[fetal growth restriction]{.smallcaps} (FGR) affects 3--10% of babies worldwide and is associated with increased risk of stillbirth, postnatal disability, and impaired motor and cognitive functions. FGR also has lifelong health implications for affected babies, with increased associated risk of hypertension, cardiovascular disease, stroke, and diabetes in adulthood ([@B1], [@B2], [@B3], [@B35]). Currently, early delivery is the only treatment for FGR, resulting in further risks of iatrogenic morbidity and mortality ([@B37]).

There are several causes of FGR, including maternal undernutrition, fetal chromosomal anomalies, and placental dysfunction, the last of these being the most common cause in Western countries. Placental dysfunction has several different phenotypic characteristics ([@B41]): *1*) the placenta often being smaller, with reduced villous area and thickened exchange barrier ([@B31]); *2*) reduced vascular complexity and abnormalities in uterine and fetoplacental blood flow ([@B25]); *3*) lower activity of nutrient transporters in the syncytiotrophoblast microvillous and basal plasma membranes ([@B36]); and *4*) abnormal placental cell turnover, typified by excessive apoptosis and attenuated proliferation ([@B7], [@B22]).

Normal fetal growth is dependent on interplay between fetal, placental, and maternal factors. One family of growth factors, the insulin-like growth factors (IGF-I and IGF-II) and their cell surface receptors (IGF-IR and IGF-IIR), is known to be critical regulators of this growth ([@B17]). In humans, circulating total IGF-I in fetal cord blood correlates with birth weight, and mutations in the IGF-I or IGF-IR genes result in severe FGR ([@B18]). In mice, ablation of IGF-I or IGF-II genes or elimination of IGF-IR begets severe growth restriction and/or perinatal lethality ([@B30]). Using the placental-specific IGF-II-knockout mouse (P0), in which a placental-specific promoter of IGF-II has been deleted, the additional importance of placentally derived IGF-II on placenta and fetal weight has been verified ([@B10]). IGF-I and IGF-II are mediators of placental cell turnover, proliferation, survival, and differentiation in vitro and modulate fetal growth in utero by promoting nutrient transfer and placental blood flow ([@B16], [@B26], [@B33], [@B42]). Studies in guinea pigs have confirmed the importance of IGF-I in nutrient partitioning from mother to fetus and the role of maternal IGF-II in enhancing placental development and function ([@B38], [@B40]).

Although the roles of IGF-I, IGF-II, and IGF-IR signaling in the human and animal placenta are affirmed, the significance of IGF-IIR in mediating IGF signaling is less well defined. Evidence for signaling through this receptor is accumulating ([@B14], [@B15]), but classically IGF-IIR has been considered a clearance receptor, trafficking excess IGF-II to lysosomes for degradation. This concept is exemplified in IGF-IIR knockout mice, which exhibit fetal and placental overgrowth ([@B45]), as a presumed consequence of surplus IGF-II overactivating fetal IGF-IR.

The development of treatments for FGR is dependent on suitable animal models in which they may be tested, and a number of models have been characterized in sheep ([@B4]), guinea pig ([@B6], [@B28]), and rat ([@B43]). Recent work on gene knockout and transgenic mouse models of pregnancy disease suggests that these may be particularly useful ([@B12]). One such model is the endothelial nitric oxide synthase knockout (eNOS^−/−^) ([@B23]), which may be considered a more amenable model for FGR. eNOS^−/−^ fetuses are 10% smaller than their wild-type (WT) counterparts, and this is associated with abnormalities in placental transporter activity and dysregulated utero- and fetoplacental blood flow, similar to observations in human FGR ([@B11]).

Although systemic administration of IGF-I or -II may be an appealing approach to address placental insufficiency, ubiquitous tissue expression of IGF receptors may cause off-target effects. A possible alternative is to enhance the bioavailability of endogenous IGF already present at the materno-fetal interface. Theoretically, this should be achievable by blocking IGF-IIR. Previously, we have described methods of influencing IGF-II signaling in vitro by siRNA knockdown of IGF-IIR and by the use of an IGF-IIR-specific agonist, \[Leu^27^\]IGF-II, to block IGF-II removal and optimize its availability ([@B21]). In vitro, this approach encourages signaling through IGF-IR, thereby enhancing mitogenesis and cell survival in placental villous explants. A previous in vivo study administering \[Leu^27^\]IGF-II to normal pregnant guinea pigs at midgestation demonstrated modified placental morphology, enhanced exchange capacity, and, most importantly, significantly increased fetal weight at term (whereas IGF-II alone did not) ([@B39]). Here, we hypothesized that the novel treatment of both normal and FGR mice with \[Leu^27^\]IGF-II would potentially increase IGF-II, therefore enhancing fetal growth in WT litters and improving weights in the growth-restricted fetuses of pregnant eNOS^−/−^ knockouts.

METHODS {#sec1}
=======

 {#sec1-1}

### Animals and administration of \[Leu^27^\]IGF-II. {#sec1-1-1}

Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986 under Home Office Licence No. PPL40/3385. The Local Ethical Review Process of the University of Manchester approved all protocols. eNOS^−/−^ were obtained from Jackson Laboratories (strain B6.129P2-Nos3tm1Unc/J). Homozygous eNOS^−/−^ mice were mated, and the presence of a copulation plug was denoted as day 0.5 of pregnancy. C57/BL6J mice, the eNOS^−/−^ background strain, were used as WT mice. Animals had free access to food (Beekay Rat and Mouse Diet; Bantin & Kingman, Hull, UK) and water and were maintained on a 12:12-h light-dark cycle at 21--23°C. On embryonic day (E)12.5, females were anesthetized using isoflurane inhalation and a 100-μl miniosmotic pump (200D; Alzet) surgically inserted subcutaneously in the upper dorsum. Miniosmotic pumps had previously been prepared to deliver vehicle (0.1 M HCl) or 1 mg·kg^−1^·day^−1^ \[Leu^27^\]IGF-II (human recombinant protein; GroPep) in 0.1 M HCl for 6 days at a flow rate of 0.51 μl/h. \[Leu^27^\]IGF-II was delivered to 26 C57/BL6J dams (199 fetuses), and 25 C57/BL6J dams (190 fetuses) received vehicle. In a separate experiment, 11 eNOS^−/−^ dams (74 fetuses) were treated with \[Leu^27^\]IGF-II, and 11 eNOS^−/−^ dams (86 fetuses) were given vehicle. All animals were euthanized on E18.5 by Schedule 1 procedure in accordance with the UK Animals (Scientific Procedures) Act 1986. Fetuses and placentas were blotted and weighed. Placentas were halved and fixed in 4% (vol/vol) neutral-buffered formalin for histological assessment. In animals in which radioactivity had not been used, maternal and fetal serum were collected and frozen in liquid nitrogen.

### Fetal weight distribution curves. {#sec1-1-2}

Fetal weight histograms were constructed as described previously ([@B12]), and a nonlinear regression (Gaussian distribution) analysis was performed. The 5th and 95th percentile weights were calculated as (−Z critical value × SD) + mean for the former and (+Z critical value × SD) + mean for the latter, where Z critical value = 1.645 and SD = standard deviation.

### Unidirectional materno-fetal clearance of ^14^C-MeAIB across the intact placenta. {#sec1-1-3}

The clearance across the placenta of ^14^C-methylaminoisobutyric acid (MeAIB), a nonmetabolizable substrate for the placental amino acid transporter system A, was measured across the intact placenta on E18.5 using an adaptation of the method of Flexner and Pohl (1941), as described previously ([@B5]). Following infusion of ^14^C-MeAIB, exsanguination of the dam occurred between 1 and 5 min postinfusion. Fetuses were rapidly collected and assessed for total radiolabel accumulation and compared with a maternal plasma ^14^C-MeAIB disappearance curve generated from current and historical in-house data. This was constructed from dams on E18.5 (*n* = 109, group data for C57/BL6J) and fitted to a one-phase exponential decay model (*r*^2^ \> 0.6), as described previously ([@B12]). ^MeAIB^Kmf (μl·min^−1^·g^−1^ placenta) was calculated as $$\text{Kmf} = \frac{\text{Nx}}{\text{W}{\int\limits_{0}^{x}\text{Cm}}\left( t \right)\text{d}t}$$

where Nx = total radiolabel accumulation (disintegrations/min) by the fetus at x min after injection of radiolabel into the maternal vein, W = placental wet weight (g) and equals the time integral of radioisotope concentration in maternal plasma (disintegrations·min^−1^·μl^−1^) from 0 to x min (taken from the maternal plasma ^14^C-MeAIB disappearance curve).

### Enzyme-linked immunosorbent assays. {#sec1-1-4}

ELISAs were performed according to the manufacturers\' protocols. Human IGF-II ELISAs to detect \[Leu^27^\]IGF-II were performed at a 1:10 dilution (detection range 0.02--9 ng/ml, intra- and interassay coefficient of variation = 12.7 and 18.0%, respectively; Mediagnost).

