The development of type 2 diabetes results from an interaction of hereditary factors and environmental factors. This study aimed to investigate the contribution of interrelatedness to the risk of developing type 2 diabetes in an isolated Dutch population.th and 21st century was constructed. In a case-control study, effects of interrelatedness and the risk of type 2 diabetes were estimated with Kinship Coefficients (KCs). Relative risks in first, second, and third degree relatives and spouses of inhabitants with type 2 diabetes were compared to matched controls.A genealogical database from inhabitants living on the former island Urk between the 14Patients with type 2 diabetes were more interrelated, expressed by a higher KC compared to controls . First, second and third degree relatives had an increased risk of developing type 2 diabetes. Second degree relatives had a similar risk,1.7 (1.5-2.0) as third degree relatives,1.8 (1.5-2.2). Spouses of patients with diabetes had a 3.4 (2.7-4.4) higher risk of developing type 2 diabetes.Interrelatedness was higher among inhabitants with type 2 diabetes compared to controls. This differences extended beyond the nuclear family, thereby supporting the hypothesis that interrelatedness contributed to the development of type 2 diabetes on Urk. However, the size of this effect was small and the patterns of risk in first, second and third degree relatives suggested that factors other than interrelatedness were the main contributors to the development of type 2 diabetes on Urk. Both environmental factors and hereditary factors contribute to the development of type 2 diabetes. On a population level, environmental factors can to a large extent explain the dramatic rise in prevalence of type 2 diabetes. During the Second World War, type 2 diabetes was virtually non-existent in the Netherlands  and the An increased familial aggregation of type 2 diabetes has been reported in various aggregation studies. Familial aggregation is a reflection of a variety of processes; ranging from genetic influences, family environment, intra-uterine and epigenetic processes . In pareAn alternative method of studying the contribution of heredity factors to the development of a disease is a pedigree study. This design has been used before to study the contribution of heredity to the development of rheumatoid arthritis on Iceland . In pediThis pedigree study was performed in a formerly isolated fisherman’s population living on the island Urk between the 14th and 21st century ,21. WithFor many centuries, the population on Urk have lived in isolation. Before 2001, immigration was limited and often temporarily; it was not until 1938 that the island of Urk was connected to the mainland by a dike. The inhabitants of Urk share a strong identity, both in religious and social aspects. The majority of the approximately 48,930 inhabitants who live and have lived on Urk up to 2001 descended from a small group of founders. A plague epidemic in 1637 killed the majority of the population; only 151 survived.In 1990, the general practitioners on Urk observed an increased age-adjusted prevalence of type 2 diabetes, at that time 3.1% compared to 1.8% in the overall Dutch population ,21. TherThe genealogy information was traced with the use of vital records, parish records, municipality records and other available documentation. All inhabitants were given an identification number, date of birth and death, and identifiers of father and mother. This resulted in a combined cohort of 48,930 inhabitants who were born, married or died in the municipality of Urk. The database included all inhabitants of Urk up to 2001 going back to the year 1300. From the 151 plague survivors in 1637, 105 (69.5%) were definitely identified through death certificates. The remaining 46 were not identified as survivors and were (most likely) included in the database through other records, for example through marital records.The founders of all individual ancestries of all inhabitants, including those with T2DM and the controls, were identified. A founder was defined as a person in the pedigree from whom no parent could be specified.The study was conducted in accordance with the regulations and laws regarding studies and privacy that applied before 2001 and in accordance to the principles expressed in the in the Declaration of Helsinki. The data needed for this study were extracted by the present owner of the database , and after de-identification provided to the researchers. A special commission appointed by the local community gave permission for this study. The presented research on data from Urk was carried out before it became mandatory, as from 2011 in the Netherlands, to submit all research proposals including non-WMO related research  for assessment to a medical ethical committee (METC). Before this decision, there was no obligation in the Netherlands to formally submit non-WMO related research to a METC. Therefore, according to Dutch rules and laws, no formal METC rules were infringed upon when using the anonymized Urk data. The METC of the Isala Hospital, the Netherlands, has retrospectively approved that this study was not WMO related, see Cases were defined as patients with type 2 diabetes known to the general practitioners who lived on Urk in the period from 1996 to 2000. The diagnosis of type 2 diabetes was based on criteria as described in the national guidelines of the Dutch College of General Practitioners from that time period . All patCase were matched (1:1) according to the absence of type 2 diabetes, concordance of gender, birth year, birth month and number of parents and grandparents in the database. Matching for the number of parents and grandparents was done to increase comparability regarding missing and available genealogical data and degree in which ancestries originated from Urk.For the analysis of heredity of type 2 diabetes, kinship coefficients (KCs) were calculated. The KC is defined as the probability that a random allele from two persons is inherited from a common ancestor . The proThe risk-ratio’s for relatives of persons with type 2 diabetes were derived from the number of relatives with type 2 diabetes divided by the total number of relatives, divided by the prevalence in the population. The calculation of risk-ratio’s of type 2 diabetes in relatives, were restricted to relatives born in the period between 1900 and 1960, since the majority of persons in the Netherlands develop type 2 diabetes at an older age. A difference in risk for siblings compared to genetically equally related first-degree relatives points to effects other than hereditary factors, for example effects of shared environment, that explain the increased risk of developing type 2 diabetes. The same holds true for an increased risk ratio in spouses.Three sensitivity analyses were carried out. The first aimed to control for shared environment in the nuclear family; by calculating KCs after excluding first- and second-degree related pairs. The second aimed to give insight in the degree of interrelatedness, by excluding all unrelated pairs. The third investigated whether a possible increased risk for developing type 2 diabetes in spouses was actually caused by increased inbreeding; by calculating the mean KC between spouses.t-test with estimation of separate variances through U statistics was used. An exact calculation method was developed to calculate the variance of the mean of all pairs, using the theory of U statistics. Testing the risk-ratio was done using the 95% confidence interval for likelihood ratiosThe KC is the sum of probabilities that two persons share the same identical allele, using all possible disjoint pathways in the genealogy database. The KC is calculated with a path-searching algorithm where all possible connections between pairs are weighted inversely proportional to 0.5 to the power k, where k is the length of the path. There are several pathways within the genealogy database that theoretically connect two persons, the mean KC was calculated by averaging the KC of all possible pair-wise combinations of inhabitants. To test for differences in relatedness among the inhabitants with and without type 2 diabetes. The mean KCs were calculated for all inhabitants with type 2 diabetes. These were compared with the mean KC for the matched control group. For testing relatedness in cases compared to controls a The total number of persons with type 2 diabetes in the period 1996 to 2000 known to their primary care physicians was 602. In the group with type 2 diabetes, 49 inhabitants could not be connected to another person through a common ancestor, compared to 78 inhabitants in the matched control group.t-test: t = 29·5; p<0·000). The slope of the fit-line . Meaning that common founders were present more frequently in ancestries of inhabitants with type 2 diabetes than in controls.From the persons born in the period between 1900 and 1960, 7.9%  descended from parents outside Urk. A total of 24% of inhabitants with type 2 diabetes were completely unrelated. A total number of 1145 founders were found for the combined groups of inhabitants with type 2 diabetes and controls. From the 1145 founders, 150 were founder for the group with type 2 diabetes only, 432 were founders for the control group only, and 563 were founder for both groups. The mean year of birth for founders was 1678 AD . The common founders were found on average more often in the ancestries of inhabitants with type 2 diabetes than in controls. None of the founders was identified as being an excessively more prominent founder in the group with type 2 diabetes. The difference in percentages in which the common founders were ancestors of both groups was significant (-3) and (3.7x10-3), respectively, The median interrelatedness was a seventh-degree relationship (range 5–13). The interrelatedness among inhabitants with type 2 diabetes was higher compared to the matched control group, mean KC (7.1x10-3), all first- and second-degree related pairs (4.6x10-3) and all unrelated inhabitants (7.6x10-3), was lower in the control group compared to inhabitants with type 2 diabetes. After excluding unrelated inhabitants, the significant difference in mean KC remained present in inhabitants with type 2 diabetes compared to controls. Matching for number of parents and grandparents in the database did not result in relevantly different results.The results from the three sensitivity analyses were consistent. The KC after excluding all first-degree related pairs (4.8x10-3 (SD 0.2x10-3), which was comparable with the mean KC of 3.9x10-3 (SD 0.2x10-3) of the spouses of controls.The mean KC between the 417 spouses of inhabitants with type 2 diabetes was 4.1x10The risk-ratios for first-degree related siblings and offspring, for second-degree related uncles/aunts and nephew/nieces as well as for third-degree related cousins were significantly increased, In this historically isolated Caucasian population, inhabitants with type 2 diabetes were more interrelated than matched controls. The risk of developing type 2 diabetes was increased in first degree relatives. The increased risk of type 2 diabetes extended beyond the nuclear family. The extent to which interrelatedness contributed to the risk of developing type 2 diabetes on Urk was small. The similar risk in second and third degree relatives and the high risk in spouses suggest that factors other than interrelatedness explain the increase in prevalence of type 2 diabetes on Urk.To our knowledge, this is the first study in a historically isolated population with many centuries of genealogic data that investigated the relationship between familial aggregation and type 2 diabetes. Previous familial aggregation studies on type 2 diabetes mostly focused on first-degree relatives and were subject to a risk for incomplete case ascertainment or over-reporting ,16,23,24The risk-ratio in siblings for developing type 2 diabetes was substantially higher compared to offspring, despite their equal probability of sharing a random allele. The difference in risk of developing type 2 diabetes between equally related offspring’s and siblings could be explained by factors other than hereditary factors ,25. For There was an interesting similarity in risk of type 2 diabetes in family members beyond the nuclear family of inhabitants with type 2 diabetes. The risk ratios for type 2 diabetes in cousins compared to nephews and nieces were more or less comparable, despite that they have a different probability of sharing a random allele. A possible explanation for this difference could be a difference in exposure to (family) environmental factors. This hypothesis is consistent with the recently proposed intergenerational prevention framework for type 2 diabetes, with emphasis on exposure to environmental factors in early life as risk factors for developing type 2 diabetes and as a basis for new type 2 diabetes prevention strategies .The remarkably high risk-ratio in spouses suggested that environmental factors in later life also influenced the risk of type 2 diabetes. An increased risk of developing diabetes in spouses was reported before . PossiblStrengths of this study were the inclusion of a total population and the matched case-control design. The main limitation was the incompleteness of the genealogy database; not all ancestry lines extended to the survivors of the plague epidemic. Furthermore, it was impossible to validate all connections between persons, and to obtain information on illegitimate births or false paternity. Incomplete knowledge of ancestry lines could have resulted in an underestimation of the degree of relatedness. Fortunately, the distribution of missing data was not different among inhabitants with and without type 2 diabetes making relevant differences in missing genealogical data unlikely. The mean KCs in inhabitants with type 2 diabetes compared to controls, matched for age, gender and the number of (grand) parents in the database supported the validity of the main results. Furthermore, the construction and verification of the database took a considerable amount of time and no clinical data were available after 2001.In conclusion, based on 7 centuries of familial aggregation in an isolated population, interrelatedness contributed to the development of type 2 diabetes. Although other factors than interrelatedness probably were the main contributors to the risk of type 2 diabetes on Urk. These results could potentially be of importance when developing type 2 diabetes prevention strategies.S1 File(PDF)Click here for additional data file.


