Vitis inclusive of different species, rootstocks, wild and cultivated subspecies. A distinct DNA barcode made up by a different number of amplicons, was attributed to each of the different accessions. TBP data were compared with those obtained, with the use of an internationally validated set of six SSR markers. Genetic relationships among the different accessions, dendrogram distributions, correlation values and polymorphic index values (PICs) were definitively comparable when not in favor of TBP. Such an experimental consistency is based upon a genomic organization of the multiple members of the β-tubulin gene family, the targets of TBP-mediated amplification, that is conserved in Vitis as in any other plant species. The TBP amplicons can actually be used as a useful source of sequence polymorphisms for generating primer pairs capable of identifying specific cultivars in a simple assay. An example for the identification of the ‘Sangiovese’ cv. is reported. More generally, these data are discussed in terms of the actual advantages that the introduction of the TBP method in the field of grape characterization and genotyping can provide.The TBP (Tubulin-Based-Polymorphism) method, based on a nuclear ILP (Intron-Length-Polymorphism) molecular marker, has been used for genotyping 37 accessions of the genus  Vitis, accounting for about 70 different species [Vitis can be further separated in two subgenera, subg Muscadinia with only two species and subg Vitis. This latter comprises all the remaining wild species including rootstocks, and Vitis vinifera, the species from which the cultivated grapes Vitis vinifera ssp. sativa and its closest wild relative Vitis vinifera ssp. silvestris originated [Vitis and its subsequent distribution all over the world remains somewhat controversial [Vitis vinifera seeds and old winery in the Mediterranean area, with Eastern Turkey as the likely primordial centre of diffusion, consistently dated around the middle of the eighth millennium B.C. [The grape family (Vitaceae) has an isolated position in the Tree of Life for flowering plants (Angiosperms) but relates to a large monophyletic clade called Rosids that contains other economically important families like legumes (Fabaceae) . The gra species , 7, is p species , 5. Accoiginated . Both thiginated . Althougoversial , reportsium B.C. , 10, 11.ium B.C. . In factium B.C. .Vitis, has been used for long time to classify grape species and accessions. It utilizes qualitative parameters to characterize the plant. The efficiency and the importance of this historical method was at last codified by international institutions  allowing to efficiently describe plant morphology of species and cultivars [Ampelography, that is the traditional scientific method based on the description of the morphological characters of the members of the genus ultivars , 15. Ampultivars . They arultivars , 18. In ultivars , 20. In ultivars –23, phenultivars , 25, aroultivars –28, stilultivars  have allultivars .Despite these existing methods, the genetic characterization of grape had the greatest improvement and development in the last two decades. The most important molecular markers applied to the classification of grapevine accessions have been RFLPs (Restriction Fragment Length Polymorphism), RADPs (Random Amplified Polymorphic DNAs), AFLPs (Amplified Fragment Length Polymorphisms), retrotransposon-based markers and microsatellites . By far, SSR markers are now preferred for their high level of polymorphism, reproducibility and codominant features . Since 2V. vinifera coding DNA sequence (CDS) averages to 213 bp but sizes expansion driven by TE activities, a process that is accelerated by domestication, may lead to intron of several thousand bp in length. Jiang and Goertzen [Vitis, at both species and subspecies level, in consideration of some advantages that their use may provide. In fact, ILP markers are neutral, co-dominant, stable, specific since they are tagged to selected genes, successfully applicable at different taxonomical levels and highly transferable among plant species [Nuclear DNA-based intron length polymorphism (ILP) markers have never been applied to the genotyping of grapevine and yet, in principle, they could perform successfully because of the high level of heterozygosity, resulting from an elevated outcrossing rate . In addiGoertzen  report oGoertzen  reportedGoertzen , 43. Thu species . The ass species . If targ species . In addi species , 49.Vitis. Application has involved wild species, rootstocks, and either table grape or wine making cvs. TBP data are compared to those obtained, on the same gDNA, with the use of the six SSR sequences of the internationally evaluated set of minimal standard markers. The comparison between the two methods offers several arguments of discussion.The purpose of this work was thus to verify if the TBP method could successfully be adopted for genotyping accessions and cvs of the genus Vitis  with different origin (Asia and America); 2 additional species (V. riparia NY 245 F15 and V. rupestris) and their 3 related hybrids  commonly employed as rootstocks in grape cultivation; 18 cvs of V. vinifera ssp sativa and their derived clones, including both table grape and wine making accessions. This latter group also includes: an intraspecific cross resulting between ‘Riesling italico’ and ‘Pinot noir’; 3 cvs of V. vinifera ssp silvestris from Tuscany , Apulia (‘Sinni 1’), in Italy, and Germany (‘Guemuld 104–64’), respectively. Eight of the 37 analyzed accessions , Italy (44°98' N and 9°09' E). The accessions were identified according to the collection name  and seleFor each accession, the genomic DNA was individually extracted from three distinct young leaves of different individual plants and then bulked together as representative of the accession. The DNA isolation method is reported by Lodhi et al.  with somFifty ng of genomic DNA was used as a template for TBP 1st intron amplification. The PCR reaction was performed in 30 μl according to Bardini et al. . Primers™ 1200 LIZ® Size Standard (Thermo Fisher Scientific) and 17,82 μl Hi-Di™ Formamide (Thermo Fisher Scientific) to a final volume of 20 μl. After denaturation at 95°C for 5 minutes, the samples were loaded on the Applied Biosystems® 3500 Genetic Analyser (Thermo Fisher Scientific). The CE separation was performed using an 8-capillary array of 50 cm trough the POP-7TM polymer, setting the following instrument specifications: 60°C, injection time 3 seconds, run time 5100 seconds, injection voltage 10 kV and run voltage 8.5 kV. The data collection was performed using the 3500 Series Data Collection Software v. 2.0. (Thermo Fisher Scientific). The data were analysed by the Gene Mapper Software v. 5.0 tools (Thermo Fisher Scientific) and summarized in a Microsoft Office Excel file.Capillary Electrophoresis TBP (CE-TBP) was performed as described in Gavazzi et al.  with thewww.ncbi.nlm.nih.gov/). The primers were manually designed and the reverse oligo was labelled in 5’ position with a FAM fluorophore (Thermo Fisher Scientific). The PCR reaction on genomic DNA, was performed in 20 μl, containing 20 ng DNA template, 1X Taq DNA Polymerase Master Mix providing 2.0 mM MgCl2 , 0.5 μM of each primers  and deionized water . PCR conditions were: 4 minutes initial denaturation at 94°C followed by 35 amplification cycles  and final extension step of 30 minute at 72°C. Quantity and quality of the amplified DNA samples were assessed on 2% (w/v) agarose gel. 2 μl containing 100–200 pg of amplified labeled DNA were mixed with 0.22 ul of GeneScan™ 500 LIZ™ Size Standard (Thermo Fisher Scientific) and 17.78 μl Hi-Di Formamide to a final volume of 20 μl. After denaturation at 95°C for 5 minutes, the samples were loaded on the Applied Biosystems® 3500 Genetic Analyser. CE separation was performed using an 8-capillary array of 50 cm trough the POP-7TM polymer, setting the following instrument specifications: 60°C, injection time 5 seconds, run time 1680 seconds, injection voltage 10 kV and run voltage 15 kV.Sequence information was recovered by cloning and sequencing the amplification products obtained by TBP. The isolated target sequences were aligned by BioEdit Sequence Alignment Editor for Window (Version 7.2) and verified according to the sequence information available on NCBI database  was used to estimate amplicons sizes. Electrophoresis was performed in 1X TBE buffer at 1500 V for 3 hours. At the end, the amplified fragments were visualized by a silver-staining procedure, according to Bassam et al. [Glycine max gDNA) were also included in the analysis.25 ng of genomic DNA was used as a template for PCR amplification. Six microsatellite markers were chosen as a core set according to the international grape genetics community . The PCRm et al.  protocolThe amplified fragments generated by both the TBP and the SSR markers were scored and used to produce a presence/absence matrix. The genetic similarity values were estimated, among the analysed genotypes, using the NTSYSpc 2.1 software, according to the Nei and Li coefficient , 57. Denst intron obtained through the amplification of genomic DNA extracted from the 37 different grapevine accessions of Vitis β-tubulin genes distributes quite sharply in two major groups (I and II in V. vinifera accessions (Group 1) from all the other species (Group 2) with few exceptions (see below and V. berlandieri x Colombard (BE) resulting from the interspecific hybridization of V. berlandieri and V. vinifera. Two subspecies of V. vinifera ssp. silvestris (SU and SS) also cluster within this subgroup.A binary matrix resulting from the score for presence/absence of the TBP amplified products was used to obtain the dendrogram of V. vinifera ssp. sativa, the two species of North American origin (LO and AR) cluster together with V. amurensis (AM), from Asia, as the most isolated subgroup of the tree. In a separated subgroup the rootstocks species (RI and RU) give rise to a recognizable cluster together with the derived hybrids (42 and K5). On the contrary of the two other V. vinifera ssp. silvestris (SU and SS), SC segregates in Gr. 2 although in a rather isolated branch.Subgroup 1b contains different clusters that separate, in a quite orderly way, all the remaining wine making cvs: one group of French origin (‘Cabernet’ and ‘Merlot’); a couple of cvs from Lombardy (‘Barbera’ and ‘Croatina’); one group from Piedmont (‘Nebbiolo’ and ‘Moscato’), the ‘Pinot Noir’ samples (PN5 and PN1) and the two cvs originated by them: IRP2  and Chardonnay (‘Pinot noir’ x ‘Gouais blanc’). With reference to Gr 1, the correlation between the data of the binary matrix and dendrogram elaboration is highly significant with r = 0.86742. Concerning Gr.2, that includes only species distinct from A correlation value r = 0.86416, obtained from comparing the raw data of the matrix and the derived dendrogram, was calculated for all the 37 species analyzed with TBP. This value is highly significant and higher than the corresponding value of r = 0,82118 we obtained by analyzing the same samples with the standard set of the six internationally recognized SSR markers .The two dendrograms obtained either by using TBP or the set of SSR markers, show confirmations and differences. These latter are mainly restricted to the group of table grapes that appear more spread in the SSR-based dendrogram.As already anticipated, our sample size included 8 accessions that were previously characterized by the use of the internationally recognized, standard set of 6 selected SSRs markers . With thV. vinifera cvs whereas, when using the SSRs set, is more closely associated to the two rootstocks (K5 and 42) clearly separated from the main cluster, in both dendrograms. On the same line of comparison, we have also evaluated the level of polymorphism present among the 37 grape samples by calculating the PIC values for both the TBP and the SSR loci  through the plant Genome database browser (http://www.plantgdb.org/VvGDB) and relevant to this study is reported in Reliability of the information based on the application of the TBP marker has been also assessed in comparison with the genomic data released by the whole genome shotgun project that used ‘Pinot Noir’ (PN40024) as the cv. of reference because of its high level of homozygosis. Nucleotide sequence information, retrieved from the The capillary electrophoresis TBP variant (CE-TBP) was used as the method of comparison. Performed under denaturing condition, CE-TBP provides more accurate molecular data then PAGE separation with reference to the actual sizes of the amplified fragments . HoweverTBP offers the advantageous possibility of developing highly specific molecular probes that can be used for cultivar recognition and wine and must authentication. The high level of allelic polymorphism present in each grapevine accession provides the basis for this strategy. As a proof of principle, we have developed a simple end point PCR approach for the easy recognition of the ‘Sangiovese’ cv. (SGM), the most largely cultivated Italian wine making grape from which prestigious brands such as Brunello di Montalcino are obtained. In order to develop a simple assay we concentrated our interest on the allelic polymorphism uncovered by the CE-TBP method around 680 bp sizes, the peak corresponding to entry 8 in PN40024 . In addiVitis genotyping. 