In the current study, we aimed to investigate the effects of alveolar decortication on local bone remodeling, and to explore the possible mechanism by which decortication facilitates tooth movement.Forty rabbits were included in the experiment. The left mandible was subjected to decortication-facilitated orthodontics, and the right mandible underwent traditional orthodontics as a control. The animals were sacrificed on the days 1, 3, 5, 7 and 14, after undergoing orthodontic procedures. Tooth movement was measured by Micro-CT, and the local periodontal tissues were investigated using H&E, Masson's trichrome and tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of genes related to bone remodeling in the alveolar bone were analyzed using real-time PCR.On days 3, 5, 7 and 14, tooth movement was statistically accelerated by decortication (P < 0.05) and was accompanied by increased hyperemia. Despite the lack of new bone formation in both groups, more osteoclasts were noted in the decorticated group, with two peak counts (P < 0.05). The first peak count was consistent with the maximum values of ctsk and TRAP expression, and the second peak counts accompanied the maximum nfatc1 and jdp2 expression. The increased fra2 expression and the ratio of rankl/opg also accompanied the second peak counts.Following alveolar decortication, osteoclastogenesis was initially induced to a greater degree than the new bone formation which was thought to have caused a regional acceleratory phenomenon (RAP). The amount of steoclastogenesis in the decorticated alveolar bone was found to have two peaks, perhaps due to attenuated local resistance. The first peak count in osteoclasts may have been due to previously existing osteoclast precursors, whereas the second may represent the differentiation of peripheral blood mononuclear cells which came from circulation as the result of hyperemia. Alveolar decortication-facilitated orthodontics is a category of new introduced techniques which combines alveolar decortications with orthodontic treatment , 2. It iAlveolar decortication has been widely used in orthodontics. It was first used to facilitate the correction of maxillary protrusion in 1931 and, since that time, to accelerate tooth movement , 4. NumeIn traditional orthodontics, both bone absorption by compression and formation by tension contribute to the tooth movement , 13. HowWe know that the first step of tooth movement is bone resorption on the pressure side. In order to find out what happens in the beginning stages after decortication and eliminate interference factors, we focused primarily on osteoclasts in this study. We hypothesized that decortication accelerates osteoclastogenesis in rabbit local alveolar bone early in orthodontic tooth movement, and that this is associated with tooth movement .Decortication-facilitated animal models were established in forty female New Zealand white rabbits, 5 months old and weighing between 3.4 and 5 kg, all consistent with our previous study . RabbitsFollowing the protocol of previous studies , 12, 15,When the animals were sacrificed, at the appropriate time point, the mandibles were immediately isolated and imaged at a resolution of 50 microns with high-resolution Micro-CT, the Inveon MM CT , as in previous studies . Samples3 each), each containing the first molar and surrounding alveolar bone, were dissected for each time point. The samples were fixed in 10% buffered formalin at 4°C for 48 hours, rinsed with sterile saline, decalcified in 10% ethylenediamine tetra-acetic acid  for 2 months with daily replacement and embedded in paraffin. Five-micron thick sections were cut in the mesial-distal direction of the first molar. The sections were stained with hematoxylin and eosin (H&E). One section from each time point was stained with H&E, another section was stained with Masson’s trichrome stain to visualize the newly formed bone [Three mandibular segments . The primers for the osteoclastogenic markers in various stages , as well as regulators (rankl/opg) are listed in The expression of genes related to osteoclastogenesis markers were investigated. Five mesial alveolar bones from the first molar at each time point from each group were separated, and the total RNA was isolated with TRIZOL Reagent . A total of 2 μg of total RNA was reverse-transcribed into cDNA using the Superscript First-Strand Synthesis System (Invitrogen) following the manufacturer’s protocol. Reactions were conducted in a 20-μl reaction mixture using the ABI 7500 real-time PCR system (ABI). The expression of genes related to bone metabolism was normalized to GADPH expression, expressed as 2All assays were repeated at least three times for each sample. Statistical analysis was performed using SPSS 18.0 . T-tests were performed to evaluate the differences in data between the experimental and control groups. P < 0.05 was considered statistically significant.The tooth movement distance was measured with Micro-CT . The disThe periodontal tissues were investigated with H&E staining in both groups on the days 3 and 7 after the procedure. Hyperemia of the periodontal ligament and the proximal alveolar wall was noted, particularly on day 3 . The hypNew bone formation in the tensile sides were visualized with Masson's trichrome staining. In the periodontal tissues on the tensile sides, where osteogenesis tends to occur after 14 days , none ofThe periodontal tissues in the compressed sides, where osteoclastogenesis is frequently observed , were inFurthermore, the number of osteoclast count exhibited two peaks in the experimental groups: the first peak was on the day 3, after which the count fell close to its minimum value on the day 5. There was a subsequent rise from day 7 through day 14, when the count reached a second peak .Isolation of mRNA from the compressed alveolar bone was performed and was analyzed for genes related to bone remodeling. In the experimental groups, the mRNA levels of trap and ctsk (cathepsin), markers reflecting osteoclast activity , 20, incThe mRNA levels of nfatc1 (nfat transcription complex 1) and jdp2 (jun dimerization protein 2), which reflect osteoclast differentiation , 22, werSimilarly, in the experimental groups, the rankl/opg ratio, a positive indicator of osteoclastogenesis , was iniIn this study, alveolar decortication-facilitated orthodontics and traditional orthodontics were performed in New Zealand white rabbits . As in pAs the decortications resulted in accelerated tooth movement, the compressed alveolar bone of the experimental groups developed significantly more osteoclasts relative to the controls Figs  and 5. HFurthermore, the number of newly formed osteoclasts exhibited two peak counts—on the days 3 and 14 post-surgery Figs  and 5. TThe mRNA levels of ctsk and TRAP reached their maximum values simultaneously with the first osteoclast peak on day 3 . Ctsk anDecortication led to hyperemia that persisted until day 7 , when thThe rankl/opg ratio, which is proportionate to the amount of osteoclastogenesis , was alsIn this study, animal models of decortication-facilitated orthodontics were established for accelerated tooth movement, consistent with previous studies , 6, 12.


The pressor response to laryngoscopy is known to be exaggerated in patients with severe preeclampsia.The aim of the present study was to compare the efficacies of continuous intravenous (IV) infusion of nitroglycerine, IV hydralazine, or sublingual nifedipine in modifying cardiovascular responses to endotracheal intubation, in women with severe preeclampsia undergoing cesarean delivery under general anesthesia.A total of 120 patients undergoing cesarean delivery were randomly divided into 3 groups, each receiving one of the following drugs before intubation: 5 µg/min nitroglycerine administered by continuous IV infusion ; a 10-mg capsule of nifedipine deposited sublingually ; or 5–10 mg hydralazine intravenously . Heart rate (HR), systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) were simultaneously recorded in the mother at pre-induction, pre-intubation, and at 1, 3, 5, and 10 min after intubation.In contrast to those in group NIF and group H, the patients in group NTG showed no significant increases in HR, SAP, DAP, or MAP after intubation, compared to baseline. The incidence of hypotension was significantly greater in group NIF than in group H or group NTG .In patients with severe preeclampsia undergoing cesarean delivery, a continuous IV infusion of nitroglycerine was able to attenuate the cardiovascular response to intubation to a greater extent than the use of sublingual nifedipine or IV hydralazine, without significant adverse effects on the newborn. The pressor response to laryngoscopy and tracheal intubation is a very important issue in hypertensive pregnant patients, involving an increase in systemic and pulmonary arterial pressures and pulmonary capillary wedge pressure -4.In addition, the stress of laryngoscopy can be associated with an increase in intracranial pressure, with the risk of cerebral hemorrhage and cardiac failure with pulmonary edema. Consequently, there is an increased risk of morbidity and mortality of both mother and baby . In ordeNitroglycerin has also been administered for rapid perioperative treatment of maternal hypertension . LongmirTherefore, to test our hypothesis, we conducted the present study, in which we compare the efficacies of continuous IV infusion of nitroglycerine, intravenous hydralazine, or sublingual nifedipine in blunting the cardiovascular effects of laryngoscopy and tracheal intubation, in women with severe preeclampsia undergoing cesarean delivery under general anesthesia.After obtaining institutional approval and informed consent, 120 patients with severe preeclampsia and gestational age of more than 20 weeks, who presented for elective or urgent cesarean delivery under general anesthesia, were enrolled in this randomized double-blind study. The patients had contraindications for spinal anesthesia. All patients had BP ≥ 160/110 and met the defining criteria of severe preeclampsia according to the American College of Obstetrics and Gynecology . CriteriPresuming a 10% rate of maternal hypotension using hydralazine  comparedAs candidate patients were enrolled in the study, they were randomly allocated to 1 of 3 groups according to the agent to be used for attenuation of the pressor response to laryngoscopy: hydralazine , nitroglycerine , or nifedipine . Allocation was performed in a double-blind fashion using a sealed envelope technique. Group H received 5–10 mg hydralazine intravenously. The initial dose of hydralazine was 5 mg, with further doses of 10 mg at intervals, according to the protocol recommended by the American College of Obstetrics and Gynecology . Group NThe study solutions were prepared by an anesthesiologist who took no further part in the study. The anesthesiologists who evaluated the patients were not aware of their group allocation. All other individuals involved in the study, including the assisting nurses and the data analysts, were also blinded to the group allocations and the drugs administered throughout the trial. Patients were premedicated with 10 mg metoclopramide and 50 mg ranitidine intravenously, 30 min before induction of anesthesia. Monitors included pulse oximetry for oxygen saturation (SpO2); electrocardiogram (ECG); end-tidal carbon dioxide (EtCO2); and noninvasive automatic BP monitoring for systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), and heart rate (HR).After pre-oxygenation for 5 min, patients were given the study drugs. One hundred seconds after receiving the study drug, anesthesia was induced with 5 mg/kg thiopental sodium given over 10-15 s, and an assistant applied cricoid pressure from the time of induction of anesthesia until the airway was secured. Laryngoscopy and tracheal intubation were performed 60 s after giving 1. 5 mg/kg succinylcholine, thus ensuring that the time from administration of the study drug to tracheal intubation was 3 min. Anesthesia was maintained with 50% nitrous oxide in oxygen and 1. 