Seventeen eyes of nine patients with FEVR who had Rdrag or Rfolds were retrospectively studied. Eyes were classified into four groups according to the severity of the retinal alterations: Group 1, without Rdrag or Rfolds (5 eyes); Group 2, with Rdrag (4 eyes); Group 3, with Rfolds (6 eyes); and Group 4, with Rfolds in which all major retinal vessels were involved (2 eyes). The amplitudes of all ERG components and the implicit times of the photopic a- and b-waves and 30-Hz flicker responses were decreased or prolonged as the severity of the retinal alterations increased (P < 0.01). The photopic negative response was most severely affected and nearly undetectable in all eyes in Groups 3 and 4, although the other ERG components were detectable in all eyes in Group 3 and one eye in Group 4. These results suggest the decrease of retinal functions was correlated with the degree of severity of Rdrag and Rfolds in eyes with FEVR. In addition, the function of the retinal ganglion cells appears to be more severely affected compared with the others.We evaluated the retinal function of retinal dragging (R Because the neurons giving rise to the different ERG components have been identified, it is possible to determine which type of neuron is altered by a specific disease processdrag and Rfolds in eyes with FEVR. We also determined whether there was a significant correlation between the severity of the retinal morphological alterations and to the degree of electrophysiological alterations of the different components of the full-field ERGs in eyes with FEVR. To accomplish this, we recorded full-field ERGs from 17 eyes of nine patients with FEVR and analyzed the different components of the scotopic and photopic ERGs.The purpose of this study was to determine which neural elements were affected by RThe protocol for this study was approved by the Institutional Review Board of the National Center for Child Health and Development, and they adhered to the tenets of the Declaration of Helsinki. This study was a retrospective observational case series.drag or Rfolds in at least in one eye and had undergone complete ophthalmic examinations under general or local anesthesia. The diagnosis of FEVR, Rdrag, and Rfolds was made according to the findings reported2drag, and/or Rfolds. Our ophthalmic examinations included ophthalmoscopy, fundus photography, fluorescein angiography (FA), and full-field ERGs. Fundus photography and FA were performed with the TRC-50LX  and RetCam 3  cameras.We studied nine patients with FEVR who had RThe full-field ERGs were recorded with a contact lens electrode, and the ERGs were elicited by white light-emitting diodes (LED) built into the contact lens electrode. The stimulus duration, intensity, and the background intensity were controlled by the electrical current delivered to the LEDs by a specially designed LED driver 1315The pupil was fully dilated with a combination of topical 0.5% tropicamide and 0.5% phenylephrine hydrochloride. The cornea was anesthetized by topical 0.4% oxybuprocaine hydrochloride before the contact lens electrode was inserted. The reference and ground electrodes were attached to the forehead and earlobe, respectively. The signals were amplified and bandpass filtered between 1 and 300 Hz .For controls, ERGs were recorded from 44 normal eyes under general anesthesia or local anesthesia. The mean ± standard deviation age of the controls was 7.5 ± 8.2 years, and they were one eye from normal eyes and the fellow normal eyes of unilateral peripapillary staphyloma, morning glory syndrome, and optic nerve hypoplasia. The 5 to 95 percentile amplitudes and implicit times for all ERG components of these normal eyes were used as the control values.drag and Rfolds; Group 2, with Rdrag only; Group 3, with Rfolds only; and Group 4, with Rfolds in which all major retinal vessels were affected. Representative fundus photographs and FAs of these four groups are shown in folds.The eyes were classified into four groups according to the degree of morphological alterations of the retina: Group 1, without RThe significance of the correlations between the fundus grading (Groups 1 to 4) and the amplitudes and implicit times of each ERG component was determined by Spearman’s rank correlation coefficient.folds in the nasal retina was excluded. The number of eyes in each group was: Group 1 had five eyes (29.4%); Group 2 had four eyes (23.5%); Group 3 had six eyes (35.3%); and Group 4 had two eyes (11.8%). All of the Rdrag and Rfolds existed in the temporal areas of the retina.Seventeen eyes of nine patients with FEVR were studied. The age at the time of the examinations ranged from 0.7 to 18.0 years . One eye with RRepresentative full-field ERGs from a normal control and from each of the groups are shown in drag to Rfolds eyes, i.e., from Group 1 to 4, was significantly correlated with the amplitudes of the b-waves of the rods, a- and b-waves and oscillatory potentials of the combined rod-cone responses, the a- and b-waves, PhNR, and PhNR/b-wave of the cone responses, and the 30-Hz flicker responses (P < 0.01). The increase in the severity from Rdrag to Rfolds eyes was also significantly correlated with the implicit times of the a- and b-waves of the cone responses, and the 30-Hz flicker responses (P < 0.01).Statistical analyses showed that the grade of severity from Rfolds was reported especially in the outer areas of the Rfolds in contrast to the rapid perfusion of the vessels within the Rfoldsfolds , which were determined by fundus and angiographic findings, was significantly correlated with the amplitudes of all ERG components. These results suggest that the depression of retinal function over the entire retina increases as the degree of RThe degree of the retinal abnormalities (Groups 1 to 4) was significantly correlated with the implicit times of the cone a- and b-waves, but were not correlated with the implicit times of the other ERG components. For example, the implicit times of the rod b-wave and combined rod-cone b-wave fell within the normal range in five of six eyes in Group 3, whereas the implicit times of the cone a-and b-waves were delayed beyond the normal range in more than one-half of the six eyes in Group 3 and the implicit times of 30-Hz flicker responses were delayed in all eyes of Group 3. The exact reason for such different degrees of abnormal implicit times of the different ERG components is unclear, but it might be due to different functional impairments of the rod and cone systems in eyes with FEVR.drag and Rfolds than the other retinal layers. This agrees with our recent optical coherence tomographic findings that the bundling of retinal nerve fiber occurs in the early stage in eyes with Rdrag, and the main components of the Rfolds are the retinal nerve fibersOne of the interesting findings was found in the PhNRs. Our results showed that the amplitudes of PhNR and the ratio of the PhNR/b-wave were significantly decreased in three of four eyes in Group 2 and the amplitude of the PhNR were completely undetectable in all eyes in Groups 3 and 4, whereas the amplitudes of all other components were still detectable in all eyes of Group 3 and one eye in Group 4 . Because15There current study had some limitations in this study. The number of study eyes were relatively small and the developmental effect depends on the patient age in retinal structure and ERG components needs to be considered.folds which may explain the reduced vision even in eyes with relatively mild Rdrag. Thus, it would be more important to focus on the retinal ganglion cells when treating eyes with Rdrag and Rfolds.In conclusion, our electrophysiological findings showed that the amplitudes and implicit times of the ERG components were decreased or prolonged with the severity of the morphological retinal alterations in eyes with FEVR. In addition, the function of the ganglion cells might be more impaired than that of other retinal cells in eyes with RHow to cite this article: Yaguchi, Y. et al. Electroretinographic effects of retinal dragging and retinal folds in eyes with familial exudative vitreoretinopathy. Sci. Rep.6, 30523; doi: 10.1038/srep30523 (2016).


