Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein.

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

White lupin (Lupinus albus L.), the neglected multipurpose crop: Its production and utilization in the mixed crop-livestock farming system of Ethiopia

A survey was conducted to generate holistic information on the production and utilization of local white lupin in two lupin growing districts, namely, Mecha and Sekela, representing mid and high altitude areas, respectively in North-western Ethiopia. During the survey, two types of participatory rural appraisal (PRA) techniques, namely, individual farmer interview (61 farmers from Mecha and 51 from Sekela) and group discussion (with 20 farmers from each district) were employed. There are significant differences (P<0.05) between the two study districts for the variables like total land holding, frequency of ploughing during lupin planting, days to maturity, lupin productivity, and number of days of soaking lupin in running water. However, there are no significant differences (P>0.05) between the two study districts for the variables like land allocated for lupin cultivation, lupin seed rate, lupin soaking at home, lupin consumption per family per week and proportion of lupin used for household consumption. The use of the crop as livestock feed is negligible due to its high alkaloid content. It is concluded that the local white lupin in Ethiopia is a valuable multipurpose crop which is being cultivated in the midst of very serious shortage of cropland. Its ability to maintain soil fertility and serve as a source of food in seasons of food scarcity makes it an important crop. However, its bitter taste due to its high alkaloid content remains to be a big challenge and any lupin improvement strategy has to focus on minimizing the alkaloid content of the crop.

Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis

Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

Effectiveness of Fluorescence-based Methods in Monitoring Progression of Noncavitated Caries-like Lesions on Smooth Surfaces

Although there has been a significant decrease in caries prevalence in developed countries, the slower progression of dental caries requires methods capable of detecting and quantifying lesions at an early stage. The aim of this study was to evaluate the effectiveness of fluorescence-based methods (DIAGNOdent 2095 laser fluorescence device [LF], DIAGNOdent 2190 pen [LFpen], and VistaProof fluorescence camera [FC]) in monitoring the progression of noncavitated caries-like lesions on smooth surfaces. Caries-like lesions were developed in 60 blocks of bovine enamel using a bacterial model of Streptococcus mutans and Lactobacillus acidophilus . Enamel blocks were evaluated by two independent examiners at baseline (phase I), after the first cariogenic challenge (eight days) (phase II), and after the second cariogenic challenge (a further eight days) (phase III) by two independent examiners using the LF, LFpen, and FC. Blocks were submitted to surface microhardness (SMH) and cross-sectional microhardness analyses. The intraclass correlation coefficient for intra- and interexaminer reproducibility ranged from 0.49 (FC) to 0.94 (LF/LFpen). SMH values decreased and fluorescence values increased significantly among the three phases. Higher values for sensitivity, specificity, and area under the receiver operating characteristic curve were observed for FC (phase II) and LFpen (phase III). A significant correlation was found between fluorescence values and SMH in all phases and integrated loss of surface hardness (ΔKHN) in phase III. In conclusion, fluorescence-based methods were effective in monitoring noncavitated caries-like lesions on smooth surfaces, with moderate correlation with SMH, allowing differentiation between sound and demineralized enamel.

Different bacterial models for in vitro induction of non-cavitated enamel caries-like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non-cavitated enamel caries-like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4-20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion-type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non-cavitated enamel caries-like lesions. The optimal period for inducing caries-like lesions was 8 days.

Relecture jakobsonienne de la distinction saussurienne langue/parole. De la constitution d'un concept à l'acceptation d'un objet donné

The Roman Jakobson’s criticism of the Saussurean langue/parole distinction, in his La théorie saussurienne en retrospection, is based on the acceptance of a given object in Bachelard’s sense: the language (langage) as a complex unity which we only have to analyze. It contains no theorization of langue as a norm. On the contrary, the Saussurean langue/parole distinction is at the same time epistemological and theoretical: It amounts to delimiting an object in the whole language (langage) and to theorizing the dimension of communication which is inherent to human language. Thus, the Saussurean distinction appears to be at the foundation of the theorization of langue, which founds linguistics, but whose necessity is dissimulated, in a linguistic theory like Jakobson’s, by the language(langage) as a given object and the langue/parole problematics following from it.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

Genome characterization, prevalence and distribution of a Macula-like virus from Apis mellifera and Varroa destructor

Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae (Tymovirales; Tymoviridae), a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV).

