Cerebral white matter abnormalities on term MRI are a strong predictor of motor disability in children born very preterm. However, their contribution to cognitive impairment is less certain.Examine relationships between the presence and severity of cerebral white matter abnormalities on neonatal MRI and a range of neurocognitive outcomes assessed at ages 4 and 6 years.The study sample consisted of a regionally representative cohort of 104 very preterm (≤32 weeks gestation) infants born from 1998–2000 and a comparison group of 107 full-term infants. At term equivalent, all preterm infants underwent a structural MRI scan that was analyzed qualitatively for the presence and severity of cerebral white matter abnormalities, including cysts, signal abnormalities, loss of white matter volume, ventriculomegaly, and corpus callosal thinning/myelination. At corrected ages 4 and 6 years, all children underwent a comprehensive neurodevelopmental assessment that included measures of general intellectual ability, language development, and executive functioning.At 4 and 6 years, very preterm children without cerebral white matter abnormalities showed no apparent neurocognitive impairments relative to their full-term peers on any of the domain specific measures of intelligence, language, and executive functioning. In contrast, children born very preterm with mild and moderate-to-severe white matter abnormalities were characterized by performance impairments across all measures and time points, with more severe cerebral abnormalities being associated with increased risks of cognitive impairment. These associations persisted after adjustment for gender, neonatal medical risk factors, and family social risk.Findings highlight the importance of cerebral white matter connectivity for later intact cognitive functioning amongst children born very preterm. Preterm born children without cerebral white matter abnormalities on their term MRI appear to be spared many of the cognitive impairments commonly associated with preterm birth. Further follow-up will be important to assess whether this finding persists into the school years. Central to reducing the longer-term morbidities associated with very preterm birth is the need to identify early neonatal markers of risk. Children born very preterm now represent almost 2% of all live births in the U.S. and other developed countries, To date, efforts to identify early perinatal markers of later neurodevelopmental risk have been somewhat disappointing, with most factors correlating only modestly with outcome. One potential exception is the presence and severity of cerebral white matter abnormalities on term magnetic resonance imaging (MRI). These diffuse white matter abnormalities affect between 50–70% of very preterm infants and include: white matter volume loss; ventriculomegaly; white matter signal abnormality; thinning of the corpus callosum; and delayed myelination. However, some caution is warranted. First, with the exception of Iwata, To address these limitations, the present study draws on prospective longitudinal data from a representative cohort of children born very preterm and a comparison group of their full-term peers to assess the cognitive, language, and executive functioning outcomes of very preterm born children from 4 to 6 years. In particular, we examined the extent to which neonatal white matter abnormalities on MRI were predictive of later impairment across a range of neurocognitive outcomes assessed at multiple time points, thus, allowing for an assessment of the stability of associations across time/age. We hypothesized that increasing severity of white matter abnormalities at term equivalent would be associated with increasing risks of impairment across all outcomes/ages, with these associations persisting after taking into account other medical and social risk factors.n = 3), 98% were followed up at age 4 and 97% at age 6. In addition, one child was excluded due to blindness and another due to missing MRI data, leaving a final sample of 104 children at age 4 and 102 children at age 6.Study participants consisted of two groups of children. The first group was a regional cohort of 110 children born very preterm (≤32 weeks gestation) who were consecutively admitted to a level III Neonatal Intensive Care Unit (NICU) at Christchurch Women’s Hospital  over a two-year period (November 1998–December 2000). In total, 92% of eligible infants were recruited. Excluding deaths (n = 32), moved overseas (n = 9), refusals (n = 9), and agreed but couldn’t attend clinic appointment/s within the 2-week assessment window due to illness or family circumstances (n = 19). No significant differences were found between recruited and nonrecruited term born infants on measures of gestational age, birth weight, socioeconomic status, or race. In addition, comparison of the socioeconomic profile of recruited families with regional census data n = 108). Data for one additional child was excluded due to incompleteness. Written informed consent was obtained from all parents/guardians and all procedures were approved by our regional Health and Disability Ethics Committee. The second group of study children, recruited at age 2 years, comprised a sample of 113 full-term born (37–41 weeks gestation) children. These children were identified from hospital birth records for the same period by alternately selecting, in a forwards and backwards fashion, the second child listed in the delivery schedule. As a group, they were matched to the very preterm cohort for gender, delivery hospital, and date of birth. Of those identified, 62% were recruited at age 2. Reasons for nonparticipation included untraced (n = 26/106); 2) mild abnormalities ; 3) moderate abnormalities ; or 4) severe abnormalities . Given the small number of children with severe white matter abnormalities, children in the moderate and severe white matter abnormalities groups were combined for the purposes of this analysis.All very preterm born children underwent structural MR imaging at term equivalent age (39–41 weeks gestation). Unsedated infants were settled, wrapped, and placed in a Vac Fix bean bag and then scanned using a 1.5-tesla General Electric Signa System  with previously documented sequences. SD) below the comparison group mean and severe delay was defined by a score that was more than 2 standard deviations below the comparison group mean. Study measures included in this analysis were as follows.At corrected ages 4 (±2 weeks) and 6 (±4 weeks), all children underwent a comprehensive multidisciplinary assessment of their general cognitive ability (IQ), language development, and executive functioning skills. At age 4, independent testers administered each measure, while at 6, one administered the IQ and language measures and another administered the executive functioning test battery. All assessments were done blind to child perinatal history and past cognitive performance. Clinical impairment was defined on the basis of the score distributions of our regionally representative comparison group. r = .89–.92). At ages 4 and 6 (corrected), children’s intellectual ability was assessed using a short form of the revised Wechsler Preschool and Primary Scales of Intelligence (WPPSI-R) r = .90) with other language measures. At age 4 (corrected), the preschool version of the Clinical Evaluation of Language Fundamentals (CELF-P) Children’s executive function abilities were assessed using a battery of age appropriate tasks administered at both follow-up time points. At age 4, this battery consisted of four tasks: 1) Tower of Hanoi (planning and problem solving) At corrected ages 4 and 6 years, very preterm born children performed less well than full-term born children across all measures of intellectual ability, language, and executive functioning. As shown in SD below control mean) and 3.3 times more likely to meet criteria for severe delay/impairment (>2 SD below control mean). By age 6, similar to intelligence, risks of executive function delay/impairment in the very preterm born group had tended to increase, with relative risks for any delay being 2.6 (41% v. 16%) and for severe delay being closer to 4 (22% v. 6%).With respect to risks of delay, at age 4, very preterm born children were 2.6 times more likely to be classified as having any cognitive delay (35% v. 13%) and 5 times more likely to be severely delayed (10% v. 2%) than full-term children. Relative risks at age 6 were generally similar, with closer to half of very preterm born children showing any delay (49% v. 17%) and around 1 in 6 severe cognitive delay (16% v. 4%). Fairly consistent rates of language delay were evident, with very preterm born children being twice (31% v. 15%) as likely as full-term children to have any delay and 3–4 times (9% v. 3%) more likely to have severe delay at ages 4 and 6 years. Finally, in terms of performance on the two executive task batteries, at 4 years, very preterm born children were almost twice as likely to show any delay (>1 SD below control mean) and severe delay (>2 SD below control mean) on measures of intelligence (p<.0001), language (p≤.003), and executive functioning (p<.0001). Specifically, children in the full-term control group obtained the highest scores and had the lowest risk of any or severe delay. In contrast, across the three very preterm born groups, as the severity of white matter abnormalities increased there were corresponding declines in children’s mean scores and risks of clinically significant delay at ages 4 and 6.Closer examination of group contrasts showed the following:Relative to the full-term group, very preterm born children with moderate-to-severe neonatal white matter abnormalities had the most impaired neurocognitive performance. At ages 4 and 6, they were 3.3–5.6 times more likely to meet criteria for any delay and 3.4–16.6 times more likely to be subject to severe delay. Specifically, 74% had cognitive delay at ages 4 and 6, 63% had language delay at age 4 and 50% at age 6, and 58–63% showed impaired performance on the executive function task battery. Risks of severe intellectual (32–37%), language (26–28%), and executive functioning (32%) impairment were also high and