Reducing soft-tissue shrinkage artefacts caused by staining with Lugol's solution.

Diffusible iodine-based contrast-enhanced computed tomography (diceCT) is progressively used in clinical and morphological research to study developmental anatomy. Lugol's solution (Lugol) has gained interest as an effective contrast agent; however, usage is limited due to extensive soft-tissue shrinkage. The mechanism of Lugol-induced shrinkage and how to prevent it is largely unknown, hampering applications of Lugol in clinical or forensic cases where tissue shrinkage can lead to erroneous diagnostic conclusions. Shrinkage was suggested to be due to an osmotic imbalance between tissue and solution. Pilot experiments pointed to acidification of Lugol, but the relation of acidification and tissue shrinkage was not evaluated. In this study, we analyzed the relation between tissue shrinkage, osmolarity and acidification of the solution during staining. Changes in tissue volume were measured on 2D-segmented magnetic resonance and diceCT images using AMIRA software. Partial correlation and stepwise regression analysis showed that acidification of Lugol is the main cause of tissue shrinkage. To prevent acidification, we developed a buffered Lugol's solution (B-Lugol) and showed that stabilizing its pH almost completely prevented shrinkage without affecting staining. Changing from Lugol to B-Lugol is a major improvement for clinical and morphological research and only requires a minor adaptation of the staining protocol.

Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers.

Since the discovery of microRNAs (miRNAs), circulating miRNAs have been proposed as biomarkers for disease. Consequently, many groups have tried to identify circulating miRNA biomarkers for various types of diseases including cardiovascular disease and cancer. However, the replicability of these experiments has been disappointingly low. In order to identify circulating miRNA candidate biomarkers, in general, first an unbiased high-throughput screen is performed in which a large number of miRNAs is detected and quantified in the circulation. Because these are costly experiments, many of such studies have been performed using a low number of study subjects (small sample size). Due to lack of power in small sample size experiments, true effects are often missed and many of the detected effects are wrong. Therefore, it is important to have a good estimate of the appropriate sample size for a miRNA high-throughput screen. In this review, we discuss the effects of small sample sizes in high-throughput screens for circulating miRNAs. Using data from a miRNA high-throughput experiment on isolated monocytes, we illustrate that the implementation of power calculations in a high-throughput miRNA discovery experiment will avoid unnecessarily large and expensive experiments, while still having enough power to be able to detect clinically important differences.

The dimensions of the tarsal sinus and canal in different foot positions and its clinical implications.

This study presents a reference for the dimensions of the tarsal sinus and canal in healthy adults in different foot positions to facilitate understanding of the kinematics of the subtalar joint, the effect of an implant, and other clinical issues. In a 3D CT stress test on 20 subjects, the right foot was forced into a neutral and eight different extreme foot positions while CT scans were obtained. The bones were segmented in the neutral foot position. The kinematics of the bones in the extreme positions were determined relative to the neutral position. The dimensions of the tarsal sinus and canal were calculated by determining the radii of the maximal inscribed spheres at 20 equidistant locations along an axis in 3D surface models of the tali and calcanei in each foot position. The radii were small on the medial side and increased laterally. Medial from the middle, the radii were small and not significantly different among the various foot positions. At the lateral side, the dimensions were affected mainly by eversion or inversion and less by dorsiflexion or plantarflexion. The pattern was reproducible among subjects, but there were between-subject differences. The dimensions are mostly determined by rotation in the frontal plane. A pivot point was found medial from the middle. These data serve as a reference and model for predicting the effect of sinus implants and understanding such clinical problems as sinus tarsi syndrome. Between-subjects differences have to be taken into account. Clin. Anat. 30:1049-1057, 2017. © 2017 Wiley Periodicals, Inc.

Unexpected regulation of miRNA abundance during adaptation of early-somite mouse embryos to diabetic pregnancy.

