Vitamin D is well recognized for its essentiality in maintaining skeletal health. Recent research has suggested that vitamin D may exert a broad range of roles throughout the human life cycle starting from reproduction to adult chronic disease risk. Rates of vitamin D deficiency during pregnancy remain high worldwide. Vitamin D deficiency has been associated with an increased risk of fertility problems, preeclampsia, gestational diabetes, and allergic disease in the offspring. Vitamin D is found naturally in only a few foods thus supplementation can provide an accessible and effective way to raise vitamin D status when dietary intakes and sunlight exposure are low. However, the possibility of overconsumption and possible adverse effects is under debate. The effect of vitamin D supplementation during pregnancy and early life on maternal and infant outcomes will be of particular focus in this review. While the beneficial effects of vitamin D supplementation with respect to reducing the risk of rickets has been recognized for close to 100 years, there is increasing concern that rickets in young children is reemerging in numerous countries . In the Currently, it is recognized that vitamin D deficiency can be demonstrated in numerous ethnic populations and locations around the world. However, the prevalence of vitamin D deficiency is a subject of intense debate, in part due to differences of opinion regarding what actually constitutes a “deficiency”. Illustrative of the above, are the markedly different circulating vitamin D concentration cut-offs for the risk of vitamin D deficiency, insufficiency, and sufficiency levels that have been identified by the U.S. Institute of Medicine (IOM) and the U.S. Endocrine Society . While t3. Differences in the degree of skin melanin can modify UVB-induced production of vitamin D by over 90%, putting dark skinned individuals at higher risk for a deficiency. Increased time spent indoors, dermatologist recommendations of daily sunscreen application, geographical location (northern latitude) and seasonal changes (winter) can reduce exposure to UVB radiation and subsequently lower vitamin D synthesis leading to lower vitamin D levels . Co. Coet altoxicity . In a ree period . Howeverin vitro fertilization (IVF) has enabled insight into fertility research. Many observational studies have linked higher maternal vitamin D status to an increase in rates of both clinical pregnancy (sonographic presence of a heartbeat following IVF) and live birth . Th. Th91]. tfeeding . Both dotfeeding . The sinA one-time, oral bolus of vitamin D is an effective way to treat vitamin D deficiency as 25(OH)D levels quickly rise following supplementation. However, the question of toxicity risk following a large, bolus should be considered. Recommendations for the upper limit for vitamin D intake ranges from 3200 to 10,000 IU . It is net al., designed a study to determine an oral dosage of vitamin D that would achieve a 25(OH)D level of 75 nmol/L or greater in 97.5% of newborn, breastfed infants by three months of age [3) for 11 months [3/day is four times the IOM recommendations for newborns. This high-dose group was discontinued early because 93% of infants had plasma 25(OH)D levels ≥ 250 nmol/L, which was reported to be associated with hypercalcemia . By 3 months, 97% of infants from any of the four supplement groups achieved 25(OH)D levels above ≥ 50 nmol/L with no differences between groups. Differences among the groups were observed when looking at the number of infants achieving serum 25(OH)D levels ≥ 75 nmol/L. By three months, a higher percentage of infants in the 1200 and 800 IU/day groups reached blood levels ≥ 75 nmol/L (92% and 81% respectively) compared to 400 IU/day (55%). Higher vitamin D supplement arms were able to sustain higher infant vitamin D serum levels at 12 months when compared to 400 IU/day, which agrees with a study by Grant et al. [3 in newborns, as hypercalcemia and hypercalciuria are potential adverse effects.Gallo s of age . Healthy1 months . The 160t et al. . Howevervs. the recommended one drop per day) of the supplement to the infant for two months. In addition, the amount of vitamin D in the supplement was threefold higher than what was listed on the label. It was estimated that the infant was receiving 50,000 IU daily for a two-month duration. After 89 days off of the supplement, 25(OH)D levels lowered to 54 ng/mL and normal growth and developmental milestones were being reached, however, nephrocalcinosis was not reversible. Levels as high as 470 ng/mL have been reported in infant toxicity cases and have life-long consequences, especially adverse renal function, or even death [Although numerous cases of newborn vitamin D toxicity have been reported over the past 80 years, toxicity is rare when compared to the number of newborns receiving vitamin D supplementation ,96,97,98en death , thus gen = 3210) [Appropriate Vitamin D dietary recommendations are a subject of intense debate . With la = 3210) . A recen = 3210) . Their c2 levels and successfully raise 25(OH)D in vegetarians or non-dairy consuming individuals [3 in the yolks from 50 IU to over 2000 IU per egg without causing harm to the hen or egg quality [Unlike folic acid fortification of grain products, vitamin D fortification has primarily focused on dairy products, potentially missing certain populations due to cultural practices or perceived or actual lactose intolerance. Many public health officials have proposed the need for enrichment of other foods. Mushrooms grown under UVB light can increase vitamin Dividuals . Hens fe quality .The Food and Drug Administration (FDA) of the United States has proposed changes to the nutrition facts label that is found on most food packages. Among the changes, is the requirement that manufacturers declare the amount of vitamin D on the label, as it is one of the new “nutrients of public health significance” based on NHANES dietary intake data . VitaminWhile the essentiality of adequate vitamin D during pregnancy is not disputed, controversy exists over the recommended dietary intake levels, the mathematical approaches to calculating that level, and the endpoints used to determine requirements; the concentrations of circulating 25(OH)D that denote deficiency, insufficiency and adequacy status; and whether race, body weight or other factors should be taken into account when making dietary recommendations, Similarly, it is not clear what vitamin D supplementation regimens are most effective during pregnancy; whether mother, child or both should be supplemented; and whether there are long-term consequences of supplementation. What is clear is that additional randomized, clinical trials that address these issues are warranted.


Functional mitral regurgitation (FMR) is frequent in patients with heart failure (HF). It develops as a consequence of left ventricle (LV) geometry alterations, causing imbalance between increased tethering forces and decreased closing forces exerted on the mitral valve apparatus during systole.FMR is known to change at rest and during effort, due to preload–afterload changes, myocardial ischemia, and/or LV dysfunction. Despite optimized medical therapy, an FMR can be responsible of shortness of breath limiting quality of life and decompensation. In this report, we present a case of dynamic FMR treated with MitraClip.MitraClip implantation is a successful and innovative opportunity for HF patients with FMR. A baseline transthoracic echocardiogram (TTE) was normal. In August 2011, she was referred to our HF clinic because the TTE documented an initial LV dysfunction (LV ejection fraction [LVEF] 45%), moderate mitral regurgitation (MR), and increased systolic pulmonary artery pressures (SPAP 50 mmHg). In the hypothesis of chemotherapy-induced cardiotoxicity, betablocker and angiotensin converting enzyme (ace)-inhibitor were started. In the meantime, total regression of lymphoma was documented.From January 2012, symptoms gradually worsened with shortness of breath in New York Heart Association (NHYA) class III. A TTE documented a further decrease of LVEF (30%), moderate FMR, massive tricuspidal regurgitation (TR), and dilated inferior vena cava. In November 2012, a dobutamine-stress-echocardiography (DSE) showed no myocardial ischemia nor contractility reserve. In March 2013, a left heart catheterization evidenced normal coronary vessels, LV dilatation with markedly depressed LVEF, and increased LV filling pressure (20 mmHg). A 6-minute walking test documented low performance (120 m), but no peripheral desaturation. Betablocker and ace-inhibitor were titrated and diuretics added.2, regurgitant volume [RV] 13 > 28 mL, color area of MR 6.3 > 11.2 cm2, mitral annulus 27 > 32 mm, VC 4 > 6 mm), probably due to an increase in the tethering forces, with a significant increase in SPAP (33 > 61 mmHg) ; the TTE showed a further LV dilatation with LVEF 45%, moderate FMR (vena contracta [VC] 4 mm), and moderate TR. Medical therapy was optimized with ivabradine. As the woman still complained dyspnea at rest, in the hypothesis of a dynamic FMR, a cycle-stress-echocardiography was performed. It was stopped at 50 watts for dyspnea, without signs of ischemia. A consistent increase of FMR, from moderate to severe, was observed (effective regurgitant orifice area [EROA] 0.1 > 0.2 cmThe follow-up assessment at 40 days showed improvement in 6MW distance (120 > 150 m) and a better result of the echo-bike (up to 100 W) with no MR increase during effort Figure . TTE at 3 Significant changes of the degree of MR in relation to physical activity are not predictable nor related to the degree of MR at rest5 but they are associated to both morphofunctional changes of the mitral valve apparatus and the dynamic variations of ventricular dyssynchrony.7 Although some patients have little dynamic variations, about 30% of them show an important increase in MR and SPAP during exercise. Dynamic change of MR in the case of severe LV dysfunction and little contractility reserve is crucial in the pathogenesis of impaired exercise tolerance. The increase in afterload and in the MR degree determines a reduction in anterograde flow and an increase in SPAP clinically manifested by the onset or worsening dyspnea during physical activity9 or acute pulmonary edema in adverse hemodynamic conditions.10 In HF with preserved ejection fraction, dynamic increase in MR and SPAP have been demonstrated during decompensation.11The Euro Heart Survey of the European Society of Cardiology showed that MR is present in 80% of HF patients, and that in 50% of them it is greater than moderate.10Dynamic changes of MR can be finely investigated with exercise echocardiography, which exactly reproduces the physiological conditions. DSE is not adequate in this setting as it usually determines a reduction in MR.2 has an important prognostic value, as well as an increase in the RV and SPAP,9 since they contribute to a worse prognosis with increased mortality and morbidity.12An increase in EROA ≥ 0.13 cmThe identification of dynamic components in the development of severe MR during exercise can be helpful to guide surgical strategy in ischemic patients candidate for coronary surgery, in order to avoid progressive LV dilation or dysfunction. Less obvious might seem the meaning of a dynamic MR in a patient not candidate for surgery. It seems arduous to consider a surgical correction of an isolated mild to moderate FMR at rest, moreover in patients at high surgical risk. Nevertheless, we often face patients as the one we reported: despite optimized drug therapy, symptoms significantly limit quality of life and frequent episodes of decompensation occur.13 The procedure is currently consolidated, with widely demonstrated safety and efficacy in more than 15,000 cases in the world. In current clinical practice, this procedure is mainly performed in patients with HFrEF and FMR, usually older than 70-years old, with comorbidities and high surgical risk.17 Data from real-world registries show the effectiveness of MitraClip implantation in terms of improvement of exercise tolerance and quality of life, reduction of rehospitalization for HF, and a trend in mortality reduction compared with medical therapy.18 Anyway, ongoing randomized trials comparing MitraClip with standard medical therapy  will definitely assess the exact clinical role of the procedure results. The case we presented has peculiar pathophysiological and technical aspects: recognition of the increased effort related to MR and SPAP as the main cause of symptoms and possible poor outcome, decision to correct the dynamic valvular defect with MitraClip implantation, and difficulty to obtain  an accurate view of the regurgitant jet. Collaboration with the cardio-anesthesiologist during the procedure was crucial, and the use of noradrenaline was effective in evoking a degree of MR similar to the one obtained during echo-bike. This case represents, in our opinion, a clear example of the heart team importance in a very challenging HF setting. To our knowledge, this is the first case in literature of a successful MitraClip implantation in FMR whose dynamic appearance significantly limits quality of life.Nowadays, the possibility to correct MR with percutaneous implantation of the MitraClip is a valid alternative to surgery, in selected cases.