### Immunohistochemistry. {#sec1-1-5}

Freshly harvested placental tissues were fixed in 4% (vol/vol) neutral-buffered formalin at 4°C overnight, washed in phosphate-buffered saline (PBS), embedded in paraffin wax, and cut into 5-μm serial sections. These were microwaved in 0.1 M sodium citrate buffer for antigen retrieval, and then endogenous peroxide activity was blocked by placing the slides in methanol containing 0.4% (vol/vol) HCl and 0.5% (vol/vol) hydrogen peroxide for 30 min. Tissue sections were washed three times in 0.05 M PBS and then incubated with 5% BSA to block any nonspecific binding sites. Sections were incubated with polyclonal rabbit anti-IGF-II (UK 1:100; Abcam, Cambridge), rabbit anti-IGF-IR (UK 1:100; Fisher Scientific-UK, Loughborough, UK), rabbit anti-IGF-IIR (1:100; R & D Systems, Abingdon, UK), or rabbit IgG (minus primary antibodies, for negative controls; DakoCytomation, Ely, UK) overnight at 4°C. Sections were washed (3 × 5 min with PBS) and then incubated with secondary antibody (biotinylated goat anti-rabbit IgG; 1:500 DakoCytomation) for 1 h at room temperature, followed by avidin-peroxidase (5 μg/ml in 0.125 M Tris·HCl, pH 7.6; Sigma-Aldrich, Gillingham, UK) for 30 min at room temperature. Slides were washed in Tris-buffered saline and incubated for 1--5 min with 0.05% (wt/vol) diaminobenzidine and 0.015% (vol/vol) hydrogen peroxide (Sigma-Aldrich). Tissue sections were counterstained with hematoxylin and visualized by light microscopy (BX41; Olympus UK, London, UK).

### Statistical analysis. {#sec1-1-6}

Generalized linear mixed-model analysis with Sidak posttest was used to determine the effect of drug treatment on fetal weight of pups from litters of varying size (*P* \< 0.05 was deemed statistically significant). Data were tested for normality following a D\'Agostino & Pearson normality test and subjected to unpaired *t*-test or Mann-Whitney *U*-test accordingly. Variance within groups was determined using an *F*-test of equality of variances (*P* \< 0.05). Correlations were determined by linear regression analysis. All statistical analyses were carried out using GraphPad Prism 5.03 software.

RESULTS {#sec2}
=======

 {#sec2-1}

### The IGF-II axis in murine placenta. {#sec2-1-1}

Previous studies have documented the presence of IGF-II and IGF-IIR mRNA ([@B18], [@B33], [@B44]), but protein localization has been less well defined. Here, we show that IGF-II is localized throughout the decidua, the junctional zone, including giant cells and glycogen cells, and labyrinth, including trophoblast and vessel endothelium ([Fig. 1](#F1){ref-type="fig"}, *A* and *D*). IGF-IR is expressed by giant cells, trophoblast, and glycogen cells in the junctional zone ([Fig. 1*B*](#F1){ref-type="fig"}) and is also present within labyrinthine trophoblast and the surrounding vessels and maternal sinuses ([Fig. 1*E*](#F1){ref-type="fig"}). IGF-IIR displayed a similar localization to IGF-IR, including glycogen cells, trophoblast, labyrinthine trophoblast, and endothelium ([Fig. 1](#F1){ref-type="fig"}, *C* and *F*).

![IGF-II axis localization in murine placenta. *A* and *D*: IGF-II was localized throughout the decidua, junctional zone \[giant cells (arrow) and glycogen cells; *A*\] and labyrinth (trophoblast, arrowhead; vessel endothelium, dashed arrow; *D*). *B* and *E*: IGF-I receptor (IGF-IR) was present throughout the junctional zone (giant cells, arrowhead; trophoblast, thin arrow; glycogen cells, thick arrow; *B*) and labyrinthine trophoblast (arrowhead), with strong expression surrounding vessels and maternal sinuses (dashed arrows) (*E*). *C* and *F*: IGF-IIR displayed a similar localization to IGF-IR, including glycogen cells (thick arrow), trophoblast, (black arrowhead in *C*), labyrinthine trophoblast (black arrowheads in *F*), and endothelium (dashed arrow). Negative staining (minus primary antibody) controls for junctional zone and labyrinth regions also included. Bars, 100 μm.](zh10241574890001){#F1}

### \[Leu^27^\]IGF-II treatment reduces fetal and placental weight variations in WT mice. {#sec2-1-2}

The delivery of \[Leu^27^\]IGF-II to the maternal circulation was confirmed by analyzing serum from treated and untreated dams using an ELISA specific for human IGF-II; only the samples from WT and eNOS^−/−^ mothers that had received \[Leu^27^\]IGF-II contained detectable levels of hIGF-II (WT levels: 42.89 ± 11.05 ng/ml, *n* = 4; eNOS^−/−^: 28.47 ± 13.91 ng/ml, *n* = 3; means ± SE).

Treatment of WT mice with \[Leu^27^\]IGF-II did not alter litter size (7.3 ± 0.4 vs. 7.8 ± 0.3, *n* = 25 and 25, respectively; means ± SE) or number of fetal resorptions (0.52 ± 0.17 vs. 0.69 ± 0.17). Although \[Leu^27^\]IGF-II did not affect mean fetal weight, population frequency distribution curves revealed that the distribution of weights was narrowed (kurtosis = 0.85 vs. 0.03), with an increase in the number of fetuses close to the mean weight (131 vs. 112 pups within 1 SD) ([Fig. 2*A*](#F2){ref-type="fig"}). Concomitantly, treatment reduced fetal (*P* = 0.028) and placental weight variations (*P* = 0.003), as assessed by *F*-test of equality of variance ([Fig. 2](#F2){ref-type="fig"}, *B* and *C*). Treatment also halved the number of pups with fetal weights below the 5th centile of the control population (skewness = 1.14 vs. 1.22) from 6.3 (*n* = 11) to 2.5% (*n* = 5). The number of pups with fetal weights above the 95th centile also decreased from 4.7 (*n* = 9) to 3.0% (*n* = 6), in line with a normalization effect. Fetal/placental weight ratios, as a measurement of placental efficiency, were unchanged (\[Leu^27^\]IGF-II-treated ratio = 15.00 ± 0.229, vehicle-treated ratio = 14.94 ± 0.404; means ± SE).

![\[Leu^27^\]IGF-II\'s effects on fetal weight variation and no. of fetal growth-restricted (FGR) pups in normal mice. Leu^27^: *n* = 26 pregnancies, 199 fetuses; vehicle: *n* = 25, 180 fetuses. *A*: C57 population distribution curves for individual fetal weights of \[Leu^27^\]IGF-II- (solid line) and vehicle-treated mice (dashed line). Treatment halved the number of pups with \<5th centile fetal weights from 6.3 to 2.5% (skewness = 1.14 vs. 1.22) and reduced the number of \>95th centile animals from 4.7 to 3%. Individual fetal and placental weights showed reduced variation in treated animals (when tested by *F*-test of equality of variances). *B* and *C*: fetal (*P* = 0.028; *B*) and placental weights (0.0032; *C*).](zh10241574890002){#F2}

### \[Leu^27^\]IGF-II improves fetal weight in large litters in WT mice. {#sec2-1-3}

Generalized linear mixed-model analyses were performed to assess the effects of different variables, including litter size and \[Leu^27^\]IGF-II treatment, on fetal and placental weights. The mean weight of both placentas and fetuses from litters with the largest number of pups ([@B10]) was lower that than of litters with four pups (*P* \< 0.05; [Fig. 3](#F3){ref-type="fig"}, *A* and *B*). Treatment with \[Leu^27^\]IGF-II increased the mean weight of the fetuses (*P* \< 0.05), but not placentas, from large litters; however, neither parameter was affected when litter size was small. Consequently, fetal/placental weight ratios were increased only in large litters (*P* \< 0.05; [Fig. 3*C*](#F3){ref-type="fig"}).

![Mean weight/ratio for litters according to size of litter (nos. of pups) and generalized linear mixed-model analysis of \[Leu^27^\]IGF-II effects. *A*: mean placental weight decreased with increased no. of pups/litter (10 or 9 vs. 4 pups, *P* \< 0.05). \[Leu^27^\]IGF-II had no effect on mean placental weight. *B*: mean fetal weight decreased with increased no. of pups/litter (10 vs. 4 pups, *P* \< 0.05). \[Leu^27^\]IGF-II increased mean fetal weight in largest litters (10 pups). *C*: fetal/placental weight ratio increased in large litters (9 vs. 4, *P* \< 0.05). \[Leu^27^\]IGF-II increased this ratio in the largest litters (10 pups). \**P* \< 0.05.](zh10241574890003){#F3}

### Placental amino acid transport is inversely correlated with fetal weight following maternal \[Leu^27^\]IGF-II treatment in WT mice. {#sec2-1-4}

To assess the impact of \[Leu^27^\]IGF-II on placental function, unidirectional materno-fetal clearance of ^14^C-MeAIB (^MeAIB^Kmf) across the placenta, a measure of in vivo system A amino acid transporter activity, was investigated. Activity was not significantly altered following \[Leu^27^\]IGF-II treatment; however, like the variation in birth weight, there was significantly less variation in clearance between the litters of treated animals (*F*-test *P* = 0.05; [Fig. 4*A*](#F4){ref-type="fig"}). System A amino acid transporter activity was inversely correlated with placental weight in both \[Leu^27^\]IGF-II- and vehicle-treated animals, with smaller pups exhibiting increased transfer per gram placenta ([Fig. 4*B*](#F4){ref-type="fig"}). An inverse correlation between fetal weight and system A amino acid transporter activity per gram placenta was observed only in the \[Leu^27^\]IGF-II-treated group (*P* \< 0.01; [Fig. 4*C*](#F4){ref-type="fig"}).

![System A amino acid transporter activity in \[Leu^27^\]IGF-II- (*n* = 7, 51 fetuses) and vehicle-treated (*n* = 8, 60 fetuses) C57 mice. *A*: unidirectional ^14C^MeAIB transfer per gram placenta (kmf) (median ± interquartile range). Variation was significantly reduced in \[Leu^27^\]IGF-II-treated mice (*P* = 0.05). *B*: transfer (kmf) was inversely correlated with placental weight in both groups (\[Leu^27^\]IGF-II: *P* = 0.011, *r*^2^ = 0.198; vehicle: *P* = 0.0007, *r*^2^ = 0.186). *C*: transfer (kmf) is inversely correlated with fetal weight in \[Leu^27^\]IGF-II-treated animals only (*P* \< 0.01, line of best fit presented).](zh10241574890004){#F4}

### \[Leu^27^\]IGF-II treatment reduces the number of growth-restricted fetuses in eNOS^−/−^ mice. {#sec2-1-5}

In eNOS^−/−^ mice, \[Leu^27^\]IGF-II did not affect mean litter fetal or placental weight but significantly increased individual fetal weights (*P* = 0.0263; [Fig. 5*A*](#F5){ref-type="fig"}) and reduced the number of eNOS^−/−^ fetuses below the eNOS^−/−^ population 5th centile by threefold \[8.8 (*n* = 5) vs. 2.6% (*n* = 1)\]. Treatment also reduced the number below the 5th centile of the WT distribution by 9% (35--44%) ([Fig. 5*B*](#F5){ref-type="fig"}). Placental weights and the variation of fetal weights around the population mean were unchanged (\[Leu^27^\]IGF-II-treated placental weight = 0.0854 ± 0.0012, vehicle-treated placental weight = 0.0873 ± 0.0015; means ± SE).

![\[Leu^27^\]IGF-II\'s effects on fetal weight in endothelial nitric oxide synthase (eNOS) mice (means ± SE). *A*: population distribution curves for individual \[Leu^27^\]IGF-II-treated eNOS (solid line), vehicle-treated eNOS (dashed line), and vehicle-treated wild-type (WT) (C57; gray line) mice. Treatment reduced the no. of \<5th centile eNOS fetuses and the no. of \<5th centile fetuses of the C57 population. *B*: individual fetal weights were increased (*P* = 0.0263).](zh10241574890005){#F5}

DISCUSSION {#sec3}
==========

The importance of IGF-II signaling in mouse fetal growth is well described, with both total and placental specific IGF-II knockout transgenic animals associated with impaired fetal and placental development ([@B9], [@B10]). Since systemic administration of exogenous IGF-II may have off-target effects, we investigated an alternative approach to optimize fetal growth via constant \[Leu^27^\]IGF-II infusion in an effort to enhance endogenous IGF-II availability. As reported in the guinea pig ([@B32]), we have shown that it is possible to deliver a constant infusion of the IGF-IIR-specific analog \[Leu^27^\]IGF-II systemically to pregnant mice, without impact on litter size or resorptions. Moreover, with apparent benefits to fetal weight, specifically with regard to the most vulnerable (smallest) pups within the litters in normal and growth-restricted mouse pregnancies, this study holds implications for the further understanding and optimization of fetal growth in utero.

To manipulate the IGF axis at the cellular level, we first confirmed the presence of its constituent components within the mouse placenta. Endogenous IGF-II as well as both IGF receptors, IGF-IR and IGF-IIR, were present throughout the murine placenta, including labyrinthine cells, where nutrient exchange occurs. These findings complement those of others, who have described similar IGF-II mRNA and protein distributions, alongside colocalization of IGF-IR and IGF-IIR principally in mouse trophoblast and vascular endothelium ([@B44]). Therefore, theoretically at least, enhancing endogenous IGF-II is an attractive strategy to stimulate placentally driven fetal growth.

We demonstrated that constant subcutaneous infusion of \[Leu^27^\]IGF-II into WT pregnant mice from midgestation onward reduces the number of fetuses with weights below the 5th centile of the study population. Although fetal sex difference was not considered, we showed \[Leu^27^\]IGF-II to have its greatest effect in preferentially increasing fetal weights within the smallest, lightest WT fetuses. Because normal animals would be expected to exhibit optimal fetal growth, it could be reasoned that any beneficial effects of treatment might be restricted to fetuses of greater in utero compromise, for example, those under nutritionally stressed circumstances. Therefore, we concluded that pups in large litters compete with varying effectiveness for maternal resources, producing a range of weights in the offspring, and any positive impact on smaller pups can restrict intake by larger pups in the same litter, thus normalizing fetal weights. Within our data, a general inverse relationship between litter size and fetal and placental weights was noted. In the absence of direct changes in placental weight, we envisage this enhancement to be through an exaggeration in functional capacity.

Previous work in WT and P0 knockout mice has shown that an increased placental efficiency, as shown by an increased fetal/placental weight ratio, is associated with increased ^MeAIB^K~mf~ ([@B8], [@B9], [@B10]). This suggests that increased placental system A amino acid transporter activity at least partially underpins the increased placental efficiency. Therefore, we investigated the relationship between ^MeAIB^K~mf~ and placental and fetal weight in the presence and absence of \[Leu^27^\]IGF-II by analysis of unidirectional materno-fetal clearance of MeAIB (^MeAIB^Kmf) across placentas as a measure of system A amino acid transport activity. Our data show a strong inverse correlation between ^MeAIB^K~mf~ and placental weight in both the presence and absence of the hormone analog; this is consistent with previous studies linking an increase in placental efficiency with enhanced system A activity. Interestingly, although there was no correlation between ^MeAIB^K~mf~ and fetal weight, which was expected since the increased placental efficiency would tend to drive fetal growth to a constant level across a litter, there was an inverse correlation in the \[Leu^27^\]IGF-II treated animals. This latter result is intriguing but difficult to decipher. With the relative interplay between placental and fetal weights uncertain, even in WT mice, the uncoupling of this relationship by \[Leu^27^\]IGF-II by either its effects on amino acid transport or a shift in allocation of resources is a possibility, but one requiring further investigation.

In support of our findings in the mouse, Sferruzzi-Perri et al. ([@B39]) have previously demonstrated beneficial effects of \[Leu^27^\]IGF-II infusion in pregnant guinea pigs from midgestation onward. Resulting increases in fetal weight were attributed to enhanced placental functional activities, with increased system A activity and glucose transfer per gram of placenta. Consistent with the concept that \[Leu^27^\]IGF-II can normalize (and arguably optimise) fetal growth, we demonstrated a reduction in litter variability in system A transport in the mouse, mirroring its impact on placental and fetal weights and potentially explaining it.

In light of improved fetal weights in constitutively small offspring of WT mice, we investigated for the first time the effects of maternal \[Leu^27^\]IGF-II infusion in a pregnant mouse model of FGR (eNOS^−/−^). \[Leu^27^\]IGF-II increased eNOS^−/−^ fetal weights, reducing numbers below the 5th centile, akin to that of wild-type mice. Although \[Leu^27^\]IGF-II failed to restore mean fetal weights to WT levels, the clinical relevance of such an outcome should not be overlooked, as preferential growth in the smallest offspring could increase fetal weights above critical centiles for both viability and in utero programming, thereby having a significant impact on fetal, neonatal, and longer-term well being in humans. In defining mechanisms, it is suggested that \[Leu^27^\]IGF-II, like IGF-II, may promote uterine angiogenesis and vascular remodelling ([@B24], [@B39]), improving blood flow and nutrient delivery. Although not assessed in this study, the eNOS^−/−^ mouse exhibits altered uterine vascular function, which may be improved through \[Leu^27^\]IGF-II exposure ([@B41]).

There are a number of signaling mechanisms by which \[Leu^27^\]IGF-II may elicit either maternal or fetal effects. Previous work by our group ([@B21], [@B27]), using human placental explants, indicates that likely saturation of IGF-IIR by \[Leu^27^\]IGF-II may enhance endogenous IGF-II bioavailability and increase signaling through IGF-IR, augmenting its well-documented growth-promoting properties. Within that study, decreased IGF-IIR expression increased an IGF-II-mediated impact on mitosis and placental cell survival. Alongside these observations and the knowledge that IGF-IIR knockout mice elicit fetal overgrowth, these findings imply a role for IGF-IIR blockade in stimulating tissue growth via increasing IGF-II bioavailability and in turn IGF-IR activation ([@B29]). Furthermore, the effects on maternal adiposity described by Sferruzzi-Perri et al. ([@B39]), which were similar to those observed previously following IGF-I (but not IGF-II) treatment, also support the concept of endogenous IGF-II displacement to enhance its interaction with IGF-IR. In addition, IGF-IIR may itself have direct signaling capabilities, rather than acting exclusively as a clearance receptor ([@B21]). Further in vitro studies have demonstrated the capacity of \[Leu^27^\]IGF-II signaling via IGF-IIR to stimulate activation of MARK3/1, resulting in trophoblast migration and cell survival ([@B32]). Therefore, although signaling pathways within pregnant mice remain uncertain, both direct and indirect IGF receptor interactions may be balanced within the placenta to ensure appropriate placental and fetal growth.

In summary, we have defined a reduction in FGR pups in WT and eNOS^−/−^ litters exposed to a maternal exogenous analog of IGF-II. Under normal circumstances, we propose that the IGF axis is already optimized for any given fetus, and as such \[Leu^27^\]IGF-II-treatment has little or no positive effect on normal mice, as IGF-IIR is saturated. By contrast, in compromised, inadequately growing fetuses (i.e., smaller pups where IGF-II is potentially restricted), \[Leu^27^\]IGF-II acts to promote availability of endogenous IGF-II, restoring placental sufficiency and standardizing fetal growth, rescuing the FGR phenotype.

GRANTS {#sec4}
======

We acknowledge the support of the Castang Foundation in funding this project, as well as an Medical Research Council (MRC) Programme Grant (Sibley G0802770/92495). M. R. Dilworth is supported by a Career Development Fellowship award from the MRC (Grant No. MR/K024442/1).

DISCLOSURES {#sec5}
===========

The authors have nothing to disclose.

AUTHOR CONTRIBUTIONS {#sec6}
====================

J.C.C., J.D.A., C.P.S., M.W., and I.P.C. conception and design of research; J.C.C. and I.P.C. performed experiments; J.C.C., M.R.D., J.D.A., and I.P.C. analyzed data; J.C.C., J.D.A., C.P.S., M.W., and I.P.C. interpreted results of experiments; J.C.C., M.R.D., and I.P.C. prepared figures; J.C.C. and I.P.C. drafted manuscript; J.C.C., J.D.A., C.P.S., M.W., and I.P.C. approved final version of manuscript; M.R.D., J.D.A., C.P.S., M.W., and I.P.C. edited and revised manuscript.

We are very grateful to the staff of the Biological Services Facility, University of Manchester, for their assistance in animal husbandry. We also thank Louise Mayall for assistance in the laboratory.


Q:

Arduino: Lightweight compression algorithm to store data in EEPROM

I want to store a large amount of data onto my Arduino with a ATmega168/ATmega328 microcontroller, but unfortunately there's only 256 KB / 512 KB of EEPROM storage.
My idea is to make use of an compression algorithm to strip down the size. But well, my knowledge on compression algorithms is quite low and my search for ready-to-use libraries failed.
So, is there a good way to optimize the storage size?

A:

You might have a look at the LZO algorithm, which is designed to be lightweight. I don't know whether there are any implementations for the AVR system, but it might be something you could implement yourself.
You may be somewhat misinformed about the amount of storage available in EEPROM on your chip though; according to the datasheet I have the EEPROM sizes are:
ATmega48P: 256
ATmega88P: 512
ATmega168P: 512
ATmega256P: 1024
Note that those values are in bytes, not KB as you mention in your question. This is not, by any measure, a "shitload".

A:

AVRs only have a few kilobytes of EEPROM at the most, and very few have many more than 64K Flash (no standard Arduinos do).
If you are needing to store something and seldom modify, for instance an image, you could try using the Flash as there is much more space there to work with.  For simple images, some crude RLE encoding would go a long way.
Compressing anything more random, for instance logged data, audio, etc, will take a tremendous amount of overhead for the AVR, you will have better luck getting a serial EEPROM chip to hold this data.  Arduino's site has a page on interfacing with a 64K chip, which sounds .  If you want more than that, look at interfacing with a SD card with SPI, for instance in this audio shield

A:

A NASA study here (Postscript)
A repost of  1989 article on LZW here
Keep it simple and perform analysis of the cost/payout of adding compression. This includes time and effort, complexity, resource usage, data compressibility, etc.


/*
 * Copyright 2014 Red Hat, Inc.
 *
 * All rights reserved. This program and the accompanying materials
 * are made available under the terms of the Eclipse Public License v1.0
 * and Apache License v2.0 which accompanies this distribution.
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 * The Eclipse Public License is available at
 * http://www.eclipse.org/legal/epl-v10.html
 *
 * The Apache License v2.0 is available at
 * http://www.opensource.org/licenses/apache2.0.php
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 * You may elect to redistribute this code under either of these licenses.
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package io.vertx.ext.auth.mongo;

import io.vertx.codegen.annotations.VertxGen;

/**
 * Password hash salt configuration.
 * @deprecated Please use the provided generic PHC strings at the column level
 */
@VertxGen
public enum HashSaltStyle {

  /**
   * Password hashes are not salted
   */
  NO_SALT,

  /**
   * Salt is in a separate column for each user in the database
   */
  COLUMN,

  /**
   * Salt is NOT stored in the database, but defined as external value like application preferences or so
   */
  EXTERNAL
}


Q:

Strange behavior with NSMutableArray

Here is a code from my application:
NSMutableArray *firstPhotoVoters = post.firstPhotoVoters;
NSMutableArray *secondPhotoVoters = post.secondPhotoVoters;

ALog(@"First voters: %@", firstPhotoVoters);
ALog(@"Second voters: %@", secondPhotoVoters);

for (Post* _post in posts) {
    if ([_post.objectId isEqualToString:post.objectId]) {
        [_post.firstPhotoVoters removeAllObjects];
        [_post.secondPhotoVoters removeAllObjects];

        ALog(@"Count: %d", firstPhotoVoters.count);

        ALog(@"Updated post: %@", _post);
    }
}

The two ALog statements (which is a macro for NSLog) dumps the array elements that exist in firstPhotoVoters and secondPhotoVoters arrays. However, inside the for loop the two arrays appear all empty. firstPhotoVoters.count is always zero. What am I missing here?
Thank you.

A:

You've just emptied the array and then you log it. It should be outside the if-statement.


[ "$FREETZ_KEEP_AVM_UCLIBC" == "y" ] && return 0

[ "$FREETZ_AVM_UCLIBC_0_9_28" == "y" ] && version="0.9.28"
[ "$FREETZ_AVM_UCLIBC_0_9_29" == "y" ] && version="0.9.29"
[ "$FREETZ_AVM_UCLIBC_0_9_32" == "y" ] && version="0.9.32"
[ "$FREETZ_AVM_UCLIBC_0_9_33" == "y" ] && version="0.9.33.2"
[ "$FREETZ_AVM_UCLIBC_1_0_14" == "y" ] && version="1.0.14"

[ -z "$version" ] && error 1 "FREETZ_AVM_UCLIBC is not configured"

echo1 "removing uClibc-${version} files"
rm_files "${FILESYSTEM_MOD_DIR}/lib/*${version}*"
[ "${FREETZ_AVM_HAS_INNER_OUTER_FILESYSTEM}" == "y" ] && rm_files "${FILESYSTEM_OUTER_MOD_DIR}/lib/*${version}*"

echo1 "removing uClibc links"
for link in \
  ld-linux.so* \
  ld-uClibc.so* \
  ld.so* \
  libc.so* \
  libcrypt.so* \
  libdl.so* \
  libld-uClibc* \
  libm.so* \
  libnsl.so* \
  libpthread.so* \
  libthread_db.so* \
  libresolv.so* \
  librt.so* \
  libubacktrace.so* \
  libutil.so* \
  ; do
	rm_files "${FILESYSTEM_MOD_DIR}/lib/$link"
	rm_files "${FILESYSTEM_MOD_DIR}/usr/lib/$link"

	if [ "${FREETZ_AVM_HAS_INNER_OUTER_FILESYSTEM}" == "y" ]; then
		rm_files "${FILESYSTEM_OUTER_MOD_DIR}/lib/$link"
		rm_files "${FILESYSTEM_OUTER_MOD_DIR}/usr/lib/$link"
	fi
done

echo1 "linking ld.so.1"
ln -sf ld-uClibc-${FREETZ_TARGET_UCLIBC_VERSION}.so ${FILESYSTEM_MOD_DIR}/lib/ld.so.1


Next, the northeastern Italian town of Vò, near the provincial capital of Padua. On March 6, all 3,300 people of Vò were tested, and 90 were positive, a prevalence of 2.7%. Applying that prevalence to the whole province (population 955,000), which had 198 reported cases, suggests there were actually 26,000 infections at that time. That’s more than 130-fold the number of actual reported cases. Since Italy’s case fatality rate of 8% is estimated using the confirmed cases, the real fatality rate could in fact be closer to 0.06%.

Looking at the U.S., Dr. Bendavid and Dr. Bhattacharya focused on the NBA to get a better sense of virus prevalence and its fatality rate.

“The best (albeit very weak) evidence in the U.S. comes from the National Basketball Association,” they wrote. “Between March 11 and 19, a substantial number of NBA players and teams received testing. By March 19, 10 out of 450 rostered players were positive. Since not everyone was tested, that represents a lower bound on the prevalence of 2.2%.”

“The NBA isn’t a representative population, and contact among players might have facilitated transmission,” the professors continued. “But if we extend that lower-bound assumption to cities with NBA teams (population 45 million), we get at least 990,000 infections in the U.S. The number of cases reported on March 19 in the U.S. was 13,677, more than 72-fold lower.”

“These numbers imply a fatality rate from Covid-19 orders of magnitude smaller than it appears,” Dr. Bendavid and Dr. Bhattacharya stated. The discrepancies from highly-cited epidemiological models is due to two factors, they explained:

First, the test used to identify cases doesn’t catch people who were infected and recovered. Second, testing rates were woefully low for a long time and typically reserved for the severely ill.

“Together, these facts imply that the confirmed cases are likely orders of magnitude less than the true number of infections,” said Dr. Bendavid and Dr. Bhattacharya, while epidemiological modelers have yet to adequately adapt their estimates to reflect these significant factors.

The virus is clearly highly transmissible, they explained, with the number of infections reportedly doubling every three days. “An epidemic seed on Jan. 1 implies that by March 9 about six million people in the U.S. would have been infected,” the doctors said. But as of March 23, CDC numbers showed there were 499 Covid-19 deaths in the nation.

“If our surmise of six million cases is accurate, that’s a mortality rate of 0.01%, assuming a two week lag between infection and death,” the doctors noted. “This is one-tenth of the flu mortality rate of 0.1%. Such a low death rate would be cause for optimism.”

COVID-19 is not a “nonissue,” they warned, highlighting the strain it places on our medical system. They also underscore that we do not the true infection rate in the U.S., though recent developments are helping us get closer to accurately determining it.

The doctors conclude by suggesting that “universal quarantine may not be worth the costs it imposes on the economy, community and individual mental and physical health” and urging evaluation of the impact of the current lockdowns.

Related: Prevention Expert: Data Shows Our Fight Against Coronavirus May Be Worse Than The Disease


TG Tubular Net Bandage for securing dressings and light compression, as well as suspension and extension. The bandage remains firmly in position without creasing or cutting into the skin. 67% cotton, 33% viscose and may be sterilized up to 120°C.


export default () => 'Index page';


From: Cammiluna

I've had a kissing gourami named Kirby for the years of 1996-2003, and his personality... changed a bit over the years. He was first in a 10-gallon tank and got along with everyone. Then I babysat someone's goldfish in the tank (Meeko), and while Kirby DID chase Meeko whenever he got lazy, he never inflicted any damage aside a few tail fin nips.

