Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi

This study was focused on obtaining the complete gene sequence of the toxR gene in V. harveyi by using toxR-targeted PCR to amplify 5' and 3' regions flanking the 576-bp Vibrio harveyi (NBRC 15634) toxR gene fragment previously amplified using degenerate PCR. To obtain the 5' flanking sequences, a forward PCR primer (VhtoxRpv) was designed based on known sequences upstream of toxR in V. parahaemolyticus and V. vulnificus. The reverse primer (VctoxR2R) was based on the sequence of the 576-bp Vibrio harveyi toxR fragment. The resulting 750-bp amplicon was sequenced, providing the 5' sequences of the V. harveyi (NBRC 15634) toxR gene. The 3' flanking region was amplified using a primer pair toxRS1 and toxRS2 based on V. parahaemolyticus and V. vulnificus toxR and toxS, resulting in a 900-bp amplicon that contained the remaining 3' sequences of the V. harveyi NBRC 15634 toxR. This paper reports, for the first time, a complete 882-bp nucleotide sequence for toxR in Vibrio harveyi. Sequence analysis and alignment revealed that the complete toxR gene in V. harveyi shares 87% sequence similarity with toxR of V. parahaemolyticus, 84% similarity with V. fluvialis, 83% with V. vulnificus and partial sequence of V. campbellii. The phylogenetic trees revealed wider divergence in toxR compared to 16S rRNA genes, so that V. harveyi could easily be distinguished from V. campbellii and V. parahaemolyticus.     

The Role of Decision Support System (DSS) in Prevention of Cardiovascular Disease: A Systematic Review and Meta-Analysis

Background

The potential role of DSS in CVD prevention remains unclear as only a few studies report on patient outcomes for cardiovascular disease.

Methods and Results

A systematic review and meta-analysis of randomised controlled trials and observational studies was done using Medline, Embase, Cochrane Library, PubMed, Amed, CINAHL, Web of Science, Scopus databases; reference lists of relevant studies to 30 July 2011; and email contact with experts. The primary outcome was prevention of cardiovascular disorders (myocardial infarction, stroke, coronary heart disease, peripheral vascular disorders and heart failure) and management of hypertension owing to decision support systems, clinical decision supports systems, computerized decision support systems, clinical decision making tools and medical decision making (interventions). From 4116 references ten studies met our inclusion criteria (including 16,312 participants). Five papers reported outcomes on blood pressure management, one paper on heart failure, two papers each on stroke, and coronary heart disease. The pooled estimate for CDSS versus control group differences in SBP (mm of Hg) was - 0.99 (95% CI −3.02 to 1.04 mm of Hg; I² = 0; p = 0.851).

Conclusions

DSS show an insignificant benefit in the management and control of hypertension (insignificant reduction of SBP). The paucity of well-designed studies on patient related outcomes is a major hindrance that restricts interpretation for evaluating the role of DSS in secondary prevention. Future studies on DSS should (1) evaluate both physician performance and patient outcome measures (2) integrate into the routine clinical workflow with a provision for decision support at the point of care.     

Interaction of C60 fullerene with lipids

Unsaturated lipids when exposed to air at room temperature undergo a slow autoxidation. When fullerene C₆₀ was dissolved in selected lipids (ethyl oleate, ethyl linoleate, linseed oil and castor oil) the spectrophotometric analysis shows that the oxidation is concentrated to C₆₀ which is converted to an epoxide C₆₀O. Thus, fullerene C₆₀ displays antioxidant activity not only when dissolved in unsaturated lipids but also, more generally, when dissolved in unsaturated solvents subjected to autoxidation like, for example, in cyclohexene. The behaviour of C₆₀ in ethyl oleate has been compared with that of the known antioxidant TMPPD (N,N′,N,N,′-tetramethyl-p-phenylenediamine) in ethyl oleate. The mechanism of the antioxidant action of C₆₀ in lipids has been proposed. The kinetics of C₆₀ oxidation in lipids was determined spectrophotometrically both at room temperature in the dark and under UV irradiation. The oxidized products derived from C₆₀ photo-oxidation in lipids have been identified.     

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy

Aims

Activation of Ca²⁺/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods and Results

In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion

These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.     

Habitat‐specific demography,source‐sink dynamics,and the niche of a common shrub in a heterogeneous and fluctuating environment

The occurrence of a species in habitats of varying quality connected through migration can only be understood by detailed investigation of itsdemography. In the Chihuahuan Desert, the common shrub Flourensia cernua is found in both productive and unproductive areas. In the former, both growing and senescent populations are regularly found, while in the latter a low density scattered population persists indefinitely. While precipitation (and its annual stochastic variation) is the same in both habitats, their geomorphological differences produce a sharp difference in the availability of the limiting resource, water. This produces different population dynamics in F. cernua, but also radically different plant communities. Counterintuitively, the low‐resource habitat (LR) supports a scattered, slightly increasing or stable population that coexists with its neighbors and acts as exporter of seeds (source population). In contrast, the high‐resource habitat (HR) allows sporadic recruitment of locally dense patches that tend towards extinction (sink population). The latter is accounted for by the increasing dominance of the grass Pleuraphys mutica. The different dynamics and regulatory mechanisms in each habitat allow the species to occupy a wider distribution than it would have in their absence. The higher abundance of F. cernua in the sink habitat, together with its consequences on community composition and dynamics, questions the idea proposed in the literature that a sink population lives outside its fundamental niche. The study provides support to the notion that the ecological niche of a species cannot be completely characterized by its requirements (e.g. as they relate to physiology), but must also include the complex demographic responses to a spatially and temporally variable environment, which may often include substandard conditions. For the niche concept to retain its usefulness, it must incorporate the demographic response of populations to spatially and temporallyvariable resource supply.     

