We examine the relationship between birth-to-birth intervals and a variety of mid- and long-term cognitive and socioeconomic outcomes, including high school GPA, cognitive ability, educational attainment, earnings, unemployment status, and receiving government welfare support. Using contemporary Swedish population register data and a within-family sibling comparison design, we find that neither the birth interval preceding the index person nor the birth interval following the index person are associated with any substantively meaningful changes in mid- or long-term outcomes. This is true even for individuals born before or after birth-to-birth intervals of less than 12 months. We conclude that in a contemporary high-income welfare state, there appears to be no relationship between unusually short or long birth intervals and adverse long-term outcomes.The online version of this article (doi:10.1007/s13524-017-0550-x) contains supplementary material, which is available to authorized users. Demographers analyzing fertility have always examined the timing and spacing of subsequent births, given that it is one of the central determinants of fertility. Moreover, demographers have shown that short birth intervals and high sibling density are associated with a large number of negative outcomes in both childhood and adulthood  has recommended that after a live birth, parents should wait at least 24 months before trying to conceive again WHO . This adIn the current study, we examine the relationship between birth intervals and high school GPA, cognitive ability, and educational attainment, earnings, unemployment status, and welfare receipts. Previous research on the long-term consequences of short birth intervals and high sibling density has generally focused on school test scores, cognitive ability, and schooling transitions, but not on earnings, unemployment, or welfare receipts. As a result, our study reevaluates the relationship between birth spacing and educational and cognitive outcomes, and extends previous research by examining long-term socioeconomic outcomes. Given the volume of research on birth order and family size, surprisingly few studies have examined the long-term consequences of birth intervals  and parental health. It is well established that parental SES is related to offspring socioeconomic attainment  how maternal physiological condition affects the prenatal development of the child, and (2) how social and financial conditions within the family after birth affect subsequent development. Physiological explanations for why a short preceding birth-to-birth interval should impact long-term offspring development center on maternal nutrient depletion King , such asMaternal nutrient depletion is also a function of breast-feeding and the time spent breast-feeding. Breast-feeding increases the length of birth intervals by inducing lactational amenorrhea Trussell . Breast-primigravid mothers (mothers for whom it is their first pregnancy) have worse perinatal outcomes than later-borns  high school GPA measured at the end of compulsory school; (2) cognitive ability in early adulthood; and outcomes measured at or around age 30, including (3) years of education, (4) earnings, (5) unemployment, and (6) receiving government welfare support. In the data that we have access to, several of these outcome variables are available for specific birth cohorts only. We therefore use three samples of data: individuals born during 1960–1981, 1965–1977, and 1982–1990. A key administrative register that we use in this study is the Swedish multigenerational register, which allows us to link individuals to their parents and all their biological siblings. This study is based on a population of sibling groups in which neither parent had any children with a third partner. Thus, none of the individuals included in our analysis have any half-siblings. Although this data restriction conditions on union stability, it offers distinct advantages when studying birth intervals: increasing the degree of genetic similarity of the siblings and increasing the likelihood that parental investment is primarily focused on the siblings within the families under study.In our analyses, we compare the results from within-family sibling fixed-effect models and between-family linear regression models. We explain those models in detail in the following section on statistical analyses, but this modeling approach also has implications for our data selection. Because the fixed-effect approach requires variance in the groups in which the comparisons are conducted, we necessarily exclude only-children, but we also exclude sibling groups with two children, given that these sibling groups contain only one birth interval. Furthermore, we exclude sibling groups with twins or other plural births because it is not possible to distinguish the impact of birth intervals from the presence of a twin on long-term outcomes in these families. In studying the effect of birth intervals on each of the six outcomes that we address, we perform separate analyses examining the importance of the length of the preceding birth interval as well as the subsequent birth interval.In the analyses examining the importance of the preceding birth interval, we necessarily exclude firstborn individuals because for those individuals there was no preceding interval. Thus, our analysis population for the analyses on the preceding birth interval is second-born and later-born children in sibling groups with at least three children. In our analyses examining the importance of the subsequent birth interval, we necessarily exclude last-born individuals because they do not have any sibling born after them. Details about how we reach our analytical sample can be seen in Table birth-to-birth interval—that is the time, in months, from one live birth to another. We categorize the length of the birth interval into 16 categories: 6-month periods from a minimum of 6 months to 96 months or longer. Figures The measure for the birth interval that we use in this study is the The data on GPA are taken from grades from the ninth and final year of compulsory education. We study individuals born between 1982 and 1991. Students are typically 15 or 16 years old at the end of the ninth grade. The system for assigning grades in the Swedish high school system has changed several times over the past decades, so we limit our analyses to a period when the grade system stayed constant: 1998–2007. Thus, we studied cohorts born in 1982–1991, who were aged 16 between 1998 and 2007. During this period, students could receive one of four grades for each subject: pass with special distinction, pass with distinction, pass, or fail. To construct an overall score, each grade was assigned a numerical score: pass with special distinction = 20, pass with distinction = 15, pass = 10, and fail = 0  scale. The subtest scores were summed to obtain an overall score and transformed onto a stanine scale with a mean of 5 and a standard deviation of 2.To examine educational attainment, we use data on cohorts born between 1960 and 1981. We examine the number of years of educational attainment achieved by age 30, measured in the year when they turn age 30. This measure is based on the number of years that correspond to the specific level of education achieved by age 30 and may not always reflect the actual number of years that an individual spent in the educational system.We examine income for individuals born between 1960 and 1981. We study logged mean yearly income for ages 29–31. We look at mean income for this specific age range to reduce the impact of short-term volatility in income on our measurement. The values are derived from Swedish administrative tax registers and include all declared income during a year from work, excluding capital gains and government benefits of all kinds. The income is inflation adjusted with the year 2000 as a reference year.försörjningstöd) are means-tested benefits for individuals with no other source of income (or a very low income) and are concentrated among highly disadvantaged individuals.Our measure of whether the individual was unemployed or received social welfare is also derived from administrative tax registers on payment of these benefits. As with the variable for income, we study individuals born between 1960 and 1981 to measure whether a given individual received unemployment benefits or social welfare payments at any time between the ages of 29 and 31. The covariate is in both cases a binary variable indicating whether the index person received any amount of either unemployment benefits or social welfare. To receive unemployment benefits, the individual must have been registered as previously employed. Thus, individuals outside the labor market are not eligible for unemployment benefits. Social welfare benefits  a between-family comparison and (2) a within-family comparison examining the relationship between the preceding birth interval and the outcome variable; and (3) a between-family comparison and (4) a within-family comparison examining the relationship between the subsequent birth interval and the outcome variable, using a different population for the analyses on the preceding and subsequent intervals:andyij is the outcome for individual i in sibling group j on any of the six outcomes that we study. In Model 1, we use a regular linear regression—meaning, a between-family comparison—to examine the relationship between PBIij  and control for birth order, maternal age, birth year, and sibling group size. PBIij is entered into the model as a series of 16 dummy variables based on six-month categories for the length of the preceding birth interval. In Model 1, our analysis population is second-born and later-born children in sibling groups with at least three children; that is, we exclude firstborns because they have no value for the length of the preceding interval.where j and remove the control for sibling group size because that is adjusted for in the fixed-effect approach. We use the same analysis sample for Model 2 as we use in Model 1.In Model 2, we introduce the sibling fixed effect αSBIij, which is a variable for the length of the subsequent interval for individual i in sibling group j. SBIij is entered into the model as a series of 16 dummy variables based on six-month categories for the length of the subsequent birth interval. In Model 3, our analysis population is all siblings in sibling groups with at least three children, with the exception that last-born children are excluded because they do not have a value for a subsequent birth interval.Model 3 is similar to Model 1 in that we use