bluuebunny/crossref_metadata_2025 huggingface dataset:
Methods to monitor classical protein‐tyrosine phosphatase oxidation
Authors: Karisch Robert , Neel Benjamin G.
Published: May 2012

Reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), act as intracellular second messengers in many signaling pathways. Protein‐tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pKa. This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS‐inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling.
Laforin is required for the functional activation of malin in endoplasmic reticulum stress resistance in neuronal cells
Authors: Zeng Li , Wang Yin , Baba Otto , Zheng Pan , Liu Yang , Liu Yan
Published: May 2012

Mutations in either EPM2A, the gene encoding a dual‐specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease in humans. Lafora disease is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration and accumulation of poorly branched glycogen inclusions, called Lafora bodies or polyglucosan bodies, within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of Lafora disease remains unknown. Here, we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its Lafora disease‐causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin‐positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild‐type malin nor its mutants elicit endoplasmic reticulum stress, although the mutants exaggerate the response to endoplasmic reticulum stress. Overexpressed laforin impairs the polyubiquitination of malin while it recruits malin to polyglucosan bodies. The recruitment and activities of laforin and malin are both required for the polyglucosan body disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin–malin complex plays a critical role in disrupting Lafora bodies and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in Lafora disease.

Structured digital abstract

Malin
 physically interacts;with Laforin and GS1 by pull down (View interaction)

GS1
 and Laforin colocalize by fluorescence microscopy (View interaction)

Laforin
 physically interacts with Malin by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
New insights into redox response modulation in Fanconi’s anemia cells by hydrogen peroxide and glutathione depletors
Authors: Cuccarolo Paola , Viaggi Silvia , Degan Paolo
Published: May 2012

Fanconi’s anemia (FA) patients face severe pathological consequences. Bone marrow failure, the major cause of death in FA, accounting for as much as 80–90% of FA mortality, appears to be significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However, 20–25% of FA patients develop malignancies of myeloid origin. A survival strategy for bone marrow and hematopoietic cells under selective pressure evidently exists. This study reports that lymphoblastoid cell lines derived from two FA patients displayed significant resistance to oxidative stress induced by treatments with H2O2 and various glutathione (GSH) inhibitors that induce production of reactive oxygen species, GSH depletion and mitochondrial membrane depolarization. Among the various GSH inhibitors employed, FA cells appear particularly resistant to menadione (5 μm) and ethacrynic acid (ETA, 50 μm), two drugs that specifically target mitochondria. Even after pre‐treatment with buthionine sulfoximine, a GSH synthesis inhibitor that induces enhanced induction of reactive oxygen species, FA cells maintain significant resistance to these drugs. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may indicate the survival strategy that is adopted in FA cells undergoing transformation. The study of redox and mitochondria regulation in FA may be of assistance in diagnosis of the disease and in the care of patients.
Unraveling the thread of nature’s tapestry: the genetics of diversity and convergence in animal pigmentation
Authors: Kronforst Marcus R. , Barsh Gregory S. , Kopp Artyom , Mallet James , Monteiro Antónia , Mullen Sean P. , Protas Meredith , Rosenblum Erica B. , Schneider Christopher J. , Hoekstra Hopi E.
Published: May 2012

Animals display incredibly diverse color patterns yet little is known about the underlying genetic basis of these phenotypes. However, emerging results are reshaping our view of how the process of phenotypic evolution occurs. Here, we outline recent research from three particularly active areas of investigation: melanin pigmentation in Drosophila, wing patterning in butterflies, and pigment variation in lizards. For each system, we highlight (i) the function and evolution of color variation, (ii) various approaches that have been used to explore the genetic basis of pigment variation, and (iii) conclusions regarding the genetic basis of convergent evolution which have emerged from comparative analyses. Results from these studies indicate that natural variation in pigmentation is a particularly powerful tool to examine the molecular basis of evolution, especially with regard to convergent or parallel evolution. Comparison of these systems also reveals that the molecular basis of convergent evolution is heterogeneous, sometimes involving conserved mechanisms and sometimes not. In the near future, additional work in other emerging systems will substantially expand the scope of available comparisons.
Specifying protein kinase C functions in melanoma
Authors: Denning Mitchell F.
Published: May 2012