2% isoflurane. Atracurium was used for muscle relaxation. EtCO2 was maintained at 33–40 mmHg throughout surgery.After delivery of the baby, analgesia was provided by 0. 1 mg/kg morphine, and 20 IU IV Syntocinon in 1 L Ringer’s lactate was infused to initiate uterine contraction. HR, SBP, DBP and MBP, ECG in lead II, and SpO2 were simultaneously recorded in the mother at pre-induction, pre-intubation (2 min after giving test drug), and at 1, 3, 5, and 10 min after intubation. During the peri-intubation period, occurrence of any type of arrhythmia, or cardiovascular complications (such as hypo- or hypertension) was noted. Hypertension was defined as an increase in BP to more than 120% of baseline values, and when BP was reduced to less than 80% of the baseline, it was considered as hypotension. After completion of surgery, the residual effects of neuromuscular blockade were reversed with 0. 05 mg/kg neostigmine and 0. 02 mg/kg atropine. Apgar scores were evaluated at 1, 5, and 10 min. The person recording the data was blinded to the study drug administered. Statistical analysis was performed using SPSS version 16. 0. MBP was taken as DBP plus 1/3 × (SAP-DAP). Statistical comparisons among the groups were performed using 2-way analysis of variance (ANOVA), followed by an unpaired t-test with Bonferroni’s correction. Hemodynamic responses to induction and intubation in a given group were analyzed using a repeated-measurements ANOVA (1-way ANOVA) followed by a paired t-test with Bonferroni’s correction. Continuous variables are presented as mean ± SD. Ordinal variables are presented as numbers (%). The Chi-squared test was applied to categorical data with Yates’ correction. Alternatively, when any expected number was less than 5, Fisher’s exact method was used. P < 0. 05 was considered the minimum level of statistical significance.Demographic characteristics, parity, baseline (preoperative) hemodynamic data, duration of laryngoscopy, and Cormack-Lehane grades of direct laryngoscopy were comparable between the 3 groups . HR was SAP was significantly lower at 1, 3, and 5 min after endotracheal intubation in group NTG compared with group NIF (P < 0. 05) . In addiMAP was significantly lower at 1, 3, and 10 min after endotracheal intubation in group NTG compared with group H (P < 0. 05) . PatientThe incidence of hypotension was significantly greater in group NIF compared with group H or group NTG . There were 2 patients in group H who developed arrhythmia, while no patients in group NIF or group NTG had arrhythmia (P > 0. 05). There was no significant difference between the 3 groups regarding gestational age, birth weight, and Apgar scores . Our study showed that nitroglycerine, administered by continuous infusion during the peri-induction period of general anesthesia, was more effective than hydralazine or nifedipine in preventing increases in HR, SAP, DAP, and MAP after laryngoscopy and intubation, in patients with severe preeclampsia undergoing cesarean delivery.Direct laryngoscopy and tracheal intubation cause an increase in BP and HR . This caOur study showed that hydralazine was ineffective for attenuation of the stress caused by laryngoscopy during induction of general anesthesia. This may be due to the slow onset and variable duration of action of this drug, as well as compensatory tachycardia, which makes it difficult to titrate its action against the hypertensive response , 30. HilNifedipine is one of several agents that have been used for the reduction of BP in severe preeclampsia. As a calcium channel blocker, it causes peripheral arterial vasodilatation . NifedipIt has been shown that modes of administration of nifedipine that avoid first-pass metabolism in the gastrointestinal tract, such as use of a sublingual perforated capsule, result in greater bioavailability, and a faster accumulation and more stable concentration of nifedipine in serum than methods that do not avoid first-pass metabolism  . In addiThe finding that the nifedipine-treated group showed both greater MAP variability and a greater maternal HR response than the nitroglycerine-treated group after laryngoscopy and tracheal intubation implies that, under the present study conditions, sublingual nifedipine induces a greater variability in the arterial baroreflex system, through mechanisms that cannot be ascertained by the present study. A study by Fenakel et al.  indicateIn conclusion, the present study found that, in women with severe preeclampsia who are managed with controlled extracellular volume expansion and MgSO4 loading and maintenance doses, a continuous IV infusion of nitroglycerine was able to attenuate the pressor response to laryngoscopy and intubation to a greater extent, faster, and more precisely than the use of sublingual nifedipine or IV hydralazine, without significant adverse effects on the baby. Limitations of our study are the lack of a proper control group and incomplete data on the potential adverse effects of hydralazine, nitroglycerine, or nifedipine on the fetus and newborn. Although more research must be done, we believe that nitroglycerine infusion may provide safe and effective prophylaxis for patients with severe preeclampsia undergoing cesarean delivery under general anesthesia, in attenuating hemodynamic responses to laryngoscopy and tracheal intubation.


Proteins of the major intrinsic protein (MIP) family, or aquaporins, have been detected in almost all organisms. These proteins are important in cells and organisms because they allow for passive transmembrane transport of water and other small, uncharged polar molecules.We compared the predicted amino acid sequences of 20 MIPs from several algae species of the phylum Heterokontophyta (Kingdom Chromista) with the sequences of MIPs from other organisms. Multiple sequence alignments revealed motifs that were homologous to functionally important NPA motifs and the so-called ar/R-selective filter of glyceroporins and aquaporins. The MIP sequences of the studied chromists fell into several clusters that belonged to different groups of MIPs from a wide variety of organisms from different Kingdoms. Two of these proteins belong to Plasma membrane intrinsic proteins (PIPs), four of them belong to GlpF-like intrinsic proteins (GIPs), and one of them belongs to a specific MIPE subfamily from green algae. Three proteins belong to the unclassified MIPs, two of which are of bacterial origin. Eight of the studied MIPs contain an NPM-motif in place of the second conserved NPA-motif typical of the majority of MIPs. The MIPs of heterokonts within all detected clusters can differ from other MIPs in the same cluster regarding the structure of the ar/R-selective filter and other generally conserved motifs.We proposed placing nine MIPs from heterokonts into a new group, which we have named the LIPs (large intrinsic proteins). The possible substrate specificities of the studied MIPs are discussed.The online version of this article (doi:10.1186/1471-2164-15-173) contains supplementary material, which is available to authorized users. Escherichia coli[The major intrinsic proteins (MIPs) , or aquachia coli and aquachia coli were thechia coli. Plant achia coli–9. The tchia coli. Certainchia coli, 5, 11. chia coli, 13: e.gchia coli. The porchia coli. It was chia coli. HoweverDiatoms are unicellular, phototrophic, eukaryotic organisms that are present in all marine and freshwater habitats. They originated as a result of double endosymbiosis followed by long-term (240 million years) evolution –18, whicIn this study we investigate MIPs from Chromista, phylum (Infrakindom) Heterokontophyta . The length of the predicted SarMIP is 286 a.a. [Pseudo-nitzschia multiseries[Fragilariopsis cylindrus[Ten MIP genes were found in the complete genome sequences for the diatoms , TpMIP2  and T. o, ToMIP2  , as well as from Aureococcus anophagefferens, AaMIP1, AaMIP2, AaMIP3, and AaMIP4 (class Pelagophyceae).The database dedicated to Major Intrinsic Proteins MIPdb  containsThe purpose of the present study was to compare the predicted a.a. sequences of 20 MIPs from these algae with MIP sequences from a wide variety of organisms. We show that MIPs from heterokonts belong to different subfamilies, and nine of them merge into a new Large intrinsic protein (LIP) subfamily, which is closely related with the SIP subfamily  and the We used the MIPdb to find homologues of the 20 MIPs from heterokonts. This database contains 8429 MIPs belonging to 11 groups (subfamilies): 577 AQPes, 1150 AQPps, 363 GLAes, 1827 GLAps, 1052 GLPps, 192 NIPs, 661 PIPs, 42 SIPs, and 375 TIPs, as well as 1053 predicted MIPs. There are 1137 sequences that are unclassified MIPs. MIPdb contains 16 MIPs from heterokonts, 15 of them belonging to the unclassified group and one of them (EsPIP) belonging to AQPe , a glyceroporin (EcGlpF), and NIPs from rice and maize . It has been showed that these four NIPs are transporters of silicic acid , 35. AmoThe results of the phylogenetic analysis Figure  demonstrP. tricornutum, and AaMIP2 from the A. anophagefferens clustered within a large clade that includes GLAp, GLAe, and GLPp with a bootstrap support of 58%.Three sequences  from T. pseudonana, ToMIP2 from T. oceanic, and AaMIP3 from A. anophagefferens clustered with unclassified MIPs from bacteria with bootstrap supports of 52% and 100%, respectively.The sequences of TpMIP1 from E. siliculosus clustered with plant PIPs with a bootstrap support of 79%. Nine sequences  constituted a separate clade with a bootstrap support of 98%. Their closest relatives are the plant SIPs and MIPC from green algae. AaMIP4 from A. anophagefferens clustered with MIPE from green algae with a bootstrap support of 71%. Only one sequence of PtMIP4 did not reliably belong to any clade. EsAQP and EsPIP clustered with CcPIP4;1 and CcPIP4;2 from green algae. AaMIP2, PtMIP1, PtMIP2, and PtMIP3 clustered with GIPs from green algae as a sister clade with a bootstrap support of 58%.Two sequences (EsAQP and EsPIP) from the brown alga Of the 20 heterokonts’ MIPs studied  in place of the cysteine (C) at position LE1. The LE1 position in the filters of EsMIP, NgMIP, PtMIP5, PnmMIP, FcMIP, AaMIP1, SarMIP, TpMIP2, and ToMIP1, as well as a majority of the filters of the sister clade, was occupied by a proline (P). Unlike the other MIPs, their LE2 positions are occupied by leucine (L) or isoleucine (I). Hence, EsMIP, NgMIP, PtMIP5, PnmMIP, FcMIP, AaMIP1, SarMIP, TpMIP2, and ToMIP1 differed from the other MIPs in this feature. The H2 strand of the filter includes tryptophan (W)/tyrosine (Y) in these nine proteins, which is a more typical feature of GLPp, GLAp, and NIPs that are not able to transport silicic acid  from the diatom A. anophagefferens also belongs to the GlpF-like intrinsic proteins, as revealed by the phylogenetic analysis  form a separate clade adjacent to the SIP clade of plants and the MIPC clade of green algae. Ishibashi et al.  concludeOn the basis of the above evidence, we suggest a new phylogenetic clade, LIPs, which includes nine proteins: EsMIP, NgMIP, PtMIP5, PnmMIP, FcMIP, AaMIP1, SarMIP, TpMIP2, and ToMIP1 .(DOCX 44 KB)Additional file 1:


