Adaptive laboratory evolution for strain engineering.

Complex phenotypes, such as tolerance to growth inhibitors, are difficult to rationally engineer into industrial model organisms due our poor understanding of their underlying molecular mechanisms. Adaptive evolution circumvents this issue by exploiting the linkage between growth rate and inhibitor resistance to select for mutants with enhanced tolerance. In order to aid experimentalists in the design and execution of adaptive laboratory evolution, we present detailed protocols for batch, continuous, and visualizing evolution in real-time (VERT) approaches to this technique.

Complete Genome Sequence of Mycoplasma hyorhinis Strain SK76.

Mycoplasma hyorhinis is a eubacterium belonging to the Mollicutes class and is responsible for porcine respiratory and arthritic diseases. It is also the major contaminant of mammalian tissue cultures in laboratories worldwide. Here, we report the complete genome sequence of M.

Double knockout pigs deficient in N-glycolylneuraminic acid and galactose α-1,3-galactose reduce the humoral barrier to xenotransplantation.

Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Methods: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs).

The epidemiology and clinical characteristics of young children hospitalized with respiratory syncytial virus infections in Guatemala (2007-2010).

Background: There have been few population-based studies from Central America on respiratory syncytial virus (RSV) infections in young children. We report population-based incidence rates and describe epidemiological and clinical characteristics of children <5 years old hospitalized with RSV infections in Guatemala.

Methods: Prospective, active hospital-based surveillance for acute respiratory infections in children <5 years old was conducted at 3 hospitals in Guatemala from November 2007 through July 2010.

Porphyromonas gingivalis strain specific interactions with human coronary artery endothelial cells: a comparative study.

Both epidemiologic and experimental findings suggest that infection with Porphyromonas gingivalis exacerbates progression of atherosclerosis. As P. gingivalis exhibits significant strain variation, it is reasonable that different strains possess different capabilities and/or mechanisms by which they promote atherosclerosis.

Disruption of the S41 peptidase gene in mycoplasma mycoides capri impacts proteome profile, H(2)O(2) production, and sensitivity to heat shock.

Members of the Mycoplasma mycoides cluster are among the most virulent of the mycoplasmas, causing worldwide economically significant diseases of cattle and goats. A distinguishing phenotype among the members of the cluster is the ability to degrade casein. The MMCAP2_0241 gene, an S41 peptidase, confers the proteolytic phenotype in Mycoplasma mycoides subsp.

Investigation of an outbreak of rickettsial febrile illness in Guatemala, 2007.

Objectives: We describe an outbreak of spotted fever group (SFG) rickettsiosis that occurred in 2007 in a farming community in southeastern Guatemala. We identified 17 cases of an acute febrile illness, among which 10, including two fatalities, were confirmed or probable cases of rickettsial disease (case-fatality proportion 12%).

Methods: PCR, a microimmunofluorescence assay (IFA), and Western blotting were performed on patient samples, and PCR was performed on ticks.

The imprint of the extragalactic background light in the gamma-ray spectra of blazars.

The light emitted by stars and accreting compact objects through the history of the universe is encoded in the intensity of the extragalactic background light (EBL). Knowledge of the EBL is important to understand the nature of star formation and galaxy evolution, but direct measurements of the EBL are limited by galactic and other foreground emissions. Here, we report an absorption feature seen in the combined spectra of a sample of gamma-ray blazars out to a redshift of z ∼ 1.

Symptoms and risk factors associated with first urinary tract infection in college age women: a prospective cohort study.

Purpose: We identified epidemiological risk factors for the initial urinary tract infection in females of college age compared to age matched controls.

Materials And Methods: We performed a prospective cohort study from July 2001 to January 2006 at the student health care facility at our institution. A total of 180 women experiencing a first urinary tract infection were compared to 80 asymptomatic women with no urinary tract infection history who served as controls.

Host genetic background impacts disease outcome during intrauterine infection with Ureaplasma parvum.

Ureaplasma parvum, an opportunistic pathogen of the human urogenital tract, has been implicated in contributing to chorioamnionitis, fetal morbidity, and fetal mortality. It has been proposed that the host genetic background is a critical factor in adverse pregnancy outcome as sequela to U. parvum intra-amniotic infection.

Risk factors for preeclampsia in women from Colombia: a case-control study.

Background: Preeclampsia (PE) is a multi-causal disease characterized by the development of hypertension and proteinuria in the second half of pregnancy. Multiple risk factors have been associated with the development of PE. Moreover, it is known that these risk factors vary between populations from developed and developing countries.

An outbreak of Klebsiella pneumoniae late-onset sepsis in a neonatal intensive care unit in Guatemala.

Background: Gram-negative bloodstream infections are an important cause of neonatal mortality. In October 2009, we investigated a Klebsiella spp outbreak in a neonatal intensive care unit in Guatemala.

Methods: Probable cases were defined as a Klebsiella spp isolated from blood in neonates aged <28 days in the neonatal intensive care unit between October 1 and November 10, 2009; confirmed cases were identified as Klebsiella pneumoniae.

Effects of alkyl groups in the rate determining step of the Baeyer-Villiger reaction of phenyl alkyl ketones: a quantum chemistry study.

In this work, we have studied the substituent effect of several alkyl groups in the rate-determining step of the catalyzed Baeyer-Villiger (BV) reaction of phenyl alkyl ketones with performic (PFA) and trifluoroperacetic (TFPAA) acids, using quantum chemistry methods. Our results reveal that the substituent effect is more pronounced in the migration step barriers than in the corresponding addition step; that could change the rate-determining step (RDS) of the reaction, as observed in the oxidation of phenyl tert-butyl ketone with both peracids. In addition, the effect of the acid/peracid pairs used is also analyzed.

