Hypertension is an increasing health issue in sub-Saharan Africa where malaria remains common in pregnancy. We established a birth cohort in Nigeria to evaluate the early impact of maternal malaria on newborn blood pressure (BP).timing of malaria parasitaemia as ‘No Malaria’, ‘Malaria during pregnancy only’ or ‘Malaria at delivery’, and parasite density as low (<1000 parasites/µl of blood) and high (≥1000/µl).Anthropometric measurements, BP, blood films for malaria parasites and haematocrit were obtained in 436 mother-baby pairs. Women were grouped to distinguish between the ≤0.005). In babies of mothers who had ‘malaria at delivery’, their SBPs adjusted for other confounders were lower respectively by 4.3 and 5.7 mmHg/kg compared with ‘malaria during pregnancy only’ or ‘none’. In contrast the mean newborn systolic (SBP) and diastolic BPs (DBP) adjusted for birth weight were higher by 1.7 and 1.4 mmHg/kg respectively in babies whose mothers had high compared with low parasitaemia.Prevalence of maternal malaria parasitaemia was 48%, associated with younger maternal age (p<0.001), being primigravid (p = 0.022), lower haematocrit (p = 0.028). High parasite density through pregnancy had the largest effect on mean birth indices so that weight, length, head and mid-upper arm circumferences were smaller by 300 g, 1.1 cm, 0.7 cm and 0.4 cm respectively compared with ‘No malaria’ (all pAs expected, prenatal malarial exposure had a significant impact on fetal growth rates. Malaria at delivery was associated with the lowest newborn BPs while malaria through pregnancy, which may attenuate growth of the vascular network, generated higher newborn BPs adjusted for size. These neonatal findings have potential implications for cardiovascular health in sub-Saharan Africa. Hypertension is now a public health and economic problem in Sub-Saharan Africa with a prevalence up to 33% in urban areas in Nigeria In numerous global studies, adult risk of hypertension and other chronic disease is associated with LBW In newborns, their blood pressure (BP) correlates with birth weight Therefore, we tested the hypothesis that BP at birth would be higher and birth size would be smaller in babies whose mothers had malaria in pregnancy, defined by its timing during pregnancy and/or at delivery and the magnitude of parasite density. This in turn may affect the rate of BP rise through childhood and set the scene for hypertension in later life.Ethical approval was obtained from the joint University of Ibadan / University College Hospital Ethics committee and the University of Manchester Ethics committee. The study protocol and the rationale for the study were explained carefully in appropriate language, most commonly Yoruba or English, with questions answered as needed and written informed consent was obtained from all participants. After the delivery of their babies, another written informed consent was also obtained for the participation of their babies in the study.A semi-urban community, Yemetu-Adeoyo, Ibadan in Southwest Nigeria, where malaria transmission is perennial, was the site for the study. Families come from a range of socioeconomic backgrounds. The local community hospital, Adeoyo Maternity Hospital (AMH), the oldest maternity hospital in Nigeria dating from 1927, provides primary and secondary medical care with over 4000 deliveries each year.Healthy women aged 18–45 years presenting at AMH before 36 weeks gestation and residing within the catchment area of the study centre for at least 2 years were recruited. Singleton babies born at ≥37 weeks gestation were included.At booking, women were tested for sexually transmitted infections and HIV and those positive were excluded and those with chronic diseases such as hypertension and diabetes. Preterm deliveries, babies with known syndromes, metabolic defects, congenital abnormalities or severe birth trauma were excluded.Women were enrolled over one year to cover both wet and dry seasons.In the year, 3496 women booked for ante-natal care, but many were not planning their delivery at AMH so 659 women were eligible, of which 624 were recruited but 161 still did not deliver at AMH; thus the final cohort included 463 mother-baby pairs. Of these, 27 were excluded due to 4 (0.9%) maternal deaths, 11 (2.4%) still births, 5 (1.1%) miscarriages and 7 (1.5%) neonatal deaths, leaving 436 pairs. There were no significant differences in the socio-demographic and clinical data of excluded women.Standard operating procedures (SOPs) were developed. Informed consent, using forms translated into Yoruba, was taken at booking, then demographic, obstetric, family and health details, malarial history and use of antimalarial drugs was recorded. All women were issued with prescriptions for sulphadoxine-pyrimethamine (SP) for Intermittent Preventive Therapy (IPT) for malaria according to standard hospital practice.Standardized measures of anthropometry and BP were taken at every antenatal visit until delivery, weight to the nearest 0.1 kg , height on a stadiometer to the nearest 0.1 cm, both without shoes, according to the SOP and training video.2 areas. Total leucocyte number was calculated as 50× the number of cells counted.At each visit throughout pregnancy, 2 ml of blood was obtained in EDTA tubes for haematocrit, leukocyte count and blood film for MP. For haematocrit, a capillary tube was filled to about 80% with blood, sealed with a flame and centrifuged for 5 minutes at 10,000 revolutions per minute. The haematocrit value was read with a heamatocrit reader. For the leucocyte count, anticoagulated blood at a one in 20 dilution in turk's solution was mixed for about 1 minute and placed in the counting chamber. The sample was left to settle, and the white cells were counted using a microscope (10× objective lens) in the four outer 1 mmBlood films were prepared, stained with 3% Giemsa at pH 7.2 and examined for malaria parasites (MP) under light microscopy. Thick smears were recorded as negative only after 200 high-power microscope fields had been scanned. In those with malaria, absolute parasite counts were determined as previously reported For quality control, 30% of negative samples and 40% of positive samples were re-examined by two different trained microscopists.Women were followed up until delivery based on routine ante-natal practice, determined by gestational age. Repeat MP blood films were obtained every visit and at delivery when a film was also prepared from cord blood. The placenta was weighed, turned to the maternal surface, cotyledons exposed and 1 ml of blood obtained from the intervillous space for a placental malarial blood smear. Detailed delivery information was recorded.Babies were weighed naked to the nearest 0.1 kg, length was measured from crown to heel on an infant stadiometer to the nearest 0.1 cm and occipito-frontal circumference (OFC) around the widest circumference of the head using a non-stretchable tape to the nearest 0.1 cm. Skinfold thicknesses  were measured using Holtain calipers on the left side to the nearest 0.1 mm. Measurements were obtained in duplicate or triplicate if disagreeing by >15%. All babies were examined within 72 hours of birth.Maternal and newborn BPs were taken according to a standard protocol and SOP. Before the BP reading, the woman was comfortably seated and relaxed with the back and arm supported, the legs uncrossed, for at least 5 minutes and not moving or speaking. Her upper arm was supported at the level of the heart with no tight clothing constricting the arm. The measurement was carried out on the left arm with validated Datascope BP monitor using appropriate-sized cuffs .Before performing the BP reading, the baby was comfortably lying on the mother's lap for at least 5 minutes, and many times they were asleep. The measurement was again done with the Datascope BP monitor, specifically validated for infants, using appropriate newborn cuffs on the left arm. Babies were measured within 72 hours of life. In both mother and child, measurements were repeated three times at least one minute apart and the mean of the last two readings used for analysis.Three nurses trained in anthropometry and BP methods, based on the WHO manual (1995) and SOPs, carried out all measurements throughout the study on the same equipment. Inter-observer and within-observer error were minimized through 3-monthly refresher training sessions.Anaemia was defined as packed cell volume (PCV)<30%.Malaria was defined as asexual blood stages of Plasmodium falciparum in peripheral blood or placenta of the pregnant women or cord blood at delivery. All visits during pregnancy and at delivery were taken into account.For this study, women were first grouped into 2 categories:‘No Malaria’ - no parasites detected throughout pregnancy or at delivery (n = 225).‘Malaria present’ - parasites present at least once during pregnancy and/or at delivery (n = 211).timing of malaria through all visits in pregnancy and at delivery:Women with malaria were then stratified to distinguish between the ‘Malaria during pregnancy only’ - presence of malaria parasites at least once during pregnancy but not at delivery (n = 138).‘Malaria at delivery’ - mothers with parasites present in the placenta and/or their peripheral blood sample at delivery and/or in the cord blood (n = 73).To examine effects of parasite load during pregnancy and at delivery, parasite density was classified into low (<1000 parasites/µl), or high (≥1000/µl) based on the highest density recorded at any time point'.Data were analysed using SPSS version 14 . Socioeconomic index scores were based on occupations and educational attainment of both parents on scales I to V, as previously All women recruited had at least 2 antenatal clinic attendances, 94% attended 3 times, 80% four times and 63% five times before delivery. The median (range) durations of the pregnancy at booking and at delivery were 28 (12–36) and 39 (37–42) weeks respectively with 28% of women being primigravida.timing, 138 (31%) had malaria parasitaemia at some time during pregnancy only and 73 (17%) at delivery.Parasitaemia was present at least once in pregnancy and/or delivery in 211 of the 436 recruited mothers . Classified by 2 = 5.276, p = 0.022) and associated with nearly a 3-fold increase in risk of having malaria .56% of those with parasitaemia were primigravid, so malaria and first pregnancy were significantly associated .About half of the women reported the use of preventive measure such as chemoprophylaxis / insecticide spray or coil / bed nets / netted windows but these were not associated with protection from malaria (p>0.05). Social class and maternal malaria parasitaemia were also not associated , they were younger  and more likely to be anaemic than those without parasitaemia Table 1.There were no differences in other clinical characteristics such as pregnancy duration at booking, weight, height, body temperature, SBP and DBP in women with and those without malaria Table 1. and Anthropometric variables and skinfolds of infants born to women with malaria parasitaemia were globally lighter than those of women without  and 3a.parasite density, birth weight, length, OFC, and MUAC of newborns born to women with high parasitaemia were smaller by 300 (95% CI 100–400)gm, 1.1 (0.5–1.6)cm, 0.7 (0.3–1)cm and 0.4 (0.2–0.6)cm respectively compared with those without parasitaemia  and DBP (p = 0.049) than those with parasitaemia Table 2.In contrast when evaluating the effect of parasite density through pregnancy , mean SBIn regression analyses, newborn SBP was independently associated with birth weight and gestational age and inversely associated with birth