The protein kinase C (PKC) family of serine/threonine protein kinases is a heterogeneous group of enzymes receiving and integrating signals involved in both normal melanocyte biology and melanoma pathology. Alterations in PKC enzyme expression and activation contribute to the malignant phenotype of melanoma in both oncogenic and tumor suppressive roles. Delineating the diverse and often context‐dependent functions of PKC enzymes in melanocyte/melanoma biology is key to capitalize on these kinases as drug targets. This review summarizes several of the diverse functions of PKC in melanocyte and melanoma biology with a focus on PKC enzyme regulation and function.
A comparative analysis of the carbon intensity of biofuels caused by land use changes
Authors: Njakou Djomo Sylvestre , Ceulemans Reinhart
Published: May 2012

Worldwide land is a limited resource and its use for the production of biofuels and other agricultural products can impact greenhouse gas emissions (GHG). Several models and approaches have been used to assess the direct (dLUC) and indirect land use change (iLUC) carbon intensity – i.e. the amount of CO2 emitted per unit of biofuel produced – of biofuels, but their outcomes diverge significantly. This analysis of 15 studies published between 2008 and 2010 (i) summarized and compared models and approaches used to estimate the dLUC and iLUC carbon intensities of biofuels, and (ii) assessed the mechanisms that led to the variation in the outcomes. The data show that the dLUC carbon intensity ranged from −52 to 34 g CO2 MJ−1, whereas the iLUC ranged from 0 to 327 g CO2 MJ−1 for bioethanol depending on the feedstock, on the type of land used or displaced and on the amortization period. The total LUC carbon intensity of bioethanol was found to be −29% to 384% of that of gasoline. This means that in some cases, LUC could potentially alter the GHG benefits of biofuels. Standardizing assumptions, carbon stock changes and methodologies for estimating the dLUC and iLUC carbon intensity will ensure more consistency and meaningful comparisons across studies in the future. This might then enable policy makers to make better justified judgments on the sustainability of biofuels.
Gastric emptying, intestinal absorption of electrolytes and exercise performance in electrolyte‐supplemented horses
Authors: Lindinger Michael I. , Ecker Gayle L.
Published: May 2012

New Findings

What is the central question of this study?

The study addresses whether electrolyte supplementation of horses one hour prior to exercise improves hydration and exercise performance.

What is the main findings and its importance?

We used radiotracer methodologies to identify that 3L was the minimum amount of electrolyte supplement required to maintain hydration and replace plasma electrolytes lost by sweating. By maintaining hydration and plasma electrolyte concentrations sub‐maximal exercise duration was extended.

Horses lose considerably more electrolytes through sweating during prolonged exercise than can be readily replaced through feeds. The present study tested an oral electrolyte supplement (ES) designed to replace sweat electrolyte losses. We measured gastric emptying of 3 litres of ES (using gamma imaging of 99Tc‐sulfide colloid), the absorption of Na+ and K+ from the gastrointestinal tract using 24Na+ and 42K+, and the distribution of these ions in the body by measuring radioactivity within plasma and sweat during exercise. Three litres of ES emptied from the stomach as fast as water, with a half‐time of 47 min, and appeared in plasma by 10 min after administration (n= 4 horses). Peak values of plasma 24Na+ and 42K+ radioactivity occurred at 20–40 min, and a more rapid disappearance of K+ radioactivity from plasma was indicative of movement of K+ into cells (n= 3 horses). In a randomized crossover experiment (n= 4 horses), 1 h after administration of placebo (water), 1 or 3 litres of ES containing 24Na+, horses exercised on a treadmill at 30% of peak oxygen uptake until voluntary fatigue. The 24Na+ appeared in sweat at 10 min of exercise, and when horses received 3 litres of ES the duration to voluntary fatigue was increased in all horses by 33 ± 10%. It is concluded that an oral ES designed to replace sweat ion losses was rapidly emptied from the gastrointestinal tract, rapidly absorbed in the upper intestinal tract and rapidly distributed within the body. The ES clearly served as a reservoir to replace sweat ion losses during exercise, and administration of ES prior to exercise resulted in increased duration of submaximal exercise.
A role for nitric oxide within the nucleus tractus solitarii in the development of muscle mechanoreflex dysfunction in hypertension
Authors: Leal Anna K. , Murphy Megan N. , Iwamoto Gary A. , Mitchell Jere H. , Smith Scott A.
Published: May 2012