E. coli, L. monocytogenes, and S. aureus in PBS were undetectable after 60 s of treatment at 80 kVRMS in air, while ACP treatment of the contaminated meat model required post-treatment refrigeration to retain antimicrobial effect. The nutritive components in the meat model exerted a protective effect during treatment, where 300 s ACP exposure yielded a maximum reduction of 1.5 log using a high oxygen atmosphere, whilst using air and high nitrogen atmospheres yielded lower antimicrobial efficacy. Furthermore, an ROS assay was performed to understand the protective effects observed using the meat model. This revealed that nutritive components inhibited penetration of ROS into bacterial cells. This knowledge can assist the optimization of meat decontamination using ACP technology where interactions with all components of the food matrix require evaluation.Atmospheric cold plasma (ACP) is a non-thermal technology, effective against a wide range of pathogenic microorganisms. Inactivation efficacy results from plasma generated reactive species. These may interact with any organic components in a test matrix including the target microorganism, thus food components may exert a protective effect against the antimicrobial mode of action. The effect of an in-package high voltage ACP process applied in conjunction with common meat processing MAP gas compositions as well as bacteria type and meat model media composition have been investigated to determine the applicability of this technology for decontamination of safety challenges associated with meat products.  In the food production chain, meat safety remains a major industrial challenge presented by the emergence of pathogens with low infectious doses, increased virulence, resistance to antibiotics and cross-contamination or recontamination of foods, food contact surfaces and water  is widely used in the food industry to extend shelf-life and avoid contamination and weight loss  has been documented for microbial inactivation in a number of food products and the underlying mechanisms of the bactericidal effect have been demonstrated to result partly from cell-lethal reactive species generation  was obtained from the National Collection of Type Cultures of the Health Protection Agency . Strains were selected to present both Gram positive and Gram negative foodborne challenges and to facilitate comparison with other studies. Strains were maintained as frozen stocks at −70°C in the form of protective beads, which were plated onto tryptic soy agar  and incubated overnight at 37°C to obtain single colonies before storage at 4°C.7 CFU ml−1 were prepared in PBS, 3 or 12% BE, and then used for inactivation of bacteria in planktonic state and ROS studies. Prior to ACP treatment, aliquots of 100 μl per well cell suspensions in PBS, 3 or 12% BE were dispensed into the wells of a microtiter plate for planktonic samples.Cells were grown overnight (18 h) by inoculating an isolated colony of each bacteria in 12% Beef Extract  at 37°C. Cells were harvested by centrifugation at 10,000 rpm for 10 min. The cell pellet was washed twice with sterile phosphate buffered saline . The pellet was re-suspended in PBS and the bacterial density was determined by measuring the absorbance at 550 nm using McFarland standard . Finally, cell suspensions with a concentration of 107 CFU ml−1 into the wells of 96 well microtiter plates .Bacterial biofilms were produced by adding 200 μl of 12% BE bacterial suspension with a cell density of 1.0 × 10The plates were incubated at 37°C for 48 h. After 24 h of incubation the supernatant from each well was replaced with fresh BE, with further incubation for 24 h. After incubation, the BE containing the suspended bacterial cells was removed and wells rinsed three times with sterile deionized water and dried, leaving only bacterial biofilms for further investigation. Negative controls were obtained by using BE without inoculation. Prior to each experiment, the microtiter plates containing biofilms were air dried for 60 min.RMS under atmospheric pressure. A polypropylene container, which acted as a sample holder and a dielectric barrier, was placed between the two perspex dielectric layers. The overall distance between the two electrodes was kept constant (3 cm) for all experiments.The DBD ACP system used in this study has been described by Han et al.  and its 2 + 70% N2 (gas 1) and 30% CO2 + 70% O2 (gas 3), the required working gas was filled into a sealed and pre-vacuumed package using a flow regulator at a controlled flow rate of 0.5 L min−1 for 1 min. To assess the effect of voltage on inactivation efficiency, bacterial samples were treated with either 60, 70, or 80 kVRMS, over a range of treatment times . Bacterial samples in PBS were exposed to shorter treatment times . After treatment, samples were stored at either room temperature or 4°C for 24 h to assess the effect of ACP generated reactive species during post-treatment storage time . Air (gas 2) was used as the working gas for generation of ACP. For the gas mixtures of 30% COOzone concentrations generated inside the sealed packages were measured using GASTEC gas tube detectors  immediately after treatment and also after 24 h storage.DCFH-DA  is a cellular assay probe widely used for fluorescence detection of intracellular ROS  at a final concentration of 5 μM in PBS for 15 min at 37°C. Two hundred micro liter aliquots of each sample were transferred into 96 well fluorescence microplate wells  and measured by a Synergy™ HT Multi-Mode Microplate Reader (BioTek Instruments Inc.) at excitation and emission wave lengths of 485 and 525 nm , which were further plated on TSA. In order to obtain low microbial detection limits, 1 ml and 0.1 ml of the treated sample was spread onto TSA plates as described by EN ISO 11290-2 method . The limit of detection was 1.0 logTo quantify the effects of ACP treatment on biofilms, PBS (100 μl) was added into the wells containing biofilms. The 96 well plate was then sonicated for 5 min to detach the bacterial cells into the solution using a water bath sonicator . PBS cell suspensions from each well for corresponding samples were mixed to obtain an average surviving bacterial population, serially diluted in MRD and surface plated on TSA.7 CFU ml−1 with four decimal dilutions of the suspensions performed in the microtiter plate. Samples were ACP treated for 60, 120, and 300 s and stored at 4°C for 24 h post-treatment. Twenty micro liter aliquots from each well were analyzed using plate count. The microtiter plates were then placed in a microplate spectrophotometer  set at 37°C. Absorbance readings were taken at 600 nm every 30 min for 48 h. The OD values were used to quantify the growth rate, μmax and the lag phase, λ, of all the bacteria studies using the following equation  of the inoculum level i, chosen as the time at which the sample in the well reaches an OD600 of 0.2  at which an OD600 of 0.2 is observed, Ni is the number of organisms per ml of the inoculum at time zero, and μmax is the maximum specific growth rate (h−1). Nturb was determined through separate experiments. The goodness of fit of these curves was validated experimentally. The kinetic parameters (μmax and λ) were calculated by performing a regression analysis on the data correlating TTD with log (Nturb/N0) using equation 1 for each replicate separately . Means were compared using analysis of variance (ANOVA) using Fisher's Least Significant Difference-LSD at the 0.05 level.RMS) on ACP efficacy against key meat pathogens grown as biofilms using a meat model medium was investigated .As before, the inactivation efficacy increased with treatment time in PBS Table , where a2+70% N2 samples and no ozone was measured inside these packages . After ACP treatment, storage at 4°C maintained population densities at the reduced level, while the recovery at 37°C nullified the microbicidal effect. To assess the effect of ACP treatment following storage, a cell recovery modeling study was performed. The effect of environmental factors on the lag phase and growth rate of foodborne pathogens has been widely reported, including environmental pH, osmolality, temperature and sterilization treatment (Mackey and Derrick, max was found to be ACP treatment time dependent, with E. coli showing more significant changes of μmax than the Gram positive bacteria. The cell damage caused by ACP treatment resulted in a longer recovery time during refrigerated storage. A similar trend is reported in Millan Sango et al. (E. coli. With this effect of temperature in the cell recovery model, refrigerated storage is recommended in combination with ACP treatment in developing industrial applications.Three pathogens of concern were evaluated in this study, with interesting trends emerging. Some comparison of ACP inactivation effects on different bacteria has been previously reported (Ermolaeva et al., o et al.  for E. cMAP is commonly used in the packaging of meat products to extend product quality and shelf-life. The efficacy of ACP treatment for inactivation of meat contaminants was therefore investigated not only in air but also in two modified atmospheres commonly used for packed meat products. The highest inactivation levels were achieved with high oxygen content gases Tables , 7. The S. aureus is well-known as a multi-resistant bacteria, however, its strong resistance was only observed in biofilm results here, with similar reductions to L. monocytogenes and E. coli in liquid (Tables L. monocytogenes showed higher sensitivity than E. coli and S. aureus in PBS but equivalent survival in BE media, which indicates the more critical role of the nutrient environment and availability on protection or recovery. Among the parameters studied, the target properties, including planktonic or biofilm and nutritive composition in media, were crucial for ACP inactivation. While a strong interactive effect of treatment time and gas composition was observed from PBS planktonic models, the influence of these parameters were minimized by the protection of surface properties.In this study, three meat pathogens were evaluated. d Tables –6. L. moOverall, the process and system parameters of ACP treatments were found to govern the inactivation efficacy by generating different amounts of reactive species. The nutrient content available at meat surfaces could