length and maternal height. Malaria parasitaemia timing, specifically at delivery was also a determinant of infant SBP Table 7.This study illustrates the continuing impact of malaria in otherwise healthy pregnant Nigerian women, almost half of whom (48%) were affected. The rates confirm recent reports in pregnant Nigerian women For those women reporting the use of preventive measures against malaria, including the 53% who said they used chemoprophylactic drugs, there was no difference in malaria parasitaemia frequency. Previous findings have shown that netted windows, insecticide sprays, mosquito repellent and insecticide-treated nets were effective in protecting against malaria In the suburban women in this study, malaria parasitaemia was not associated with social class, similar to a Kenyan report In this study, diagnosis of malaria was based on microscopy of peripheral, placental and cord blood samples. Malaria in pregnancy still presents diagnostic challenges and has relied mainly on microscopy in studies in sub-Saharan Africa. Microscopy, though valuable, requires well-trained and skilled staff Babies of mothers with parasitaemia were globally smaller, most marked among babies of mothers with high parasite density during pregnancy. Similarly, mean birth weights in Burkina Faso, Tanzania, Mali and Pakistan for babies born to mothers with malaria in pregnancy were lower by 105 gm Birth weight is the single most important determinant of neonatal and infant survival and health, and malaria reduced all growth parameters, Some of the mechanisms linking small birth size and adult hypertension are poor maternal nutrition and maternal iron deficiency anaemia, which reduces vascular elasticity In this cohort we found that babies whose mothers had malaria parasitaemia at delivery had lower SBP and DBP . This obOverall, malarial load through pregnancy had the greatest impact on birth size . Thus thduring pregnancy leads to general growth restriction and smaller birth size and relatively higher BP for size. The more limited vascular tree of lighter infants may not be able to meet end-organ oxygen and nutritional demand without reactive peripheral vasoconstriction and higher BPs over time. Marginally but progressive higher BPs in infancy and early childhood may result, leading to a higher risk of hypertension in later life. Recurrent post-natal malaria will intermittently alter peripheral blood flow, but again at the expense of optimal supply to particular organs, hence restricting growth. The balance between the overall size of the fetus' vascular tree, how well particular organs grow during pregnancy, notably the renal glomeruli with their afferent and efferent arterioles, and continuing environmental hazards  or opportunities  will determine vascular performance, now measurable by pulse wave velocity This leads to the question of how these two contrasting observations on BP related to timing and parasite density are mediated. Our hypothesis is that in these LBW babies, placental parasitaemia is associated with significant inflammation in the placental and infant arterial tree leading to more acute placental changes and significant infant vascular dilatation as an initial protective mechanism and hence lower BP at birth in those with parasitaemia at delivery. In contrast, parasitaemia There is a high incidence of malaria and anaemia in this apparently healthy cohort of pregnant women, particularly the younger mothers and primigravids. Malaria in pregnancy adversely affects birth size, with high parasite density during pregnancy having the greatest impact on all growth parameters and being associated with higher BP corrected for weight. Follow-up studies to extend these observations into early childhood and to provide a better understanding of the influence of maternal malaria on BP in this cohort are underway.


Regardless of the root cause of these unwanted effects, understanding the scope of a drug’s activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a “minipool” composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug−target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug−target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields “hits” that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl−positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi  Despite enormous spending increases in pharmaceutical research and development, the number of new drugs approved each year has remained nearly constant during the past 60 years . Among ti.e., DHFR) gene expression is reduced by short hairpin RNA (shRNA) knock-down, cells become hypersensitive to methotrexate treatment  comprise additional variables.RNA interference is a reliable and efficient approach to modulate gene expression in mammalian cells. It is also a powerful technique for identifying putative drug targets by knocking down mRNA, subsequently reducing protein expression and observing the resulting cell fitness in the presence of drug  our compilation of experimental profiles provides a foundation for future clustering and pattern matching informatics studies that can be applied to less well-characterized compounds. We expect that these results will illuminate some of the biology that underlies the enormous variability in patient drug response and that this simple robust protocol can be adopted and adapted for different cellular pathways.http://www.atcc.org) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) + 10% fetal bovine serum (FBS) + penicillin/streptomycin (P/S) and incubated at 37° and 5% CO2.A549 cells (human lung adenocarinoma) were obtained from ATCC . Hairpins were selected based on the reported knockdown efficiency, as measured by real-time polymerase chain reaction (PCR) by TRC (Table S7). The resulting pool contains 1098 shRNA lentivirus plasmids that target 368 human genes. Equal amounts of plasmid DNA were combined, and the pooled shRNA plasmids, including lentivirus packaging and viral envelope plasmids, were mixed and transfected into HEK293T packaging cells using FuGENE reagent (Roche). Forty-eight hours after transfection, media containing lentivirus shRNA particles were collected, aliquoted and stored at −80° as described (http://www.broadinstitute.org/rnai/public/resources/protocols).Three hairpins were selected for each of 368 human genes from The RNAi Consortium (TRC) lentiviral libraries (7) were infected with the lentiviral shRNA minipool at a multiplicity of infection of 0.3−0.4. After 2 d of selection in 2 μg/mL puromycin-containing medium to eliminate uninfected cells, puromycin-resistant A549 cells were amplified for an additional 2 d, resulting in a total of 4 × 107 cells. The transduced A549 cells were split into aliquots of 1.5 × 106 cells each and frozen for future screens. Frozen aliquots of the A549 with the integrated shRNA minipool collection are available upon request.A549 cells (3 × 1025) was used for subsequent minipool screens  then in RPMI, with the final DMSO concentration not exceeding 1%. After 72 hr of incubation, cell viability was measured with a sulforhodamine B (SRB) viability assay . Briefly screens .5 cells/well in 2 mL of DMEM + FBS + P/S such that each hairpin was represented at least 125 times per well. The cells from the first generation of growth were saved as the “T0” sample and stored at −80°. Two hours after seeding, drug (at IC25) was added to fresh cells. Cells were incubated for 72 hr and trypsinized. Then, 1.65 × 105 cells/well were reseeded in 2 mL of DMEM + FBS + P/S + drug, whereas 5−8 × 105 cells were collected and stored at −80° for subsequent genomic DNA extraction. Each screen comprised seven consecutive 3 d time points and was performed in triplicate.Infected A549 cells were seeded in six-well plates at 1.65 × 10The genomic DNA of each individual sample was extracted using a QiaExtractor DNA robot (QIAGEN) in a 96-well format according to manufacturer’s instructions. DNA was normalized such that, on average 1 ug was obtained and 50% was used in each barcode PCR. PCR amplification of shRNA was performed using indexed Illumina PCR primers to incorporate both the Illumina adapter sequences and indexing sequences, with Platinum Pfx polymerase kit (Invitrogen) as previously described , followed by taking the antilog of the counts. To assess the combined impact of shRNA knock-down and drug treatment on cell growth, a linear model was fit to the observed triplicate data for each hairpin as follows. Briefly, we fit a generalized linear model with Poisson distribution and log link function using the glm function in R (v.2.15.0). Normalized read counts were treated as the response variable, number of population doublings as the first predictor variable, and drug treatment and doublings as an interaction term. This scoring approach assumes (correctly) a common intercept on the y-axis (starting read counts) for both the vehicle control and the drug treatment, and allows for missing data when necessary. Hairpins were ranked according to the fitted interaction parameter as a measure of the effect of drug treatment while controlling for the effect of the hairpin treatment. Hairpins with interaction values that are 2 SD from the screen mean  were considered as primary hits.Raw read counts were shifted up by 100 to remove zero counts and converted to logFile S1).Specific target genes in A549 cells were silenced by lentiviral shRNA as described previously. Three days after drug treatment, cells were harvested, washed, and stained with fluorescein isothiocyanate−conjugated annexin V and propidium iodide according to manufacturer’s instructions (BD 556419). Cells undergoing apoptosis were detected by phosphatidylserine externalization  using two-color flow cytometry analysis on a BD FACS Calibur. A step-by-step protocol is provided as an appendix , ChEMBL (http://www.ebi.ac.uk/chembldb/), and MATADOR (http://matador.embl.de). The resulting 368 human drug targets were then grouped according to their Gene Ontology designation. Gene Ontology analysis showed that the final list of targets belong to diverse pathways including signaling, protein/DNA metabolism, transport, receptor, cell cycle, DNA damage, and repair .The genes comprising our shRNA minipool library were selected on the basis of their previous characterization as human drug targets, 25 .We prioritized 50 drugs of the 640 present in the Biomol FDA library for our screen against the mini-pool library. For the purposes of this study, we divided these drugs into two classes (antiproliferatives and nonantiproliferatives). Together this set covers a broad range of therapeutic indications, including immunosuppressive agents, antipsychotic, anti-cholesterol, anticoagulant, anti-inflammatory, antidiabetic, and cancer chemotherapeutics . For eac25 was a suitable dose in that it maintained drug pressure while exhibiting minimal variation in inhibition after each splitting during the 21 d screens. This value is similar to the IC15-30 used in many large-scale yeast screens  in the sequence count number for each hairpin was found between daughter populations  but more importantly, a very high correlation (r = 0.947 and r = 0.932) was also found between the mother and both daughter populations . Additionally, a closer analysis revealed that only 25 shRNAs (of 1098) were missing from our collection and that most of these were lost immediately after the initial infection step . Thus, freezing aliquots after the initial infection provides a uniform source of cells for screening. These results encouraged us to screen our minipool of 1098 shRNAs targeting 368 human genes, against 50 selected drugs at IC values between 