Spatial variations in river water quality were related to numerous anthropogenic and natural factors. Urban land use was found to be the most important explanatory variable for BOD5, CODMn, TN, DN, NH4+-N, NO3−-N, DO, pH and TP. The animal husbandry output per capita was an important predictor of TP and turbidity, and the gross domestic product per capita largely determined spatial variations in EC. The remaining unexplained variance was related to other factors, such as topography. Our results suggested that pollution control of animal waste discharge in rural settlements, agricultural runoff in cropland, industrial production pollution and domestic pollution in urban and industrial areas were important within the Cao-E River basin. Moreover, the percentage of the total overall river water quality variance explained by an individual variable and/or all environmental variables (according to RDA) can assist in quantitatively identifying the primary factors that control pollution at the watershed scale.Understanding the primary effects of anthropogenic activities and natural factors on river water quality is important in the study and efficient management of water resources. In this study, analysis of Variance (ANOVA), Principal component analysis (PCA), Pearson correlations, Multiple regression analysis (MRA) and Redundancy analysis (RDA) were applied as an integrated approach in a GIS environment to explore the temporal and spatial variations in river water quality and to estimate the influence of watershed land use, topography and socio-economic factors on river water quality based on 3 years of water quality monitoring data for the Cao-E River system. The statistical analysis revealed that TN, pH and temperature were generally higher in the rainy season, whereas BOD The deterioration of river water quality has become a primary environmental concern due to unsustainable anthropogenic activities; the demand for freshwater has been rapidly increasing in many developing countries, especially in China Table S1. However, comprehensive applications of different statistical methods to evaluate the effects of environmental variables on water quality have not been fully explored in river studies in China. Furthermore, few studies have used RDA analysis to quantitatively evaluate the effects of watershed characteristics on the overall river water quality.Recently, Pearson correlations have been widely employed to determine the relationships between environmental variables and river water quality The Cao-E River is located upstream of the Qiantang Estuary, which is a major riverine system in Zhejiang Province, China, and is the primary source of industrial, agricultural and domestic water supplies In this study, ANOVA, Pearson correlation analysis, MRA, PCA and RDA were applied to investigate the effects of sub-watershed land use, topography and socio–economic factors  on river water quality at the watershed scale for the Cao-E River system in a GIS environment. The objectives of this study were threefold: (1) to examine the temporal and spatial variations in water contamination within the study area, (2) to investigate the relationships between the river water quality parameters and land use, topography and socio-economic factors, and (3) to identify the primary pollution sources to estimate the possible sources that affect the water quality parameters in the Cao-E River basin. The results can be helpful for water conservation in the Cao-E River basin, and they provide a valuable tool for water quality agencies to develop assessment strategies for effective water quality management and rapid solutions for water pollution problems at the watershed scale.No specific permits were required for the described field studies; the sampling did not cause any disturbance to the environment or to the protected species at the sampling sites.The Cao-E River  is located in Zhejiang Province, East China. The Cao-E River is one of the main tributaries of the Qiantang Estuary, which flows into the East China Sea. The river system contains a main stream and several major tributaries  at the specific sampling sites were triple more than the standard deviation for total sampling sites. The samples were collected once a month between 9:00 am and 16:00 pm. Moreover, the water samples were collected at approximately 0.3 m below the water surface in the central stream, placed into plastic bottles (2.5 L), transported to the laboratory and stored at 0∼4°C for subsequent chemical analysis. The chemical measurements were performed in the laboratory within 24 h after collecting the water samples. Moreover, dissolved oxygen (DO), water temperature (T), pH and electrical conductivity (EC) were measured when the water samples were collected with a hand-held multi-parameter instrument . The turbidity was measured with a hand-held Turbiquant 1000IR . The following chemical and biological water quality parameters were measured: chemical oxygen demand (CODMn), 5-day biochemical oxygen demand (BOD5), total nitrogen (TN), total phosphorus (TP) and dissolved phosphorus (DP), which were measured using standard methods 4+-N) content was measured using an Astoria analyzer system  after filtration through a 0.45-µm filter . The nitrate nitrogen (NO3−-N) content was measured from the absorbance of the sample at 220 nm (A220) and 275 nm (A275) using a visible and ultraviolet spectrophotometer  after filtration through a 0.45-µm filter; the results were determined by the following relationship: A220 - 2×A275 The stream water samples  were collected from July 2003 to June 2006 at 20 sampling sites throughout the watershed . SpecifiLatitude and longitude were measured using a hand-held GPS. The catchment boundaries were delineated on a 30-m spatial resolution digital elevation model (DEM); the stream network was represented using the Soil and Water Assessment Tool (SWAT) model. ArcGIS 9.3 Desktop GIS software was used to calculate the watershed characteristics for each sampling site. All datasets were converted to a common digital format (WGS 1984) and a common coordinate system . Hydrologic units were not solely defined by the watershed above an individual point The socioeconomic data, including land use change, the gross domestic product, animal husbandry output and human population in the watershed, were obtained from the Shengzhou (SZ), Xinchang (XC), Shangyu (SY) and Shaoxing (SX) County Statistical Yearbooks for the period 2003–2006 and were adjusted with some typical investigation in several towns within the watershed. The temporal variations in land use were considered to be minimal. The annual changes in forest areas and water areas were less than 0.05%, the annual decrease rate in cropland areas was less than 1% and the annual increase rate in urban areas was less than 2.5%., respectively, for the entire watershed and throughout the study period. Thus, Landsat Thematic Mapper imagery from 2006 was used to map the land cover in the study area p variables  that are interrelated or correlated to various degrees into a new dataset containing p new orthogonal and uncorrelated variables; these variables are called the principal components (PCs) p original variables . Additional PCs up to p can be formulated in a similar manner. The variances of the PCs are the eigenvalues; the coefficients or weights, are the eigenvectors extracted from the covariance or correlation matrix. In the example PCA, the correlation matrix was used in all cases. The goal of PCA is that the first k PCs (where k<<p) retain most of the information in all of the p original variables, which effectively reduces the practical dimensionality of the dataset. More specifically, if the correlations are high among many of the original variables, the first few PCs contain (or explain) a large percentage of the total variance and may be used to describe multivariate patterns or water quality variations across the watershed nearly as well as using the complete set of p original variables. These patterns are often related to specific sources of contamination Analysis of variance (ANOVA) was used to compare river water quality variations between different seasons  via the Student-Newman-Keuls multiple-range test (P<0.05). Relationships among the considered variables were tested using Pearson's coefficient with statistical significance set at P<0.05. Major gradients and principal patterns in the water quality data between wet and dry seasons were detected using principal component analysis (PCA). PCA transforms a dataset consisting of Table S2. The statistical analyses, except RDA, were completed using the SAS 9.1 software for Windows.The stepwise regression analysis was performed to determine the environmental or socio-economic factors  that best explained the spatial variability of an individual river water quality variable 5, TN, DO, pH, T and turbidity exhibited significant temporal variations (p<0.10). Moreover, TN, pH and T were generally higher in the rainy season (April to September), whereas higher values for BOD5, DO and turbidity occurred in the dry season (October to March). Most of the water quality parameters, except pH and T, exhibited considerable spatial variations. BOD5, CODMn and NH4+-N were higher in urban areas compared with surrounding rural areas, whereas DO exhibited a reverse response to the extent of urbanization. TN, DN, NO3−-N, TP and DP were higher in the lower part of the basin where more land has been developed and were lower in the upper-eastern part of the Cao-E River basin. Turbidity and EC were higher in the lower part of the basin . Temporal and spatial variations in the physico-chemical parameters are shown in 5, CODMn, TN, DN, NH4+-N, and T. The PCA of the wet season water quality data showed that the eigenvalues for the 2 most significant factors were 8.442 and 1.817, accounting for 78.9% of the total variance. Factor 1 accounted for 64.9% of the total variance and was positively correlated with BOD5, CODMn, TN, DN, TP and DP  explained 87.8% of the total variance Table 3.3−-N, TP and DP . Moreover, UB was positively correlated with BOD5, CODMn, TN, DN, NH4+-N, TP, DP and EC  and negatively correlated with DO and pH . FRT was positively correlated with DO and pH  and exhibited a negative correlation with BOD, COD, TN, DN, NH4+-N, NO3−-N TP, and DP . Furthermore, SLPmean was positively correlated with DO  and negatively correlated with CODMn, TN, DN, NH4+-N, NO3−-N, TP, DP, T, turbidity and EC . The elevation was positively correlated with DO  and negatively correlated with CODMn, TN, DN, NO3−-N, TP, DP, turbidity and EC . Additionally, GDPpc exhibited a positive correlation with COD, TN, DN, NO3−-N, TP, DP, turbidity, and EC . AHOpc was positively correlated with TN, NO3−-N, TP, DP, turbidity and EC . Lastly, HPd exhibited a negative correlation with DO and pH  and a positive correlation to all other river water quality parameters  , which was positively correlated with UB and HPd and negatively correlated with FRT and SLPmean  and the RDA analysis  results, urban areas play a pivotal role in influencing water quality . Topography largely regulated the river water quality parameters. Most related studies have shown that higher SLPmean and/or ELV lead to higher erosion rates, which subsequently increase the rate at which particulate matter enters a water body mean and ELV were both negatively correlated with TN, DN, NO3−-N, TP and DP; SLPmean exhibited a highly significant negative correlation with NO3−-N values . Similar results were published regarding water quality spatial variations in the Han River basin in South Korea Topographical factors played important roles in explaining spatial variations in river water quality within the Cao-E River basin  Pollution control for animal waste discharge in rural settlements was important in the study area. Moreover, agricultural runoff, industrial pollution and domestic pollution in urban and industrial areas were also important factors within the Cao-E River basin.The percentage of total overall river water quality variance explained by individual variables and/or all environmental variables in the study area (as determined using RDA) can assist in quantitatively identifying primary pollution control factors at the watershed scale.This study improved our understanding of the anthropogenic activities and natural factors that affect river water quality and can assist in the design of efficient strategies for controlling river water pollution at the watershed scale. Moderate-resolution remote sensing data were adopted in this study; future investigations will require high-resolution DEM maps and additional land use classes to better evaluate the effects of specific environmental variables on overall river water quality.Table S1Summary of commonly used statistical methods on pollution source identification in recent years.(DOC)Click here for additional data file.Table S2Application summary of 5 statistical methods in this study.(DOC)Click here for additional data file.