Structural studies of proteinase entrapment by human alpha 2-macroglobulin using 3-D electron microscopy

Human a2 -macroglobulin ( a2 M; homotetramer, Mr 720 kDa) is an essential scavenger of proteinases in the serum. Each of its four subunits has a ‘bait region’, with cleavage sequences for almost all endo-proteinases, an unusual thiol ester moiety and a receptor-binding domain (RBD). Bait region cleavage in native a2 M ( a2 M-N) by a proteinase results in rapid thiol ester breakage, with a large-scale structural transformation, in which a2 M uniquely entraps the proteinase in a cage-like structure and exposes receptor-binding domains for rapid endocytosis. Transformed a2 M ( a2 M-TR) contains up to two proteinases, which remain active to small substrates. 3-D electron microscopy is optimally suited to study this unusual structural change at resolutions near (1/30) Å−1. ^ The structural importance of the thiol esters was demonstrated by a genetically-engineered a2 M, with the cysteines involved in thiol ester formation mutated to serines, which appeared structurally homologous to a2 M-TR. This demonstrates that the four highly labile thiol esters alone maintain the a2 M-N structure, while the ‘closed trap’ formed by a2 M-TR is a more stable structural form. ^ Half-transformed a2 M ( a2 M-HT), with cleaved bait regions and thiol esters in only two of its four subunits, provides an important structural link between a2 M-N and a2 M-TR. A comparison with a2 M-N showed the two proteinase-entrapping domains were above and below the plane bisecting the long axis. Both a2 M-N and a2 M-TR consist of two dense, oppositely twisted strands with significant interconnections, indicating that the structural change involves a rotation of these strands. In a2 M-HT these strands were partially untwisted with large central openings, revealing the manner in which the proteinase enters the internal cavity of a2 M. ^ In reconstructions of a2 M-N, a2 M-HT and a2 M-TR labeled with a monoclonal Fab, the Fabs were located on distal ends of each constitutive strand, demonstrating an anti-parallel arrangement of the subunits. Separation between the top and bottom pairs of Fabs was nearly the same on all structures, but the pairs were rotated about the long axis. Taken together, these results indicate that upon proteinase cleavage the two strands in a2 M-N separate. The proteinase enters the structure, while the strands re-twist to encage it. In a2 M-TR, which displays receptor-binding arms, more than two subunits are transformed as strands in the transformed half of a2 M-HT were not separated. ^

The Fern-like seed-plants of the carboniferous flora

By D. H. Scott

Caracterización de tres suelos en Hidalgo y Querétaro, México: disponibilidad y daño tóxico por arsénico en garbanzo (Cicer arietinum L.)

La presencia de arsénico (As) en suelos permite su migración hacia cultivos como el garbanzo (Cicer arietinum L.). En este trabajo se comparan contenidos de As en tres suelos de dos estados de la República Mexicana, y su acumulación en C. arietinum L. Los suelos resultaron moderadamente alcalinos, no salinos, reductores intermedios y con potenciales zeta (pZ) que indican suspensiones coloidales moderadamente estables. Con moderados contenidos de humedad y texturas franco- arcillosas, densidad aparente, capacidad de campo, agregados estables y capacidad de intercambio catiónico, significativamente diferentes y semejantes en velocidad de infiltración y espesor del horizonte A. En materia orgánica, carbono y nitrógeno son significativamente diferentes y con bajos contenidos. Existen diferencias importantes entre semillas de C. arietinum L. certificadas o no en su capacidad de germinación y desarrollo de raíces. El efecto genotóxico del As en raíces de C. arietinum L. se apreció por inducción de micronúcleos, reducción de 2,8 veces la división celular de muestras tratadas con agua con As, respecto de muestras control. Se apreció incremento de As de 9,5 veces en plántulas germinadas en suelo de El Salitre, que en suelo de Bella Vista, indicativo de migración del As. La suma de efectos de concentración de As en suelos y agua se incrementa 15,3 veces. Entre suelos de Bella Vista y Querétaro la correlación fue 2,9 veces mayor. En controles positivos los IBAs resultan 3 veces mayores que en las pruebas experimentales.