largely confined to this group.Very preterm born children with mild white matter abnormalities on neonatal MRI were, in general, at lower risk of adverse neurocognitive outcomes than very preterm children with moderate-to-severe white matter abnormalities, and at higher risk than very preterm born children without white matter abnormalities and full-term children. Compared to the full-term group, they were 1.7–3.0 times more likely to show any delay and 1.8–3.6 times more likely to exhibit severe delay on measures of IQ, language, and executive functioning. Most of the problems in this group were in the mild (26–52%) as opposed to the severe (3–20%) range.p>.05). Mean score differences were generally small, as were relative risks for any (RR = 1.2–1.7) and severe delay (RR = 1.6–3.2).No significant differences were found between very preterm born children without white matter abnormalities and full-term comparison children on any of the outcome measures assessed at either 4 or 6 years , reduced white matter volume , cystic abnormality , ventricular dilation  and thinning of the corpus callosum/reduced myelination  were all associated with IQ. White matter signal abnormality , reduced white matter volume , thinning of the corpus callosum/reduced myelination  and cystic abnormality  were also correlated with later language function. Finally, a similar, but generally weaker, pattern of associations was evident for executive functioning at ages 4 and 6 .Extending on these findings, linear associations between the individual MRI scale scores used to assess white matter abnormalities were examined in relation to each outcome. A generally consistent pattern of findings emerged across measures, although associations tended to be stronger at age 4 than at age 6. White matter signal abnormality , and race.p<.05) and 6  whereas being born small for gestational age increased risks of cognitive delay at age 6 . A more consistent and stronger pattern of associations emerged for social risk factors, with lower family socioeconomic status , low maternal education , and being born to a single mother  placing children at increased neurocognitive risk. Examination of the cumulative effects of these social risk factors showed that each additional risk factor increased the likelihood of an adverse cognitive outcome by 1.7 fold at age 4 (p = . 01)and 1.6 at age 6 (p = .003).Across these analyses, few medical risk factors were correlated with outcome. Antenatal corticosteroid exposure in very preterm born children appeared to be protective for general cognitive delay at both ages 4 , cerebral white matter abnormalities remained a significant independent predictor of neurocognitive outcome across all measures. The odds of later cognitive, language, and executive function delay for very preterm children with mild white matter abnormalities were reduced from 1.9–5.3 to 1.8–4.0, while for very preterm children with moderate-to-severe white matter abnormalities they were reduced from 5.7–18.6 to 4.5–15.5 after covariate adjustment.To examine the extent to which the presence and severity of cerebral white matter abnormalities on term MRI made an independent net contribution to the prediction of subsequent cognitive risks at ages 4 and 6 years, the associations shown in SD below control mean) compared to just over 1 in 8 of full-term children . At age 6, a similar pattern of results was observed. Although there was some suggestion that the proportion of very preterm children subject to intellectual and/or executive function delay may have increased somewhat, with between 40–50% meeting criteria for any delay and 15–20% severe delay. This small upward trend may reflect tester or measurement effects, or may well suggest a tendency for these domain impairments to become increasingly apparent with age and the transition to school. Further follow-up using repeated measures and analysis methods that take account of measurement error will be important to help clarify this issue.Findings confirm that preschool and early school-aged children born very preterm are at high risk of neurocognitive delay and/or impairment, To date, most follow-up studies in very preterm born children have tended to focus on the risks of severe impairment and/or disability. However, given the substantial numbers of very preterm survivors with mild cognitive impairment, this group appears to be as important. These less severe but clinically significant impairments will impede children’s early school progress and have cumulative impacts on achievement and the need for remedial support. They are also likely to be more responsive to treatment. Therefore, targeting these children during the important preschool and early school years may be more effective strategy to reduce the morbidities associated with very preterm birth than focusing solely on only those with the most severe impairments. Recognition of milder impairments is also consistent with the secular trend for a reduction in major cystic white matter lesions that traditionally have been associated with more severe disability. Instead, an increasing number of children will likely experience milder forms of white matter abnormality that will result in high-incidence, low-severity conditions  that compromise classroom learning. SD and<−2 SD) and severe (>−2 SD) delay, and particularly severe delay. Very preterm born children with mild white matter abnormalities also had increased risks of neurocognitive delay relative to their full-term peers, but their difficulties tended to be of a less severe nature.Key to reducing longer term risks associated with very preterm birth is the need for a better understanding of the neuropathological mechanisms that place very preterm born children at neurocognitive risk. This issue was the central focus of this paper. Across all outcome measures assessed at both time points, clear linear relationships were found between the presence and severity of cerebral white matter abnormalities defined qualitatively on neonatal MRI and children’s later risks of both any and severe neurocognitive delay. Specifically, findings suggested that very preterm infants who are spared cerebral white matter abnormalities can be expected to have similar levels of preschool and early school age cognitive functioning as their full-term peers with respect to general intelligence, language, and executive functioning . In contrast, very preterm born children with white matter abnormalities on term MRI were at increased risk of delays, with risks increasing with the severity of neonatal white matter abnormalities. Those with moderate-to-severe white matter abnormalities were at greatest risk of both mild  children with no-to-mild and moderate-to-severe white matter abnormalities had lower mean Bayley-III cognitive and language scores than full-term children at age 30 months (N = 85). This finding differs somewhat from the present study, which failed to find any differences between very preterm born children with no cerebral white matter abnormalities and full-term control children. This may reflect the high-risk extremely preterm sample and/or the grouping of no and mild white matter abnormality cases together. The second study to include a full-term comparison group focused on a series of domain specific executive functioning outcomes and reported a similar pattern of findings. These findings extend on previous within-group analyses showing that cerebral white matter abnormalities on term MRI are associated with increased risks of severe cognitive delay at age 2 The composite nature of the qualitative rating system used to classify white matter abnormalities makes it difficult to speculate confidently on the neural mechanisms underlying these neurocognitive impairments. Examination of the individual white matter abnormality subscales in relation to neurocognitive outcomes suggested that all were contributing to later risk, but that white matter signal abnormalities and volume loss may be particularly relevant. Very preterm born children with periventricular white matter signal abnormalities have been shown to be characterized by less mature fiber tract development and organization, particularly in those fibers tracking through the internal capsule and nearby regions. p = .07). Irrespective of the modeling approach used, consistent with other studies, The importance of cerebral white matter abnormalities as a predictor of neurocognitive delay for very preterm born children was further reinforced after extensive covariate adjustment for gender, other neonatal medical factors, and family social risk. White matter abnormality severity remained a significant independent predictor of later intellectual, language, and executive function delay. The only exception was executive function delay/impairment at age 6 which was reduced to marginal significance (mild white matter abnormality OR = 2.8, moderate-to-severe white matter abnormality OR = 5.3, The strengths of this study include the prospective longitudinal design, the unselected sample of children born very preterm in our region, inclusion of a regionally representative comparison group, assessment of neurocognitive outcome using multiple measures at multiple time points, the use of blinded independent testers, and our high sample recruitment and retention. However, the study was limited by the use of an abbreviated rather than full WPPSI-R at ages 4 and 6 years. This approach was adopted to minimize sample member over-burden and to allow a wider range of measures to be collected. A second limitation was that we did not administer the same language measure at both ages. Receptive and expressive language was assessed at age 4, whereas only receptive skills were assessed at age 6.Nonetheless, findings indicate that neonatal white matter abnormalities on MRI at term represent an important predictor of persistent neurocognitive problems during the preschool and early school years. Conventional MRI at term equivalent is likely to provide useful information to assist with post discharge planning and monitoring when interpreted alongside other influences on child cognitive development such as family social circumstances and functioning.


