Lymphatic vessels arise from specialized angioblasts within a venous niche.

How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the 'angioblast-to-lymphatic' transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.

Downregulation of dystrophin expression in pupae of the whitefly Bemisia tabaci inhibits the emergence of adults.

The whitefly Bemisia tabaci is a major pest to agriculture. Adults are able to fly for long distances and to colonize staple crops, herbs and ornamentals, and to vector viruses belonging to several important taxonomic groups. During their early development, whiteflies mature from eggs through several nymphal stages (instars I to IV) until adults emerge from pupae. We aim at reducing whitefly populations by inhibiting the emergence of adults from nymphs. Here we targeted dystrophin, a conserved protein essential for the development of the muscle system in humans, other animals and insects. We have exploited the fact that whitefly nymphs developing on tomato leaves feed from the plant phloem via their stylets. Thus, we delivered dystrophin-silencing double-stranded RNA to nymphs developing on leaves of tomato plantlets with their roots bathing in the silencing solution. Downregulation of dystrophin expression occurred mainly in pupae. Dystrophin silencing induced also the downregulation of the dystrophin-associated protein genes actin and tropomyosin, and disrupted F-actin. Most significantly, the treatment inhibited the emergence of adults from pupae, suggesting that targeting dystrophin may help to restrain whitefly populations. This study demonstrates for the first time the important role of dystrophin in the development of a major insect pest to agriculture.

Bread wheat milling behavior: effects of genetic and environmental factors, and modeling using grain mechanical resistance traits.

KEY MESSAGE: Genetic (Pinb-D1 alleles) and environment (through vitreousness) have important effects on bread wheat milling behavior. SKCS optimal values corresponding to soft vitreous or hard mealy grains were defined to obtain the highest total flour yield. Near-isogenic lines of bread wheat that differ in hardness, due to distinct puroindoline-b alleles (the wild type, Pinb-D1a, or the mutated forms, Pinb-D1b or Pinb-D1d), were grown in different environments and under two nitrogen fertilization levels, to study genetic and environmental effects on milling behavior. Milling tests used a prototype mill, equipped with two break steps, one sizing step, and two reduction steps, and this enabled 21 individual or aggregated milling fractions to be collected. Four current grain characters, thousand grain weight, test weight, grain diameter, and protein content, were measured, and three characters known to influence grain mechanical resistance, NIRS hardness, SKCS hardness index, and grain vitreousness (a character affecting the grain mechanical behavior but generally not studied). As expected, the wild type or mutated forms of Pinb-D1 alleles led to contrasted milling behavior: soft genotypes produced high quantities of break flour and low quantities of reduction flour, whereas reverse quantities were observed for hard genotypes. This different milling behavior had only a moderate influence on total flour production. NIRS hardness and vitreousness were, respectively, the most important and the second most important grain characters to explain milling behavior. However, contrary to NIRS hardness, vitreousness was only involved in endosperm reduction and not in the separation between the starchy endosperm and the outer layers. The highest flour yields were obtained for SKCS values comprised between 30 and 50, which corresponded either to soft vitreous or hard mealy grains. Prediction equations were defined and showed a good accuracy estimating break and reduction flours portions, but should be used more cautiously for total flour.

Interactions of hydrophilic quantum dots with defect-free and defect containing supported lipid membranes.

Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges.

Colorectal cancer in young patients in Israel: a distinct clinicopathological entity?

PURPOSE: This study was designed to characterize the entity of colorectal cancer (CRC) in young patients and to evaluate whether it has any unique epidemiological or clinicopathological features. METHODS: The study population consisted of all consecutive young (≤50 years old at diagnosis) patients with CRC who were diagnosed during the years 1997-2007 and were treated at our institution, and a matching group of patients (>50 years at diagnosis). The medical files of these patients were reviewed, and the epidemiological, clinical, and pathological features of both groups were compared. RESULTS: There were 406 patients: 203 in each group. The features of the older group were typical for patients with CRC, but the younger group showed female predominance, different ethnic composition, prevalence of family history of cancer and hereditary CRC syndromes, and lower incidence of polyps. The incidence of left-sided tumors and advanced stages (III-IV) at diagnosis was higher in the younger patients. Mucinous/signet ring histology, grade, stage, lymphatic and vascular invasion were all predictive of survival, whereas age was not. CONCLUSIONS: Colorectal cancer in young patients was found to display a cluster of unique characteristics but fewer than previously reported and young age by itself was not found to impact patient outcome.

