Evidence for an evolutionary antagonism between Mrr and Type III modification systems

The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.     

Activation of the Salmonella typhimurium Mrr protein

The Mrr protein of Escherichia coli K12 is a cryptic type IV restriction endonuclease with specificity for methylated DNA. Recently it was discovered that endogenous activation of E. coli Mrr could be triggered by high pressure stress, resulting in the generation of double strand breaks in the host chromosome and concomitant induction of the SOS response. In this report we focused on Mrr activity of Salmonella Typhimurium LT2, and although we surprisingly found no evidence of high pressure induced activation, a large number of constitutively activated Mrr mutants could be isolated when the mrr gene was routinely cloned in an expression vector. Analysis of several spontaneous mutants revealed different single mutations that rendered the Mrr protein constitutively active. Moreover, a spontaneous S. Typhimurium mutant could be isolated that displayed an increased basal SOS induction because of a point mutation in the chromosomal mrr gene. Based on these findings the physiological role of Mrr in the cell is discussed.     

Cellular localization and dynamics of the Mrr type IV restriction endonuclease of Escherichia coli

In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.     

Ancient DNA from Nubian and Somali wild ass provides insights into donkey ancestry and domestication

Genetic data from extant donkeys (Equus asinus) have revealed two distinct mitochondrial DNA haplogroups, suggestive of two separate domestication events in northeast Africa about 5000 years ago. Without distinct phylogeographic structure in domestic donkey haplogroups and with little information on the genetic makeup of the ancestral African wild ass, however, it has been difficult to identify wild ancestors and geographical origins for the domestic mitochondrial clades. Our analysis of ancient archaeological and historic museum samples provides the first genetic information on the historic Nubian wild ass (Equus africanus africanus), Somali wild ass (Equus africanus somaliensis) and ancient donkey. The results demonstrate that the Nubian wild ass was an ancestor of the first donkey haplogroup. In contrast, the Somali wild ass has considerable mitochondrial divergence from the Nubian wild ass and domestic donkeys. These findings resolve the long-standing issue of the role of the Nubian wild ass in the domestication of the donkey, but raise new questions regarding the second ancestor for the donkey. Our results illustrate the complexity of animal domestication, and have conservation implications for critically endangered Nubian and Somali wild ass.     

Obesity and Pulmonary Function in African Americans

BackgroundObesity prevalence in United States (US) adults exceeds 30% with highest prevalence being among blacks. Obesity is known to have significant effects on respiratory function and obese patients commonly report respiratory complaints requiring pulmonary function tests (PFTs). However, there is no large study showing the relationship between body mass index (BMI) and PFTs in healthy African Americans (AA).ObjectiveTo determine the effect of BMI on PFTs in AA patients who did not have evidence of underlying diseases of the respiratory system.MethodsWe reviewed PFTs of 339 individuals sent for lung function testing who had normal spirometry and lung diffusion capacity for carbon monoxide (DLCO) with wide range of BMI.ResultsFunctional residual capacity (FRC) and expiratory reserve volume (ERV) decreased exponentially with increasing BMI, such that morbid obesity resulted in patients breathing near their residual volume (RV). However, the effects on the extremes of lung volumes, at total lung capacity (TLC) and residual volume (RV) were modest. There was a significant linear inverse relationship between BMI and DLCO, but the group means values remained within the normal ranges even for morbidly obese patients.ConclusionsWe showed that BMI has significant effects on lung function in AA adults and the greatest effects were on FRC and ERV, which occurred at BMI values < 30 kg/m2. These physiological effects of weight gain should be considered when interpreting PFTs and their effects on respiratory symptoms even in the absence of disease and may also exaggerate existing lung diseases.     