Protected areas are increasingly considered to play a key role in the global maintenance of ecosystem processes and the ecosystem services they provide. It is thus vital to assess the extent to which existing protected area systems represent those services. Here, for the first time, we document the effectiveness of the current Chilean protected area system and its planned extensions in representing both ecosystem services  and biodiversity. Additionally, we evaluate the effectiveness of protected areas based on their respective management objectives. Our results show that existing protected areas in Chile do not contain an unusually high proportion of carbon storage (14.9%), agricultural production (0.2%) or biodiversity (11.8%), and also represent a low level of plant productivity . Proposed additional priority sites enhance the representation of ecosystem services and biodiversity, but not sufficiently to attain levels of representation higher than would be expected for their area of coverage. Moreover, when the species groups were assessed separately, amphibians was the only one well represented. Suggested priority sites for biodiversity conservation, without formal protection yet, was the only protected area category that over-represents carbon storage, agricultural production and biodiversity. The low representation of ecosystem services and species’ distribution ranges by the current protected area system is because these protected areas are heavily biased toward southern Chile, and contain large extents of ice and bare rock. The designation and management of proposed priority sites needs to be addressed in order to increase the representation of ecosystem services within the Chilean protected area system. Ecosystem services, the benefits that humans derive from ecosystems, are vital for sustaining human well-being The provision of key ecosystem services can present trade-offs  making their conservation within the same areas challenging There is a particular paucity of data for appropriate evaluation of protected area effectiveness in capturing ecosystem services in poor and developing countries This study analyses for the first time to what extent the current and suggested integrated protected area system represent selected ecosystem services of Chile. Specifically, the representation of three ecosystem services - plant productivity, carbon storage, agricultural production - and biodiversity is assessed under three protection scenarios. These scenarios capture the current status and medium-term projections for the protected area system. Given the large extent of pristine forest ecosystem remaining, we would expect that Chilean protected areas tend to represent high levels of net primary production, carbon storage, and biodiversity, but tend to exclude agricultural production. We address three main questions: 1) How are the three ecosystem services and biodiversity distributed across Chile?; 2) To what extent do the three protection scenarios represent the chosen ecosystem services and biodiversity?; and 3) How effective are Chilean protected area categories in representing ecosystem services and biodiversity?integrated protected area system. Current protected areas, PSBC and private protected areas datasets were obtained from the Chilean Ministry of Environment in vector format. Current protected areas datasets are freely available at the Chilean Ministry of Environment web site (ide.mma.gob.cl). Considering the entire set used in this study (n = 510), the average size of protected areas was 40,218 ha, varying from a minimum of 0.64 ha to a maximum of 3,677,849 ha. All of the seven protected area groups, except PSBC and private protected areas, are listed under an IUCN category In order to assess the effectiveness of the current Chilean protection system we considered all areas with statutory protection. These are the protected areas belonging to SNASPE, which comprises 33 national parks, 49 national reserves, and 16 natural monuments. We also included nature sanctuaries (n = 31), and lands protected by the Chilean Ministry of National Heritage (n = 18) . SNASPE Following a similar approach to that of Pliscoff and Fuentes-Castillo – Scenario 1 . Thus, a total of 246 carbon zones with unique carbon stock values were compiled based on the IPCC Tier-1 methods.We calculated carbon storage by combining an estimate of above and below ground vegetation biomass and a soil organic carbon (SOC) dataset (in kg C). Aboveground vegetation data were obtained following the IPCC GPG Tier-1 method for estimating vegetation carbon stocks using the global default values provided for above ground biomass Soil carbon density data were obtained from the most recent soil carbon database, the Harmonized World Soil Database (HWSD) version 1.1 We used these datasets to create a final carbon storage estimation at 1.25×1.25 km resolution.Plant productivity (PP) patterns were determined using the Normalized Difference Vegetation Index (NDVI), which has been widely used for this purpose 2 resolution Global MODIS (TERRA)  dataset, available for 2000–2010. For each pixel we calculated the average of the annual NDVI mean for the 10 year period.Monthly NDVI composites were obtained from the 1 kmhttp://faostat3.fao.org/home/index.html), thus obtaining USD of agricultural production per pixel.Agricultural production was calculated as the sum of gross production (USA dollar) for 2000. In order to generate a fine resolution layer, a spatial disaggregation process was carried out, in which a coarse resolution dataset is ‘disaggregated’ in a finer and related resolution dataset. Specifically, the agricultural production layer was calculated as follows (i) We multiplied the harvested area of 32 major crops (i.e. proportion of a grid cell that has been harvested for a specific type of crop)  at 10 kmThe Chilean biodiversity dataset comprised four taxonomic groups: mammals (n = 113), birds (n = 364), amphibians (n = 58) and vascular plants . Distribution maps for mammals, birds and amphibians that occur in Chile were obtained from the IUCN Global Mammal Assessment, BirdLife International and the Global Amphibian Assessment, respectively. All these are freely available at the IUCN Red List web site Each taxonomic group was analysed separately, using a 1 km × 1 km grid resolution.A spatial overlap analysis was used to calculate the representation of each ecosystem service and of biodiversity within the Chilean protected area system. The three protection scenario covers were overlapped with each ecosystem service and biodiversity layer, and the spatially coincident coverage extracted. However, as ecosystem service layers and biodiversity were mapped in different units, their representation was calculated in distinct ways as follows:– The units of carbon storage and agricultural production layers are the total amount of carbon (kg) and USD production, respectively, per pixel. Thus, the representation of these two ecosystem services was calculated as the sum of all those pixels that fell within protected areas.– The PP captured was estimated from the average of the NDVI values of those pixels that fell within protected areas. As NDVI varies according to vegetation type, we calculated a weighted average in accordance with the proportion of total area of each vegetation type found within the protected area system. Thus, the resulting NDVI average is representative of the extents of different vegetation types within the protected area system. Vegetation types found within protected areas were Forest, Shrubland, Steppe, Wetland, Crop, Peatland and Bare areas . For com– The representation of biodiversity was calculated as the summed proportion of species’ ranges that fell within the protected area coverage. This was calculated per taxon and for all species together.We divided the percentage of each of the measures of ecosystem services and biodiversity contained within each scenario and protected area category by the percentage land area covered by that particular scenario and category The bulk of carbon storage, net primary production and agricultural production were located in the south-central zone of Chile . Areas wThe proportion of stored carbon varied from a low of 14.9% in Scenario 1, which increased to 19.0% and 19.9% in Scenario 2 and Scenario 3, respectively . In the The current protected areas capture 13.9%, 18.2%, 20.7% and 8.9% of mammal, bird, amphibian and plant ranges respectively. Amphibians was the only group well represented, with a ratio slightly higher than one (1.03). All species’ representation levels increased substantially in Scenario 2 (i.e. when PSBC sites were included), capturing this time 18.9%, 23.0%, 27.1% and 14.7% of mammal, bird, amphibian and plant species’ ranges respectively. In Scenario 2 only amphibian representation ratio was above one (1.07). When Private Protected Areas were included in Scenario 3, representation levels increased by approximately 1% for all species groups, with only that of amphibians’ being well represented . When spCarbon storage was well represented only by PSBC (4.08 times as much as would be expected for the area). The other protected area categories, except private protected areas, had values slightly below 1 . All proPrevious assessments of the effectiveness of the Chilean protected area system have focused exclusively on biodiversity First, as forest coverage, as well as protected areas, is concentrated in southern Chile, we would have expected a higher representation of carbon storage . The c. Second, the underrepresented crop production found within the existing protected areas suggests that these are displacing or avoiding areas of agricultural production, which could reasonably be argued as reflecting their effectiveness. Chilean protected areas conserve a significant proportion of untransformed landscape, facing the challenge of displacing human activities beyond their boundaries. What is not clear however is whether the low agricultural activity within protected areas is due to the management strategy of conserving these lands intact, or because the spatial bias of protected areas towards southern regions renders them unsuitable for agriculture.Third, while the largest coverage by protected areas is concentrated in the Austral Chilean zone (44°–56° S), our results show that the highest species richness areas are located in the central (28°–36° S) and south-central (36°–43° S) zones of Chile , which iAdding PSBC and private protected areas to the current protected area system (i.e. Scenarios 2 and 3) enhances the representation of ecosystem services and total biodiversity . This inWhen protected areas were evaluated based on their management objective categories, our results showed that no existing protected area category with statutory protection represents the level of ecosystem services and biodiversity one would have expected based on their coverage, except the Natural Monument category that over-represented biodiversity . This ovThe existing Chilean protected area network does not perform well in representing all biodiversity groups together, but achieves a good representation of amphibians. Also, its provision of ecosystem services is poor. It is highly likely that this gap would need to be addressed principally by the expansion of the coverage of the protected area system. In this regard we suggest two measures. First, a re-evaluation of the already suggested new sites for the integrated protected area system, as these do not significantly increase ecosystem service representation. Second, a systematic assessment plan of current conservation management objectives and strategies, in order to enhance ecosystem service supply by existing protected areas.Figure S1Distribution maps of species richness for four taxonomic groups at 12 grid resolution.km a) Amphibians, b) Mammals, c) Birds and d) Plants.(TIF)Click here for additional data file.Table S1Protected area categories used in this study, and their associated management strategies defined under the International Union for Conservation of Nature (IUCN) regulatory framework.(DOC)Click here for additional data file.Table S2Summary of values used in calculating agricultural production .(DOC)Click here for additional data file.Table S3Average of NDVI values and coverage characteristics of different vegetation types in Chile.(DOC)Click here for additional data file.Table S4Biodiversity representation by species group in the five management categories and the suggested sites for the new integrated protection system (PSBC and Private protected areas). A ratio of >1 indicates that a particular group is over-represented relative to what would be expected for its area; values <1 indicate under-representation. ‘All management strategies’ refers to the area covered by all the seven categories. PA: Protected Area; PSBC: Priority sites for biodiversity conservation.(DOC)Click here for additional data file.Table S5Land use cover within the current Chilean protected areas system (Scenario 1).(DOC)Click here for additional data file.Table S6Land cover within each of the five management categories and the suggested sites for the new integrated protection system (PSBC and Private protected areas). PA: Protected Area; PSBC: Priority sites for biodiversity conservation.(DOC)Click here for additional data file.