Maternal diabetes is one of major causes of congenital malformations in offspring, but the underlying mechanism is still unclear. MiRNAs play an important role in transcriptional and post-transcriptional regulation of gene expression. However, no miRNA expression profiling of hyperglycemic offspring are thus far available. Female mice were made diabetic with streptozotocin, treated with slow-release insulin tablets, and mated. MiRNA expression profiling with Next Generation Sequencing on the SOLiD5 platform was performed on 8 control and 5 hyperglycemic embryonic day (ED)8.5 and 9 control and 6 hyperglycemic ED9.5 embryos. Differential expression was analyzed with the Wald test. On ED8.5, the abundance of expressed miRNAs was similar in control and hyperglycemic ED8.5 embryos. The spectrum of expressed miRNAs had not changed in ED9.5 embryos, but the abundance of most miRNAs increased ∼5-fold in control embryos. However, hyperglycemic D9.5 embryos were unable to mount this increase in prevalence. Only 3 miRNAs were differentially expressed in control and hyperglycemic ED9.5 embryos, but their putative target genes were underrepresented in the Jackson database of genes causing cardiovascular or neural malformations.

Augmented mandibular bone structurally adapts to functional loading.

Long-term changes in trabecular bone structure during the 10 years following onlay grafting with simultaneous mandibular implant placement were studied. Extraoral radiographs of both mandibular sides in eight patients were taken regularly. Bone structure was analysed using a custom-written image analysis program. Parameters studied were trabecular area and perimeter and marrow cavity area and perimeter. After skeletonisation of the trabecular network, the number of end points and branching points, skeleton length, and branch angle were determined. The observed structural changes agree with the development of a more complex and more delicate or fine osseous structure. The bone shows more trabecular branching. All changes are most pronounced in the graft spongiosa, but are also found in the graft cortex and in the original mandible. The mean trabecular branch angle becomes more horizontal. The applied technique can be used to analyse long-term changes in the architecture of bone grafts. Changes found in the graft architecture correspond to changes expected after functional adaptation to loading.

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

Three-dimensional measurement and visualization of morphogenesis applied to cardiac embryology.

Volume growth and proliferation are key processes in heart morphogenesis, yet their regionalization during development of the heart has been described only anecdotally. To study the contribution of cardiomyocyte proliferation to heart development, a quantitative reconstruction method was designed, allowing the local mapping of this morphogenetic process. First, a morphological surface reconstruction is made of the heart, using sections stained specifically for cardiomyocytes. Then, by a comprehensive series of image processing steps, local three-dimensional (3D) information of proliferation is obtained. These local quantitative data are then mapped onto the morphological surface reconstruction, resulting in a reconstruction that not only provides morphological information (qualitative), but also displays local information on proliferation rate (quantitative). The resulting 3D quantitative reconstructions revealed novel observations regarding the morphogenesis of the heart.

The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements, contains mRNA-instability elements and is involved in glutamine sensing.

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.

Image processing and analysis program for measurement of bone density changes in reference and follow-up standardized extraoral oblique lateral cephalometric radiographs of the mandible.

OBJECTIVES: The aim of this study was to develop an image processing and analysis program for peri-implant bone density measurements of the mandible using extraoral radiographs, which includes a correction for the variable projection of the soft tissues of the face. METHODS: The measurement procedure is based on pairs of reference and follow-up extraoral oblique lateral cephalometric radiographs (OLCRs) of patients with endosseous implants in the anterior part of the atrophic edentulous mandible. The procedure consists of image acquisition, correction for radiographic variation using an aluminium wedge (i.e. film exposure and development) and transformation of the grey values into aluminium-equivalent values. After correction for variation in the projection of the soft tissues of the face using internal calibration fields, the actual peri-implant bone density measurements are performed. RESULTS: The soft tissue projection correction significantly reduces the variation between radiographs owing to the position of the soft tissues. CONCLUSIONS: It is concluded that the described image processing and analysis program, in combination with extraorally made OLCRs, is a valuable technique for measurement of peri-implant bone density changes of the mandible. With minor adaptations, the program can be used for other semi-edentulous patients.

[Onlay grafts in combination with endosseous implants in severe mandibular atrophy. A prospective radiological study]. / Botopbouwen in combinatie met implantaten bij sterke atrofie van de onderkaak. Een prospectief röntgenologisch onderzoek.