Mutational analysis and a structural model of methyl-directed restriction enzyme Mrr

The Mrr protein of Escherichia coli K12 is a cryptic Type IV restriction endonuclease whose activity appears to be triggered by high pressure stress. In this report we used high pressure to isolate and analyze several Mrr mutants, and generated a new structural model of the Mrr protein. The activity of a number of spontaneous and strategically constructed Mrr mutants is discussed in the light of this model, providing a first insight into the structure-function relationships of the Mrr enzyme.     

Contact‐free endoscopic photoacoustic sensing using speckle analysis

Photoacoustic endoscopy (PAE) is an emerging imaging modality, which offers a high imaging penetration and a high optical contrast in soft tissue. Most of the developed endoscopic photoacoustic sensing systems use miniaturized contact ultrasound transducers or complex optical approaches. In this work, a new fiber‐based detection technique using speckle analysis for contact‐free signal detection is presented. Phantom and ex vivo experiments are performed in transmission and reflection mode for proof of concept. In summary, the potential of the technique for endoscopic photoacoustic signal detection is demonstrated. The new technique might help in future to broaden the applications of PAE in imaging or guiding minimally invasive laser procedures.      

Synthetic adiponectin-receptor agonist,AdipoRon, induces glycolytic dependence in pancreatic cancer cells

Obesity creates a localized inflammatory reaction in the adipose, altering secretion of adipocyte-derived factors that contribute to pathologies including cancer. We have previously shown that adiponectin inhibits pancreatic cancer by antagonizing leptin-induced STAT3 activation. Yet, the effects of adiponectin on pancreatic cancer cell metabolism have not been addressed. In these studies, we have uncovered a novel metabolic function for the synthetic adiponectin-receptor agonist, AdipoRon. Treatment of PDAC cells with AdipoRon led to mitochondrial uncoupling and loss of ATP production. Concomitantly, AdipoRon-treated cells increased glucose uptake and utilization. This metabolic switch further correlated with AMPK mediated inhibition of the prolipogenic factor acetyl coenzyme A carboxylase 1 (ACC1), which is known to initiate fatty acid catabolism. Yet, measurements of fatty acid oxidation failed to detect any alteration in response to AdipoRon treatment, suggesting a deficiency for compensation. Additional disruption of glycolytic dependence, using either a glycolysis inhibitor or low-glucose conditions, demonstrated an impairment of growth and survival of all pancreatic cancer cell lines tested. Collectively, these studies provide evidence that pancreatic cancer cells utilize metabolic plasticity to upregulate glycolysis in order to adapt to suppression of oxidative phosphorylation in the presence of AdipoRon.Subject terms: Pancreatic cancer, Cell growth, Biologics, Pancreatic cancer     

Microbiology of siljo,a traditional Ethiopian fermented legume product

Siljo was prepared by thoroughly cooking powdered horsebean (Vicia faba) in safflower (Carthamus tinctorius) extract into a semi-solid slurry and then adding to it untreated black mustard (Brassica nigra) powder after cooling to 50°C. The black mustard powder was the source of starter microorganisms, with Lactobacillus acidophilus, L. plantarum and L. delbruekii initiating and later dominating the fermentation process. The pH of the fermenting mass dropped to 4.5 within 36 h and reached 4.0 at 168 h. Aerobic mesophilic bacteria and lactic acid bacteria were each present at about 1×10¹⁰ c.f.u./ml after 36 h of fermentation but Enterobacteriaceae were never detected. The dominant aerobic mesophilic flora consisted of Micrococcus, Bacillus and Lactobacillus spp. Crude protein, crude fat and ash increased slightly during the fermentation, with final values of around 28%, 25% and 7%, respectively, but there was a marked increase in protein availability and concentration during the fermentation.T. Mehari is with the Department of Biochemistry, Physiology and Pharmacology, Faculty of Veterinary Medicine, Addis Ababa University, P.O. Box 32, Debre-Zeit, Ethiopia, M. Ashenafi is with the Department of Basic Sciences, Awassa College of Agriculture, Addis Ababa University, P.O. Box 5, Awassa, Ethiopia.