Assessment of health effects of a forest environment is an important emerging area of public health and environmental sciences.To demonstrate the long-term health effects of living in a forest environment on subclinical cardiovascular diseases (CVDs) and health-related quality of life (HRQOL) compared with that in an urban environment.This study included the detailed health examination and questionnaire assessment of 107 forest staff members (FSM) and 114 urban staff members (USM) to investigate the long-term health effects of a forest environment. Air quality monitoring between the forest and urban environments was compared. In addition, work-related factors and HRQOL were evaluated.2, NOx, SO2, CO, PM2.5, and PM10 in the forest environment were significantly lower compared with those in the outdoor urban environment. Working hours were longer in the FSM group; however, the work stress evaluation as assessed by the job content questionnaire revealed no significant differences between FSM and USM. HRQOL evaluated by the World Health Organization Quality of Life-BREF questionnaire showed FSM had better HRQOL scores in the physical health domain.Levels of total cholesterol, low-density lipoprotein cholesterol, and fasting glucose in the USM group were significantly higher than those in the FSM group. Furthermore, a significantly higher intima-media thickness of the internal carotid artery was found in the USM group compared with that in the FSM group. Concentrations of air pollutants, such as NO, NOThis study provides evidence of the potential beneficial effects of forest environments on CVDs and HRQOL. Specifically, living in a forest environment lowers cholesterol concentrations, blood pressure (BP), and pulse rates, increases levels of natural killer cell activity, has beneficial effects on cardiovascular and metabolic parameters, improves psychological wellbeing, enhances human immune function, decreases sympathetic nerve activity, and enhances parasympathetic nerve activity in the human body The objectives of this study were (1) to compare the measurements of cardiovascular parameters and health-related quality of life (HRQOL) in people working in forest and urban environments and (2) to demonstrate the forest environment has superior air quality compared with that in an urban environment.This research has been approved by the 37th meeting  of Research Ethics Committee of the National Taiwan University Hospital. The committee is organized under, and operates in accordance with, the Good Clinical Practice guidelines and governmental laws and regulations. The study participants have provided their written informed consent before received a series of detailed examinations and questionnaires.Because the staff members in forest environment  were 160 in total, excluding those of clinical diabetes and documented cardiovascular diseases, we determined sample size (n = 100) for healthy volunteers in forest and urban groups and sent 120 invitations to staff members for each environment for this study. All participants provided informed consent for our study before examination. Finally, the study participants included 114 urban staff members (USM) working in an urban environment in Taipei city and 107 forest staff members (FSM) working in an experimental forest of National Taiwan University, Nantou County, Taiwan. The FSM group or USM group included participants who had worked in the forest or urban environment for more than 1 year.All participants received a series of detailed examinations and questionnaires, including baseline examination, oral glucose tolerance test (OGTT), HRQOL questionnaires, job stress assessment, cardio-ankle vascular index (CAVI), and carotid artery IMT assessment. The health examination and questionnaires (including QOL questionnaire and job stress assessments) were conducted during the initial spring season from January 31, 2013 to February 25, 2013 in Taipei and Nantou County, Taiwan.Forest and urban environmental samples were collected from the Xitou experimental forest of National Taiwan University, and an interior office of a commercial building in Taipei, respectively. The Xitou experimental forest, located in central Taiwan, covers 2,349 ha and ranges in elevation from 500 to 2,025 m. The mean relative humidity and temperature data from 2010 to 2012 were 89% and 17°C, respectively, as indicated by the Xitou monitoring station of the Central Weather Bureau by using Finger Stick Capillary Dried Blood Spots. Plasma glucose and serum levels of cholesterol, triglycerides, and low- and high-density lipoprotein cholesterol (LDL-C and HDL-C) were measured using an auto-analyzer . Small dense LDL-C also was measured by commercial kit .BP was measured twice after at least 5 min of rest in a sitting position. The systolic BP used in the analyses was the average of two measurements. In both groups, a standard 75-g OGTT was performed after an overnight fast of at least 10 h, with measurements from blood samples at fasting via an ante-cubital vein, and at 30 min, 1 h, 90 min, and 2 h post-challenge plasma glucose 3, TECO 49, API 400A: based on the principle of UV absorption), a nitrogen oxide analyzer , a sulfur dioxide analyzer , a PM10 monitor , temperature, and relative humidity probe . CO, O3, NO, NO2, NOx, SO2, PM10, temperature, and relative humidity measurements were recorded daily every minute during the examination period. PM2.5 was collected by a Harvard impactor  Instruments for forest and urban environmental monitoring were set in the Xitou experimental forest and in an interior office of a commercial building (indoor environment) in Taipei. Each monitoring system comprised a carbon monoxide analyzer , an ozone analyzer . ABI and 4-limb blood pressure were also measured. The vascular screening system was placed in a quiet and independent room in the Xitou natural education area. CAVI and ABI for each participant were conducted at least twice. The test-retest reliability of CAVI and ABI in the Taiwanese population has been demonstrated as excellent Carotid atherosclerosis in the common carotid artery was assessed by measuring carotid IMT, using a high-resolution B-mode, GE Vivid i ultrasound system , equipped with a 3.5–10 MHz real-time B-mode scanner. Details concerning the methods of carotid IMT measurements have been reported previously The brief Taiwanese version of the World Health Organization Quality of Life (WHOQOL-BREF) The general characteristics, OGTT, environmental factors, working hours, and job stress scores were compared between the groups FSM and USM. Continuous variables were expressed as the mean ± standard deviation and binary variables were expressed in percentage. We used t-test to test the group mean difference of the characteristic variable if the variable is continuous and normally distributed. The Chi-square test was applied for categorical data.Data regarding the difference between FSM and USM including CAVI, ABI, and maximum and mean values of IMT at CCA, ICA, BULB, and mean IMT  were compared using fitted regression models with the adjustment of age, gender, fasting sugar, SBP, BMI, LDL-C, and habits of smoking and alcohol. Work stress scores and HRQOL scores also were compared between two groups in regression models with the adjustment of age, gender, fasting sugar, SBP, BMI, LDL-C, smoking, and alcohol in multiple regression models. Data analysis was performed with SAS statistical software .General characteristics among participants in the FSM and USM are summarized in OGTT results are presented in The working hours, job stress scores, and WHOQOL-BREF domain scores are presented in 2, NO, NO2, NOx, CO, O3, and PM10), temperature, and relative humidity are presented in 2, NO, NO2, NOx, CO, PM10, temperature, and relative humidity factors between the urban (indoors and outdoors) and forest environments. The forest environment had higher O3 concentration than the indoor urban environment, however there were no significant differences between of forest environment (23.1 ppb) and outdoor urban environments (25.4 ppb). The mean concentration of PM2.5 in the forest environment was lower than in the urban environment (indoor and outdoor). Despite a non-significant trend for PM2.5 between the forest and indoor urban environments, the mean concentration of PM2.5 in the outdoor urban environment was 37.2±24.0 µg/m3, which was higher than in the forest environment (7.2±3.9 µg/m3), with a significant difference (p<0.028).The mean concentration of air pollutants  in forest environments compared to urban (indoor and outdoor) environments. In a previous study, we have proved that short-term O3 exposure had a significant adverse effect on aortic stiffness by CAVI measurement in young mail-carriers 3 level in forest environment were not lower than that in urban environment, the potential hazardous effect arose from O3 exposure should be taken into account. This partially explains why there are no significant differences in CAVI measurement between the two groups in our study, even though all air quality indicators other than O3 were better in forest environment. Because CAVI measurements can be affected by short-term air pollution exposure 3 concentration in forest environment. There is a need to continuously monitor the concentration of O3 and the changes in different seasons. As for PM10 and PM2.5, the mean concentration in the outdoor urban environment was also higher than those in the forest environment.Urban air pollution associated with CVDs morbidity and mortality has been well documented It is commonly recognized that PM consists of soil dust, nanoparticles, industrial emissions, sea salts, smog particles, and combustion particles from vehicle sources The concept of QOL complements the WHO definition of health as “not only the absence of disease and infirmity but also the presence of physical, mental, and social well-being” This study has several strengths and limitations, as follows: this is the first study to demonstrate the long-term health effects of a forest environment on subclinical CVDs and HRQOL compared with those of an urban environment. Even though the study indicated the potential beneficial health effects of living in a forest environment on CVDs and HRQOL, we indeed cannot infer the better HRQOL and subclinical CVDs in workers living in forest environment because of their better air quality by real-time monitoring of air pollutants in forest compared with those of an urban environment.First, the CV effects of changes in seasons have not been considered in this paper. Seasonal variation of cardiovascular events may be linked to the changes of cardiovascular and endocrine/metabolic markers in different seasons Fourth, the sample size is small, and we may not have had the statistical power to detect a significant effect on the CAVI and OGTT after 2 h post-challenge plasma glucose, and job stress with different variables. Therefore, increasing the number of samples for detailed studies of their significant differences is very important in the future.Even though the preliminary results report the first detailed survey and environmental monitoring during early spring. The ongoing study of this project will explore the seasonal changes in the health effects of both FSM and USM groups by 4-seasons’ field environmental monitoring, and follow-up health examinations to corroborate and provide important evidence on the health effects of a natural environment as an alternative therapeutic option for CVDs.In conclusion, this study indicated the potential health effects on subclinical marker of cardiovascular disease, in terms of CIMT and subjective HRQOL in workers living in forest environment. A large-scale and cohort study in peoples living in forest comparing to living in urban environments should be warranted.