20 and 30%.To test whether the freezing and thawing affected the representation of our hairpin collection in the A549 cells, we compared two sets of infected cells. The first set contains cells collected immediately after the original infection (“mother” population). The second set represents cells from two stock vials used in all our screens, obtained after infection, puromycin selection, amplification, and one freeze-thaw cycle (“daughter” populations 1 and 2). For each sample, genomic DNA was extracted from 1 × 10Figure S3). To directly address what fraction of the minipool library was depleted in the absence of drug, we analyzed the sequence count numbers for each hairpin and found that less than 7% of the minipool library (73/1098 shRNAs) was toxic in A549s .To estimate the number of cytotoxic hairpins in the A549 cell background, we calculated the hairpin representation after 12 and 21 doublings, when the cells were cultured in the presence of DMSO, methanol, or a no-vehicle control. We first compared how well different screens correlated with one another. As anticipated, greater correlations were observed after 12 doublings compared to 21 doublings for all three culture conditions, reflecting a continuing dynamic loss and amplification of shRNAs overtime. A good correlation (0.72 < r < 0.85) was observed between different doubling times compared with T0, attesting to the moderate level of toxicity conferred by the expression of hairpins . This format allowed a single person to test 50 drugs in a few weeks. After drug treatment, cells were collected every 3 d and maintained in culture in drug over 21 d, being split every third day. Cell counts were obtained at each subculture step. Once the final time point was collected, the shRNAs from each time point were amplified via PCR from gDNA as described . A totale.g., r ≥ 0.8, day 21, Figure S5). We quantified the abundance of each shRNA in the presence or absence of drug. A linear fit was applied to each of the three shRNAs for each gene . The difference between DMSO controls (dashed line) and drug treatment (solid line) is designated as the “interaction value,” a term that reflects the abundance of shRNAs in the population during the course of drug treatment. Any shRNA that renders a cell hypersensitive is characterized by a negative interaction value. Based on their interaction values (Table S2) and their linear plots, a list of potential synthetic lethal genes for each of the 50 tested drugs was then compiled (Table S3).For each drug, the triplicate screens showed a high correlation at each time point (25 = 8.70 μM) compared with the nonantiproliferative drugs , further validating the doses we selected and highlighting that A549 is an appropriate cell line to test and validate our initial pipeline.Drugs were classified according to their known antiproliferative and nonantiproliferative properties in cultured human cells. As anticipated, our assay was more sensitive for antiproliferative compounds (average ICTable S4). A total of 56% of targets were identified using antiproliferative compounds, whereas only 17% of reported targets were identified with nonantiproliferatives. This enrichment for antiproliferatives reflects the nature of the screen and the growth readout which we used. By acting on a target whose activity is crucial for the cell survival, antiproliferative agents induce toxicity at low concentrations. In contrast, nonantiproliferative agents modulate targets whose function is not necessarily essential for cell growth, and, consequently, greater concentrations are required to induce a detectable growth defect. The results also highlight the need to tailor the assay for nonantiproliferative drugs by, for instance, selecting a non growth-based assay. Despite a greater risk of false-positive results that could arise from using relatively high doses of drug, the study of nonantiproliferative compounds can also prove useful in understanding secondary activities (Table S5 and Table S6).Among the 50 screened drugs, 28 have targets that are reported in the literature. We identified the targets for 11 of these drugs (40%) in the top hits from our screens , suggesting that DPP4 may be an additional target of metformin.Metformin is an oral diabetic drug that decreases insulin resistance and is prescribed as a first-line treatment according to most type 2 diabetes mellitus guidelines  which, taken together, suggests that the consequence of disrupting signaling is often the induction of apoptosis. This suggests that in addition to detecting growth inhibition by perturbing new potential drug targets, the data can provide insight regarding general pathways required for viability. These data are summarized in Gossypol is a natural polyphenolic compound derived from seeds, foliage, and roots of cotton plants and was first described as an endocrine disruptor . After 3 d of exposure to 5 μM of gossypol, the parent A549 cells and control A549 cells with an integrated RFP hairpin showed 81% and 77% survival, respectively, compared with cells cultured without drug , consistent with our initial dosing experiments. In addition, only 2 hairpins strongly reduced the cell viability in their own , one of which (HUWE1) was already found toxic in our screen . The shRNA validation was based on the median absolute deviation statistical method , consistent with previous reports , a finding in contrast to the published reports that HUWE1 depletion shows a reduced degradation of MCL1, less apoptosis and an increase in cell survival  by stabilization of the MCL1-HUWE1 complex; (2) by inducing apoptosis through inhibition of the anti-apoptotic protein MCL1 via HUWE1; and (3) by arresting cancer cells in G1 phase presumably via HDAC2 inhibition .Table S5 and Table S6) as well as a detailed, step-by-step protocol in the Appendix. Focusing our shRNA library on protein targets for which drugs already exist has the potential for uncovering new therapeutic applications of such compounds. Translating this experimental method and data analysis routine to other cell lines and particular pathways will allow such screens to be performed using the cell culture expertise that already exists in many laboratories.In this study, we report the development and application of a sensitive, cost-effective assay to facilitate drug mode of action studies. To demonstrate the utility of this method, we screened shRNAs and drugs based on known drug−target interactions. From 50 selected drugs with diverse activities, 28 have targets that are reported in the literature. We identified 40% of known targets, several potential novel targets and related drug pathways The data for all screens is available (


Very little is known about complementary and alternative medicine (CAM) use by older adults in Germany. The aim of this study was to investigate the use of CAM and other health promoting substances  by older adults of at least 70 years of age.A cross-sectional questionnaire study was conducted among persons of ≥70 years from metropolitan Berlin and rural parts of Brandenburg, Germany. Recorded were: demographics, current use of CAM, medical diagnoses, users’ opinions and preferences.A total of 400 older adults, living as ‘self-reliant’ (n = 154), ‘home care service user’ (n = 97), or ‘in nursing home’ (n = 149), and with the legal status ‘without guardian’ (n = 355) or ‘with guardian’ (n = 45) were included . Any type of CAM used 61.3% of respondents ; 3.0% used drug-interaction causing preparations. Usage was based on recommendations , own initiative (27.3%), and doctors’ prescription (25.8%). Participants with legal guardian took almost solely prescribed dietary supplements. Of the others, only half (58.7%) informed their general practitioner (GP) of their CAM use. Participants expected significant (44.9%) or moderate (37.1%) improvement; half of them perceived a good effect (58.7%) and two-thirds (64.9%) generally preferred a combination of CAM and conventional medicine. More than half (57.9%) stated that they could neither assess whether their CAM preparations have side effects, nor assess what the side effects might be. Strongest predictors for CAM use were two treatment preferences  and the type of health insurance ; against CAM use two subjective assessments predicted .Older German adults frequently use CAM. They perceived it as an effective complement to conventional medicine, but are not sufficiently informed about risks and benefits. Germany has a very long tradition of complementary and alternative medicine (CAM). Many older adults have lifelong experience with herbal medicine and other home remedies due to unavailable conventional care during their childhood. CAM therapies are often used as self-care to enhance wellbeing, to prevent and to cure illnesses . HoweverPolypharmacy is also a problem in the geriatric care in Germany and poses a risk for side effects and drug interactions. While seniors at the age of 60 years take 2–3 prescribed medications daily, the number increases among those over 80 years to more than 4–5 drugs per day ,10.Therefore we investigated the use of CAM and other health promoting substances  by older adults of at least 70 years, taking care to include under-researched areas such as rural areas or nursing homes.Our survey solicited information regarding which form(s) of CAM is used and how its use is subjectively assessed, as well as medical context information. We asked for all natural products, drugs and therapies that were taken for treatment or prevention of diseases, this included not only drugs but also medically applied herbal teas and juices. To get the best possible representation of real-life conditions, we included older adults living with a variety of needs for care, living in either a metropolitan or a rural area, and with or without a legal guardian.We conducted a cross-sectional questionnaire study from November 2010 through July 2012. Participation was anonymous and voluntary; participants expressed their agreement through completion of the questionnaire. For those under legal guardianship, the guardian provided legal consent. The study protocol was approved by the ethics committee of the Charité Universitätsmedizin Berlin .Older adults at least 70 years of age living in the states of Berlin (entire city) and Brandenburg  were approached through care service providers, nursing homes, community clubs of older adults, or directly through the distribution of questionnaires in mailboxes of senior residential facilities (Berlin only). The care service providers and nursing homes were selected from the phone book and contacted in alphabetical order. The older adults lived in their own homes, either self-reliant or assisted by a home care service, or in a retirement or nursing home. Both home care service users and nursing home residents included older adults with, as well as without, a legal guardian, resulting in 5 study arms Figure . Care seThe questionnaire asked for social and demographic data. In this study we also included non-medical health promoting substances such as vitamins or teas under CAM. We recorded for CAM preparations: Name and dosage, reason for the application, on what basis the decision for their use was made, and information regarding participants’ general practitioners (GPs’) knowledge about their patients’ CAM usage. Further items covered the use of non-pharmacological treatments, and, for participants without legal guardians, their subjective opinions and assessments about CAM: expectations towards, experience with, and perceived effects and risks, as well as the generally preferred treatment.Due to a lack of data about