Arabinoxylan content and grain tissue distribution are good predictors of the dietary fibre content and their nutritional properties in wheat products.

Wheat millstreams and wheat-based foods (pasta, biscuits and bread) enriched or not in dietary fibre with fractions extracted from wheat grains, have been characterized either for their total dietary fibre content (TDF) and their arabinoxylan (AX) content. A strong correlation (r2 = 0.98) is observed between the AX and TDF contents indicating that AX can be used to estimate TDF content in wheat products. Moreover, by adding a previous step including enzymatic hydrolysis with a xylanase, a functional evaluation of DF is proposed based on the amount of AX released by the enzyme. Xylanase hydrolysable AX are likely also released by microbiota's enzymes in the gut and therefore an indicator for the proportion of fermentable DF in grain fractions and wheat-based foods (pasta, biscuits and bread). This assay opens the door for simple characterization of qualitative attribute of cereal DF.

Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases.

Gradients of ammonium sulfate in liposomes [(NH4)2SO4]lip. > [(NH4)2SO4]med. were used to obtain 'active' loading of amphipathic weak bases into the aqueous compartment of liposomes. The loading is a result of the base exchange with the ammonium ions. This approach was applied to encapsulate anthracyclines and acridine orange inside the liposomes at very high efficiency (> 90%). Doxorubicin was accumulated in the aqueous phase of the liposomes where it reached a level as high as 100-fold the doxorubicin concentration in the remote loading medium. Most of the intraliposomal doxorubicin was present in an aggregated state. The active entrapment and loading stability were dependent on liposome lipid composition, lipid quality, medium composition and temperature, as well as on the pKa and hydrophobicity of the base. The ammonium sulfate gradient approach differs from most other chemical approaches used for remote loading of liposomes, since it neither requires preparation of the liposomes in acidic pH, nor to alkalinize the extraliposomal aqueous phase. The stability of the ammonium ion gradient is related to the low permeability of its counterion, the sulfate, which also stabilizes anthracycline accumulation for prolonged storage periods (> 6 months) due to the aggregation and gelation of anthracycline sulfate salt.

Spontaneous transfer between phospholipid bilayers of dehydroergosterol, a fluorescent cholesterol analog.

The spontaneous interbilayer transfer of dehydroergosterol, a fluorescent cholesterol analog, was examined using small unilamellar phospholipid vesicles. The kinetic data were best fit by an equation of the form Aexp (-kt) + B. Qualitatively, the general trend of the half-time for transfer and the base values (B) obtained for dehydroergosterol resemble the corresponding values obtained in the earlier studies of cholesterol transfer. However, quantitative differences, which reflect the molecular structure of the sterol, were observed. Acrylamide quenching performed on the donor vesicles at different stages of the transfer indicated that a time-dependent organization of DHE within the vesicles occurs.

Airway CD8(+) T cells induced by pulmonary DNA immunization mediate protective anti-viral immunity.

Vaccination strategies for protection against a number of respiratory pathogens must induce T-cell populations in both the pulmonary airways and peripheral lymphoid organs. In this study, we show that pulmonary immunization using plasmid DNA formulated with the polymer polyethyleneimine (PEI-DNA) induced antigen-specific CD8(+) T cells in the airways that persisted long after antigen local clearance. The persistence of the cells was not mediated by local lymphocyte proliferation or persistent antigen presentation within the lung or airways. These vaccine-induced CD8(+) T cells effectively mediated protective immunity against respiratory challenges with vaccinia virus and influenza virus. Moreover, this protection was not dependent upon the recruitment of T cells from peripheral sites. These findings demonstrate that pulmonary immunization with PEI-DNA is an efficient approach for inducing robust pulmonary CD8(+) T-cell populations that are effective at protecting against respiratory pathogens.