This went on for four years (I've grown so attatched to Meeko that I was allowed to keep him). During those four years, my dad gave me fish ranging from angel fish to tiger barbs and other gourami, and they've all attacked my kisser a lot, but he never did anything back to them. Kirby was a pansy, surely. Particularly, my kisser, goldfish, and an iridescent shark by the name of Boomer roomied together for 4 years. The later 2 years they grew so large I had to get a 20-gallon tank for them. The shark grew too big for that and kept rampaging around the tank every few minutes, crashing into the goldfish and impaling him until he died from his injuries.

I know this topic is about Kissing Gourami, but my kisser and the shark got along PERFECTLY for four years, but on the morning after my goldfish died, Kirby Ripped the flesh off the shark (whose twice his size) and mauled him until he died. I was afraid to buy any new fish for the tank for a while so he was on his own and I put him back in a 10-gallon tank since my dad wasn't around to help me with maintenance anymore. A year later I was given some new brown gouramis for the tank and they picked on my kisser all the time. He once again did nothing to fight back, and he remained friendly again until he died from eating poisoned roaches that drowned in his tank.

I have a new kisser now after a 3-year haitus from raising fish. He's about the size of a thumb and gets along with my two catfish quite well. Based on my experiences with Kirby and the new guy, A good setup for a community tank with Kissers can use a lot of balance. Kissing Gouramis like the mid and top waters (mostly mid), so bottom-dwellers like Catfish will do fairly well in the tank. Kirby never bothered my catfish at all to times where I forgot those catfish exist in my tank sometimes. Also, I think Kissers get annoyed with slow/lazy fish, especially if they occupy his height level of the tank (hence why my goldfish was chased around for four years), so make sure his mid-water roomates are active and speedy. Smaller fish can probably do well as long as they're speedy, even Neon tetras. It may not work for every kisser, but this balance worked for my gang for many years.


Improving the implementation of cost recovery for health: lessons from Zimbabwe.
In the current debate over health financing policy in developing countries, governments are increasingly focusing on cost recovery--having patients pay part or all of their health care costs--as a way to mobilize more resources for health, improve equity by selectively charging the wealthy, and increase efficiency by encouraging reinvestment of fee revenues into cost-effective primary care. Zimbabwe offers an important example of a country with a tradition of levying fees in government health facilities, but where enforcement became lax in the 1980s. In 1991, policymakers resolved to resuscitate and strengthen cost recovery, as part of a broader economic reform program. This paper discusses the strengths and weaknesses of Zimbabwe's cost recovery system, its potential for improvement, and the obstacles to change in revising the fee structure and billing and collection procedures. It argues that cost recovery can help to achieve Zimbabwe's health objectives, but only in conjunction with other measures to redirect public spending to essential public health and clinic care and improve the efficiency of government services. The paper finds that during the 1980s, the fee schedule became badly misaligned with actual medical care costs and created distortions in patient referral patterns. Billing and collection were also weak, because of deficiencies in personnel and information systems and lack of incentives for revenue generation. The paper concludes that if key steps were taken to raise the collections-to-billings ratio, recover fees from privately-insured patients, and adjust fees in line with medical cost inflation, recoveries could increase fourfold, from 5% to 20% of government spending for clinical care. At the same time, access to government health services for the poor could be maintained by improving exemption procedures.


Q:

Result from CTE query not sorted by Level, why?

Update:
I asked the question because the result does not look like how it is executed... which is explained here:
http://msdn.microsoft.com/en-us/library/ms186243(v=sql.105).aspx
Well I have another question...
If I do not need the same parents and the Level, is it possible to return one parent only once using the CTE (not the select after it) or maybe some other sql?
===========================
Desired result is like:
......
    54          4
    **** the above is anchor member, the numbers are correct
    4           1
    1           0
    2           1
    36          35
    35          8
    8           1
    54          12
    12          1
    11          1
    3           1

===========================
I am using a recursive query to find out all the parents from a Hierarchy table, for items from Items table; I thought the result should be by Level, but it is not... I know I can sort it using order by, I just think the output itself should be ordered by Level because the Recursive Memeber is run by Level, right?
            WITH Result(ItemID, ParentID, Level)
            AS
            (
                --get the anchor member from tbItems
                SELECT itemID, itemParentID, 0 AS Level
                FROM   tbItems  WHERE
                    approved = 0
                UNION ALL
                --recursive member from tbHierarchy
                SELECT h.hierarchyItemID, h.parentItemID, Level + 1
                FROM tbHierarchy AS h
                INNER JOIN
                    Result AS r
                        ON
                        h.hierarchyItemID = r.ParentID

            )
            SELECT *
            FROM Result

the Result is:
    ItemID      ParentID    Level
    ----------- ----------- -----------
    7           3           0
    11          2           0
    18          11          0
    19          11          0
    21          54          0
    31          2           0
    33          36          0
    34          36          0
    35          36          0
    36          36          0
    38          2           0
    39          2           0
    40          2           0
    54          4           0
    **** the above is anchor member, the numbers are correct
    4           1           1
    1           0           2
    2           1           1
    1           0           2
    2           1           1
    1           0           2
    2           1           1
    1           0           2
    36          35          1
    35          8           2
    8           1           3
    1           0           4
    36          35          1
    35          8           2
    8           1           3
    1           0           4
    36          35          1
    35          8           2
    8           1           3
    1           0           4
    36          35          1
    35          8           2
    8           1           3
    1           0           4
    2           1           1
    1           0           2
    54          12          1
    12          1           2
    1           0           3
    11          1           1
    1           0           2
    11          1           1
    1           0           2
    2           1           1
    1           0           2
    3           1           1
    1           0           2

A:

The data in a database are not ordered. If you do not put an order by, you cannot be sure of the order of the output.
Never think the database will do things the way you think, 90% of the time, it's wrong. The objective of database is to understand your query and find the fastest way to find the solution, and it's often not the way you think.
Depending of your editor, but sometimes you can ask it to explain the plan used to find the solution, it might give you some explanation of how the result is obtained.


1. Field
One or more embodiments described herein relate to a touch sensing apparatus and method.
2. Description of the Related Art
Touch screens have been used in a variety of devices including mobile phones and smartphones. Touch screens may be classified as resistive, capacitive, ultrasonic, and infrared according the way they operate. Capacitive touchscreens have excellent durability and long life, and also supports multi-touch functioning.
A capacitive touch screen detects the position of a touch (e.g., the finger of a user) based on a change in capacitance. Some touch screens can detect sliding inputs. However, capacitive touch screens cannot detect a change in capacitance (and thus a touch) caused by a stylus pen. Thus, electro-magnetic resonance (EMR) sensor pads have been installed in touch screen in order to detect touches from an EMR stylus pens.


Q:

Avoid Empty Element Tag in Simple Framework output

What is the correct way to avoid "empty element tags" when serializing required elements?
Example:
@ElementList(name="rpc", required=true)
public ArrayList<FunctionRequest> getRequestedFunctions() {
    return requestedFunctions;
}

An empty list will result in a single XML element in the referred "empty element tag" style:
<rpc/>

What I actually need is the following representation:
<rpc></rpc>

Edit: I need a solution for Lists of different types! For example, there might also be List<String>, List<int>, List<WhateverClass>, ... PLUS different name attributes of the annotation which is "rpc" in the example.
Thanks for your help.
Solutions
I will first present the solution I've actually implemented and that I'm using in my project. After that I will present a proposal of changes which could be added directly to the Simple code.
My solution I actually use:
Since I did not want to define the XML elements by hand and create a Converter for each List I've got in my program, I decided to use the existing annotations of my classes, as well as the name attributes of the annotations. My solution will work for classes that shall be serialized if they use getters and setters! It is currently bound to classes that use the Attribute and Text annotations, only!
To be more flexible I created a "base" class RequestListConverter. It has two protected methods prepareMethodList and writeRequest. prepareMethodList will go through all methods of a given class using reflection and creates a method-annotation-map. writeRequest will then write a single object of the type given to method prepareMethodList to the OutputNode of Simple that is given in the write method of the Converter interface.
public class RequestListConverter {
    private HashMap<Method, Object> curMethodAnnotationMap = new HashMap<Method, Object>();

    @SuppressWarnings("rawtypes")
    protected void prepareMethodList(Class targetClass) {
        /*
         * First, get the annotation information from the given class.
         *
         * Since we use getters and setters, look for the "get" methods!
         */

        Method[] curMethods = targetClass.getMethods();
        for (Method curMethod : curMethods) {
            String curName = curMethod.getName();

            // We only want getter methods that return a String
            if (curName.startsWith("get") && (curMethod.getReturnType() == String.class)) {
                Attribute curAttrAnnotation = curMethod.getAnnotation(Attribute.class);
                Text curTextAnnotation = curMethod.getAnnotation(Text.class);

                if (curAttrAnnotation != null) {
                    curMethodAnnotationMap.put(curMethod, curAttrAnnotation);
                } else
                if (curTextAnnotation != null) {
                    curMethodAnnotationMap.put(curMethod, curTextAnnotation);
                }
            }
        }
    }

    protected void writeRequest(OutputNode curNode, Object curRequest) throws Exception {
        for (Map.Entry<Method, Object> curMapEntry : curMethodAnnotationMap
                .entrySet()) {
            if ((curMapEntry.getKey() == null)
                    || (curMapEntry.getValue() == null)) {
                continue;
            }

            Method curMethod = curMapEntry.getKey();

            Attribute curAttrAnnotation = null;
            Text curTextAnnotation = null;

            if (curMapEntry.getValue() instanceof Attribute) {
                curAttrAnnotation = (Attribute) curMapEntry.getValue();
            } else if (curMapEntry.getValue() instanceof Text) {
                curTextAnnotation = (Text) curMapEntry.getValue();
            } else {
                continue;
            }