Evidence suggests that the muscle mechanoreflex, a circulatory reflex that raises blood pressure and heart rate (HR) upon activation of mechanically sensitive afferent fibres in skeletal muscle, is overactive in hypertension. However, the mechanisms underlying this abnormal reflex function have yet to be identified. Sensory input from the mechanoreflex is processed within the nucleus tractus solitarii (NTS) in the medulla oblongata. Within the NTS, the enzymatic activity of nitric oxide synthase produces nitric oxide (NO). This centrally derived NO has been shown to modulate muscle reflex activity and serves as a viable candidate for mediating the mechanoreflex dysfunction that develops in hypertension. We hypothesized that mechanoreflex dysfunction in hypertension is mediated by abnormal alterations in NO production in the NTS. Mechanically sensitive afferent fibres were stimulated by passively stretching hindlimb muscle before and after blocking the endogenous production of NO within the NTS via microdialysis of the NO synthase inhibitor l‐NAME (1 and 5 mm) in normotensive Wistar–Kyoto rats and spontaneously hypertensive rats (SHRs). Changes in HR and mean arterial pressure in response to stretch were significantly larger in SHRs compared with Wistar–Kyoto rats prior to l‐NAME dialysis. Attenuating NO production via l‐NAME in normotensive rats recapitulated the exaggerated cardiovascular response to stretch observed in SHRs. Dialysing l‐NAME in SHRs further accentuated the increases in HR and mean arterial pressure elicited by stretch. These findings support the contention that reductions in NO production within the NTS contribute to the generation of abnormal cardiovascular control by the skeletal muscle mechanoreflex in hypertension.
Mitogen‐activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II
Authors: Vento Peter J. , Daniels Derek
Published: May 2012

Angiotensin II (Ang II) acts on central angiotensin type 1 (AT1) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT1 receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II‐induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II‐induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen‐activated protein (MAP) kinases (ERK1/2), which play a role in Ang II‐induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar1,Ile4,Ile8‐Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II‐induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein‐independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.
Lipopolysaccharide enhances FcɛRI‐mediated mast cell degranulation by increasing Ca2+ entry through store‐operated Ca2+ channels: implications for lipopolysaccharide exacerbating allergic asthma
Authors: Yang Chengbin , Mo Xucheng , Lv Jingzhang , Liu Xiaoyu , Yuan Meichun , Dong Ming , Li Li , Luo Xinping , Fan Xinmin , Jin Zhe , Liu Zhigang , Liu Jie
Published: May 2012

Lipopolysaccharide (LPS) can exacerbate asthma; however, the mechanisms are not fully understood. This study investigated the effect of LPS on antigen‐stimulated mast cell degranulation and the underlying mechanisms. We found that LPS enhanced degranulation in RBL‐2H3 cells and mouse peritoneal mast cells upon FcɛRI activation, in a dose‐ and time‐dependent manner. Parallel to the alteration of degranulation, LPS increased FcɛRI‐activated Ca2+ mobilization, as well as Ca2+ entry through store‐operated calcium channels (SOCs) evoked by thapsigargin. Blocking Ca2+ entry through SOCs completely abolished LPS enhancement of mast cell degranulation. Consistent with functional alteration of SOCs, LPS increased mRNA and protein levels of Orai1 and STIM1, two major subunits of SOCs, in a time‐dependent manner. In addition, LPS increased the mRNA level of Toll‐like receptor 4 (TLR4) in a time‐dependent manner. Blocking TLR4 with Cli‐095 inhibited LPS, increasing transcription and expression of SOC subunits. Concomitantly, the effect of LPS enhancement of Ca2+ mobilization and mast cell degranulation was largely reduced by Cli‐095. Administration of LPS (1 μg) in vivo aggravated airway hyperreactivity and inflammatory reactions in allergic asthmatic mice. Histamine levels in serum and bronchoalveolar lavage fluid were increased by LPS treatment. In addition, Ca2+ mobilization was enhanced in peritoneal mast cells isolated from LPS‐treated asthmatic mice. Taken together, these results imply that LPS enhances mast cell degranulation, which potentially contributes to LPS exacerbating allergic asthma. Lipopolysaccharide increases Ca2+ entry through SOCs by upregulating transcription and expression of SOC subunits, mainly through interacting with TLR4 in mast cells, resulting in enhancement of mast cell degranulation upon antigen stimulation.
Individual differences in the cardiovascular responses to tonic muscle pain: parallel increases or decreases in muscle sympathetic nerve activity, blood pressure and heart rate
Authors: Fazalbhoy Azharuddin , Birznieks Ingvars , Macefield Vaughan G.
Published: May 2012

