Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism

Using an experimental approach, we investigated the RNome of the pathogen Staphylococcus aureus to identify 30 small RNAs (sRNAs) including 14 that are newly confirmed. Among the latter, 10 are encoded in intergenic regions, three are generated by premature transcription termination associated with riboswitch activities, and one is expressed from the complementary strand of a transposase gene. The expression of four sRNAs increases during the transition from exponential to stationary phase. We focused our study on RsaE, an sRNA that is highly conserved in the bacillales order and is deleterious when over-expressed. We show that RsaE interacts in vitro with the 5′ region of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex. A previous report showed that RsaE targets opp3B which is co-transcribed with opp3A. Thus, our results identify an unusual case of riboregulation where the same sRNA controls an operon mRNA by targeting two of its cistrons. A combination of biocomputational and transcriptional analyses revealed a remarkably coordinated RsaE-dependent downregulation of numerous metabolic enzymes involved in the citrate cycle and the folate-dependent one-carbon metabolism. As we observed that RsaE accumulates transiently in late exponential growth, we propose that RsaE functions to ensure a coordinate downregulation of the central metabolism when carbon sources become scarce

Essays on Environmental and Resource Economics

This dissertation consists of three applied essays on environmental and resource economics. The essays study consumer and local government responses to exogenous shocks: randomized environmental messages, variation in international mineral prices and a widely publicized food-borne disease outbreak. In particular, this dissertation focuses in three broad areas: (i) investigating whether environmental messages can increase energy efficient technology adoption by poor populations and understanding the characteristics of the individuals who respond to these messages, (ii) testing whether increases in municipal income translate into additional environmental investments and policies in developing countries and, (iii) studying the role of food borne disease outbreaks on consumer purchases and preferences

ARE Update Volume 17, Number 1

1. Foodborne Disease Outbreaks and Consumer Purchases.2. Demand Growth and Commodity Promotions for Fresh Hass Avocados.3. China’s Growing Role in Agricultural Trade

DNA structure-specific priming of ATR activation by DNA-PKcs.

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs

Is muscle and protein loss relevant in long‐term fasting in healthy men? A prospective trial on physiological adaptations

Abstract Background Fasting is attracting an increasing interest as a potential strategy for managing diseases, including metabolic disorders and complementary cancer therapy. Despite concerns of clinicians regarding protein catabolism and muscle loss, evidence‐based clinical data in response to long‐term fasting in healthy humans are scarce. The objective of this study was to measure clinical constants, metabolic, and muscular response in healthy men during and after a 10 day fast combined with a physical activity programme. Methods Sixteen men (44 ± 14 years; 26.2 ± 0.9 kg/m2) fasted with a supplement of 200–250 kcal/day and up to 3 h daily low‐intensity physical activity according to the peer‐reviewed Buchinger Wilhelmi protocol. Changes in body weight (BW) and composition, basal metabolic rate (BMR), physical activity, muscle strength and function, protein utilization, inflammatory, and metabolic status were assessed during the 10 day fast, the 4 days of food reintroduction, and at 3 month follow‐up. Results The 10 day fast decreased BW by 7% (−5.9 ± 0.2 kg, P < 0.001) and BMR by 12% (P < 0.01). Fat mass and lean soft tissues (LST) accounted for about 40% and 60% of weight loss, respectively, −2.3 ± 0.18 kg and −3.53 ± 0.13 kg, P < 0.001. LST loss was explained by the reduction in extracellular water (44%), muscle and liver glycogen and associated water (14%), and metabolic active lean tissue (42%). Plasma 3‐methyl‐histidine increased until Day 5 of fasting and then decreased, suggesting that protein sparing might follow early proteolysis. Daily steps count increased by 60% (P < 0.001) during the fasting period. Strength was maintained in non‐weight‐bearing muscles and increased in weight‐bearing muscles (+33%, P < 0.001). Glycaemia, insulinemia, blood lipids, and blood pressure dropped during the fast (P < 0.05 for all), while non‐esterified fatty acids and urinary beta‐hydroxybutyrate increased (P < 0.01 for both). After a transient reduction, inflammatory cytokines returned to baseline at Day 10 of fasting, and LST were still lower than baseline values (−2.3% and −3.2%, respectively; P < 0.05 for both). Conclusions A 10 day fast appears safe in healthy humans. Protein loss occurs in early fast but decreases as ketogenesis increases. Fasting combined with physical activity does not negatively impact muscle function. Future studies will need to confirm these first findings

FANCD2 Binds MCM Proteins and Controls Replisome Function upon Activation of S Phase Checkpoint Signaling

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis

Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency

International audienceSynthetic vectors such as cationic polymers and cationic lipids remain attractive tools for non-viral gene transfer which is a complex process whose effectiveness relies on the ability to deliver a plasmid DNA (pDNA) into the nucleus of non-dividing cells. Once in the cytosol, the transport of pDNAs towards the nuclear envelope is strongly impaired by their very low cytosolic mobility due to their large size. To promote their movement towards the cell nucleus, few strategies have been implemented to exploit dynein, the microtubule’s (MT’s) motor protein, for propagation of cytosolic pDNA along the MTs towards the cell nucleus. In the first part of this review, an overview on MTs, dynein, dynein/virus interaction feature is presented followed by a summary of the results obtained by exploitation of LC8 and TCTEL1 dynein light chain association sequence (DLC-AS) for non-viral transfection. The second part dedicated to the adenoviral protein E3-14.7K, reports the transfection efficiency of polyplexes and lipoplexes containing the E3-14.7K-derived P79-98 peptide linked to pDNA. Here, several lines of evidence are given showing that dynein can be targeted to improve cytosolic pDNA mobility and accumulate pDNA near nuclear envelope in order to facilitate its transport through the nuclear pores. The linkage of various DLC-AS to pDNA carriers led to modest transfection improvements and their direct interaction with MTs was not demonstrated. In contrast, pDNA linked to the P79-98 peptide interacting with TCTEL1 via a cytosolic protein (fourteen seven K-interacting protein-1 (FIP-1)), interaction with MTs is evidenced in cellulo and transfection efficiency is improved