provide protective effects against ACP treatment with reduced inactivation levels due to the decreased ROS levels. Moreover, Gram positive and negative bacteria showed significant differences in the quantities of ROS detected, implying different damaging patterns, although ACP had similar antimicrobial efficacy against both. Additionally, maintaining refrigerated storage temperature will maintain the microbial reductions achieved by ACP treatment. These results indicate both the potential and the limitations of the use of ACP for inactivation of microbiological challenges to meat safety and can help to develop and optimize treatment strategies. The further development of HV ACP for meat decontamination can be determined according to the characteristics of the biological target, the matrix and/or surface characteristics in addition to equipment design.PB, LH, VV, PC, and DB conceived and designed the experiments; LH, DZ, DS, CH, AP performed the experiments; PB, LH, DB, VV, DS wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NRI) between canopy trees and seedlings in forest understory with that between canopy trees and seedlings in forest gaps. We found that phylogenetic relatedness generally increases from seedlings through saplings and treelets up to canopy trees, and that phylogenetic relatedness does not differ between seedlings in forest understory and those in forest gaps, but phylogenetic turnover between canopy trees and seedlings in forest understory is lower than that between canopy trees and seedlings in forest gaps. We conclude that tree species tend to be more closely related from seedling to canopy layers, and that forest gaps alter the seedling phylogenetic turnover of the studied forest. It is likely that the increasing trend of phylogenetic clustering as tree stem size increases observed in this subtropical forest is primarily driven by abiotic filtering processes, which select a set of closely related evergreen broad-leaved tree species whose regeneration has adapted to the closed canopy environments of the subtropical forest developed under the regional monsoon climate.Investigating patterns of phylogenetic structure across different life stages of tree species in forests is crucial to understanding forest community assembly, and investigating forest gap influence on the phylogenetic structure of forest regeneration is necessary for understanding forest community assembly. Here, we examine the phylogenetic structure of tree species across life stages from seedlings to canopy trees, as well as forest gap influence on the phylogenetic structure of forest regeneration in a forest of the subtropical region in China. We investigate changes in phylogenetic relatedness (measured as NRI) of tree species from seedlings, saplings, treelets to canopy trees; we compare the phylogenetic turnover (measured as β Understanding how the phylogenetic structure of a forest changes as individual plant development proceeds within the forest and what role forest gaps play in driving phylogenetic community structure of forest regeneration is crucial to understanding the assembly processes that shape forest structure and species composition –3. WithiDifferent size classes of tree stems within a primary (or old-growth) forest stand may be considered as a putative proxy for forest temporal dynamics , 18–19; 2) due to the monsoon climate primarily caused by the uplift of the Qinghai-Tibetan Plateau [The subtropical zone, which is a transitional zone between tropical and temperate zones, is narrow in all regions of the world except in China where it covers a large area , mi Plateau , althougPrevious studies have shown that environment is a major determinant of phylogenetic structure of communities , 30–32. In this study, corresponding to the four expectations noted above, we intend to test the following four predictions for a forest in subtropical China: (1) Phylogenetic relatedness of tree species increases  in this humid subtropical region. Local annual mean temperature is 15.3°C, with January minimum temperature being −6.8°C and July maximum temperature being 38.1°C; annual mean precipitation recorded from 1958 to 1986 was 1964 mm [Castanopsis eyrei  and Schima superba  and is a typical EBLF. All individuals of tree species with diameter at breast height (DBH) ≥ 1 cm were identified, measured, and georeferenced during the summer of 2005. Detailed descriptions for the reserve and FDP can be found in Zhu et al. [Our study site was located at the Gutianshan National Nature Reserve  in Zhejiang Province, China. The reserve is ~81 km 1964 mm ) of ever 1964 mm . The foru et al. . The Adm2, 100–200 m2, 200–300 m2, and 300–500 m2 [The FDP experienced a severe ice-storm damage in 2008 , leading0–500 m2 –48. WithFor each forest gap, we set up a sampling plot which included all area within the radius of 30 m from the center of the forest gap. This 30-m distance threshold was chosen to avoid density-dependent effects within a distance of 20 m  on one hP < 0.05 in all cases, Kendall’s τ test for correlations; see One hundred and twenty one (85.2%) of the 142 angiosperm tree species in the entirety of the 24-ha Gutianshan FDP were found in the 32 sampling plots, although the sampling plots covered only less than 37.7% of the total area of the FDP. The relative abundance of tree species in major clades  in the 32 sampling plots was consistent with that in the entire FDP not only for the combination of all tree stems of sapling, treelets, and canopy trees but also for each of the three groups of tree stems  and Zheng [The 24-ha Gutianshan FDP comprises 144 tree species, 142 of which are angiosperms. These species were described as either trees or small trees in nd Zheng  and are nd Zheng , 51) to nd Zheng  and thusnd Zheng .The phylogeny used in this study was pruned from the phylogeny constructed for the Gutianshan FDP based on three common barcoding genes . This psample is the observed MPD, MPDrandom is the expected MPD of randomized assemblages, and sdMPDrandom is the standard deviation of the MPD for the randomized assemblages. Randomized (null) assemblages were generated by randomly drawing species from the species pool to replace the species in the sample. A positive NRI value indicates that MPD is lower than expected by chance  and that phylogenetic clustering of species occurs; a negative NRI value results when the observed MPD is greater than expected by chance  and thus indicates phylogenetic evenness or overdispersion. For each NRI value, 999 randomized assemblages were generated. Species were weighted by their abundance  within each assemblage. For each sampling plot, we calculated NRI separately for seedlings in the forest gap, seedlings under the forest canopy, saplings, treelets, and canopy trees. In addition to calculating NRI for each assemblage of seedlings with DBH < 1 cm, we also calculated NRI for each assemblage of seedlings with height < 50 cm, which is a stricter definition for seedlings (see [Net relatedness index (NRI) is a commonly used index to quantify phylogenetic relatedness e.g., , 51, 54), 5451, 5ngs (see ). NRI wangs  and between those in either habitat and canopy trees, we utilized the βemblages  with speemblages :βNRI=−1hylocom; ). A highocom 4.2 .We used Wilocoxon signed-rank test to determine statistic significance for pairwise comparisons . We usedP < 0.001, Cochran-Armitage test for trend for either seedlings in forest gaps, saplings, treelets and trees or seedlings in forest understory, saplings, treelets and trees; P > 0.1, Kendall’s τ coefficient for correlations between forest gap sizes and the two phylogenetic measures of seedlings in forest gaps; see NRI values for seedlings in forest understory and in forest gaps were not significantly different from those expected by chance . NRI valNRI between seedlings and canopy trees was significantly greater than zero regardless of whether seedlings under forest canopies or in forest gaps were considered  are the dominant species forming the canopies of forests in this subtropical region [Although 44% of the 142 angiosperm tree species included in our study site  are deciduous species, nearly all canopy trees are evergreen broad-leaved species. The relative abundance of trees with DBH ≥ 10 cm in our study site was 78.1% for evergreen broad-leaved trees, 15.4% for deciduous broad-leaved trees, and 6.5% for coniferous trees. These characterize a typical feature of the primary  EBLFs in this region. Many of the canopy tree species are congeners, confamilials, or taxa in closely related families. For example, of the 80 evergreen broad-leaved tree species found in our study site, ten species are in Lauraceae and nine species are in Fagaceae, eight of which belong to l region . Our stuPrevious studies have shown that phylogenetic structures of early successional forest communities are often more clustered than primary or old-growth forest communities in tropical regions , 54, 61 In sum, our study shows that phylogenetic relatedness generally increases from seedlings through saplings and treelets up to canopy trees in the humid subtropical forest that we investigated. Our study also shows that phylogenetic relatedness does not differ between seedlings in forest understory and those in forest gaps, but phylogenetic turnover between canopy trees and seedlings in forest understory is lower than that between canopy trees and seedlings in forest gaps. Evergreen broad-leaved forests of subtropical regions are an important type of forests not only in China  but alsoS1 Fig(DOC)Click here for additional data file.S2 FigCastanea henryi, Cerasus discoidea, Euonymus centidens, Fraxinus chinensis, Houpoea officinalis, Ilex formosana, Ilex litseifolia, Maclura tricuspidata, Mallotus apelta, Meliosma rigida, and Trema cannabina var. dielsiana.The following species in the phylogeny are additional to Liu et al.’s (2013) phylogeny: (DOC)Click here for additional data file.S3 FigG stands for seedlings of < 50 cm tall in forest gaps, NG for seedlings of < 50 cm tall in forest understory. See (DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Tableψ, seedling is defined as individual with DBH < 1 cm; φ, seedling is defined as individual with height < 50 cm. See (XLS)Click here for additional data file.S3 TableSee (XLS)Click here for additional data file.