We recently showed that acute muscle pain, induced by bolus intramuscular injection of hypertonic saline, causes a sustained increase in muscle sympathetic nerve activity (MSNA) and a modest increase in blood pressure and heart rate. However, it is not known whether long‐lasting (tonic) pain, which more closely resembles chronic pain, causes a sustained increase in MSNA and blood pressure. We tested this hypothesis by recording MSNA in 12 healthy subjects. Tonic pain was induced for ∼60 min by slow intramuscular infusion of hypertonic saline (7%) into the ipsilateral tibialis anterior muscle. Pain was sustained at a tolerable level (5/10 to 6/10 on a visual analog scale). Seven subjects showed progressive increases in mean MSNA amplitude during tonic pain, increasing to 154 ± 17% (SEM) at 45 min and remaining essentially constant for the duration of the infusion. In these subjects, blood pressure and heart rate also increased. Conversely, for the other five subjects MSNA showed a progressive decline, with a peak fall of 67 ± 11% at 40 min; blood pressure and heart rate also fell in these subjects. We conclude that tonic muscle pain has long‐lasting effects on the sympathetic control of blood pressure, causing a sustained increase in some subjects yet a sustained decrease in others. This may have implications for individual differences in the cardiovascular consequences of chronic pain.
Noxious somatic stimuli diminish respiratory–sympathetic coupling by selective resetting of the respiratory rhythm in anaesthetized rats
Authors: Korim Willian S. , Egwuenu Eyitemi , Fong Angelina Y. , McMullan Simon , Cravo Sergio L. , Pilowsky Paul M.
Published: May 2012

Noxious somatic stimulation evokes respiratory and autonomic responses. The mechanisms underlying the responses and the manner in which they are co‐ordinated are still unclear. The effects of activation of somatic nociceptive fibres on lumbar sympathetic nerve activity at slow (2–10 Hz) and fast frequency bands (100–1000 Hz) and the effects on respiratory–sympathetic coupling are unknown. In anaesthetized, artificially ventilated Sprague–Dawley rats under neuromuscular blockade, ensemble averaging of sympathetic activity following high‐intensity single‐pulse stimulation of the sciatic nerve revealed two peaks (∼140 and ∼250 ms) that were present at similar latencies whether or not slow or fast band filtering was used. Additionally, in the slow band of both lumbar and splanchnic sympathetic nerve activity, a third peak with a very slow latency (∼650 ms) was apparent. In the respiratory system, activation of the sciatic nerve decreased the expiratory period when the stimulus occurred during the first half of expiration, but increased the expiratory period if the stimulus was delivered in the second half of the expiratory phase. The phase shifting of the respiratory cycle also impaired the respiratory–sympathetic coupling in both splanchnic and lumbar sympathetic nerve activity in the subsequent respiratory cycle. The findings suggest that noxious somatosympathetic responses reduce the co‐ordination between respiration and perfusion by resetting the respiratory pattern generator.
Autonomic neuroimmunomodulation in chagasic cardiomyopathy
Authors: Ribeiro Machado Marcus Paulo , Dias da Silva Valdo Jose
Published: May 2012

Chagas disease is an endemic parasitic disease, caused by the flagellate protozoan Trypanosoma cruzi, with a high prevalence in Latin America. During its chronic phase, chronic chagasic cardiomyopathy is the most apparent clinical form, affecting 25–30% of patients. This clinical form may present as congestive heart failure, thromboembolic phenomena, cardiac arrhythmias and sudden death. Pathological findings in the heart include mononuclear inflammatory infiltrate, focal myocarditis, epicarditis and neuroganglionitis, associated with variable focal fibrosis and widely variable autonomic dysfunction. The immune–inflammatory response has been considered to be the cause of the autonomic dysfunction, which may trigger life‐threatening arrhythmias and sudden death. In the last few years, several reports in the literature have described the marked role played by the autonomic nervous system in the modulation of the immune–inflammatory response in some experimental models of infectious, ischaemic and autoimmune diseases. However, nothing is known about this autonomic neural modulation of the immune response in Chagas disease. In the present report, we discuss several sets of evidence suggesting that changes in the autonomic drive directed towards the heart could modify blood and tissue parasitism, as well as inflammatory infiltration, in chagasic cardiomyopathy. The pathogenic implications of these potential neural immune manipulations are also discussed.
Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto–Kakizaki rats
Authors: Salem K. A. , Adrian T. E. , Qureshi M. A. , Parekh K. , Oz M. , Howarth F. C.
Published: May 2012