Cyprinus carpio L.) at concentrations representative for commercial fish feeds. Experimental feeding with 352, 619 or 953 μg DON kg−1 feed resulted in unaltered growth performance of fish during six weeks of experimentation, but increased lipid peroxidation was observed in liver, head kidney and spleen after feeding of fish with the highest DON concentration. These effects of DON were mostly reversible by two weeks of feeding the uncontaminated control diet. Histopathological scoring revealed increased liver damage in DON-treated fish, which persisted even after the recovery phase. At the highest DON concentration, significantly more fat, and consequently, increased energy content, was found in whole fish body homogenates. This suggests that DON affects nutrient metabolism in carp. Changes of lactate dehydrogenase (LDH) activity in kidneys and muscle and high lactate levels in serum indicate an effect of DON on anaerobic metabolism. Serum albumin was reduced by feeding the medium and a high dosage of DON, probably due to the ribotoxic action of DON. Thus, the present study provides evidence of the effects of DON on liver function and metabolism.Deoxynivalenol (DON) frequently contaminates animal feed, including fish feed used in aquaculture. This study intends to further investigate the effects of DON on carp ( Fusarium fungi, but also fungal species, such as Myrothecium, Cephalosporium, Verticimonosporium and Stachybotrys /EC) [−1 . These id higher . Lipid ad higher , inadequd higher ,11,12, pd higher ,14,15 and higher . Evidenc 56 days , whereby[−1 feed .Cyprinus carpio), as an aquaculture species of high global relevance, is needed to guarantee fish health and a safe product for human nutrition.In the last few years, many studies have been focused on fish health interactions with dietary management. In aquaculture, fish nutrition is critical, and the applied feed contributes up to more than 60% of the total production costs . Thus, nThe aim of the present study was therefore the evaluation of various DON feed dosages on the growth performance and health status of juvenile carp. The present study is a part of a feeding study in which stress and immune responses already have been evaluated and published elsewhere . The anaThe experimental diets were prepared as shown in −1 dry matter).The nutritional compositions of experimental diets are given in After four weeks of feeding, two tanks containing six fish each were sampled, whereas two similar treated tanks containing 12 fish were fed the uncontaminated diet for a further two weeks and sampled thereafter. The growth performance, serum parameters and biochemical composition of all fish were analysed.Feeding carp the experimental diets at a restricted daily basis of 2% of body weight resulted in increased fish body weights after four weeks . CompareFurthermore, condition factors were not different between treatment groups after four weeks of feeding and also after recovery for additional weeks . The finet al. [Reduced intake of DON-contaminated feed leading to reduced weight gain was reported in mice , but notet al.  suggest max and luminosity values -stained sections revealed significant differences between the condition of the liver tissue of control fish and DON-treated fish . DON-treobserved .−1 DON in the same study led to fatty infiltration in liver. Histological changes, including fat deposition, have also been found in DON-treated carp in the present study. Liver damage was probably caused by the occurrence of oxidative stress (as indicated by the lipid peroxidation assay) together with apoptotic loss of cell integrity and disturbance of nutrient metabolism. Liver damage also often leads to the occurrence of increased liver fat content, as is reported for fish recovering from being fed the high-dose DON feed for four weeks  showed differences in the kidneys after four weeks of DON feeding and also after additional two weeks of recovery but not in liver and spleen . During p = 0.065). Still, a significant relationship between the toxin concentrations in the experimental feeds and lipid content was found .Investigation on whole body composition revealed that the fat content in whole body homogenates of fish treated with the medium- and high-dose DON diet was increased compared to control fish , whereasAfter two additional weeks of recovery, all DON-treated fish showed lower lipid levels than the control fish . These rp = 0.002). Moreover, a significant relationship between the toxin concentrations in the experimental feeds and the lipid and ash content was found  in fish after the recovery phase. This led to the investigation of similar parameters in samples of liver tissue and white dorsal musculature to allow a possible explanation for these observations. In the recovered fish, the ash content correlated with the lipid content negatively  for control fish of 24.6 versusCVs of 92.2, 42.3 and 51.0 for fish treated with the low-, medium- and high-dose, respectively, while the CVs for all fish in the recovery phase ranged from 9.5 to 23.2). ALT activity also correlated with the mycotoxin concentrations in feed . This indicates damage to liver tissue, as has already been observed in carp under chemical stress [Liver glucose, lactate and ascorbate concentrations were not influenced by DON feeding and did not correlate with toxin concentrations in the experimental feeds. Although ascorbic acid has been shown to prevent the hemolytic action of DON on rat erythrocytes to some extent , ascorbal stress . In contA considerable amount of the fish consists of white musculature, which shows low levels of myoglobin and is mostly used for burst swimming . SwimminAs expected, the lipid content in carp muscle was found to be low in the present study, and it was not influenced by DON feeding. Different crossbreds of common carp showed lipid contents in the fillet of up to 9.9%, with mirror carps showing 2.4% lipid in fillet . Low musThe significant difference in the dry matter of white muscle of fish fed the low-dose diet compared to the control fish cannot be explained at the moment. Reductions of dry matter in the musculature can be caused by the depletion of tissue nutrient contents, which are compensated for by increasing the water content . HoweverMuscle glucose shows no differences of the DON treatment of fish. The glucose concentration in red musculature has been reported to be rather independent of blood glucose levels , but theAscorbate levels in white muscle samples strongly depend on the supply via the diet and have been reported to be low in fish musculature . This coOur study demonstrates a large variation of biochemical serum parameters, which are known to be affected by many endogenous and exogenous factors, such as age, health condition, nutrition or stress, including chemical stress ,49.Glucose levels in the experimental fish in the present study were comparable to values for unstressed carp reported previously ,49. GlucSerum lactate levels in control fish are comparable to values in unstressed fish in other studies ,50. HoweTotal protein content in all carp used in the present study was slightly lower in most cases compared to previously reported values in other studies ,49. Fromp = 0.012, 0.022 and 0.025, respectively). Moreover, after the same time period, albumin or lactate concentrations in serum correlated with the toxin concentration in the experimental feeds . Similar results have also been obtained for the recovery phase for the correlation of ALT and LDH with the toxin concentrations previously applied . Furthermore, glucose or lactate concentrations in serum correlated with the toxin concentration in the experimental feeds . Thus, the activity of serum ALT and LDH together with lactate concentrations seems to be a sensitive indicator of the fish responses to DON. The symptoms of carp exposed to DON resemble the situation of freshwater snakehead fish, Channa punctatus, that were treated with sublethal concentrations of a carbamate pesticide, leading to increases of LDH and decreased SDH activity in several organs and hyperglycaemia and hyperlactaemia, which suggested that anaerobic metabolism was favored [In contrast to their activity in liver tissue , the act favored .Fusarium toxin contamination of these diets. For the preparation of experimental feeds, all ingredients listed in −1, medium dose, 619 μg kg−1, and high dose, 954 μg kg−1, final feed, respectively) [Ingredients were chosen so that no cereals were included in the experimental diets to exclude cereal-based ctively) . The preCarp were raised from eggs in our facilities and used for the experiments at 12–16 cm in total length. The fish were kept at a 16 h light/8 h dark photoperiod at 25 ± 0.2 °C (mean ± SD) and acclimatized to the tanks that were integrated into a flow-through system providing 6 L fresh and conditioned water per hour per tank prior to the experiments for three weeks.All chemicals were obtained from Sigma-Aldrich , unless indicated otherwise.The prepared pellets (4 mm in diameter) were given at 2% of body mass once every day to juvenile carp, which were separated into four different feeding groups  with 6 fish each in quadruplicate 54-L tanks for each treatment. The maintenance of optimal rearing conditions  was controlled during the entire experiments . All expn = 6 fish per treatment level per sampling day). Tissues were automatically processed , and at least six sections per fish (3 μm-thick) were mounted on microscope slides and stained with haematoxylin and eosin (HE). Additionally, sections were stained with PAS or Prussian blue to analyze glycogen content and to detect changes in tissue iron content related to erythrocyte turnover. For histopathological examination (Nikon Eclipse 400 microscope), sections were examined in detail at 400× magnification. Quantitative analyses, as described below, were conducted on digital images, which were taken with a Nikon DXM 1200 F digital camera and Nikons ACT-1 software V2.63. Damage to liver tissue was estimated by histopathological scoring in 10 HE-stained sections per fish using semi-quantitative assessments of the severity  according to the suggestions of Zodrow et al. [max. Luminosity can be deduced from the histogram settings in Adobe Photoshop. RGBmax was achieved by lightening the green and blue channels in the RGB space. Chrominance was calculated by subtracting the minimal RGB value  from the RGBmax (Adobe® Photoshop® CS3 Extended version 10.1).Histological assessments were conducted on liver , 20% (w/v) trichloric acid (TCA), 9.1 μL mL−1 hydrochloric acid (37%),0.06% (w/v) BHT and 866.9 μL distilled water. Thereafter, samples were incubated at 70° C for 90 min, cooled to room temperature and centrifuged at 16,000× g for 15 min at room temperature. In parallel, standards containing 0 to 3200 nM malondialdehyde (MDA) were prepared. Optical densities of all samples were read at 532 nm . Aliquots of the tissue homogenate were also used for the lactate dehydrogenase (LDH) assay and protein determinations. The latter were conducted using the bicinchoninic acid (BCA) assay (Sigma), according to the manufacturer`s protocol. The activity of LDH in tissue homogenates and serum samples was measured according to Bergmeyer [−1 in Tris-NaCl solution ) were mixed with 33 μL pyruvate solution (9.76 mmol L−1 in Tris-NaCl solution). The reaction was started by the addition of a 20 μL sample, and an absorption decrease at a wavelength of 339 nm was recorded for 10 min .Tissue samples were homogenated in 19 volumes PBS containing 0.1% (w/v) butylated hydroxytoluene (BHT), homogenized using an UltraThurrax  for 10 s and centrifuged for 10 min at 10,000× g at 4 °C . The supernatant was used for the TBARs assay  with theergmeyer . In shorFish from each group were killed by an overdose of anaesthetic, cut into small pieces with scissors and blended. Homogenates were then dried at 105 °C for 24 h and dry mass was noted. Fat content in dried homogenates was analysed in duplicate by petroleum ether extraction using a Soxhlet apparatus .The nutritional status of snap-frozen liver and muscle samples was analysed using different methods, as follows. To amounts of 100 to 250 mg, 1 mL distilled water was added. Liver samples were homogenated with an UltraThurrax  for 10 s, and the muscle samples were homogenized manually by using a glass potter . For the determination of total lipid content, samples were extracted with chloroform-methanol (2:1) containing 0.01% BHT as an antioxidant, according to the method of Bligh and Dyer , followe2-saturated atmosphere. The bomb is placed in a water bath, which absorbs the heat generated during the combustion; the increase in the temperature of water relates to the heat generated during the combustion. The device is calibrated with a standard compound, benzoic acid . Obtained values (in Joules g−1 dry matter) were converted to values relative to the fresh weight of fish (% g fish−1).The energy content of dried homogenates was analysed using an IKA C 200 bomb calorimeter. By this method, the dried sample is wrapped in combustible paper and placed in a sealed iron bomb, where its explosive combustion is unleashed by an electric flash, under an Oet al. [o-phenylenediamine . Fluorescence emission values were recorded at 450 nm immediately using a plate reader  with excitation at 345 nm. Standard curves were prepared with ascorbate diluted to 14 different concentrations ranging from 0 to 150 μM using the methanol-water mixture containing DPTA.Ascorbate concentrations were analyzed in medium samples and cell extracts according to the method by Vislisel et al. . Tissue −1 o-dianisidin, 4 U mL−1 glucose oxidase and 2.54 purpurgallin units of peroxidase (from horseradish) per mL. Plates were incubated at 37 °C for 30 min, and the reaction was stopped by the addition of 100 μL 12 N sulphuric acid to each well. Optical densities were read at 540 nm .Serum was immediately prepared from blood samples taken with heparinised syringes. Serum samples were stored at −80°C until analyses. Tissue homogenates were centrifuged at 10,000 × g for 10 min, and the supernatant was used for the analysis of glucose and lactate. Glucose was analysed according to the glucose oxidase method. Therefore, samples were mixed with sodium acetate buffer  containing 0.1 mg mL−1).Lactate in serum and tissue samples was determined according to Maughan  using hyet al. [Total protein contents were analysed from a diluted serum sample using the bicinchoninic acid (BCA) assay (Sigma), according to the manufacturer’s protocol. Albumin was determined using the bromocresol green (BCG) method, as described by Doumas et al. , with thet al. [Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from samples of 20 μL of serum or tissue homogenate were determined according to the modified methods described by Casillas et al.  after inet al.  using frData are presented as the mean ± standard error of the mean (SEM), unless indicated otherwise. Coefficients of variance were calculated as shown in Equation (4).p-value of <0.05 was accepted as being statistically significant.The effects of the treatments were determined by the comparison of treatment groups to controls using non-parametrical Mann-Whitney U-tests (SPSS 9.0 for Windows). Relationships between parameters were evaluated using Spearman correlation tests. A The histological alterations in carp livers of DON-treated fish suggest that the fish may face a metabolic crisis caused by tissue damage. The results indicate that oxidative stress leading to lipid peroxidation is involved in the detrimental effects of DON feeding. Taken together, the chemical body composition of the experimental fish in the present study was influenced by the abovementioned factors and gives strong evidence for an influence of DON on the nutritional status of carp. Similar metabolic disorders, including impairment of hepatic metabolism of fats due to oxidative stress, have also been observed in pesticide-treated carp . Neverth


Bats are natural reservoirs for a spectrum of infectious zoonotic diseases including the recently emerged henipaviruses (Hendra and Nipah viruses). Henipaviruses have been observed both naturally and experimentally to cause serious and often fatal disease in many different mammal species, including humans. Interestingly, infection of the flying fox with henipaviruses occurs in the absence of clinical disease. The extreme variation in the disease pattern between humans and bats has led to an investigation into the effects of henipavirus infection on the innate immune response in bat cell lines. We report that henipavirus infection does not result in the induction of interferon expression, and the viruses also inhibit interferon signaling. We also confirm that the interferon production and signaling block in bat cells is not due to differing viral protein expression levels between human and bat hosts. This information, in addition to the known lack of clinical signs in bats following henipavirus infection, suggests that bats control henipavirus infection by an as yet unidentified mechanism, not via the interferon response. This is the first report of henipavirus infection in bat cells specifically investigating aspects of the innate immune system. Bats have been identified as the natural reservoir hosts of many human and animal pathogens of medical importance, including a spectrum of infectious zoonotic agents such as Ebola virus Henipavirus, are highly pathogenic viruses that are harboured in pteropodid bats. HeV has occasionally been observed to transmit to and cause disease in horses and then have the ability to infect humans Hendra virus (HeV) and Nipah virus (NiV), from the genus Tadarida brasiliensis) and the Mvi/It . However, neither cell line is susceptible to henipavirus infection . Therefore there was a need to establish Pteropus alecto cell lines which, coupled with the development of molecular immunological tools, provide the necessary foundation to further investigate infection by henipavirus in its natural reservoir P. alecto cell lines have been established There are two commercially available bat cell lines, Tb1-Lu , specifically for the purpose of understanding the