Commercial fibrin sealants are not equivalent in a rabbit liver-resection model which quantitatively evaluates hemostasis and formation of adhesions.

A rabbit partial liver resection model was used to determine the hemostatic effectiveness of a new fibrin sealant. Persistent bleeding, with a mean bleeding time of 372 s and blood loss of 18 ml, from a resected lobe of the liver was achieved after rabbits in the untreated control group had been infused continuously with unfractionated heparin over 20 min with 0.2 IU/ml at a rate of 1 ml/min. Spraying the resected surface with the new fibrin sealant, Quixil, reduced bleeding to < 1 ml and the post-resection bleeding times was 25 s. Bleeding time, blood loss and the volume of sealant used in the rabbit model were inversely correlated with the thrombin concentration in the sealant. In direct comparisons with Tissucol and Beriplast, Quixil was associated with the shortest bleeding times, the lowest volume of sealant used and the lowest score of abdominal adhesions.

Functional correlates with left-right asymmetry of visual evoked potentials in stroke patients: modeling and experimental results.

OBJECTIVE: To determine the correlation between a clinical measure of function in patients after first stroke and left-right scalp amplitude of visual evoked potentials using a theoretical model of the head. DESIGN: A random sample of first-stroke patients underwent routine function measurement and investigation of left-right scalp potential asymmetry. Results of the encephalographic tests were compared with those of a healthy subject. To examine the effect of the conductivity in the damaged area on the potential asymmetry, numerical calculations were performed on a model, with four concentric circular compartments representing the brain, cerebrospinal fluid layer, skull, and scalp. The damaged region was modeled as a circular section. SETTING: Neurologic rehabilitation ward of a major rehabilitation hospital and university-affiliated biomedical engineering laboratory. PATIENTS: Four men aged 58 to 71 years, 3 with brain thrombosis and 1 with hemorrhagic stroke. The patients were admitted for rehabilitation an average of 3 weeks after the stroke and stayed for an average of 137 days. Damage was confined to the right brain in all cases; three of the patients had neglect syndrome and/or sensory disturbances. A healthy subject without stroke was also examined. MEASURES: Function was measured with the Functional Independence Measure (FIM) at 48 to 72 hours from admission and during the last week before discharge. Functional gain was calculated by subtracting the FIM admission score from the discharge score. Left-right scalp visual evoked potential amplitude was studied with flash stimuli according to the 10-20 international system and a theoretical model of the head based on two-dimensional computed tomography images; the volume conductor equation was solved numerically using the finite volume method. Left-right potential asymmetry and the damaged-region-to-brain-area ratio were calculated and correlated with the FIM values by linear regression analysis. Negative asymmetry indicates that the activity in the right damaged hemisphere is lower than in the undamaged one. RESULTS: A negative correlation was noted between the FIM score on admission and the left-right scalp potential amplitude asymmetry, and between the FIM gain and the damaged-region-to-brain-area ratio obtained from the computed tomography image. Asymmetry was negative in the thrombotic patients and positive in the hemorrhagic one. The healthy subject showed nonsignificant asymmetry. CONCLUSION: A relationship might exist between the left-right asymmetry of the scalp visual evoked potential and both the damaged-region-to-brain-area ratio and the functional outcome of rehabilitation in poststroke patients. The modeling study shows that the left-right asymmetry is most likely the result of changes in the conductivity at the damaged area, which, in turn, are probably associated with patient functional status and evolution. Further validation in larger groups of patients and normal subjects is needed before these parameters can serve as useful indices for clinical purposes.

Fraction of cholesterol undergoing spontaneous exchange between small unilamellar phosphatidylcholine vesicles.

The kinetics of the spontaneous exchange of [3H]cholesterol between small unilamellar vesicles of phosphatidylcholine has been reexamined. Although first-order exchange kinetics were observed (k = 0.0117 min-1), in good agreement with previous studies, about 20% of the total cholesterol was found to be nonexchangeable in the 8-h time frame of the experiments. The size of this nonexchangeable pool was found to depend on the type of phospholipid and the temperature. It seems probable that the two pools of cholesterol defined in these experiments reflect the complex phase structure of the cholesterol-phosphatidylcholine vesicles.