regular linear regression, but we substitute the variable for the preceding interval with j, and remove the control for sibling group size because that is adjusted for in the fixed-effect approach. We use the same analysis sample for Model 4 as we use in Model 3. We regard Models 2 and 4 as an improvement on Models 1 and 3, respectively, given that the sibling comparison approach used in Models 2 and 4 minimizes residual confounding from unobserved factors that are shared by siblings. To this end, we are much better able to isolate the net effect of birth intervals on the multiple long-term outcomes that we study.In Model 4, we again introduce the sibling fixed effect, αTable The results for the relationship between birth intervals and high school GPA are shown in Fig. However, the results from the fixed-effects analyses—the within-family comparison—tell a different story. Here we see few statistically significant differences by the length of the birth interval, and that the point estimates are far smaller, indicating a difference of no more than 1 or 2 GPA points. Indeed, even where the preceding birth interval was extremely short—less than 12 months—we find no statistically or substantively significant difference in the GPA mark achieved when comparing siblings within the same family. This finding suggests that the results in the between-family analyses are largely driven by differences across families in factors related to both birth timing as well as the educational performance of the children. We consider the results from the sibling comparison model to be a much more accurate representation of the relationship between birth spacing and high school GPA at age 16 because that sibling comparison model allows us to minimize residual confounding that may predict both the length of birth intervals as well as long-term outcomes, such as high school GPA.>96 months. In the within-family analysis, however, we again see very little difference in the impact of the subsequent birth interval. Some of the differences are statistically significant, but the point estimates never show a difference in the GPA of greater than 2.5 points no matter how long or short the interval until the birth of the subsequent sibling, which is small in substantive terms.The results from Models 3 and 4  are shown in the right panel of Fig. The results for the relationship between birth intervals and cognitive ability for men are shown in Fig. The results for the effect of the subsequent birth interval are shown in the right panel of Fig. The results from analyses examining educational attainment at age 30 are shown in Fig. The results for the length of the subsequent interval—meaning, the length of time until the birth of the sibling after the index person—are also very similar to the previous analyses. The between-family analyses show that an interval until the birth of the next youngest sibling of only 6–12 months resulted in a disadvantage of approximately 0.5 years less education relative to an interval of 25–30 months. Educational attainment also declined monotonically as the length of the subsequent birth interval increased from 31 months up to 96 months or longer, although the association is weaker than the association with a particularly short birth interval, and the difference is never greater than 0.1 years of education by age 30. As with our previous analyses, the within-family comparison shows that the length of the subsequent birth interval makes little difference for educational attainment, although especially long intervals of 91 months or longer are associated with a small significant decrease in educational attainment. Because these sibling comparison models adjust for unobserved confounding much more effectively, we argue that they more accurately represent the relationship between birth intervals and educational attainment at age 30.The results for the relationship between birth intervals and logged income averaged over ages 29–31 are shown in Fig. The results from the within-family analysis tell a different story. The sibling fixed-effect model shows that a particularly short preceding interval is advantageous and is associated with an increased income of 4.9 % relative to the reference category of 25–30 months. However, given that the rest of the point estimates in the within-family comparison analysis are not statistically significant and hover around zero, we do not think that this is a strong signal of any real advantage associated with a short birth interval.The results for the analyses examining the relationship between subsequent birth intervals and logged mean income over ages 29 to 31 are shown in the right panel of Fig. The results in Fig. The results for the relationship between the subsequent birth interval and the predicted probability of unemployment show that relative to the reference category, both particularly short and particularly long intervals are associated with an increased risk of unemployment. For subsequent birth intervals of 6–12 months, the predicted probability of unemployment, relative to when the interval is 25–30 months, is .02 higher. The association with the probability of unemployment is slightly lower if the interval is 31–36 months and then roughly increases as the length of the interval increases from 37 months. The results from the within-family comparison again show that after adjusting for within-family factors that remain constant, the length of the subsequent birth interval does not matter for the long-term predicted probability of unemployment for the index person.The results for the relationship between birth intervals and the predicted probability of receiving government welfare support between the ages of 29 and 31 are shown in Fig. The results from the analyses examining the relationship between the probability of receiving government welfare support and the subsequent birth interval for the between-family analysis show that a very short interval is associated with a higher probability of receiving welfare (.035) relative to the reference category. Intervals between 31 and 66 months show a marginal decrease. However, the within-family comparison shows that the length of the interval does not seem to have any substantial or significant impact on the probability of receiving government welfare support.For all the preceding analyses, for all outcomes, we also ran additional models restricting the analysis to sibling groups with three children to understand whether the between-family results are driven by the inclusion of very large families. Those robustness checks are fully consistent with the main results presented here and are available on request from the authors. Further additional analyses in which we included individuals from two-child sibling groups in our between-family analyses also produced results very similar to those presented in this article


The impact of ultrashort nanopulse on cellular membrane is of biological significance and thus has been studied intensively. Different from cell study, this ex vivo study aims to investigate the biological effects of nanosecond pulsed electric field (nsPEF) on an independent multimembrane parasite, human hydatid cyst, to observe the unique influence of nanopulse on macromembrane structure, permeabilization, and biochemistry. The 300 ns nsPEF was delivered on an experimental model of single human hydatid cyst ex vivo with eight different parameters. Then pathological changes during 7 days of 48 parasite cysts were followed up after nsPEF. The laminated layer, the germinal layer, the protoscolex, and cyst fluid were evaluated by the morphological, pathological, and biochemical measurements. The parameter screening found that nsPEF can damage hydatid cyst effectively when the field strength is higher than 14 kV/cm. When nsPEF is higher than 29 kV/cm, nsPEF destroy hydatid cyst completely by collapsing the germinal layer, destructing protoscolices, and exhausting the nutrition. Echinococcus granulosus is a zoonotic disease worldwide. It is highly prevalent in stock raising areas such as Middle East and Australia [ Echinococcus granulosus occurrence is high in northwest China, becoming a serious public health and economic problem in Xinjiang, China [ustralia . It is australia  which caustralia . China australia . The recg, China , and an  Echinococcus tapeworms. The most common type, cystic echinococcosis, is caused by E. granulosus. Human are accidentally infected by ingesting parasite eggs from definitive hosts  by oral-fecal route. Parasite eggs travel in human digestive system, hatch in duodenum, grow up as larvae, and pass through intestinal wall into to liver with blood stream. About 75% cases affect liver [Hydatid disease is caused by thect liver . It can Current treatments against hydatid disease include chemotherapy and surgery. It is difficult for the antiparasite medicine to penetrate into the cystic echinococcosis to kill the protoscolices due to multiple layers of cyst . TherefoNowadays the surgery approaches changed gradually from being destructive to constructive. The minimally invasive treatment provides excellent cure rates with minimal morbidity and mortality . PAIR  acellular connective tissue, (2) germinal layers, (3) viable protoscolices, and (4) metabolic cyst fluid, making itself an ideal independent biological system. Whether and how nsPEF affect hydatid cyst viability and cyst permeability are unknown. Our hypothesis is that hydatid cyst is a good ex vivo multimembrane model to demonstrate the visible morphological and biofunctional changes caused by nsPEF compared with tiny cellular model or suppositional simulation. This initial investigation ex vivo traced cyst morphological changes and fluid biochemistry changes after nsPEF treatment. Echinococcus granulosus. Hydatid cyst is of a semipermeable structure that allows the nutrition ingredients to pass from the host to the parasite but it can prevent macromolecules from entering the cyst.In this study, 48 single hydatid cysts were chosen as the experimental model to investigate the biological effect of nsPEF onIn this experimental study, the eight different nsPEF treatment parameters were screened. The 300 ns nsPEF was delivered at frequency 1 Hz with different field strengths and different pulse numbers. The effect of nsPEF on laminated layer, the germinal layer, the protoscolex, and cyst fluid were observed. After nsPEF treatment, the morphology, pathology, and biochemical changes were followed up for 7 days to evaluate the efficacy after nsPEF ablation. This study tried to illuminate the possible mechanism and provide the feasible treatment strategy for