rex mothers. Evidence is presented here that the site of Rex action is the ribosomal RNA gene cluster (the bb locus) only; no other heterochromatin is affected. The Rex locus may be useful in studying regulation of rRNA-gene copy number, mitotic chromosome behavior and heterochromatic function.

Rex-induced recombination implies bipolar organization of the ribosomal RNA genes of Drosophila melanogaster.

Rex-induced mitotic recombination was used to determine whether nucleolus organizers can pair in both inverted and noninverted orientations. Two target chromosomes, each duplicated for the rDNA region, were exposed to maternal Rex activity. Recombination in one orientation should yield deletion of the material between the two nucleolus organizers, recombination in the other orientation should yield inversion of the same material. Both products were recovered from both target chromosomes. The generality of using Rex-mediated recombination for analysis of the rDNA is considered.

Overexpression of sarcoplasmic reticulum Ca(2+)-ATPase improves cardiac contractile function in hypothyroid mice.

OBJECTIVE: Prolonged cardiac contraction and relaxation in hypothyroidism are in part related to diminished expression of the gene coding for the calcium pump of the sarcoplasmic reticulum (SERCA2a). Therefore, we examined whether or not transgenic SERCA2a gene expression in mice may compensate for the cardiac effects of hypothyroidism. METHODS: SERCA2a mRNA and protein were analyzed from hearts of euthyroid and hypothyroid mice of wild-type or SERCA2a transgene status. Contractile function was studied in isolated left ventricular papillary muscles. RESULTS: We found significant decreases of SERCA2a mRNA and protein levels in hearts of hypothyroid wild-type mice in comparison with euthyroid wild-type mice (controls). Papillary muscles from hypothyroid wild-type mice showed significant increases in time to peak contraction and relaxation times compared with controls. In contrast, SERCA2a mRNA and protein levels were significantly higher in hypothyroid SERCA2a transgenic mice than in hypothyroid wild-type mice. The transgene led to a functional improvement by compensating for the prolonged contraction and relaxation of papillary muscles. CONCLUSIONS: Our murine model of hypothyroidism revealed decreases in SERCA2a gene expression accompanied by prolonged contraction and relaxation of papillary muscles, and an improvement of the contractile phenotype due to compensated SERCA2a gene expression in SERCA2a transgenic mice.

Altered cardiac phenotype in transgenic mice carrying the delta337 threonine thyroid hormone receptor beta mutant derived from the S family.

The heart has been recognized as a major target of thyroid hormone action. Our study investigates both the regulation of cardiac-specific genes and contractile behavior of the heart in the presence of a mutant thyroid hormone receptor beta1 (T3Rbeta1-delta337T) derived from the S kindred. The mutant receptor was originally identified in a patient with generalized resistance to thyroid hormone. Cardiac expression of the mutant receptor was achieved by a transgenic approach in mice. As the genes for myosin heavy chains (MHC alpha and MHC beta) and the cardiac sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) are known to be regulated by T3, their cardiac expression was analyzed. The messenger RNA levels for MHC alpha and SERCA2 were markedly down-regulated, MHC beta messenger RNA was up-regulated. Although T3 levels were normal in these animals, this pattern of cardiac gene expression mimics a hypothyroid phenotype. Cardiac muscle contraction was significantly prolonged in papillary muscles from transgenic mice. The electrocardiogram of transgenic mice showed a substantial prolongation of the QRS interval. Changes in cardiac gene expression, cardiac muscle contractility, and electrocardiogram are compatible with a hypothyroid cardiac phenotype despite normal T3 levels, indicating a dominant negative effect of the T3Rbeta mutant.

Cardiac ion channel expression and contractile function in mice with deletion of thyroid hormone receptor alpha or beta.

Cardiac myocytes express the two thyroid hormone receptors (T(3)Rs), T(3)Ralpha and T(3)Rbeta. However, which isoform contributes to specific, T(3)-induced alterations of cardiac function remains unclear. Here, we used individual T(3)R isoform knockout (KO) mice to study the effects of T(3)Ralpha and T(3)Rbeta in the heart. Our findings indicate that potassium channel genes that code for K(+) channels involved in action potential repolarization, like KV 4.2 and minK, are T(3)Ralpha targets. Both are markedly regulated by thyroid status. The recently identified cyclic nucleotide-gated channels, HCN2 and HCN4, are targets of T(3)Ralpha and are unchanged in a euthyroid T(3)Rbeta KO. However, these transcripts respond markedly to altered T(3) signaling concomitant with bradycardia in T(3)Ralpha KO and hypothyroid animals, as well as tachycardia in hyperthyroid T(3)Rss KO mice. SERCA2a and myosins are T(3) regulated and were also targets of T(3)Ralpha, and the papillary muscles of alphaKO animals showed a slowed rate of force development. Because of the absence of significant cardiac effects in euthyroid T(3)Rss KO mice, we determined messenger RNA levels for both T(3)Ralpha and T(3)Rss in the heart. We found that T(3)Rss is present at a 1:3 ratio to T(3)Ralpha1. We conclude that the cardiac phenotype regulated by T(3) is predominantly mediated by T(3)Ralpha and that the lack of T(3)Ralpha cannot be compensated by T(3)Rss in the heart.

The thyroid hormone receptor-beta-selective agonist GC-1 differentially affects plasma lipids and cardiac activity.