Effect of sphingomyelin composition on the phase structure of phosphatidylcholine-sphingomyelin bilayers.

In this study, we examine the phase behavior as well as lateral diffusion and percolation in the region of coexisting gel and fluid phases in binary mixtures composed of dimyristoylphosphatidylcholine and one of two totally synthetic D-erythro-sphingomyelins (having either C16 or C24 acyl chains, both having similar gel to liquid-crystalline phase transition temperatures). This study stems from the uniqueness of sphingomyelins having gel to liquid-crystalline transition temperatures in the range of physiological interest, and the fact that more than 50% of the naturally occurring sphingomyelin species have a chain mismatch. The presence of sphingomyelin in biological membranes can thus be expected to give rise to a complex phase structure. Fluorescence recovery after photobleaching, differential scanning calorimetry, and electron microscopy are used to show that, despite similarity in the temperature range of the gel to liquid-cystalline phase transition of the two sphingomyelins, the two differ in their phase structure. Also they differ to a large extent in their mixing with dimyristoylphosphatidylcholine. Dimyristoylphosphatidylcholine and C16 sphingomyelin mix nearly ideally, with the percolation threshold locus lying close to the liquidus on the phase diagram. In contrast, the C24 sphingomyelin and dimyristoylphosphatidylcholine mix nonideally, with the percolation threshold locus lying close to the solidus. In addition, mixtures containing C24 sphingomyelin have a complex thermotropic behavior which may be related to the observation that these dispersions contain several types of particles, some of which are not multilamellar vesicles. These studies suggest that the degree of sphingomyelin chain mismatch is an important factor in determining lateral organization in the membrane.

Liposomes containing Candida albicans ribosomes as a prophylactic vaccine against disseminated candidiasis in mice.

This study describes the development of prophylactic anti-Candida vaccine which combines the advantages of Candida albicans ribosomes as antigen(s) and of liposomes as carrier/adjuvant with minimal side-effects, which could be suitable for human use. The liposomal vaccine was composed of C. albicans ribosomes and the lipids dimyristoyl phosphatidyl choline (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG) (9:1 molar ratio). Some of the vaccines contained Lipid-A (LA) as an additional adjuvant. The vaccine was prepared by two methods: (I) dehydration by lyophylization of small liposomes in the presence of the Candida ribosomes (DRV method); (II) colyophylization of DMPC/DMPG (9:1 mole ratio) in tertiary butanol with aqueous dispersion of Candida ribosomes. In both cases a dry powder was obtained which was rehydrated to form large multilamellar vesicles. The efficacy of the vaccines in mice was tested by their protectivity against a challenge with C. albicans, as assessed by survival rate, induction of cell mediated immunity (measured by delayed type hypersensitivity-DTH) and anti-Candida antibody titer. Unimmunized mice and mice vaccinated by ribosomes supplemented with incomplete Freund's adjuvant (IFA) were used as controls. The results indicated that the liposomal vaccines were effective at least as the IFA-based vaccine. The study indicates the feasibility of developing an efficacious anti-Candida vaccine for human use.

Dependence on phospholipid composition of the fraction of cholesterol undergoing spontaneous exchange between small unilamellar vesicles.

Spontaneous cholesterol exchange between small unilamellar vesicles comprised of different phospholipids and their binary mixtures has been studied in order to understand the factors involved in the establishment and maintenance of intracellular cholesterol distributions. Exchange was performed from neutral donor vesicles containing different cholesterol concentrations, traces of [3H]cholesterol, and [14C]cholesteryl oleate as a nonexchangeable marker. The acceptor vesicles, in 10-fold excess, had the same composition, but 15 mol % phosphatidylglycerol was included to permit chromatographic separation. Data were best fitted by a single exponential and a base value. In donor vesicles containing only one phospholipid, the kinetic rate constants agreed with data reported previously; however, the base values were larger than the expected equilibrium value of 9.09%. The size of this nonexchangeable pool and the exchange rate were found to depend on the type of phospholipid. In binary phospholipid donor systems, well above the transition temperatures of the lipid components, the exchange parameters were preferentially closer to those of one component according to the order POPC greater than DMPC greater than DPPC greater than bovine brain SPM.