the further preclinical studies.The experimental protocol was approved by the Ethical Committee of the First Affiliated Hospital of Xinjiang Medical University (approved project number: 20141217003). The experiments have been conducted according to the principles expressed in the Declaration of Helsinki.The experiment design was illustrated in The parasite cysts were collected from naturally infected human hydatid cysts during an open surgery in sterile operation room at the First Affiliated Hospital of Xinjiang Medical University. The sample collection written informed consent forms and picture release agreement had been signed in advance. The single cysts were separated. The 48 single cysts of 1 cm with thin wall, clear fluid, and transparent capsule were chosen for nsPEF treatment.The hydatid cysts were cultured in the 6-well culture plate. Total 5 mL RPMI1640 medium containing 10% fetal bovine serum was added. The plate was kept in 37°C 5% CO2 incubator. After nsPEF treatment, the cyst morphological changes were checked every day under the converted light microscope.The pulse generator was made in Leibniz Institute for Plasma Science, Germany, based on the same Blumlein design and treatment parameters described previously . The paiThe single hepatic hydatid cyst was removed completely and put into the culture plate and treated immediately by nsPEF ex vivo. The nsPEF generator and electrodes were made by Xinjiang Nanosecond Pulsed Power Institute.The 300 ns nsPEF was delivered 1 Hz with four different nsPEF electric field strengths  and two pulse numbers (50 and 100 pulses), as shown in After the nsPEF treatment, the 48 cysts were observed under the microscope  and the result was recorded by a camera . Images were quantitatively analyzed according to the collapse rate. The collapse rate is .μm slide sections, and stained with hematoxylin and eosin, and pictures of them were taken under light microscopy to evaluate tissue structure and pathological changes.On the 7th day, the specimens were fixed with paraformaldehyde and glutaraldehyde, respectively. The ultrastructural changes were observed by H&E stain and SEM, respectively. Briefly, for ultrastructure observation by scanning electron microscope, the separated protoscolices were fixed in 2.5% glutaraldehyde 4°C for overnight and then put in 2% OsO4 for 24 hours, followed by dehydration in series of ethanol. After coating with gold, pictures of the samples were taken on a scanning electron microscope  at the Electron Microscope Center, Xinjiang Medical University. For H&E stain, the separated hydatid cysts were fixed in 10% formalin and then embedded in paraffin, cut into 5 Before nsPEF treatment and on the 7th day after nsPEF treatment, the cysts were cut open and the cyst fluid was aspirated for the biochemistry analysis. The basic nutrition markers for parasite survival  were tested by automatic biochemical analyzer AU2700, . The instrument was calibrated by the quality-control liquid before testing the hydatid cyst fluid. The biochemical results in nsPEF-treated group (under the previously proved effective parameter as 29 kV/cm and 100 pulses) were compared with the control group (cysts without any nsPEF treatment but maintained for the same time period). The self-parallel blank control was also set up to deduct the effect of culture time and background noise.P < 0.05 by Student's t test.All data are present mean ± SEM from 3 independent experiments. Data were analyzed with SPSS 17.0 software. The comparison was considered statistically different when The cyst wall has multiple layers and provides the physical defense for parasites. A single hydatid cyst was observed under light microscope. The outermost layer is a thin connective tissue of host origin, pericyst; the middle layer, laminated membrane; and the inner layer, germinal layer were hard to be differentiated, but the hydatid cyst contents such as cyst fluid, protoscolices, and daughter cysts were visible (P < 0.05), indicating the dose effect of nsPEF. The nsPEF at 29 kV/cm and 100 pulses was the most effective parameter which was then used for the further experiment.To quantitatively describe the result in Normal hydatid cysts are fluid-filled and with a uniformly thin and smooth wall to round shape. To better clarify the nsPEF caused bioeffects, we derived human hydatid cysts, kept ex vivo, and analyzed the morphological changes taking place in response to the exposure to 8 different nsPEF treatment parameters. We identified 48 living hydatid cysts freshly removed from hydatid patients, each with a single parasite cyst of 1 cm in diameter. During 7-day follow-up period, all nsPEF-treated cysts were histologically evaluated. The cyst wall morphological changes were visualized by microscopy and evaluated by image analysis. nsPEF induced morphological changes indicating damage of multiple membranes. These findings indicate that nsPEF exerts time-dependent changes in the cyst wall, which may contribute to parasite dysfunction and death.H&E stain confirmed that nsPEF treatment disrupted the continuity of cyst wall which was made of laminated layer and germinal layer . As showSEM confirmed that nsPEF treatment destroyed protoscolices. As shown in All together 48 single different hydatid cysts were included in the morphological study, SEM is too time- and labor-consuming to get statistical analysis. Only the protoscolex with the most intensive nsPEF treatment (#8 parameter in P < 0.05) while, after nsPEF treatment, the pH drop became even more sharply (P < 0.001). Notably, for each nutrition component, the serum equivalent from human host was also tested. The protein in the hydatid cyst fluid was lower than 1% of that in the human host serum (parasite 0.5 g/L versus host serum protein 48 g/L), glucose 20% (parasite 1-2 mmol/l versus host serum 6.3 mmol/L). The pH values were similar (parasite 7.35 vesrus host 7.45), indicating that, before nsPEF treatment, parasite has set up its own relatively independent metabolism environment inside the cyst to keep the long-term symbiotic relationship with human host. After nsPEF, the nutrition production cycle and metabolism balance in the inner environment were broken.Because cyst fluid makes up the inner environment for parasite and exchanges nutrition with the host, an understanding of how the essential components of cyst fluid change after nsPEF treatment will help to predict the parasite survival. Three basic biochemical, markers, protein, glucose, pH, are the basic nutritional elements. They can reflect the parasite metabolism. After nsPEF treatment they were significantly lower than those in control group . To deduA detailed understanding of dose effect of nsPEF on hydatid cyst facilitates development of treatment strategies for hydatid disease. The hydatid cyst is also a good close system to study the biological effect of nsPEF. So this study is of medicine and mechanism importance.In general, when patients have the higher parasite load, the more severe hydatid disease symptoms will occur in human. The host defense fights to reduce the parasite load to lower levels. Human patients generally fail to eliminate the protoscolices completely. The germinal layer makes the parasite production continue. In this study nsPEF targets two of the parasite defenses by (1) destructing the hydatid cyst wall so that the parasite's protective fence is broken and (2) destroying the protoscolex so that the germinal sources are damaged.In accordance with germinal activity, World Health Organization classified the cystic echinococcosis as CE1–CE5 with their specific ultrasound image characters : CE1, a The benzimidazoles such as mebendazole and albendazole have been developed as antihydatid disease drugs recommended by WHO . Some nePreviously high-energy ultrasound focused knife, radio frequency ablation, and cryoablation techniques have been tried in hepatic hydatid cysts –16. ThesNanosecond pulse ablation is a novel method of ablation technique that is independent of heat accumulation . It applEchinococcus granulosus eggs were spread into environment from infected dog feces. The eggs can stay alive in soil, water, and vegetable and survive snow and freezing conditions. The eggs can survive at least a year in the environment as they are highly resistant to environmental stress. Egg survival time is increased in damp conditions . In this experiment parasite cyst was kept in an ex vivo cell culture system with suitable temperature liquid and nutrition which ensure the viability.Our hypothesis is nsPEF can disrupt the hydatid cyst wall and change the multiple cyst layers, leading to the killing effect on protoscolices in the germinal layer and facilitate the membrane permeability for drug penetrating into the cyst. in vivo research [First, this study set up an ex vivo model for nsPEF biological effect investigation. Due to the complexity of the hydatid cyst, it is hard to set up theoretical explanation by mathematical models without the knowledge of the pathological structure. The hydatid cyst model elucidates its efficacy on ablating hydatid cysts with quantitative experimental parameters. Another independentresearch  confirmeSecond this study tried different treatment parameters. The pulse duration was fixed as 300 ns; the frequency was fixed as 1 Hz. The field strengths were changed  as well as the pulse number (50 and 100 pulses). The data showed a dose effect. The higher electric field and more pulse numbers lead to more significant killing parasite effect. 29 kV/cm and 100 pulses were the effective treatment. The nsPEF showed a killing effect on the protoscolices when the field strength was higher than 14 kV/cm. The killing rate increased in accordance with the increase of the voltage. The morphological change of the protoscolices was significant. When the electric field intensity increased up to 29 kV/cm, the protoscolices were totally destroyed. The stratum corneum lamellar structure of the parasite cyst was destroyed. The stratum and outer layer were broken accordingly. Several typical characteristics of nsPEF, such as voltage amplitude and the number of pulses, will affect the effect of ablation therapy. This ex vivo study provides the preliminary dose reference for further clinical study.Third, this study proved that nsPEF has the following advantages: nsPEF treatment did not burn the hydatid cyst; the collagen fibers and other connective tissue components in the hydatid cyst were not denatured. in vivo are needed to determine its contribution in parasite eradication.The biological effects of nanosecond pulse electric field have been intensively investigated , 27–30.


