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Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for β-Cell Regeneration

Xu, Jin; Cui, Jiaxi; Del Campo, Aranzazu; Shin, Chong Hyun

The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of β-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in β-cell regeneration . PMID:26845333

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.

Amado, S; Simões, M J; Armada da Silva, P A S; Luís, A L; Shirosaki, Y; Lopes, M A; Santos, J D; Fregnan, F; Gambarotta, G; Raimondo, S; Fornaro, M; Veloso, A P; Varejão, A S P; Maurício, A C; Geuna, S


Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration . The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

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Research Article
Dentin reactions to caries are misinterpreted by histological “gold standards” [version 1; referees: 1 approved, 2 approved with reservations]
Priscila Florentino Silva, Danilo Augusto de Holanda Ferreira, Kássia Regina Simões Meira, Franklin Delano Soares Forte, Ana Maria Barros Chaves, Frederico Barbosa de Sousa
Priscila Florentino Silva, Danilo Augusto de Holanda Ferreira, [...] Kássia Regina Simões Meira, Franklin Delano Soares Forte, Ana Maria Barros Chaves, Frederico Barbosa de Sousa
PUBLISHED 16 一月 2014
Department of Morphology, Health Science Center, Federal University of Paraiba, Cidade Universitária, Paraiba, 58051-900, João Pessoa, Brazil Laboratory of Microscopy and Biological Image, Health Sciences Center, Federal University of Paraiba, Cidade Universitária, Paraiba, 58051-900, João Pessoa, Brazil Department of Clinical and Social Dentistry, Health Sciences Center, Federal University of Paraiba, Cidade Universitária, Paraiba, 58051-900, João Pessoa, Brazil


Dentin reactions to caries, crucial for pathogenesis and for the determination of the severity of caries lesions, are believed to be reasonably detected by stereomicroscopy (SM) and polarized light microscopy in quinoline (PLMQ), but accuracies are not available. Here, stereomicroscopy of wet (SW) and dry (SD) ground sections of natural occlusal caries lesions resulted in moderate (0.7, for normal dentin) and low accuracies (< 0.6, for carious and sclerotic dentin) as validated by contrast-corrected microradiography. Accuracies of PLMQ were moderate for both normal (0.71) and carious dentin (0.71). The hypothesis that detection of dentin reactions by SM and PLMQ would be influenced by the contrast quality of micrographic images was rejected. Dentin reactions were scored by SW, SD, PLMQ, and three types of microradiographic images with varying contrast qualities and each technique was compared against the one that resulted in the highest number of scores for each dentin reaction. Large differences resulted, mainly related to the detection of sclerotic dentin by both SW and SD, and normal and carious dentin by PLMQ. It is concluded that contrast-corrected microradiography should be preferred as the gold standard and SM and PLMQ should be avoided, but the relationship of PLMQ with dentin mineralization deserves further investigation.


sclerotic dentin, dentin, dental caries, histopathology, diagnosis, stereomicroscopy, microradiography

Corresponding Author(s)
Frederico Barbosa de Sousa ( [email protected] )
Grant information: The first author received a master degree scholarship from CNPq (Brazilian Ministry of Science, Innovation and Technology).


Dentin reactions to caries are crucial for the pathogenesis and severity determination of caries lesions. Since caries is mainly a demineralization process, the high ratio of X-ray absorbance between calcium and the chemical elements of the organic content, and the fact that the density of the mineral content is higher (more than 2 times) than that of the organic content, radiography with microscopic resolution (microradiography, MR) is considered as a highly reliable gold standard for detecting variations in dentin mineral content. Also widely accepted as gold standards for dentin reactions are stereomicroscopy (SM; commonly referred as histology) and (to a lesser extent) polarized light microscopy with quinoline as the immersion medium (PLMQ). The acceptance of SW, the currently most used “gold standard”, is based on studies reporting opaque and translucent dentin under SM related to radiolucent and radiopaque dentin, respectively. It is lacking, however, accuracy in numbers. To our knowledge, data regarding the accuracy of SM are available from only one study that included unerupted teeth and no data on translucent/sclerotic dentin. In addition, early studies with MR reported cases classified as translucent dentin by transmitted light microscopy (where the interaction of light with dentin is similar to that under SM) that were then classified as demineralized dentin by the use of MR.

Currently the only evidence for the detection of dentin reactions by PLMQ is only qualitative. Thus, research into the accuracy of SM and PLMQ is needed. Regarding the use of MR as a gold standard, it must be considered that images of microradiographic plates taken using transmitted light microscopy are commonly biased by the effect of heterogeneous illumination, which is inversely proportional to the objective magnification. Heterogeneous illumination is expected to influence judgment of brightness (a procedure required for diagnosis from MR images), possibly including bias when other techniques are validated using MR.

The aim of this study was three fold: to test the accuracy of both SM and PLMQ in detecting dentin reactions to natural caries; to test the hypothesis that elements of accuracy are influenced by the quality of the microradiographic image contrast; and to test the hypothesis that SM, PLMQ and MR (regardless of image contrast quality) detect dentin reactions equally.