the prevalence of the use of alternative medical drugs in the studied population we assumed a prevalence of 50% of the use of CAM drugs in in the studied population. With an accuracy of 10% for a two-sided confidence interval of prevalence at the 95% level, 96 participants would be needed. In order to achieve this precision, 96 questionnaires would be needed for each of the four groups of older adults: living self reliantly, receiving homecare, living in nursing homes or having a legal guardian. We anticipated that 60% of the self reliant living older adults would return the questionnaires, in all others a return rate of 80% of the questionnaires. Thus the number of self reliant older adults to be contacted was n = 160, for all other groups n = 120. The total number of issued questionnaires thus was n = 520.The answers from the questionnaires were entered into an electronic database (MS Access™) and checked for plausibility and quality. Descriptive analyses were calculated, and patient groups, stratified by CAM use  were compared using chi-square  and t-test (continuous data). For participants without legal guardians, the effect of variables on CAM use was estimated with a multiple logistic regression. The variables were selected on the basis of the calculated p-values and frequencies, as well as an expert’s opinion. Several potential predictor models were tested before the final logistic model was calculated. SPSS 19.0 and higher (© SPSS/IBM) and Statistical Analysis Systems 9.3 (© SAS Institute) were used for randomization and all analyses.We contacted 33 care service providers  and 19 nursing homes , of which 6 care service providers  and 6 nursing homes  participated. Of the 761 questionnaires that were issued to older adults without legal guardian or their nurses, and the 47 questionnaires issued to nurses of older adults with legal guardian, a total of 418 (51.7%) were returned; 400 were entered into the final analysis  used at least one CAM preparation Table . Both hiThe CAM preparations most frequently used by older adults without legal guardians are listed in Table 3 (22.2%), vitamin B12 (20.0%), folic acid (13.3%), calcium (6.7%), magnesium (2.2%), and iron (2.2%) and only in 2.2% the herbal preparation, valerian. CAM users without legal guardians expected a marked (44.9%) or moderate (37.1%) improvement of their conditions; 11.7% did not state their expectations. More than half (58.7%) of the users experienced a good effect, 27.4% only a minor effect, and 6.0%, no effect.Of the older adults with legal guardians, 88.9% used CAM. In 92.9% of cases, their GPs prescribed the preparations but only 51.3% documented the reason. Users took predominantly dietary supplements: Vitamin DCAM usage was in 31.3% based on recommendations  or as a result of one’s own initiative (27.3%). Only a quarter (25.8%) of the total CAM uses were prescribed by medical or nonmedical practitioners (in Germany ‘Heilpraktiker’) . It is unclear whether they can be transferred to other German states. The recruitment of older adults with legal guardians was less successful than expected. The additional work for their nurses  and possibly other concerns established an entry barrier that resulted in less than the 100 required participants; thus not all planned statistics could be calculated.In all groups it is possible that some older adults listed all of their diseases as reasons for CAM use. Also, results from nursing homes may have been influenced by the fact that some of the homes offered CAM treatments, which may be interpreted as a distortion of the results or as part of the environment. A few respondents who were much younger than the intended inclusion age made it necessary to relax the strict intention-to-treat approach and exclude these untargeted extreme outliers.A comparison of our results with the existing research is difficult because of the small body of literature and its different eligibility criteria. For Germany, only one anonymous questionnaire survey by Büssing et al. exists , which iAnother German survey of the general population included persons aged 18 to 79 years . It alloA general German population survey by the Allensbach Institute  also fouWorldwide, only 5 studies on CAM use in residential care settings appear to have been published . Their rA cross-sectional survey by Cherniack et al.  with 421Another cross-sectional analysis by Cohen et al.  from a gA randomized subsample (n = 1099) of the 2000 wave of the Health and Retirement Study  answeredIn our study we found none of the predictors for, or correlations with, the use of CAM preparations or therapies that had been seen in some of the more or less differing populations cited above , but we In this first study that included participants in living situations involving various degrees of independence we found a high rate of users of CAM preparations and dietary supplements among German older adults. Self-reliant older adults primarily use dietary supplements, herbal medicines and external preparations. For the most part they follow recommendations by pharmacists, friends or relatives or make their own decisions, whereas older adults with legal guardians or a high need for care take prescribed vitamins and minerals. General practitioners were insufficiently informed about CAM usage. Older adults perceived CAM as an effective complement to conventional medicine, but were not sufficiently informed about risks.The authors declare that there are no conflicting interests regarding the publication of this article. This study was funded by the Karl and Veronica Carstens Foundation .KS participated in study concept and design, data collection, statistical analysis, data interpretation, and manuscript drafting. SB did the statistical analysis and participated in data interpretation and manuscript drafting. MT participated in study concept and design, data interpretation, and manuscript drafting. CMW participated in study concept and design, data interpretation, and manuscript drafting. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/14/38/prepub


Adding propagules (source) to a degraded site (recipient) is a common way of manipulating secondary succession to restore diversity and services formerly provided by forests. However, heretofore no study has considered the effect of “successional distance” between source and recipient site. Four sites in the Shilin karst area of SW China were treated as different states along a secondary successional sere: grass, shrub, young secondary forest, and primary forest. Ten 1 m ×1m soil quadrats in the grass, shrub and young forest sites were replaced with 10 cm deep soil sources from corresponding later successional stage(s) in January 2009. Woody plant seed germination was monitored in the first year and seedling survival was monitored until the end of the second year. At the end of 2010, 2097 seeds of woody plants belonging to 45 taxa had germinated, and 3.9% of the seedlings and 7.8% of the species survived. Germination of most species was sensitive to ambient light . Soil source and recipient site had a significant effect on the total number of seeds and number of species that germinated, and on the percentage of seedlings that survived through the end of the second year. Closer successional stages between recipient site and soil source had higher seed germination and seedling-survival percentages. However, a transition threshold exists in the young forest state, where seeds can germinate but not survive the second year. Our results, although based on an unreplicated chronosequence, suggest that successional distance between soil sources and recipient sites affect forest recruitment and restoration in degraded karst of SW China. Worldwide demands for practices to restore diversity and services formerly provided by forests are increasing. These practices include removing human disturbance  to facilitate natural succession, and human intervention to accelerate and influence the successional trajectory of recovery –3. NaturAdding seeds via the soil seed bank (SSB) or from collected seeds ,8 is a cSuccession is a key factor in vegetation dynamics . Both th However, no study has considered the effect of successional distance of the source and the recipient site on recruitment and restoration. Surface soil of later successional stage communities contains seeds representing those successional stages, the persistent seeds of plants of earlier successional stages and a superior germination substrate . ReplaciDoes the soil source from a later successional stage favor the establishment and abundance of plant communities at earlier successional stage recipient sites? Does the successional distance between the soil source and recipient site affect seed germination and seedling survival? In other words, does the same soil source have different effects on different earlier successional stage recipient sites? Similarly, do the soil sources from different successional stages have different effects on the same recipient site? 2 with underlying Permian carbonate rocks, of which more than 400 km2 has developed into karst and related landforms. Altitudes range from 1600 to 2200 m above sea level. Mean annual precipitation is 968 mm (at 1680 m), 80% of which falls between May and October. Mean annual temperature is 16.2 °C, mean maximum temperature 20.7 °C (July), and mean minimum temperature 8.2 °C (January) [This study was conducted at Shilin Stone Forest Geographical Park , Yunnan Province, southwest China, a karst geo-park famous for rugged limestone landscapes. This park is approximately 900 kmJanuary) . The priJanuary) . Over thFour sites covered with grass, shrubs, young secondary forest and primary forest were selected for this study. All four sites are located on the same karst rock base, and have similar geographical features. Thus, we suggest the vegetation cover at these four sites can be treated as different states along the secondary successional trajectory. A preliminary study (three 20 m × 20 m plots) showed that there are 633 ± 29 (ind./ha ± SD) stems (DBH ≥3 cm), with DBH = 10.7 cm ± 7.4 (average ± SD) from 24 species at the primary forest site, 375 ± 39 (ind./ha± SD) stems (DBH ≥3 cm), with DBH = 7.1 cm ± 4.6 (average± SD) from 12 species at the young forest site, and there were a few individual (DBH <3 cm) trees that had sprouted at the shrub site. Grasses mixed with a few shrubs dominated at the grass site . We treaSoil was collected from the surveyed primary forest, young forest and shrub sites, but not from the grass site because we are only concerned with the effect of soil sources from later successional stages. Thirty 1m ×1m soil sample quadrats in the primary forest site, 20 at the young forest site and 10 at the shrub site were established. In January, 2009, all soil and litter to a depth of 10 cm were collected from each quadrat, put into plastic bags, and transferred to a concrete platform, where soil from each site was combined and hand mixed. Then, the total amount of source soil from each site was weighed and divided evenly into 30 parts from the primary forest site, 20 parts from the young forest site and 10 parts from the shrub site. By mixing and re-dividing the soils, we aimed to reduce the variation caused by the difference among the parts of the same soil sources since most seeds are unevenly distributed on the forest floor. We refer to soils containing the soil seed banks as the ‘soil source’; we used soil sources from the primary forest (state 4), young forest (state 3) and shrub (state 2) sites. Plants, soils, and stones to a depth of 10 cm were removed from 30 1 m ×1m plots in the grass site, 20 in the shrub site and 10 in the young forest site (quadrats in the young forest and shrub sites were reused). We refer to these as recipient site. Soil sources from later successional stages were transferred to a plot at sites with an earlier vegetative successional stage, so that the following soil transfers and successional distance were made: Distance across one state: primary forest to young forest, young forest to shrub, and shrub to grass; Distance across two states: primary forest to shrub, young forest to grass; andDistance across three states: primary forest to grasst) and dead seedlings (NDt) of each species in each plot. After recording their number, the dead seedlings were removed to prevent recounting. We transferred 10 replicates of each soil source at each recipient site, making a total of 30 plots at the grass site, 20 at the shrub site, and 10 at the young forest site. The successional distance between soil source and recipient site may be caused by the soil itself and viable seeds within the soil. All plots in the same site were numbered and randomly selected to receive different soil sources. A map showing the distribution of plots was prepared for future identification. A thin layer of rice straw was placed on each surface treatment to prevent moisture loss. The straw was removed on April 6-10, 2009, before the beginning of the rainy season in May, to monitor germination. Only seedlings of woody species were identified and recorded (herb seedlings were discarded). Seedlings were recorded at half- month intervals from June to September 2009, and then at 1-month intervals from October 2009 to the end of 2010. At each observation time (t) we counted live seedlings (NA Red light (R), far-red light (FR), and R:FR ratios at the soil surface in each plot were measured using a 660/730 nm quantum sensor, and photosynthetically active radiation (PAR) was measured with a 400–700 nm quantum sensor  between 12:00–14:00 on cloud-free days in September 2009, December 2009, and February 2010. Volumetric soil moisture content (%) (VSMC) at a depth of 10 cm was measured with a PR2 sensor  at the same time as light measurements were taken. t(x) as the total number of seeds germinated of species x up to time t, NSt(x) as the total seedlings of species x at time t, NAt(x) as live seedlings of species x at time t and NDt(x) as dead seedlings of species x at time t. Thus, the total number of seedlings of a species at a plot at time t was:The total number of germinated seeds per species, and their survival percentage are considered in this study. We denote TSt+1(x) may be greater than, less than or equal to NAt(x) (dead seedlings were taken out after recording at time t). Thus, the total number of seeds germinated at time t+1 is:Some new seedlings may emerge, and some seedlings may die at time t+1, thus, NATSt+1(x)=TSt(x)+(NSt+1(x)-NAt(x)) if NAt+1(x)>NAt(x)(new seedlings emerged)Or TSt+1(x)=TSt(x) if NAt+1(x)≤NAt(x)( no new seedlings emerged) t(x) to the total number of seeds germinated was the germination rate at time t. The total number of seeds germinated in a plot was the sum of seeds of each species that germinated. Thus, for species x in plot i, the total number of seeds germinated was the TS(x) at the end of 2009, and the percentage of TSt(x) to the total number of seeds germinated was taken as the percentage survival of species x at time t (SR t(x)), and the proportion of live seedlings (species) to the total number of seeds (species) germinated in a plot at time t as the survival percentage of seedlings (species) of this plot. For most species, germination stopped, and death of seedlings began before the end of 2009. The proportion of NARepeated measures ANOVA was performed in the statistical package SPSS 13.0 with recording time as the repeated measures (within subject) factor and recipient site as between-subjects factors to test the significance of light, and soil water content. The total number of seeds, total number of species identified at the end of 2009, survival percentage of the total number of seedlings and the total number of species were used to test differences among the three soil sources and three recipient sites by two-way ANOVA. Where main effects were significant (P<0.05), Duncan’s multiple range tests were used to compare differences in means of seed germination or seedling survival between soil sources or between recipient sites. The relationships between R, FR, R:FR, PAR and VSMC and total emerging seedlings of the species and between environment variables and the survival percentage at the end of second year of the species were analyzed with Canonical Correspondence Analysis (CCA) (CANOCO 4.0). Prior to analysis, seed and environmental data were log(x+1) transformed. To examine the significance of the canonical axis of the partial CCA, Monte Carlo permutation tests were conducted with 199 permutations under a reduced model. The results were displayed as a CCA bi-plot of species and environmental variables.Cyclobalanopsis glaucoides, Neolitsea homilantha, Pistacia weinmannifolia, Pistacia chinensis, Albizia julibrissin, and Olea yunnanensis had the largest number of germinated seeds. Shrub seeds accounted for 26.0% of the total, of which Sophora davidii, Zanthoxylum scandens, Rhamnus leptophylus, Dodonaea viscose, and Myrsine africana had the largest number of germinated seeds. Liana seeds only accounted for 14.0% of the total (Appendix). Two thousand and ninety-seven seeds from 45 woody plant taxa had germinated at the three recipient sites by the end of December 2009: 13 (28.3%) tree species, 17 (37.0%) shrub species and 9 (19.6%) different woody lianas. Tree seeds accounted for 59.3% of the total, of which Cyclobalanopsis glaucoides accounted for the largest portion of seedlings germinated from the young forest soil source, and Sophora davidii dominated the soil seed bank from the shrub soil source. A total of 38 species emerged from the primary forest soil sources at all recipient sites, 30 species from the young forest soil sources and 14 species from the shrub soil sources. More seeds and species of tree and woody lianas germinated from forest soil sources (both primary forest and young forest) than from shrub soil source (Appendix). The shrub soil source contained more shrub seeds than forest soil sources. June to September was the main germination period for all three soil source types at each of the three recipient sites. The number of seeds and species increased linearly from June to December 2009 P<0.001, .-2 ±10.6) than from primary forest soils (40.5 m-2 ±8.6). A significantly higher number of species and seeds germinated in the primary forest soil source that was transferred to the shrub site than to the young forest or grass sites. Seed density and richness per square meter varied among soil sources and among recipient sites . The recRadiation at the three measurement times  did not Five canonical correlations were obtained between seed germination environmental variables R, FR, R:FR ratios, PAR and water content and the total number of seeds that germinated with CCA. The Monte Carlo permutation test (with 199 permutations) both on the first axis  and on all axes  were highly significant. Canonical axis 1 and axis 2 explained 50.7% and 24.5% of the cumulative percentage variance of species-environment relations, respectively. Axis 1 was strongly correlated with PRA (-0.958), FR (-0.952) and R (-0.937). The weighted correlation coefficient between axis 1 and R:FR was also high (-0.750) . These dNeolitsea homilantha, T3, Pistacia chinensis), two liana species , and two shrub species  species were not sensitive to light and soil water.The optima of many species, including six of the eight tree species , two of the five lianas  and two of the five shrubs  were sensitive to the change in light variables PAR, R, FR and R:FR . A higheSurvival percentage of seedlings and of species decreased 50% by the end of 2009 (12 months after soil transfers), and this decrease continued through 2010 . SeedlinAt the end of 2010, 3.9% of seedlings, and 7.8% of species were still alive. No seedlings survived at the young forest recipient site. Only 2.65% of seedlings and 11.33% of species survived at the grass recipient sites and 7.72% and 6.49% at the shrub recipient sites . Soil soCyclobalanopsis glaucoides (16%) and Olea yunnanensis (13.2%) had the highest survival percentage. Survival percentage of the other major tree germinants, Pistacia chinensis, Albizia julibrissin, Pistacia weinmannifolia was < 2%. Only a few shrub seedlings survived, including Rhamnus leptophylus, Campylotropsis polyantha, and liana seedlings from one species, Dalbergia mimosoides.Degraded ecosystems may take an ordered continuum route   or a non“Successional distance” between the recipient site and soil source had a significant effect on the seed germination and survival when the soil source was added. Soil transferred from state 3 (young forest) to recipient sites at earlier successional states - state 1 (grass), and state 2 (shrub) - were better than soil sources from state 4 (primary forest) in promoting seedling recruitment because they contained more seeds  and provEnvironmental cues that can affect seed germination in natural environments include fluctuations in light quality ,22, R:FRConditions for survival were more stringent than conditions for germination. In a thorough review on seed addition, Clark et al.  found th A transition threshold controlled by biotic interaction usually exists in the successional process of highly degraded ecosystem . In our There are weaknesses and potential confounding factors in this study’s design. Soil replacement by a later successional soil source will yield a new combination of seeds and environmental factors, which will in-turn, affect germination and seedling survival. We expected the outcomes of soil replacement to be influenced only by the successional distance between soil source and recipient site. However, other minor factors, i.e. alteration of the soil profile through mixing, and the change of the vertical distribution pattern of seeds may also contribute to seed germination and seedling survival. In addition, a 10 cm deep layer soil-source may not fully function as the original soil in the later successional state. Our conclusions are somewhat weakened because we use an unreplicated chronosequence.Our findings suggest that soil seed banks in forests in karst areas in SW China, together with their soil substrates can provide woody seedlings for initiating species turnover of degraded forest land. The successional distance between the stage of soil source and the stage of recipient site strongly influence seed germination the first year and seedling survival the second year. These results also suggest that soil sources from secondary forest, and not necessarily primary forest, can satisfy the need for successional manipulation, thus making soil replacement economically and practically preferable since secondary forests are more common and easier to access, and may be spatially closer to the degraded land than primary forest. Consequently, we may save more primary forests. Our results also imply that restoration designs should consider transition threshold states and environmental factors, such as light and soil water. Restoration success at secondary forests could be improved by the implementation of open canopy windows to increase light illumination, and success at grass lands by adding water since both light and soil moisture will affect germination and seedling survival. Appendix S1(DOC)Click here for additional data file.