4 of these SSRs  are still present in the group of 20 and 3 of them in the subgroup of 9.SSRs undoubtedly represent a very useful molecular tool that, either alone or in combination with additional approaches such as ampelography, use of retrotransposons or nucleotide sequencing, can assess genetic diversity in cultivated grape, wild species and rootstocks, solve homonymy and synonymy issues, identify hybrids parentage, defining the internal genetic relationships among genotypes of large germplasm collections. The robustness of the systems of identification based on SSRs has been progressively implemented by augmenting the number of SSRs that are concomitantly used rather than increasing the length of their core, repeated unit. Of lately, there seems to be a general agreement on the use of a set of 20 SSRs for providing the most reliable information . Laucou Since we were aiming at verifying the level of reliability of TBP with respect to an SSR-based approach, we used the same 6 SSRs reference panel for genotyping those cvs that were in common between our and the international consortium study. As a matter of fact we acted as an additional external laboratory producing SSR data perfectly consistent with those reported by the International consortium thus providing a correct ground for comparison. Although very powerful, as reported, microsatellites remain anonymous markers subjected to a high degree of genetic hyper-mutability that, based on impaired DNA mismatches repair, is likely independent from the stabilization of morphological features and traits of agronomical relevance. It is under this view that some difference observed by comparing the dendrograms derived either from the TBP or the SSR markers can likely be explained. In addition, due to some variability in size determination that may occur in different laboratories, depending on different reagents, instruments and PCR conditions, reference alleles for each microsatellite must be designated to strengthen inter-laboratory data comparability. Moreover, the amount of data generated by the use of SSRs in large size samples is relevant and requires adequate software and computer sciences facilities to be processed, conditions not always available in research laboratories. Given all this, we investigated if a completely different molecular marker based upon Intron Length Polymorphism (ILP) could represent a valid option to grape genotyping and classification by providing reliable information, comparable to that obtained with the SSRs, with a reduced operation time and no need for handling a massive amount of data.V. vinifera, as inferred by the whole genome sequence, stored in the VvGDB , grouped the 37 analyzed samples in a way that is comparable to SSR's if not more consistent with respect to some taxonomic and agronomic trait. In fact, the TBP method clusters together the majority of the grapevine species and rootstocks (group 2) while they are scattered across the tree when using the SSRs set. Table grapes group together with TBP but separately when the SSRs are used and this occurs even with the 34 SSRs set . Both melora spp., 64. TBPVitis accessions including rootstocks, genotyping grapevine collections and developing variety-specific assays.Finally, an additional advantage that can be attributed to TBP is the design and production of primer pairs that are specific for any cv. or accessions of interest. In fact each TBP-amplified band may potentially contain a distinguished sequence information. Working on the amplified bands that, in the different wine-making cvs of this study, correspond to the β-tubulin entry 8 of the VviGDB, we have designed and used a couple of primers capable, in a simple PCR reaction, to amplify a doublet that is specific for ‘Sangiovese’ (SGM), the grapevine cv. more widely cultivated in Italy, used for making the most appreciated brands of wine. This is just an example that can be followed up by the preparation of additional and numerous variety specific assays, useful for cv. recognition rather than must characterization. Referring to this latter, and wine in perspective, the possibility of amplifying a short size diagnostic fragment, as shown for SGM, is of importance in consideration of the difficulty that is encountered in DNA isolation, typically degraded and present in low abundance. In conclusion, this paper provides a series of data and evidence in favor of the argument that TBP is a useful, rapid, simple, reliable molecular tool that can be used, either alone or in combination with other markers, for different purposes that may include establishing the genetic relationships within the grape family Vitaceae, barcoding wild and cultivated S1 FigDendrogram of the 37 grapevine accessions based on SSR marker analysis. The bar at the bottom of the figure provides a scale, from 0 to 1, of the estimated similarity. The numbers next to each node represent the bootstrap value (1000 replicates).(TIF)Click here for additional data file.S2 FigExon nucleotide sequences amplified by TBP are aligned using rice OsTub4 as reference. The sequence corresponding to the primer pair (Fex1 and Rex1) is shown at the two opposite ends while an arrows indicate the position of intron1.(TIF)Click here for additional data file.S3 FigThe tubulin exon portions shown in (TIF)Click here for additional data file.S1 TableComparison of the six SSR alleles size obtained in this study (indicated by * in bold character) with those reported by the ten laboratories (lab. 1–10) of the International consortium , on the (XLSX)Click here for additional data file.


Mesenchymal stem cells (MSCs) are ideal candidates for treatment of diseases. Amniotic membranes are an inexpensive source of MSCs (AM-MSC) without any donor site morbidity in cell therapy. Adipose tissue derived stem cells (ASCs) are also suitable cells for cell therapy. There is discrepancy in CD271 expression among MSCs from different sources. In this study, the characteristics of AM-MSC and ASCs and CD271 expression were compared. Adult adipose tissue samples were obtained from patients undergoing elective surgical procedure, and samples of amniotic membrane were collected immediately after caesarean operation. After isolation and expansion of MSCs, the proliferation rate and viability of cells were evaluated through calculating DT and MTT assay. Expression of routine mesenchymal specific surface antigens of MSCs and CD271 was evaluated by flow cytometry for both types of cells. The growth rate and viability of the MSCs from the amniotic membrane was significantly higher compared with the ASCs. The low expression of CD14 and CD45 indicated that AM-MSC and ASCs are non hematopoietic cells, and both cell types expressed high percentages of CD44, CD105. The results revealed that AM-MSC and ASCs expressed no CD271 on their surfaces. This study showed that amniotic membrane is a suitable cell source for cell therapy, and CD271 is a negative marker for MSCs identification from amniotic membrane and adipose tissue. In addition, immune-modulatory properties of MSCs make these cells as the primary choice for allo and xeno transplantation.11Mesenchymal stem cells are derived from bone marrow,12 However, researchers direct their efforts to find a suitable marker to ensure their isolation. Some studies show that CD271 (LNGFR) is one of the most selective markers for MSCs isolation from bone marrow.13 Cuevaz et al. showed that amount of CD271+ cells in ASCs correlates inversely with donor age.14 Iorocha et al. revealed that CD 271+ and CD105+ population are highly rich in molecular marker of early osteo and adipo progenitors.15Multi differentiation potential, adherence to plastic and specific surface antigen expression  and absence of  are three criteria defining MSCs in culture.et al. published that CD 271 is a specific marker for the isolation of MSCs from bone marrow.13 However, Battula et al. reported that CD271 is not a suitable marker for MSCs isolation from chorionic membrane.16 Torales et al. proposed the use of single marker, CD271 for isolation of MSCs.17 The present study set to introduce a suitable source of MSCs with high proliferation rate, and to find an appropriate marker for MSCs characterization. To achieve these, MSCs properties of adult adipose tissue and amniotic membrane were evaluated.Quirici Three samples of adult adipose tissue were obtained from adult patients aged 45 and 60 years old who underwent the elective surgical procedure, and samples of amniotic membrane were collected from women donors immediately after caesarean operation with informed consents and ethical approval committee of the Tabriz University of Medical Sciences. 4), Total cell yield=(Cells/ml)×. Under sterile condition, the samples were washed three times with phosphate buffered saline (PBS) containing 1% penicillin/streptomycin ; the samples were cut into 1-2 mm thick. For enzymatic digestion, the pieces were transferred to conical tubes containing 2.5% collagenase 1  and were then shaken in water bath for 1 hour at 37°C. After digestion, for neutralizing the collagenase enzyme, the same volume of working culture medium  containing 10% FBS  and 1% penicillin/streptomycin  were added to the cell suspension. The digested samples were centrifuged at 1600 rpm/5 min, and then cell pellet was obtained. The cells were counted under an invert microscope before plating cells in culture flasks as follows: Cells/ml=(Number of viable cells)/. Afterwards, 1 ml of trypsin/EDTA solution was added to culture flasks, and the flasks were incubated at 37°C for 1 minute. One milliliter of working DMEM was added to the flasks for neutralizing of trypsin/EDTA. Then, the cells were centrifuged at 1600 rpm/5 min and counted for adipose and amniotic membrane cells. DT was calculated by the following formula, while N0 is the number of seeded cells at time t0, and Nt is the number of taken cells at time t, and T represents the culture period:For expansion, the cells were plated at 7-10 ×10At first, adipose and amniotic membrane cells were cultured in 96-well plate (3000 cells per well). After 24 hours, 300 µl pure DMEM medium and 20 µl of MTT solution  were added onto the cells. After four hours, 100µl of dimethyl sulfoxide  was added onto the crystals. In a dark room, the color absorbance was recorded by ELISA reader at 540 nm.5 cells from each sample were washed two times with PBS containing 1% BSA and then were incubated, in appropriate combination, with 3 µl of the following fluorochrome conjugated monoclonal antibodies: CD44-FITC, CD105-PE, and CD45-FITC, CD14-PE, and CD271-PE for 30 min in a dark room. After staining, the cells were washed with PBS containing 1% BSA and centrifuged and the cell pellet vertexes with 500 µl of PBS/1% BSA were evaluated using BD instrument. To analyze the expression of specifics surface antigens, 10The viability, proliferation rate during expansion period, expression of positive markers , negative markers , and expression of CD271 on MSCs surface derived from adult adipose tissue and amniotic membrane were compared. The data were analyzed using WIN MDI software. Statistical analyses were performed using Graph Pad Prism. Mean comparison was carried out via t-test analysis of variance; P<0.05 was considered significant.Cell expansion cultures showed that the confluence reached in the MSCs derived adult adipose tissue flasks on the seventh day, and in the AM-MSCs flasks, this was on day 4 for the same number of seeded cells . The DT Also, MTT assays showed more proliferation rate cells in AM-MSCs compared with ASCs; these differences were statistically significant (P<0.05) between donors . The ana18,19 Common limitation of MSCs in cell therapy is the low number of cells with low proliferation rate. Therefore, finding the suitable cell source is the major challenge in recent years. In this study, we concentrated on the proliferation rate and viability of MSCs from fetal material. To evaluate the efficiency of these cells in cell therapy, we compared their proliferation rate, viability, and expression of specific surface markers and CD271 expression at AM-MSCs with ASCs. The findings showed that the growth rate and viability of AM-MSCs as a fetal tissue were significantly higher than ASCs. Therefore, MSCs from fetal tissue are more suitable for therapeutic purposes compared with adult stem cells. Previous studies have not presented similar results regarding the comparison of fetal material and adult tissues.20MSCs are the best candidates for disease treatment.21 Hence, a new cell source was presented, overcoming the problem of low number of MSCs for cell therapy. Another limitation of the use of MSCs in clinical application is finding a suitable marker for characterization of MSCs. There are studies which show that CD271 (LNGFR) is one of the most selective markers for isolation of MSCs from bone marrow.22,23However, the current study worked on MSCs derived from bone marrow and placenta stem cells, showing no significant difference in the cell number and growth kinetics in MSCs of these sources.