            String curValue = null;
            try {
                // Try to invoke the getter
                curValue = (String) curMethod.invoke(curRequest);
            } catch (IllegalAccessException | IllegalArgumentException
                    | InvocationTargetException e) {

                // The getter method seems to need any argument, strange! Skip
                // this!
                continue;
            }

            // If the method has an Attribute annotation, then ...
            if (curAttrAnnotation != null) {
                boolean curAttrRequired = curAttrAnnotation.required();
                String curAttrName = curAttrAnnotation.name();

                /*
                 * IF the returned method value is NULL THEN IF if the attribute
                 * is required THEN throw a NullPointerException, ELSE skip the
                 * attribute
                 */
                if (curValue == null) {
                    if (curAttrRequired) {
                        throw new NullPointerException(
                                "The required attribute " + curAttrName
                                        + " returned NULL!");
                    } else {
                        continue;
                    }
                }

                // The attribute will be added as XML text now
                curNode.setAttribute(curAttrName, curValue);
            } else
            // If the method has a Text annotation, then ...
            if (curTextAnnotation != null) {
                // we only need to store it for later string creation
                curNode.setValue(curValue);
            }
        }

        curNode.commit();
    }
}

Based upon this class, I created - for example - the class SetRequestListConverter. It implements Simple's Converter interface, so it provides the methods read which is not implemented and write which gets the List that might have elements or might be empty.
The example shows the implementation of a Converter of the class SetRequestList. It extends the previously introduced base RequestConverter class and implements the Converter typed to the SetRequestList.
public class SetRequestListConverter extends RequestListConverter implements Converter<SetRequestList> {

    @Override
    public SetRequestList read(InputNode newNode) throws Exception {
        return null;
    }

    @Override
    public void write(OutputNode newNode, SetRequestList newValue) throws Exception {
        if (newValue.requests.isEmpty()) {
            newNode.setValue("");
            return;
        }

        this.prepareMethodList(SetRequest.class);

        /*
         * Now we can go through all SetRequests and call the methods
         * to build the XML attributes and to get the element value (i.e. parameters)
         */

        for (SetRequest curRequest : newValue.requests) {
            OutputNode curNode = newNode.getChild("set");

            this.writeRequest(curNode, curRequest);
        }
    }
}

The used SetRequestList is a simple class that holds an ArrayList<SetRequest>. This is needed to hide the fact that this actually is an ArrayList.
@Root
@Convert(SetRequestListConverter.class)
public abstract class SetRequestList {
    protected ArrayList<SetRequest> requests = new ArrayList<SetRequest>();

    public void add(T newRequest) {
        requests.add(newRequest);
    }
}

This class can then be used like this:
public class ClassToSerialize {
    private SetRequestList requestedSets = new SetRequestList();

    @Element(name="get", required=true)
    public SetRequestList getRequestedSets() {
        return requestedSets;
    }

    @Element(name="get", required=true)
    public void setRequestedSets(SetRequestList newRequestedSets) {
        requestedSets = newRequestedSets;
    }
}

The generated XML of SetRequestList containing elements will look like that:
<get>
    <set someAttribute="text" anotherAttribute="bla">Some Text</set>
    ...
</get>

The generated XML of SetRequestList being empty will look like that:
<get></get>

Yay, exactly what I needed PLUS the fact that I can go on and use annotations in the SetRequest or whatever classes! No need to do (re)define the XML structure again!
Code proposal for Simple
Attention: This only is a solution proposal and has not been tested!
I looked through the source code of Simple and found that the Formatter class is actually writing start and end tags, as well as the empty element tag. It is created by handing over a Format object. Simple's Javadoc describes the Format class as follows:

The Format object is used to provide information on how a generated XML document should be structured.

So it could be extended with the information if empty element tags should be created or not. For this I added the private variable useEmptyEndTag and the appropriate getter and setter methods. The variable will be initialized with true inside the constructor. If the empty end tag style is not what should be created, you can set it after creating a Format object by using myFormat.setUseEmptyEndTag(false).
The Formatter class is enhanced by a new private variable holding the given Format object to be able to access the set parameters at the appropriate code location. The empty end tag is written inside writeEnd. Have look at the official source code to see the original code. Here is what my proposal is to avoid empty element tags:
public void writeEnd(String name, String prefix) throws Exception {
    String text = indenter.pop();

    // This will act like the element contains text
    if ((last == Tag.START) && (!format.isUseEmptyEndTag())) {
        write('>');
        last = Tag.TEXT;
    }

    if (last == Tag.START) {
        write('/');
        write('>');
    } else {
        if (last != Tag.TEXT) {
            write(text);
        }
        if (last != Tag.START) {
            write('<');
            write('/');
            write(name, prefix);
            write('>');
        }
    }
    last = Tag.END;
}

This proposal is the better solution - in my eyes - and I hope it will be added to the Simple's sourcecode.

A:

As baraky has written before, you can solve the part with the <rpc></rpc> tags with a Converter like posted here: Prevent inclusion of empty tag for an empty ElementList in an ElementListUnion.
There's a comment on the ExampleConverter where this special part is done.
Moreover to get the name attribute see here: How do you access field annotations from a custom Converter with Simple?
So what you need is a Converter-implementation for your class. For types (int, String etc.) check out Transformer-class.


Q:

reverse chunks in a file

My basic Java problem is this: I need to read in a file by chunks, then reverse the order of the chunks, then write that out to a new file.  My first (naive) attempt followed this approach:

read a chunk from the file.
reverse the bytes of the chunk
push the bytes one at a time to the front of a results list
repeat for all chunks
write result list to new file.

So this is basically a very stupid and slow way to solve the problem, but generates the correct output that I am looking for.  To try to improve the situation, I change to this algorithm:

read a chunk from the file
push that chunk onto the front of a list of arrays
repeat for all chunks
foreach chunk, write to new file

And to my mind, that produces the same output.  except it doesn't and I am quite confused. The first chunk in the result file matches with both methods, but the rest of the file is completely different.
Here is the meat of the Java code I am using:
FileInputStream in;
FileOutputStream out, out2;

Byte[] t = new Byte[0];
LinkedList<Byte> reversed_data = new LinkedList<Byte>();
byte[] data = new byte[bufferSize];
LinkedList<byte[]> revd2 = new LinkedList<byte[]>();

try {
in = new FileInputStream(infile);
out = new FileOutputStream(outfile1);
out2 = new FileOutputStream(outfile2);
} catch (FileNotFoundException e) {
    e.printStackTrace();
    return;
}
while(in.read(data) != -1)
{
    revd2.addFirst(data);
    byte[] revd = reverse(data);
    for (byte b : revd)
    {
        reversed_data.addFirst(b);
    }
}
for (Byte b : reversed_data)
{
    out.write(b);
}
for (byte[] b : revd2)
{
    out2.write(b);
}

At http://pastie.org/3113665 you can see a complete example program (a long with my debugging attempts). For simplicity I am using a bufferSize that divides evenly the size of the file so all chunks will be the same size, but this won't hold in the real world.  My question is, WHY don't these two methods generate the same output?  It's driving me crazy because I can't grok it.

A:

You're constantly overwriting the data you've read previously.
while(in.read(data) != -1)
{
    revd2.addFirst(data);
    // ignore byte-wise stuff
}

You're adding the same object repeatedly to the list revd2, so each list node will finally contain a reference to data filled with the result of the last read. I suggest replacing that with revd2.addFirst(data.clone()).


After nearly a decade of working as a congressional press secretary, Kevin Long found himself in Colombia as a U.S. Department of Defense communications subcontractor, teaching local military law enforcement personnel how to handle the media. While dining out one evening, Long watched a "SportsCenter" clip in which Kevin Garnett spoke about an NBA playoff game in terms of having "weapons" and "ammunition" and being ready to "go to war." "They were making a big deal out of his comments, because there had just recently been a school shooting. I turned to the guy next to me and said, 'I ought to go home and train athletes how to talk to the media,' " says Long, who has been doing just that for the past four years. "That's how MVP Sports Media Training was born - in a bar in Bogotá." To date, Long has visited more than 30 college campuses (and a few professional athletes), videotaping a dozen mock interviews in a day and critiquing individual performances. The role-playing "reporter" recently took time to answer a few of Paul Steinbach's questions.

Q: Did your work on the Hill translate to your current calling? A: Politics and sports are not that different. You're dealing with people who are generally very successful, at the top of their field, but it hasn't meant that they've had to deal with the media that much. Athletes practice their sport to get better at it, but they never practice talking to reporters. How can they expect to get any better at it if they don't practice? I use that logic with them, and it seems to really hit home.

Q: What are common mistakes made by student-athletes? A: One, they're not comfortable or confident in front of a microphone, because they don't think they know what to say. I tell them, "Look, you are the subject-matter expert. When you walk in, you know more about basketball than anybody else in the room, except maybe the coach or one of your teammates sitting next to you." The second thing they don't understand is that when they've said enough, it's enough said. Most of the time when athletes say something that is taken out of context or that they didn't mean to say, it's because they've answered the question and they continue to ramble on.

Q: Is there one interview setting that's tougher than others? A: I think the toughest challenge for an athlete or coach is the interview when they're walking off the field at halftime or right after the game, because they're wrought with emotion. You have to put yourself in a different mentality before you start speaking to reporters if you want to come through with the desired message and deliver it with some poise and grace.

Q: Who might serve as a media-savvy role model? A: The best, without a doubt - and he is levels above everyone else - is Tiger Woods. He's very competitive in everything he does, and I think he wants to be the best at doing interviews. That comes across in the way he prepares himself, delivers his comments and structures his answers to questions. I talk to student-athletes about his skills, but I tell them, "Look, he didn't get this way naturally. He learned it." I'm told he's done weeks and weeks of media training to get to where he is.