Visualizing evolution in real-time method for strain engineering.

The adaptive landscape for an industrially relevant phenotype is determined by the effects of the genetic determinants on the fitness of the microbial system. Identifying the underlying adaptive landscape for a particular phenotype of interest will greatly enhance our abilities to engineer more robust microbial strains. Visualizing evolution in real-time (VERT) is a recently developed method based on in vitro adaptive evolution that facilitates the identification of fitter mutants throughout the course of evolution.

Visualizing evolution in real time to determine the molecular mechanisms of n-butanol tolerance in Escherichia coli.

Toxicity of products or feedstock components poses a challenge in the biocatalyst-based production of fuels and chemicals. The genetic determinants that are involved in increased resistance to an inhibitor form the adaptive landscape for the phenotype; so in order to engineer more robust biocatalysts, a better understanding of the adaptive landscape is required. Here, we used an adaptive laboratory evolution method called visualizing evolution in real time (VERT) to help map out part of the adaptive landscape of Escherichia coli tolerance to the biofuel n-butanol.

Highly regio- and stereoselective Diels-Alder cycloadditions via two-step and multicomponent reactions promoted by infrared irradiation under solvent-free conditions.

Infrared irradiation promoted the Diels-Alder cycloadditions of exo-2-oxazolidinone dienes 1-3 with the Knoevenagel adducts 4-6, as dienophiles, leading to the synthesis of new 3,5-diphenyltetrahydrobenzo[d]oxazol-2-one derivatives (7, 9, 11 and 13-17), under solvent-free conditions. These cycloadditions were performed with good regio- and stereoselectivity, favoring the para-endo cycloadducts. We also evaluated the one-pot three-component reaction of active methylene compounds 20, benzaldehydes 21 and exo-2-oxazolidinone diene 2 under the same reaction conditions.

Angiogenic imbalance and plasma lipid alterations in women with preeclampsia from a developing country.

Background: An imbalance between anti-angiogenic factors (e.g. soluble vascular endothelial growth factor receptor-1 (s-FLT1) and soluble endoglin (s-Eng)) and pro-angiogenic factors (e.

Fast gated EPR imaging of the beating heart: spatiotemporally resolved 3D imaging of free-radical distribution during the cardiac cycle.

In vivo or ex vivo electron paramagnetic resonance imaging (EPRI) is a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. However, applications of EPRI have been limited by long projection acquisition times and the consequent fact that rapid gated EPRI was not possible. Hence in vivo EPRI typically provided only time-averaged information.

Nutritional status among women with pre-eclampsia and healthy pregnant and non-pregnant women in a Latin American country.

Aims: Pre-eclampsia (PE) is one of the leading causes of maternal and perinatal morbidity and mortality worldwide. It has been proposed that, among other risk factors, the nutritional status of women can lead to the endothelial dysfunction that characterizes this entity. The aim of the present study was to compare the nutritional status of women with PE with healthy pregnant and non-pregnant women.

Rotavirus disease burden among children <5 years of age--Santa Rosa, Guatemala, 2007-2009.

Objectives: To assess the burden of rotavirus disease in Guatemala, in view of the recent introduction of a national rotavirus vaccination programme.

Methods: We examined data from an active, facility-based surveillance system in Santa Rosa, Guatemala, from October 2007 through September 2009 among children <5years of age presenting to the hospital or ambulatory clinics with diarrhoea (≥3 loose stools in 24 h during the last 7 days). Demographic and epidemiological data were collected, and specimens were tested for rotavirus via enzyme immunoassay.

Short report: Molecular insights for Giardia, Cryptosporidium, and soil-transmitted helminths from a facility-based surveillance system in Guatemala.

We molecularly characterized samples with Giardia, Cryptosporidium, and soil-transmitted helminths from a facility-based surveillance system for diarrhea in Santa Rosa, Guatemala. The DNA sequence analysis determined the presence of Giardia assemblages A (N = 7) and B (N = 12) and, Cryptosporidium hominis (N = 2) and Cryptosporidium parvum (N = 2), suggestive of different transmission cycles. All 41 samples with soil-transmitted helminths did not have the β-tubulin mutation described for benzimidazole resistance, suggesting potential usefulness in mass drug administration campaigns.

Low usage of government healthcare facilities for acute respiratory infections in guatemala: implications for influenza surveillance.

Background: Sentinel surveillance for severe acute respiratory infections in hospitals and influenza-like illness in ambulatory clinics is recommended to assist in global pandemic influenza preparedness. Healthcare utilization patterns will affect the generalizability of data from sentinel sites and the potential to use them to estimate burden of disease. The objective of this study was to measure healthcare utilization patterns in Guatemala to inform the establishment of a sentinel surveillance system for influenza and other respiratory infections, and allow estimation of disease burden.

The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems.

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied.

Detection of pulsed gamma rays above 100 GeV from the Crab pulsar.

We report the detection of pulsed gamma rays from the Crab pulsar at energies above 100 giga-electron volts (GeV) with the Very Energetic Radiation Imaging Telescope Array System (VERITAS) array of atmospheric Cherenkov telescopes. The detection cannot be explained on the basis of current pulsar models. The photon spectrum of pulsed emission between 100 mega-electron volts and 400 GeV is described by a broken power law that is statistically preferred over a power law with an exponential cutoff.

Gene targeting and cloning in pigs using fetal liver derived cells.

Background: Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig.