A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2

The human pathogen, Vibrio cholerae, belongs to the 10% of bacteria in which the genome is divided. Each of its two chromosomes, like bacterial chromosomes in general, replicates from a unique origin at fixed times in the cell cycle. Chr1 initiates first, and upon duplication of a site in Chr1, crtS, Chr2 replication initiates. Recent in vivo experiments demonstrate that crtS binds the Chr2-specific initiator RctB and promotes its initiator activity by remodeling it. Compared to the well-defined RctB binding sites in the Chr2 origin, crtS is an order of magnitude longer, suggesting that other factors can bind to it. We developed an in vivo screen to identify additional crtS-binding proteins and identified the global transcription factor, Lrp, as one such protein. Studies in vivo and in vitro indicate that Lrp binds to crtS and facilitates RctB binding to crtS. Chr2 replication is severely defective in the absence of Lrp, indicative of a critical role of the transcription factor in licensing Chr2 replication. Since Lrp responds to stresses such as nutrient limitation, its interaction with RctB presumably sensitizes Chr2 replication to the physiological state of the cell

Sequestration of CO2 by red mud with flue gas using response surface methodology

Energy demand is projected to increase throughout the globe in near future which necessitates the development of carbon capture and storage (CCS) technologies to avoid the harmful impacts of climate change. Direct mineral carbonation of alkaline solid wastes is one such technology that provides the benefit of being a permanent, cost effective and a promising on-site option, while improving the environmental quality of the waste. The present work attempts to study direct mineral carbonation of red mud (RM) with CO2 (concentration as in flue gas) by wet and dry route employing response surface methodology (RSM). Maximum reduction of about 18.5% in the concentration of CO2 was attained in wet carbonation conditions (65.1 ºC, 42.1 bar, L/S ratio of 0.53, 1 h) and the sequestration capacity of RM was found to be 15.5 g of CO2/kg, correspondingly. The models were found to be statistically significant (R2 value > 98.5%). Temperature and reaction time were found to influence the dry route. Meanwhile in wet route, temperature, pressure, liquid to solid (L/S) ratio and reaction time were all significant. The results demonstrated that RM could significantly sequester CO2 even from flue gas. The carbonation process was evidenced using Fourier-Transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM)

Super-enhancer associated core regulatory circuits mediate susceptibility to retinoic acid in neuroblastoma cells

Peer reviewed: TrueAcknowledgements: We gratefully thank Prof. Deborah Tweddle for providing the neuroblastoma cell lines. We thank Prof. Jason Carroll, and Shakhzada Ibragimova for helpful discussions. Finally, we thank Divinn Lal, Dr. Sathishkumar Ramaswamy, and Maha ELNaofal from Al Jalila Genomics Center for their help. We would like to acknowledge the support of the Mohammed Bin Rashid University of Medicine and Health Sciences and Al Jalila Foundation.Neuroblastoma is a pediatric tumour that accounts for more than 15% of cancer-related deaths in children. High-risk tumours are often difficult to treat, and patients’ survival chances are less than 50%. Retinoic acid treatment is part of the maintenance therapy given to neuroblastoma patients; however, not all tumours differentiate in response to retinoic acid. Within neuroblastoma tumors, two phenotypically distinct cell types have been identified based on their super-enhancer landscape and transcriptional core regulatory circuitries: adrenergic (ADRN) and mesenchymal (MES). We hypothesized that the distinct super-enhancers in these different tumour cells mediate differential response to retinoic acid. To this end, three different neuroblastoma cell lines, ADRN (MYCN amplified and non-amplified) and MES cells, were treated with retinoic acid, and changes in the super-enhancer landscape upon treatment and after subsequent removal of retinoic acid was studied. Using ChIP-seq for the active histone mark H3K27ac, paired with RNA-seq, we compared the super-enhancer landscape in cells that undergo neuronal differentiation in response to retinoic acid versus those that fail to differentiate and identified unique super-enhancers associated with neuronal differentiation. Among the ADRN cells that respond to treatment, MYCN-amplified cells remain differentiated upon removal of retinoic acid, whereas MYCN non-amplified cells revert to an undifferentiated state, allowing for the identification of super-enhancers responsible for maintaining differentiation. This study identifies key super-enhancers that are crucial for retinoic acid-mediated differentiation.</jats:p