Activation of the auditory cortex (AC) by a given sound pattern is plastic, depending, in largely unknown ways, on the physiological state and the behavioral context of the receiving animal and on the receiver's experience with the sounds. Such plasticity can be inferred when house mouse mothers respond maternally to pup ultrasounds right after parturition and naïve females have to learn to respond. Here we use c-FOS immunocytochemistry to quantify highly activated neurons in the AC fields and layers of seven groups of mothers and naïve females who have different knowledge about and are differently motivated to respond to acoustic models of pup ultrasounds of different behavioral significance. Profiles of FOS-positive cells in the AC primary fields , the ultrasonic field (UF), the secondary field (AII), and the dorsoposterior field (DP) suggest that activation reflects in AI, AAF, and UF the integration of sound properties with animal state-dependent factors, in the higher-order field AII the news value of a given sound in the behavioral context, and in the higher-order field DP the level of maternal motivation and, by left-hemisphere activation advantage, the recognition of the meaning of sounds in the given context. Anesthesia reduced activation in all fields, especially in cortical layers 2/3. Thus, plasticity in the AC is field-specific preparing different output of AC fields in the process of perception, recognition and responding to communication sounds. Further, the activation profiles of the auditory cortical fields suggest the differentiation between brains hormonally primed to know (mothers) and brains which acquired knowledge via implicit learning . In this way, auditory cortical activation discriminates between instinctive (mothers) and learned  cognition. Mammals including humans can perceive acoustic messages in a given behavioral context as meaningful, if the heard sound patterns match the context, and sound and context are congruent with the physiological state of the individual. The evaluation of sounds in the auditory system leads to the representation of acoustic object properties in the auditory cortex  were involved. Animals were kept in plastic cages (26.5 × 20 × 14 cm) at 22°C and at a 12 h light-dark cycle (light on at 7 h). Food and water were available ad libitum. The experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and were approved by the appropriate authority .House mice  aged 8–12 weeks were used. Two groups of primiparous mothers and five groups of virgin females  and virgin females (group C and G) with 5 days of pup-caring experience (PE) for releasing a preferred approach to the sound source  and shaped to tone burst series (3 bursts per second) of the desired burst duration . Sound pressure levels were adjusted to 80 dB for the 50 kHz tones and 66 dB for the 20 kHz tones  to provide about equal intensity of 60 dB above perceptual threshold for both frequencies Ehret,  in the cc-Fos expression started 1 week after the surgery, at the earliest.In seven mice  the position of the left-side AC with its five fields  equipped with a CCD camera  and evaluation software (Lucia measurement 5.0). The left side AC of the seven electrophysiologically mapped mice was localized in the frontal sections by the alcian-blue mark (5x objective). To delimit the dorsoventral elongation of the auditory cortical fields in the sections, scale-edited models of the area of AII-AAF-UF and AI-DP Figure  were madWith the stereotaxic relationships of the electrophysiological recording sites to the alcian-blue reference point at the AI-AAF border, this border was localized at average −2.92±0.08 mm posterior of the bregma point of the skull  from the non-tonotopically organized fields UF, AII and DP. The counts were assigned to layers 2/3, 4, 5, or 6 of the cortex which can be distinguished by their cytoarchitecture  was used to test for differences in data between the experimental groups of animals (independent variable). In the text, the respective F and DF  values are indicated in cases statistically significant differences were detected. In the figures concerning ANOVA results, the shown p-values indicate the results of the post-hoc Tukey-test.Statistical tests were run with SigmaPlot (version 11.0) software. Since the data in our samples were normally distributed (Kolmogorov-Smirnov test) they were plotted as means with standard deviations and compared with parametric tests. In all tests, the given level of significance was α ≤ 0.01 (two-tailed) in order to identify only major differences. For statistical comparisons between measures from the left and right hemisphere of animals of a given experimental group or of other dependent data , the paired c-Fos expression . Virgins with 5 days PE were also, but less than the mothers, engaged in maternal behavior. Virgins with 1 day PE were maternal at a low rate, independent of the type of sound and at significantly lower rates than the mothers , and anesthetized virgins with 5 days PE (group G) were unable to show any behavior  but was significantly larger in the left compared to the right hemisphere . The dorsoventral extent of the AC was determined about 500 μm caudal of the AI-AAF border. FOS-positive cells were also found over a larger extent in the left compared to the right hemisphere . Statistically significant differences between the experimental groups did not occur .The rostrocaudal extent of FOS-positive cells in the total AC did not differ among the experimental groups  and the animals in the groups A–F . This peak (P) occurred in AI and AAF of group A–F animals at an average rostrocaudal position of 47% of the total rostrocaudal extent of AI and AAF, respectively, away from the AAF-AI border , we calculated the average values from these IPIs in AI and AAF separately for each group and hemisphere of the awake animals and plotted the average IPI counts/section together with the respective average peak counts/section in Figure t-test for each hemisphere and experimental group, p < 0.01, or p < 0.001). Figure p > 0.2) and, therefore, averaged the IPI counts from AI and AAF of both hemispheres and all awake animals (groups A–F) with the resulting grand average background (13.5 FOS-positive cells/section) for the awake AI and AAF  and were very similar to the grand average background of groups A–F . The average AAF IPIs of group G were in both hemispheres significantly smaller  than the IPIs of the awake animals, smaller than the AI IPIs of group G , and smaller than the grand average background of groups A–F  did not show peak areas in AI or AAF. Therefore, all counts of FOS-positive cells from this group were taken as IPI or background. The average values for each hemisphere AI and AAF are plotted in Figure p > 0.2) without differences between the hemispheres . These numbers did not differ from the peak values neither from AI or AAF  in the respective groups but were significantly higher than the respective values in the IPIs . Virgin females hearing the 20 kHz tones (group F) had smaller numbers of FOS-positive cells in UF than the awake animals in the other groups . Further, the UF counts of group F animals were very similar to their IPI counts both for AI and AAF  and significantly smaller than the AI  and AAF  peak counts of the awake animals of groups A–E. The anesthetized females of group G had significantly smaller numbers of FOS-positive cells in UF than the awake animals in all the other groups .The ultrasonic field (UF) had no peaks in the number of FOS-positive cells along the rostrocaudal extent in any hemisphere of all 49 animals. Therefore, we calculated the mean number of FOS-positive cells from the 10 evaluated sections separately for both hemispheres of each animal. Then, we averaged these numbers over all animals within an experimental group and plotted these cell counts/section in Figure In summary, 50 kHz tones activated the UF significantly more than 20 kHz tones, however only in awake animals. Unexpectedly, in awake animals 50 kHz tones led to a local peak of activation above the general background in the middle of both AI and AAF. Compared to awake and behaving animals, anesthesia eliminated the peak activation in AI and AAF and significantly reduced the background activation in AAF and UF.av) was subtracted from its average UF value separately for each layer and the sum of all layers, and this difference (UF—IPIav) was averaged across all animals of a given group. Since the anesthetized animals of group G did not show a P area in AI and AAF and only reduced labeling below the grand average background in UF . There was no difference of IPI labeling in AI (both hemispheres) between the awake (A–F) and the anesthetized (G) animals . Significant differences occurred, however, in AAF where group G animals had lower values in the sum of layers  and in layer 2/3 . The average cell counts above the respective backgrounds in the layers 4, 5, and 6 were all close to zero  when the animals heard 50 kHz tones (groups A–E) did not only concern layer 2/3 but also layers 5 and 6. Generally larger numbers of FOS-positive cells were counted in layers 5 and 6 in the UF (UF—IPIav) compared to the numbers in the respective layers of the peak areas in AI and AAF  when the respective values from all animals of groups A-E were combined as shown in Figure t-test, p < 0.01) in the sum of layers and in layer 2/3 in the P areas of AI/AAF compared to UF. In addition, 20 kHz stimulation in the experimental group F led to significantly smaller numbers of FOS-positive cells  in the sum of layers and in layer 2/3 in UF compared to 50 kHz stimulation in the other groups .The results of the layer-related labeling patterns Figure  show:  activated cortical layers 2/3, 5, and 6 in the UF of all awake animals irrespective of the meaning of the sounds to the listening animals. The peak of labeling in AI and AAF in all groups of awake animals was due to increased activation only in layers 2/3. 