Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A,X-linked adrenoleukodystrophy,X-linked hydrocephalus and incontinentia pigmenti loci on Xq28

Preimplantation genetic diagnosis (PGD) first consisted of the selection of female embryos for patients at risk of transmitting X-linked recessive diseases. Advances in molecular biology now allow the specific diagnosis of almost any Mendelian disease. For families with an identified X-linked recessive disease-causing mutation, non-specific diagnosis by sex identification can be considered as a sub-standard method, since it involves the unnecessary disposal of healthy male embryos and reduces success rate by diminishing the pool of embryos eligible for transfer. The most telomeric part of the X-chromosome long arm is a highly gene-rich region encompassing disease genes such as haemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti. We developed five single-cell triplex amplification protocols with microsatellite markers DXS1073, DXS9901 (BGN), G6PD, DXS1108, DXS8087 and F8C-IVS13 located in this Xq terminal region. These tests allow the diagnosis of all diseases previously mentioned providing that the genetic material allowing the identification of the morbid allele can be obtained. The choice of the microsatellite set to use depends on the localisation of the gene responsible for the diagnosed pathology and on the informativity of the markers in particular families. Single-cell amplification efficiency was assessed on single lymphocytes. Amplification rate of the different markers ranged from 89–97% with an allele drop out rate of 2–19 %. So far PGD has been carried out for three carrier females at risk of transmitting X-linked adrenoleukodystrophy, X-linked hydrocephalus and hemophilia A. The latter one is now pregnant.     

Capreomycin is active against non-replicating M. tuberculosis

Background

Eradication of Helicobacter pylori is an important objective in overcoming gastric diseases. Many regimens are currently available but none of them could achieve 100% success in eradication. Eugenol and cinnamaldehyde that are commonly used in various food preparations are known to possess antimicrobial activity against a wide spectrum of bacteria.

Aim

The present study was performed to assess the in vitro effects of eugenol and cinnamaldehyde against indigenous and standard H. pylori strains, their minimum inhibitory concentrations (MICs) and time course lethal effects at various pH.

Methods

A total of 31 strains (29 indigenous and one standard strain of H. pylori ATCC 26695, one strain of E. coli NCIM 2089) were screened. Agar dilution method was used for the determination of drug sensitivity patterns of isolates to the commonly used antibiotics and broth dilution method for the test compounds.

Results

Eugenol and cinnamaldehyde inhibited the growth of all the 30 H. pylori strains tested, at a concentration of 2 μg/ml, in the 9th and 12th hours of incubation respectively. At acidic pH, increased activity was observed for both the compounds. Furthermore, the organism did not develop any resistance towards these compounds even after 10 passages grown at sub-inhibitory concentrations.

Conclusion

These results indicate that the two bioactive compounds we tested may prevent H. pylori growth in vitro, without acquiring any resistance.     

Interactions among mycorrhizae, atmospheric CO2 and soil N impact plant community composition

We examined plant community responses to interactions between arbuscular mycorrhizal (AM) fungi and availability of atmospheric CO₂ and soil N. Communities of 14 plant species were grown in mesocosms containing living or killed AM fungal inoculum, ambient or elevated atmospheric CO₂ and low or enriched soil N. After one growing season, significantly different plant communities existed in the different treatments. Plant species richness was lowest in +N mesocosms and highest in +AM + CO₂ mesocosms. At ambient CO₂, AM fungi reduced richness but at elevated CO₂ they increased it. This was caused by changes in mortality rates of several C₃ forbs and may suggest that CO₂ enrichment ameliorates the carbon cost of some AM symbioses. Soil moisture was higher in +CO₂ mesocosms but +AM counteracted this effect. These results suggest that AM symbioses may be important mediators of plant community responses to anthropogenic CO₂ and N enrichment.     

A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8(+) T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8(+) T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8(+) T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.     

Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers

E1-deleted adenoviral recombinants most commonly based on the human serotype 5 (AdHu5) have been shown thus far to induce unsurpassed transgene product-specific CD8(+) T cell responses. A large percentage of the adult human population carries neutralizing Abs due to natural exposures to AdHu5 virus. To circumvent reduction of the efficacy of adenovirus (Ad) vector-based vaccines by neutralizing Abs to the vaccine carrier, we developed E1-deleted adenoviral vaccine carriers based on simian serotypes. One of these carriers, termed AdC68, expressing a codon-optimized truncated form of gag of HIV-1 was shown previously to induce a potent transgene product-specific CD8(+) T cell response in mice. We constructed a second chimpanzee adenovirus vaccine vector, termed AdC6, also expressing the truncated gag of HIV-1. This vector, which belongs to a different serotype than the AdC68 virus, induces high frequencies of gag-specific CD8(+) T cells in mice including those pre-exposed to AdHu5 virus. Generation of an additional E1-deleted adenoviral vector of chimpanzee origin allows for sequential booster immunizations with heterologous vaccine carriers. In this study, we show that such heterologous prime boost regimens based on E1-deleted adenoviral vectors of different serotypes expressing the same transgene product are highly efficient in increasing the transgene product-specific CD8(+) T cell response. They are equivalent to sequential vaccinations with an E1-deleted Ad vector followed by booster immunization with a poxvirus vector and they surpass regimens based on DNA vaccine prime followed by a recombinant adenoviral vector boost.     

Variation in paternity in the field cricket Teleogryllus oceanicus: no detectable influence of sperm numbers or sperm length

Recent attention has focused on the role that sperm competitionmay play in the evolution of sperm morphology. Theoretical analysespredict increased sperm size, decreased sperm size, and no changein sperm size in response to sperm competition, depending onthe assumptions made concerning the life history and functionof sperm. However, although there is good evidence that spermmorphology varies widely within and between species, the adaptivesignificance of this variation has not been examined. Here wedocument significant intraspecific variation in sperm lengthin the field cricket, Teleogryllus oceanicus. Sperm length didnot influence the rate of migration of sperm from the spermatophoreto the female's spermatheca. We performed sperm competitiontrials in which we varied the numbers of sperm transferred byeach of two males that differed in the length of sperm theyproduced. Neither sperm length nor the number of sperm transferredinfluenced paternity. The same results were obtained using twodifferent methods for assigning paternity. The distributionof paternity across a female's mates was highly variable, withfrequently one, or more in the case of females mated to fourmales, principal sire. There were no mating order effects onpaternity. These data show that sperm do not mix randomly inthe female's spermatheca. We discuss several alternative explanationsfor the patterns of paternity observed.     

Production,Fate and Pathogenicity of Plasma Microparticles in Murine Cerebral Malaria

In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.     

Synthesis and Mechanistic Studies of a Novel Homoisoflavanone Inhibitor of Endothelial Cell Growth

Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.