Salusin-α attenuates inflammatory responses in vascular endothelial cells

Atherosclerosis accounts for numerous cardiovascular diseases, and cytokines have a critical role in acceleration or suppression of disease. Salusin-α presents a new class of bioactive peptides that can have anti-atherogenic properties. Therefore, the effects of salusin-α on the expression of some pro- and anti-inflammatory cytokines and on TNF-α-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) were examined. The involvement of the NF-κB pathway in effects of salusin-α in HUVECs was checked using Bay 11-7082 as an NF-κB inhibitor. The mRNA expression of pro-inflammatory cytokines including IL-6, IL-8, and IL-18 and anti-inflammatory cytokine IL-1Ra was assessed by real-time PCR. The protein levels of cytokines were measured by the ELISA method. Salusin-α suppressed both mRNA and protein expression of pro-inflammatory cytokines and induced mRNA and protein expression of IL-1Ra in HUVECs. Salusin-α suppressed TNF-α-induced inflammatory responses in HUVECs. The down-regulatory or up-regulatory effects of salusin-α on expression of cytokines could not be influenced by Bay 11-7082 pretreatment. Our findings indicate anti-inflammatory effects of salusin-α and suggest a novel peptide-based therapeutic strategy for atherosclerosis.     

Cryptochrome Mediates Light-Dependent Magnetosensitivity of Drosophila's Circadian Clock

Since 1960, magnetic fields have been discussed as Zeitgebers for circadian clocks, but the mechanism by which clocks perceive and process magnetic information has remained unknown. Recently, the radical-pair model involving light-activated photoreceptors as magnetic field sensors has gained considerable support, and the blue-light photoreceptor cryptochrome (CRY) has been proposed as a suitable molecule to mediate such magnetosensitivity. Since CRY is expressed in the circadian clock neurons and acts as a critical photoreceptor of Drosophila's clock, we aimed to test the role of CRY in magnetosensitivity of the circadian clock. In response to light, CRY causes slowing of the clock, ultimately leading to arrhythmic behavior. We expected that in the presence of applied magnetic fields, the impact of CRY on clock rhythmicity should be altered. Furthermore, according to the radical-pair hypothesis this response should be dependent on wavelength and on the field strength applied. We tested the effect of applied static magnetic fields on the circadian clock and found that flies exposed to these fields indeed showed enhanced slowing of clock rhythms. This effect was maximal at 300 μT, and reduced at both higher and lower field strengths. Clock response to magnetic fields was present in blue light, but absent under red-light illumination, which does not activate CRY. Furthermore, cry^b and cry^OUT mutants did not show any response, and flies overexpressing CRY in the clock neurons exhibited an enhanced response to the field. We conclude that Drosophila's circadian clock is sensitive to magnetic fields and that this sensitivity depends on light activation of CRY and on the applied field strength, consistent with the radical pair mechanism. CRY is widespread throughout biological systems and has been suggested as receptor for magnetic compass orientation in migratory birds. The present data establish the circadian clock of Drosophila as a model system for CRY-dependent magnetic sensitivity. Furthermore, given that CRY occurs in multiple tissues of Drosophila, including those potentially implicated in fly orientation, future studies may yield insights that could be applicable to the magnetic compass of migratory birds and even to potential magnetic field effects in humans.     

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

Reproducibility of swallow-induced cortical BOLD positive and negative fMRI activity

Functional MRI (fMRI) studies have demonstrated that a number of brain regions (cingulate, insula, prefrontal, and sensory/motor cortices) display blood oxygen level-dependent (BOLD) positive activity during swallow. Negative BOLD activations and reproducibility of these activations have not been systematically studied. The aim of our study was to investigate the reproducibility of swallow-related cortical positive and negative BOLD activity across different fMRI sessions. We studied 16 healthy volunteers utilizing an fMRI event-related analysis. Individual analysis using a general linear model was used to remove undesirable signal changes correlated with motion, white matter, and cerebrospinal fluid. The group analysis used a mixed-effects multilevel model to identify active cortical regions. The volume and magnitude of a BOLD signal within each cluster was compared between the two study sessions. All subjects showed significant clustered BOLD activity within the known areas of cortical swallowing network across both sessions. The cross-correlation coefficient of percent fMRI signal change and the number of activated voxels across both positive and negative BOLD networks were similar between the two studies (r ≥ 0.87, P < 0.0001). Swallow-associated negative BOLD activity was comparable to the well-defined "default-mode" network, and positive BOLD activity had noticeable overlap with the previously described "task-positive" network. Swallow activates two parallel cortical networks. These include a positive and a negative BOLD network, respectively, correlated and anticorrelated with swallow stimulus. Group cortical activity maps, as well as extent and amplitude of activity induced by volitional swallowing in the cortical swallowing network, are reproducible between study sessions.     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Oxidative toxicity in diabetes and Alzheimer’s disease: mechanisms behind ROS/ RNS generation

Reactive oxidative species (ROS) toxicity remains an undisputed cause and link between Alzheimer’s disease (AD) and Type-2 Diabetes Mellitus (T2DM). Patients with both AD and T2DM have damaged, oxidized DNA, RNA, protein and lipid products that can be used as possible disease progression markers. Although the oxidative stress has been anticipated as a main cause in promoting both AD and T2DM, multiple pathways could be involved in ROS production. The focus of this review is to summarize the mechanisms involved in ROS production and their possible association with AD and T2DM pathogenesis and progression. We have also highlighted the role of current treatments that can be linked with reduced oxidative stress and damage in AD and T2DM.     