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There are numerous commercialized household carts for carrying household items, such as groceries, beverages and some items of wearing apparel. These prior art household carts have frequently included a pair of wheels mounted on a basket. A handle, frequently having an inverted U shape, usually extends from the basket and, in certain configurations, telescopes with respect to the basket. Some of the baskets are foldable, for ease of storage in automotive vehicles and residences. The typical prior household carts have limited volumetric and weight capacity. In addition, they are not usually adapted to carry elongated clothing articles, such as pants, suits or skirts, without folding and/or compressing the articles against each other. Since several of these articles of clothing are frequently on hangers while transported to a residence from a cleaning establishment, the present household carts are rarely used by householders for such articles. Certainly, a householder does not want his or her clothing folded and compressed after being commercially pressed. The prior art household carts have generally been incapable of carrying substantial loads, such as kitchen, laundry room or basement "white" appliances. Also, the prior art household carts have generally been difficult to manage on steps or street curbs.
Because of these problems, the prior art household carts have met with only limited success. Consequently, the vast majority of householders transport household items between their automotive vehicles and residence by hand. This frequently necessitates several back and forth trips. When a householder transports clothing articles, such as topcoats, full length dresses, suits, pants and skirts, from an automotive vehicle to a residence, the householder usually grasps a large number of hangers and slings the clothing over his or her shoulder, causing significant hand pain. In addition, the clothing articles are compressed together and the expense of commercial pressing is, to a certain extent, wasted. There is, to our knowledge, no currently available household cart capable of carrying garments on hangers, simultaneously with carrying other items, such as groceries, particularly heavy items such as cases of beverages. Further, many household carts of which we are aware are relatively expensive and difficult to maneuver.
It is, accordingly, an object of the present invention to provide a new and improved, relatively inexpensive and highly flexible household cart having the capability of carrying many different types of household items.
Another object of the invention is to provide a new and improved relatively inexpensive household cart having a handle which can be positioned in several different positions on the cart.
A further object of the invention is to provide a new and improved household cart that can be easily stowed in a trunk (i.e., a boot in the United Kingdom) of an automobile.
An additional object of the invention is to provide a new and improved household cart including a handle having plural operative positions and which can be stowed on the cart so the cart can be easily stored in a relatively small volume, such as an automobile trunk or behind a door or in a closet of a residence.
An added object of the invention is to provide a new and improved household cart including a handle selectively having relatively short and long lengths, selectively enabling the cart to (1) be easily stowed and (2) carry relatively large clothing apparel items, such as topcoats, full length dresses, suits, pants and skirts.
A further object of the invention is to provide a new and improved household cart which can easily be wheeled about a kitchen and is arranged so items to be stored in kitchen receptacles, such as cabinets and refrigerators, can easily be removed from the cart, without bending.
An additional object of the invention is to provide a new and improved household cart having a handle for carrying household items, wherein the handle is positioned relative to wheels of the cart to minimize the likelihood of cart tipping.
A further object of the invention is to provide a new and improved household cart having a base for securely carrying relatively heavy articles, such as beverage cases and watermelons, as well as heavier household articles such as refrigerators, and having a handle for carrying other household items.


Developer Community

Developer Blog

PostgreSQL + WAL-E + Cloudfiles = Awesome

If you're a big PostgreSQL fan like I am, you may have heard of a tool called
WAL-E. Originally developed by Heroku, WAL-E is a tool for efficiently sending
PostgreSQL's WAL (Write Ahead Log) to the cloud. In addition to Heroku, WAL-E
is now used by many companies with large PostgreSQL deployments, including
Instagram.

Let's unpack what that means. If you've ever set up replication with PostgreSQL
you're probably familiar with the WAL. Essentially there are two parts to
replication and backup in PostgreSQL, the "base backup" and the WAL. Base
backups are a copy of your database files that can be taken while the database
is running. You might create base backups every night, for example. The WAL is
where PostgreSQL writes each and every transaction, as they happen. When you
run normal replication, the leader will send its log file to the followers as
it writes it.

Instead of just using a simple socket to communicate, WAL-E sends these base
backups and WAL files across the internet with the help of a cloud object
store, like Cloudfiles (or any OpenStack Swift deployment). This gives you the
advantage that, in addition to just being replication, you have a durable
backup of your database for disaster recovery. Further, you have effectively
infinite read scalability from the archives, you can keep adding more followers
without putting more stress on the leader.

With the help of WAL-E's primary author, Daniel Farina, we recently added
support for OpenStack Swift to it. It's not yet in a final release, but if
you're interested in checking it out, read on!

Setting it up

The first thing to do to get this running is to spin up two cloud servers, one
for your PostgreSQL leader, and another for the follower (you can do it with
one server that has both the leader and the follower, but where's the fun in
that?). You can spin up your servers with either the control panel, or one of
our many SDKs. Here I'm booting up two 1GB performance cloud servers.


BP #6: Cops Parking Illegally

Some of the topics I covered in my other blog posts were kind of serious, so this time I’m going to talk about something that in the larger scheme of things isn’t that bad.

Down in Miami, Florida, a U.S. active-duty Army major, Glen Carr, has reported over 100 cases of Miami police cars parked illegally in the city. Each report contains photographic proof that the car is indeed parked illegally. I have to give this guy some respect just on what he says about the subject, “It’s not like I’m against them. I just want them to be better at what they do. I feel they don’t hold each other accountable.”

Again, this issue pales in comparison to some of the other things that are currently happening in the world. However, that doesn’t mean it shouldn’t be completely ignored. Comparing this to Kant’s concept of the “Good Will,” this example can be used to highlight the important difference between doing things for duty and doing things from duty.

To explain this distinction further, acting for duty means that you would be acting not because the action is correct or “good,” but because you have some other inclination or motivation to do so. The police officers that have parked their cars illegally are acting for duty, using their power in a way that isn’t inherently good, but because it provides them convenience in their daily life.

Acting from duty means that you are acting because the action is the right thing to do, and you have no ulterior motive or inclination. If these police officers had to park illegally in order to help somebody, that would be an action from duty. Major Carr is trying to show us that these officers who are parked illegally aren’t acting from duty because their actions aren’t done solely for “good”.

This is a very small concern in the grand scheme of things, but it’s not one that takes a lot of effort to resolve. I applaud Major Carr for bringing this issue to light because it shows an abuse of power that sometimes goes unnoticed. Again, a quote from Major Carr that shows why this issue should be resolved, “I was able to show my daughter, hey, they were wrong; the police can be wrong too. But they have to be accountable.”

Thank you for reading this, if you have any questions for comment feel free to reply below!


Q:

Why does this. or this. in Parent Class have no effect? Java

I am wondering why this.(...) has no effect in this scenario. The following is a Task of my Exam:
class PARENT {
    public int x;
    public PARENT(int a)    {this.x = a;}

    public int getX()   {return this.x;}
    public int getY()   {return this.getX();}
}

class CHILD1 extends PARENT {
    public int x;
    public CHILD1(int b) {super(b); this.x = 2 * b;}
}

class CHILD2 extends PARENT {
    public CHILD2(int c)    {super(c);}

    public int getX()   {return 5;}
}

public class ThisTestMain {
    public static void main(String[] args)  {
        PARENT PP = new PARENT(10);
        PARENT PC1 = new CHILD1(100);
        PARENT PC2 = new CHILD2(1000);
        System.out.println(PP.getY());
        System.out.println(PC1.getY());
        System.out.println(PC2.getY());

        CHILD1 CC = new CHILD1(10);
        System.out.println(CC.getY());
    }
}

The output is:
10
100
5
10

My question now is why at System.out.println(PC1); the output is NOT 200. Because when I debug the code in IntelliJ I can see that this has the reference
CHILD1@799 and the Object can see the values x and PARENT.x.
At this point why is getX() choosing PARENT.x and not CHILD1.x?
By overriding methods this has also no effect. In this case for example System.out.println(PC2); uses always the getX() in CHILD2, no matter if you write in the getY() Method return this.getX(); or return getX();.
Can someone summerize the system behind that? Maybe also considering super? Thanks!

A:

In CHILD1 you have no overridden methods.
So when you call getY() it then calls getX() from the PARENT. The method from the PARENT has no idea about variables in its child classes.
You must override method getX() in CHILD1 to get access to the variable CHILD1.x.


U.S. Senate Committee Considers Impact of 340B Program in Helping Hospitals Serve Their Communities in the Face of High Drug Prices

May 15, 2018

The U.S. Senate Health, Education, Labor and Pensions (HELP) Committee held a second hearing on the 340B Drug Pricing Program to examine its federal oversight and consider avenues to strengthen program integrity and promote transparency.

The HELP Committee previously heard testimony from advocates representing safety net hospitals, community health centers, health system pharmacists, and the pharmaceutical industry during a March hearing.

The 340B program allows health care organizations that care for large numbers of uninsured and low-income patients, serve rural communities, and offer vital services to cancer patients and children to purchase outpatient drugs at discounted prices. The program serves as a crucial tool for hospitals to stretch scarce resources to better serve vulnerable patients and communities across the commonwealth, particularly as the price of prescription drugs places an increasing financial burden on patients and hospitals.

During the hearing today, officials from the U.S. Department of Health and Human Services Office of the Inspector General (OIG) and the U.S. Government Accountability Office (GAO) provided testimony on the conclusions and recommendations laid out in oversight reports.