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation–contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation–contraction coupling in the hearts of early onset (8–10 weeks of age) type 2 diabetic Goto–Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real‐time RT‐PCR; shortening and intracellular Ca2+ were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non‐fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0.03 versus 0.80 ± 0.11; Kcna4, 0.79 ± 0.25 versus 1.90 ± 0.26; and Kcnj2, 0.52 ± 0.07 versus 0.78 ± 0.08) in GK ventricle compared with control ventricle. The amplitude of ventricular myocyte shortening and the intracellular Ca2+ transient were unaltered; however, the time‐to‐peak shortening was prolonged and time‐to‐half decay of the Ca2+ transient was shortened in GK myocytes compared with control myocytes. The results of this study demonstrate changes in expression of genes encoding various excitation–contraction coupling proteins that are associated with disturbances in myocyte shortening and intracellular Ca2+ transport.
Diversity of Gene Cassette Promoters in Class 1 Integrons from Wastewater Environments
Authors: Moura Alexandra , Jové Thomas , Ploy Marie-Cécile , Henriques Isabel , Correia António
Published: May 2012

The diversity of gene cassette promoters in class 1 integrons was investigated in 47 strains isolated from wastewaters. The weak PcW and PcH1 variants predominated, suggesting that, similar to clinical environments, high rates of gene cassette recombination, rather than high expression of gene cassettes, have been preferentially selected in wastewaters.
Quantitative Estimation of the Viability of Toxoplasma gondii Oocysts in Soil
Authors: Lélu Maud , Villena Isabelle , Dardé Marie-Laure , Aubert Dominique , Geers Régine , Dupuis Emilie , Marnef Francine , Poulle Marie-Lazarine , Gotteland Cécile , Dumètre Aurélien , Gilot-Fromont Emmanuelle
Published: May 2012

Toxoplasma gondii
oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples.
Proteomic Phenotyping of Novosphingobium nitrogenifigens Reveals a Robust Capacity for Simultaneous Nitrogen Fixation, Polyhydroxyalkanoate Production, and Resistance to Reactive Oxygen Species
Authors: Smit Anne-Marie , Strabala Timothy J. , Peng Lifeng , Rawson Pisana , Lloyd-Jones Gareth , Jordan T. William
Published: May 2012

Novosphingobium nitrogenifigens
Y88
T
(Y88) is a free-living, diazotrophic
Alphaproteobacterium
, capable of producing 80% of its biomass as the biopolymer polyhydroxybutyrate (PHB). We explored the potential utility of this species as a polyhydroxybutyrate production strain, correlating the effects of glucose, nitrogen availability, dissolved oxygen concentration, and extracellular pH with polyhydroxybutyrate production and changes in the Y88 proteomic profile. Using two-dimensional differential in-gel electrophoresis and tandem mass spectrometry, we identified 217 unique proteins from six growth conditions. We observed reproducible, characteristic proteomic signatures for each of the physiological states we examined. We identified proteins that changed in abundance in correlation with either nitrogen fixation, dissolved oxygen concentration, or acidification of the growth medium. The proteins that correlated with nitrogen fixation were identified either as known nitrogen fixation proteins or as novel proteins that we predict play roles in aspects of nitrogen fixation based on their proteomic profiles. In contrast, the proteins involved in central carbon and polyhydroxybutyrate metabolism were constitutively abundant, consistent with the constitutive polyhydroxybutyrate production that we observed in this species. Three proteins with roles in detoxification of reactive oxygen species were identified in this obligate aerobe. The most abundant protein in all experiments was a polyhydroxyalkanoate granule-associated protein, phasin. The full-length isoform of this protein has a long, intrinsically disordered Ala/Pro/Lys-rich N-terminal segment, a feature that appears to be unique to sphingomonad phasins. The data suggest that Y88 has potential as a PHB production strain due to its aerobic tolerance and metabolic orientation toward polyhydroxybutyrate accumulation, even in low-nitrogen growth medium.
Identification of a Novel Dihydrodaidzein Racemase Essential for Biosynthesis of Equol from Daidzein in Lactococcus sp. Strain 20-92
Authors: Shimada Yoshikazu , Takahashi Masayuki , Miyazawa Norihiro , Abiru Yasuhiro , Uchiyama Shigeto , Hishigaki Haretsugu
Published: May 2012