antagonism of the interferon pathways following henipavirus infection in P. alecto cells. We previously reported that interferon production is antagonized in human cells following infection with henipaviruses, while the interferon signaling pathway remains functional Most viruses, including the henipaviruses, express one or more interferon antagonist proteins which suppress host interferon production and/or signaling pathways. In this study we have conducted P. alecto as described in Crameri et alP. alecto lung cells immortalized with SV40T), PaFe , PaFeT  and PaKi . All bat cells were maintained in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 HAM (Sigma), which also contains 15 mM HEPES, NaHCO3, pyridoxine and L-glutamine, with 10% bovine calf serum , 100 U/ml penicillin, 100 µg/mL streptomycin and 1.25 ug/ml of amphotericin B (Invitrogen). All cells were maintained at 37°C with 5% CO2. Human epidermoid carcinoma cells (HEp-2) were maintained in modified Eagle's Minimal Essential Medium (EMEM) supplemented with 10% BCS (Hyclone), 100 U/ml penicillin, 100 µg/mL streptomycin and 1.25 ug/ml of amphotericin B (Invitrogen).Bat cells used for this study were previously generated from The henipavirus stocks used in this study were derived from the following isolates: Hendra virus/Australia/Horse/1994/Hendra (HeV), Nipah virus/Malaysia/Human/1999/PKL (NiV-M) and Nipah virus/Bangladesh/Human/2004/Rajbari R1 (NiV-B). All work with live Hendra and Nipah viruses was carried under BSL-4 conditions at the Australian Animal Health Laboratory (AAHL), Geelong, Australia.P. vampyrus (available in the Ensembl database and the GenBank trace file archive using BLAST on NCBI). BLAST searches and the analysis tools contained within the software package Clone Manager 9 Professional Edition  were used to identify and analyse each gene. Nucleotide and protein sequence alignments were performed with sequences from a selection of available mammalian species and used to generate a mammalian consensus sequence. These consensus sequences were used to design oligonucleotide primers to direct the cloning and sequencing of the P. alecto genes of interest. The real-time primer sequences for ISG54 are as follows: ISG54F CTACGCCTGGGTCTACTATCAC and ISG54R AATTGCCAGTCCGGAGGAG. The primer sequences for GAPDH, IFN-α and IFN-β Oligonucleotide primers for amplification of GAPDH, IFN-α, IFN-β, ISG54 and ISG56 were designed from the sequence data of 6 cells were infected with a multiplicity of infection (MOI) of 10 TCID50/cell with HeV, NiV-M or NiV-B, and at 3 h post infection (pi), the cells were harvested for RNA extraction . For interferon signaling analysis, approximately 0.2×106 cells were infected with HeV, NiV-M or NiV-B (MOI 10). At 24 h pi, the cells were treated with 1000 U of Universal Type I Interferon (PBL). Universal Type I interferon is an IFN-α hybrid constructed from recombinant human IFN-α A and human IFN-α D (Human Interferon α A/D). 3 h post interferon treatment, the cells were harvested, and total RNA isolated. Random primed cDNA was reverse transcribed using Superscript® III Reverse Transcriptase (Invitrogen), and quantitative Real Time PCR was performed using SYBR Green  in an ABI 7900 or 7500. The PCR cycling conditions were as follows: one cycle at 95°C for 20 secs and 40 cycles of 95°C for 3 secs and 60°C for 30 secs. Individual mRNA transcripts were assayed in duplicate, and CT values were used to calculate the relative fold changes in each gene. GAPDH mRNA levels were used to normalize samples.For interferon production analysis, approximately 0.2×10HEp-2 cells were infected at an MOI of 1 for 24 h, and the PaLuT02 cells were infected at an MOI 10 for 24 h. Cell lysates were prepared and analysed as previously described The bat cells  were infected with either HeV, NiV-M or NiV-B at an MOI of 10 for 24 h. Immunofluorescence was undertaken as previously described Virus infection induces many responses in cells, the most rapid of which include the secretion of type I and III interferons. Stimulation of bat cells with poly I∶C results in the induction of type I (IFN-α and IFN-β) and type III interferons (IFN-λ) In addition to type I interferons, the IFN-λ genes (type III interferons), IL-29 and IL-28B were investigated following henipavirus infection . FollowiThe effect of henipavirus infection on the interferon signaling pathway in PaLuT02 cells was investigated by stimulation with exogenous interferon in order to circumvent the effects of viral antagonism on the interferon production pathway. As there are limited bat immunology reagents available, a universal recombinant human interferon alpha hybrid (PBL) was used. Cells from a variety of mammalian species have been demonstrated to respond to Universal Interferon, including human, monkey, mouse, bovine, rat, cat, pig, rabbit, guinea pig and hamster P. alecto cell lines. Cells were infected with HeV in order to determine whether a block in interferon signaling is universal or unique to the PaLuT02 cells. Primary fetus and kidney cells (PaFe and Paki), and cloned, immortalised fetus and lung cells (PaFeT and PaLuT02) were infected with HeV for 24 h at a high MOI and were treated with Universal Interferon (1000 U) for 3 h prior to harvesting for real time assays  cells showed a decreased level of P, V and W expression in P. alecto cell lines compared with human cells . This contrasted with that reported from transfected cells expressing P gene derived proteins in vivo in bats. Following this study, investigation into the effect of henipavirus infection on immune cells, such as dendritic cells, will also be undertaken.In addition to suppression of interferon production, the interferon signaling pathway was also antagonized in bat cell lines following infection as demonstrated by the absence of an ISG response. This block in interferon signaling was observed in multiple cell lines derived from several different organs. Although the block is not 100% following HeV infection, we hypothesize that this is due to a small percentage of uninfected cells, and not leaky activation of the interferon signaling pathway. This hypothesis is reflected in the level of infected cells seen by immunofluorescence on duplicate samples at the point of RNA extraction. The antagonism of the interferon signaling pathway in all cell types suggests that the block in interferon signaling is not cell type specific, and is a universal response that would be expected to occur P. alecto STAT1. We previously determined that henipavirus proteins only antagonise the interferon signaling pathway in human cells as an artefact of overexpression systems, not during in vitro infection studies Although interferon induction in human cell lines is inhibited by henipavirus infection, treatment with exogenous interferon restores the ISG response These results highlight a difference in henipavirus-host interactions between bats  and humans . Transcription and expression of ISGs results in the generation of an antiviral state in cells, preventing infection and replication of virus in neighbouring cells. Blocking both the interferon production and signaling pathways is the optimal mechanism for a virus to counteract the host response. If the production pathway is blocked, no interferon is secreted. However, professional antigen presenting cells such as dendritic cells are programmed to produce large amounts of interferon, which may not be prevented by viral interferon antagonist proteins. Blocking the signaling pathway in this situation is insurance, as the virus can block the effect of interferon. Blocking both pathways is considered to give the virus a strong advantage over the host and would be expected to allow for improved infection in the host where this is possible. The ability of henipavirus to block interferon induction and signaling in bat cells is surprising given the asymptomatic nature of henipavirus infection in bats compared with humans, which have an intact signaling response but generally fatal response to infection. This would suggest that bats control henipavirus infection by an as yet unidentified mechanism, not via the interferon response. Given the importance of the interferon response in controlling viral replication in other mammals, this result is significant, providing evidence for differences in the antiviral response of bats compared to humans.In this study, we have shown that henipaviruses block interferon production in bat cells. We have also demonstrated differences in the ability of the henipaviruses to block the interferon signaling pathway in bat cells compared with human cells and this block is not due to an increased level of viral protein expression. We hypothesize that the interferon response is not responsible for the differences in the susceptibility of bats and humans to henipavirus infection. Identification of the mechanism by which bats control viral infections has the potential to direct research in the development of new, broadly active antiviral strategies. This study has added to the reported investigations into virus-bat interaction and bat immunology in general.