Obsessive-compulsive disorder (OCD) is among the most debilitating psychiatric disorders. Comorbid autism spectrum disorder (ASD) or autistic traits may impair treatment response in OCD. To identify possible neurostructural deficits underlying autistic traits, we performed white matter tractography on diffusion tensor images (DTI) and assessed autistic trait severity using the Autism-Spectrum Quotient (AQ) in 33 OCD patients. Correlations between AQ and the DTI parameters, fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were examined in major white matter tracts that were suggested to be altered in previous OCD studies. We found a negative correlation between AQ and FA and positive correlations between AQ and MD, AD and RD in the left uncinate fasciculus using age, Beck Depression Inventory, Yale-Brown Obsessive-Compulsive Scale, intelligence quotient and medication as covariates. However, we could not detect the significant results between AQ and all DTI parameters when adding gender as a covariate. In addition, in the ASD comorbid group, FA in the left uncinate fasciculus was significantly lower than in the non-ASD comorbid group and MD and RD were significantly higher than in the non-ASD group. These results did not survive correction for multiple comparisons. In ASD, the socio-emotional dysfunction is suggested to be related to the alteration of white matter microstructure in uncinate fasciculus. Our results suggest that variations in white matter features of the left uncinate fasciculus might be partly explained by autistic traits encountered in OCD patients. Obsessive-compulsive disorder (OCD) is the fourth most common psychiatric disorder , with anAccording to the National Institute for Health and Care Excellence (NICE) treatment guideline, adults with OCD should be offered cognitive behavior therapy (CBT), selective serotonin reuptake inhibitor (SSRI) therapy, or combined SSRI/CBT treatment. However, OCD appears to have a low remission rate and a high probability of recurrence. In a 3-year perspective follow-up of SSRI-treated OCD patients, only 65% achieved partial remission and the relapse probability was 60% . BejerotASD is characterized by persistent deficits in social communication and interaction across multiple contexts, as well as restricted, repetitive patterns of behavior, interests, and activities that usually emerge during early development . EstimatThese high rates of psychiatric comorbidity in ASD may be due to shared etiology, the social disadvantages of autism, or overlapping diagnostic criteria . Jacob ePrevious findings of neuroimaging studies have been reported in both OCD and ASD. In OCD, functional and structural neuroimaging studies suggest that there are abnormalities in the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), thalamus, and striatum, and the cortico–striato–thalamo–cortical (CSTC) circuit's dysfunction is central . MenziesIn addition to functional neuroimaging studies, diffusion tensor imaging (DTI) studies have suggested white matter (WM) alterations in OCD. DTI measures WM microstructure by quantification of the directionality and coherence of water diffusion. Tissues with highly regular fiber orientation have high fractional anisotropy (FA), while those with less regular fibers have low FA . FA is tIn the volumetric studies to evaluate macrostructure in WM tracts, increased WM-volume was also reported in arcuate and uncinate fasciculus (UF) . On the These relationships between OCD and ASD were also examined in several neuroimaging studies. Ecker et al.  reportedThirty-three patients  from the Chiba University Psychiatric Imaging Database were enrolled as subjects. All were outpatients of Chiba University Hospital. Diagnosis of OCD was confirmed by trained psychiatrists according to the psychosis subsections of the Structured Clinical Interview for DSM-IV Axis I Disorders, Research Version, Patient Edition (SCID-I/P) . We asse2; number of excitations: 1; bandwidth: 31.25 kHz; inversion time: 420 ms; acceleration factor: 2). DTI data were acquired using a single-shot echo-planar sequence with the following parameters: TE = 61.1 ms, TR = 8,500 ms, matrix size = 128 × 128, imaging resolution = 1.875 × 1.875 × 2 mm3, band width = 250 kHz, number of motion-probing gradient (MPG) directions = 30, b-value = 1,000 s/mm2, number of acquisitions = 2, acceleration factor = 2.All subjects underwent DTI and T1-weighted MRI using a 3T scanner equipped with a 32-channel phased-array head coil . Images were collected by 3D fast spoiled gradient-echo (FSPGR) sequence  (www.fmrib.ox.ac.uk/fsl/), and a priori probabilities of WM pathways were computed and mapped the selected initial control points from the template space to individual diffusion space. Moreover, fitting of the ball-and-stick model of diffusion (DTI and T1-weighted images of each subject were processed in FreeSurfer 5.3.0 (ard.edu)  using thard.edu)  with a pard.edu) . In imagard.edu) . Subsequard.edu) . In addiiffusion  was perfiffusion  locationiffusion .U-tests or Fisher's exact test. Correlations between AQ scores and DTI parameters  were investigated in target WM tracts . Head motions during DTI scans were evaluated by two-sample t-test after test of normality in average translation and average rotation, percent of bad slices, and drop out scores. The partial correlation coefficient was obtained using age, BDI, Y-BOCS, FIQ, medication, and gender as control variables. The differences of WM features were investigated between OCD patients divided into two groups according to ASD diagnosis using Mann-Whitney U-tests. The significance level was set at p < 0.05 (two-sided). Y-BOCS total score were used as a nuisance covariate in order to adjust for the effect of OCD severity in WM features.All statistical calculations were performed using SPSS software . The difference of clinical and demographic data between ASD and Non-ASD group were investigated using Mann-Whitney p = 0.057) was shown but the larger head rotation was shown in OCD comorbid with ASD group (p = 0.049). No percent of bad slices were shown in both groups. Drop out scores were one in both groups (Table Average translation and average rotation in head motion in with or without ASD group were 0.880 ± 0.288 mm and 0.006 ± 0.003 degrees and 0.563 ± 0.115 mm and 0.004 ± 0.001) degrees, respectively. No significant difference in head translation  and FA in the left cingulum angular bundle , and significant positive correlations were observed between AQ score and MD, AD, and RD in the left UF , MD in the right cingulum cingulate gyrus , AD in the right cingulum angular bundle . However, correlation analysis did not survive the multiple comparisons (Holms method). We also added gender as a covariate because gender should be adapted to control variables for their effect on WM integrity  was significantly lower, and MD  and RD  were significantly higher than in the non-ASD comorbid group in the left UF. These results could clarify that the ASD traits in OCD might affect WM features in this pathway.The differences of WM features in the left UF were investigated between OCD patients divided into two groups according to ASD diagnosis using Mann-Whitney We observed a negative correlation between AQ scores and FA and positive correlations between AQ scores and MD, AD, and RD in the left UF, FA in left cingulum angular bundle, MD in right cingulum cingulate gyrus, AD in right cingulum angular bundle using age, BDI score, Y-BOCS, medication and FIQ as covariates Figure , and sigVon Der Heide et al.  proposedIn previous studies of ASD, UF is associated with socio-emotional functioning . Normal Our study has some limitations. First, our sample size may not be large enough to detect less robust correlations between autistic traits and DTI parameters. Second, the majority of OCD patients took medications, but medication may introduce additional behavioral or neurostructural heterogeneity, thereby obscuring other DTI-AQ associations . Third, Despite these limitations, this is the first study to examine the correlations between AQ and the DTI parameters, FA, MD, AD, and RD in major white matter tracts in OCD. We found correlations between autistic trait severity and the extent of left UF features in OCD patients. The AQ was negatively related to FA in left UF, and positively related to MD, AD, and RD in left UF. These results suggest that variations in WM features of the left UF might be partly explained by autistic traits encountered in OCD patients.MK, YH, and AN wrote the manuscript. MK and YH conducted the experiments and analyzed the data. AN recruited subjects and conducted assessments. KM acquired the imaging data. KA, FO, SN, YM, and MI arranged the conditions for the experiment, and ES supervised this study. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus anthracis—a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cell. However, growth of ΔapeX mutants in iron-depleted medium was not affected, and virulence in mice was not attenuated. Instead, petrobactin components were determined to be exported through a different protein, which enables iron transport sufficient for growth, albeit with a slightly lower affinity for iron. This is the first report to identify a functional siderophore exporter in B. anthracis and the in vivo functionality of siderophore components. Moreover, this is the first application of LAESI-MS to sample a virulence factor/metabolite directly from bacterial culture media and cell pellets of a human pathogen. Bacillus anthracis requires iron for growth and employs the siderophore petrobactin to scavenge this trace metal during infections. While we understand much about petrobactin biosynthesis and ferric petrobactin import, how apo-petrobactin (iron free) is exported remains unknown. This study used a combination of bioinformatics, genetics, and mass spectrometry to identify the petrobactin exporter. After screening 17 mutants with mutations of candidate exporter genes, we identified the apo-petrobactin exporter (termed ApeX) as a member of the resistance-nodulation-cell division (RND) family of transporters. In the absence of ApeX, petrobactin accumulates inside the cell while continuing to export petrobactin components that are capable of transporting iron. Thus, the loss of ApeX does not affect the ability of B. anthracis to cause disease in mice. This has implications for treatment strategies designed to target and control pathogenicity of B. anthracis in humans. Since inthesis” ), and pend FatE) , 14.In general, and relative to siderophore import systems, export systems of siderophores remain underdescribed. Currently, the mechanism of iron-free petrobactin export has not been identified. In described systems from other