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.     

Pastoralism in Northern Peru during Pre-Hispanic Times: Insights from the Mochica Period (100–800 AD) Based on Stable Isotopic Analysis of Domestic Camelids

Llama (Lama glama) and alpaca (Vicugna pacos) are the only large domesticated animals indigenous to the Americas. Pastoralism occupies a fundamental economic, social and religious role in Andean life. Today, camelid livestock are confined to the ecozone of the puna (above 3,500 masl), while their presence on the Pacific coast during pre-Hispanic times is attested by archaeological skeletal remains. This study aims to document herding practices on the northern Peruvian coast during the Early Intermediate Period (200 BC-600 AD) by gaining insights into diet, location of breeding and mobility of archaeological camelids from the funerary and ritual contexts of two Mochica sites, Uhle Platform in Huacas de Moche and El Brujo. The three first early years and the long-term life histories of the animals were documented by the combined bulk analysis of bone collagen (δ¹³C_col and δ¹⁵N_col) and bone structural carbonate (δ¹³C_bone and δ¹⁸O_bone) and the serial analysis of structural carbonate of molar tooth enamel (δ¹³C_enamel and δ¹⁸O_enamel). Mochica camelids were bred in the low and/or middle valleys, unlike their modern counterparts, who are restricted to highland puna C₃ pastures. Archaeological camelids had diverse and complex life histories, usually with substantial maize foddering. An ontogenetic switch in diet and possible residential mobility during the course of life were identified for some specimens. Although the inference of geographic origin from δ¹⁸O_bone and δ¹⁸O_enamel values was limited because of the lack of understanding of the influence of environmental and biological factors, tooth enamel analysis has great potential for exploring camelid herding practices and Andean pastoralism. Our study suggested that Mochica herders adapted their practices to the difficult lowland environment and that herding practices were varied and not restricted to breeding at higher altitudes. The role of maize in different aspects of the economic life of the Mochicas is also underlined.     

Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice

The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.     

A broad mercury resistant strain of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> secretes pyoverdine under limited iron conditions and high mercury concentrations

The Pseudomonas putida FB1, known as a broad-spectrum mercury resistant strain, becomes yellow-green due to the secretion of pyoverdine (PVDs) under limited iron conditions and high mercury concentrations. Different modified Nelson’s media were obtained by adding mercury, iron, and the complexing agent nitrilotriacetic acid to demonstrate that the strain produces only the highest concentrations of PVDs due to the induction with 25 µM Hg²⁺. An amount of 250 mg PVDs was purified from the supernatant of 1 litre culture. The various forms of PVDs were characterized using different techniques such as fluorescence spectroscopy, high performance liquid chromatography coupled with high resolution mass spectrometry, and scanning electron microscope equipped with energy dispersive X-ray analyser. A set of “in vivo” experiments demonstrated that additions of Hg²⁺ to the cultures from 10 to 25 µM Hg²⁺ stimulate an over secretion of PVDs suggesting that the toxic cation strongly reduces the availability of apo-PVDs, because the complex mercuric-pyoverdine is very stable at neutral pH, and hinder the formation of PVDs-Fe(III).     

Complementary antiviral efficacy of hydroxyurea and protease inhibitors in human immunodeficiency virus-infected dendritic cells and lymphocytes

Dendritic cells are susceptible to human immunodeficiency virus (HIV) infection and may transmit the virus to T cells in vivo. Scarce information is available about drug efficacy in dendritic cells because preclinical testing of antiretroviral drugs has been limited predominantly to T cells and macrophages. We compared the antiviral activities of hydroxyurea and two protease inhibitors (indinavir and ritonavir) in monocyte-derived dendritic cells and in lymphocytes. At therapeutic concentrations (50 to 100 microM), hydroxyurea inhibited supernatant virus production from monocyte-derived dendritic cells in vitro but the drug was ineffective in activated lymphocytes. Concentrations of hydroxyurea insufficient to be effective in activated lymphocytes cultured alone strongly inhibited supernatant virus production from cocultures of uninfected, activated lymphocytes with previously infected monocyte-derived dendritic cells in vitro. In contrast, protease inhibitors were up to 30-fold less efficient in dendritic cells than in activated lymphocytes. Our data support the rationale for testing of the combination of hydroxyurea and protease inhibitors, since these drugs may have complementary antiviral efficacies in different cell compartments. A new criterion for combining drugs for the treatment of HIV infection could be to include at least one drug that selectively targets HIV in viral reservoirs.