In both experimental groups, passive mechanical loading had a sparing effect of muscle weight independent on nutritional status. Thus, this study confirms the strong impact of the mechanical silencing associated with the ICU condition in triggering CIM, overriding any potential effects of caloric intake in triggering CIM. In addition, the positive effects of passive mechanical loading on muscle fiber size and force generating capacity was not affected by the nutritional status in this study. However, due to the small sample size these pilot results need to be validated in a larger cohort.The muscle wasting and loss of specific force associated with Critical Illness Myopathy (CIM) is, at least in part, due to a preferential loss of the molecular motor protein myosin. This acquired myopathy is common in critically ill immobilized and mechanically ventilated intensive care patients (ICU). There is a growing understanding of the mechanisms underlying CIM, but the role of nutritional factors triggering this serious complication of modern intensive care remains unknown. This study aims at establishing the effect of nutritional status in the pathogenesis of CIM. An experimental ICU model was used where animals are mechanically ventilated, pharmacologically paralysed post-synaptically and extensively monitored for up to 14 days. Due to the complexity of the experimental model, the number of animals included is small. After exposure to this ICU condition, animals develop a phenotype similar to patients with CIM. The results from this study show that the preferential myosin loss, decline in specific force and muscle fiber atrophy did not differ between low  The most frequent neuromuscular disorders underlying muscle weakness in intensive care unit (ICU) patients is critical illness myopathy (CIM). CIM is characterized by reduced muscle membrane excitability and a preferential loss of the molecular motor protein myosin. A number of different descriptive names have been given to this disorder, such as acute quadriplegic myopathy, thick filament myosin myopathy, acute myopathy of severe asthma and myopathy of intensive care ,2. In 19Initially, CIM was thought to be a rare event in the ICU; we now know that neuromuscular dysfunction is found in up to 30% of the general ICU population and in 70%–80% of certain sub-groups ,5,6. Thei.e., absence of mechanical loading related to muscle contraction or weight bearing . The use of a unique experimental ICU model allows for detailed studies of skeletal muscle in mechanically ventilated, deeply sedated, pharmacologically paralyzed and extensively monitored rats for several days. Using this model, we have recently shown in a time-resolved analysis that complete mechanical silencing per se is a dominating factor triggering the preferential myosin loss, atrophy and loss of specific force (SF) in fast- and slow-twitch muscles and muscle fibers in limb skeletal muscles  [Energy =112 × (rat weight in kg)[kcal/d] This energy is recommended to be ingested in the form of fats, carbohydrates and proteins; broken down as: 20% fat (3 g), 75%carbohydrates (8.5 g), 5% protein (0.6 g).Animals were maintained in a protein fluid balance: (1) Intra-arterial solution: Alfa Cobra toxin , Potassium, Oxacillin , Ringers , Water; (2) Intra-venous solution: Oxacillin (Fresenius Kabi), Ringers, Water, Glucose  and Intralipid, (Fresenius Kabi).In both instances, the LC and EC caloric groups were given the same intra-arterial solution, the intra-venous solution differs depending on caloric intake and individual rat’s body weights.The experimental model has previously been described in detail ,30 and mThe left leg (loaded leg) of the LC (n = 3) and EC (n = 2) groups were activated 12 hours per day, using a mechanical lever arm that produced a continuous passive maximal ankle joint flexion-extension of 180° at a speed of 13.3 cycles per minute and the right leg served as the unloaded internal control. Controls underwent no mechanical loading on either leg.To analyze the function of myofilament proteins in cells with an intact filament lattice, but without the confounding effects of intercellular connective tissue or protein heterogeneity of whole muscle or multicellular preparations, we measured the force generating capacity of single muscle fibers.The tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius and soleus muscles were dissected from the loaded left leg and the unloaded right leg immediately after euthanasia. One half of the soleus and EDL muscles were quickly frozen in liquid propane cooled by liquid nitrogen, and stored at −160 °C for future analyses. The other half of the soleus and EDL muscle were dissected into bundles. Specimens were placed in relaxing solution at 4 °C and bundles of approximately 50 fibers were dissected free and tied with surgical silk to glass capillary tubes at slightly stretched lengths. They were then treated with skinning solution  for 24 hours at 4 °C, after which they were transferred to −20 °C. The bundles were detached from the capillary tubes and treated with sucrose, a cryoprotectant, within 1–2 weeks for long-term storage, snap frozen in liquid nitrogen-chilled propane and stored at −180 °C .On the day of experiment, bundles were de-sucrosed; transferred to a 2.0 M sucrose solution for 30 minutes and subsequently incubated in solutions of decreasing sucrose concentrations (1.5–0.5 M) and finally kept in a skinning solution at −20 °C.A single fiber was removed from the muscle bundle and was placed between two connectors, at a length of 1 to 2 mm long. One connector leads to a force transducer , and the other to a lever arm system  ,35. The 2+, 20 imidazole, 7 EGTA, 14.5 creatine phosphate and KCl to adjust the ionic strength to 180 mM and pH to 7.0. The concentrations of free Ca2+ were 10−9.00 M (relaxing solutions) and 10−4.50 M (activating solution), expressed as pCa . Apparent stability constants for Ca2+-EGTA are corrected for temperature (15 °C) and ionic strength (180 mm). The computer program Fabiato [2+-EGTA buffering capacity. This solution was identical to the relax solution except that the EGTA concentration is reduced to 0.5 mm, which results in a more rapid attainment of steady-state force during subsequent activations.For the mechanical recordings, relaxing and activating solutions (in mM) contained 4 Mg-ATP, 1 free Mg Fabiato  is used P0/CSA).Force was measured by slacking the fiber once steady-state isometric force was reached at pCa 4.5. Seven slacks were performed for each fiber and maximum force was calculated as the difference between the maximal steady-state isometric force in activating solution and the resting force measured in the same segment while in relaxing solution. Maximal force production was normalized to CSA . A muscle fiber was accepted and included in the analyses if the sarcomere length of a single muscle fiber changed by <0.10 μm between relaxation and maximum activation, and maximal force changed by <10% from first to seventh activation .After mechanical recordings, each skinned fiber was placed in sample buffer  in a plastic micro-centrifuge tube and stored at −180 °C for subsequent electrophoretic analysis ,38. MyHCSoleus and EDL 10-μm cryo-sections were dissolved in 100 μL urea buffer  after centrifugation and heating (90 °C for 2 minutes). The samples were run on 12% SDS-PAGE and gel bands corresponding to actin and myosin were identified and quantified as previously described [p < 0.05 was considered statistically significant.Means and standard error of the mean was calculated according to standard procedures. One way-analyses of variance (ANOVA) were used when comparing multiple groups and p < 0.05) and eucaloric  groups  groups . There w) groups . In both) groups .p < 0.05 and *** p < 0.001), as well as in soleus, loaded and unloaded between Ctl and EC. Furthermore there was a statistically significant difference (p < 0.05) between the unloaded and loaded leg of those who received LC and EC in the slow-twitch soleus but not in the fast-twitch EDL  and were analyzed; 100 soleus fibers expressing the type I MyHC isoform and 100 EDL fibers expressing IIx or IIb MyHC isoforms were included in the experimental groups. Muscle fiber CSA and SF measurements were carried out in both the loaded and unloaded leg of each animal.A detailed analysis of fiber CSA and force generating capacity , p < 0.001) and force generating capacity (p < 0.001) irrespective of muscle fiber MyHC isoform expression (p < 0.001), irrespective of muscle fiber MyHC isoform expression  was observed in fiber CSA between EC and LC groups  in single muscle fibers and myosin:actin ratios were not affected in either the slow-twitch soleus or the fast-twitch EDL muscles in animals exposed to a mimicked ICU setting over a period of 10–14 days. Irrespective of caloric intake, animals developed a phenotype considered characteristic of CIM, i.e., a preferential myosin loss, fiber atrophy and decreased specific force. Furthermore, the caloric intake did not have an additional effect on the improvement in muscle fiber size or force in response to passive mechanical loading. Thus, indicating that caloric intake is of less importance compared with e.g., mechanical signaling in the pathogenesis of CIM in ICU patients. Although these results are of significant scientific importance and clinical interest, it is important that these results be validated in a larger cohort in future studies.The major findings from this study is that low vs. fast-twitch limb muscles [The reduced preferential myosin loss in response to passive mechanical loading, irrespective of caloric intake, in the slow-twitch soleus but not in the fast-twitch EDL is in accordance with our previous observations . This mu muscles .Skeletal muscle serves as a major protein reservoir in the critically ill patients. During prolonged critical illness muscle strength and function are compromised, which in turn can have a detrimental effect on recovery and more specifically in weaning patients from the ventilator . The trei.e., ‘mechanical silencing’ of skeletal muscle is a dominating factor triggering the specific myopathy (CIM) associated with the ICU condition. Furthermore, the loss of muscle mass and function can be attenuated by early mechanical loading [In a previous experimental and clinical study from our group ,11,12, w loading . The resNon-depolarizing post-synaptic neuromuscular blockade has been suggested to be an important factor triggering CIM, but in our previous studies using a porcine ICU model we have not observed a significant difference in muscle structure and function in pigs exposed to mechanical ventilation and deep sedation with or without neuromuscular blockade .i.e., parenteral vs. enteral, has been debated for many years. In our model parenteral feeding was the means used to administer nutrition, but enteral nutrition is the preferred route of administration for critically ill patients [The unique experimental model used in this project mimics the ICU condition well. Unfortunately, this model is technically demanding and involves extensive monitoring 24 hours per day during the 10–14 day experimental period. This has limited the number of animals included in the LC and EC groups, which we acknowledge and suggest that a confirmatory study is needed. Thus, small differences in muscle size and function related to caloric intake may have gone undetected in this study. However, we suggest from the present results that such differences are negligible compared with the effects of mechanical silencing in inducing the CIM phenotype as well as the positive effects caused by passive mechanical loading. Furthermore, the most appropriate type and timing of feeding for ICU patients, patients ,50, howepatients ,24. ThusAlthough artificial nutrition plays an important role for energy metabolism in critically ill ICU patients, the present results show that caloric intake is not an important factor in triggering CIM or in the improvement associated with passive mechanical loading during the ICU condition.