pathogens, siderophore export across the cell membrane can occur through dedicated proteins from transporter families, including the ABC, multifacilitator superfamily (MFS), and resistance-nodulation-cell division (RND) family .Mycobacterium smegmatis exochelin exporter ExiT, the Salmonella enterica salmochelin exporter IroC, and the Pseudomonas aeruginosa pyoverdine exporter PvdE  Thus, we developed a bioinformatics-based protocol to identify petrobactin exporter candidates. From this list, markerless deletion mutants were made and screened for their ability to grow in iron-limiting medium and to export petrobactin. To screen our mutants, we adapted laser ablation electrospray ionization mass spectrometry (LAESI-MS) for detection of bacterial metabolites, specifically petrobactin. Previous studies have used complex sample extraction techniques coupled with analytical methods, including thin-layer chromatography or mass spectroscopy, to screen for the presence or absence of petrobactin in a sample  analysis. The majority of LAESI applications to date have focused on mass spectrometry imaging of plant and animal tissues, although recent studies have used LAESI to sample and identify bacterial metabolites from roadkill and soybean microbiomes (–LAESI is an ambient ionization technique for mass spectrometry that uses an infrared laser and relies on the water present in the sample as a makeshift matrix to promote ion formation . The enerobiomes –36. Thusliquid chromatography–high-resolution electrospray ionization mass spectrometry (LC-HRESIMS). Through our novel application of LAESI-MS we identified the apo-petrobactin exporter (ApeX), a member of the RND-type transporter family, and observed the export of petrobactin components in its absence. We also show that these siderophore components can bind and transport sufficient iron for growth and are therefore relevant to disease in a murine model of inhalational anthrax.Using LAESI-MS for high-throughput, high-accuracy analysis for petrobactin detection, we directly demonstrate petrobactin in the cells and supernatant of predicted exporter mutants. These results were confirmed using B. anthracis Sterne growth under iron-limiting conditions exist, including growth in mammalian blood, within macrophages, and in iron-depleted medium (IDM) (–B. anthracis genome was queried for homologues of known siderophore exporters (multidrug and toxic compound extrusion (MATE) family transporters  –39. Nextxporters , 21, 25 xporters . The retsporters . The MATter spp. . We focuB. anthracis Sterne mutant unable to export petrobactin would exhibit both reduced growth under iron-limiting conditions and reduced levels of petrobactin in culture medium. Accordingly, each of the mutants was screened for the ability to grow in IDM and for the presence of the petrobactin catechol component 3,4-dihydroxybenzoate . During growth of wild-type (WT) B. anthracis, 3,4-DHB can be measured in the culture medium IDM beginning at 3 h postinoculation (p.i.) using the Arnow’s assay, which detects catechols  for the detection of petrobactin in our sample supernatants (+) peak at m/z 719.21. We further improved the LAESI peak shape to provide increased resolution and allowed more laser pulses to be averaged per MS scan by increasing the maximum injection time to 100 ms, which synchronized the LAESI pulse and the MS scan rate. By keeping the number of pulses per well at 20 with a frequency of 10 Hz, we obtained at least 4 data points per well. Each mutant is therefore represented by two time points from three independent growth curves, with technical replicates performed in triplicate.To address whether our mutants were exporting intact petrobactin and/or petrobactin components , culturrnatants , 14. Thirnatants . During 600]) and presented as a percentage of the wild type. We began by screening a selection of eight mutants, primarily consisting of double and triple deletions (asb negative-control mutant (no petrobactin biosynthesis) and the Δ1302 Δ2407 double mutant (RND-type transporters) (1642 Δ3296 mutant (MATE-type transporters) in The processed data were normalized to the sample cell density  . As an eporters) , we incl600) and presented as percentage of wild-type petrobactin levels. As with the culture medium, Δ1642 Δ3296 cell pellet petrobactin levels were similar to wild-type levels, and the Δasb mutant contained no petrobactin in the cell pellet. However, Δ1302 Δ2407 cell pellets accumulated petrobactin to levels greater than 400% of the wild-type level  to determine which transporter(s) was responsible for petrobactin export. As with the double mutant, culture medium and cell pellets were analyzed by LAESI-MS for the presence of intact petrobactin molecules. Δ1302 culture medium and cell pellets contained wild-type-like levels of petrobactin  rescued export of petrobactin into the culture medium  vector control matched previous data  and redous data . Collectasb, Δ2407(pAH001), and Δ2407 p2407 mutants by LC-HRESIMS. Five-hour 25-ml IDM cultures were dried down, and the pellets were resuspended in methanol and analyzed. These data matched the LAESI-MS data, with petrobactin being detected in wild-type and Δ2407 p2407 culture media but absent from Δ2407(pAH001) and Δasb media  The Δ2407, wild-type, and Δasb strains were grown in IDM for 4 h and stained for cell permeability. Data are presented as a ratio of live to dead cells and are the average and standard deviation from three independent experiments measured in triplicate. Statistical significance was determined by two-way ANOVA with a Tukey’s multiple comparison posttest. ****, P ≤ 0.0001; ns, not significant. (B) The Δ2407, wild-type, and Δasb strains in IDM were supplemented with either 0, 50, 100, or 150 µM iron chelator 2,2-dipyridyl. Their growth was measured by OD600 every hour for 5 h. Data are representative of 3 independent experiments with triplicate measurements. Download FIG S1, TIF file, 0.3 MB.The ΔCopyright © 2017 Hagan et al.2017Hagan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2407 mutant growth in IDM. To test this, we both supplemented growth of the Δasb petrobactin biosynthesis mutant in IDM with Δ2407 spent medium and tested for the ability of the Δ2407 mutant to import iron using gallium supplementation. Thus, the Δasb mutant was supplemented with spent culture medium from the Δ2407, wild-type, or Δasb strain  resembles iron in both charge and size and can be imported by siderophores for incorporation as an enzymatic cofactor. However, while iron improves growth under iron-limiting conditions, Ga3+ inhibits bacterial growth since its redox potential is more limited than that of iron  petrobactin-based components can assist in iron transport.Petrobactin is composed of a central citrate moiety bearing two spermidine arms, each capped by a 3,4-DHB moiety . To idens tested . This piasbA, ΔasbB, ΔasbC, ΔasbD, ΔasbE, and ΔasbF) had impaired growth in IDM  hadfunction . See Tab10.1128/mBio.01238-17.2FIG S2 asb mutant. (A) Individual ΔasbA, ΔasbB, ΔasbC, ΔasbD, ΔasbE, and ΔasbF petrobactin biosynthesis mutants, the wild type, and the Δasb mutant were grown in IDM. (B) The strains were assayed for the export of catechols at 5 h. (C) Culture medium from each of the mutants in panel A was filter sterilized at 5 h postinoculation and used to supplement the growth of the Δasb strain in IDM. (D) The difference in growth at 5 h was calculated as the percentage of increased growth relative to the Δasb mutant in IDM alone. Growth curves (A and C) are representative of three independent experiments, and data (B and D) are compiled from three independent experiments. Error bars indicate standard deviations, and statistical significance was determined by two-way ANOVA with a Dunnett’s multiple comparison posttest. ****, P ≤ 0.0001; ns, not significant. Download FIG S2, TIF file, 0.7 MB.Petrobactin components from truncated biosynthesis are exported and supplement growth of the ΔCopyright © 2017 Hagan et al.2017Hagan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the asb mutant in IDM. The B. anthracis Δasb strain was grown in 15 ml of fresh IDM supplemented with either 10 ml of spent medium from each asbA, -B, -C, -D, -E, or -F mutant, the Δasb mutant, or the wild type , we filter sterilized culture medium from each of the mutants at 5 h postinoculation to supplement the growth of the Δild type . To quanild type . Supplemasb and Δ2407 mutants were filter sterilized at 5 h postinoculation and frozen at −80°C. The supernatants were lyophilized to dryness, resuspended in methanol, and assayed by LC-HRESIMS. Data obtained from LC-HRESIMS were queried for the presence of petrobactin and the projected petrobactin components listed in asb control (data not shown). The identities and sources of the petrobactin components exported by the Δ2407 mutant remain to be confirmed.To characterize the exported petrobactin components, spent IDM culture media from the wild type or ΔB. anthracis Sterne, we took advantage of the receptor for iron-petrobactin, FpuA. The B. anthracis ΔfpuA mutant strain biosynthesizes and secretes petrobactin but grows poorly in IDM due to an inability to take up iron-bound petrobactin , petrobactin accumulates in the cell, while corresponding components are exported through an unknown mechanism -like transporter, which has 12 transmembrane domains and is ubiquitous across cell types. RND-type transporters frequently function as trimers and interact with other proteins to form export complexes that transport a variety of ligands. Our initial list of candidate genes included those encoding two RND-like transporters, GBAA_2407 and GBAA_1302. Both were identified in a PSI-BLAST search of the B. anthracis genome with the M. tuberculosis mycobactin exporter Mmpl4. In M. tuberculosis, Mmpl4 coordinates with a periplasmic accessory protein, Mmps4, to export and recycle mycobactin in a process that also involves a last step in mycobactin biosynthesis (Marinobacter genus (data not shown), members of whom also produce petrobactin, also revealed sequence similarity to transmembrane helices 2 to 7 . (C) Overlays of Phyre2 protein predictions indicate some structural conservation between Mmpl4 (purple) and ApeX (gray) transmembrane domains but none in the extracellular (pore) domains, and ApeX is not predicted to have a docking domain. Download FIG S4, TIF file, 1.5 MB.Predicted structure of ApeX. (A and B) Phyre2 predicted structure of ApeX. Homology between Mmpl4 and ApeX is strongest in two areas: (i) transmembrane helices 2 through 7 (TM1 to -7), extracellular beta sheets 3 to 6, and extracellular helices 4 through 7 (pink and dark pink) and (ii) transmembrane helices 8 through 12, extracellular beta sheets 7 and 8, and extracellular helices 9 