Diagnosis of occlusal caries lesions using ICDAS II

Forty three erupted third molars with various stages of natural occlusal caries were collected from volunteers who signed consent terms (as approved by the Ethical Committee of the Federal University of Paraiba; certificate of ethical appreciation number 4125.0.000.126-10). All teeth were gently cleaned with 1% hypochlorite solution (Vetec, Brazil), mounted in a wax base and surrounded by a rubber dam isolator prior to analysis of their occlusal surfaces using the ICDAS II scoring system. Before obtaining final ICDAS scores for analysis, examiners were calibrated using a sub-set of the whole sample. Thirty occlusal sites were scored by two calibrated examiners (Kappa’s intra-examiner’s scores of 0.9 and 0.89, and 0.85 for inter-examiner agreement), with a one week interval, in order to test intra- and inter-examiner reproducibilities. Final scores were those obtained by a consensus between examiners. In nine teeth, two sites on the occlusal surface were selected, yielding a total of 52 occlusal caries lesions.

Ground section preparation

All teeth were cut longitudinally to their crowns (through their occlusal surfaces) using a diamond disc mounted (Kavo Sorensen, Brazil) in a low-speed handpiece under water irrigation, so that a section of the selected site with a given IDCAS score was obtained. All cuts were then ground using a customized metallic (brass) lapping jip and silicon carbide paper (granulations of 240–1200) under water irrigation to achieve a final thickness of ~100 ± 20 μm. Prepared ground sections (n = 52) were kept in a 0.02% sodium azide aqueous solution until examination.

Calibration of examiners of ground sections and selection of histological sites

At each ground section, histological sites (area of ~150 μm × 150 μm) presenting suggestive signs of normal, carious, or sclerotic dentin were selected. All examinations of ground sections (SM of wet and dry samples, three types of MR images, and PLMQ) were performed by the same two examiners, whose intra and inter-examiner reproducibilities were determined (using Kappa’s statistics) from their scores of all histological sites (of all samples) from each technique obtained with a one week interval. Examiners agreed on the final scores by consulting with each other.

Histological sites were selected from the outer half of the dentin layer, including the area adjacent to the deepest enamel lesion; at least one dentin reaction type per sample was included where possible. Cases were included in the sample when two sites had the same type of dentin reaction detected by SM, while showing different types of dentin reactions when detected using MR. Thus, up to 6 histological sites were selected per sample, yielding a total sample size of 168 sites.

Stereomicroscopy (SM)

SM (10× magnification) with reflected light was used to analyze ground sections under two conditions: wet (SW) and dried (SD; after exposure to 25ºC and 50% relative humidity for 2 hours). Temperature and relative humidity were measured just adjacent to the samples. Digital photomicrographs (digital camera Nikon D80) of wet and dried samples were obtained. Dentin reactions were scored as normal, carious discolored (white/yellow/brown), and translucent (“sclerotic”).

Microradiography (MR)

All samples were mounted in a microradiographic plate (resolution of 2000 lines/mm; AGHD plates, Microchrome Technology, San Jose, USA) and exposed to X-rays in a PCBA Inspector (tungsten anode filtered with a 0.25 mm-thick beryllium window, GE, Germany) for 25 minutes using 40 keV and 0.25 mA. Digital photomicrographs of the microradiographic plate were obtained in a transmitted light microscope (2× objective) under different conditions:

Condition 1: using the condenser aligned according to the principles of Kohler illumination for low magnification objectives;

Condition 2: no condenser and using a light shaping filter (Luminit, USA) above the field diaphragm.

The possible scores for dentin reactions using MR were: normal dentin, demineralized (radiolucent) dentin, and hypermineralized (highly radiopaque; sclerotic) dentin. Digital images were analysed (using the freeware program ImageJ, NIH, USA) with the following contrast conditions:

Image obtained with aligned condenser, without any adjustment of brightness and contrast from ImageJ, and no light shaping filter (NFNBC image);

Image obtained with aligned condenser, no light shaping filter, but with adjustment of brightness and contrast from ImageJ (NFBC image);

Image obtained without a condenser, with both a light shaping filter and adjustment of brightness and contrast from ImageJ (FBC image).

Such conditions created an ordinary scale of heterogeneous illumination of the field of view. Images without light shaping filter (NF) and FBC images presented a Gaussian normalized light intensity (R = 0.87 for both) across the field of view with heights of 0.13 and 0.05 (lower heterogeneity), respectively. Brightness and contrast adjustment (according to a consensus from both examiners) allowed this difference to be easily detected by the naked eye.

Polarized light microscopy in quinoline (PLMQ)

Ground sections were dried at room temperature for 24 hours, immersed in quinoline (Vetec, Brazil) for 24 hours, and then were positioned with the dentin tubules at – 45º on the stage of a polarizing microscope (Axioskop, Carl Zeiss, Germany) equipped with a Red I filter, 2× objective, and digital camera (Nikon D80, Japan). Dentin reactions were scored as either negatively (carious) or positively birefringent (normal). Since the technique of PLMQ is not intended to diagnosis dentin sclerosis, no diagnosis of sclerotic dentin was attempted. Color digital images were split into color channels using ImageJ, resulting in a sharp demarcation of negatively and positively birefringent areas.

We tested the accuracies of the SW, SD, and PLMQ techniques for detecting dentin reactions using the FBC MR image as the gold standard in all cases. Total positive (TP), total negative (TN), false positive (FP), and false negative values (FN) were obtained and used to calculate accuracy (AC) from:

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