The placement of endosseous implants in combination with iliac crest onlay grafting of the anterior mandible is one of the treatment modalities for extreme atrophy of the mandible. The remodelling of these onlay grafts was studied using standardized extraoral oblique lateral cephalometric radiographs (OLCRs). A group of 8 patients was used in this prospective study. The measurements obtained from the OLCRs indicated the existence of the following stages in the process of remodelling of the bone grafts: 1. decrease in thickness and radiographic density of the (upper) cortex of the graft, predominantly during the first half year after grafting; 2. no significant changes in the radiographic density of the upper spongeous part of the graft; 3. a mean decrease of approximately 25% in the overall thickness of the graft, particularly during the first half year; and 4. an increase in the radiographic density of the lower part of the spongeous bone in the second half year after grafting. It is concluded that the remodelling of the graft has a predictable pattern in time. Densitometric measurement using standardized OLCRs can be a useful tool to evaluate quantitative changes of bone grafts to the mandible. The treatment described can be used on very strict indications only.

Melatonin generates an outward potassium current in rat suprachiasmatic nucleus neurones in vitro independent of their circadian rhythm.

The present study investigated the membrane mechanisms underlying the inhibitory influence of melatonin on suprachiasmatic nucleus (SCN) neurones in a hypothalamic slice preparation. Perforated-patch recordings were performed to prevent the rapid rundown of spontaneous firing rate as observed during whole cell recordings and to preserve circadian rhythmicity in SCN neurones. In current-clamp mode melatonin (1 microM or 1 nM) application, in the presence of agents that block action potential generation and fast synaptic transmission, resulted in a membrane hyperpolarisation accompanied with a decrease in input resistance in the majority of SCN neurones (71-86%). The amplitude of the hyperpolarisation was not found to be significantly different between circadian time 5-12 and 14-21. In voltage-clamp mode melatonin (1 microM or 1 nM) induced an outward current accompanied with an increase in membrane conductance. The current was found to be mainly potassium driven with voltage kinetics resembling those of an open rectifying potassium conductance. Investigations into the signal transduction mechanism revealed melatonin-induced inhibition of SCN neurones to be sensitive to pertussis toxin but independent of intracellular cAMP levels and phospholipase C activity. The present study shows that melatonin, at night-time physiological concentrations, reduces the neuronal excitability of the majority of SCN neurones independent of the time of application in the circadian cycle. Thus in vivo melatonin may be important for circadian time-keeping by amplifying the circadian rhythm in SCN neurones, by lowering their sensitivity to phase-shifting stimuli occurring at night.

Overexpression of arginase alters circulating and tissue amino acids and guanidino compounds and affects neuromotor behavior in mice.

Arginine is an intermediate of the ornithine cycle and serves as a precursor for the synthesis of nitric oxide, creatine, agmatine and proteins. It is considered to be a conditionally essential amino acid because endogenous synthesis only barely meets daily requirements. In rapidly growing suckling neonates, endogenous arginine biosynthesis is crucial to compensate for the insufficient supply of arginine via the milk. Evidence is accumulating that the intestine rather than the kidney plays a major role in arginine synthesis in this period. Accordingly, ectopic expression of hepatic arginase in murine enterocytes by genetic modification induces a selective arginine deficiency. The ensuing phenotype, whose severity correlates with the level of transgene expression in the enterocytes, could be reversed with arginine supplementation. We analyzed the effect of arginine deficiency on guanidine metabolism and neuromotor behavior. Arginine-deficient transgenic mice continued to suffer from an arginine deficiency after the arginine biosynthetic enzymes had disappeared from the enterocytes. Postweaning catch-up growth in arginine-deficient mice was characterized by increased levels of all measured amino acids except arginine. Furthermore, plasma total amino acid concentration, including arginine, was significantly lower in adult male than in adult female transgenic mice. Decreases in the concentration of plasma and tissue arginine led to significant decreases in most metabolites of arginine. However, the accumulation of the toxic guanidino compounds, guanidinosuccinic acid and methylguanidine, corresponded inversely with circulating arginine concentration, possibly reflecting a higher oxidative stress under hypoargininemic conditions. In addition, hypoargininemia was associated with disturbed neuromotor behavior, although brain levels of toxic guanidino compounds and ammonia were normal.

Preliminary report: prescription of prism-glasses by the Measurement and Correction Method of H.-J. Haase or by conventional orthoptic examination: a multicenter, randomized, double-blind, cross-over study.