and 10 (green). Blue indicates conserved residues, and dark pink indicates regions homologous in a PSI-BLAST search of ApeX against the Copyright © 2017 Hagan et al.2017Hagan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the apeX, and neither did a PSI-BLAST search of Mmps4 against the B. anthracis genome reveal homologous proteins to Mmps4. While there is a small 60-residue hypothetical protein downstream of ApeX (GBAA_2408), it has an unknown function and is smaller than the 140-residue Mmps4. Given that petrobactin can be fully synthesized in vitro by supplying purified enzymes AsbA to -E with 3,4-DHB, citrate, and spermidine, we hypothesize that an accessory protein is unlikely. However, whether petrobactin biosynthesis occurs in the cell cytoplasm by transient interactions or if the biosynthetic enzymes form a discrete complex remains unknown. It is possible that an accessory protein facilitates biosynthesis by anchoring a complex to the cytoplasmic side of the cell membrane next to the exporter.It remains unclear whether ApeX requires additional accessory proteins to facilitate petrobactin export. However, we did not identify a candidate within an operon with B. anthracis to retain the ability to retrieve sufficient iron for growth. This phenomenon has been described in E. coli siderophore systems  to rescue growth of the Δasb nutant was unexpected  or by reduction (step 3b)  . The citstep 3b) . If 3,4-f growth  (our expE. coli (ExPEC) increased component export but reduced virulence  and generated by allelic exchange, as described by Janes and Stibitz (B. anthracis spores at 37°C for 72 h (600) of 0.05 and grown at 37°C with hourly measurements for 5 to 6 h. Strains containing the complementation plasmid were grown in the presence of 10 μg/ml chloramphenicol. Media and chemicals were purchased from Fisher Scientific or Sigma-Aldrich.The strains used are described in  Stibitz . Complem Stibitz . All necfor 72 h . Spores for 72 h . Twenty-10.1128/mBio.01238-17.5TABLE S1 B. anthracis Sterne 34F2 used in this work. Download TABLE S1, DOCX file, 0.01 MB.Strains of Copyright © 2017 Hagan et al.2017Hagan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01238-17.6TABLE S2 TABLE S2, DOCX file, 0.01 MB.Primers used to generate mutant strains used in this work. Download Copyright © 2017 Hagan et al.2017Hagan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 600.The Arnow’s catechol assay was used to measure catechol rings in IDM . BeginniB. anthracis Sterne cultures grown in IDM for 4 h. The cells were pelleted for 1 min at 13,000 rpm, washed once, and resuspended in an equal volume of sterile phosphate-buffered saline. One-hundred-microliter aliquots were distributed into three wells of a 96-well black, clear-bottom microtiter plate. An equal volume of 2× Cyto9-propidium iodide was added to each well, and then the mixture was incubated in the dark for 15 min. Florescence was measured at 530 nm and 630 nm  in a SpectraMax M2 spectrophotometer. Data are presented as a LIVE/DEAD ratio of 530/630.Cell viability was measured using the LIVE/DEAD BacLight kit (Invitrogen). Briefly, 300 µl aliquots were taken from 4 in 0.1% nitric acid at both 0 and 2 h postincubation, and the second was left untreated. They were grown at 37°C for 4 h. Data are presented as percentage of growth of the untreated control at 4 h: (OD600 of treated at 4 h/OD600 of untreated at 4 h) × 100.In IDM, each strain was used to inoculate two flasks. One was supplemented with 20 µM GaSO600 of 0.01. Spent culture media were obtained from IDM cultures grown as indicated above but which had their culture media collected at 5 h, filter sterilized with a 0.22-µm-pore filter, and stored at −80°C. The culture medium-supplemented cultures were grown at 37°C for 5 h. Data are presented as the percentage of increased growth relative to the fresh IDM-only control at 5 h: [(OD600 of treated at 5 h − OD600 of untreated at 5 h)/OD600 of untreated at 5 h] × 100.For each strain, the appropriate number of flasks containing 15 ml IDM plus 10 ml filter-sterilized spent IDM were inoculated at identical starting optical densities at no less than an OD600 of 0.05 (WPA biowave CO9000 cell density meter) and supplemented with the indicated concentrations of either 2,2′-dipyridyl, citrate, spermidine, or 3,4-dihydroxybenzoate. Stock solutions were suspended in water and filter sterilized. Cultures were grown at 37°C with shaking for 5 h. Data are presented as the percentage of increased growth relative to the IDM-only control at 5 h: [(OD600 of treated at 5 h − OD600 of untreated at 5 h)/OD600 of untreated at 5 h] × 100.Three milliliters of IDM was inoculated with the appropriate strain for a starting ODm/z ratios (Thermo Fisher Scientific).For analysis, culture media and pellets were collected from cultures grown in IDM for 4 and 5 h. Culture media were separated from the cells by centrifugation and then frozen at −80°C until analysis. Pellets were washed in an equal volume of fresh IDM and frozen at −80°C. After thawing at room temperature, the pellets were resuspended in 30 µl of water for analysis. Twenty microliters of each sample was plated in triplicate wells of shallow 96-well plates and subjected to laser-based ablation. Here, the sample is heated by irradiation from a 2,940-nm infrared laser, leading to vaporization of molecules from the surface. The process initiates a dynamic equilibrium between energy deposition and consumption of the vaporization process. Any shift toward larger deposition of energy (such as reentry of the vaporized droplets) results in expulsion of a second plume, which is primarily responsible for ablation efficiency. During method development, the data obtained from the first well positions of a 96-well plate varied greatly compared to other columns. A possible cause for this phenomenon is air currents created by movement of the LAESI stage, which might disrupt the ion sampling. To offset this observed stage effect, we left the initial wells in each column either blank or filled with water. The ESI mass spectrograph was obtained using a Thermo Fisher LTQ XL mass spectrometer, containing an atmospheric pressure ionization stack with a tube lens and skimmer, three multipoles, a single linear trap configuration, and a set of 2 electron multipliers with conversion dynodes. The mass spectrometer was connected to a Protea LAESI DP-1000 instrument with an ESI electrospray emitter for ambient ionization. The collected data points were exported to Gubbs Mass Spec Utilities  and procg for 1 min, and the supernatant was used for analysis. Reverse-phase HPLC was performed using Luna 5-µm C18(2) 100-Å, 100- by 2-mm HPLC column and a solvent gradient from 30% water (plus 0.1% formic acid) to 90% methanol (plus 0.1% formic acid) over 16 min.For final confirmation of our findings from LAESI-MS, we acquired HRESIMS spectra using an Agilent 6520 quadrupole time of flight (qTOF) mass spectrometer equipped with an Agilent 1290 HPLC system at the Life Sciences Institute core facility. For this analysis, cultures were grown in IDM for 5 to 6 h, and 20 ml of culture medium was collected by filter sterilization through a 0.22-µm-pore filter and then stored at −80°C. Culture media were dried down (Labconco freeze dry system), and the pellet was resuspended in 1 ml of ACS-grade methanol. Undissolved particulates were removed by centrifugation at 5,000 × 5 spores as described by Heffernan et al.  were infected intratracheally with 1.5 × 10n et al. . Prior t


Birth weight and length have seasonal fluctuations. However, it is uncertain which meteorological element has an effect on birth outcomes and which timing of pregnancy would explain such effect. Therefore, the purpose of this study was to examine temperature effects during pregnancy and which timing of pregnancy has effects on size at birth.n = 3267). Meteorological data such as temperature, precipitation, relative humidity, and daily sunshine hours during pregnancy were observed at the nearest observatory site of Bangladesh Meteorological Department.A large, randomized, controlled trial of food and micronutrient supplementation for pregnant women was conducted in Matlab, Bangladesh (MINIMat Study), where women were enrolled from November 2001 to October 2003. The fetal growth data which included the size at birth and information of their mothers were obtained (P < 0.001). Increased temperature during the last month of pregnancy was significantly related with increased birth length with adjustment for gestational weeks and the season at birth, and remained significant with further adjustments for precipitation, sex of infants, maternal early-pregnancy BMI, parity, and education status of the mother (P < 0.01). On the other hand, increased temperature at mid-gestation was associated with increased birth weight (P < 0.05).Infants born in colder months (November–January) were shorter than those born in hot and dry, and monsoon months (mean (SD) of birth length was 47.5 cm (2.2) vs. 47.8 cm (2.1) vs. 47.9 cm (2.1) respectively; These findings suggest that temperature affects both birth weight and length. The more temperature increased at the last month of pregnancy, birth length became longer. For birth weight, the temperature at mid-pregnancy affected in a positive way.The online version of this article (doi:10.1186/s41182-016-0041-6) contains supplementary material, which is available to authorized users. Climate change has become a public health priority due to a huge amount of evidence on global warming and the impact of human activities . The chaA well-nourished mother is crucial for proper fetus development and adequate birth outcome . The devSeveral studies found the seasonal variations on birth outcome, and some of those have also examined the association between birth size and meteorological factors –18. EffeLBW is a major determinant of mortality and morbidity in neonates, infancy, and childhood. The incidence of LBW in the world is 15%, most of which are born in developing countries . Yasmin Matlab is located about 50 km southeast of Dhaka, the capital city of Bangladesh. The area is a typical rural and riverine delta area of Bangladesh. The most common livelihoods are rice cultivation and fishing, and around 85% of the population is Muslim. The International Center for Diarrhoeal Disease Research, Bangladesh  has been maintaining a health and demographic surveillance system in Matlab since 1966. icddr,b and collaborative partners have been conducting randomized trials aiming to improve maternal nutrition through food and micronutrient supplementation in this area since 2001, the Maternal and Infant Nutrition Intervention Study at Matlab (MINIMat study) .In brief, the MINIMat study consists of three