Conventional diffusion imaging techniques are not sufficiently accurate for evaluating glioma grade and cellular proliferation, which are critical for guiding glioma treatment. Diffusion kurtosis imaging (DKI), an advanced non-Gaussian diffusion imaging technique, has shown potential in grading glioma; however, its applications in this tumor have not been fully elucidated. In this study, DKI and diffusion weighted imaging (DWI) were performed on 74 consecutive patients with histopathologically confirmed glioma. The kurtosis and conventional diffusion metric values of the tumor were semi-automatically obtained. The relationships of these metrics with the glioma grade and Ki-67 expression were evaluated. The diagnostic efficiency of these metrics in grading was further compared. It was demonstrated that compared with the conventional diffusion metrics, the kurtosis metrics were more promising imaging markers in distinguishing high-grade from low-grade gliomas and distinguishing among grade II, III and IV gliomas; the kurtosis metrics also showed great potential in the prediction of Ki-67 expression. To our best knowledge, we are the first to reveal the ability of DKI to assess the cellular proliferation of gliomas, and to employ the semi-automatic method for the accurate measurement of gliomas. These results could have a significant impact on the diagnosis and subsequent therapy of glioma. Gliomas are the most common type of intra-axial brain tumors. They have different cellular origins and are divided into four grades according to the World Health Organization (WHO) criteria . AccuratConventional diffusion weighted imaging (DWI) of three orthogonal directions and diffusion tensor imaging (DTI) are not sufficiently accurate in evaluating the glioma grade –6 and Kib value) in more than 15 nonlinear directions, the kurtosis metrics (including mean kurtosis (MK), axial kurtosis (Ka) and radial kurtosis (Kr)) and conventional diffusion metrics (including mean diffusivity (MD), axial diffusivity (Da), radial diffusivity (Dr) and fractional anisotropy (FA)) are obtained simultaneously. Ka is parallel to the main direction of diffusion, Kr is perpendicular to the main direction of diffusion, and MK is the average kurtosis of all diffusion directions [Diffusion kurtosis imaging (DKI) is an advanced non-Gaussian diffusion imaging technique that can be used to account for this deficiency. It provides a more accurate model of diffusion for quantifying the deviation from a Gaussian distribution, which is known as kurtosis . By acqurections . Ka mighrections . As an erections –14. Addirections , 15. Thurections , traumatrections , neoplasrections , neuroderections , 20, andrections . AccordiGliomas comprise a heterogeneous group of tumors characterized by increased microstructural complexity and heterogeneity, especially for higher grade gliomas or gliomas with higher cellular proliferation, which might impede proton diffusion and lead to higher non-Gaussianity and increased kurtosis , 14. AltIn this study, we assessed and compared the value of the kurtosis metrics  and conventional diffusion metrics  in grading gliomas and also evaluated the correlation between these metrics and the Ki-67 labeling index (Ki-67 LI). Our results demonstrate that the kurtosis metrics are more promising imaging markers in grading, and also had great potential in the prediction of cellular proliferation in gliomas.DKI and DWI scans were performed on 102 patients with suspected glioma between July 2012 and June 2014. Of these 102 patients, 23 were excluded because they failed to undergo resection or biopsy, and another 5 were excluded because their lesions were histopathologically confirmed to be non-gliomas. Ultimately, a total of 74 patients were included in this study, and all of them underwent tumor resection. According to WHO criteria, 3 had grade I glioma, 31 had grade II, 19 had grade III, and 21 had grade IV. Other detailed information for the patients is shown in t-test. The results demonstrated that the kurtosis metrics were significantly higher in the HGGs compared with the LGGs . In contrast, MD and ADC were significantly lower in the HGGs compared with the LGGs . However, FA did not differ significantly between the two groups . Therefore, all metrics except FA were able to well distinguish HGGs from LGGs.The kurtosis and conventional diffusion metric values in the solid region of the tumor and contralateral normal-appearing white matter (NAWM) are reported as the mean and standard deviation in Table P < 0.001; Ka: 0.589 ± 0.087 for grade II, 0.725 ± 0.119 for grade III, 0.877 ± 0.115 for grade IV, P < 0.001; Kr: 0.367 ± 0.084 for grade II, 0.491 ± 0.075 for grade III, 0.588 ± 0.077 for grade IV; P < 0.001); the differences between each pair of grades were also significant in multiple comparisons (P < 0.05). This observation was also the case for MD and ADC among grade II, III and IV gliomas  as well as between each pair of grades in multiple comparisons (P < 0.05). However, FA did not show significant differences among grade II, III and IV gliomas . As the tumor grade increased, the kurtosis metrics showed an increasing trend; in contrast, a decreasing trend was demonstrated for MD and ADC, whereas no obvious trends were found for FA, as shown in Figure In addition to differentiating between HGGs and LGGs, the differences among grade II, III and IV gliomas were further compared using one-way ANOVA, and Student-Newman-Keuls tests were used for the multiple comparisons. The kurtosis metrics were significantly different among grade II, III and IV gliomas  curves. The area under the curve (AUC), optimal cut-off value, and corresponding sensitivity and specificity for all metrics used to differentiate between LGGs and HGGs, between grade II and III gliomas, and between grade III and IV gliomas are reported in Table P < 0.05 for all).The AUCs of MK, MD, ADC and FA were further compared because these four metrics are the representative metrics. The results shown in Table P < 0.001), between grade II and III gliomas , and between grade III and IV gliomas ; whereas the other metrics were not included in the stepwise multiple logistic regression model in all differentiations.Furthermore, a stepwise multiple logistic regression analysis of MK, MD, ADC and FA was performed to find the most significant metric for the differentiations, and the results demonstrated that MK was a significant predictor positively associated with the glioma grade in the differentiations between HGGs and LGGs . The difference in Ki-67 LI among grade II, III and IV gliomas was also significant (P < 0.001), which was also the case for Ki-67 LI between each pair of grades (P < 0.05). The Ki-67 LI was higher for higher grade gliomas.The gliomas from 66 patients were subjected to additional immunohistochemistry examination to detect Ki-67 expression. The Ki-67 LI for different grade gliomas are reported as the mean and standard deviation in r = 0.623, P < 0.001; Ka: r = 0.629, P < 0.001; Kr: r = 0.597, P < 0.001), as well as for MD  and ADC . In contrast, FA had no obvious correlation with Ki-67 LI . Corresponding scatter diagrams are shown in Figure As the prediction of cellular proliferation is valuable in the evaluation of tumor behavior, response to therapy and prognosis, the correlations between Ki-67 LI and each metric were evaluated using Pearson correlation analysis. Significant correlations were found between Ki-67 LI and the kurtosis metrics , 25 with diffuse astrocytoma (grade II), 16 with anaplastic astrocytoma (grade III), 1 with gliomatosis cerebri (grade III) and 21 with glioblastoma (grade IV). The results obtained from subgroup analysis were basically consistent with those of the whole glioma group analysis, and these results are shown in P < 0.001 for all). The kurtosis metrics, MD and ADC were significantly different among grade II, III and IV gliomas , and the differences between each pair of grades were also significant in multiple comparisons  with the exception of MD in the differentiation between grade III and IV astrocytic tumors. The kurtosis metrics increased but MD and ADC decreased as the tumor grade increased. However, FA did not show significant differences between each pair of glioma grades .The kurtosis metrics were significantly higher (but MD and ADC were significantly lower) in the HGGs compared with the LGGs . Moreover, the results of the stepwise multiple logistic regression analysis of MK, MD, ADC and FA also demonstrated that MK was a significant predictor positively associated with the glioma grade in all differentiations , whereas the other metrics were not included in the stepwise multiple logistic regression model.Of all the ROC curves, the ROC of the kurtosis metrics exhibited the maximal AUCs and optimal sensitivity and specificity in all differentiations. MD and ADC showed lower ones, followed by FA. The AUC of MK was significantly higher than that of MD, ADC and FA in differentiating between LGGs and HGGs as well as between grade II and III gliomas; the AUCs of MD and ADC were significantly higher than that of FA in differentiating between LGGs and HGGs . In contrast, FA had no obvious correlation with Ki-67 LI (P > 0.05).Additional immunohistochemistry of Ki-67 was performed for the gliomas from 58 patients, and significant correlations were found between Ki-67 LI and each kurtosis metric, MD or ADC , but their AUCs, sensitivity and specificity were lower than those of the kurtosis metrics. These findings are basically in agreement with those of previous researchers except for the results of MD, because previous researchers found that MD could not be used to distinguish between grade II and III gliomas , 15. TheBy comparing the AUCs of ROC curves of the kurtosis and conventional diffusion metrics combined with the stepwise multiple logistic regression analysis of MK, MD, ADC and FA, the kurtosis metrics were shown to be the most promising imaging markers for grading gliomas in the present study. These occurred because a series of changes caused by higher grade gliomas greatly increased the heterogeneity and complexity of the microstructure of tissues and cells , 15, 23.In addition, our study assessed the correlations between these metrics and the cellular proliferation of glioma. Ki-67 was chosen as a proxy for evaluating cellular proliferation because Ki-67 detection can be performed easily and routinely. Moreover, Ki-67 detection is considered one of the most reliable methods for evaluating cellular proliferation and for providing information on tumor behavior and on response to treatment and prognosis . In the In contrast, FA was decreased at all grades and also all levels of Ki-67, perhaps because diffusion is restricted to a similar degree in all directions in the solid region of the tumor. A different tumor grade or Ki-67 level only changes the sphere size of the diffusion range, but does not change its shape. In short, FA is of low value in evaluating both glioma grade or cellular proliferation.Investigating the relationship between glioma grade and DKI measurements has been covered in multiple previous works , 14, 15.b values in 25 nonlinear directions rather than 5 and 3 b values in more than 25 nonlinear directions, as reported in previous studies [In acquiring DKI data, we used 2  studies , 15. Thi studies . This me studies –32, acut studies  and brai studies , and mos studies , 35 have studies , 15. TheIn conclusion, normalized kurtosis metrics in the solid region of the tumors were better diagnostic factors in distinguishing HGGs from LGGs and identifying grade II, III