Studies on N2-fixing bacteria associated with the salt-tolerant grass,Leptochloa fusca (L.) Kunth

Summary N₂-fixing bacteria were isolated from the rhizosphere of naturally grown salt tolerant grass (Leptochloa fusca). A broad spectrum of diazotrophs was found to be associated with the roots ofL. fusca. the systematic position of the three isolates, NIAB-1, C-2 and Iso-2 was determined by morphological, biochemical and mol % (G+C) DNA contents. Two isolates were identified asKlebsiella pneumoniae (NIAB-1) andBeijerinckia sp. (Iso-2).¹⁵N enrichment studies confirmed the nitrogen fixing ability of the isolates. The effects of different levels of combined nitrogen (NO ₃ ^– & NH ₄ ⁺ ), pH (5.5–9.0) and salt (NaCl) on nitrogenase activity of the isolates were determined at various time intervals. All isolates exhibited nitrogenase activity even in the presence of 5 mmol/l NO ₃ ^– or NH ₄ ⁺ in a semi-solid medium after 24 h of growth. Maximum nitrogenase activity was observed at alkaline pH and all isolates were able to tolerate up to 3% NaCl in the medium.

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Resumen Se han aíslado bacterias fijadoras de N₂ en la rizosfera del hábitat natural de la graminea halófilaLeptochloa fusca. Un amplio espectro de diazotrofos se encontró asociado con las raíces deL. fusca. La posición sistemática de tres aíslados: NIAB-1, C-2 y Iso-2 se determinó utilizando sus características morfológicas, bioquímicas y el % (G+C) molar del ADN. El aíslado NIAB-1 se identificó comoKlebsiella pneumoniae y el aíslado Iso-2 comoBeijerinckia. sp. Estudios mediante¹⁵N confirmaron la habilidad fijadora de N₂ de los aíslados. Se determinaron periodicamente los efectos de distintos niveles de nitrógeno combinado (NO₃ ^– y NH₄ ⁺), pH (5.5–9.0) y sal (NaCl) en la actividad nitrogenásica de los aíslados. Todas las cepas aísladas mostraron actividad nitrogenásica incluso en presencia de 5mmol/l de NO₃ ^– y NH₄ ⁺ en un medio semisólido desqués de 24 h. de crecimiento. La actividad nitrogenásica máxima se observó a pH alcalino y todos loa aíslados eran capaces de tolerar hasta 3% de NaCl en el medio.

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Résumé Des bactéries fixatrices de l'azote ont été isolées à partir de la rhizosphère de l'herbe halotoléranteLeptochloa fusca développée dans les conditions naturelles. Il a été constaté qu'un large spectre de diazotrophes est associé aux racines de la plante. La position taxonomique de 3 souches isolées, NIAB-1, C-2 et Iso-2, a été déterminée par des critères morphologiques et biochimiques et par le pourcentage de (G+C) de l'ADN. Deux souches on été identifiées commeKlebsiella pneumoniae (NIAB-1) etBeijerinckia sp. (Iso-2). Les études d'enrichissement en¹⁵N ont confirmé l'aptitude des souches à fixer l'azote. les effets de différents niveaux d'azote combiné (NO₃ ^– et NH₄ ⁺), de pH (5.5–9.0) et de sel (NaCl) sur l'activité nitrogénasique des souches ont été déterminés à divers intervalles de temps. Toutes les souches présentent une activité nitrogènase après 24 h de croissance en milieu semi-solide, et cela même en présence de 5 mmol/l de NO₃ ^– ou NH₄ ⁺. L'activité nitrogènase maximum est observée à pH alcalin, et toutes les souches tolèrent jusqu'à 3% de NaCl dans le milieu.

     

Metagenomics analysis of the fecal microbiota in Ring-necked pheasants (Phasianus colchicus) and Green pheasants (Phasianus versicolor) using next generation sequencing

Pheasant reintroduction and conservation efforts have been in place in Pakistan since the 1980 s, yet there is still a scarcity of data on pheasant microbiome and zoonosis. Instead of growing vast numbers of bacteria in the laboratory, to investigate the fecal microbiome, pheasants (green and ring neck pheasant) were analyzed using 16S rRNA metagenomics and using IonS5TMXL sequencing from two flocks more than 10 birds. Operational taxonomic unit (OTU) cluster analysis and phylogenetic tree analysis was performed using Mothur software against the SSUrRNA database of SILVA and the MUSCLE (Version 3.8.31) software. Results of the analysis showed that firmicutes were the most abundant phylum among the top ten phyla, in both pheasant species, followed by other phyla such as actinobacteria and proteobacteria in ring necked pheasant and bacteroidetes in green necked pheasant. Bacillus was the most relatively abundant genus in both pheasants followed by Oceanobacillus and Teribacillus for ring necked pheasant and Lactobacillus for green necked pheasant. Because of their well-known beneficial characteristics, these genus warrants special attention. Bird droppings comprise germs from the urinary system, gut, and reproductive sites, making it difficult to research each anatomical site at the same time. We conclude that metagenomic analysis and classification provides baseline information of the pheasant fecal microbiome that plays a role in disease and health.