GAO indicated it will be issuing new reports in the coming months about:

The use of contract pharmacies, and financial arrangements with contract pharmacies and third party administrators

Program oversight by the Health Resources and Services Administration and audits

How discounts under the program are passed on to patients

Senators participating in the hearing noted the importance of having data and independent analysis to inform their policy decisions. At the same time, these senators also acknowledged that financial analyses may not reflect the true impact of policy changes on hospitals’ ability to serve their communities.

In addition to providing financial assistance to patients unable to afford their prescriptions, Pennsylvania hospitals have reported using savings from the 340B program to:

Fund other medical services, such as obstetrics, diabetes education, oncology services, and other ambulatory services

Establish additional outpatient clinics to improve access

Create new community outreach programs

Offer free vaccinations for vulnerable populations

The 340B program is subject to significant policy discussion in Washington:

Pennsylvania hospitals have raised concerns with a newly implemented payment policy that significantly undercut the benefit of the program by cutting payment by nearly 30 percent for certain drugs purchased through the 340B program. HAP has supported efforts by national hospital groups to overturn the payment policy through a legal challenge and legislation, H.R. 4392.

The U.S. House Energy & Commerce Committee issued a report that calls for significant changes to the structure of the program.

The Trump Administration unveiled a drug pricing blueprint last week. This week, the administration issued a policy statement and request for information (RFI) on ways to lower drug prices and reduce out-of-pocket costs for consumers. The RFI specifically asked for feedback on further changes to outpatient payments for hospitals participating in the 340B program.

HAP has urged federal policymakers to recognize that taking action on shortsighted policies, such as reducing benefits of the 340B Drug Pricing Program, does nothing to address the underlying causes of continually increasing drug pricing. In fact, it actually makes it harder for hospitals to manage high drug costs and help patients.

HAP continues to advocate for Congress to:

Safeguard the long-term sustainability of the 340B Drug Pricing Program

Solidify the important benefits of the program, which play a key role in helping hospitals balance the growing burden of ever increasing drug costs

Promote program reforms that enhance transparency and accountability without placing onerous hardships on hospitals providing care to vulnerable patient populations


Background
==========

This case is of interest to clinicians and sonographers as it highlights the utility of the paravertebral imaging window. Paravertebral imaging is a useful addition to conventional diagnostics, facilitating best practice through innovative and interesting imaging for patients undergoing echocardiography evaluation.

Case presentation
=================

A 62-year-old female presented to her local district general hospital with sudden onset of severe central chest pain (rated by the patient as 10/10 in severity) radiating to the scapula and abdomen. At this time, she was hemodynamically stable with a heart rate of 85 bpm and respiratory rate of 16 resps/min. Her blood pressure was 155/50 mmHg on the right and 148/80 mmHg on the left.

Investigation
=============

She underwent chest x-ray and computed tomography (CT) ([Fig. 1A](#fig1){ref-type="fig"} and [B](#fig1){ref-type="fig"}), which confirmed a widened mediastinum and a type A acute aortic dissection. She was transferred to our institute and underwent an urgent ascending aorta and hemi-aortic arch replacement. During recovery, she was referred for a transthoracic echocardiogram (TTE) for the assessment of aortic valve competency and pericardial effusion. Figure 1(A) CT demonstrating a dissection flap consistent with an aortic dissection, pericardial and pleural effusions; lines mark the distance to both the paravertebral window and the conventional transthoracic window. (B) Chest x-ray showing a widened mediastinum; (C) and (D) paravertebral echocardiographic images demonstrating a pericardial effusion (1), a dissection flap in the descending aorta (2) and the left ventricle (3).

Echocardiographic examination was complicated by a narrow anterior-posterior dimension and small acoustic windows, making parasternal and apical images technically challenging with suboptimal quality. Additional paravertebral images (PV) were taken from the right and left posterior lung fields in the fifth intercostal space just lateral to the spine with the imaging notch directed to 12 o'clock and toward the spine. Images from the lung fields clearly demonstrated the presence of a pleural and pericardial effusion, normal left ventricular systolic function and a dissection flap in the descending aorta with excellent image quality obtained ([Fig. 1C](#fig1){ref-type="fig"} and [D](#fig1){ref-type="fig"}).

Treatment and outcome
=====================

Surgical intervention included ascending aorta and hemi-aortic arch replacement (as outlined earlier).

Discussion
==========

Imaging from the paravertebral (PV) position has long been recognised as useful in identifying pleural effusions ([@bib1], [@bib2]). However, the utility of this imaging technique beyond the scope of pleural effusion is not well known and is thus under-utilised ([@bib3], [@bib4]). The current case demonstrates three important points regarding the application and usefulness of paravertebral echocardiography.

Firstly PV imaging has the ability to provide superior definition of the descending aorta ([@bib5], [@bib6]). Due to the close proximity between vessel and acoustic window, the distance traveled by the ultrasound signal is reduced. Consequently, there is less attenuation of the ultrasound beam and enhanced definition of the structures of interest. [Figure 1A](#fig1){ref-type="fig"} showed the relationship between the position of the descending aorta relative to both the conventional transthoracic window and the PV acoustic window. In this case, PV imaging additionally facilitates the identification of a dissection flap in both a short and long axis view, a significant abnormality if yet undetected ([Fig. 1C](#fig1){ref-type="fig"} and [D](#fig1){ref-type="fig"}). Current literature suggests that TTE does not perform well in the diagnosis of acute aortic dissection of the descending aorta with specificity and sensitivity reported at as low as 71% and 74% respectively ([@bib7]). This underscores the value of PV echocardiography with better morphological characterisation of the aorta and suggests this neglected imaging window should be considered a standard view when aortic dissection is suspected, improving sensitivity and further assisting with correct diagnosis ([@bib3]).

Secondly, in the presence of pleural effusion, image quality is further enhanced by decreased absorption. This is as a consequence of the lower viscosity of the lungs and reduced reflection as a result of limited variation in acoustic impedance between media ([@bib8]). As a result, paravertebral echocardiography provides superior characterisation of the pericardium and associated effusions. Cardiac tamponade is a frequent complication of aortic dissection occurring in one of five patients ([@bib9]). [Figure 1B](#fig1){ref-type="fig"} and [C](#fig1){ref-type="fig"} demonstrate the echogenic pericardial effusion with clear borders defining the pericardial space. These alternative images further assist in distinguishing between pleural and pericardial effusions, which in practice often proves challenging when conventional TTE images do not yield optimal visualisation of the descending aorta.

Finally, the current case also supports the use of PV imaging in the assessment of left ventricular systolic function. From this window, in the presence of a pleural effusion, a short axis view of the left ventricle can reliably be obtained. This view offers the potential to evaluate systolic function, by examining both global and regional myocardial abnormalities ([@bib8]). A thorough assessment of left ventricular systolic function is vital for the postoperative patient, and the PV window can further assist in providing additional high-quality information.

PV echocardiography plays an important role in providing enhanced image quality and offers a useful approach for the identification of aortic dissection, pericardial and pleural effusions and left ventricular systolic function. We suggest this imaging approach may prove valuable as a routine adjunct to conventional transthoracic imaging.

Declaration of interest
=======================

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this case report.

Funding
=======

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Patient consent
===============

Written informed consent has been obtained from the patient in regard to publication of the submitted article and accompanying images. .

Patient's perspective
=====================

It was interesting to see the pictures and although, with no medical expertise, I needed them explaining to me, I can see that they are incredibly clear. I hope this education and learning advances the use of imaging work in this area.

Author contribution statement
=============================

All authors have contributed equally to the writing and revisions of this manuscript. The named physician of the patient is one of the authors and has thus given permission for this case to be written up and published.


Q:

Why are Stiefel-Whitney classes not represented in de Rham cohomology?

Stiefel-Whitney classes of a real vector bundle $(E, \pi, M)$ are defined  axiomatically in singular cohomology $H^*(M, \mathbb{Z}/2\mathbb{Z})$ (compare wiki).
Now various books state, that Stiefel-Whitney classes are not represented in de Rham cohomology (e.g. "The Stiefel Whitney classes are not de Rham cohomology classes" compare click).
Why is this the case? Naivly I would think, that the composition of the coefficient homomorphism with the de Rham iso $H^*(M, \mathbb{Z}/2\mathbb{Z}) \rightarrow H^*(M, \mathbb{R}) \rightarrow H_{dR}(M)$ should map Stiefel-Whitney classes on classes of de Rham cohomology satisfying the axioms of Stiefel-Whitney classes. Why
does this fail? Can you give a reference?

A:

There is no (nonzero) map $f : H^*(M; \mathbb{Z}/2\mathbb{Z}) \to H^*(M; \mathbb{R})$ as you claimed. The field $\mathbb{Z}/2\mathbb{Z}$ has characteristic $2$, but the field $\mathbb{R}$ has characteristic $0$. In $H^*(M; \mathbb{Z}/2\mathbb{Z})$, all the elements $\alpha$ satisfy $2 \alpha = 0$. So under your map $f$ you get $2 f(\alpha) = 0$, but over $\mathbb{R}$ you can divide by $2$ and you get $f(\alpha) = 0$...
More abstractly, the universal Stiefel-Whitney class $w_n$ lives in $H^n(\operatorname{Gr}_n(\mathbb{R}^\infty); \mathbb{Z}/2\mathbb{Z})$, the $n$th cohomology group of the Grassmanian of $n$-subspaces in $\mathbb{R}^\infty$. All the other Stiefel-Whitney classes $w_n(\xi)$ are pullbacks of this one. So if you wanted "de Rham" Stiefel-Whitney classes, you'd be looking at $H^n(\operatorname{Gr}_n(\mathbb{R}^\infty); \mathbb{R})$. But for example $\operatorname{Gr}_1(\mathbb{R}^\infty)$ is the infinite-dimensional real projective space $\mathbb{RP}^\infty$, and $H^1(\mathbb{RP}^\infty; \mathbb{R}) = 0$.