20 kHz tones activated layers 2/3 more in AI/AAF than in UF, but did not lead to a significant number of labeled cells above background in layers 4, 5, and 6 of AI/AAF. Anesthesia significantly reduced labeling in all layers of UF, but only in layer 2/3 of AAF.Counts of FOS-positive cells in the non-primary fields AII and DP are shown in Figure t-test, p > 0.1 in each case). In contrast to the primary fields, 50 kHz tones activated AII differently depending on who was listening . The most striking group characteristics . Also the virgins with 5 days PE hearing the 50 kHz short tones (group D) and the virgins with 1 day PE hearing the 50 kHz long tones (group E) had significantly smaller numbers of FOS-positive cells than group B mothers . The smallest numbers of FOS-positive cells in AII occurred in the mothers hearing the 50 kHz long tones (group A), the anesthetized virgins hearing the same sounds (group G), and the awake virgins hearing the 20 kHz tones (group F). Their numbers were even below the grand average background, significant for the sum of all layers and layer 2/3 and 5, the latter only in group A .In AII, possible left-right differences in the numbers of FOS-positive cells were analyzed in each experimental group separately for the sum of all layers and each layer and no statistical differences were obtained . Hemisphere differences were not present in the other groups . In general , mothers and virgins with 5 days of PE (groups A–D) had significantly higher counts of FOS-positive cells than virgins with only 1 day of PE  and anesthetized animals (group G), irrespective of the sound they were stimulated with . This difference was present for the sum of labeled cells in all layers and layer 2/3, and for layers 5 and 6 of the left hemisphere  in the sum of all layers and in layer 2/3 . Anesthetized virgins (group G) had smaller numbers of FOS-positive cells in both hemispheres of DP  compared to the grand average background .In DP, hemisphere differences were noted for mothers and virgins with 5 days of PE hearing the 50 kHz long tones , i.e., there were significantly higher counts of FOS-positive cells in the left than in the right hemisphere for the sum of all layers, and for all individual layers of the mothers and layers 2/3 and 6 of the virgins  and virgin females (groups C–F), i.e., the processing of the different ultrasound models led to a large difference in activation of AII of the mothers but not of the virgins. The AII of anesthetized virgins was less activated, mainly in layers 2/3 and 5, compared to awake virgins hearing the same sounds. Activation of DP was especially high in the left brain hemisphere of group A and C animals. In addition, DP (at least of the left hemisphere) was highly activated in animals with high or medium maternal motivation  in the numbers of highly activated cells all over AI, AAF, and UF both with regard to the total numbers of activated cells and their distribution across the cortical layers Figures  did not On top of the background labeling, wriggling call models, whether meaningful or not, led to peaks of FOS-positive cells at the tonotopically correct positions in AI and AAF of awake animals with an increment of average 6 cells/section . Unexpectedly and in mismatch of tonotopic representation, ultrasounds are processed locally in AI and AAF without significant activation, however, of cortical output layers 5/6 in all respective groups (A–E). In addition, the background activation across all layers in these primary fields relates to levels of arousal and motor activity of the animals reflecting their internal state , however only in the presence of sounds. Hence, hypothesis 6 is not confirmed for primary AC, strengthening hypothesis 2 not only for primary AC in general but also specifically for processing in the cortical layers.Another striking result of our study concerns the group-specific activation in AII, separating mothers and virgins Figure . TherefoAII activation demonstrates an important difference depending on how knowledge about coherence of ultrasound and context has been acquired (hypothesis 3). Hormonal and sensory priming during pregnancy and giving birth in the mothers . Anesthetized animals (group G) were not motivated at all resulting in a low number, below the grand average background, of FOS-positive cells Figure . VirginsPerception of the meaning of ultrasounds by adult mice in the pup-caring context initiates adequate action, i.e., orienting toward the sound source Ehret, . DirecteThe left AC of all experimental groups was activated over a significantly larger area than the right AC supporting hypothesis 1 Figure . The lefGE conceived the work. GE and DG designed the study. DG and SS performed the experiments and analyzed the data. GE and DG wrote and SS revised the paper. GE, DG, and SS approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing. Even with the rapid developments in DNA sequencing technologies that enable the production of gigabases of sequence within a few days  of thewheat NimbleGen 40k unigene microarray, was hybHybridization and washing of the NimbleGen 17k 1BL ISBP arraywere performed according to manufacturer's procedure except that the hybridization time was extended to 120 hours. The arrays were dried by centrifugation at 1,200 × g for 1 min.The arrays were scanned using the InnoScan 900AL scanner . Data were extracted from scanned images using the NimbleScan 2.5 Software (Roche NimbleGen Inc.) that allows for automated grid alignment, extraction and generation of data files.et al. [et al. [For the wheat NimbleGen 40k unigene microarray,the normalization of the MTP pool data was done using the methods developed by Rustenholz et al. ,16. Two The normalization of the sorted 1BL chromosome data was done using automated script developed with the R software. A lowesP-value threshold of 0.05 were performed. All markers with at least 75% of their probes above this threshold and with a P-value below 0.05 were considered positive for a pool.For the 1BL ISBP NimbleGen array,the normalization was performed using automated scripts developed with the R software. The background intensity was estimated using the median of the intensities of the empty spots and subtracted fromthe intensity of each spot. After a log2 transformation, linear-lowess normalization was performed. Data were then subtracted by the median of the total spot intensity and divided by the standard deviation. For each probe,two thresholdswere calculated:the mean + 2 × the standard deviation and a Student's t-test at a -25, a tolerance of 12, and a MTP addresses that explain all the true positive pools; two overlapping BACs with one fingerprint matching a third BAC, included in a contig other than the two overlapping BACs, at a cut-off of 1e-25, a tolerance of 12, and a MTP addressing these three BACs that explain all the real positive pools;and a pair of overlapping BACs with one fingerprint matching a BAC in a second pair of overlapping BACs, included in a contig other than the two first overlapping BACs, at a cut-off of 1e-25, a tolerance of 12, and a MTP addressing these fourBACs that explain all the true positive pools. TheDSMP.pl script is available upon request from the corresponding author.A home-made Perl script, called DSMP.pl was developed for the deconvolution of molecular marker screening data produced on the three-dimensionalBAC pools of the MTP. Essentially, the script checks if two overlapping BACs of the physical map explain all the positive pools and can alsodeconvolute three others types of results: two non-overlapping BACs from two different contigs with fingerprints matching at acut-off of 1eThe molecular marker screening data produced on the plate pools of the whole 1BL BAC library were deconvoluted with the Elephantsoftware and the default parameters.Briefly,et al. [The Roche454 sequencing of the 1BL sorted chromosome was performed by Wicker et al. . They prSequences were analyzed using Basic Local Alignment Search Tool (BLAST) software . BLASTN Oryza sativa) [B.distachyon[Sorghum bicolour) peptides [B. distachyon gene identified.BLASTXanalyses of the 39,179 unigenes sequences represented on the wheat NimbleGen 40k microarray against the databases of all rice ( sativa) , B.distaistachyon and sorgpeptides  were perB.distachyon. We also checked with the latest release of the wheat unigene build (version 59) for further redundancy compared to the build version 55 that was used to build the wheat NimbleGen 40K unigene microarray[B.distachyon, and were grouped in the same cluster of the new build version 59, only one unigene was kept on the 1BL physical map.To eliminate redundancy in the unigene set, we used information from orthologous genes in rice and icroarray. When unet al. [TBLASTXanalyses of the coding region of 128 rice genes corresponding to 64 ancient duplicated genes between rice chromosome 1 and 5 as defined by Muratet al. were perfThe GenomeZipper ,37 of chB.distachyon  against three reference genomes of ion v1.2,), rice  and sorion v1.2,) were peThe corresponding orthologous genes were anchored to the marker backbone via bi-directional blast hits. Genes without marker association were located according to their position in the corresponding reference genomes. The obtained 1BL linear ordered gene map was then expanded using 1BL repeat filtered contigs, wheat ESTs ( v1.19) , and wheThe ordering of genes along chromosome 1BL was based first on the ordering of the physical contigs and second on the position of the genes within contigs. Each 1BL gene was assigned to one or several overlapping BACs using the information from the 40k unigenes NimbleGen array.The position of each gene in each contig was estimated based on the average of the consensus band map coordinate of the BAC(s) containing the gene. When several genes were assigned to the same BAC(s), the order was based on the synteny information when available or randomly chosen if not. The position of the genes with a clear position relative to their neighbor or with synteny information was consideredto have high confidence. The position of the genes with unclear placement with their neighbor genes or with discrepancy between synteny information and position in the contig was consideredto have low confidence.B. distachyon for the GenomeZipper and the 1,161 1BL unigenes andall genes with the same orthologous genes in one or more of the three cereals were considered as common genes.Figure To compare the two virtual gene orders, we identified the common genes. BLASTN  analysesA genome browser of the physical map of the wheat chromosome 1BL is available from the Unité de Recherche Génomique Info website . All theBAC: bacterial artificial chromosome; BLAST: Basic Local Alignment Search Tool; bp: base pair; cM: centimorgan; COS: conserved orthologous set; Elephant: electronic physical map anchoring tool; EST: expressed sequence tag; FPB: FingerPrint Background removal; FPC: FingerPrinted Contigs; Gb: gigabase; kb: kilobase; ISBP: insertion site-based polymorphism; ITMI: International Triticeae Mapping Initiative; IWGSC: International Wheat Genome Sequencing Consortium; LD: linkage disequilibrium; LTC: Linear Topological Contig; MAGIC: multi-parent advanced generation inter-cross; Mb: megabase; MTP: minimal tiling path; QTL:quantitative trait loci; RFLP: restriction fragment length polymorphisms; SSR: single sequence repeats; TE: transposable elements; WGP: Whole Genome Profiling; WGS: whole genome shotgun.The authors declare that they have no competing interests.RP carried out the physical mapping, the anchoring of the physical map in the deletion bin and genetic maps, the comparative genomic analysis, the gene space analysis, and drafted the manuscript. EP supervised the physical mapping and the anchoring. IB produced all the PCR marker data and the hybridization on the wheat NimbleGen 40k unigene microarray and analyzed the hybridization results. PS produced all the genetic maps. FC participated in the development of the DMSP.pl Perl script for anchoring. CL developed the 1BL NimbleGen 17K ISBP microarray, produced the hybridization data and analyzed the hybridization results. HŠ, JS and JD sorted the DNA of the wheat chromosome 1BL and produced the 1BL BAC library. AB, SV and HB handled the 1BL BAC library and produced the three-dimensionalMTP pools. ZF and AK participated in the assembly of the physical map with LTC. FC, FM and SS produced the high-information-content fingerprints of the whole 1BL BAC library. MM and KM produced the GenomeZipper of the wheat chromosome 1BL and participated in the comparison of the two virtual gene orders. CF acquired the funding, supervised all the analyses and drafted the manuscript.Information on the 5,489 markers assigned to the physical map of wheat chromosome 1BL. Table listing the marker name, the marker type, the names of the deletion bin,the ID of the BAC clones andthe name of the physical contigsto which the marker is assigned,the position (in cM) of the markerson the Chinese Spring ×RenanF2 genetic map,the order of the geneand the level of confidence in this order, the unigene length, the ID of the orthologous genes in rice, sorghum and Brachypodium and the gene islands number for the 1,161 genes assigned to the physical map of the wheat chromosome 1BL.Click here for fileFeatures of the 616 contigs of the final LTC physical map of the wheat chromosome 1BL. Table listing the contig name, the number of clones, the contig size, the total number of markers, the number of genes, the name of the deletion bin to which the contig is assigned, the position on the 1BL neighbor genetic map and the number of the ordered contigs.Click here for fileGenomeZipper of wheat chromosome 1BL. Table listing the marker name, the position on the genotyping by sequencing genetic map, the ID of the orthologous genes in rice, sorghum and Brachypodium, the ID of the full-length cDNA matching the marker or the orthologous genes, the wheat Illumina reads matching the marker or orthologous gene and the ID of the wheat ESTs matching the marker or orthologous gene.Click here for file


Dental materials in general are tested in different animal models prior to the clinical use in humans, except for bleaching agents. 2O2 gel in the pulp tissue during in-office bleaching of rats’ vital teeth. To evaluate an experimental rat model for comparative studies of bleaching agents, by investigating the influence of different concentrations and application times of H2O2 gels, respectively, for 5, 10, 15, 30, or 45 min (n=10 rats/group). Ten animals were untreated (control). The rats were killed after 2 or 30 days, and the maxillae were examined by light microscopy. Inflammation was evaluated through histomorphometric analysis with inflammatory cell count in the coronal and radicular thirds of the pulp. Fibroblasts were also counted. Scores were attributed to odontoblastic layer and vascular changes. Tertiary dentin area and pulp chamber central area were measured histomorphometrically. Data were compared by analysis of variance and Kruskal-Wallis test (p<0.05). The right and left maxillary molars of 50 Wistar rats were bleached with 20% and 35% H After 2 days, the amount of inflammatory cells increased in the coronal pulp occlusal third up to the 15-min application groups of each bleaching gel. In the groups exposed to each concentration for 30 and 45 min, the number of inflammatory cells decreased along with the appearance of necrotic areas. After 30 days, reduction on the pulp chamber central area and enlargement of the tertiary dentin area were observed, without the detection of inflammation areas. The rat model of extracoronal bleaching showed to be adequate for studies of bleaching protocols, as it was possible to observe alterations in the pulp tissues and tooth structure caused by different concentrations and application periods of bleaching agents. Some of the changes, such as inflammation and bleeding, reversed after 60 days2O2 gel resulted in necrosis near to the pulp horns in rats2O2 gel on human premolars did not cause pathological changes in the dental pulpin vivo model analyzed, as well as the bleaching protocols employed, determined different results.An increase in vascular permeability depending on the duration of the bleaching procedures has been observed in rats’ incisors2O2, and consequently more damage to pulp tissues2O2 damage to pulp tissues. This model will enable the evaluation of new dosages, formulations and concentrations of bleaching agents that arise in the market, in addition to the evaluation of potential therapeutic agents that may be used to minimize the damage caused by H2O2 to the pulp tissue, in different application protocols,Thus, the lesser thickness of enamel and dentin in teeth of rats might allow greater penetration of H,The choice of rats was due to the ease of standardization and control of these animals, and the possibility of performing other tests2O2 gel during in-office bleaching of rats’ vital teeth. It was hypothesized that: (I) the H2O2 in bleaching gel is capable of penetrating pulp tissue and causing greater damages with increasing time of application and H2O2 concentration; (II) pulp tissue is capable of recuperating from the damages caused by H2O2 after long periods of time.Therefore, the purpose of this study was to characterize an experimental animal model for comparative studies of bleaching agents, by investigating the influence of different concentrations and application times of HSixty male Wistar rats (180-200g) were used in this study. The animals were housed in a temperature-controlled environment (22°C±1°C) on a standard light–dark schedule with unrestricted access to food and water. The experimental protocol was approved by the Ethics Committee (CEUA 2013-01253) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health .2O2 , respectively, for 5, 10, 15, 30, or 45 min (n=10 rats/group). Ten animals (controls) did not receive any treatment.The rats were anesthetized with intramuscular injections of ketamine  and xylazine . The right and left molars in every animal were bleached with 20%  and 35% HAnimals were killed with an overdose of the anesthetic solution 2 or 30 days after the bleaching sessions. Their bilateral maxillae were separated, dissected, and fixed in a 10% buffered formalin solution for 24 h. The specimens were decalcified in a 10% ethylenediaminetetraacetic acid (EDTA) solution for three months, and then dehydrated in a graded ethanol series, embedded in paraffin, cut into 6-μm sagittal cross-sections, and stained with hematoxylin and eosin (H&E).The serial histological sections of each specimen were selected from the point where the mesial root of the first molar was seen in all its longitudinal extension.2 field in each third of the pulp tissue of each specimen, examined under light microscopy .The coronal pulp was divided into occlusal, middle, and cervical thirds and the radicular pulp was divided into cervical, middle, and apical thirdsScores were attributed to the odontoblastic layer in each third of the pulp tissue, as follows: 1- intact odontoblastic layer; 2- disorganized odontoblastic layer; or 3-disruption of the odontoblastic layer. Scores for vascular changes were also assigned as follows: 1- normality; 2- increase in the number of blood vessels; or 3- necrosis.The mean central area of the pulp chamber was measured by image processing software  . With thAfter the application of the Kolmogorov-Smirnov test of normality, the data obtained in counts of inflammatory cells and fibroblasts were submitted to two-way analysis of variance and Tukey’s test for intergroup comparisons at the significance level of 5% (p<0.05). The scores obtained in the analysis of odontoblastic layer and vascular changes were submitted to Kruskal-Wallis and Dunn’s tests (p<0.05). The values obtained in the mean central area of the pulp chamber were submitted to Kolmogorov-Smirnov test of normality and one-way analysis of variance (p<0.05).The dental pulp of the control animals exhibited well-defined acellular and cell-rich layers under an intact odontoblastic layer and an even distribution of cells, blood vessels, and extracellular matrix structures .This group exhibited no inflammatory infiltrate. The dental pulp appeared similar to that of the control group. The odontoblastic layer was intact and the blood vessels showed normal characteristics. The cementum, periodontal ligament, alveolar bone, and other supporting structures also seemed normal .This group did not exhibit a considerable amount of inflammatory cells. There was a reduction in the amount of fibroblasts in the occlusal and middle thirds of the coronal pulp. The odontoblastic layer was partially disorganized in the occlusal third, and there was an increase in the number of blood vessels in the occlusal and middle thirds of the coronal pulp .There was an increased amount of inflammatory cells in the occlusal and middle thirds of the coronal pulp, the amount of fibroblasts was reduced, and