and IV gliomas; the kurtosis metrics also offered great potential to noninvasively predict the cellular proliferation of gliomas in the studied cohort of patients. Compared with conventional diffusion metrics, the kurtosis metrics show greater potential as imaging markers for the accurate demonstration of the microstructural changes caused by increasing glioma grade and cellular proliferation, which might allow more accurate diagnosis and optimal therapy for glioma patients.This prospective study was approved by the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology and abided by the statement of ethical standards. Informed consent was obtained from every patient prior to inclusion in our study. The following inclusion criteria were applied in this study: 1) patients who were suspected of having conditions of a) primary cerebral glioma and b) recurrent gliomas untreated for more than 6 months prior to MRI based on conventional radiologic findings; 2) patients who underwent routine MRI, DWI and DKI in the same scanner; and 3) patients with tumors that were histopathologically confirmed as cerebral gliomas by subsequent resection or biopsy. The following exclusion criteria were applied: 1) patients who rejected surgery or underwent surgery more than 4 weeks after DKI; and 2) patients with motion artifacts.All patients underwent magnetic resonance imaging (MRI) prior to surgery with a 3.0T GE MR 750 system .2). Two images of b0 were acquired, and b values of 1,250 and 2,500 s/mm2 were applied in 25 uniformly distributed directions. The acquisition time was 5 minutes 45 seconds.DKI used a spin-echo echo-planar imaging (SE-EPI) diffusion sequence for image acquisition . Images with and without 3 orthogonal directional motion-probing gradients  were obtained simultaneously. The acquisition time was 42 seconds.DWI images were also acquired using a SE-EPI sequence , transverse T2 fast spin echo (T2-FSE) and transverse T2 fluid-attenuated inversion recovery (T2-FLAIR). The following acquisition parameters were applied: TR = 2,992 ms, TE = 24 ms, TI = 869 ms, NEX = 1, matrix = 320 × 320 for T1-FLAIR; TR = 4,599 ms; TE = 102 ms; NEX = 2; matrix = 320 × 224 for T2-FSE; TR = 8,000 ms; TE = 160 ms; TI = 2,100 ms; NEX = 1; matrix = 256 × 256 for T2-FLAIR. The section thickness, spacing, section number and FOV of all routine sequences were 5 mm, 1.5 mm, 20 and 240 × 240 cmAll the sequences had the same scan coverage. The scan plane paralleled the line combining anterior and posterior commissure, and the range covered the entire brain.The DKI data were processed using Diffusional Kurtosis Estimator , and the DWI images were processed using the image calculator of ImageJ . Metric maps were calculated, including MK, Ka, Kr, MD, FA and ADC.Before delineating the ROI, the image resolution, number of slices and FOV of enhanced T1-FLAIR, T2-FSE and ADC maps should be changed to match the DKI metric maps. Although the scan matrix, slice thickness, spacing and FOV were different in enhanced T1-FLAIR, T2-FSE, DWI and DKI, the image resolutions ultimately generated by the scanner was 512 × 512, 512 × 512, 256 × 256 and 256 × 256, respectively, due to interpolation; in addition, these four sequences had the same scan coverage. Thus, the image resolution of enhanced T1-FLAIR, T2-FSE and ADC was first resized to 240 × 240 (pixels were resized to 1 mm × 1 mm to match the DKI metric maps); the number of slices was changed to 43 without interpolation; and the canvas size was adjusted to 256 × 256 due to the difference in FOV. All these protocols were finished in ImageJ, as shown in ROIs over the solid region of the tumor and NAWM were semi-automatically delineated using the wand tool in ImageJ by an experienced neuroradiologist who was blinded to the histological diagnosis. To determine the inter-observer reproducibility, another neuroradiologist used the same method to delineate the ROIs in 30 randomly selected patients. Semi-automatic delineation was achieved by setting the optimal threshold range and proper combination of ROIs, including “AND”, “OR” and “XOR”. First, a proper threshold range of signal intensity was set for each subject to ensure that the red color covered the area of interest as completely as possible but did not cover the surrounding structures. Next, the wand tool was used to automatically delineate the connective pixels as a ROI. If the ROI created covered the entire area of interest, then the ROI delineation was finished; otherwise, for example, if it only covered a portion of the area, additional ROIs covering the residual portions were created using another threshold range to perform the combination of ROIs using “OR”. In certain cases, “AND” (for selecting a common area of two or more ROIs) and “XOR” (for selecting different area of two or more ROIs) were also applied to flexibly draw the ROIs, depending on the situation. Example delineations of the ROIs are shown in Figure Average MK, Ka, Kr, MD, FA and ADC values were calculated for each ROI. The values in the solid region of the tumor were normalized to the corresponding values in the contralateral NAWM of each patient to eliminate whole-brain variations between individuals .To ensure a match of the location between histopathology and MR imaging, we first determined the area of the solid tumor preoperatively via MR images, and then requested the neurosurgeon to obtain the corresponding tissue during surgery for the further histopathological analysis. The nature and grade of the tumor were determined according to the 2007 WHO classification . Immunohhttps://www.medcalc.org/, version 11.4.2.0). The inter-observer variability of measurements in 30 randomly selected patients was evaluated using the intra-class correlation coefficient. The independent-samples t-test was used to compare the differences in metrics in the solid region of the tumor and Ki-67 LI between HGGs and LGGs. One-way ANOVA was further employed to compare the differences among grade II, III and IV gliomas, and the Student-Newman-Keuls tests were used for multiple comparisons. The ROC curves were applied to evaluate the diagnostic efficiency of each metric in grading gliomas and to determine the optimal cut-off values. The Z test was applied to compare the differences in AUCs among MK, MD, ADC and FA. A stepwise multiple logistic regression analysis of MK, MD, ADC and FA was also performed to find the most significant metric for differentiating between each pair of glioma grades. The Pearson correlation analysis was used to evaluate the correlation between Ki-67 expression and each metric. A default alpha level of 0.05 was used for all tests, and all the tests were two-tailed.The data were analyzed using SPSS software  and MedCalc software (


Fabry disease (FD) is a lysosomal disorder leading to progressive systemic involvement, including microvascular damage that leads to neurological and cardiovascular disorders. We hypothesize that the latter could be documented at an early stage by performing a microcirculation study with nailfold capillaroscopy and evaluation of Raynaud phenomenon.The objective was to measure the prevalence of Raynaud phenomenon and nailfold capillaroscopic abnormalities in FD.This cross-sectional study included a standardized questionnaire and a nailfold capillaroscopy that assessed previously reported patterns in FD , and was conducted on 32 Fabry patients and 39 controls. Capillaroscopic photographs were reviewed by 2 independent blinded investigators.P < 0.001), of whom none were males (P < 0.001). Raynaud phenomenon was concomitant or before the occurrence of pain in the extremities in 42% of Fabry patients.Twelve Fabry patients (38%) suffered from Raynaud phenomenon, 5 were males , compared with 2 controls (13%) (P = 0.016).More ramified capillaries were significantly observed in Fabry patients  than in controls (5/39, 13%, Secondary Raynaud phenomenon should lead to screening for FD, especially in men. By extension, in high-risk populations for FD, the presence of Raynaud phenomenon and ramified capillaries should be assessed. GLA gene, belongs to X-linked lysosomal disorders. The progressive accumulation of neutral glycosphingolipids, particularly globotriaosylceramide, within lysosomes leads to a multiorgan disease.2 The “classic” severe phenotype, including systemic involvement, has an estimated incidence of 1 in 117,000 in the general population.3 This figure is probably underestimated, as suggested by newborn screening and by the discovery of atypical variants with late-onset isolated renal or cardiac manifestations.7 A diagnosis is frequently delayed for many years because of non-specific signs.8Fabry disease (FD), secondary to a deficit in α-galactosidase A caused by abnormalities in the 11 especially if it is started before the onset of complications, hence the importance of early screening of these patients. One of the earliest and common symptoms is pain in the extremities,1 classically attributed to neurologic involvement .14 FD also induces vasculopathy of small vessels by accumulation of neutral glycosphingolipids in smooth muscular and endothelial cells, which affect the microcirculation.16 Raynaud phenomenon results from a disorder of vascular thermoregulatory control mechanisms, related to an increased sympathetic activity affecting the microcirculation and to endothelial dysfunction. This is more pronounced in the case of secondary Raynaud phenomenon.17Enzyme-replacement therapy is available and decreases morbidity,Although nailfold capillaroscopy assesses small vessels and Raynaud phenomenon is secondary to dysregulation of vascular homeostasis, little research has been done on these subjects in FD.18 followed by another case report of ramified capillaries in 1994,19 and a study of 8 members of a family who had FD in 2006, which reported 37% of ramified capillaries.20 Wasik et al,21 in a matched case–control study with 25 Fabry patients  in 2009, reported 72% of dystrophic capillaries, with a higher incidence of Raynaud phenomenon in Fabry patients, especially male patients (20%). Costanzo et al,22 in 2014, supported these results and reported 52.6% of dystrophic capillaries in a study of 19 Fabry patients, compared with 0% in 19 matched controls.The first identification of dystrophic capillaries in FD occurred in 1993 with the description of 3 case reports,The objective of this study was to measure the prevalence of Raynaud phenomenon and nailfold capillaroscopic abnormalities in a larger cohort of Fabry patients.We performed a cross-sectional study during a patient-support association meeting. This meeting, organized by the FD French patients association in June 2013, brought together already diagnosed Fabry patients from all over France. Diagnoses were made by experienced clinicians in FD on patients presenting clinical signs or familial history of the disease with a biological confirmation . The subjects fulfilled the following inclusion criteria: being aged ≥18 years, and had given their written informed consent after oral information was supplied by the investigators.23 of the 3 last fingers of each hand  to assess the following parameters, according to the previously reported patterns observed in FD:dystrophic capillaries (particularly bushy and ramified capillaries),avascular fields,irregular architecture,dilatation of capillaries,giant capillaries,hemorrhage, anddensity of capillaries (per mm).The subjects viewed a slideshow focusing on Raynaud phenomenon, describing its 3 phases with photographs. They then completed a standardized self-assessment questionnaire, including age, gender, medical