TF, P589S), hemochromatosis (HFE) C282Y and (H63D)], iron biochemical variables  and apolipoprotein E (APOE) gene variants in 139 Alzheimer's disease (AD), 27 Mild Cognitive Impairment (MCI), 78 Parkinson's disease (PD) patients and 139 healthy controls to investigate mechanisms of iron regulation or toxicity. No difference in genetic variant distributions between patients and controls was found in our Italian sample, but the stratification for the APOEε4 allele revealed that among the APOEε4 carriers was higher the frequency of those carriers of at least a mutated TF P589S allele. Decreased Tf in both AD and MCI and increased Cp:Tf ratio in AD vs. controls were detected. A multinomial logistic regression model revealed that increased iron and Cp:Tf ratio and being man instead of woman increased the risk of having PD, that increased values of Cp:Tf ratio corresponded to a 4-fold increase of the relative risk of having MCI, while higher Cp levels were protective for PD and MCI. Our study has some limitations: the small size of the samples, one ethnic group considered, the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest the hypothesis that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk.Deregulation of iron metabolism has been observed in patients with neurodegenerative diseases. We have carried out a molecular analysis investigating the interaction between iron specific gene variants [transferrin ( Specifically, an excessive iron accumulation has been reported in neuritic plaques, neurofibrillary tangles and in specific brain areas of patients with AD  proteins seem to play a key role. These proteins, encoded by the TF and HFE genes . The C1 variant is the most prevalent (95%) and its encoding polymorphic gene has two allelic variants that produce transferrin C2 . Both TF C2 (rs1049296) and the 2 HFE, C282Y (rs1800562) and H63D (rs1799945) gene variants, have been investigated as potential risk factors for neurodegenerative disorders. Studies analyzing the association between HFE mutations and AD came to different conclusions regarding the hypothesis that H63D mutation may anticipate the disease onset in sporadic AD   gene risk factor is reported herein.The brain iron content increases with advancing age % 68, age mean ± standard deviation 63 ± 12 years), 139 AD , 27 MCI  and 78 PD patients . All individuals included in this study are Caucasian. The study was approved by the local IRB and all the subjects involved or legal guardians signed their informed consent.APOE genotyping was performed according to establish methods . Genotyping of SNPs rs1800562, rs1799945, rs1049296 were achieved by the TaqMan allelic discrimination assays from Applied Biosystems Inc. (Foster City CA). The predesigned SNPs genotyping assays ID are C_1085595_10, C_1085600_10, C_7505275_10 (Applied Biosystems). The total reaction volume per well was 20 μL, including 5 ng genomic DNA, 1 μL TaqMan SNP genotyping assay (containing two PCR primers and two dye (VIC or FAM) labeled TaqMan MGB probes) and 10 μL TaqMan Universal PCR Master Mix (Applied Biosystems), according to the manufacturer's manual. PCR was performed at 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. The samples were amplified, read, and analyzed using the ABI Prism 7900HT Sequence Detection Systemand ABI Prism SDS 2.4 software. Two blank controls in each 96-well-plate were used for the assay quality control. *1.25) and multiplying by 100. The ferritin was analyzed by immunoturbidimetry assay enhanced with latex ]. All biochemical measures were automated on a Cobas Mira Plus  and performed in duplicate.Patients' fasting blood samples were collected in the morning and serum was rapidly stored at −80°C. Ceruloplasmin (Cp) was analyzed by immunoturbidimetry assay  Wolf, . Iron wat-test and the chi-square (χ2) test, or a non parametric test when appropriate, were used to compare separately each diagnosis  group vs. healthy controls. The effects of both age and sex were considered in all statistical analyses. Diagnosis groups were compared using ANCOVA models adjusting for genetic variable as well as age and sex. A Multinomial Multiple Logistic Regression model was applied, considering the control group as the reference category, to evaluate the effects of the biological and genetic variables on the probability of being AD or PD or MCI. All tests were interpreted at the 0.05 level of significance. SPSS 16.0 software was used for all statistical analyses.Demographic and clinical characteristics in our patient and control samples were described either in terms of mean ± SD if quantitative, or in terms of proportions. Student's HFE gene and P589S (rs1049296) TF gene mutations in order to test their association with AD, MCI and PD. Gene frequencies were in Hardy-Weinberg equilibrium in each group.We screened participants for C282Y (rs1800562), H63D (rs1799945) HFE gene variants among AD, MCI and PD subjects vs. healthy controls, though a C282Y mutation higher frequency approached significance in the MCI group  = 4.055]; in particular only the estimated parameter for AD diagnosis resulted significative , a significant simple effect of age was evident too . The simple effect of diagnosis was significant on Cp:Tf ratio  but it was due to difference between MCI and controls . Also the simple effect of age  were significant on Cp:Tf ratio.We measured iron, transferrin, ceruloplasmin, and calculated the Cp:Tf ratio and %Tf saturation in the whole study population. Some of these variables were not normally distributed and so we applied a Mann-Whitney non parametric test and these data are presented as median (min-max) in Table HFE on iron concentrations  and, always on iron concentrations, a significant simple effect of sex , age [p = 0.028 F = 4.876]. The analysis reconfirmed the previous results on Cp:Tf ratio while the effect of HFE was not significant (p > 0.20). The effect of age was significant  on the Tf values and also the effect of diagnosis factor . Finally, we observed the only significant effect of sex factor on Cp concentrations . The probability of having AD, PD or MCI was estimated with a multinomial logistic regression model considering the diagnosis as the dependent variable and the biochemical and genetic markers, as well as sex and age, as the independent variables and the control group was set as the reference category. These statistical analyses confirmed age as the main risk factor: the increase of age (1 year) increased of 14% the risk of being AD . Concerning PD, increased of 1 μg/dL of iron , of Cp:Tf ratio  as well as being man instead of woman  resulted in a higher risk of having PD, while the increase of Cp concentration (1 mg/dL) was a protection factor . In MCI, we observed that increasing values of Cp:Tf corresponded to a 4-fold increase of the relative risk of having MCI , and conversely that higher Cp levels resulted as a protection factor .Then, we again performed the ANCOVA analysis taking into account factors like diagnosis, age, sex and genetic variants and their possible interaction on concentration of biochemical variables under study. The analysis revealed a significant influence of the interaction between sex and HFE and TF genes on the risk of having neurodegenerative disorders, in particular AD, PD and MCI even though separately. The results reported are often controversial and this inconsistency suggests that, beside the complex genetic etiology, additional interactions can contribute to the disease, as for example, environmental factors, age, sex, and metal ion involvement or oxidative stress. Considering the current literature, we examined the contribution of HFE and TF mutations in PD, AD and MCI testing if single gene variants have an impact on iron metabolism. Even though our results don't support the hypothesis that these mutations are a genetic risk factor for Italian cohort they have been reported to have a strong effect on the disease risk in several populations  to Fe (III) allows iron binding to Tf and its mobilization from iron tissue storage. More specifically, the result that increased Cp concentrations are a protecting factor for PD is in line with the evidence that CP mutations have been associated with increased deposits of iron in SN  results in an increased absorption of iron in men. A “disease effect” seems essential to explain this relationship, since increased iron levels cannot be ascribed to the HFE change alone, but rather it is caused by the synergy between being man and HFE change on iron levels, sustaining previous results (Fargion et al., A result worthy of interest is the effect of interaction between sex and Our study has some limitations that need to be taken into account: the small size of the sample, and specifically the small number of MCI subjects included; one ethnic group considered; the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk.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.