combined intervention trials in a group of undernourished pregnant women of rural Bangladesh. All women who were identified as pregnant in the ongoing demographic surveillance system area of icddr,b were recruited for the study. Pregnant women were identified within 6–8 weeks of conception through monthly home visits, and their pregnancies were confirmed by two times of urine tests and ultrasonography. Pregnant women were enrolled in a 2-year period from November 2001 to October 2003. A total of 5880 women were identified as eligible for the study. Of these women, 1444 were excluded because 504 refused to participate. A further 604 women had to be excluded due to having a fetus whose GA >14 weeks exceeded the limits for the study and 217 no longer having a viable fetus by ultrasound, 112 migrated out from the study area, and 8 excluded for other reasons. Of the 5580 pregnant women, 4436 women were successfully enrolled in the MINIMat study. Among infants of these women, birth weight of 3298 infants was measured. Of these infants, the number of singleton birth was 3267 born from May 2002 to June 2004 and were considered as the current study population. In the present study, the mean person-years of follow-up was 0.71 year or 8.6 months.The information for mothers contained maternal weight, height, age, last menstrual period (LMP) by ultrasonography, formal education years, and parity. Maternal weight and height were measured at enrollment, and other information was collected during home visits. The study data contained the information of infants such as birth weight, birth length, sex, date of birth, and gestational week at birth. Birth weight was measured by using beam scales  that were precise to 10 g. Birth length was measured to the nearest 0.1 cm with locally constructed wooden length board. These measurements were taken within 72 h after birth by study staff. The birth weight and birth length were adjusted by the timing of the measurements after birth .The information of meteorological elements was observed at the nearest observatory station in Chandpur, located about 10 km from the central Matlab, by the Bangladesh Meteorological Department. The meteorological data between January 2001 and December 2004 was obtained.The mean daily temperature, precipitation, and relative humidity were calculated from the data collected every 3 h. Ten time points during pregnancy were selected . The MINIMat study was scheduled for enrollment at 8–10 weeks and three subsequent clinic visits at 14, 19, and 30 weeks of gestation. However, pregnant women came for ultrasound examinations at slightly varied timings. To have maximum participation, the study allowed to examine the women any time before the next examination schedule. Apart from the fixed timings , the effect of temperature was tested to have additional time points of two points in each pregnancy trimester between 8 and 30 weeks, i.e., 8 and 12 weeks in the first trimester, 19 and 24 weeks for the second trimester, and 28 and 30 weeks for the third trimester. Close to births, test points were considered in 2-week intervals up to 6 weeks from the birth point , since 7% of the children were preterm born and other children also vary for birth timing (varied for gestation weeks at birth). The 2-week moving average of each meteorological element was calculated for each point from daily average data. For example, average temperature at 8 weeks of gestational age was calculated by using moving average of temperature at 7 and 8 weeks (up to the end of weeks) of gestation. Moving averages for other points were also calculated in the same manner except the point at birth. As for the temperature at birth, the 2-week moving average before the birth was used. Fetus development is a steady growth process. To observe slight effect of temperature, we need an incubation period to assess the effect of temperature on fetal growth and birth outcome. However, we have also done sensitivity analyses with 1-week moving average, which showed a smaller effect size compared to the 2-week moving average  at early-pregnancy was also examined. Seasonal and monthly differences of birth weight or length were examined by one-way ANOVA. Simple regression analyses for birth weight or length and temperature or precipitation at each gestation point were conducted. Multivariable regression analyses for these associations were conducted in separate models for each gestation points with adjustments for gestational week at birth, season at birth, sex of infant, parity, early-pregnancy BMI, and education level of mothers, and further with precipitation at the same gestation week. Gestation week at birth, parity, early-pregnancy BMI, and precipitation were incorporated as continuous variables as they were distributed normally. Infant sex, season at birth (three seasons), and mother’s education years  were categorical variables in the multivariable models.For examining non-linear association, the study tested by incorporating polynomial terms of the temperature into the models. The study checked the quadratic models of the temperature and birth weight and birth length, and then found that beta coefficients of temperature did not significantly appear as compared to those in linear regression models. Therefore, the study used linear models.2), as nearly one third of the study participants were undernourished (BMI <18.5). P value of <0.05 at two-sided test was considered as statistically significant. IBM SPSS  was used for statistical analyses.Stratified analyses of the mean temperature at each gestation point for birth weight or length were given by maternal early-pregnancy BMI . Of infants, 1663 (51%) were male. The mean (range) birth weight was 2693 (1059–4249) g. About 30.5% of infants were born with low birth weight (LBW); however, 80% of those infants had full-term delivery (born after 37 weeks of gestation). The number of winter newborn was 1564 (48%), one-third of infants were born in the monsoon season, and the rest of the infants were born in the hot and dry season.Table The means of temperature, precipitation, and birth size among seasons are shown in Table P = 0.001). Infants who were born in hot and dry season tended to be heavier; however, it was not statistically different (P > 0.05). A seasonal pattern of birth length was seen. Infants who were born in winter tended to be shorter than infants who were born in the other two seasons  . Birth weight was also correlated with gestation week at birth , parity , education level , and early-pregnancy BMI .Birth weight and birth length were strongly correlated (P < 0.05). After adjustment for gestational week and season at birth, the temperature at 12 to 19 weeks of gestation had significant positive associations with birth weight (P < 0.05). When did further adjustment for sex of infant, early-pregnancy BMI, mother’s education, and parity, the significant association with birth weight was only seen in the temperature at 19 weeks of gestation. After adding the precipitation for adjustment, as temperature (1 °C) increased at 19 weeks of gestation, birth weight was increased up to 4.7 g.Results of liner regression analyses for birth weight are shown in Table 2, the low temperature during the last month of pregnancy was associated with the lower birth weight (P < 0.05) after adjustment for sex of infant, early-pregnancy BMI, mother’s education level, parity, and precipitation. The temperature at 6 weeks before birth was associated with both groups (P < 0.05). When the temperature at 6 weeks before birth was increased, birth weight was increased in infant whose mother had lower BMI, while it was decreased in infant whose mother had higher BMI.Table r = 0.518; P < 0.001), parity , education level , and early-pregnancy BMI . Birth length was also significantly correlated with the mean of overall temperature , and precipitation .Birth length was correlated with gestation week at birth (Table Multicollinearity was examined and variance inflation factor (VIF) for all the models of birth length and weight were less than 2, indicating no serious collinearity impact. Supplementary tables were provided with VIF for models of birth lengths for 6, 4, and 2 weeks before birth and at birth  vs. 47.8 cm (2.2); P < 0.001). By stratified analyses with adjusted for gestation weeks and season at birth, temperature of the last month of gestation was significantly associated with birth length for both groups (P < 0.05). After adjusted for the sex of infant, early-pregnancy BMI, education level, parity, and precipitation on previous model, correlations were almost unchanged (Table P < 0.05). When the temperature increased at 8 weeks of gestation, birth length was decreased.The newborns whose mother’s BMI was less than 18.5 kg/med Table . Birth lIn this cohort study, we have observed associations between birth size and temperature at various timings of pregnancy, which was supported by previous study findings –18. PresExposure in bright sunlight during pregnancy identified as a significant determinant of height , and thiPresent study also found increasing temperature at 6 weeks before birth is associated with reduced birth weight particularly for children of well-nourished women and improved birth weight of children of undernourished women, which has been supported by previous study . That stIncreased temperature at birth was associated with adverse effect on birth weight observed in a Greek study . This asThe placental weight in the second trimester was a strongly significant predictor of birth weight . An animMalnourished mothers are more likely to give birth to LBW, IUGR, and preterm infants, as seasonality and direct heat exposure are also associated with the same adverse birth outcome , 19. TheOne of the limitations of this study was that factors which could affect birth size such as maternal dietary information and maternal vitamin D status during pregnancy were not available. Mothers who participated in the MINIMat study were randomized into six supplementation groups to receive different nutrition supplements during pregnancy. Nevertheless, the effect on birth weight and birth length by supplementation groups was not observed in the MINIMat study ; consequThe present study found the significant effect of temperature during pregnancy on birth size. As temperature increased at the last month of pregnancy, birth length was longer. However, the effect varies by the mother’s nutritional status. The present study suggests that pregnant women, particularly whose BMI is less than 18.5, should avoid colder temperatures in late pregnancy. Several other aspects are associated with fetal growth, but it could be reasonable for pregnant women to avoid cold temperature during mid and late pregnancy.