Pointwise transinformation (PTI) provides a quantitative nonlinear approach to spatiotemporal synchronization patterns of the rhythms of coupled cortical oscillators. We applied PTI to the waking and sleep EEGs of 21 healthy sleepers; we calculated the mean levels and distances of synchronized episodes and estimated the dominant frequency shift from unsynchronized to synchronized EEG segments by spectral analysis. Recurrent EEG synchronization appeared and ceased abruptly in the anterior, central, and temporal derivations; in the posterior derivations it appeared more fluctuating. This temporal dynamics of synchronization remained stable throughout all states of vigilance, while the dominant frequencies of synchronized phases changed markedly. Mean synchronization had high frontal and occipital levels and low central and midtemporal levels. Thus, a fundamental coupling pattern with recurrent increases of synchronization in the EEG (“RISE”) seems to exist during the brain's resting state. The generators of RISE could be coupled corticocortical neuronal assemblies which might be modulated by subcortical structures. RISE designates the recurrence of transiently synchronized cortical microstates that are independent of specific EEG waves, the spectral content of the EEG, and especially the current state of vigilance. Therefore, it might be suited for EEG analysis in clinical situations without stable vigilance. The EEG frequency spectrum and the distribution of EEG transients vary extensively across the wake-sleep cycle. Since most EEG waves are synchronized across several EEG leads, an expansion of the conventional single-channel evaluation to regional and global EEG coupling analysis can reveal complex intracortical interactions. Coherence and cross correlation and other linear measures have been applied effectively to the EEG –7. HowevIn contrast, in a former study of EEG synchronization during REM sleep , we founPointwise transinformation (PTI) provides an algorithm to detect and quantify dynamic coupling processes in the EEG, capturing both linear and nonlinear interactions –19. An oK-complexes, sleep spindles, and delta waves) and the spectral content of the EEG. If this pattern corresponds to a physiological mechanism which synchronizes different sites of cortical and probably also subcortical networks, the distributions of the durations of synchronized and unsynchronized EEG sections assessed by PTI should be consistent for all waveforms and states of vigilance. Taking into account the results of other EEG synchronization studies, this would imply that the mean level and the dynamic changes of cortical synchronization depend only partially on the current state of vigilance, while other synchronization patterns are characterized by a constant spatiotemporal distribution; the latter patterns should be detected by PTI. In addition, clinical situations with an instable vigilance are common. In these situations, conventional EEG parameters are subject to marked changes; in contrast, PTI as an EEG synchronization measure that is largely independent of vigilance could provide a valuable approach to measuring fundamental cortical coupling patterns.The objective of the present study was the identification and quantification of synchronization phases in the waking and NREM and REM sleep EEG using the PTI algorithm. Based on our previous studies, we expected to find a recurrent pattern of synchronization independent of both specific EEG waves and graphoelements . Exclusion criteria were the intake of substances with central nervous effects, a diagnosis of concomitant or former neurological or psychiatric disorders, and abnormal neurological, medical, or psychiatric status. All participants had given informed consent for their EEG data to be used for further evaluation.A waking EEG and a sleep EEG of each participant were evaluated. The waking EEGs had been derived in the morning after undisturbed sleep  following standard procedure . The sleep EEGs had been derived for a complete night of normal sleep in a laboratory for video-EEG polygraphy with 18 leads  and the same technical parameters. Sleep EEGs with pathological sleep profiles were excluded. All EEG derivations included electromyography (EMG) of the mental muscles, electrooculogram (EOG), and ECG by a D2 lead. The EEGs were recorded with a Schwarzer Brainlab 3.30-0.00 system and stored on hard disc. Visual sleep scoring [day”) from the waking phases in the sleep EEGs (“Wnight”). Mean PTI, mean duration of elevated synchronization phases, and mean duration of unsynchronized intervals were assessed separately for each state of vigilance . Elevated synchronization phases were defined as phases with a PTI level ≥ mean PTI + 1 standard deviation. Since absolute values of PTI depend on the selection of the embedding parameters and only relative changes of PTI were of concern for our study, PTI was normalized to 1 by maximal/minimal values.Details of the PTI algorithm are given in the appendix. PTI was calculated for all EEGs with segments of 60 s duration (15000 data points) for FP1-FP2, F3-F4, C3-C4, P3-P4, O1-O2, F7-F8, T3-T4, T5-T6, A1-A2, F3-O1, and F4-O2. Since circadian changes of the waking EEG have been described , the wak2 s intervals around all synchronized episodes were also analyzed by an FFT spectral analysis with a frequency range of 0–25 Hz and a frequency resolution of 0.5 Hz. The frequency bands were defined as delta 0.5–3.5 Hz, theta > 3.5–8 Hz, alpha > 8–12 Hz, sigma > 12–14.5 Hz, and beta > 14.5–25 Hz. The power spectrum of the 2 s interval beginning with the onset of the elevated coupling was compared to the previous unsynchronized 2 s interval. The dominant frequency shift was then defined by the frequency band with the highest increase of relative power.P < 0.05. Since 3 parameters for 11 pairs of EEG leads and 6 states of vigilance were compared, an α-adjustment for multiple comparisons was made. The false discovery rate was controlled by the Benjamini-Hochberg procedure [m hypotheses are tested, the P values are ordered as p1 ≤ p2 ≤ ⋯≤pm, and k : = max {i : pi ≤ q · i/m} is defined with q = the control level of the false discovery rate. The hypotheses H10,…, Hk0 are then rejected. All calculations were performed by a PC-based statistical program .All EEG epochs without visually identified artifacts were analyzed. The distribution of all PTI parameters could be approximated by a lognormal distribution, which we confirmed by a Kolmogorow-Smirnow test. Stage dependent differences of the PTI parameters were assessed for all patients by an analysis of variance after a logarithmic transformation. Significance was assumed for rocedure : when m day) and the night (Wnight) did not differ, these phases were summarized for all further evaluations and referred to as stage W.All persons had physiological sleep profiles with normal sleep duration, latencies, and percentages of sleep stages . A consiP < 0.05). This difference was especially prominent for the longest intervals, distinguishable by a significantly reduced p90 value (P < 0.05) for these derivations  and unsynchronized intervals  we obtained were comparable to the durations of stable microstates reported by two studies on spatiotemporal patterns of the alpha EEG , 38. SimK-complexes and is itself synchronized in corresponding regions of both hemispheres [In contrast to RISE, conventional spectral parameters vary extensively with the wake-sleep cycle –45. Visuispheres . Thus, tAlpha waves contribute to RISE in different states of vigilance. Anterior and posterior alpha patterns of the characteristic waking-alpha rhythm are seemingly interconnected via corticocortical synapses and modulated thalamic inputs ; this coBeta and gamma oscillations have been examined preferentially in the context of cognitive processes ; these fA dynamical, but qualitatively stable, pattern of recurrent EEG synchronization seems to continue throughout all physiological waking and sleep phases. RISE describes the rapid change of synchronized microstates with EEG waves across the whole spectrum as well as with state-specific EEG graphoelements, but it remains remarkably unaltered by changes of vigilance; thus, it seems to provide a persistent spatiotemporal framework for recurrent EEG synchronizations during the brain's resting state. The probable generators of RISE could be coupled corticocortical neuronal assemblies, modulated by thalamocortical and other subcorticocortical pathways; transient, but highly synchronized spatiotemporal configurations of these neuronal networks seem to alternate with phases of low synchronization. RISE needs to be evaluated for different pathological EEGs in future studies to examine its clinical relevance.