Equol is metabolized from daidzein, a soy isoflavone, by the gut microflora. In this study, we identified a novel dihydrodaidzein racemase (
l
-DDRC) that is involved in equol biosynthesis in a lactic acid bacterium,
Lactococcus
sp. strain 20-92, and confirmed that histidine-tagged recombinant
l
-DDRC (
l
-DDRC-His) was able to convert both the (
R
)- and (
S
)-enantiomers of dihydrodaidzein to the racemate. Moreover, we showed that recombinant
l
-DDRC-His was essential for
in vitro
equol production from daidzein by a recombinant enzyme mixture and that efficient
in vitro
equol production from daidzein was possible using at least four enzymes, including
l
-DDRC. We also proposed a model of the metabolic pathway from daidzein to equol in
Lactococcus
strain 20-92.
Natural Transformation of Gallibacterium anatis
Authors: Kristensen Bodil M. , Sinha Sunita , Boyce John D. , Bojesen Anders M. , Mell Joshua C. , Redfield Rosemary J.
Published: May 2012

Gallibacterium anatis
is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in
G. anatis
, we have established methods for transformation and targeted mutagenesis. The genus
Gallibacterium
belongs to the
Pasteurellaceae
, a group with several naturally transformable members, including
Haemophilus influenzae
. Bioinformatics analysis identified
G. anatis
homologs of the
H. influenzae
competence genes, and natural competence was induced in
G. anatis
by the procedure established for
H. influenzae
: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with
G. anatis
chromosomal DNA and with linearized plasmid DNA carrying
G. anatis
sequences. Both DNA types integrated into the
G. anatis
chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10
−4
transformants CFU
−1
. Transformation was also efficient with circular plasmid containing no
G. anatis
DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse
G. anatis
strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The
G. anatis
genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but
G. anatis
did preferentially take up its own DNA over that of
Escherichia coli
. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of
G. anatis
.
Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa
Authors: Rybtke Morten T. , Borlee Bradley R. , Murakami Keiji , Irie Yasuhiko , Hentzer Morten , Nielsen Thomas E. , Givskov Michael , Parsek Matthew R. , Tolker-Nielsen Tim
Published: May 2012

The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming
Pseudomonas aeruginosa
and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in
P. aeruginosa
. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive
cdrA
promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in
P. aeruginosa
strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of
P. aeruginosa
with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of
P. aeruginosa
.
Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems
Authors: Chen Dandan , Zhang Qi , Zhang Qinglin , Cen Peilin , Xu Zhinan , Liu Wen
Published: May 2012

FK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain
Streptomyces tsukubaensis
by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that
S. tsukubaensis
is amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.
Biochemical Characterization of a Novel Haloalkane Dehalogenase from a Cold-Adapted Bacterium
Authors: Drienovska Ivana , Chovancova Eva , Koudelakova Tana , Damborsky Jiri , Chaloupkova Radka
Published: May 2012

A haloalkane dehalogenase, DpcA, from
Psychrobacter cryohalolentis
K5, representing a novel psychrophilic member of the haloalkane dehalogenase family, was identified and biochemically characterized. DpcA exhibited a unique temperature profile with exceptionally high activities at low temperatures. The psychrophilic properties of DpcA make this enzyme promising for various environmental applications.
Detection of Imported Wild Polioviruses and of Vaccine-Derived Polioviruses by Environmental Surveillance in Egypt
Authors: Blomqvist Soile , El Bassioni Laila , El Maamoon Nasr Eman M. , Paananen Anja , Kaijalainen Svetlana , Asghar Humayun , de Gourville Esther , Roivainen Merja
Published: May 2012

Systematic environmental surveillance for poliovirus circulation has been conducted in Egypt since 2000. The surveillance has revealed three independent importations of wild-type poliovirus. In addition, several vaccine-derived polioviruses have been detected in various locations in Egypt. In addition to acute flaccid paralysis (AFP) surveillance, environmental surveillance can be used to monitor the wild poliovirus and vaccine-derived poliovirus circulation in populations in support of polio eradication initiatives.
Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation
Authors: Hirayama Yosuke , Sakanaka Mikiyasu , Fukuma Hidenori , Murayama Hiroki , Kano Yasunobu , Fukiya Satoru , Yokota Atsushi
Published: May 2012