+ ASCs correlated inversely with donor age, and the highest CD271 yield was found at 30-40 year-old range.14 The present inquiry proved similar results as adipose tissue samples were collected from 45 and 60 years old patients. Also, Jones et al. showed that CD271 is a crucial selective marker for enriching MSCs from bone marrow.22 Jarocha et al. suggested that CD271+ and CD105+ population had higher enrichment in markers of early osteo and adipo progenitors.15In this regard, the present results revealed that ASCs and AM-MSCs express no CD271. Another study in 2013 showed that CD271et al. observed that CD271 is not a suitable marker for the characterization of MSCs from chorine.16 Watson et al. published that CD271 is a suitable marker for characterization of MSCs from bone marrow, but failed to characterize MSCs from umbilical cord blood (UCB).24 This study presented amniotic membrane as a suitable cell source for cell therapy, also it was revealed that CD271 is a negative marker for the identification of MSCs from amniotic membrane and adipose tissue.Battula


SMPD1 mutations. Historically, ASMD has been classified as Niemann-Pick disease (NPD) types A (NPD A) and B (NPD B). NPD A is associated with a uniformly devastating disease course, with rapidly progressing psychomotor degeneration, leading to death typically by the age of 3 years, most often from respiratory failure. In contrast, the clinical phenotype and life expectancy of patients with NPD B may vary widely. Almost all patients have hepatosplenomegaly and an atherogenic lipid profile, and most patients have interstitial lung disease with progressive impairment of pulmonary function and hematologic abnormalities including cytopenias. Other common clinical manifestations include liver dysfunction, heart disease, skeletal abnormalities and growth delays. Some patients with ASMD who survive beyond early childhood have intermediate phenotypes (variant NPD B) characterized by combinations of non-neurologic and mild to severe neurologic symptoms. The physical and psychosocial burden of illness in patients with NPD B is substantial. Common symptoms include shortness of breath, joint or limb pain, abdominal pain, bleeding and bruising. The disease often leads to chronic fatigue, limited physical or social activity and difficulties in performing daily activities or work. Many patients die before or in early adulthood, often from pneumonia/respiratory failure or liver failure. Available treatments are limited to symptom management and supportive care. An enzyme replacement therapy currently in clinical development is expected to be the first treatment addressing the underlying pathology of the disease. Early diagnosis and appropriate management are essential for reducing the risk of complications. While knowledge about ASMD is evolving, more evidence about ASMD and the natural history across the disease spectrum is needed, to improve disease recognition, timely diagnosis and appropriate disease management.Acid sphingomyelinase deficiency (ASMD), a rare lysosomal storage disease, is an autosomal recessive genetic disorder caused by different  SMPD1 gene [Acid sphingomyelinase (ASM) deficiency (ASMD), a rare lysosomal storage disease, is an autosomal recessive genetic disorder caused by mutations in the PD1 gene . HistoriPD1 gene , 4. NPD PD1 gene . The terPD1 gene –8. ImporPD1 gene –11. CurrPD1 gene . More evSMPD1 [SMPD1 mutation and appears to reflect the level of residual ASM activity [ASMD, also known as NPD types A (NPD A) and B (NPD B), is a rare autosomal recessive lysosomal storage disorder caused by mutationsSMPD1 . Insuffiactivity . Patholoactivity . Infantsactivity .Patients with ASMD have been categorized historically as NPD A and NPD B based on disease severity and the presence or absence of neurologic symptoms. Patients designated as NPD A have a relatively uniform natural history characterized by severe progressive neurodegeneration in the first year of life and death typically by 3 years of age . In contSMPD1 sequence variations that do not result in loss of ASM function [Reliable estimates of ASMD incidence worldwide are currently lacking. Although ASMD is a pan-ethnic genetic disease, many mutations are private and others are found preferentially in specific ethnic groups –20, whicfunction .SMPD1 mutant alleles. Systematic genetic carrier screening for these three mutations in Ashkenazi Jewish adults suggests a carrier frequency of 1 in 80–100, which would extrapolate to an estimated two to three cases per 100,000 births in this population [SMPD1 mutation associated with NPD B suggest a rate of approximately two cases per 100,000 births, which is higher than the rate suggested by confirmed diagnoses [Another impediment to obtaining precise estimates of ASMD incidence is the potential for underdiagnosis due to lack of disease awareness. Although some estimates based on actual diagnoses place the incidence of ASMD at approximately 0.5 per 100,000 births , 23, estiagnoses .SMPD1 variants through genetic testing. For patients from populations with common known SMPD1 mutations , targeted analysis for defined pathogenic mutations can be performed, with full-length sequence analysis of SMPD1 only required if the presence of a mutation in both alleles is not confirmed by other means. In populations in which common mutations do not exist, full-length sequence analysis should always be performed.Due to the rarity of ASMD and the heterogeneity of its manifestations, ASMD diagnosis may be missed at initial presentation or occur only at advanced stages of disease progression. ASMD should be suspected in patients with hepatosplenomegaly, developmental delay and/or cherry-red maculae , inSMPD1 variants, if necessary, can be accomplished with the use of the same dried blood spots. Enzymatic and molecular analysis in noncultivated chorionic villi also is feasible and permits rapid and accurate prenatal diagnosis of ASMD [SMPD1 mutations [Newborn screening for ASMD is feasible by testing the enzymatic activity in dried blood spots , 28, and of ASMD . Geneticutations , 29.A detailed discussion of the diagnosis of ASMD is beyond the scope of this paper. However, a consensus recommendation on a diagnostic guideline for ASMD, which also provides an overview of the relevant differential diagnoses, is available .Our current understanding of the natural history and overall clinical burden of ASMD is based on single case reports, small case series and a limited number of relatively larger observational studies. A literature search in PubMed since 2004 identified 18 studies that described the natural history and/or the clinical manifestations of patients with ASMD , Brazil (n = 13), Italy (n = 8), France (n = 7) and Germany (n = 5) provided valuable insight in the spectrum of NPD B disease manifestations [CO; r = −0.306, p = 0.052) and percentage of predicted forced vital capacity , and did not correlate with exercise tolerance as measured by the 6-min walk test distance  [Unlike the fairly homogeneous NPD A phenotype, patients with NPD B have extensive phenotypic heterogeneity, including a wide range of disease manifestations and severity levels and variable rates of disease progression . A prospstations . For eac= 0.075) .9/L and most had low HDL cholesterol. Thus, as expected from the underlying pathophysiology, ASMD is associated with multi-organ disease in most patients.Similar clinical findings were documented in a Dutch and Belgian study of 25 patients with ASMD . Of the Typical findings by organ class are discussed in detail below and summarized in Fig. Splenomegaly is one of the most common disease manifestations of NPD B , 5, 36 aCO <60% of predicted value) had only mildly abnormal imaging findings [CO, which are consistent with restrictive lung disease and impaired gas exchange secondary to interstitial lung disease. A natural history study of 29 pediatric and adult patients with NPD B documented slow progression of pulmonary disease with small mean annual changes in all measures of pulmonary function [Most patients in the cross-sectional study  of 59 pafindings . Therefofunction . Overallfunction , 32.In a study of morbidity and mortality in 103 patients with NPD B carried out at a single center between 1992 and 2012, six patients had fulminant liver failure, and liver biopsies from three additional patients showed evidence of cirrhosis . Two of In the cross-sectional study of 59 patients with NPD B, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated in 51% of the patients, and total bilirubin was elevated in 33% of patients . In the SMPD1 p.(Ala359Asp) (A359D) mutation associated with moderate to severe NPD B, five (38.5%) patients developed clinically relevant liver disease due to progressive cirrhosis [In a series of 13 Chilean pediatric patients homozygous for the irrhosis . Notablyirrhosis . In summirrhosis .According to medical history, 19% of the patients in the cross-sectional study had suffered one or more bone fractures . A subsen = 6), left ventricular hypertrophy (n = 4) and conduction abnormalities (n = 6). Two-dimensional echocardiograms showed abnormalities in 29 (50%) patients, most commonly mild mitral valve regurgitation. However, two patients had previously undiagnosed moderate to severe aortic regurgitation and one patient had pulmonary hypertension [In the cross-sectional study, electrocardiogram abnormalities were present in 16 of 58 (28%) patients evaluated . These irtension .n = 58) of 10.3 was 2.3 times the upper limit of normal [r = 0.79, p < 0.0001), and the highest score  was present in a 18 year old female [Most patients in the cross-sectional study  had an af normal . In a stf normal . Mean led female . Thus, cd female .SMPD1 A359D mutations in a recent study [The cross-sectional study documented bleeding episodes in 49% of the 59 patients enrolled . The mosnt study . Presumant study .Growth data collected in the cross-sectional study  revealedAs noted above, there are patients with ASMD who do not present with the classic NPD A phenotype but have variable neurologic symptoms ranging from mild hypotonia or hyporeflexia to severe progressive neurologic abnormalities such as loss of motor function and mental deterioration , 42, 43.SMPD1 mutations are associated with an increased risk of Parkinson’s disease [Of note, a patient with NPD B who was diagnosed at age 59 and died at age 60 from severe restrictive lung disease, also had Parkinson’s disease . Althoug disease .n = 