Parkinson's disease (PD) is a complex neurodegenerative disorder that manifests through hallmark motor symptoms, often accompanied by a range of non-motor symptoms. There is a putative delay between the onset of the neurodegenerative process, marked by the death of dopamine-producing cells, and the onset of motor symptoms, creating an urgent need to develop biomarkers that may yield early PD detection. Neuroimaging offers a non-invasive approach to examining the potential utility of a vast number of functional and structural brain characteristics as biomarkers. We present a statistical framework for analyzing neuroimaging data from multiple modalities to determine features that reliably distinguish PD patients from healthy control (HC) subjects. Our approach builds on elastic net, performing regularization and variable selection, while introducing additional criteria centering on parsimony and reproducibility. We apply our method to data from 42 subjects (28 PD patients and 14 HC). Our approach demonstrates extremely high accuracy, assessed via cross-validation, and isolates brain regions that are implicated in the neurodegenerative PD process. Parkinson's disease (PD) is a devastating, progressive movement disorder affecting 7–10 million individuals worldwide  at the National Institutes of Health's (NIH's) National Institute of Neurological Disorders and Stroke to identify early stage biomarkers for PD. In the context of our study, we regard biomarkers in a general sense, defined by an NIH Biomarkers Definitions Working Group as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention,” although more specific molecular definitions have been proposed  and personally identifiable information (PII). Accordingly, this research qualifies as Research of Existing Data, Records, Specimens [Basic Exempt Criteria 45 CFR 46.101(b)(4)], and has been deemed “Not Human Subjects Research”  by NIH and Columbia University Medical Center Institutional Review Board (Protocol: IRB-AAAO0062).p < 0.001). The PD group has 13 females (46.4% of PD patients) and the control group has 8 females (57.1% of controls), reflecting a small sex difference between groups, although not statistically significant (p = 0.74). The mean Unified Parkinson's Disease Rating Scale (UPDRS) Part III (motor) score for these patients was 19.4 (standard deviation 10.2). The mean duration of disease was 7.7 years (standard deviation 3.3 years), although the duration was not calculable for 5 patients due to missing data.We consider data from 42 subjects, including 28 PD patients and 14 healthy control (HC) subjects. The data include a collection of magnetic resonance (MR) derived scans characterizing different structural and functional properties of the brain as well as demographic measures. Specifically, we use T1- weighted anatomical MRI scans, rs-fMRI, and DTI. The mean age of the subjects is 65.0 years (9.0 years standard deviation), and the subjects include 21 males and 21 females. The mean age is 61.9 years (8.7 years standard deviation) for PD patients and 71.4 years (5.8 years standard deviation) for controls , with three leading and three trailing B0 scans. The remaining 6 subjects followed a DTI protocol  comprised of 128 directions (B = 1000s/mm2), with six leading and five trailing B0 scans.All scans were captured with a Siemens Trio Tim 3T MRI scanner; the first 36 subjects were scanned with a 12 channel head coil and the remaining 6 subjects (5 PD and 1 control) were scanned with a 32 channel head coil . The structural T1 scans were acquired using MPRAGE . Echo planar imaging (EPI) was used to acquire 140 frames of rs-fMRI scans  for each subject. DTI data were captured using a biphase approach with consecutive left-to-right and right-to-left phase scans. The first thirty six subjects underwent DTI scans  comprised of 64 directions (B = 1000s/mmWe implemented standard neuroimaging preprocessing steps including voxel-based morphometry (VBM) on the anatomical T1 scan, using the VBM toolbox Gaser,  under SPt-test applied to the resting state scans shows no difference in mean temporal SNR between PD (54.4 ± 2.9) and control (53.9 ± 4.9) subjects (p = 0.92), with standard error of the mean used to express variability. Similarly, a Wilcoxon rank sum test shows no significant difference in the maximum absolute displacement over the duration of the scan between PD (1.67 mm ± 0.21 mm) and control (1.31 mm ± 0.17 mm) subjects (p = 0.53). Finally, another motion-related quantity, the average motion per TR, also does not differ significantly between PD (0.10 mm ± 0.01 mm) and control (0.08 mm ± 0.01 mm) subjects (p = 0.38).A For DTI scans, each subject's two opposing phase DTI scans were combined to estimate the susceptibility-induced off-resonance field using a method similar to that described in Andersson et al.  as impleThe first step is to determine the spatial scale for data representations. The imaging data from MRI, rs-fMRI, and DTI are acquired at a voxel level. We utilize a popular neuranatomic parcellation of the brain, the Automated Anatomical Labeling (AAL)  for each imaging modality and specify the spatial scale. Figure We perform marginal screening to reduce the 46,580 features prior to analysis by eliminating features that are unlikely to carry strong predictive power. Screening typically improves the performance and facilitates implementation of subsequent modeling by eliminating sources of noise and reducing data dimensionality. Toward our goal of attaining reproducible PD biomarkers, we perform a bootstrap screening procedure for each feature independently using logistic regression, with modality- specific screening thresholds. The bootstrap procedure isolates a set of viable markers, after accounting for sampling variability, which increases the likelihood that the identified features will emerge in other samples. Specifically, our screening rule selects features satisfying the following:B = 100 bootstrap samples indexed by b = 1, …, B to obtain a corresponding p-value pb, apply designated modality-specific thresholds p0, and determine features that are selected in at least r = 0.75 proportion of the bootstrap samples. After our screening process, we retained 24 regional VBM features (p0 = 0.2), 6 fALFF (p0 = 0.2), 225 FC estimates across the brain (p0 = 0.05), 6 regional FA measures (p0 = 0.2), and 10 SC estimates (p0 = 0.2), giving 271 features in total  represent the probability that subject i has PD, given a set of predictors, and use logistic regression to model We propose an analytic approach that uses imaging data from multiple modalities and demographic information to classify subjects as either PD patients or HCs. We present an approach that builds on elastic net with refinements to encourage parsimony and reproducibility. Let 2 penalty and the lasso (α = 1) using an L1 penalty  using an Lp ≫ n, and when the set of predictors includes some highly correlated variables, which poses a challenge for L1 penalization alone.The penalized framework, implemented here in context of a logistic model, points to the predictive ability of a specific subset of imaging and demographic variables that constitute a signature for PD in our sample. Ridge regression shrinks the coefficients and tends to draw the coefficients of correlated predictors towards each other. The lasso tends to pull many coefficients near zero, with a small subset of coefficients with larger magnitudes, therefore serving as a useful tool for variable selection. We perform covariate adjustment for demographic variables (age and sex) and scan differences (head coil) in our models. The elastic net penalty is particularly useful when B, in which to optimize . Specifically, we considerWe modify the usual optimization procedure for the tuning parameters, when necessary, to promote parsimony, accuracy, and reproducibility (see Results section for details). Our procedure defines a restricted or bounded tuning parameter space, B incorporates a lower bound on AUC as a measure of accuracy.where AUC represents the area under the receiver operating characteristic (ROC) curve. Inducing parsimony may sacrifice accuracy, so the subspace k-fold cross validation scheme for model training and testing to promote reproducibility. A typical implementation of k-fold cross validation splits the data into k groups, trains the model by fitting the data from k − 1 groups (training set), and uses the estimates obtained to predict the disease status of each subject in the remaining group . The process then rotates the training sets and validation sets until testing has been performed on each group, hence each subject. Variability is inherent in k-fold cross validation, which is not typically accounted for in practice. For example, by constructing the folds differently, one may obtain a different estimate of accuracy and detect the involvement of different predictors. To encourage the identification of neuroimaging markers of PD that are reproducible and have high predictive strength and to account for variability in the cross-validation process, we implement an iterated framework. Specifically, we implement two-fold cross-validation and repeat the process 100 times, randomly assigning subjects to folds in each iteration. This process results in 200 training samples.We evaluate accuracy using an iterated B, we aim to select the top 10% of features based on these coefficient strengths. Let M be the random variable representing the magnitude of a predictive effect at . Conceptually, imaging features βj satisfying Pr(M ≥ |βj|) ≤ τS, and which contribute to high predictive accuracy, are regarded as strong candidates for potential biomarkers. In practice, we use the cross-validation process to estimate the empirical distribution function of M and determine predictors that have the most sizable effects (on average) across 200 training samples. So for our data, we specifically seek to determine the features satisfying j th effect and ξ0.10 is defined by Pr(M ≥ ξ0.10) = 0.10.The cross-validation approach presents an important advantage in the context of our quest to identify likely PD biomarkers, allowing us to gauge the overall importance of each feature by virtue of its average predictive effect. Since the features were standardized, coefficient strengths are comparable: a larger average coefficient strength indicates a greater predictive effect. At each  in S represent the set of features satisfying the above condition for coefficient strength, i.e., M ≥ ξ0.10) = 0.10;}. Our procedure selects featuresMoreover, we track the consistency with which these predictors with sizable effects are selected for specific values  and ultimately choose features that are consistently strong across various combinations . Let B] denotes the cardinality or number of elements in set B, and I is the indicator function. We set τC = 0.90, effectively taking the features that were selected to be in set S in 90% or more of the points in B. The set of features in C are deemed to have high predictive strength, to be extremely parsimonious, to have high likelihood of emerging in other samples, and to be robust over a range of values in the tuning parameter space. These properties aid the delivery of potential PD biomarkers that can be investigated further in future research. In the application of our methods to the multimodal imaging data of PD patients and healthy controls discussed below, we explore further reductions of the set C.where the notation #, with 25 points per decade. For every  pair, we fit elastic net to half the subjects, then apply the resulting model to the other half of the subjects to predict their disease status. We control for head coil, sex, and age in the model fit. Then we swap sets of subjects and perform the operation again; i.e., two-fold cross validation. We compare the result of the predictions with true disease status to compute the ROC curve