history, cardiovascular risk factors, and manifestations of FD, enzyme-replacement therapy, and Raynaud phenomenon. They also underwent a nailfold capillaroscopyTwo independent blinded reviewers assessed the capillaroscopic photographs. A consensus was reached regarding any differences. Accompanying persons constituted the control group.This study was approved by the local ethics committee.http://datadryad.org/ with the doi:10.5061/dryad.kq04t.Data sets are available from the Dryad repository, at 2 or Fisher exact test, according to expected frequencies. Continuous variables are expressed as their means ± standard deviations, and were analyzed using Student t test. A P value of <0.05 was considered statistically significant.Statistical analyses were performed using R 3.0.3 software. Categorical variables are reported as percentages and were compared using the χp < 0.001). Patients with FD and Raynaud phenomenon all suffered from pain in the extremities, versus no pain in the controls (P = 0.011). Raynaud phenomenon was concomitant or before the occurrence of pain in the extremities in 42% of Fabry patients. Twenty-five of the 32 Fabry patients (78%)  were receiving enzyme-replacement therapy. The only nailfold capillaroscopy parameter that was statistically different was a particular major dystrophic capillary pattern (Table Seventy-one questionnaires were collected from 32 Fabry patients (45%) and 39 controls (55%). Concerning demographic data, sex ratio was statistically different, with more men in the control group Table . Data re26 in the general population. This figure is higher than the 15.3% reported in the study by Germain et al, who measured the prevalence of Raynaud phenomenon in Fabry patients.27 Among males, half of our Fabry patients had Raynaud phenomenon compared with none among the control group. According to the literature, the prevalence of Raynaud phenomenon varies from 0.5 to 16%29 in the general male population, and 2.5% to 22% in the general female population.26This study reveals a higher incidence of Raynaud phenomenon in Fabry patients (38%) than in controls (5%), versus 3–22%30 The high prevalence of Raynaud– phenomenon in FD, hardly described so far, should be considered as a cause of secondary Raynaud phenomenon. By inducing small-fiber neuropathy (both autonomic and sensitive),13 endothelial dysfunction,31 smooth-muscle-cell proliferation,32 the accumulation of neutral glycosphingolipids may impair vascular tone and favor Raynaud phenomenon. Because Raynaud phenomenon is concomitant or before the occurrence of pain in the extremities in almost half of Fabry patients, this could be, at least in part, a causal factor of this pain.Secondary Raynaud phenomenon in men is usually thought to be associated with chronic occupational exposure, especially in manual workers, which may explain the higher incidence of dystrophic capillaries in men.22 a significantly higher rate of ramified capillaries in Fabry patients was observed, particularly in males, which may reflect a more severe involvement of the microcirculation in the latter, as already identified for other involvements.1 In contrast, no significant difference was found regarding dystrophic capillaries as a whole. This discrepancy might be explained by the higher proportion of males, more susceptible to dystrophic capillaries,30 within our control group compared with the Fabry group, or it may have been because of improved microcirculation after treatment. Indeed, 78% of our Fabry patients were receiving enzyme-replacement therapy, compared with 0% in the other 2 published studies. López-Rodríguez et al20, in their study of a family of 8 members who had FD (5 of them receiving enzyme-replacement therapy), noted that the 3 patients with a normal nailfold capillaroscopy were under treatment. It would be interesting to carry out a prospective study on the diagnostics of FD to monitor any associations between capillaroscopic abnormalities and enzyme-replacement therapy.Consistent with data in the literature,1 It now seems that dystrophic capillaries, observed by nailfold capillaroscopy, and newly reported vessel tortuosity, visible on the external surface of the upper eyelid,33 enrich Fabry vasculopathy of new clinical signs that can be easily accessed in a clinical examination. These clinical signs could improve screening of people in high-risk populations for FD .1Angiokeratoma and tortuosity of the conjunctival and retinal blood vessels have been previously described.22 but the largest, study to assess nailfold capillaroscopic abnormalities in FD. The control group was constituted of family members, sharing the same genetic and/or environmental factors, potential source of confounding factors. It was not matched on age and sex. Despite this, age was not statistically different between the 2 groups. The female predominance in our Fabry patients can be explained by a higher mortality and morbidity rate in male patients before the availability of enzyme-replacement therapy,34 impeding their participation to meetings, and/or by a possible over-representation of females in the patient-support association meetings.36 This non-comparability of the control group regarding sex ratio is the main limitation of this study. Nevertheless, the predominance of males in the control group, more susceptible to dystrophic capillaries, could have revealed a more specific pattern of dystrophic capillaries in FD, namely ramified capillaries, not discovered hitherto. Moreover, the inhomogeneity of Fabry patients regarding enzyme-replacement therapy, unlike the other 2 studies, opens up new perspectives of studies that can explain some of the discrepancies encountered with previous data. Because this study was based on self-assessment questionnaire, no data on mutations were available. Therefore, no genophenotypic correlation could be done. We report the prevalence of Raynaud phenomenon and nailfold capillaroscopic abnormalities in French Fabry patients. Hence, our findings may not apply to other countries with different genetic and environmental factors.To our knowledge, this study is the third,Our data strengthen the fact that secondary Raynaud phenomenon should lead to screening for FD, especially in men. By extension, in high-risk populations for FD , the presence of ramified capillaries and Raynaud phenomenon should also be assessed.


Leiopathes glaberrima is a tall arborescent black coral species structuring important facies of the deep-sea rocky bottoms of the Mediterranean Sea that are severely stifled by fishing activities. At present, however, no morphological in vivo description, ecological characterization, age dating and evaluation of the possible conservation actions have ever been made for any population of this species in the basin. A dense coral population was reported during two Remotely Operated Vehicle (ROV) surveys conducted on a rocky bank off the SW coasts of Sardinia (Western Mediterranean Sea). L. glaberrima forms up to 2 m-tall colonies with a maximal observed basal diameter of nearly 7 cm. The radiocarbon dating carried out on a colony from this site with a 4 cm basal diameter revealed an approximately age of 2000 years. Considering the size-frequency distribution of the colonies in the area it is possible to hypothesize the existence of other millennial specimens occupying a supposedly very stable ecosystem. The persistence of this ecosystem is likely guaranteed by the heterogeneous rocky substrate hosting the black coral population that represents a physical barrier against the mechanical impacts acted on the surrounding muddy areas, heavily exploited as trawling fishing grounds. This favorable condition, together with the existence of a nursery area for catsharks within the coral ramifications and the occurrence of a meadow of the now rare soft bottom alcyonacean Isidella elongata in small surviving muddy enclaves, indicates that this ecosystem have to be considered a pristine Mediterranean deep-sea coral sanctuary that would deserve special protection. In the terrestrial environment, trees form complex ecosystems, the forests, which represent important three-dimensional habitats supporting high levels of biodiversity both within their area and in their neighborhoods . ForestsSimilarly, oceans host extraordinary examples of forest-like ecosystems, internationally identified as gardens, comparable in complexity, biodiversity, and structuring role to the terrestrial ones, but, especially in the deep-sea, entirely structured by colonial animals , 3, 4. IAntipathella subpinnata  and Parantipathes larix  [The existence of Mediterranean deep-sea black coral gardens is well known due to the fact that these species are common in the fishing bycatch , 16 and r, 1790) , 12. Mixr, 1790) , 8 and ar, 1790) , 22, whir, 1790) .Savalia and the antipatharian Leiopathes) characterized by a proteinaceous and chitinous skeleton and reaching millennial ages  [Leiopathes sp.).Another aspect that these marine ecosystems share with the terrestrial ones is the longevity of the structuring species . Similarecimens) , 27 , which tend to progressively slow down (5 μm per year) [−1) [Leiopathes species as biological archives of paleoclimatic information and environmental indicators [With the exception of the specimens from the Gulf of Mexico, showing a linear growth trend , most ofer year) , 27, whiar) [−1) . Growth ar) [−1) . Extremedicators , 31, 32.Leiopathes glaberrima . This large, arborescent species, despite it was described over two centuries ago in the Mediterranean Sea and may form vast populations starting from 200 m depth [L. glaberrima represents the type species of the Family Leiopathidae [Leiopathes, the only one of the family, comprehends 8 species [Leiopathes secunda Opresko, 1998, Leiopathes bullosa Opresko, 1998 and Leiopathes acanthophora Opresko, 1998 Leiopathes valdiviae ), three are known exclusively from the North Atlantic Ocean , while L. glaberrima is the only one reported, so far, both in the North Atlantic Ocean (Macaronesian Archipelago and Bay of Biscay) and in the Mediterranean basin. Firstly described from the Gulf of Naples [The aim of this paper was to characterize the deep-sea coral community of the Carloforte Shoal (Southwestern Sardinia) studying, for the first time, the population structure of the dominant species, the black coral  m depth , 15, is pathidae . The gen species , 34 of wf Naples , this spa Esper, 88. This f Naples , 15, 42.in vivo morphological characteristics of L. glaberrima, its habitat preferences and its associated fauna in order to define the ecological role of this species. Data have been interpreted also in a conservation key, by quantifying the fishing impact on both the rocky and surrounding soft bottom communities and through the age analysis of a black coral specimen.In this paper we elucidated the L. glaberrima collected as bycatch of fishing in the area of the Carloforte Shoal and preserved in the collection of the DISVA (University of Cagliari).The ROV Campaign was authorized by Italian Coast Guard (Compamare Cagliari). The radiocarbon dating was conducted on a colony of The study area, denominated the Carloforte Shoal is located 11 nautical miles off the Southwestern coasts of S. Pietro, a volcanic island comprehended in the Sulcis Archipelago (western Mediterranean Sea) . The Car2 in surface, with a minimum depth of 186 m, encountered along the Southwestern side of the structure  satellite imagery .The high resolution morpho-bathymetric map  was obtaAstrea exploring the Carloforte Shoal in a depth range comprehended between 186 and 210 m depth. ROV