Glycemic and insulin responses to some breakfast items in diabetic subjects

The glycaemic and insulin responses to four common breakfast items namely iddli, pongali uppuma and bread were studied in South Indian non-insulin dependent diabetic subjects and compared with 75 gms of glucose. The breakfasts- provided 300 K cals of which complex carbohydrate contributed 68-81%, proteins 12-14% and fat 8-19% of total calories, while the dietary fibre contents varied from 3.8 g to 7.4 g in 300 K ca1 portions. The results indicated that pongal, uppuma and iddli were suitable for diabetics as jiheir glycaemic responses to the test foods were low. Bread was considered unsuitable as its glycaemic response was as high as that of glucose. A number of parameters like the mode of cooking and' processing, the form of food and the differences in food constituents which affect digestion, absorption and metabolism seem to influence the glycaemic and insulin responses

Chromosome 1 licenses chromosome 2 replication in <i>Vibrio cholerae</i> by doubling the <i>crtS</i> gene dosage

<div><p>Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation of different chromosomes. In <i>Vibrio cholerae</i>, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously. The delay is due to the dependence of Chr2 initiation on the replication of a site, <i>crtS</i>, on Chr1. The mechanism by which replication of <i>crtS</i> allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra <i>crtS</i> copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated <i>crtS</i> copies have significant activity, and suggests that a role of replication is to double the copy number of the site that sufficiently increases its activity for licensing Chr2 replication. We further show that <i>crtS</i> activity promotes the Chr2-specific initiator function and that this activity is required in every cell cycle, as would be expected of a cell-cycle regulator. This study reveals how increase of gene dosage through replication can be utilized in a critical regulatory switch.</p></div

Blocking replication of Chr1 blocks Chr2 replication.

<p>(A) Scheme for blocking Chr1 replication by induction of <i>ter</i>-binding Tus protein from <i>pbad</i> promoter. (B) Time-lapse microscopy of <i>V</i>. <i>cholerae</i> (CVC3022) showing <i>ori1</i> (green) and <i>ori2</i> (red) foci when cells were grown either without inducer (panels 1–4) or with 0.2% inducer (arabinose) to induce Tus for blocking Chr1 replication (panels 5–12). Without inducer, cells were born with two <i>ori1</i> foci and one <i>ori2</i> focus (panel 1). Upon cell growth, the <i>ori2</i> focus duplicates (panel 2), followed by duplication of <i>ori1</i> before cell division (panels 3,4). With Tus induction, many cells show one <i>ori1</i> focus, indicating a block of Chr1 replication (panel 5). In most of these cells, the <i>ori2</i> focus remains single, indicating that Chr2 replication is blocked (panels 6–8). In a few cells, the <i>ori2</i> focus was seen to divide even as the <i>ori1</i> focus remained single, (panels 9–12). Scale bars, 2 μm. (C) Marker frequency analysis by qPCR. Markers analyzed were <i>ori1</i>, <i>ter1</i>, <i>ori2</i> and <i>ter2</i> of <i>V</i>. <i>cholerae</i> grown in LB in the absence (black) or presence (grey) of 0.2% arabinose for 150 min. The induction reduced the <i>ori1/ter1</i> and <i>ori2/ter2</i> values. Data represents mean ± standard error of mean (SEM) from three biological replicates, each performed in triplicate. Statistical significance was calculated using a Student’s <i>t</i>-test. (D) Histogram of <i>ori2</i> foci number per cell (x-axis) as percentages in cells that have one <i>ori1</i> focus (y-axis). The cells with no visible <i>ori2</i> focus (grey bars, ~17%) are possibly due to weak tdTomato fluorescence. Data represents mean ± SEM of percentages calculated from three biological replicates each with at least 100 cells (Σn = total cells counted = 522). (<i>E</i>) Mean length of cells with one <i>ori1</i> focus, two <i>ori2</i> foci and one or two <i>ori2</i> foci. Note that the cells with two <i>ori2</i> foci are longer, revealing that with accumulation of mass Chr2 can overcome its dependence on Chr1 replication. Error bars denote SEM.</p

Model for Chr2 replication licensing by remodeled RctB.

<p>The triggering of Chr2 replication initiation requires two copies of <i>crtS</i>. The two copies are normally produced by passage of the Chr1 replication fork across the <i>crtS</i> site (left diagram) or, can also be provided in the absence of Chr1 replication by inserting an extra copy of <i>crtS</i> (right diagram). This suggests that the product of replication but not the passage of the fork <i>per se</i> is obligatory for <i>crtS</i> function. In our experiments, the requirement for a second copy of <i>crtS</i> could be obviated by increasing RctB supply (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007426#pgen.1007426.g004" target="_blank">Fig 4</a>). This, together with the findings that to date all revertants of Δ<i>crtS</i> map in <i>rctB</i> and that <i>crtS</i> alters the DNA binding activities of RctB, lead us to propose that <i>crtS</i> activates RctB. The two <i>crtS</i> copies are apparently required to activate RctB sufficiently for initiation.</p