Thyroid hormones influence the function of many organs and mediate their diverse actions through two types of thyroid hormone receptors, TRalpha and TRbeta. Little is known about effects of ligands that preferentially interact with the two different TR subtypes. In the current study the comparison of the effects of the novel synthetic TRbeta-selective compound GC-1 with T3 at equimolar doses in hypothyroid mice revealed that GC-1 had better triglyceride-lowering and similar cholesterol-lowering effects than T3. T3, but not GC-1, increased heart rate and elevated messenger RNA levels coding for the I(f) channel (HCN2), a cardiac pacemaker that was decreased in hypothyroid mice. T3 had a larger positive inotropic effect than GC-1. T3, but not GC-1, normalized heart and body weights and messenger RNAs of myosin heavy chain alpha and beta and the sarcoplasmic reticulum adenosine triphosphatase (Serca2). Additional dose-response studies in hypercholesteremic rats confirmed the preferential effect of GC-1 on TRbeta-mediated parameters by showing a much higher potency to influence cholesterol and TSH than heart rate. The preferred accumulation of GC-1 in the liver vs. the heart probably also contributes to its marked lipid-lowering effect vs. the absent effect on heart rate. These data indicate that GC-1 could represent a prototype for new drugs for the treatment of high lipid levels or obesity.

Characterization of calcitonin- and adrenocorticotropin-producing human cloned cell lines.

We have developed three human cloned cell lines that produce immunoreactive human calcitonin (ihCT) and ACTH (iACTH) as well as exhibit characteristics of cultured neural cells. Clones HMS-41/I, -78/2, and -98/2 were developed from cell lines HeLa AV3, MBA 9812 (bronchogenic carcinoma), and SW 267 (pheochromocytoma), respectively. Karyological analysis of both the parent and the cloned cell lines confirmed the identity of HeLa AV3 and MBA 9812. When grown in serum-free media designed for culturing neural cells, the patterns of production for both ihCT and iACTH varied among the clones. The multiple patterns of hormone production suggest that the mechanisms involved in the biosynthesis, processing, and secretion of these hormones differ among the clones. The clones contain neuron-specific enolase and the putative neurotransmitters beta-alanine and gamma-amino butyric acid, and they respond to cAMP analogs by differentiating, as noted by the extension of neurites (except the HeLa-derived HMS-41/I). The iACTH extracted from cells and synthetic ACTH exhibited equivalent profiles upon isoelectric focusing. The forms of ihCT noted in cell extracts were similar to those observed in extracts of human tumor tissue. Our rabbit antiserum to hCT failed to detect ihCT in those cell extracts prepared for ACTH determination or in extracts of rat pituitaries, but it did detect CT in rat thyroids by both RIA and immunofluorescent procedures. We concluded that our antisera to hCT do not detect the precursor form of ACTH. The availability of these cloned cell lines provides model systems for examining the production of these peptide hormones and the concomitant expression of neural and endocrine characteristics.

Imaging of coronary artery microstructure (in vitro) with optical coherence tomography.

OCT achieves high-resolution and image differentiation of vascular tissues to a degree that has not been previously possible with any method except excisional biopsy. Thus, OCT represents a promising new diagnostic technology for intracoronary imaging, which could permit the in vivo evaluation of critical vascular pathology.

Optical coherence tomography of the human retina.

OBJECTIVE: To demonstrate optical coherence tomography for high-resolution, noninvasive imaging of the human retina. Optical coherence tomography is a new imaging technique analogous to ultrasound B scan that can provide cross-sectional images of the retina with micrometer-scale resolution. DESIGN: Survey optical coherence tomographic examination of the retina, including the macula and optic nerve head in normal human subjects. SETTING: Research laboratory. PARTICIPANTS: Convenience sample of normal human subjects. MAIN OUTCOME MEASURES: Correlation of optical coherence retinal tomographs with known normal retinal anatomy. RESULTS: Optical coherence tomographs can discriminate the cross-sectional morphologic features of the fovea and optic disc, the layered structure of the retina, and normal anatomic variations in retinal and retinal nerve fiber layer thicknesses with 10-microns depth resolution. CONCLUSION: Optical coherence tomography is a potentially useful technique for high depth resolution, cross-sectional examination of the fundus.

Micrometer-scale resolution imaging of the anterior eye in vivo with optical coherence tomography.

OBJECTIVE: To demonstrate a new diagnostic technique, optical coherence tomography, for high-resolution cross-sectional imaging of structures in the anterior segment of the human eye in vivo. Optical coherence tomography is a new, noninvasive, noncontact optical imaging modality that has spatial resolution superior to that of conventional clinical ultrasonography (< 20 microns) and high sensitivity (dynamic range, > 90 dB). DESIGN: Survey of intraocular structure and dimension measurements. SETTING: Laboratory. PATIENTS: Convenience sample. MAIN OUTCOME MEASURES: Correlation with range of accepted normal intraocular structure profiles and dimensions. RESULTS: Direct in vivo measurements with micrometer-scale resolution were performed of corneal thickness and surface profile (including visualization of the corneal epithelium), anterior chamber depth and angle, and iris thickness and surface profile. Dense nuclear cataracts were successfully imaged through their full thickness in a cold cataract model in calf eyes in vitro. CONCLUSIONS: Optical coherence tomography has potential as a diagnostic tool for applications in noncontact biometry, anterior chamber angle assessment, identification and monitoring of intraocular masses and tumors, and elucidation of abnormalities of the cornea, iris, and crystalline lens.