In a multicenter, randomized, double-blind, cross-over study in the Netherlands, the effectiveness of (prism-)glasses prescribed by the Measurement and Correction Method of H.-J. Haase (MKH) was compared to that of glasses prescribed by conventional orthoptic examination. Nine pairs of MKH-optometrists and orthoptists recruited patients who primarily presented with asthenopia, and each prescribed the patient (prism-)glasses. A questionnaire for asthenopia was developed that rated headache and tired eyes as 0-7 days per week and none-light-medium-severe, respectively. Light sensitivity, problems with focusing, near-work problems and burning eyes were each rated as: never-occasionally-often-always. A patient was eligible if he scored 'medium', 'often' or '5 days a week' twice; or 'medium' (etc.) once and 'light' (etc.) twice. Controls, in contrast to the patients, typically answered 'none' or 'never' to half of the complaints, but 37% of them would have passed the admission criteria. Among other criteria were: 18 to 40 years of age, horizontal angle < 4 degrees, vertical < 1.7 degrees, acuity > or = 0.8, stereopsis threshold disparity < 120". Seventy-two patients fulfilled all criteria and returned sufficient questionnaires. They wore the first glasses for six weeks, were without glasses for two weeks, and then wore the second glasses for six weeks. At the start, halfway and at the end of each 6-week period, questionnaires were filled out; 97% were returned. Only 19 of the orthoptists' glasses contained prisms (14 horizontal, 5 vertical; horizontal average of all glasses 0.49 PD, vertical 0.05 PD). Five of the orthoptists' glasses were plano. All MKH glasses contained prisms, 53 of 72 both horizontal and vertical, 18 only horizontal and one only vertical (horizontal average of all glasses 2.83 PD, vertical 0.79 PD). The starting levels of complaints were high and both glasses improved complaints dramatically. The starting levels were lower, but not significantly, in the second 6-week period and improvement was less outspoken. Because of these differences, the two periods had to be evaluated separately. The primary outcome of the study was defined as the difference between the effect of the MKH glasses and that of the orthoptists' glasses in the first and second 6-week periods. For problems with focusing, in the first 6-week period, and for tired eyes, in the second 6-week period, the difference exceeded the difference that had been defined as clinically significant (one day per week less headache or half the distance light-medium or half the distance occasionally-often), but it did not reach statistical significance. The statistical power was approximately 0.7 for demonstrating this clinically significant difference. Statistical significance was not reached in multivariate repeated measure ANOVA either. Forty-four patients preferred to keep the MKH glasses, 25 the orthoptists' glasses, including one plano. It is striking that 25% of the patients did not prefer the glasses that, according to the questionnaire, improved their complaints the most. A year after the study, the questionnaire was sent again to all patients: The levels of complaints after a year were similar to those at the end of the second 6-week period, whether they had preferred the MKH or the orthoptists' glasses, and were similar to the levels in controls. The most conspicuous finding was that both glasses improved the complaints dramatically. Apart from the prisms, other reasons could be: spherical and cylindrical correction, improved wearing comfort of the frame, placebo effect, Hawthorne effect and regression to the mean.

Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth malformation syndrome that maps to human chromosome 11p15.5, a region that harbours a number of imprinted genes. We studied the methylation status of H19 and KCNQ1OT1 (LIT1/KvDMR1) in a large series of BWS patients. Different patient groups were identified: group I patients (20%) with uniparental disomy and hence aberrant methylation of H19 and KCNQ1OT1; group II patients (7%) with a BWS imprinting centre 1 (BWSIC1) defect causing aberrant methylation of H19 only; group III patients (55%) with a BWS imprinting centre 2 (BWSIC2) defect causing aberrant methylation of KCNQ1OT1 only; and group IV patients (18%) with normal methylation patterns for both H19 and KCNQ1OT1. BWS patients have an increased risk of developing childhood tumours. In our patient group, out of 31 patients (group III) with KCNQ1OT1 demethylation only, none developed a tumour. However, tumours were found in 33% of patients with H19 hypermethylation (group I and II) and in 20% of patients with no detectable genetic defect (group IV). All four familial cases of BWS showed reduced methylation of KCNQ1OT1, suggesting that in these cases the imprinting switch mechanism is disturbed.