there was an increased amount of blood vessels. The odontoblastic layer was partially disorganized in the occlusal third .The highest number of inflammatory cells was found in the middle third of the coronal pulp in this group. There was a large reduction in the amount of fibroblasts in the occlusal third, where there was disruption of the odontoblastic layer. The amount of blood vessels increased in the occlusal and middle thirds of the coronal pulp . The radThis group showed an increased number of inflammatory cells in the cervical and middle thirds of the coronal pulp. The occlusal third showed necrotic areas. A reduction in the number of fibroblasts was observed in the cervical third of the crown. The odontoblastic layer was absent in the occlusal third and partly disorganized in the middle third of the coronal pulp. There was an insignificant amount of inflammatory cells in the cervical third of the radicular pulp .The amount of inflammatory cells in this group was not significant. The amount of fibroblasts reduced in the occlusal third of the coronal pulp, where the odontoblastic layer was partially disorganized. An increase in the number of blood vessels was observed in all areas of the coronal pulp .There was an increase in the number of inflammatory cells in the occlusal and middle thirds of the coronal pulp in this group. The amount of fibroblasts reduced in the occlusal third. The odontoblastic layer was absent in the occlusal third, and partly disorganized in the middle third of the coronal pulp. There was an increase in the number of blood vessels throughout the coronal pulp .There was an increase in the number of inflammatory cells in the occlusal and middle thirds of the coronal pulp, while the number of fibroblasts reduced. The odontoblastic layer was absent in the occlusal third, and partly disorganized in the middle third of the coronary pulp. There was an increase in the number of blood vessels throughout the coronal pulp .The number of inflammatory cells and fibroblasts reduced in the occlusal third, where necrotic areas were present. There was an increase in the number of inflammatory cells in the middle and cervical thirds of the coronary pulp. The amount of fibroblasts was still low in the middle third of the coronary pulp. The odontoblastic layer was absent in the occlusal and middle thirds of the crown. There was an increase in the number of blood vessels in the middle and cervical thirds of the coronal pulp. The occlusal third was characterized as necrotic. A small amount of inflammatory cells was found in the cervical third of the radicular pulp .There was necrosis in the occlusal third of this group, with absence of inflammatory cells and fibroblasts. The number of inflammatory cells increased in the cervical and middle thirds of the coronal pulp, and in the cervical third of the radicular pulp. The number of fibroblasts reduced in these thirds. The odontoblastic layer was absent in the occlusal and middle thirds of the crown, and partially disorganized in the cervical third. The number of blood vessels increased in the cervical third of the coronary and radicular pulp. The remaining thirds seemed normal .Thirty days after the bleaching sessions, all the specimens showed normal dental pulp. However, the central area of the pulp chamber reduced, and the tertiary dentin area increased .At 30 days, the specimens showed a gradual increase in the tertiary dentin area. Significant differences were observed between the 35%–45 min group and the other groups, except for the 35%–30 min group (p<0.05). The 20%–5 min, 20%–10 min, 20%–15 min, and 35%–5 min groups did not differ significantly from the control group (p>0.05) .Tooth bleaching is an aesthetic alternative for discolored teeth, but it has potential adverse effects that are not yet completely understoodin vitro studies have shown that ROS generated by the H2O2 of bleaching gels are capable of causing histochemical and morphological changes in enamel and dentin,A large number of In vivo studies showed cellular damage, classified as mild to severe. These include studies performed in dog teeth,,,. Cell culture studies also demonstrated cellular damage as apoptosis,ex vivo manipulated cells have significant importance in preliminary studies of bleaching agents. However, in those studies, the pulp cells are not examined as organized tissues. Teeth have vital pulp components that can prevent or hinder the H2O2 effects in pulp tissues, such as dentinal fluid, cytoplasmatic extensions,2O2,in vivo experiments are the ones that best represent the reality of bleaching effects.Studies predominantly with 2O2 gel on human premolars does not cause pathological changes in the dental pulpApplication of 38% HEven though variations of the pulp response have been shown in human teeth, our study aimed to characterize an experimental animal model of easy reproduction and standardization for the study of new bleaching agents, posology, concentrations, and application time.2O2 showed greater changes immediately beneath the region where the gel was applied2O2 gel applied for 30 or 45 min and 20% H2O2 gel for 45 min. Severe pulp damage may occur when bleaching agents are applied on the buccal surface of teeth with thin enamel and dentin,,In dog teeth, dental bleaching using 35% HThe use of rats as the experimental model presents advantages such as ease of handling, reproduction, control, predictability,,Despite the difference in enamel and dentin thickness between humans and rat teeth , they both show the same proportion of these structures2O2 gel applied for 30 or 45 min caused necrosis or a severe inflammatory response in the dental pulp, especially in the upper coronal two-thirds. Clinically, in-office bleaching with high H2O2 concentrations for 30 to 45 min in a single session is frequently associated with a high incidence of tooth sensitivityIn the present study, 35% H2O2 detected in the pulp chamber is related to the concentration and application time of the gel2O2 gel applied for 30 min, as well as 20% and 35% H2O2 gel for 45 min, was related to changes in vascular permeability2O2),2O2). Our results allow choosing a concentration and time of application for comparative analysis in the initial inflammatory process (at two days) as well as in the subsequent reparative process (at 30 days).The amount of H2O2 gel can diffuse through enamel and dentin and cause significant cell damage,In our evaluation at 30 days after bleaching, we observed that all the groups showed signs of repair. Tertiary dentin was formed to protect the dental pulp, reducing the pulp chamber central area, and inflammatory cells were absent. The groups of low concentration/application time showed significant differences from those of high concentration/application time. Studies of the effects of high concentrations of bleaching gels on pulp cell cultures have shown that products released by 35% H2O2 can penetrate the cell membrane, increase alkaline phosphatase activity, and induce apoptosis in the periodontal ligament and dental pulpHThe characterization of this experimental model does not replace human trials, but allows the knowledge of new bleaching agents mechanism of action; the comparison between protocols of bleaching; and the study of desensitizing and remineralizing agents used before and after bleaching to minimize effects on pulp tissues.2O2 gel caused immediate inflammation and accelerated aging of the dental pulp by inducing deposition of tertiary dentin, and the degree of damage increased with increasing concentration and application time of the bleaching agent.In conclusion, the rat model of extracoronal bleaching showed to be adequate for studies of bleaching protocols, as it was possible to observe alterations in pulp tissues and tooth structure caused by different concentrations and application times of bleaching agents. In-office bleaching with H


Clinical studies have shown the similarity of the spectrum of physiological effects of Selank and classical benzodiazepines, such as diazepam and phenazepam. These data suggest that there is a similar basis of their mechanism of action. To test this hypothesis we studied the effect of Selank and GABA on the expression of genes involved in neurotransmission. We analyzed the expression of 84 genes involved in neurotransmission  in the frontal cortex of rats 1 and 3 h after the administration of Selank or GABA (300 μg/kg) using real-time PCR method. We found significant changes in the expression of 45 genes 1 h after the administration of the compounds. Three hours after Selank or GABA administration, 22 genes changed their expression. We found positive correlation between the changes in genes expression within 1 h after administration of Selank or GABA. Our results showed that Selank caused a number of alterations in the expression of genes involved in neurotransmission. The data obtained indicate that Selank is characterized by its complex effects on nerve cells, and one of its possible molecular mechanisms is associated with allosteric modulation of the GABAergic system. Regulatory peptides play key roles in the formation, development, and normal functioning of the nervous system. They are not understood fully despite the accumulating experimental data in recent years. The study of their mechanisms of action is of particular interest because regulatory peptides have potential in the creation of safe drugs on their basis with specific clinical properties and direct physiological effects. One representative of this class of drugs is the synthetic regulatory peptide Selank. It was designed and produced at the Institute of Molecular Genetics, Russian Academy of Sciences, in cooperation with the V.V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences. Selank is a synthetic analog of the endogenous tuftsin molecule (the short Thr-Lys-Pro-Arg fragment of the human immunoglobulin G heavy chain), which was elongated at the C terminus via the addition of three natural L-amino acids (Pro-Gly-Pro) to improve its metabolic stability and yield a relatively longer duration  and can increase the inhibitory action of GABA, the major inhibitory neurotransmitter in the CNS. The similarity of the spectrum of physiological effects of Selank and classical benzodiazepines (such as diazepam and phenazepam) suggests that there is a