3), depression requiring anti-depressant therapies (n = 3), and psychosis (n = 1) [A recent paper analyzing the phenotype of 28 adult French patients with ASMD found a peripheral neuropathy ( (n = 1) .SMPD1 A359D mutations, five developed fulminant liver failure over an average follow-up period of 10 years [SMPD1 genotypes. In both the US [SMPD1 mutations were present, whereas the study with Chilean patients [In a single-center longitudinal US study, 29 patients with NPD B had serial evaluations over a 10-years period to evaluate disease progression . All pat10 years . The difh the US  studies,patients  includedAs noted above, mortality in NPD A is most frequently due to respiratory failure by the age of 3 years . Resultsn = 58) and chronic neurovisceral ASMD , including 78 patients who had died and seven patients who had received liver transplants because of terminal liver disease [A more recent study evaluated disease-related morbidities and the primary causes of death in 85 patients: with chronic visceral ASMD  [SMPD1 mutation (A359D) may account for most cases of NPD B and appears to be associated with a typical clinical phenotype with normal cognitive and psychomotor development but moderate to severe visceral disease manifestations, including a high incidence of clinically significant liver disease [More than 180 different variants . Becauseted gene . Consequ of ASMD . Predict of ASMD . Neverth of ASMD , 20. For fsP330) –50. Beca disease . The R61 disease . It is a disease . However disease , 52.SMPD1 mutation (W391G), neurologic manifestations were generally highly diverse but similar among relatives [SMPD1 mutation profile, other genetic factors may influence the ASMD phenotype.Evidence from two studies suggests that the Q292K mutation is strongly associated with neurologic involvement. Specifically, the Q292K mutation was present in heteroallelic or homoallelic form in 10 of 12 Czech and Slovak patients with progressive neurologic disease , and threlatives . This raInformation about the impact of ASMD on health-related quality of life (QoL) and the psychosocial burden of ASMD has to be derived from case reports, as very few studies have attempted to address these topics. Owing to the rarity and heterogeneity of ASMD, there is a lack of robust quantitative data regarding the impact of the disease on patients’ and caregivers’ QoL. This deficiency is compounded by the lack of validated disease-specific instruments to evaluate QoL in patients with ASMD, and the limitations of generic QoL instruments in providing useful quantitative data . The ecoThe devastating burden of NPD A for the affected children and their families in terms of physical and emotional impact is self-evident and reflected in testimonials by parents of children with the disease . Before A single study  assessedn = 8) and the parents (n = 9) of the adolescent patients [The human experience and psychosocial impact of the disease has been evaluated in a small number of adolescent and adult patients with NPD B  with recombinant human ASM is a potentially disease-modifying therapeutic approach currently in clinical development for the treatment of ASMD. ERT represents a known mechanism of action that has been used successfully in other lysosomal storage disorders . ResultsASMD is a rare genetic, progressive life-threatening disease with highly variable severity and disease course. NPD A has a devastating clinical course associated with onset of rapidly progressing psychomotor degeneration often shortly after the first 6 months of age. Children typically die from respiratory failure by the age of 3 years. NPD B is a chronic progressive disease, representing a spectrum of phenotypes with varying severity levels, progression rate and prognosis. NPD B is associated with a substantial burden for many patients, caused by the profoundly negative impact of its clinical manifestations on physical, mental and psychosocial well-being. However, published data regarding the impact of the disease on QoL and daily functioning are limited, mainly owing to the rarity and heterogeneity of the disease and the lack of adequate disease-specific instruments to measure QoL. Given the wide spectrum of NPD B phenotypes, including intermediate forms characterized by mild to severe neurologic defects in addition to visceral, skeletal and hematologic ASMD manifestations, a refinement of the ASMD classification system is needed. A more precise description of ASMD subpopulations may improve disease recognition at initial presentation and thus lead to more timely diagnoses. Better characterization and delineation of different ASMD subpopulation will require additional studies able to improve our current understanding of the disease and the factors that influence disease phenotype, natural history and prognosis.


Proteobacteria, Chloroflexi, Bacteroides, Firmicutes, Actinobacteria, Acidobacteria and Nitrospirae were the dominated bacterial phyla. The Zn·Fe group had an unexpectedly high proportion (75.49%) of Proteobacteria during the primordial differentiation stage, while Pseudomonas also occupied a high proportion (5.52%) in this group. These results suggested that different trace elements clearly affected morel yields and soil bacterial community structures, particularly due to the high proportions of Pseudomonas during the primordial differentiation stage.This study described the effects of elements  on the following factors in artificial morel cultivation: the characteristics of mycelia and sclerotia, soil bacterial community structures, yields and contents of microelements. The results indicated that the groups containing Mn significantly promoted mycelia growth rates, and all the experimental groups resulted in higher yields than the control (P<0.01), although their mycelia and sclerotia did not show obvious differences. It was also found that  Morchella spp (morel) is highly prized for its medicinal and nutritional qualities, such as antioxidant, anti-inflammatory and antitumor activities, as well as strengthening the immune response [response ~4. Accorresponse –6, but aIt is common practice to cover morels with soil during cultivation to support soil microbes that are responsible for the promotion of primordial differentiation and ascocarp growth. However, thus far, we have known very little about how soils affect the growth and development of morels. Nevertheless, it has been demonstrated that soil microbes are important components of soil ecosystems and that bacteria are widely distributed in soil –10. InorA few studies have reported that trace elements found in fruit bodies differ by strain types and applied fertilizers ,18,19. O4∙7H2O), manganous sulfate (MnSO4∙4H2O), ferrous sulfate (FeSO4∙7H2O) and other chemical reagents were analytically pure and purchased from local chemical reagent companies.The strain used in this study was preserved at 4°C in the authors’ laboratory . Zinc su4 (Zn), FeSO4 (Fe), MnSO4 (Mn)), three binary elemental complexes  and one ternary complex . Their final contents were 100 mg/L, while pure PDA media was used as the control group. Each group was triplicated and incubated under 18°C for 21 days. Mycelia growth and sclerotia formation were observed using a zoom stereo microscope . Mycelia growth rates were calculated using the formula reported by Liu et al (2015) [The stock culture of the experimental strain, which was preserved in a slant tube, was activated on PDA Petri dishes  and incubated under 18°C for four days. The mycelia plugs (6 mm in diameter) were sub-cultured in PDA media, which were treated with three single elements (including ZnSOl (2015)  and were2 in size. The experimental strain was reactivated using PDA media and incubated under 16~18°C for 15 days and then was transferred into the newly sterile spawn media . After 15 days of cultivation under 18°C, there were many sclerotia adhering to the glass walls. Approximately one bottle of spawn per square meter (m2) were sowed in the experimental plots and covered with fine soil (3~5 cm in depth) to support the subsequent production of morels. It was necessary to build a sunshading net (2 meters above the ground) prior to seeding, and water mist was thoroughly sprinkled on the soil until two or three days after sowing when the soil moisture content reached 22%. About twenty days later, the frost-like sclerotia of the morels were covering the soil surface. The mycelia and sclerotia were observed by collecting the frost-like soil blocks and using a zoom stereo microscope . After the collection, nutritional bags containing the same solid spawn media were added to the soil (approximately 3 bags per square meter) to rejuvenate the mycelia. There were seven experimental groups of different elements, including three single elements (6 g/m2 of each element), three binary elemental complexes (3 g/m2 of each element) and a ternary elemental complex (2 g/m2 of the element). The trace elements were resolved in 5 L of pure water, with an equal volume of pure water used as the control. They were sprayed onto the soil surfaces one month after seeding. Soil samples were collected during the primordial differentiation stage and the ascocarp growth stage .The outdoor experimental field was in Longxing Town, Chongzhou, Sichuan, P.R. China, and each experimental plot was 10 mThe soil genomic DNAs were extracted according to the previous report . The unihttp://qiime.org/). All sequence reads were trimmed and assigned to each sample based on their barcodes. High quality sequences  were used for downstream analysis. The sequences were clustered into operational taxonomic units (OTUs) at a 97% identity threshold. The aligned ITS gene sequences were used for a chimera check using the Uchime algorithm [The sequence data were processed using QIIME Pipeline–Version 1.7.0 (lgorithm . All samlgorithm .Sequence data have been deposited in the NCBI Sequence Read Archive (SRA) database with the accession number SRP093475.When the ascocarps were close to maturity, the morels were carefully collected until the end of March, and the yields of all the groups were measured in triplicate. Randomly chosen morel ascocarps from each group were prepared by the acid digestion method described in the literature , and theThe growth rates of morel’s mycelia were clearly enhanced by the different elements ; in partWhen mycelia covered approximately the whole plate (3 days after inoculation), the morphology of the mycelia were observed under a zoom stereomicroscope. The results  demonstrIt was discovered that many bridged mycelial branches  were finThe rarefaction curves were generated at a 97% identity threshold , and theProteobacteria, Chloroflexi, Bacteroidetes, Acidobacteria, Nitrospirae, Firmicutes and Actinobacteria, were found in all groups, and their total percentages ranged from 74.72% to 93.63%  to 76.92% (Zn∙Fe group).The bacterial community structure was noticeably affected by different trace elements . Seven do 93.63% . The totProteobacteria and Bacteroidetes proportions from most groups tended to be higher than those in ck, while Actinobacteria, Nitrospirae, and Acidobacteria were lower than ck. The highest proportion of Proteobacteria (32.34% on average) was found in the Zn∙Fe group , while the lowest proportion was found in the Fe group (23.14%). The highest proportion of Bacteroidetes (8.14%) was found in the Zn∙Mn group, while the lowest proportion was found in ck (4.09%); Bacteroidetes proportions from most experimental groups were higher (ranging from 9.78~99.02%) than that of ck during primordial differentiation stage. Alternatively, Actinobacteria and Nitrospirae proportions from all experimental groups were lower than ck, with the lowest proportions of these two phyla found in the Zn∙Fe group. Proportions of Acidobacteria in most groups (except Mn and Zn groups) were also lower than ck.When the bacterial community structures of the experimental groups were compared with that of the control group during the primordial differentiation stage, Proteobacteria, Bacteroidetes and Firmicutes increased for most groups during ascocarp stage, while Acidobacteria, Actinobacteria, Chloroflexi and Nitrospirae mostly decreased. The proportions of Bacteroidetes were rose across all groups during the ascocarp growth stage, an increase that ranged from 