Functional analysis of
Bifidobacterium
genes is essential for understanding host-
Bifidobacterium
interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in
Bifidobacterium longum
. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-
ΔrepA
, which lacks the plasmid replication gene
repA
, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors
repA
, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in
B. longum
105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using the
aga
gene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of
aga
. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based
aga
-complemented strain. These functional analyses revealed that
aga
is the only gene encoding a functional α-galactosidase enzyme in
B. longum
105-A.
Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion
Authors: Prince Tessa , McBain Andrew J. , O'Neill Catherine A.
Published: May 2012

Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit
Staphylococcus aureus
infection. Evaluation of the candidate probiotics
Lactobacillus reuteri
ATCC 55730,
Lactobacillus rhamnosus
AC413, and
Lactobacillus salivarius
UCC118 demonstrated that both
L. reuteri
and
L. rhamnosus
, but not
L. salivarius
, reduced
S. aureus
-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (
P
< 0.01) or simultaneously with (
P
< 0.01) infection with
S. aureus
but not when added after infection had commenced (
P
> 0.05). The protective effect of
L. reuteri
was not dependent on the elaboration of inhibitory substances such as lactic acid.
L. reuteri
inhibited adherence of
S. aureus
to keratinocytes by competitive exclusion (
P
= 0.026).
L. salivarius
UCC118, however, did not inhibit
S. aureus
from adhering to keratinocytes (
P
> 0.05) and did not protect keratinocyte viability.
S. aureus
utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (
P
= 0.03). This suggests that the protective mechanism for
L. reuteri-
mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by
S. aureus
and thus aid in the prevention of infection.
Involvement of PatE, a Prophage-Encoded AraC-Like Regulator, in the Transcriptional Activation of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933
Authors: Bender Jennifer K. , Praszkier Judyta , Wakefield Matthew J. , Holt Kathryn , Tauschek Marija , Robins-Browne Roy M. , Yang Ji
Published: May 2012

EnterohemorrhagicEscherichia coli(EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach.In silicoanalysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene,patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis inE. colishowed that the expression ofpatEis induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperonehdeAby binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.
Enhanced Tolerance to Naphthalene and Enhanced Rhizoremediation Performance for Pseudomonas putida KT2440 via the NAH7 Catabolic Plasmid
Authors: Fernández Matilde , Niqui-Arroyo José Luis , Conde Susana , Ramos Juan Luis , Duque Estrella
Published: May 2012

In this work, we explore the potential use of thePseudomonas putidaKT2440 strain for bioremediation of naphthalene-polluted soils.Pseudomonas putidastrain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation byP. putidaKT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere.Pseudomonas putidaKT2440(NAH7) efficiently expresses the Nah catabolic pathwayin vitroandin situ, leading to the complete mineralization of [14C]naphthalene, measured as the evolution of14CO2, while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.
Ammonia Inactivation of Ascaris Ova in Ecological Compost by Using Urine and Ash
Authors: McKinley James W. , Parzen Rebecca E. , Mercado Guzmán Álvaro
Published: May 2012

Viable ova of
Ascaris lumbricoides
, an indicator organism for pathogens, are frequently found in feces-derived compost produced from ecological toilets, demonstrating that threshold levels of time, temperature, pH, and moisture content for pathogen inactivation are not routinely met. Previous studies have determined that NH
3
has ovicidal properties for pathogens, including
Ascaris
ova. This research attempted to achieve
Ascaris
inactivation via NH
3
under environmental conditions commonly found in ecological toilets and using materials universally available in an ecological sanitation setting, including compost (feces and sawdust), urine, and ash. Compost mixed with stored urine and ash produced the most rapid inactivation, with significant inactivation observed after 2 weeks and with a time to 99% ovum inactivation (
T
99
) of 8 weeks. Compost mixed with fresh urine and ash achieved a
T
99
of 15 weeks, after a 4-week lag phase. Both matrices had relatively high total-ammonia concentrations and pH values of >9.24 (pK
a
of ammonia). In compost mixed with ash only, and in compost mixed with fresh urine only, inactivation was observed after an 11-week lag phase. These matrices contained NH
3
concentrations of 164 to 173 and 102 to 277 mg/liter, respectively, when inactivation occurred, which was below the previously hypothesized threshold for inactivation (280 mg/liter), suggesting that a lower threshold NH
3
concentration may be possible with a longer contact time. Other significant results include the hydrolysis of urea to ammonia between pH values of 10.4 and 11.6, above the literature threshold pH of 10.
Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds
Authors: Ismail Wael , Gescher Johannes
Published: May 2012

Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.