similar basis of their mechanism of action; that is, the allosteric modulation of GABAA receptors.Clinical studies have shown that the effect of Selank is similar to that of tranquilizers at low doses, but is not accompanied by the unwanted side effects of benzodiazepine tranquilizers such as amnesia, withdrawal, and dependence  and GABA (γ-aminobutyric acid) were dissolved to a concentration of 10 mg/ml in deionized water.n = 30) were divided into three groups: one control group (n1 = 10) and two experimental groups: Selank group (n2 = 10) and GABA group (n3 = 10). A single intranasal administration of the water solution of Selank or GABA was performed on each animal from the experimental groups (6 μl at the concentration of 300 μg to 1 kg of body weight) and the equivalent volume of deionized water was performed on each animal from the control group. Selank dose of 300 μg/kg was selected based on the data that this dose was the most effective therapy dose exerting anxiolytic action  and the statement of the ethics committee of the Institute of Molecular Genetics, Russian Academy of Sciences.The animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals  according to the manufacturer's protocol.Frontal cortex tissues obtained from each rat were pooled according to the corresponding groups and time points, resulting in six pools. Total RNA was extracted from each resulting pool of tissues using the RNeasy® Mini Kit  according to the manufacturer's protocol. First-strand cDNAs were synthesized using the RT2 Profiler™ PCR Array: CAPR11632 . Amplification was carried out on the device StepOnePlus™ Real-Time qPCR System  using the RT2 SYBR Green Mastermixes . Thermal cycling was carried out as follows: (1) 95°C for 600 s, followed by (2) 40 cycles of 15 s at 95°C and 60 s at 60°C. All reactions were repeated three times in each group for each time point.The effect of Selank and GABA on the expression of genes was studied with the help of the real-time PCR method using a Custom RTCt-values of the reference genes. Statistical data analysis of the normalized Ct-values was performed using the Relative Expression Software Tool 2009 (REST 2009) v.2.0.13, and Statistica 8.0. Genes with significant changes (p ≤ 0.05) in the mRNA level by 1.5 times or more were considered in the analysis to assess the changes in expression by the action of the test compounds.The threshold reaction cycle (Ct) values obtained for the genes under study were normalized to the We studied the effects of Selank and GABA on changes in the mRNA levels of 84 genes involved in neurotransmission in the frontal cortex of rats 1 and 3 h after the intranasal administration of the compounds. The intranasal administration of Selank was shown to be optimal for delivery of peptide molecules in the CNS  had a high threshold reaction cycle (Ct > 35), indicating a low content of mRNA in the tissue. Therefore, these genes were excluded from further analysis.Preliminary analysis showed that, among the 84 studied genes, seven genes  changed only after Selank administration, and that of 16 genes  changed only after GABA administration.Of the remaining 77 genes, summarily 45 genes showed changes in mRNA level 1 h after Selank or GABA administration , Gabrq , and Hcrt . An increase in mRNA level was noted only for two genes; the mRNA level of Drd3 increased 3.4 and 2 times after Selank and GABA administration, respectively, and the mRNA level of Gabrb3 increased 1.6 and 2.1 times after Selank and GABA administration, respectively.For 23 genes whose expression changed 1 h after administration of either compound, a decrease in the mRNA level was observed. The effect was especially pronounced for three genes: Junb and Gabrd . For the five genes Birc5, Gabra1, Gabrb1, Gabrg3, and Glul, the increase in mRNA level was no greater than two times compared with that of the control group. The genes whose expression changed only after Selank administration (1-h time point) were characterized by an increase in mRNA level, although not more than 2 times; the mRNA levels of Drd2 and Ptgs2 increased 1.6 times, and those of Drd1a and Slc6a13 increased by 2 times.Of the 16 genes whose expression changed only after GABA administration (1-h time point), 11 showed reduction in mRNA expression, in particular Abat, Adcy7, Ccnd1, Drd5, Gabre, Gabrq, HcRt, Htr3a, Prlhr, Slc32a1, Slc6a1, Slc6a11, and Slc8a3) changed only after Selank administration. The mRNA levels of five genes  changed only after GABA administration. The mRNA levels of four genes  changed after administration of either compound  was more than twice that of the genes whose expression changed after 3 h (22 genes). Most of the genes (76%) showed a decrease in mRNA level 1 h after administration of the test compounds. By contrast, the mRNA level increased in nearly almost all genes (95%) whose expression changed 3 h after administration of the test compounds , 3 h after GABA administration, the number of genes whose expression changed was reduced to only 9 (12%). In contrast to GABA, Selank affected the expression of fewer genes 1 h after the administration , but the reduction in the number of genes whose expression changed 3 h after Selank administration was smaller .Another interesting feature was revealed: at the 1-h time point; 56% of the genes exhibited changed expression regardless of the test compound, whereas the change in expression was selective for one compound for 44% of the genes. A different pattern was seen at the 3 h-time point; only 18% of the genes exhibited changed expression regardless of the test compound, whereas the change in expression of 82% of the genes was selective for one compound . By contrast, a negative correlation was observed between the changes in gene expression at the 3-h time-point after the administration of Selank or GABA .Correlation analysis was performed for those genes whose mRNA level changed significantly after administration of the test compounds. A positive correlation was observed between the changes in gene expression within 1 h after administration of Selank or GABA  altered only under the influence of Selank at the 1-h time point. Intriguing results were obtained in relation to Slc6a13, which encodes the low-affinity transporter of the GABA GAT-2. GAT-2 plays a key role in peripheral mechanisms involved in the work of GABAergic system and is also responsible for the redistribution and metabolism of drugs, which can affect the operation of the GABAergic system (Schlessinger et al., Drd2 and Drd1a encode dopamine receptors, which are associated with G-protein and are involved in the regulation of adenylate cyclase activity, thereby mediating intracellular signal transduction (Beaulieu and Gainetdinov, Drd5 expression at the 3-h time point was observed only for Selank. Drd5 encodes the dopamine receptor, which plays a key role in the formation of memory and learning processes by ensuring long-term potentiation (Beaulieu and Gainetdinov, Interestingly, the mRNA levels of four genes (Hcrt 3 h after Selank administration. This gene encodes a precursor of orexins and is involved in the regulation of the balance between sleep and wakefulness (Ohno and Sakurai, Hcrt increases after a single administration of Selank (Kolomin et al., Hcrt may be in the foundation of the normalizing effect Selank has on the balance of sleep patterns in patients with general anxiety disorders. The observed changes may also explain the lack of hypnosedative action of Selank, which is common for classical benzodiazepines, because of the shift in the balance toward wakefulness.Of particular interest is the significant change in the mRNA level of Thus, our obtained data indicate that, like other peptide drugs, Selank is characterized by its complex effects on nerve cells. One of its possible molecular mechanisms is associated with allosteric modulation of the operation of the GABAergic system. At the same time, Selank may act through other systems such as the dopamine and serotonergic systems. Further research is needed to identify all aspects of the mechanisms of action of Selank and the effects of this peptide on various neurotransmitter systems in different models.AV, TK, LA performed the experimental work. AV, MS, and PS undertook all statistical analyses and helped with their interpretation. PS, MS designed the study. AV, MS wrote the first draft of the manuscript. MS and PS contributed to the final writing of the manuscript. SL, NM was involved in revising the manuscript critically for important intellectual content. All authors contributed to and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Idiopathic normal pressure hydrocephalus (iNPH) is clinically important as a treatable gait disturbance and/or preventable dementia by shunt operation. iNPH usually occurs as a sporadic onset; however, genetic factor(s) is suggested to be involved in the pathogenesis of iNPH because of the presence of familial onset (two or more patients with NPH in a single family) of the disease, whose clinical and brain MRI features are indistinguishable from those of iNPH. Up to the present time, the genetic risk factor(s) for iNPH remains undetermined.We made a whole-genome analysis for copy number variations (CNV), using DNA from peripheral blood of 8 subjects with MRI-supported possible iNPH/AVIM and 110 healthy controls. As a result, 4 (50%) of the 8 subjects were found to have a segmental copy number loss within the SFMBT1 (Scm-like with four MBT domains protein 1) gene. The copy number loss was heterozygous, and occurred at the 12 kb region within intron 2 of the SFMBT1 gene. Such genetic change was detected in one (0.9%) of the 110 controls. Next, using qPCR, we examined 8 patients with definite iNPH, who were shunt-responsive, and found that five (63%) of the 8 patients had a copy number loss at the same locus of the SFMBT1 gene. Immunohistochemical examination of the normal human brain revealed that the SFMBT1 protein localized mainly in the arterial walls, the ependymal cells, and the epithelium of the choroid plexus, which play a crucial role in the secretion, flow, and absorption of cerebrospinal fluid.The present study suggests that a copy number loss within the SFMBT1 gene may be a genetic risk factor for iNPH. Further studies on SFMBT1 will contribute to the elucidation of molecular basis of iNPH.