25.43% to 427.24% compared with that of primordial differentiation stage for each group. However, in the Zn∙Fe group, the proportions of Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Chloroflexi were higher in ascocarp stage than primordial differentiation stage, but the group in former stage produced lower proportions of Nitrospirae and Proteobacteria.After comparing the proportions of predominant bacterial phyla in the primordial differentiation stage and the ascocarp growth stage, it was found that Proteobacteria phylum, the associated bacterial genera that was found in all groups were specially selected to also be analyzed in this study  had higher proportions of Rhodoplanes though other experimental groups (binary and ternary complexes) had lower proportions than the control group. The proportions of Geobacter and three unknown genera  were lower in the ck group than most experimental groups. The group with Zn∙Fe (No.175) had the highest proportion of Acinetobacter (23.36%), Janthinobacterium (8.18%), and Pseudomonas (5.52%) during primordial differentiation stage.Since the most predominant bacteria in the grown soil came from the is study . Three gPseudomonas was also the dominated genus in groups with Zn (5.23%), Zn∙Fe∙Mn (4.15%), Zn∙Mn (2.83%), Fe (1.79%) and Ze∙Fe (1.12%). However, the control group had the highest proportion of Geobacter (1.75%) and Rhodoplanes (1.45%) which was higher than that of most experimental groups .During the ascocarp stage, Using the weighted UniFrac distance metric, PCoA showed that 175 (Zn) had the greatest diversity, followed by 186 (Zn∙Fe∙Mn) and 182 (Mn) . The disThe relative yields of artificial morel demonstrated the following trend: Zn∙Fe>Mn>Zn>Zn∙Mn>Fe∙Mn>Zn∙Fe∙Mn>Fe>ck . The yieThus far, large-scale planting of morels in China is usually field-based; therefore, yields are greatly affected by natural conditions and soil. Although some growers can cultivate morels indoors during the normal planting season, the production cost is still very high. Because morels must be planted in soil or covered with soil, it is very important to discover how the soil supports morel growth. It is well known that there are substantial amounts of microbes and nutrient elements in soil that are necessary for morel growth. Therefore, this study selected and applied those elements which were rich in the ascocarp of morel, such as Fe, Zn and Mn, to stimulate differences in morel growth and yield while comparing their effects on morel-grown soil community structures.In this study, it was confirmed that there were not clear differences in mycelia characteristics among the control group and those treated with trace elements; they differed significantly in mycelia growth rates, yields and ascocarp mineral contents, especially in the Zn∙Mn group. The mycelia growth rates of experimental groups which contain Mn were higher than others; therefore, there was a synergetic effect between Zn and Mn. The earliest sclerotia, which is crucial to fruit body formation ,6, firstProteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, Nitrospirae, Bacteroidetes and Firmicutes. It is very interesting that Proteobacteria was found in the largest proportion at an unexpected level in the Zn∙Fe  and Zn∙Fe∙Mn groups , while the yields of these groups were also significantly higher than the control group. It has been reported that the secretions of some bacteria may promote morel growth [Proteobacteria bacteria may have contributed to the improved ascocarp yield. Further analyses verified that Pseudomonas, Geobacter and Rhodoplanes were the main bacteria in Proteobacteria. We know that some strains of Pseudomonas had bio-control properties, such as killing parasitic fungi and some phytophagous nematodes [Agaricus bisporus [Pseudomonas was the most common bacteria (5.52%) in the Zn∙Fe group , and this group also harvested the highest yield of ascocarps. Hence, we hypothesize that Pseudomonas may have such effect on morel primordial differentiation, which is the key to the formation of the fruit body throughout the morel life cycle [Geobacter is usually associated with electron transport and conducive to oxidizing organic substrates [Rhodoplanes was observed using organic compounds such as carbon sources and electron donors [This study also demonstrated that artificial morel’s soil bacterial diversity and richness were significantly affected by different elements. Seven predominant phyla were found in all groups, including l growth , perhapsematodes , as wellbisporus . Howeverfe cycle . On the bstrates , while Rn donors . These bNitrospirae plays a key role in the nitrogen cycle [Nitrospirae strains requiring nitrogen. Chlorobacteria and Nitrospirae were also reported to be the dominant bacteria in activated sludge in wastewater treatment, which can degrade ammonia-nitrogen and organic chlorides [Acidobacteria plays an important role in maintaining the pH value [Bacteroidetes is important for degrading cellulose and chitin [en cycle , its nithlorides –36. AcidpH value ,38 and ipH value ,40. Bactd chitin –44. On td chitin ,46, wherThe morel yields in this study were clearly correlated with soil bacterial diversity and richness; these differences were derived from different trace elements or their complexes. There are typically bountiful morel harvests in forests that have been subject to fire, which provides many trace elements via the ashes of trees and grasses ,48. RobbMorchella, and its average value (668.38 mg/kg) was similar to that of Craterellus odoratus [Russula virescens [Fe was the largest microelement found in odoratus . A varieodoratus . The aveirescens . The higirescens . The conirescens . Though To date, the cultivation of morel still uses semi-artificial planting. This study demonstrated that trace elements affect not only the mycelia and sclerotia growth, the yields and mineral contents of ascocarp, but also bacterial community structures. Based on this research, further studies should focus on determining which types of microbes directly or indirectly affect ascocarp yield. Many similar research achievements and breakthroughs will thrust the morel industry to a new level.S1 FigThe morphology of mycelia showed no obvious difference between the control group and treatment groups. Scale bars represent 100 μm.(TIF)Click here for additional data file.S2 FigReticular formation was formed by hyphal branching.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigDistance shows that the differences or similarities of soil bacteria community structures among different groups.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


This study aimed to evaluate bone repair in rat dental sockets after implanting nanostructured carbonated hydroxyapatite/sodium alginate (CHA) and nanostructured carbonated hydroxyapatite/sodium alginate containing 5% strontium microspheres (SrCHA) as bone substitute materials.Twenty male Wistar rats were randomly divided into two experimental groups: CHA and SrCHA (n=5/period/group). After one and 6 weeks of extraction of the right maxillary central incisor and biomaterial implantation, 5 μm bone blocks were obtained for histomorphometric evaluation. The parameters evaluated were remaining biomaterial, loose connective tissue and newly formed bone in a standard area. Statistical analysis was performed by Mann-Withney and and Wilcoxon tests at 95% level of significance.The histomorphometric results showed that the microspheres showed similar fragmentation and bio-absorbation (p>0.05). We observed the formation of new bones in both groups during the same experimental periods; however, the new bone formation differed significantly between the weeks 1 and 6 (p=0.0039) in both groups.The CHA and SrCHA biomaterials were biocompatible, osteoconductive and bioabsorbable, indicating their great potential for clinical use as bone substitutes. Implant-supported restoration has been increasingly performed by dentists for both aesthetic and functional reasons. However, when infections, pathological processes, extractions, or congenital and traumatic injuries on the maxilla and the mandible lead to bone loss, dental implant installation might not be the best opition10(PO4)6(OH)2 has been widely used as a bone substitute for approximately 80 yearsAmong these grafts, HA CaHowever, the clinically used HA is not biodegradable and remains at the implantation site for long periods4) or hydroxyl groups (OH) with carbonate (CO3) to develop a nanostructured carbonated hydroxyapatite at low temperaturesResearchers have chemically modified HA by substituting phosphate groups (PO2+)2+ increases the number of interconnected pores, promoting cell migration, interfacial bonding,Furthermore, the stability and flexibility of the HA structure enables different ionic substitutionsThus, this study evaluated histomorphometric bone repair in rat tooth sockets after implanting CHA and SrCHA synthesized at low temperatures.Animal experiments and breeding were performed according to the institutional review board (CEUA/ UFF), N°179/2012, the NIH Guide for Care and Use of Laboratory Animals and the Brazilian legislation on animal research.3. The synthesized solids were filtered and washed with deionized water  until reaching neutral pH (pH = 7). Then, the material was lyophilized in a FreeZone 1 lyophilizer  and separated on sieves according to particle sizes from 74 μm to 37 μm.The nanostructured carbonated hydroxyapatite powder and nanostructured carbonated hydroxyapatite containing strontium were prepared using a precipitation wet method with average temperature of 5°C and 6% wt CO®/Fluka Biochemika®, Buchs, Switzerland) diluted in ultrapure water  at a ratio of 15:1. The mixture was then extruded into 0.15 molar calcium chloride (0.15 M CaCl2), in which instant microsphere formation was observed. The mixture was kept at rest for 24 h until complete gelation. After this step, the microspheres were washed with ultrapure water until fully eliminating saline. Immediately after the washing, the microspheres were dried by lyophilization for 24 h and separated on a stainless steel sieve with a particle size from 425 μm to 600 μm. The microspheres were then separated into aliquots in an Eppendorf tube and sterilized with gamma radiation in a cobalt-60 irradiator (Gamma Cell) for 760 m with total dose of 15 kGy and dose rate of 19.72 Gy/m. Electron microscopy scanning of the microspheres showed similar morphology and surface texture. SrCHA presented fewer surface pores than CHA, as previously shownTo produce microspheres, the obtained powders were individually mixed with a solution of sodium alginate  with five animals for each experimental period. The sample size was based previous studies that followed the same animal protocol®, Veltbrands, São Paulo, Brazil) and sedation with 1.5 ml/kg xylazine , the antisepsis of the perioral region and oral mucosa was achieved with 2.0% and 0.12% chlorhexidine, respectively. Then, sindesmotomy and extraction of the upper right central incisor were performed with a dental explorer no. 5  and a pediatric forceps no. 151 , respectively  was administered subcutaneously every 24 h for three d since the day of the surgery.After anesthesia with intramuscular injection of 75 mg/kg ketamine hydrochloride , Itapira, São Paulo, Brazil)] to collect bone blocks containing the biomaterials and surrounding tissues. The specimens were fixed in 4% formaldehyde, decalcified in decalcification solution  for 48 h and embedded in paraffin. The blocks were cut at 5-μm thickness, stained with hematoxylin and eosin (HE) and examined by light microscopy .After the experimental period, the animals were euthanized by applying a lethal dose of thiopental 150 mg/kg [ with 5 microscopic fields under 20x augmentation with a 1-blinded examiner (SCS). In each histological slice stained with HE, 5 non-superimposing microscopic fields obtained by scanning at 20x magnification were captured in the medium third region of the socket after biomaterial implantation. With the Image-Pro Plus® 6.0 , a grid of 250 points superimposed on the area under analysis allowed the determination of the volume density of the newly formed bone, of the connective tissue and of the residual biomaterial. The 250 points superimposed on each photomicrograph were considered as 100%, so