Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Background  

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.     

S100B: a multifunctional role in cardiovascular pathophysiology

S100B, a calcium-binding protein of the EF-hand type exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures, and high level expression of S100B in transgenic mice hearts and aortic smooth muscle cells inhibit cardiac hypertrophy and the associated phenotype, arterial smooth muscle proliferation, respectively, but demonstrate increased apoptosis following α₁-adrenergic stimulation or myocardial infarction. Knocking out S100B, augmented hypertrophy, decreased apoptosis and preserved cardiac function following myocardial infarction. S100B induces apoptosis by an extracellular mechanism by interacting with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular, and extracellular, roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular disease.     

Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves

The phyllosphere, which is defined as the parts of terrestrial plants above the ground, is a large habitat for different microorganisms that show a high extent of adaption to their environment. A number of hypotheses were generated by culture-independent functional genomics studies to explain the competitiveness of specialized bacteria in the phyllosphere. In contrast, in situ data at the metabolome level as a function of bacterial colonization are lacking. Here, we aimed to obtain new insights into the metabolic interplay between host and epiphytes upon colonization of Arabidopsis thaliana leaves in a controlled laboratory setting using environmental metabolomics approaches. Quantitative nuclear magnetic resonance (NMR) and imaging high-resolution mass spectrometry (IMS) methods were used to identify Arabidopsis leaf surface compounds and their possible involvement in the epiphytic lifestyle by relative changes in compound pools. The dominant carbohydrates on the leaf surfaces were sucrose, fructose and glucose. These sugars were significantly and specifically altered after epiphytic leaf colonization by the organoheterotroph Sphingomonas melonis or the phytopathogen Pseudomonas syringae pv. tomato, but only to a minor extent by the methylotroph Methylobacterium extorquens. In addition to carbohydrates, IMS revealed surprising alterations in arginine metabolism and phytoalexin biosynthesis that were dependent on the presence of bacteria, which might reflect the consequences of bacterial activity and the recognition of not only pathogens but also commensals by the plant. These results highlight the power of environmental metabolomics to aid in elucidating the molecular basis underlying plant–epiphyte interactions in situ.     

The effect of neonatal treatment of rats with nerve growth factor on the blood pressure and structure of the mesenteric arteries.

Newborn male Wistar rats were treated with nerve growth factor daily by subcutaneous injection for 2 weeks, and control rats were treated with either cytochrome c or buffered saline. Average body weight of the treated animals was lower than that of the controls during the 2 weeks of treatment, but became similar to that of the controls thereafter. Tissue levels of norepinephrine were elevated in the brain, adrenal glands, mesenteric arteries, and vas deferens of the treated animals immediately after the treatment, but became similar in the three groups 2 weeks after the termination of the treatment. Blood pressure and heart rate were measured beginning at 4 weeks of age until 28 weeks, when the rats were sacrificed and the mesenteric arteries sampled for morphometric measurements of vessel wall dimensions. Pretreatment with nerve growth factor did not affect blood pressure, nor heart rate. Structural alteration of the three types of mesenteric arteries was also absent in the treated animals. We conclude that even though neonatal treatment of normal Wistar rats with nerve growth factor for 2 weeks induced an elevation of the norepinephrine levels in several tissues at the end of the treatment period, it was not sufficient to produce hypertension and structural alterations in the blood vessels.     

Effects of pre-, peri-, and postmyocardial infarction treatment with losartan in rats: effect of dose on survival, ventricular arrhythmias, function, and remodeling

Angiotensin receptor blockers (ARBs) reduce adverse left ventricular (LV) remodeling and improve LV function and survival when started postmyocardial infarction (MI). ARBs also reduce ventricular arrhythmias during ischemia-reperfusion injury when started pre-MI. No information exists regarding their efficacy and safety when started pre-MI and continued peri- and post-MI. We evaluated whether the ARB losartan improves the outcome when started pre-MI and continued peri- and post-MI. Male Wistar rats (n = 502) were treated for 7 days pre-MI with losartan at a high dose (30 mg.kg(-1).day(-1)), progressively increasing dose (3 mg.kg(-1).day(-1) increased to 10 mg.kg(-1).day(-1) 10 days and 30 mg.kg(-1).day(-1) 20 days post-MI), or no treatment. Ambulatory systolic blood pressure and Holter monitoring were performed for 24 h post-MI. Echocardiography was done 30 days post-MI, and LV remodeling, cardiac hemodynamics, and fetal gene expression were assessed 38 days post-MI. High-dose losartan reduced 24-h post-MI survival compared with the progressive dose and control (21.9% vs. 36.6% and 38.1%, P = 0.033 and P = 0.009, respectively). This was associated with greater hypotension in the high dose and no change in ventricular arrhythmias in all groups. In 24-h post-MI survivors, the progressive dose group had reduced mortality from 24 h to 38 days (8.5% vs. 28.6% for control vs. 38.9% for high dose, P = 0.032 and P = 0.01, respectively). Survivors of both losartan groups demonstrated improved LV remodeling, cardiac hemodynamics, preserved GLUT-4, and reduced cardiac fetal gene expression. Pretreatment with ARBs does not reduce 24-h post-MI ventricular arrhythmias or survival, and high doses increase mortality by causing excessive hypotension. In 24-h post-MI survivors, progressively increasing doses of losartan have multiple beneficial effects, including improved survival.     