Human mesenchymal stem cells (hMSCs) were seeded onto cubic scaffolds prepared from demineralized bone matrix (DBM) by three methods - static, hydrodynamic, or fibrin hydrogel-assisted seeding. The resulting cell-scaffold constructs were cultured in vitro by static flask culture or hydrodynamic culture. The initial cell density and the subsequent in vitro proliferation and alkaline phosphate activities of the constructs were analyzed. The constructs were also subcutaneously implanted in nude mice to examine their in vivo osteogenic activities. Hydrogel-assisted seeding gave the highest seeding efficiency, followed by hydrodynamic and conventional static seeding. During in vitro culture, hydrodynamic culture produced higher plateau cell densities, alkaline phosphatase (ALP) activities, and extracellular matrix production than static culture. After subcutaneous implantation in nude mice, the implants prepared by the combination of hydrogel-assisted seeding and hydrodynamic culture produced higher wet weight and bone mineral density than implants prepared by other methods. The results suggest that the hydrogel-assisted seeding can substantially increase the initial seed cell density in scaffolds. Subsequent hydrodynamic culture can promote the proliferation and osteoblastic differentiation of the seeded cells. Correspondingly, bone grafts produced by the combination of these two methods achieved the highest osteogenic activity among the three methods employed.This study aimed to study the effects of initial cell density and  The bone tissue engineering technique provides a new approach to this problem in vitro culture of cell-scaffold constructs. A dynamic fluid flow was found to positively affect the behavior of seeded cells, such as proliferation, differentiation, and migration Currently, cells are seeded primarily by static or hydrodynamic methods. In the static method, a suspension containing seeded cells is dispensed on a scaffold, followed by a period of rest to allow the cells to enter the scaffold. With this method, the initial cell density (the number of cells which attached in 3D scaffold when tissue engineering bone were preparation and without culturing in vivo or in vitro) in the scaffold can be increased by increasing the cell concentration of the suspension within a certain range, though at the expense of seeding efficiency (i.e. the percentage of cells that entered the scaffold), but cannot be further increased beyond a plateau level et al increased the initial density of human umbilical cord mesenchymal stem seeded cells in injectable bone tissue engineering constructs by using hydrogel microbeads et al seeded chondrocytes onto polyglycolid acid scaffolds under hydrodynamic conditions, and obtained intermediate initial cell densities and sustained subsequent proliferation in vitro maturation of the cell-scaffold constructs into clinically applicable grafts.A number of studies have separately exploited the advantages associated with a higher initial cell density or hydrodynamic culture Here, we investigate whether a combination of fibrin glue-assisted seeding and hydrodynamic culture in rotating wall vessel bioreactor can substantially improve the seeding efficiency and subsequent proliferation and osteoblastic differentiation. We further determined if these improvements translated into enhanced osteogenic activity in a nude mice subcutaneous implantation model. This study aims to understand the effects of the key factors of tissue engineering preparation methods, including initial cell density and hydrodynamic culture methods, in an attempt to provide experimental basis for improvement the osteogenesis performance of bone tissue engineering.Nude mice (6 weeks old) were purchased from the Laboratory Animal Center of our university. The animal experiment was approved by the ethics committee of Third Military Medical University and conducted in conformity with the ‘Guiding Principles for Research Involving Animals and Human Beings’ as adopted by The American Physiological Society.Human mesenchymal stem cells (hMSCs) derived from bone marrow of the iliac crests of young healthy volunteers were provided from Tissue Engineering Research and Development Center of The Third Military Medical University. hMSCs were isolated by density gradient centrifugation in Percoll  and adherent culture. The cells were expanded by in vitro culture and confirmed to be hMSCs by a previously published method Cubes of human demineralized cancellous bone matrix  were obtained from the tissue bank of our university and used as the scaffolds in this study. The porosity of DBM is 70% and the pore size is 300–800 µm. The DBM was prepared by a series process, as previously described by Tan et al. Four kinds of bone substitutes were constructed based on different approach of seeding and culture .7 MSCs were added into the high-aspect ratio vessel of a rotary cell culture system . The vessel was filled with 50 ml of DME/F12 culture medium  and degassed. The rotation speed was adjusted daily (18–24 rpm) to ensure that the rotating trajectories of the scaffolds would not collide with the vessel wall or converge to the center. The rotary culture system was incubated in an atmosphere of 5% CO2 and 100% relative humidity at 37°C, with daily adjustment of rotation speed and a change of medium every 48 h.Hydrodynamic seeding and hydrodynamic culture (group A): Fifty DBM scaffolds and 5.0×107 cells/ml. The suspension was added dropwise onto the DBM scaffolds (0.05 ml/scaffold). Then 0.05 ml of catalytic agent was sprayed on each scaffold, and was allowed to stand for 5 min for the glue to gelate. Then the scaffolds were added into the high-aspect ratio vessel of a rotary cell culture system, and were cultured with the same condition of group A.Hydrogel-assisted seeding and hydrodynamic culture (group B): The fibrin glue  was prepared by mixing fibrinogen and thrombin within 90 min under sterile condition. The hMSCs were mixed with a fibrin glue to form a suspension containing 2×107 cells/ml and added dropwise onto the DBM scaffolds (0.05 ml/scaffold). The seeded scaffolds were statically cultured in 24-well plates without additional medium for 2 h, then turned over to reduce the downward flow of liquid and the cell loss, and cultured in the same conditions for another 2 h. Then, each well was filled with 1 ml DMEM/F12 and incubated in an atmosphere of 5% CO2 and 100% relative humidity at 37°C, with a change of medium every 48 h.Static seeding and static culture : The hMSCs were dissociated in DMEM/F12 medium into a suspension containing 2×102 and 100% relative humidity at 37°C, with a change of medium every 48 h.Hydrogel-assisted seeding and static culture (group D): DMB scaffolds were seeded by the same hydrogel-assisted method as in the group B and were cultured statically in 24-well plates  containing DME/F12 medium (1 ml/well) and incubated in an atmosphere of 5% COTwenty four hours after seeding, the seeding efficiency of each group was analyzed, which was defined as the ratio of the number of cells existing in the scaffold to the number of cells added originally to the scaffold. After transferring bone substitutes to other wells, non-adherent cells in the well were collected by rinsing repeatedly with DMEM/F12 medium. Then, the cells attached to well bottom were digested with 0.25% trypsin plus 0.01% EDTA and collected. The cells in the supernatant and those adhering to the bottom of the well were separately counted by hemocytometer. The sum of these two portion of cells was recorded as ‘remaining cell number’ in each well. The seeding efficiency was calculated by: /initial cell number.The viabilities of cells in scaffolds were assayed at various time points . The cell-scaffold constructs were removed from their medium, rinsed with phosphate buffered saline (PBS), and placed in a 96-well culture plate. Cell viability was determined as reported 2, 37°C, 100% relative humidity). The absorbance at 405 nm was measured on a microplate reader and converted into the ALP activity against a standard curve, which was established based on the reaction of 10 ml of a p-nitrophenyl solution (Wako) and 200 ml of substrate buffer for 30 min. ALP activities were expressed as U values.The ALP activities were measured at various time points  after seeding. The cell-scaffold constructs were rinsed twice with PBS and then lysed with 0.2% Triton X-100 . The lysate was centrifuged at 600 g for 5 min and the supernatant was collected and incubated for 15 min  glutaraldehyde solution for 48 h at 4°C, washing over-night with 0.1 M PBS solution, decalcifying with 0.5 mol/L EDTA for 7 days, washing over-night with 0.1 M PBS solution, incubating in 2.5% glutaraldehyde solution for 48 h at 4°C, and post-fixing and staining with 1% osmium tetroxide for 2 h at 4°C, dehydrating in a graded series of ethanol, coating with an ultrathin gold layer. Then the specimens are observed under SEM  to assess investigate morphological features of the attached cells and ECM fibers formed on scaffolds.in vivo osteogenetic activities of scaffolds and cell-scaffold constructs were evaluated with a subcutaneous implantation model in 24 nude mice. Each mouse received four implants in its back, termed implants I–IV , cell proliferation in the scaffold increased by 10 fold (data not shown). Therefore, to ensure an equal cell density before in vivo implantation, the initial cell density for implant III was 9 times greater than implant II. Implant IV was a construct seeded with 2×107/ml hMSCs by the hydrogel-assisted method followed by static flask culture for 12 d; it was placed at the right caudal position.The nts I–IV . ImplantThe mice were killed by decapitation 4, 8, and 12 weeks after implantation . Each mouse was placed in a supine position and examined by X-ray radiography. The implants were retrieved, stripped of soft tissues, and weighted wet. They were also analyzed by dual-energy X-ray absorptiometry  for bone mineral densities.Histological analysis of the implants was undertaken at 12 weeks after surgery. Specimens from the bone defect sites were fixed with 4% paraformalclehyde for 48 hours, decalcified with 0.5 mol/L EDTA for 2 weeks, dehydrated with gradient ethanol solutions for 2 days, vitrified with dimethylbenzene, embedded in paraffin, and cut to yield 6 µm thick sections. The sections were stained with haematoxylin and eosin (H&E) for histological evaluation and examined under light microscope.Data were expressed as mean ± standard deviation. Data were analyzed by one-way analyses of variance (ANOVA) and Student–Newman–Keuls (SNK) post hoc tests using SPSS 12.0 . A p-value of less than 0.05 was considered statistically significant.The hMSCs tended to form calcium nodus after 12 d conditional culture . The hMSIn group A (dynamic seeding followed by dynamic culture), after culture for 8 h, a small number of cells were observed on the surface of the pores in the scaffolds. Then, the cells gradually increased in number and became uniformly distributed on the surface of the pores. After culturing for 5 days, the cells started to produce ECM, the cells and ECM were both uniformly distributed . In grouThe number of attached cells and density of ECM fibers in the interior of the scaffold after 14-day culture are significantly different among four groups. The number of attached cells and density of ECM fibers in group B is most and the cells and ECM were uniformly distributed in the scaffold . The celThe seeding efficiency was 32.10±0.72% in group A and 25.24±1.56% in