each point was classified and the percentage of each parameter was obtained.Histomorphometric analysis was performed to quantify the remaining biomaterial, the connective tissue loose and the bone newly formed in the standard area. The morphometric measurements were performed using the Image-Pro PlusThe results are presented as percentages, and values are presented as the mean value (±) standard deviation. The mean values and standard deviations obtained in each group were tested for normality according to D'Agostino-Pearson's omnibus test. The data did not present a normal distribution, this way both the non-parametric statistical analysis of Mann- Withney for inter-group and and the Wilcoxon for intragroup were performed using the Prism Graph Pad 6.3 software , with p≤0.05 being considered statistically significant.At the first experiment, both biomaterial groups presented the dental socket filled with fragmented biomaterial spheres surrounded by granulation tissue with remnants of blood clots, neoformed capillaries, and fibroblasts. In both groups, we observed moderate mononuclear inflammatory infiltrate as lymphocytes and macrophages between the components of the conjunctive tissue and the surroundings of partially bioabsorbed microspheres. We also observed giant cells permeating the biomaterial particles. New bone formation occurred centripetally in patches during this period and was more evident in the apical region of the socket .In both groups, we observed a discrete chronic inflammatory response, with a few giant cells close to the microspheres, which were evidently fragmented. The biomaterials were fragmented and bioabsorbed differently. The quantity of remaining material in the SrCHA group was slightly lower than that from the CHA group. Bone formation process replaced the inflammatory cell content in the first experiment at the same time that there was a decrease in mature connective tissue composed by collagen fibers. The newly formed bone characterized by the thick trabecular bone was similar in both groups and occurred near the remaining bone and in direct contact with microspheres .The histological evaluation showed that in the interstitial spaces formed between the microspheres with no bone formation had a highly vascularized loose of connective tissue after one week that showed increased organization after six weeks with newly formed bone. This angiogenesis is essential for bone regeneration because these new blood vessels provide oxygen, nutrients, and cells, all considered essential for bone formation.Histomorphometric analysis showed no significant differences after 1 (CHA=14.6±2.50 and SrCHA=18.9±1.69) and 6 weeks (CHA=16.5±2.41 and SrCHA=10.4±2.33) between groups regarding biomaterials bioabsorption . The amo2+, Mg2+, and Zn2+. This change affects the crystallinity, solubility, surface energy, and dissolution rate of the material, thus improving its bioactivity. Based on these findings, this study evaluated a promising bone substitute based on a synthesized at low temperature nanostructured carbonated hydroxyapatite containing strontiumCalcium ions in biological apatite have been partially substituted with other ions, such as SrAccording to a previous studyThe non-strontium-containing hydroxyapatite was used as control group, as we aimed to evaluate the influence of strontium on hydroxyapatite in bone repair. We did not perform the dental socket filling with blood clot, as these results have already been published with the same experimental periods,The biomaterials evaluated in this study were biocompatible, despite the presence of a limited chronic inflammatory response with giant cells that diminished with time. The giant cells, considered as foreign body type giant cells, are of monocyticphagocytic lineage and are important in the tissue repair mechanism, as they carry out cellular and tissue debris phagocytose, as well as contribute to the bioabsorption of biomaterial fragments, besides secreting cytokines that favor essential cellular events in tissue repair. The presence of multinucleated giant cells modulated by the chemical surface of the biomaterials,,,A previous study observed that at the nanoscale, the biomaterial resembled biological apatiteThe use of biomaterial microspheres has been considered preferable because the interstitial space between the implanted spheres provides macropores for tissue invasion and also because microsphere implantation can be performed with minimally invasive surgical techniques. In addition, the spheres do not have surface edges or dimensions that could lead to inflammation. To produce the microspheres, we mixed nanostructured carbonated HA powders with sodium alginate, an inert and biodegradable polymer. The histological results showed that there were interstitial spaces between the microspheres. In areas where there was no new bone formation, we observed a highly vascularized and loose connective tissue after one week that showed increased organization after six weeks. This angiogenesis is essential for bone regeneration because these new blood vessels provide oxygen, nutrients, and cells considered essential for bone formationIn the histological evaluation, we observed a greater fragmentation of SrCHA compared to the CHA group. We did not sinter the biomaterials used in this study, so they are considered low crystalline materials. However, the incorporation of strontium in the hydroxyapatite by partially replacing it with calcium changed crystallinity, morphology, lattice parameters, crystal size, stability, bioactivity, biocompatibility, and osteoconductivity of CHA. This set of physical and chemical changes alter the fragmentation and bioabsorption of biomaterials2+ has been widely used for partial substitutions of HA because of its dual ability to stimulate bone formation and reduce bone resorption. However, in our study, we observed no increase in newly formed mineralized tissue in the SrCHA group, indicating that Sr2+ did not influence the osteogenic potential of nanostructured carbonated HA, possibly because of the low Sr2+ content (1.7%) identified by atomic absorption spectrometry after synthesis and prior to the implantation, regardless of an initial theoretical Sr2+ concentration of 5%. These results are similar to those obtained by other studySr2+ associated with nanostructured carbonated hydroxyapatite are necessary to evaluate the effect of Sr2+ on the biological response.Our results suggest that both CHA and SrCHA, produced at low temperature and not sintered, were biocompatible, bioactive, ostecondutive osteoconductive, and bioabsorbable, indicating its great potential for clinical use as bone substitutes. Further studies with a higher content of Sr


To investigate independent relationships of childhood linear growth (height gain) and relative weight gain to adult cardiovascular disease (CVD) risk traits in Asian Indians.Data from 2218 adults from the Vellore Birth Cohort were examined for associations of cross-sectional height and body mass index (BMI) and longitudinal growth  in infancy, childhood, adolescence, and adulthood with adult waist circumference (WC), blood pressure (BP), insulin resistance (homeostatic model assessment-insulin resistance [HOMA-IR]), and plasma glucose and lipid concentrations.P < .001), BP , and HOMA-IR  but lower glucose concentrations (women β = −0.03 log mmol/L per SD P = .003). Greater height or height gain at all earlier ages were associated with higher adult CVD risk traits. These positive associations were attenuated when adjusted for adult BMI and height. Shorter length and lower BMI at birth were associated with higher glucose concentration in women.Higher BMI/greater conditional relative weight gain at all ages was associated with higher adult WC, after 3 months with higher adult BP, HOMA-IR, and lipids, and after 15 years with higher glucose concentrations. Taller adult height was associated with higher WC (men β = 2.32 cm per SD, women β = 1.63, both Greater height or weight gain relative to height during childhood or adolescence was associated with a more adverse adult CVD risk marker profile, and this was mostly attributable to larger adult size. Cardiovascular disease (CVD) is the leading cause of death globally, and the population incidence of CVD and related metabolic disorders is higher in low- and middle-income countries (LMICs) than in the rest of the world.4Growth patterns in early life are important predictors of adult CVD risk factors.Understanding when in childhood growth relates to later CVD risk may guide the timing of interventions to prevent disease. Identifying specific ages when linear growth and weight (soft tissue) gain predict later outcomes is complicated by the fact that serial measurements of height or weight within an individual are strongly positively correlated, and height and weight are correlated with each other. Two-way conditional growth measures, which are adjusted for prior size, and mutually adjust weight and height for each other, have been developed to overcome these limitations.The Vellore Birth Cohort includes individuals born within defined areas of Vellore town and adjoining rural villages in Tamil Nadu, India during 1969-1973.Adult follow-up took place at a median (IQR) age of 28.1 years .In selecting ages for the growth analysis, we aimed to include (in addition to birth and adulthood) infancy, childhood, and adolescence. The exact ages selected were based upon availability of data. No infant data were collected before March 1, 1971 or in December 1973, and therefore, we excluded births at these times (n = 326), leaving 1892. We also excluded 7 cohort members without birth length and 7 without adult height and weight, leaving 1878.www.jpeds.com). All measurements were converted into within-cohort age- and sex-specific z scores [(subject mean-cohort mean)/cohort SD]. Exact values at age 3 months, and age 6.5 and 15 years were then obtained by interpolation of the z scores, using the nearest measurements to that age and within 2 months for the infant value and 2 years for the childhood and adolescent values, and back-transformation to the units of measurement.Among the 1878, infant measurements were available for 1613 , childhood measurements for 1680 , and adolescent measurements for 1108   and then by additional adjustment for adult body size . Model 1 (“forward-looking” approach) addresses the question: What is the net association of size at each age with the adult outcome? Model 2 (“backward-looking” approach) addresses the question: Given that this person has achieved a particular adult BMI and height, is there any remaining effect on the outcome of size at earlier ages, or do the earlier measurements have all their effect through their contribution to adult size? Both models were adjusted for year of birth and sociodemographic variables. We used interaction tests to examine whether associations between body size and cardiovascular risk markers differed between the sexes, and because there were more statistically significant interactions than expected by chance, all analyses were stratified by sex.We constructed sex-specific and height- and weight-specific conditional variables, which are standardized residuals derived from regressing size z scores at each age on prior size measures.Conditional relative weight and height gain variables represent children's deviation from expected size based on their own prior measures and on the growth of the other children in the cohort, and can be interpreted as representing greater or less than expected soft tissue gain and linear growth respectively. For example, a child with a positive conditional relative weight at 6.5 years of age is heavier than expected given his/her current height and prior size and, thus, had a faster rate of soft tissue gain from age 3 months to 6.5 years. Again, we created “forward-looking” models adjusted only for adult age (model 1), and “backward-looking” models further adjusted for adult BMI and height (model 2). We included 907 participants who had data at all selected ages for conditional analysis. Analyses were undertaken using SPSS v 22  and Stata v 13.1 .P < .001). There were differences in the education level of the head of the household at the time of their birth, but these were small . There were no