Ultrafast precessional magnetization reversal by picosecond magnetic field pulse shaping.

Since the invention of the first magnetic memory disk in 1954, much effort has been put into enhancing the speed, bit density and reliability of magnetic memory devices. In the case of magnetic random access memory (MRAM) devices, fast coherent magnetization rotation by precession of the entire memory cell is desired, because reversal by domain-wall motion is much too slow. In principle, the fundamental limit of the switching speed via precession is given by half of the precession period. However, under-critically damped systems exhibit severe ringing and simulations show that, as a consequence, undesired back-switching of magnetic elements of an MRAM can easily be initiated by subsequent write pulses, threatening data integrity. We present a method to reverse the magnetization in under-critically damped systems by coherent rotation of the magnetization while avoiding any ringing. This is achieved by applying specifically shaped magnetic field pulses that match the intrinsic properties of the magnetic elements. We demonstrate, by probing all three magnetization components, that reliable precessional reversal in lithographically structured micrometre-sized elliptical permalloy elements is possible at switching times of about 200 ps, which is ten times faster than the natural damping time constant.

Left-right asymmetry of visual evoked potentials in brain-damaged patients: a mathematical model and experimental results.

The left-right asymmetry in the potential amplitude on the scalp was studied in poststroke patients by using flash visual evoked potential (VEP) and a numerical two-dimensional model of the head. The left-right asymmetry of the VEP was measured in three patients after thrombosis, in one after hemorrhage, and in one healthy subject. The numerical model used computed tomography images to define the different compartments of the head. The volume conductor equation for the potential distribution created by a dipole source in the occipital region was solved numerically with use of a finite volume method. Left-right asymmetry was calculated with several values of conductivity of the damaged region. The experimental results revealed a negative asymmetry in the three patients after thrombosis (i.e., the potential amplitude over the ischemic hemisphere was smaller than that over the intact hemisphere), whereas, in the patient after hemorrhage, a positive asymmetry was found. Nonsignificant left-right asymmetry was found in the healthy subject. The numerical model revealed that the electrical conductivity of the damaged tissue has a major effect on the left-right asymmetry. Negative asymmetry, such as that found for patients after thrombosis, was obtained when the conductivity of the damaged region was greater than that of the brain, whereas positive asymmetry (hemorrhage patient) was obtained when that conductivity was smaller than that of the brain. This finding indicates that the left-right asymmetry in the scalp VEP of patients after brain damage may be a result of changes in the conductivity of the volume conductor (the ischemic region) between the source and the electrodes.

Resolving the interactions between hydrophilic CdTe quantum dots and positively charged membranes at the nanoscale.

The use of quantum dot nanoparticles (QDs) in bio-applications has gained quite some interest and requires a deep understanding of their interactions with model cell membranes. This involves assessing the extent of nanoparticle disruption of the membrane and how it depends on both nanoparticle and membrane physicochemical properties. Surface charge plays an important role in nanoparticle adsorption, which is primarily driven by electrostatic interactions; yet, once adsorbed, most reported works overlook the subsequent spatial nanoparticle insertion and location within the membrane. There is therefore a need for studies to assess the mutual role of membrane and nanoparticle charge into membrane structure and stability at the nanoscale, with a view to better design and control the functionality of these nanomaterials. In this work, we have resolved the extent of the interactions between hydrophilic, negatively charged CdTe QDs and positively charged lipid bilayers. A multiscale combination of surface-sensitive techniques enabled probing how surface charge mediates QD adsorption and membrane reorganization. Increasing membrane surface charge results into a larger adsorption of oppositely charged QDs, concomitantly inducing structural changes. Hydration of the membrane hydrophobic parts by QDs goes deeper into the inner leaflet with increasing membrane charge, resulting in supported lipid bilayers with decreased nanomechanical stability.