group C, compared with 82.14±1.09% in group B and 81.53±1.37% in group D. Groups B and D were similar (p = 0.396), and the differences between all other group pairs were highly significant .In group A, the cell number remained stable during the first 3 days, followed by a continuous increase till day 14. In group B, the cell number was stable during the first 2 days, then started to increase, and attained a plateau on day 12. In group C (control group), the cell number  remainedThe ALP activities in all groups increased from day 2 to day 6 . The actSEM revealed similar results to optical microscopy. The number of cells and density of ECM fibers in group B  is most,7/ml hMSCs, followed by dynamic culture for 12 d) showed substantially higher density than the other implants, and implant I (cell-free DBM scaffold) had the lowest densities at both time points. At week 12, implant I showed a slightly higher density compared with the host soft tissue, while implant II clearly showed increased density indicating calcification. The implants III (hydrogel-assisted seeding of 1×108/ml hMSCs without further in vitro culture) and IV (hydrogel-assisted seeding 2×107/ml MSCs followed by static culture for 12 d) also showed signs of calcification, but substantially weaker than that in implant II.The radiographic densities of all implants increased from week 4 to week 12 . ImplantTwelve weeks after implantation, implant I showed a significantly lower wet weight compared with other implants . Moreover, the wet weight of implant II was statistically higher than the implants III (p = 0.008) and D (p = 0.004). Implants III and IV were similar (p = 0.770) .Twelve weeks after implantation, implant II showed a significantly higher bone mineral density than all other implants . The bone mineral density of implant I was significantly lower than the other implants . Implants III and IV were similar (p = 0.741) .Twelve weeks after implantation, implant I  showed pIn the present study, we evaluated the effects of seeding methods on seeding efficiency and initial cell density for constructing tissue-engineered bone. Compared with other synthetic bone substitutes, tissue-engineered grafts generally have superior osteogenic activities because of the incorporation of seeded cells. Various factors can influence the osteoblastic differentiation of marrow stromal cells in tissue engineering scaffolds during cultivation, including the density and spatial distribution of the seeded cells in the scaffolds Seeded cells are commonly seeded in scaffolds by static infiltration. Although convenient, this method can attain only limited cell density. Under the action of gravity force, the seeded cells are easily detached from the scaffold and became concentrated at its bottom side, thus resulting in loss of cells. Various methods have been used to promote cell penetration and minimize cell detachment Hydrogels have been combined with seeded cells to construct grafts for the repair of cartilage as well as bone In this study, we compared the seeding efficiency and initial cell density resulting from three seeding methods: fibrin hydrogel-assisted seeding, hydrodynamic seeding (simulated microgravity in RWVB), and the simple static infiltration. Microscopy, cell counting, and viability assays showed that fibrin hydrogel-assisted seeding generated a significantly higher seeding efficiency and initial cell density than the other two methods. The improvement can increase the utilization of seeded cells and is expected to increase the osteogenic activity of the resulting grafts.Fibrin glue has been clinically confirmed to be safe, biocompatible, and fully absorbable within two weeks in vitro. There is still no consensus on whether tissue-engineered bone grafts need to be cultured in vitro before implantation. Many studies have suggested that in vitro culture can allow the seeded cells to stably adhere on the scaffold and, thereby, prevent their detachment, migration, or death resulting from changes of microenvironment in vivo condition should be optimal for the growth, differentiation, and function of cells. In contrast, in vitro cultured constructs may be structurally unstable, mechanically weak, and subject to changes in tissue structure and type in vivo microenvironment, some studies also explored ectopic implantation to engineer mature, vascularized bone grafts in vivo engineered” grafts were found to have superior osteogenic activities, but the technique involves a long in vivo culture and additional damage to the patient.We further identified the effect of hydrodynamic culture on cell proliferation and differentiation in vivo microenvironment, promote mass exchange, and create appropriate mechanical stimuli. These improvements may be used to produce more mature and bioactive tissue-engineered grafts Recent development of bioreactor techniques has made it possible to better simulate the In this study, we found that when comparing static and hydrogel-assisted seeding, the statically cultured cell-scaffold constructs achieved lower plateau values. In comparison, regardless of the initial cell densities, the dynamically cultured constructs showed continued increase in cell density and became approximately two times higher than the statically cultured grafts. Furthermore, with a higher seeding efficiency and cell density by the hydrogel-assisted seeding, group B achieved plateau earlier than the group A. The ALP activities of the constructs  followedThe favourable effect of hydrodynamic culture may be attributed to three factors. First, the vortex in the bioreactor generated fluid flow in the construct, which enhanced mass transfer and improved the cell distribution 7 cells/ml, 0.05 ml), followed by hydrodynamic culture for 12 days to achieve the plateau cell number and, hypothetically, the best osteogenic activity. Its superior performance confirmed that the combination of hydrogel-assisted seeding and hydrodynamic culture is a promising protocol for tissue-engineering bone grafts.In this study, we also observed that osteogenic activity could be influenced by the initial cell number and in vitro culture methods. Ectopic osteogenesis in nude mice is a widely used method for evaluating the performance of bone substitutes. Moreover, subcutaneous implantation is a challenging model for the implants because of the lack of osteoblast progenitors in the implantation area. Twelve weeks after implantation into the subcutaneous pocket, implant I (cell-free DBM) was filled mainly by soft tissues and showed only slight increase in radiographic density, indicating its lack of osteogenic activity in this site. Implant II showed the highest osteogenic activity according to radiography, histology, wet weight, and bone mineral density. This implant was seeded by the hydrogel-assisted method (2×10in vitro culture. Therefore, a comparison between implants III and II demonstrated that the in vitro culture increased the osteogenic activity of implants. The increase may be attributed to several aspects. The in vitro culture increased the number of seeded cells, and allowed the cells to adhere more stably to the scaffold and thus prevented their detachment after implantation. The cells might also rearrange in order to more effectively interact and communicate with each other in vitro culture, which accelerated the subsequent osteogenesis in the subcutaneous pocket.Implant III showed an intermediate osteogenic activity between the implants I and II. This implant was seeded with the same number of hMSCs as implant II by the hydrogel-assisted method, and was immediately implanted without in vitro before implantation. Its inferior performance may be primarily attributed to its lower cell number as a result of the static culture, which lacked mechanical stimulation for the cells to proliferate and differentiate Similarly, implant IV also showed lower osteogenic activity than implant II. Compared with implant II, implant IV was seeded with the same number of cells but statically cultured in vitro and in vivo results suggest that hydrogel-assisted seeding can significantly increase the seeding efficiency and the initial cell density in the cell-scaffold construct. A subsequent hydrodynamic in vitro culture can significantly increase the plateau cell density. Correspondingly, bone grafts produced by the combination of these two methods can achieve the highest osteogenic activity. These findings can have a significant bearing in clinical applications and in optimizing tissue engineering strategy.In summary, both


EHP 120(4):501–507; Bellinger].Many studies report the impact of various environmental chemicals on children’s neurological development as “clinically insignificant.” But this designation, which describes the individual child, does not always reflect the broader implications of exposure for the overall population—i.e., that a mild but frequent impact could add up to a substantial population-level burden. A new analysis establishes priorities for reducing adverse neurodevelopmental impacts for U.S. children aged 5 years and younger [The author assessed the population impact of various risk factors, including exposure to environmental chemicals, on Full-Scale IQ (FSIQ) scores. FSIQ values are a useful measure for such an assessment because past studies have shown that these scores correspond to work productivity and associated lifetime earnings. FSIQ declines therefore can be used to estimate economic consequences for various risk factors.The data necessary for such an analysis, including information on both prevalence of exposure and effects, were available for only 3 environmental chemicals: lead, methylmercury, and organophosphate pesticides. The loss in FSIQ points was also calculated for a variety of medical conditions, neurodevelopmental disorders, traumatic brain injury, failure to thrive as a result of neglect and abuse, and iron deficiency. Loss of IQ points attributable to each risk factor was estimated based on meta-analyses or pooled analyses of existing data, then generalized to the 25.5 million children aged 0–5 years estimated to live in the United States, based on the prevalence of exposure to each risk factor.Preterm birth resulted in the largest population health burden, with estimated losses of more than 34 million IQ points populationwide, followed by lead exposure at 22.9 million points lost. Organophosphate pesticides and attention deficit/hyperactivity disorder both resulted in estimated losses of more than 16 million points, iron deficiency in 9.4 million points, pediatric bipolar disorder in 8 million points, autism spectrum disorders in 7 million points, brain injury in 5.8 million points, and failure to thrive in 5.3 million points. The remaining risk factors resulted in estimated cumulative IQ losses of fewer than 1 million points each. Interactions between factors certainly exist, although the current study was not able to assess these.A surprising finding was that a large fraction of the total estimated IQ loss associated with lead was contributed by children in the lower reaches of the blood lead distribution—that’s because so many children have low levels of exposure. But the author estimates that reductions in U.S. lead exposure nevertheless “saved” more than 100 million IQ points between the 1976–1980 and 2005-2006 iterations of the National Health and Nutrition Examination Survey. This suggests that similar reductions in nations where lead exposures remain high could result in dramatic improvements in neurodevelopment on a population level.