significant differences in parental weight and height. Subjects studied were similar at birth  and heavier at 3 months of age , but there were no significant differences in measurements during childhood. Subjects studied during adolescence were lighter than the subjects not studied though the differences were small.When compared with the remainder of the original cohort of 10 691 live singleton births, the 1878 men and women included in this analysis were less likely to have been born to a primiparous mother  was 28%, overweight (BMI 25-30 kg/m2) 11%, and obesity (BMI >30 kg/m2) 2%. Despite a low mean BMI, 14% of men and 17% of women had central obesity defined by the International Diabetes Federation as a waist circumference (WC) >90 cm in men and >80 cm in women.Early life and adult characteristics of the study sample are shown in As expected, length or height at all ages was positively correlated with adult height; correlations strengthened with increasing age of the earlier measurement, from r = 0.28 in men and 0.25 in women for birth length to r = 0.66 in men and 0.79 in women for 15-year height. Similarly BMI at all earlier ages correlated positively with adult BMI .Taller adult height was associated with higher WC, BP, and homeostatic model assessment-insulin resistance (HOMA-IR) in both sexes, but with lower 120-minute glucose and cholesterol concentrations in women . Higher The positive associations between earlier height and BMI and adult CVD risk markers were attenuated, and most were no longer statistically significant, after adjusting for adult height and BMI . ExceptiP < .001) but did not differ significantly in birth weight or height and weight at 3 months, 6.5, 15, or 28 years of age, any of the cardiometabolic risk markers, or rural v urban residence at birth or currently.The 907 men and women included in the longitudinal analysis were shorter at birth by 0.48 cm than the 971 not included (www.jpeds.com).The results are presented in www.jpeds.com).The strongest finding was that greater conditional relative weight gain between 15 and 28 years of age was associated with a more adverse CVD risk profile in both sexes. Greater conditional weight gain at all ages was associated with higher adult WC. Conditional relative weight gain between birth and 3 months of age was unrelated to the risk factors except for WC. Greater conditional relative weight gain between 3 months and 6.5 years of age was associated with higher SBP in both sexes. Greater conditional relative weight gain from 6.5 to 15 years of age was associated with higher triglyceride concentrations in both sexes, higher SBP and DBP in women, and higher total cholesterol in men. The positive associations of conditional relative weight gain between 15 and 28 years of age with risk factors were considerably larger in magnitude than those for conditional relative weight gain before the age of 15 years (All the above positive associations were attenuated, and most became nonsignificant after adjusting for adult size. However, greater linear growth between birth and 3 months of age remained associated with higher cholesterol and triglyceride concentrations in men, and higher DBP in women; greater linear growth between 3 months and 6.5 years of age with higher DBP in men; and greater linear growth between 6.5 and 15 years of age with higher BP and total cholesterol and triglyceride concentration in men, and DBP in women.Overall, our study shows that greater height and BMI after birth and through to adolescence are associated with higher CVD risk factors in adulthood, and these associations are largely attributable to adult height and BMI. The significant differences between males and females were mainly due to differences in the specific ages at which earlier size or growth were related to the outcomes.Strengths of the study include the use of prospectively recorded growth data and the use of conditional growth analysis, which enabled us to examine independent effects of weight and height growth during specific age periods. Conditional variables are uncorrelated, and expressing them as z scores allows direct comparison of coefficients within regression models. This offers advantages over other representations of growth, and provides more information than weight gain alone. However, we acknowledge that conditional relative weight gain cannot differentiate between lean and fat gain. Very few birth cohorts have longitudinal measurements of lean and fat tissue through childhood, but recent data from a younger South Indian cohort has shown that the adipose component of childhood weight gain contributes most strongly to associations of weight gain with CVD risk markers.Positive associations of BMI and conditional relative weight gain from midchildhood onward with adult risk markers are in agreement with recent studies from LMICsPediatricians in LMICs tend to promote weight gain during infancy because it is known to increase survival and benefit neurodevelopment. This practice has been questioned recently because of data from developed countries showing that rapid infant weight gain is associated with an increased risk of adult obesity and insulin resistance.Taller height and faster linear growth during infancy, childhood, and adolescence were associated with higher WC. Positive associations between height or height gain and BP, insulin resistance, cholesterol, and triglycerides were less consistent and mostly explained by greater adult size, although the associations of BP with linear growth 6.5-15 years of age, and of cholesterol with linear growth 0-3 months of age and 6.5-15 years of age in men remained significant after adjustment for adult size. An association of WC with height is not surprising, given that WC is a measure of frame size as well as of central adiposity. An association between BP and height in children is well described and possibly represents a physiological adaptation to perfuse a longer arterial system.Longer birth length was associated with lower glucose and cholesterol in women, and after adjusting for adult size, with lower HOMA-IR and triglycerides. Higher BMI at birth was associated with lower glucose concentration in women, and in men after adjusting for adult size, similar to previous reports that showed inverse associations between birth weight and T2DM.Monitoring childhood weight and height, and active intervention to prevent or reverse upward crossing of BMI percentiles may reduce later CVD risk. Individuals who had greater linear growth during childhood and/or became taller as adults had higher adult WC, BP, insulin resistance, and cholesterol concentrations. It is not clear whether these associations reflect an increased risk of future CVD,


Human mammary tumor virus (HMTV) is 90–95% homologous to mouse mammary tumor virus (MMTV), one of the causal agents of murine mammary tumors. HMTV (MMTV-like) sequences were reported to be present in human breast cancers from several populations with a prevalence range of 0–78%; however, the prevalence of HMTV in breast cancers from Myanmar remains unknown.Fifty-eight breast cancer samples from Myanmar women were examined in this study. DNA was isolated from formalin-fixed paraffin-embedded specimens, and HMTV envelope sequences were detected by semi-nested PCR. The sequence of the PCR products was also confirmed.Only 1.7% (1 of 58) of the breast cancers were positive for HMTV, and the sequence of PCR products was 98.9% identical to the reference HMTV sequence (GenBank accession No. AF243039). The tumor with HMTV was grade III invasive ductal carcinoma, 7.0 cm in size with lymph node metastasis .We, for the first time, investigated the presence of HMTV in Myanmar breast cancer patients. In accordance with other Asian studies, the prevalence of HMTV in Myanmar was quite low, supporting the hypothesis that Asian breast cancers have different etiologies than in Western countries, where HMTV is more prevalent. Worldwide, breast cancer is the most frequently diagnosed cancer affecting women, with an estimated 1.7 million cases and 521,900 deaths in 2012 , 4.The etiology of human breast cancer can be significantly affected by environmental factors, including viruses , 6. AmonAs with other countries, breast cancer is a leading cause of morbidity and mortality in Myanmar women . The purth edition was applied for tumor staging. The experimental protocol employed in this study was approved by the Ethics Committee of Okayama University and the Ethics Review Committee of Department of Medical Research of Yangon City (Myanmar).In this study, we employed 58 breast cancer cases diagnosed in 2015 at Myeik General Hospital  and Sakura Specialist Hospital . All hematoxylin and eosin-stained sections were reviewed by two independent pathologists. The criteria defined by the World Health Organization (2012) were used for the histopathological diagnosis and classification of breast carcinoma . NottingTwo to four 10-μm-thick sections were cut from each paraffin block, and genomic DNA was extracted using the Nucleospin DNA FFPE XS kit  according to the manufacturer’s instructions. The amount of extracted DNA was measured on a Nanodrop 1000 spectrophotometer . The A260:A280 ratio was used to measure the purity of DNA (~1.80). DNA quality was also confirmed by PCR amplification of the 268 bp β-globin gene using GH20 and PC04 primers  DNA was amplified by semi-nested PCR using the primers mt15982F and mt16267R for the first-round PCR and mt16115F and mt16267R for the second-round PCR, which yielded a final PCR product of 153-bp. DNA extracted from the 4 T1 murine breast cancer cell line was used as positive control.The list of all primers used and their positions in the genome are shown in Table xl Genetic Analyzer  at the Central Research Laboratory of Okayama University. PCR products were isolated from gels, sequenced and aligned by a BLAST search (NCBI).Sequencing was performed using an ABI3130Clinical data for the 58 breast cancer patients are shown in Table Experiments were conducted to analyze the prevalence of HMTV. Genomic DNA was extracted from each paraffin block and its quality was measured using the A260:A280 ratio >1.80) and a distinct β-globin PCR product. HMTV sequence was investigated in all 58 samples using semi-nested PCR, which revealed one case (MB14) was positive for HMTV . To the best of our knowledge, the prevalence of HMTV (MMTV-like) sequences in breast cancers has been reported for 17 countries, including the present study. Figure The prevalence of HMTV shows geographic heterogeneity. A high prevalence of HMTV was detected in North and South America, Australia and Mediterranean countries, where the range is from 12 to 78% (average 49.4%) –25. Convhttp://globocan.iarc.fr.). Breast cancer develops at earlier ages in Asia (peak incidence between 40 and 50 years) than in Western countries (peak incidence between 60 and 70 years) [Mus domesticus, having the highest numbers of endogenous MMTV proviral loci, inhabits the United States, Italy and Australia, where the prevalence of HMTV in breast cancers is high. On the other hand, M. musculus and M. castaneus, having the fewest MMTV proviral loci, ranges from central Europe east to China and Japan and from southern China to central Iran, respectively.Interestingly, Asian countries with zero or low HMTV prevalence such as Japan, Vietnam and Myanmar have low breast cancer incidence  compared with countries with high HMTV prevalence like the United States, Italy and Australia  (0 years) . These d0 years) . Mus domA question arises about the sensitivity of the detection system. False negatives may be present in zero or low prevalence reports as primers and/or reaction conditions are critical in the detection system , 38; howIt would be interesting if there was a correlation between HMTV status and clinicopathological parameters. Although several reports demonstrated causal association between these , 24, 39,This is the first study to report the prevalence of HMTV in breast cancers in Myanmar. The prevalence of HMTV in Myanmar was consistent with other Asian countries with low or zero prevalence. The frequency of infection and HMTV sequence closely resembled that from Vietnam. It appears that HMTV does not play an important role in breast cancer carcinogenesis in most Asian populations.