Identification of genes differentially expressed in dorsal and ventral chick midbrain during early Development

Background  

During the development of the central nervous system (CNS), patterning processes along the dorsoventral (DV) axis of the neural tube generate different neuronal subtypes. As development progresses these neurons are arranged into functional units with varying cytoarchitecture, such as laminae or nuclei for efficient relaying of information. Early in development ventral and dorsal regions are similar in size and structure. Different proliferation rates and cell migration patterns are likely to result in the formation of laminae or nuclei, eventually. However, the underlying molecular mechanisms that establish these different structural arrangements are not well understood.     

S100A6 is a negative regulator of the induction of cardiac genes by trophic stimuli in cultured rat myocytes

S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the alpha1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) by PDGF, PE, AII, and the prostaglandin F2alpha (PGF2alpha), induction of the S100B promoter by PE, and induction of the alpha-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and beta-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells.     

Neurocognitive Function in HIV Infected Patients on Antiretroviral Therapy

Objective

To describe factors associated with neurocognitive (NC) function in HIV-positive patients on stable combination antiretroviral therapy.

Design

We undertook a cross-sectional analysis assessing NC data obtained at baseline in patients entering the Protease-Inhibitor-Monotherapy-Versus-Ongoing-Triple therapy (PIVOT) trial.

Main outcome measure

NC testing comprised of 5 domains. Raw results were z-transformed using standard and demographically adjusted normative datasets (ND). Global z-scores (NPZ-5) were derived from averaging the 5 domains and percentage of subjects with test scores >1 standard deviation (SD) below population means in at least two domains (abnormal Frascati score) calculated. Patient characteristics associated with NC results were assessed using multivariable linear regression.

Results

Of the 587 patients in PIVOT, 557 had full NC results and were included. 77% were male, 68% Caucasian and 28% of Black ethnicity. Mean (SD) baseline and nadir CD4+ lymphocyte counts were 553(217) and 177(117) cells/µL, respectively, and HIV RNA was <50 copies/mL in all. Median (IQR) NPZ-5 score was −0.5 (−1.2/−0) overall, and −0.3 (−0.7/0.1) and −1.4 (−2/−0.8) in subjects of Caucasian and Black ethnicity, respectively. Abnormal Frascati scores using the standard-ND were observed in 51%, 38%, and 81%, respectively, of subjects overall, Caucasian and Black ethnicity (p<0.001), but in 62% and 69% of Caucasian and Black subjects using demographically adjusted-ND (p = 0.20). In the multivariate analysis, only Black ethnicity was associated with poorer NPZ-5 scores (P<0.001).

Conclusions

In this large group of HIV-infected subjects with viral load suppression, ethnicity but not HIV-disease factors is closely associated with NC results. The prevalence of abnormal results is highly dependent on control datasets utilised.

Trial registry

ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT01230580","term_id":"NCT01230580"}}NCT01230580     

The flat-plate plant-microbial fuel cell: the effect of a new design on internal resistances

Due to a growing world population and increasing welfare, energy demand worldwide is increasing. To meet the increasing energy demand in a sustainable way, new technologies are needed. The Plant-Microbial Fuel Cell (P-MFC) is a technology that could produce sustainable bio-electricity and help meeting the increasing energy demand. Power output of the P-MFC, however, needs to be increased to make it attractive as a renewable and sustainable energy source. To increase power output of the P-MFC internal resistances need to be reduced. With a flat-plate P-MFC design we tried to minimize internal resistances compared to the previously used tubular P-MFC design. With the flat-plate design current and power density per geometric planting area were increased (from 0.15 A/m² to 1.6 A/m² and from 0.22 W/m² to and 0.44 W/m²)as were current and power output per volume (from 7.5 A/m³ to 122 A/m³ and from 1.3 W/m³ to 5.8 W/m³). Internal resistances times volume were decreased, even though internal resistances times membrane surface area were not. Since the membrane in the flat-plate design is placed vertically, membrane surface area per geometric planting area is increased, which allows for lower internal resistances times volume while not decreasing internal resistances times membrane surface area. Anode was split into three different sections on different depths of the system, allowing to calculate internal resistances on different depths. Most electricity was produced where internal resistances were lowest and where most roots were present; in the top section of the system. By measuring electricity production on different depths in the system, electricity production could be linked to root growth. This link offers opportunities for material-reduction in new designs. Concurrent reduction in material use and increase in power output brings the P-MFC a step closer to usable energy density and economic feasibility.