Citrus trees that are grown, especially in the highly weathered soils of the tropics due to the inherently low nutrient availability in the soil solution. Leaf sprays containing micronutrients are commonly applied to orchards, even though the nutrient supply via soil could be of practical value. This study aimed to evaluate the effect of Zn fertilizers that are applied to the soil surface on absorption and partitioning of the nutrient by citrus trees. A greenhouse experiment was conducted with one-year-old sweet orange trees. The plants were grown in soils with different textures (18.1 or 64.4% clay) that received 1.8 g Zn per plant, in the form of either ZnO or ZnSO4 enriched with the stable isotope 68Zn. Zinc fertilization increased the availability of the nutrient in the soil and the content in the orange trees. Greater responses were obtained when ZnSO4 was applied to the sandy loam soil due to its lower specific metal adsorption compared to that of the clay soil. The trunk and branches accumulated the most fertilizer-derived Zn (Zndff) and thus represent the major reserve organ for this nutrient in the plant. The trees recovered up to 4% of the applied Zndff. Despite this relative low recovery, the Zn requirement of the trees was met with the selected treatment based on the total leaf nutrient content and increased Cu/Zn-SOD activity in the leaves. We conclude that the efficiency of Zn fertilizers depends on the fertilizer source and the soil texture, which must be taken into account by guidelines for fruit crop fertilization via soil, in substitution or complementation of traditional foliar sprays.The zinc (Zn) supply increases the fruit yield of  Zinc (Zn) deficiency is common in intensively managed citrus orchards in tropical soils and limits tree growth and fruit production . This isThe acid soils are the main soil types in Brazil, which produces nearly 40% of the world’s orange juice. Other crops, such as sugar cane, coffee and rubber tree may also respond positively to Zn management under similar conditions ,4.Early studies have suggested that the application of Zn to acid soils with a sandy texture maintains the nutrient supply for a longer period than does Zn application to the leaves of plants . However4 was used as a source of soluble Zn  grafted on ‘Sunki’ mandarin (C. sunki hort. ex Tan.) grown for 250 days in 20 dm3 pots containing a sandy loam or a clay type soil found in the main citrus growing areas of the tropics, which characteristics affect Zn availability to plants (A greenhouse experiment was conducted using sweet orange  x 100. The measurements carried out by ICP-MS showed higher accuracy, with relative error lower than 0.95%, for all Zn isotopic ratios for the certified reference material . The percentage of Zn in each plant part derived from the fertilizer source (%Zndff) and the amount of Zn used by the plants (%AZnf) were determined via isotopic dilution ; Znp is the total amount of Zn in the plant material (mg plant-1); Znf is the total amount of 68Zn that was provided by the fertilizer (1.8 g plant-1); and ZndffA is the Zn derived from the fertilizer in the plant part of the interest (mg plant-1).The ion intensities of all Zn isotopes  were measured in a mass spectrometer coupled to a plasma source (ICP-MS) with an octapole reaction system, . In order to minimize polyatomic interferences  containing 3 mM L-1 dithiothreitol, 1 mM L-1 EDTA, and 4% (w/v) polyvinylpolypyrrolidone . The extBradford  using bo-1 nitroblue tetrazolium, 0.05 mM L-1 riboflavin, and 0.3% tetramethylethylenediamine (v/v) for 30 min. After incubation, the reaction solution was removed, and the gels were rinsed with deionized water and exposed to light for several minutes to achieve photo-oxidation. The bands corresponding to the SOD activity did not undergo oxidation. Photo-oxidation was stopped via treatment with 7% acetic acid (v/v) for 15 min.The superoxide dismutase activity was determined via 12% polyacrylamide gel electrophoresis (PAGE) . Separat2O2; Fe-SOD when inactivated by H2O2 and resistant to KCN; and Mn-SOD when resistant to both compounds.The following SOD isoforms were identified according to Azevedo et al. : Cu/Zn-S2O2 (30% solution) at 25°C. The enzyme activity was determined by following the decrease in the absorbance at 240 nm, which represents the decomposition of H2O2, for 1 min. The results were expressed in M min-1 mg-1 protein-1.The catalase activity was determined via spectrophotometry . The readff in the plants, and the catalase activity using the F test . When the interaction soil*source was significant, means were compared using the Tukey test at 5%. Pearson correlation was used to describe relationships between data sets .Descriptive statistical analyses were applied to the data. A two-way ANOVA was performed to evaluate the effect of Zn-fertilizers and soil texture on the Zn in the soil and plants, the Zn4 than for those that received the less-soluble source of Zn (p<0.05). Furthermore, plant growth was 68% higher in the sandy loam soil  than in the clay soil . Within the same soil, the fruit production was 50% higher, on average, for the plants that received ZnSO4 compared to those that received ZnO  production was higher for plants that received ZnSO p<0.05, .-3  was proportional to that in leaves .Although the level of available Zn in both of the soils was approximately 54 mg dm-3 , the totoam soil . In addiL = 1220 mg kg-1) than the sandy loam one (bL = 790 mg kg-1). However, the binding energies between the metal and soil surface were similar for both, with an average value of 0.033 mg L-1 of Zn.Maximum adsorption values, which are estimates of the maximum amount of Zn that these mineral surfaces can hold, were calculated for soil types based on the Langmuir isotherm . The cladff than did the leaves due to greater DM production. The Zndff content was much higher in younger leaves than in older leaves for all of the treatments (4 application in the sandy loam soil).The roots, stems, and branches accumulated more Zneatments . However4 to the sandy loam soil provided the greatest amount of this nutrient to the different parts of the plant, with the woody organs (stem and branches) and the new leaves exhibiting Zndff>96% , fruits (R2 = 0.96) among the different fertilizers and for both of the soils were well correlated (p<0.01).The Znd leaves . The Znddff (PZndff) in the shoots of the orange trees was higher in the woody organs (stems and branches) than leaves, flowers and fruits, ranging from 58 to 73% of Zndff, mainly due to the greater DM accumulation in the plants .Following gel electrophoresis, three bands were identified for Cu/Zn-SOD, two for Mn-SOD and one for Fe-SOD. The Cu/Zn-SOD bands were the most highly expressed in the plants that were treated with ZnSOoam soil . The ban4 resulted in a higher efficiency in providing Zn to the plants, leading to an increase in the total DM  than in sandy soil (P = 50%), which explains the differences in DM production that were observedAlso relevant to the differences in the DM production of the plants that were grown in the sandy loam and clay soils  is the f-1 in this organ (p<0.01)  compared to those levels that were found in our study, in which the plants that were grown in the sandy loam soil were treated with the ZnSO4 (47 mg kg-1 in the leaves and 33 mg kg-1 in the flowers)  , suggestus soils . Furtherflowers) .-1) is associated with the greater compartmentalization and lower metal transport to the shoots, which results in the resistance or tolerance of plants to excessive concentrations in order to maintain homeostasis [dff (>99%) and the Zndff content (65 mg) were much higher in the roots than in the other organs  (Tables The high content of Zn in the roots of plants (>300 mg kgeostasis . Accordi) Tables  and 5. Fdff value in the new leaves compared to that in the older leaves  in the orange trees (Tables -1 (dff in the young leaves (-1 of Zn in the leaves of citrus plants are sufficient to ensure vigorous growth [2 leaf-1) than that of the leaves in the untreated plants (23 cm2 leaf-1).The higher solubility of ZnSOs Tables  and 5. Wables -1 , correspg leaves . Levels s growth . Howevers growth , which cs growth . The aveIn this experiment, the plants were evaluated for during one cycle of vegetative growth following fertilizer application. Besides the decreased availability of Zn that was applied to the soil because of the partition of the metal in fractions that are bound to soluble forms, complexed with organic colloids, or linked to Mn and Fe oxides , the resdff, with the exception of the flowers, which may have been due in part to the lower DM yield of these leaves in contrast to that of the other developing organs, such as the stems and branches , in the leaves; an excess of these nutrients could negatively affect the absorption and distribution of Zn in plants . For ins4, particularly when applied to the sandy loam soil due to the greater solubility of this source. The pH value of both of the soils was approximately 5.2. Consequently, the difference in the FUE was dependent on the physical and mineralogical features of those soils, which determine their adsorptive capacity (bL) (clay = 1220 mg kg-1 and sandy loam = 790 mg kg-1). The clay soil exhibited a higher bL for Zn [The fertilizer use efficiency (FUE) is defined as the amount of Zn that is recovered by the plant as a proportion of the amount of Zn supplied by the fertilizer. The FUE was higher for ZnSO4 in the sandy loam soil, respectively (4 (0.7 mg L-1 65Zn) that was applied to the leaves was absorbed by orange trees. However, despite this greater recovery of the nutrient following foliar application, the absorption of 65Zn occurred primarily during the first several days after fertilization, after which no significant absorption was detected. Under the conditions of this experiment, the time between sampling and the 68Zn fertilization of the plants was 150 days. This time period may have been insufficient to allow for more significant root absorption, which would have increased the fertilizer efficiency, particularly for the ZnO in the clay soil. This result would be supported by a previous study in which the soil application of ZnSO4 in a medium-texture Oxisol [Of the total Zn supply, the proportion of plant uptake ranged from 0.01% to 4.15% for ZnO in the clay soil and ZnSOectively . These v65Zn is redistributed to the other parts of the plant [Moreover, when Zn was applied to the soil, this mineral is distributed to all of the parts of the plant, in contrast to the application of fertilizer to the leaf, after which less than 1% of the he plant . For thihe plant .-1)   S1; . Total SAlthough an increase in Cu/Zn-SOD activity was observed, there was no increase in CAT activity. Thus, the increase in the metal level in the plant tissue  did not Thus, based on our findings, the establishment of an adequate strategy for soil application of the micronutrient for citrus and other fruit crops, in substitution to the traditional foliar sprays, should consider the effect of the soil texture and the quality of the fertilizer source. However, make it necessary to note that in the case of fertirrigated orchards, given the thermodynamic characteristics of the soil solution under drippers other issues that define the fertilizer efficiency characteristic for this system should also be considered.dff occurred mainly in the roots and woody organs, which may be important reserve organs for this nutrient. Despite the relatively low fertilizer efficiency, the nutrient requirement of the citrus plants was likely satisfied, as verified by the increased Zn contents in the leaves, the higher DM production, and the increased SOD activity.The Zn fertilizer use efficiency, based on the recovery rate in the plants, was as high as 4% when a water-soluble Zn source was used in a sandy loam soil, but it decreased to only 0.1% when a less soluble ZnO source was used on the clay soil with the highest Zn sorption capacity. Under these conditions, the accumulation of Zn