USAGE: a web-based approach towards the analysis of SAGE data. Serial Analysis of Gene Expression.

MOTIVATION: SAGE enables the determination of genome-wide mRNA expression profiles. A comprehensive analysis of SAGE data requires software, which integrates (statistical) data analysis methods with a database system. Furthermore, to facilitate data sharing between users, the application should reside on a central server and be accessed via the internet. Since such an application was not available we developed the USAGE package. RESULTS: USAGE is a web-based application that comprises an integrated set of tools, which offers many functions for analysing and comparing SAGE data. Additionally, USAGE includes a statistical method for the planning of new SAGE experiments. USAGE is available in a multi-user environment giving users the option of sharing data. USAGE is interfaced to a relational database to store data and analysis results. The USAGE query editor allows the composition of queries for searching this database. Several database functions have been included which enable the selection and combination of data. USAGE provides the biologist increased functionality and flexibility for analysing SAGE data. AVAILABILITY: USAGE is freely accessible for academic institutions at http://www.cmbi.kun.nl/usage/. The source code of USAGE is freely available for academic institutions on request from the first author.

Cytokines in aqueous humour and serum before and after corneal transplantation and during rejection.

Cytokine profiles in aqueous humour were studied in relation to corneal disease and subsequent corneal graft survival or rejection. Cytokine levels in samples obtained from eyes with clear grafts (n = 59) were all within the normal range. At the time of penetrating keratoplasty (n = 146), intraocular levels of IL-6 were increased in 38% (50/131), most markedly in eyes with previous allograft failure or herpetic stromal keratitis. The level of IL-10 was increased in 1 eye (n = 144) and of IL-4 and IFN-gamma in none. During rejection (n = 10), the levels of IL-6 in aqueous humour were increased in 75% (3/4), of IL-10 in 50% (3/6), of IL-4 in none (0/4) and of IFN-gamma in 40% (2/5). In conclusion, the levels of total protein and IL-6 were increased prior to penetrating keratoplasty in eyes with previous inflammation. These results could however not predict the final outcome of the graft. Increased intraocular levels of IL-6, IL-10 and IFN-gamma were observed during rejection.

Vasopressin increases GABAergic inhibition of rat hypothalamic paraventricular nucleus neurons in vitro.

This investigation used an in vitro hypothalamic brain slice preparation and whole cell and perforated-patch recording to examine the response of magnocellular neurons in hypothalamic paraventricular nucleus (PVN) to bath applications of vasopressin (VP; 100-500 nM). In 22/38 cells, responses were characterized by an increase in the frequency of bicuculline-sensitive inhibitory postsynaptic potentials or currents with no detectable influence on excitatory postsynaptic events. Perforated-patch recordings confirmed that VP did not have an effect on intrinsic membrane properties of magnocellular PVN neurons (n = 17). Analysis of intrinsic membrane properties obtained with perforated-patch recording (n = 23) demonstrated that all of nine VP-sensitive neurons showed a rebound depolarization after transient membrane hyperpolarization from rest. By contrast, 12/14 nonresponding neurons displayed a delayed return to resting membrane potentials. Recordings of reversed inhibitory postsynaptic currents with chloride-loaded electrodes showed that responses to VP persisted in media containing glutamate receptor antagonists but were abolished in the presence of tetrodotoxin. In addition, responses were mimicked by vasotocin [Phe(2), Orn(8)], a selective V(1a) receptor agonist, and blocked by [beta-Mercapto-beta, beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2), Arg(8)]-VP (Manning compound), a V(1a)/OT receptor antagonist. Neither [deamino-Cys(1),Val(4),D-Arg(8)]-VP, a selective V(2) receptor agonist, nor oxytocin were effective. Collectively, the results imply that VP acts at V(1a) receptors to excite GABAergic neurons that are presynaptic to a population of magnocellular PVN neurons the identity of which features a unique rebound depolarization. Endogenous sources of VP may be VP-synthesizing neurons in suprachiasmatic nucleus, known to project toward the perinuclear regions of PVN, and/or the magnocellular neurons within PVN.