was equipped with a digital camera , a strobe (Nikon SB 400), a high definition video camera (Sony HDR-HC7), a navigation camera , and 3 jaw grabbers. The ROV hosted also a depth sensor, a compass, and three laser beams providing a 10-cm scale for the measurement of the frames area and size of organisms. The ROV was equipped with an underwater acoustic tracking position system  providing detailed records of the tracks along the seabed.Video and photo footage was gathered by a ROV “Pollux” during two surveys conducted in October 2011 and September 2013 on board of the Research Vessel 2 of explored area (frames on average 3 m2). Each frame was then elaborated with the software ImageJ  in order to calculate the density of each target species (N° colonies m-2 ± Standard Error). Target species were considered as all the arborescent, habitat-forming anthozoans including scleractinians, alcyonaceans, antipatharians and zoanthids.Four video transects were carried out on the Carloforte Shoal in three distinct habitats: 1) mud , 2) bench terraces , and 3) plateau  . Seven hL. glaberrima. Measures of height and width were gathered also for Isidella elongata. Other information, such as associated fauna and the in vivo aspect of the black coral colonies, has been registered. Finally, the fishing impact, based on the number of frames showing lost gears (N° impacted frames) and their direct damages on the colonies (N° of entangled colonies) [ROV-Imaging technique was employed also to define the morphometric characteristics of the black coral colonies  in order study the population structure of olonies) , has beeL. glaberrima were collected in the study area and preserved in 4% formaldehyde and ethanol 95° for morphological analyses together with samples collected in two other localities . The entire data set was considered: data, sqr(x)transformed, tested with a Bray-Curtis similarity measure, were distributed homogenously with n = 451, 166, and 110 frames, respectively, for BT, M, and P. Moreover, to find out whether there was a significant difference in relative abundance between the three areas of the bank for the dominant target species and for the total coral abundance, a Kruskal-Wallis test was performed . These analyses were performed using PAST for Windows version 1.91 .L. glaberrima (n = 155), normally distributed , were used to apply, with Excel, two linear regression analyses and were tested with a two-tailed Pearson’s correlation. Data of height and width of I. elongata (n = 109), not normally distributed, were tested with a Spearman’s correlation.Data of height, width and basal diameter of L. glaberrima and I. elongata (in terms of height classes of the colonies).Size-frequency distribution curves were also obtained to express the size structure of the populations of L. glaberrima with a 4 cm basal diameter and a 20 cm long stem (ahead of the rupture) recently collected as fishery bycatch in the area of the Carloforte Shoal and preserved in the collection of DISVA, University of Cagliari, was used for radiocarbon dating. Analyses were carried out by AMS (Accelerator Mass Spectrometry) at CEDAD (Centre for Dating and Diagnostics), University of Salento, Italy [One colony of o, Italy .14C/12C and 13C/12C isotopic ratios. From the measured isotopic ratios conventional radiocarbon ages were then calculated according to [Two sub-samples were selected for the analyses from the core of the base and the apex of the specimen . Selecterding to .Conventional radiocarbon ages were then calibrated to calendar years by using the MARINE09 calibration curve valid for marine data  and the Callogorgia verticillata , Acanthogorgia hirsuta Gray, 1857, Bebryce mollis Philippi, 1842, Eunicella cavolinii , Isidella elongata , the antipatharians L. glaberrima, P. larix, and Antipathes dichotoma Pallas, 1766.The bottom of the Carloforte Shoal hosted a plurispecific megabenthic assemblage with almost 1670 specimens of 8 target coral species counted in the examined frames : the alcL. glaberrima  , B. mollis (5%), A. hirsuta (3%), P. larix (3%) and A. dichotoma (1%) with densities ranging from 0.02 to 0.13 colonies m-2. Moreover, the massive sponge Pachastrella monilifera Schmidt, 1868 counted here almost 630 specimens for an average density of 0.5 ± 0.04 specimens m-2.The three considered habitats showed significative differences in terms of species relative abundance Figs. , 6 (ANOS2) Figs. , 6. In tL. glaberrima and C. verticillata . These two species showed significatively lower abundances than on the bench terraces   , E. cavolinii (2%) and B. mollis (1%), with abundances lower than 0.05 colonies m-2. Moreover, about 100 specimens of P. monilifera were recorded, corresponding to 0.35 ± 0.05 colonies m-2, significatively lower than on the bench terraces .In the second habitat, the rocky, silted plateau, the coral community was dominated by a mixed assemblage of y) Figs. , 6. The I. elongata  that has never been reported in the other two habitats   Figs. , 6.-2), than along the plateau and on the bench terraces  .Overall, the total coral abundance is significatively lower in the muddy area  (-1) (-1). Fertile polyps are distributed in patches along the branchlets: each fertile polyp contains less than 50 gametes present at various stages of maturation with sperm cysts up to 230 μm in diameter, while eggs up to 260 μm in diameter.The skeleton of the branchlets presents small, conical spines (0.04–0.06 mm high), organized in 3–4 longitudinal rows, and spaced apart 0.6–1.4 mm (on average 1–2 spines mm-1) . Spines -1) . Adult p-1) (-1) . Alterna-1) (-1) . On the -1) (-1) . In thisCnidome comprehends spirocysts (abundant in tentacles and mouth) , p-mastiL. glaberrima showed a typical heterogeneous distribution, with colonies aggregated in small patches, particularly along the bench terraces . Peaks of about 8 colonies m-2 were observed in some frames. Considering the total number of counted colonies in the frames, it is possible to hypothesize the occurrence of over 2600 black coral colonies along the entire recorded video transects.In the Carloforte Shoal, es Figs. , 6. The L. glaberrima may represent a substrate for the development of numerous encrusting organisms, in particular hydroids (Sertulariidae), sponges, bryozoans, zoanthids, and polychaetes living on the dead portions of the colonies  with the exception of the crab Anamathia rissoana  (35 specimens counted), often observed in a pairs on the colonies , Macroramphosus scolopax , Zeus faber Linnaeus, 1758, Lappanella fasciata , Trachurus sp., and Benthocometes robustus , the latter being one of the most characteristic of this coral species. A deep-sea school of the jellyfish Pelagia noctiluca , with 51 specimens counted entrapped in the ramifications of the corals  were observed in the area, moving on the muddy bottom near the rocky elevations and within the coral branches   Figs. ; 7C; 8b.The size frequency distribution of the heights is a curve with a maximum peak in the class of 45–50 cm. The population, however, shows also other three minor peaks, corresponding to the size classes 90–95, 120–125 and 160–165 cm and a long tail of colonies over 200 cm of height .L. glaberrima are listed in L. glaberrima (diameter of 4 cm) from the study area was thus dated to 40 ± 55 AD (Anno Domini), which corresponds to an age of 1973 years before the date of the test (2013).Data for the conventional radiocarbon dating carried out on a sample of I. elongata  and Desmophyllum dianthus  occasionally reported on dead branches of deeper colonies [L. glaberrima hosts a nursery area [S. canicula may locally represent a common component of fishing stocks in the Mediterranean Sea [P. noctiluca within the coral ramifications is in agreement with the supposed late summer-autumn vertical migration of this species recently hypothesized for nutrients-rich areas, such as canyons [This species, due to its arborescent corallum and its tendency of forming dense aggregations, fits in the definition of habitat-forming species, an organism able to three-dimensionally structure the environment and enhance its ecological functionality . The blacolonies . Moreovecolonies , the habery area . Despitenean Sea , it is nnean Sea . However canyons . The jelL. glaberrima, characterized by the presence of an un-usually large isorhiza. This nematocyst closely resembles in shape the large penicilli E (16–26 x 9–10 μm) identified for the zoanthid Savalia savaglia  by [L. glaberrima. This isorhiza is not the largest known for black corals: Pseudocirrhipathes mapia Bo & Bavestrello, 2009 and the related species Allopathes desbonni  and Aphanipathes verticillata Brook, 1889 are known to possess a large basitrich isorhiza (35 x 5 μm) possessing a strong taxonomic value [This work describes for the first time the cnidome of 1819) by , whetheric value .L. glaberrima. In the Mediterranean Sea, this datum was mainly reported for shallow waters alcyonaceans [A. subpinnata and P. larix [L. glaberrima juvenile colonies on the Carloforte Shoal can partially be ascribed to the difficulty in identifying small colonies among arborescent adults in the video frames. However, in consideration of the high number of observed colonies, it is plausible that the population is well represented by at least four distinct cohorts of colonies attributable to a discontinuous recruitment and that the low abundance of juveniles may reflect an overall stable population of a slow growing organism.This work describes for the first time also the structure of a population of onaceans , 68, andP. larix , 12. In Leiopathes species from deeper oceanic regions [I. elongata, another deep-sea anthozoan showing slow growth rates [For the first time the presence of millennial organisms was described in the Mediterranean Sea. Comparable ages were already reported for  regions , 26, 28,th rates  and a loth rates .I. elongata surviving in the small enclaves of soft bottom nearby the rocks. Due to its occurrence on incoherent grounds populated by commercial shrimps, this alcyonacean was, among the deep-sea species, one of the most commonly reported in the past as part of the coral bycatch [I. elongata has been reported also along the western Sardinian platform slope [I. elongata may therefore survive in the shallowest or deepest ranges of the species distribution, for example in shallow water refuges of muddy bottoms protected by trawling activities (as in the Sardinian case) or in grounds where trawling is forbade, for example on slope grounds or below 1000 m depth [It seems highly probable that the persistence of the Carloforte Shoal biocoenosis is due to the rough topography of the region characterized by numerous rocky elevations sufficiently high to avoid trawling and limiting long line fishing . This si bycatch . Known trm slope . Howeverrm slope , 55, 73 rm slope  and, todIn the last decades, the Mediterranean shallow waters have experienced deep changes generally attributed to anthropogenic pollution and global warming . These cBlack coral gardens represent long-term biological archives of inestimable value , 27, 28 I. elongata on compact muds, were added to the OSPAR (Oslo and Paris Conventions for the Protection of the Marine Environment of the North-East Atlantic) “List of threatened and/or declining species and habitats” in 2007 [Coral aggregations have now been internationally identified as special ecological features that require protection under the Convention of Biological Diversity . More sp in 2007 . Due to  in 2007  recommen in 2007 ). Despit in 2007 , in the S1 Dataset(XLS)Click here for additional data file.