Objective. To assess the incidence of adverse pregnancy outcome in native and nonnative Dutch women with pregestational type 2 diabetes (T2D) in a multicenter study in The Netherlands. Methods. Maternal characteristics and pregnancy outcome were retrospectively reviewed and the influence of ethnicity on outcome was evaluated using independent t-test, Mann-Whitney U-test, and chi-square test. Results. 272 pregnant women (80 native and 192 non-native Dutch) with pregestational T2D were included. Overall outcome was unfavourable, with a perinatal mortality of 4.8%, major congenital malformations of 6.3%, preeclampsia of 11%, preterm birth of 19%, birth weight >90th percentile of 32%, and a Caesarean section rate of 42%. In nonnative Dutch women, the glycemic control was slightly poorer and the gestational age at booking somewhat later as compared to native Dutch women. However, there were no differences in incidence of preeclampsia/HELLP, preterm birth, perinatal mortality, macrosomia, and congenital malformations between those two groups. Conclusions. A high incidence of adverse pregnancy outcomes was found in women with pregestational T2D, although the outcome was comparable between native and non-native Dutch women. This suggests that easy access to and adequate participation in the local health care systems contribute to these comparable outcomes, offsetting potential disadvantages in the non-native group. Pregestational diabetes mellitus comprises both type 1 and type 2 diabetes mellitus. Pregestational type 1 diabetes mellitus is clearly associated with an increased incidence of adverse maternal, fetal, and neonatal outcome , and sevThe Netherlands can be considered as a representative developed European country with ethnic minorities reflecting economic and social history . Data frA multicentre, retrospective study was performed involving seven large hospitals  spread around the Netherlands. All women with pregestational type 2 diabetes are referred to hospital care in the Netherlands. 272 women in whom a singleton pregnancy progressed beyond 20 weeks of gestation and who delivered between January 1997 and August 2009 were included in an anonymised database and subsequently evaluated. The diagnosis pregestational type 2 diabetes was accepted when patients were anti-GAD antibody negative and/or had never experienced a keto-acidotic episode and the diabetes was being managed with diet alone or oral blood glucose-lowering agents and/or insulin. In the latter case, the diagnosis of type 2 diabetes was accepted when treatment with insulin was initiated >6 months after the initial diagnosis.Patients were divided into native and nonnative Dutch groups, which was representative for the socioeconomic history of the Netherlands. Patients in the latter group were of North-African, Hindu, Afro-Caribbean, Asian, or other nonnative origin.We retrospectively reviewed all charts and recorded maternal characteristics , duration of diabetes, presence of chronic complications, and preconceptional treatment of diabetes. To assess the potential impact of ethnicity on pregnancy outcome, the origin of the pregnant women was classified as (1) native Dutch and (2) nonnative Dutch. The size of the different ethnic groups  was too small for adequate statistical analysis of these specific ethnicities. HbA1c (mmol/mol) values were recorded and median HbA1c was calculated in the periods one year before pregnancy and during the first, second, and third trimesters. 9/L, elevated liver enzymes , and haemolysis characterised by serum lactic dehydrogenase level >600 U/L [Preeclampsia was defined as a diastolic blood pressure ≥90 mmHg on two occasions at least four hours apart in the second half of pregnancy in previously normotensive women and de novo albuminuria (≥300 mg/24 h) . In wome>600 U/L . PretermOnly major congenital malformations, defined as abnormalities that were fatal, required major surgery or resulted in severe organ malfunction or cosmetic defects were recorded and classified as related to the cardiovascular, central nervous, urogenital system, or other systems. Perinatal mortality was defined as fetal loss after 22 weeks of gestation or neonatal loss during the first 28 days after delivery. Large for gestational age (LGA) was defined as a birth weight above the 90th percentile corrected for gestational age, sex, and parity . Q1–Q3) in case of a skewed distribution. Categorical results were expressed as percentages. The appropriate (non)parametric tests  were used to compare differences between groups for continuous data and the chi-square or Fisher's exact test for categorical variables. A P value <0.05 was considered as statistically significant. All statistical analyses were performed using PASW for windows version 18.0 .Continuous parameters were expressed as mean ± SD when normally distributed and as median . Data from 36 women from one of the centres have been published before .P = 0.829). The median gestational age (GA) at first booking was significantly higher in the nonnative group compared to the native group (11.5 (8.0–19.0) versus 8.0 (6.0–10.3) weeks; P < 0.001). The clinical duration of diabetes was generally short. There was a significant difference in the use of medication between the two groups: about half of the native women were on insulin, whereas about three-quarters of the nonnative women were on oral glucose-lowering drugs or diet only at presentation (P < 0.001). The glycemic control was less favourable in nonnative women as compared to native women before and during pregnancy , although in itself quite reasonable.Baseline data of the women are shown in Pregnancy outcome of the total study population and between native and nonnative Dutch women is shown in Perinatal Mortality. 13 perinatal deaths (4.8%) occurred in the total study group, five fetal and eight neonatal deaths (n = 1), premature rupture of the membranes (n = 2), tension pneumothorax (n = 1), or remained unexplained (n = 1). No differences in perinatal mortality reflecting maternal ethnicity were found.l deaths . Four  infants of the total study population; the incidence in native and nonnative Dutch women was similar. Major congenital malformations involved the cardiovascular system (n = 7), the central nervous system (n = 4), urogenital system anomalies (n = 2), or other structures (n = 4). Three of these women were on oral glucose-lowering drugs during the first trimester.Large for Gestational Age. 85 (31.3%) infants were large for gestational age in the total study population, with no differences between native and nonnative Dutch women.In contrast to our hypothesis, we showed no differences in pregnancy outcome between native and nonnative Dutch women with pregestational type 2 diabetes, with the exception of a higher CS rate in native Dutch women. However, a high incidence of adverse pregnancy outcomes in the complete group of women with pregestational type 2 diabetes living in the Netherlands was found.Pregnancy outcome of women with pregestational type 2 diabetes has been studied before and our results are generally in accordance with those reports , 7–9. AcThe only parameter which differed between native and nonnative Dutch women with pregestational type 2 diabetes was the higher incidence of a (primary) CS in the native group. This difference might partly be due to a higher incidence of maternal overweight, which according to US papers is related to the CS incidence , 23. It The proportion native/nonnative Dutch  was not representative for the normal Dutch population . FirstlyNevertheless, the pregnancy outcome of all studied women still seems worse than the outcome of the general population. According to our study, ethnicity appears not to be involved in the development of this adverse outcome. Possibly, other factors play a pathophysiological role in this process and offer opportunities to improve this adverse outcome. Firstly, general awareness of the necessity of pregnancy planning in women with type 2 diabetes may be less than optimal in The Netherlands, since these women are generally seen in long-term community care and only referred to secondary care when already pregnant. Unfortunately, we were unable to report about the preconceptional care of the studied women, due to the retrospective nature of our study. Therefore, a prospective study about the effects of preconceptional care and pregnancy planning on pregnancy outcome in women with type 2 diabetes is needed. Secondly, postprandial spikes of the blood glucose levels contribute to a high-normal or a minimally elevated, but acceptable (42–53 mmol/mol), HbA1c level . NowadayLimitations of our study are the retrospective nature, the limited size of the total study population and of the different ethnic groups, and the possibility of selection bias. Only 272 pregnancies were studied from seven large hospitals covering a representative area in The Netherlands, but possibly not fully representative, since four centres are tertiary care centres and three are larger secondary care centres. This might have led to a selection bias, since patients with a poor obstetrical history are referred to tertiary centres. In conclusion, this study has demonstrated that ethnicity seems not to be a major issue in the cause of pregnancy complications in women with pregestational type 2 diabetes with easy access to medical care. The results are still not approaching the pregnancy outcome of healthy, nondiabetic women. More prospective research is needed to validate these results and to pay more attention to possible causative factors like preconceptional care, avoiding postprandial spikes of the blood glucose and socio-economic status, in the development of adverse pregnancy outcome in women with pregestational type 2 diabetes.