Aquila chrysaetos canadensis) in three geographic regions of western North America to conduct a preliminary study of the genetics of the North American subspecies, and to provide data for United States Fish and Wildlife Service (USFWS) decision-making for golden eagle management. We used a combination of mitochondrial DNA (mtDNA) D-loop sequences and 16 nuclear DNA (nDNA) microsatellite loci to investigate the extent of gene flow among our sampling areas in Idaho, California and Alaska and to determine if we could distinguish birds from the different geographic regions based on their genetic profiles. Our results indicate high genetic diversity, low genetic structure and high connectivity. Nuclear DNA Fst values between Idaho and California were low but significantly different from zero (0.026). Bayesian clustering methods indicated a single population, and we were unable to distinguish summer breeding residents from different regions. Results of the mtDNA AMOVA showed that most of the haplotype variation (97%) was within the geographic populations while 3% variation was partitioned among them. One haplotype was common to all three areas. One region-specific haplotype was detected in California and one in Idaho, but additional sampling is required to determine if these haplotypes are unique to those geographic areas or a sampling artifact. We discuss potential sources of the high gene flow for this species including natal and breeding dispersal, floaters, and changes in migratory behavior as a result of environmental factors such as climate change and habitat alteration. Our preliminary findings can help inform the USFWS in development of golden eagle management strategies and provide a basis for additional research into the complex dynamics of the North American subspecies.Understanding the genetics of a population is a critical component of developing conservation strategies. We used archived tissue samples from golden eagles ( An understanding of genetic diversity and structure is vital for developing conservation strategies for species of concern . GeneralAquila chrysaetos) are Holarctic in distribution and some studies have evaluated genetic diversity and structure for the species [A. c. canadensis) occurs, Katzner et al. [Golden eagles , has primary authority for the management of golden eagles in the US. Some golden eagles are harvested by Native Americans and falconers ; current management also allows for additional “take” of golden eagles under certain circumstances. The Eagle Act defines “take” as “…pursue, shoot, shoot at, poison, wound, kill, capture, trap, collect, or molest or disturb…”. Recently, the USFWS began to permit a limited unintentional “take” of eagles at energy projects , but witGenetic data can help identify subpopulations and are useful for designing conservation and management plans ,15,34,35We investigated population genetic variation and structure among sampled golden eagles from three geographic areas in western NA using a combination of mitochondrial DNA (mtDNA) D-loop sequences and microsatellite loci. We used archived blood samples from birds that spent the summer (breeding) season in Idaho/eastern Oregon, in California, and in Alaska and samples of eagles wintering in Idaho that represented both migrants and year-round residents. Our research goals were to: 1) investigate the extent of gene flow among golden eagles from these three geographic areas, 2) determine if summer resident eagles from different geographic locations in western NA can be distinguished based on their genetic profiles and 3) determine if the natal origin of winter residents in Idaho can be determined from their genetic profiles. We discuss our results as initial data for understanding the genetics of the subspecies, as a consideration for USFWS in evaluating the effects of permitting actions, and in development of conservation and management plans that must account for complex lifetime movements of individuals.n = 45), tissue (n = 3), feathers (n = 9) and fecal material (n = 1). In addition, we used blood samples from eagles that wintered in Idaho (n = 9) to determine if we could assign them to geographic summer locations based on our results from known origin birds. DNA was extracted from blood, tissue and feather samples using a modified DNeasy Blood and Tissue Kit protocol  [The samples from golden eagles were collected by the authors or were contributed  ,37, and n = 6) and California , were tested for amplification success and variability in birds from Idaho .® Terminator v3.1 Cycle Sequencing Kit. Sequencing products were cleaned using the BigDye® Xterminator™ Purification Kit according to the manufacturer’s protocol . Sequences were run on a 3130xl Genetic Analyzer  and analyzed using Sequencher 4.7 . Sequences were aligned by eye in Sequencher 4.7 (Gene Codes Corporation), and the number of haplotypes was determined using the filter redundant taxa option in MacClade 4.08 [www.ncbi.nlm.nih.gov/genbank/).The sample of western golden eagles was also assessed using mitochondrial DNA (mtDNA) sequencing of the D-loop region . A ~415bp fragment of the D-loop region was amplified using the primers GOEA_CR1L and GOEA_CR595H . PCR proade 4.08 . Golden ade 4.08  were dows [The number of alleles, observed and expected heterozygosity and the probability of identity (PID and PIDsibs) were calculated for each microsatellite locus using Gimlet 1.3 . The eigs ) were pes .A median joining network was drawn using Network 5 . Included in the network were sequences from an additional 6 eagles that summered in Alaska .ST in Arlequin 3.5 for both the microsatellite loci and the D-loop haplotype data [The samples from Alaska were excluded from population differentiation analyses due to small sample size. The degree of genetic differentiation between California and Idaho summer resident golden eagles was assessed by calculating pairwise Fype data ,45.To evaluate whether individuals can be classified into genetic groups based on their genetic signature, we used an assignment test approach as implemented in the software Geneclass 2 ,47, plusTwenty-five of the 36 microsatellite loci amplified successfully and produced bands in the correct size ranges  and all 6 samples from Alaska provided by Domenech and Sherrington. Eight mtDNA D-loop haplotypes were detected  and Idaho populations .Haplotype (h) and nucleotide (π) diversity was similar between California (h = 0.74 ± 0.064 SD), (π = 0.0025 ± 0.0019 SD) and Idaho (h = 0.68 ± 0.085 SD), (π = 0.0023 ± 0.0018 SD) and lower for Alaska (h = 0.49 ± 0.18 SD), (π = 0.0013 ± 0.0013 SD). Tajima’s D and Fu’s FST value calculated from the microsatellite data between Idaho and California was low but significantly greater than zero  indicating that the two populations are not in panmixia, but genetic differentiation is low between the Idaho and California golden eagle populations. The pairwise FST value calculated from the D-loop haplotype data between Idaho and California was also low and not significantly greater than zero .The pairwise FST values less than 0.03 [The assignment test implemented in Geneclass 2 correctly assigned an individual eagle to its sampling region of origin 60% of the time. This suggests there is not enough of a difference between allele frequencies in the California and Idaho populations to confidently assign individuals to their sampling population with the current number of loci. The results from program STRUCTURE indicate that the value of K with the greatest log likelihood is one when analyzing the Idaho and California samples only, and we obtained the same result when the 6 samples from Alaska were included . Programhan 0.03 . Three oResults of the AMOVA showed that 97% of the mtDNA haplotype variation was within the geographic populations while 3% of the haplotype variation was among populations . Using gSimilar to other work on raptors in NA ,12,13, wS using mtDNA sequence data from eagles in Idaho and California provide no evidence for recent population expansion or bottlenecks.Our findings of low genetic structure and high connectivity are consistent with earlier preliminary comparisons of mtDNA and nDNA structure of 44 eastern and 25 western NA golden eagles that found no evidence for population substructure and shared mtDNA haplotypes across these two regions . HoweverST levels (0.03) also indicated high gene flow. Our study adds to the growing understanding of golden eagle genetics in NA and similar to Doyle et al.’s recent work [In contrast to our findings, Sonsthagen et al.  were ablent work , furtherGenetic research can contribute to understanding the extent to which animals rely on resources and face threats beyond their natal area . Some diNatal dispersal is integral to genetic structure, species demography, and conservation . In IdahBreeding dispersal by adults also could contribute to gene flow among our sample populations. Unsuccessful golden eagle breeders can disperse  and reprUnderstanding the role that floaters and other immigrants play in the population dynamics at a locale is difficult to do without knowledge of the genetics of that portion of the population. Undetected replacement of breeders can mask mortality in breeding locales . For exaGolden eagles can encounter multiple risks  across S1 File(DOCX)Click here for additional data file.