Bladder cancer is a cause of considerable morbidity worldwide. Electromotive Drug Administration is a method that combines intravesical chemotherapy with local electric field application. Electroporation has been suggested among other mechanisms as having a possible role in the therapy, so the goal of the present study was to investigate the electric fields present in the bladder wall during the treatment to determine which mechanisms might be involved.Electromotive Drug Administration involves applying intravesical mitomycin C with direct current of 20 mA delivered through a catheter electrode for 30 min. For numerical electric field computation we built a 3-D nonhomogeneous patient specific model based on CT images and used finite element method simulations to determine the electric fields in the whole body.2.Results indicate that highest electric field in the bladder wall was 37.7 V/m. The mean electric field magnitude in the bladder wall was 3.03 V/m. The mean magnitude of the current density in the bladder wall was 0.61 A/mThe present study shows that electroporation is not the mechanism of action in EMDA. A more likely explanation of the mechanism of action is iontophoretic forces increasing the mitomycin C concentration in the bladder wall. Bladder cancer is a cause of considerable morbidity and mortality worldwide. The intensive care and treatment of bladder cancer represents a considerable burden to patients and society . Intraveex vivo model that concentrations of MMC may be increased in all the layers of the bladder wall, when the drug was delivered with EMDA. Clinical studies where EMDA/MMC was applied showed lower recurrence rates than MMC alone, and equivalent efficacy for the treatment of high-risk bladder tumors when compared to BCG  is a method that combines intravesical chemotherapy with local electric field application. d to BCG  with fewIt has been proposed that EMDA leads to the combination of several electro-molecular interactions; iontophoresis, electroosmosis/electrophoresis, and electroporation . All theThe objective of this study is to perform calculations of the electric field present in the bladder wall during EMDA treatments, and in that way estimate what biological mechanisms may be at play when the tissue is exposed to this particular electric field.in-silico representation of a human body during the delivery of an EMDA treatment. This was done by reconstructing the anatomical shape of the body and the relevant tissues from CT images and then using a computational approach to determine the relevant electric quantities in the tissues: electric field and current density. These quantities were then analyzed to get an insight into the possible mechanisms of EMDA.The main goal of the following steps was to build an ®) that delivers a controlled electric current of 0–30 mA/0–55 V DC, which is passed between electrodes: (1) the active intravesical electrode is a silver spiral integrated to the tip of a specifically designed transurethral catheter, (2) two dispersive ground electrodes, placed in the lower abdominal skin. The operator sets the active electrode’s polarity and current intensity on the generator.EMDA is administered by a battery powered current generator  at a rise rate of 50 mA/s. The total treatment time is 30 min. This setup is shown in Different tissues have different electrical properties. To be able to accurately compute the electrical quantities in the body we need to differentiate between these tissues and assign them their appropriate properties. Abdominal CT images of a patient treated with EMDA were segmented into the following tissues  using ITK-Snap version 2.4 . The patA radiologist verified the segmentation. The pixel size in the segmentation was 0.781 mm, while the slice separation was 3 mm. In total 72 slices were segmented, corresponding to 14 slices below the bladder and 3 slices above the top edge of the external electrodes.A 3-D model of the patient’s anatomy was constructed in COMSOL Multiphysics version 5.2 . The outside of the body was constructed using a planar contour method using an algorithm written in Matlab  . The tisWe used the stationary finite element method solvers in COMSOL Multiphysics to solve the Laplace equation for electric potential in the 3-D model of the patient’s anatomy. The model was discretized using a free tetrahedral mesh, which consisted of 2,779,405 elements. We used the conductive media DC interface in COMSOL Multiphysics to run a static simulation of the applied current density and electric fields inside the body. The electrodes were driven with a current-source terminal boundary condition, while the skin was taken into account only at the skin-electrode interface, by using a built-in thin resistive layer boundary condition in COMSOL. The results were extracted on a grid with data points positioned at the centers of the image elements in the original anatomical CT scans.Since the electrodes were driven by a current source (20 mA), it is possible to compute the resulting electrode voltages with the data from the manufacturer’s specifications. The voltage between the two electrodes was 13.1 V, which is within the generator specification and corresponds well to the expected voltage. Unfortunately, the generator does not specify the output voltage at the time of the treatment so the exact value used in the treatment is not available for the validation of the model.Electric fields in the whole body were extracted. The highest magnitude of electric field in the simulations was 271 V/m, in the subcutaneous fat tissue near the surface electrodes. The maximum values in the bladder wall were 71.9 V/m. The mean and SD of electric field magnitude in the bladder wall were 6.0 V/m and 7.1 V/m, respectively.2. The largest current density in the bladder wall was 14.4 A/m2. Mean current density in the bladder wall was 1.2 A/m2, with the standard deviation being 1.4 A/m2. The highest current density in all other tissues was 35.6 A/m2, however this value was found in a single voxel and may be due to numerical error. The current density is mostly perpendicular to the bladder, although the current density is much higher at the anterior part than at the posterior as would be expected based on electrode positioning The largest current density was found around the catheter electrode: 134 A/mMax = 1.5ER, where E is the electric field strength to which the cell of radius R is exposed. Based on the size of the cells used by Pucihar et al. in their experiments, the highest transmembrane voltage at 100 ms pulses was approximately 150 mV. The superficial layer of the bladder contains polyhedral cells of up to 250 µm in diameter  is a novel and interesting treatment modality in use for the treatment of cutaneous tumors and in clinical trials for tumors in internal organs (Our study shows that electroporation is l organs . Also fol organs . These dBased on numerical computational modelling based on realistically dimensioned models of EMDA treatment, we suggest that iontophoretic forces are predominant in this treatment. Electrode placements could mean that the anterior wall of the bladder receives a higher treatment field than other parts of the bladder. Also, due to the presence of a small air pocket in the bladder, the location directly above the air pocket receives a reduced treatment. Based on the numerical results alternate electrode positions should be considered if the original location of the excised tumor is not on the anterior bladder wall.


The remanent magnetization with different applied electric field along [100] and [01-1] directions was achieved, which showed the non-volatile remanent magnetization driven by an electric field. The two giant reversible and stable remanent magnetization states were obtained by applying pulsed electric field. This can be attributed to the piezostrain effect originating from the piezoelectric substrate, which can be used for magnetoelectric-based memory devices.We reported the non-volatile electric field-mediated magnetic properties in the half-metallic Heusler alloy Co The non-volatile electric-field-driven remanent magnetization along [100] and [01-1] directions was achieved, and the two giant reversible and stable remanent magnetization states are obtained by applying pulsed electric field  direction as 0°, whereas, the [01-1] direction as 90° . Figure E = 5 kVcm−1. Compared with the result as shown in Fig. −1. The magnetic easy axis returns to [100] direction when the applied electric field continues to decrease to − 5 kVcm−1 as shown in Fig. −1, the easy magnetization direction of in-plane remanent magnetization curve is along the direction [100]. The value of relative remanent magnetization (Mr/Ms) is significantly smaller than 1, which indicates a part of the magnetic moment not coherent arrangement. With increasing electric field to + 2.5 kVcm−1, the magnetic anisotropy decreases. When continuing to increase the electric field to + 5 kVcm−1, the in-plane magnetic anisotropy reappears. Compared with the remanent magnetization curve at − 0 and + 5 kVcm−1, the easy axis rotates 90°, which is consistent with the result of hysteresis loops as shown in Fig. Hσ) in the CFA/PMN-PT heterostructure. The magnetic anisotropy of the CFA/PMN-PT heterostructures is affected by the combination of Hσ and Hk [The magnetic hysteresis loops of the CFA/PMN-PT heterostructure were measured along the direction of [100] and [01-1] under applied electric fields of ± 0 and ± 5 kVcm−1 . The eleσ and Hk .Fig. 2a–M-E curves were measured by sweeping the electric field from + 10 to − 10 kVcm−1 in Fig. −1) is different from + 10 kVcm−1 demonstrated by the capital letters A and E in Fig. −1 demonstrated by B and F, which is the residual stress from the PMN-PT substrate. Residual polarization state is the remanence of the 0 kVcm−1 state, which is derived from the PMN-PT substrate residual stress, and not the same at + 10 and − 10 kVcm−1. It is consistent with the residual strain of strain curve in Fig. In order to verify the piezostrain effect induced by the electric field, the remanent magnetization with the applied electric field in the [01-1] and [100] directions was measured. We measured the change of remanent magnetization by sweeping the electric field after removing the saturation magnetic field 1200 Oe in the [100] and [01-1] directions, respectively. The asymmetric butterfly-like remanent magnetization versus applied electric field is obtained. We can determine that the remanence of the CFA/PMN-PT heterostructure is responsive to an electric field shaped as a butterfly; the −1) with the electric field in the [100] and [01-1] directions, in order to reflect the non-volatile control of the electric field. It can be found that the change of the remanence with the electric field also shows a change in the shape as a loop-like, and the remanence of the sample shows a good non-volatile, which is from the remnant polarization stress under the positive and negative electric fields, as shown in Fig. We have carried out experiments on the relationship between the remanence in the unsaturated polarization state (± 5 kVcm−1 are applied across the sample in the [100] and [01-1] directions. Firstly, the magnetic field is set up to 1200 Oe and reduced to 0 subsequently. Then the pulsed electric field is first stuck at ± 5 kVcm−1 in the [100] direction and reduced to 0 subsequently with results of the two residual polarization states demonstrated by the capital letters A and B in Fig. −1 was also observed as other residual polarization states C and D in Fig. −1 and reduced to 0 subsequently, we can see that the remanence is relatively large immediately, and when it is applied to 5 or 10 kVcm−1 and reduced to 0 subsequently, the remanence is significantly reduced; this phenomenon and the value of Mr/Ms are consistent with the results of Fig −1 and then instantly removed in the [01-1] direction, respectively. In summary, the remanence of Co2FeAl/PMN-PT heterogeneous is stress control and thus realizing the multistate remanence under the pulsed electric field, which can be used for polymorphic storage.For magnetic memory application, the non-volatile remanence in the pulsed electric field was achieved, as shown in Fig. −1. Additionally, the piezostrain-mediated non-volatile stable remanent magnetization reversal in the two directions is observed under positive and negative pulsed electric fields, which can be used for magnetic storage [In summary, the non-volatile electric field-mediated magnetic properties in the CFA/PMN-PT heterostructure are investigated at room temperature. The striped domain structure was obtained by the MFM measurement in the CFA film. The magnetic anisotropy was modulated by the electric field. The result measured by rotating-angle VSM demonstrates piezostrain-mediated non-volatile 90° magnetic easy axis rotation at − 0 and + 5 kVcm storage , 33.