and associated AUC value. Finally, we perform this operation 100 times at every  in the grid and record the average AUC and various statistics on the model coefficients for each of the 271 features.We applied the methods above to our multimodal imaging data. We consider a 51 × 151 grid of elastic net tuning parameters, with α ∈  and λ ∈ [10The resulting average AUC values in the  grid are shown in Figure B = { | p ≤ 75, AUC ≥ 0.9}, for the tuning parameters to induce parsimony . Figure From the previously described elastic net with repeated two-fold cross-validation, Figure C includes 24 features, each of which is consistently among the most predictive for at least 90% (τC = 0.9) of the operating points in B. The features are listed in Table Considering all operating points in the search area, the resulting set The 24 features contribute extremely strong predictive power. Using logistic regression, still controlling for head coil, sex, and age, one can achieve perfect separation between PD patients and HC using subsets of as few as three of these multimodal imaging features. In fact, out of all possible three-feature models, three of them achieve perfect separation between the groups, and comprise an aggregate of eight separate features. The three models and the associated map of features are presented in Figure p-value falls below the designated modality-specific threshold across the 200 samples. Features that pass the threshold in 75% or more of the 200 cross validation samples are regarded to pass univariate screening in our sensitivity analysis.Performing univariate screening as a separate step prior to cross validation of the elastic net could potentially impact variable selection and classification performance. To examine this possibility for our analysis, we performed univariate screening using the same 200 (2x100) cross validation training samples constructed in the elastic net stage. Note that for this sensitivity analysis, we did not additionally implement our bootstrap procedure within each of the cross validation samples, given the computational cost. We track the number of times each feature's corresponding Nearly all (23 of 24) features that emerged from the elastic net stage in our original analysis were also selected by this revised cross-validation univariate screening. The excluded feature, the functional connectivity between the right frontal inferior orb and the middle temporal lobe, fell just below our threshold by being selected in only 73% of the 2 × 100 cross-validation trials. Thus our findings suggest that the final panel of 24 features is not substantially impacted by the decoupling of screening and cross validation, perhaps buffered by the addition of our bootstrap screening procedure which accounts for sampling variability.High dimensional prediction and classification methods are subject to inflated measures of accuracy for the particular data set under consideration, resulting in findings that are not reproducible on independent data sets. We take measures to minimize the risk of overfitting and to assess the potential influence of overfitting in our analysis. In particular, we perform iterated two-fold cross-validation in the elastic net procedure with 271 variables as outlined above. To assess the presence and potential influence of overfitting, we conduct a null randomization experiment in which we combine all subjects and then randomly assign the subjects to one of two groups, with the group sizes matched to the actual sizes of the PD and control groups in our experimental data. In our null data, one would not expect to observe systematic between-group differences given the random mixing of PD and control subjects. We repeat our analysis on the null data and, as expected, obtain an ROC curve that roughly tracks a 45° line (chance), giving no indication of inflated accuracy due to our methods.We note that this null hypothesis verification was performed only within the cross-validated elastic net analysis, using the 271 features that had previously passed screening and hence been deemed to be strongly associated to disease status. Given the massive number of features relative to the number of subjects in the analysis, it is entirely possible that other sets of features could be found, which have high explanatory affinity for random (perhaps meaningless) subject groupings. We assume that the PD (vs. control) labels reflect true manifestation of disease and thus that the identified features are strong candidates for PD biomarkers.Our analysis provides a broad multimodal view of prevailing alterations in PD, which serve as accurate and reliable predictors. We identify 24 neural manifestations of PD, which contribute extremely strong predictive power in these subjects Table  and are Bilaterally, the middle temporal pole (MTP) exhibited strong discriminatory power and consistency. For PD patients, the right MTP shows increased FC with the rIFG pars triangularis (rIFG-PT) and with the right middle temporal gyrus (MTG). The left MTP exhibits decreased FC with the left thalamus in PD patients and increased FC the right superior temporal sulcus (STS). The OFC, which is linked to inhibitory control, exhibits functional connections with several regions that are predictive of PD. Right inferior areas of the OFC show increased FC bilaterally with the MTG. The left middle areas of the OFC show increased FC with the left hippocampus. The left superior OFC exhibits increased FC in PD patients with the left insula, the left inferior parietal region, and the left superior temporal pole; and decreased FC with the right STS. Our analysis also reveals increased FC between the left medial superior frontal gyrus, which includes the supplementary motor area (SMA) and the preSMA, and the left ACC. These FC alterations all yield strong power to dissociate PD patients from controls.PD symptoms have been linked to FC between the pars triangularis and the orbito-frontal cortex, specifically with FC shown to be positively associated with the Movement Disorder Society (MDS) Unified Parkinson's Disease Rating Scale (UPDRS), part II, entitled Motor Aspects of Experiences of Daily Living  and the posterior cingulate cortex (PCC) and between the right amygdala and both the left lingual gyrus and the right angular gyrus. In contrast to our work examining specific connections between pairs of brain regions, graph-theoretic approaches seek to characterize whole brain topological properties of brain networks . AAL regions are predominantly composed of gray matter and contain relatively less white matter (median across regions and subjects is roughly 15%). As such, we may have excluded potentially predictive markers from DTI-related measures (FA or SC) sampled from regions dominated by white matter. Also, the features extracted from the multimodal imaging data reflect particular characteristics at a selected spatial scale. Some data reduction is necessary, e.g., to limit the data from generating billions of features. We cannot determine in advance, which spatial scale will extract maximal information for the purpose of dissociating PD patients from controls.FB conceptualized methodology and guided evolution of the research. FB also wrote the majority of the paper. DD processed the imaging data and performed statistical analyses on the results. DD contributed to the data and methods section and generated tables and figures for the paper. DH managed data collection and provided expertise in Parkinson's disease research and imaging. DH provided thoughtful comments and corrections throughout the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is an increasing prevalence of childhood asthma and allergic diseases in mainland of China. Few studies investigated the indoor dampness, ventilation and cleaning habits and their interrelationship with childhood asthma and allergic diseases. A large-scale cross-sectional study was performed in preschool children in Urumqi, China. Questionnaire was used to collect information on children’s health, home dampness and ventilation/cleaning (V/C) habits. Multiple logistic regressions were applied to analyze the associations between childhood asthma/allergic diseases and each sign of home dampness, dampness levels, each V/C habit and total V/C scores. The associations between dampness and health were further performed by strata analyses in two groups with low and high V/C scores. Totally 4618(81.7%) of 5650 children returned the questionnaire. Reports on home dampness were most common for water condensation on windows (20.8%) followed by damp beddings (18.0%). The most common ventilation measure was the use of exhaust fan in bathroom (59.3%), followed by daily home cleaning (48.3%), frequently putting beddings to sunshine (29.9%) and frequently opening windows in winter (8.4%). There were positive associations between the 6 signs of home dampness and children’s health particularly the symptoms last 12 months. By comparing with the reference dampness level (dampness scored 0), both the low dampness (scored 1~2) level and the high dampness level (scored 3~6) showed significantly increasing associations with childhood symptoms. There were crude negative associations between V/C habits and childhood health but not significant adjusting for home dampness levels. The risks of home dampness on children’s health were lower in the group with higher V/C score but the differences were not statistically significant. Home dampness is a potential risk factor for childhood asthma and allergic symptoms in preschool children in Urumqi, China. No significant effects were observed for ordinary home ventilation and cleaning habits in reducing the risks of home dampness on childhood asthma and allergic diseases in Urumqi, China. Asthma, one of the most common chronic respiratory disorders in children, has become a severe public health problem. Although the prevalence has been reported toward stabilization or even decreasing in previously high-prevalence countries ,2, the IAs a complex disease, asthma has a broad spectrum of environmental influencing factors. Over the past decades, rapid industrial and transitional economic changes have taken place in the mainland of China. The residential indoor environment has been dramatically changing. New residential buildings are constructed in a more tight style lack of good ventilation . Air conIndoor dampness has now been viewed as one potential risk factor for human health around the world, especially for children who spend a substantial fraction of time indoors . DampnesTo reduce or remove indoor dampness, there are different measures including opening windows, using exhaust fan in kitchen/bathroom, putting beddings/mattress to sunshine and home cleaning. To our knowledge, opening windows or using exhaust fan in kitchen/bathroom is helpful in reducing the indoor air humidity and increasing the fresh air supply , 17. PutIn this study, a large-scale cross-sectional survey on childhood asthma and allergic diseases in relation to home dampness and ventilation/cleaning habits was performed in children aged 1 to 8 years in Urumqi, northwest of China. With the typical temperate continental arid/semiarid climate, Urumqi's winter lasts as long as 5–6 months with severely cold climate . In the Written consents were obtained from participating parents before the study. This study was approved by the Ethic Committee of School of Public Health, Fudan University, China.This study is part of a national epidemiological study in 10 cities in China on childhood asthma and allergies in association with home environment-China, Children, Homes and Health . The curIn this study, children were selected by stratified random cluster sampling in day care centers in Urumqi. Among the total 7 administrative areas in Urumqi , 2–4 day care centers in each administrative area were randomly selected. Parents or guardians of all children in each day care center were invited to participate in the questionnaire survey. They were asked to answer the questionnaire either at home or in schools and return the questionnaire within 3 days. Finally, 18 day care centers were included and 5650 children received the questionnaires (aged 1–8 years old). More details regarding subject selection have been published previously .The whole questionnaire included 4 parts of questions on children’s demographical information, childhood and parental asthma and allergic diseases, signs of home dampness and ventilation/cleaning habits.The childhood asthma and allergic diseases were asked by using the core questionnaire in the ISAAC study . They weThe information on both home dampness and ventilation/cleaning habits was collected by using questionnaire similarly in the studies in Sweden , BulgariHome dampness was evaluated by 6 signs of dampness: mold spots, damp stains, damp beddings, water leakage, water condensation on windows and moldy odor. For mold spots and damp stains, it was asked “Have you noticed any visible molds/damp stains on the floor, walls or ceiling in the children’s sleeping room?” (Yes vs. No); For damp beddings: “Have you noticed that your beddings or mattress are affected with dampness in the last 12 months?” (Yes vs. No); For water leakage: “Have you noticed any flooding or other kinds of water damages in the rooms in the last 12 months?” (Yes vs. No); For water condensation on windows: “Have you noticed any condensation or moisture occurred inside and at the bottom of windowpanes in the children’s sleeping room in the winter?” (>5 cm vs. ≤5 cm); For moldy odor: “Have you perceived moldy odor in your dwelling in the last 3 months?”