The study included 20 neonates . The Large Intestine 4 acupuncture point  was stimulated by a microlaser needle  for 5 minutes, bilaterally. All neonates underwent polygraphic recording during undisturbed daytime sleep, including heart rate (HR), peripheral oxygen saturation (SpO2), and measurement of nasal flow. Using NIRS, rcSO2 was measured continuously. Cerebral fractional tissue oxygen extraction (cFTOE) was calculated. We did not observe any significant changes in SpO2 and HR values during the whole observation period. However, there was a significant decrease in rcSO2 (P = 0.003) within postintervention period, accompanied by a significant increase in cFTOE (P = 0.010) in postintervention period.Laser acupuncture (LA) becomes more and more relevant in neonates and infants. With near-infrared spectroscopy (NIRS), a continuous and noninvasive measurement of tissue oxygenation is possible. Aim was to investigate, whether the application of LA was associated with any changes in regional cerebral oxygen saturation (rcSO Hegu) or Liv 3 (Taichong) induced specific patterns of brain activity in adults [There is an increasing interest in complementary and alternative medicine (CAM) in particular in herbal medicine, homeopathy, and traditional Chinese medicine (TCM) to treat the pediatric population –3. TCM hn adults  and chiln adults . This brn adults . In 1978n adults  firstly n adults  and concn adults , 23. HowPreterm and term newborns admitted to the Neonatal Intensive Care Unit of the University Hospital of Graz scheduled for sleep studies were included. Infants were excluded with known genetic anomalies or major malformations, pathology in cranial ultrasound or abnormal neurological examination, elevated bilirubin levels, suspected intrauterine infection, and suspected sepsis or septic shock. Infants >28 weeks gestation were also excluded if they had any need for respiratory support or oxygen during study phase. Infants <28 weeks gestation were only included if they required <30% oxygen or had only needed continuous positive airway pressure. The university ethics committee approved the study, and parental written informed consent was obtained.Measurements were performed after 2 hours of undisturbed daytime sleep in supine position using a Babytherm 8000 incubator . During the measurements no medication was administered; the ambient temperature and humidity were kept constant. The infant was comforted with a pacifier if needed, and the eyes were protected with an eye shield  as previously described .Heart rate, oxygen saturation, and breathing movements were recorded. Cerebral oxygenation was measured using NIRO 300 . The optodes were placed on the left side of the forehead with an interoptode distance of 4 cm and a sampling rate of 2/s. Light shielding was performed with a slim cap. 2Hb), deoxyhemoglobin (HHb) concentration, and regional oxygen saturation ”). Measurement of rSO2 was performed using the Spatially Resolved Spectroscopy (SRS) method, in which the tissue absorption coefficient is determined from the regionally dependent weakening of light. A detector especially developed for the SRS method registers the emitted near-infrared light from the light source into tissue and allowed rSO2 calculation using the SRS algorithm. All parameters were stored within a multichannel system.The NIRO 300 continuously measures changes in the oxyhemoglobin (OHegu) was selected. LI 4 (Hegu) is located in the Large Intestine Meridian in the middle of the 2nd metacarpal bone on the radial side  and a radiation time of 5 min resulted in an power density of about 1,5 J/cm² per acupuncture point [The laser needle used for acupuncture provides continuous laser light with a wavelength of 685 nm and an output power of 10 mW per laser needle. An output power of 10 mW (diameter 500 re point .Hegu). Once the baby fell asleep, the fixed laser needle was activated for 5 minutes and left undisturbed on the child's LI 4 (Hegu), bilaterally (for at least 10 minutes). Before LA was performed, the skin at the acupuncture point was disinfected, and the laser needles were fixed to the skin with a special adhesive tape, bilaterally at LI 4 , heart rate (HR), regional cerebral oxygen saturation (rcSO2) were calculated for the two baseline periods, as well as for the 10 intervention periods. Cerebral fractional tissue oxygen extraction (cFTOE) was calculated for each period ((SpO2 − rcSO2)/SpO2) [2, HR, rcSO2, and cFTOE within the intervention period compared to preintervention and postintervention periods using a linear mixed model with a fixed effect for time and a first order autoregressive covariance structure. A P value of < 0.05 was considered as a statistical significance. The statistical analyses were performed using IBM SPSS Statistics .The prospective protocol consisted of two baseline periods, each lasting 5 minutes, one before (preintervention period), one after intervention (postintervention period). To depict dynamic changes during intervention, the intervention period was divided into 10 periods, each lasting 30 seconds. Mean values of peripheral oxygen saturation (SpO2)/SpO2) . Data ar6/7 to 406/7 weeks and a birth weight of 690 to 3680 g. Mean (range) measurements were performed on day 22 (11–68) after birth. The study group encompassed 20 neonates with a gestational age ranging from 262 and HR values during the whole observation period. However, there was a significant decrease in rcSO2 (P = 0.003) within postintervention period, accompanied by a significant increase in cFTOE (P = 0.010) in postintervention period  in postintervention period, (ii) significant increase in cerebral fractional tissue oxygen extraction (cFTOE), and (iii) no changes at all in peripheral oxygen saturation or heart rate during the whole observation period. This is the first study to demonstrate changes in cerebral tissue oxygenation in association with LA in term and preterm infants. There is an increasing interest in complementary medical treatment of infants and newborn; however, the evidence is scare. A recent meta-analysis demonstrated that  acupunct2, which did not show any significant changes either. Therefore, we hypothesize that the observed significant changes in rcSO2 and cFTOE were due to changes in regional cerebral tissue perfusion and oxygen extraction. Our findings suggest that there was a decrease in local arterial blood supply after discontinuation of LA, expressed in a decrease of rcSO2. This was accompanied by an increase in regional tissue oxygen extraction, expressed in an increase of cFTOE. Furthermore, we hypothesize that the increase of local cerebral tissue perfusion may have been a slow process, and that may be the reason why there were no significant changes measureable during LA. There are some similarities to reported results of LA in adults [2Hb and TOI after needle and laser acupuncture in adult patients [Oxygen delivery is the product of blood flow and oxygen content. In neonates, the cardiac output is dependent on HR. In our study, there were no significant differences in HR, which leads us to assume that there were no differences between both groups as regards to blood flow. The oxygen content of arterial blood supplying the brain equates SpOn adults , 30. Litpatients . Furtherpatients . (Hegu), which is considered to be one of the most effective acupuncture points for general pain control, was used in the current study. Manual acupuncture applied to LI 4 (Hegu) activates the sympathetic and parasympathetic nervous systems in healthy individuals [Hegu) bilaterally resulted in a more immediate effect than unilateral stimulation [Hegu) has been used to treat infantile colic [Hegu) induces specific patterns of brain activity in adults and children during manual acupuncture, electroacupuncture, and LA [LI 4 ividuals . Stimulamulation . LI 4  the results could be more significant, especially during LA . But based on our recent published data about the changes of the skin temperature in preterm infants undergoing laser acupuncture , it seem2 during postintervention period. This was accompanied by a significant increase in cFTOE. This was in contrast to SpO2 and HR, where no changes could be observed. Therefore, we hypothesize that observed changes were due to changes in regional cerebral perfusion and oxygen supply. This is the first study to demonstrate changes of cerebral tissue oxygenation caused by laser acupuncture in term and preterm neonates.There was a significant decrease in rcSO