(Never vs. Sometimes/often).damp, scored 0, none of the dampness signs reported), the group with a low dampness level  and the group with a high dampness level .The answer ‘yes’ to each of the 6 questions on the dampness signs was coded as 1, while the answer ‘no’ was coded as 0 . The dampness score for each subject was calculated by adding the coded numbers of the 6 signs of dampness (the scores ranged 0–6). Based on the dampness scores, the subjects were classified into 3 groups: the reference group with no signs of dampness (RefVentilation/cleaning (V/C) habits were assessed by four aspects: (1) Frequently opening windows in winter: “Do you open the windows at night when the children sleep in winter season?” ; (2) Frequently putting beddings to sunshine: “Do you put your beddings/mattress to sunshine in the sunny days?” ; (3) Daily home cleaning: “Do you clean the children’s sleeping room every day?” (Yes vs. No); (4) Bath exhaust fan: “Do you use exhaust fan in the bathroom?” (Yes vs. No).V/C, referring to 0–1 positive answer out of the 4 questions) and the high-score V/C group .The answer of either ‘yes’ or ‘frequently’ to each of the 4 questions on V/C habits was coded as 1 while the answer ‘no’ or ‘never’ was coded as 0. The V/C score for each subject was calculated by adding the coded numbers of the 4 questions on V/C habits abovementioned (the scores ranged 0–4). Based on the V/C scores, the subjects were classified into 2 groups: the low-score V/C group , the 4 ventilation/cleaning habits individually and the 2 categorical V/C levels (LV/C and HV/C), respectively. Both the crude model (Model I) and the adjusted model (Model II) were used to analyze the associations. The crude model was set up by controlling for children’s age, gender, ethnicity, history of PAA, ETS and dwellings’ location. The adjusted model was set up by further controlling for the V/C score levels (LV/C and HV/C) in the analyses on each dampness sign, dampness levels and childhood health, or by further controlling for dampness levels  in the analyses on each V/C habit and V/C score levels and childhood health. The association trend was tested statistically between the Ldamp, Hdamp and the Refdamp level. Finally, stratified analyses in subgroup with low and high V/C scores (LV/C and HV/C) were performed on the associations between childhood asthma and allergic diseases and dampness levels . The interaction effect between dampness levels and V/C scores was tested. In all analyses, two-tailed tests and a 5% significant level was applied if no special statement and a 10% significant level was applied for interaction effect analysis. The association was expressed as odds ratios (ORs) with 95% confidence interval (95% CI). All statistical analyses were performed by SPSS 16.0 .Chi-square test was used to compare the prevalence of childhood asthma and allergic diseases between groups with and without history of PPA, ETS and the dwellings’ location . The same method was used to compare the proportion of each sign of dampness between LTotally, 4618 out of 5650 children (response rate 81.7%) returned the questionnaire. The average age was 4.6 ± 0.9 years (min-max 1–8 years old). Among them, 53.7% were boys and the Han nationality  people accounted for 82.4%. Most subjects lived in the urban areas (85.2%). More than half of subjects (59.2%) were exposed to home ETS and 14.2% of parents had a history of asthma/allergic diseases.More than 40% of children reported on rhinitis ever (46.4%) or current rhinitis (42.7%) and 25.3% of children had current wheeze . The repdamp group, scored 0), 30.9% had a low level of dampness  and 7.2% had a high level of dampness . On ventilation/cleaning habits, 40.4% of subjects had 0~1 positive answer out of the 4 V/C habits and were classified into the Lv/c group, while 59.6% of subjects had 2~4 positive answers and were classified into the Hv/c group. Comparing the positive reports on each sign of dampness between LV/C and HV/C groups, it was found that the 6 signs of dampness were generally more reported in the Lv/c group. There was a statistically significant difference between the Lv/c and Hv/c group on mold spots (P<0.001), damp stains (P <0.001) and water leakage (P = 0.004). Comparing the frequency distribution of the 3 dampness levels  between the Lv/c and Hv/c groups, it showed that the proportion of the Hdamp was higher in the Lv/c group while the proportion of the Ldamp was lower in the Hv/c group (P = 0.002).For home dampness , nearly There were generally positive associations between each sign of dampness and childhood asthma and allergic diseases or symptoms by multiple regression analyses in Model I and Model II . Most asdamp, both Ldamp and Hdamp levels showed positive associations with childhood asthma and allergic symptoms either as a continuous variable .In comparison with the Refvariable  or a catvariable . As a caThere were generally negative associations between each V/C habit and childhood asthma and allergic diseases or symptoms in the crude model . The negdamp, Ldamp and Hdamp) and childhood asthma and allergic diseases or symptoms were further analyzed in two strata with Lv/c and Hv/c levels. The results showed that, there were more significantly positive associations between home dampness and childhood symptoms in the Lv/c group in comparison with the Hv/c group andthe OR levels were higher as well (Pinteraction>0.1).The associations between home dampness levels  in Urumqi. Home dampness was one potential risk factor for childhood asthma and allergic symptoms, while the protective effects of ordinary home ventilation/cleaning habits were not clear in Urumqi, China.Dampness is a common phenomenon in the world. An estimated 17%-24% of homes in the Nordic countries, 37% in Canada and 50% in USA manifest signs of indoor dampness such as visible moulds on walls, floor or ceilings or water leakage –35. In tThere are several factors related with building dampness including the poor ventilation, high humidity and the inappropriate construction design, operation, maintenance and the use of buildings . Mold spAs on respiratory health in children, home dampness has been suggested as one potential risk factor for asthma and respiratory symptoms in children (e.g. wheeze and rhinitis) , 37. In Good ventilation/cleaning habits, particularly good ventilation habits were related with lower dampness in our study, suggesting that developing or improving home ventilation/cleaning habits might be beneficial in reducing the adverse effects by home dampness in Urumqi, China. Close to 60% of parents (59.3%) reported using exhaust fan in bathroom, less proportion of parents had the habits of frequently putting beddings to sunshine (29.9%) and even less on frequently opening windows in winter season (8.4%). However, opening windows as a measure of natural ventilation, together with the use of exhaust fan in bathroom as a measure of mechanical ventilation, helps to increase the interior ventilation, while putting beddings to sunshine and daily home cleaning are beneficial in reducing the microbial growth or allergen accumulation. There is evidence showing that a sufficient flow by ventilation either in natural or mechanical type is necessary to remove dampness and moisture in indoor air to reach the acceptable levels for occupants’ health .In perspectives of the health effects of ventilation/cleaning habits, negative associations between ventilation/cleaning habits and childhood health were observed in crude models. However, these negative associations disappeared after controlling for home dampness levels. It suggested that in environment with dampness or potential pollution due on dampness, common ventilation or cleaning habits might have limited effects in removing the adverse health effects by dampness. There was a quasi-experimental designed study in clinically diagnosed allergy patients reported that after bedding control (including putting bedding to sunshine), there was a significant decrease of the frequency of dyspnea . In thisv/c and Hv/c levels, particularly for rhinitis ever and current wheeze. By comparing the ORs of these associations in the two groups, higher risks were observed in the group with Lv/c level but not reaching statistical significance. One recent review on respiratory tract symptoms and asthma in relation to the remediating buildings damaged by dampness and mould showed that, there was moderate-quality evidence that asthma-related symptoms and respiratory infections were improved after the intervention such as cleaning/repairing all relevant causes of mould or dampness, improving ventilation, removing damaged materials and replacing them with new ones [The positive associations between dampness and childhood asthma and allergic diseases were observed in both subgroups with Lnew ones . This coThere are some strengths and limitations in this study. A large sample size with high response rate (81.7%) ensured the good representativeness of the aimed population at this age. No prior information was collected on participating children’ health status, dampness conditions and ventilation/cleaning habits which further reduced the sample selection bias. Parents or other guardians like grandparents of all children in the recruited kindergartens completed the questionnaire and most of parents  lived together (in one bedroom) with their children. This indicated that the reported home dampness by parents could well reflect the dampness situation in children’s living environment. Self-reported symptoms based on questionnaire alone and lack of clinical diagnosis may cause information bias to some extent. However, the core questions on asthma and allergic diseases were the same in the ISAAC study , 4 whichIn conclusion, asthma and allergic symptoms were common in preschool children in Urumqi, China. Home dampness could have adverse effects on children’s asthmatic symptoms. Developing good home ventilation/cleaning habits might benefit in reducing the dampness levels in this area. The improvement of childhood asthmatic symptoms by increasing the ordinary home ventilation/cleaning levels was not clear which needs further investigation in this area.