Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.     

Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same beta subunit has been demonstrated.     

Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Delta gpd2Delta mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Delta gpd2Delta mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Delta1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Delta1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Delta1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Delta gpd2Delta mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Delta gpd2Delta mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.     

Inducer-dependent phenotypic divergence in an embryonal-carcinoma cell line

Abstract. In monolayer cultures, embryonal carcinoma (EC) cells from the cell line Nulli-SCCI can be induced to differentiate at high efficiency by exposure to either retinoic acid or hexamethylenebisacetamide. Depending on which inducing agent is used, two distinct differentiated phenotypes result. These phenotypes resemble two of the earliest differentiated derivatives of the cells of the inner cell mass of a mouse blastocyst, i.e., parietal and visceral endoderm. Both differ from EC cells as well as from each other on the basis of their morphology, antigenic expression, secretion of plasminogen activator, and protein-synthetic profiles.     

CX3CR1 Signaling on Monocytes Is Dispensable after Intracerebral Hemorrhage

Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6C^hi CCR2⁺ monocytes have been shown to contribute to acute injury after intracerebral hemorrhage. The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6C^lo CX3CR1⁺ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6C^lo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1⁺ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.     

Impact of an Ivermectin Mass Drug Administration on Scabies Prevalence in a Remote Australian Aboriginal Community

Background

Scabies is endemic in many Aboriginal and Torres Strait Islander communities, with 69% of infants infected in the first year of life. We report the outcomes against scabies of two oral ivermectin mass drug administrations (MDAs) delivered 12 months apart in a remote Australian Aboriginal community.

Methods

Utilizing a before and after study design, we measured scabies prevalence through population census with sequential MDAs at baseline and month 12. Surveys at months 6 and 18 determined disease acquisition and treatment failures. Scabies infestations were diagnosed clinically with additional laboratory investigations for crusted scabies. Non-pregnant participants weighing ≥15 kg were administered a single 200 μg/kg ivermectin dose, repeated after 2–3 weeks if scabies was diagnosed, others followed a standard alternative algorithm.

Principal Findings

We saw >1000 participants at each population census. Scabies prevalence fell from 4% at baseline to 1% at month 6. Prevalence rose to 9% at month 12 amongst the baseline cohort in association with an identified exposure to a presumptive crusted scabies case with a higher prevalence of 14% amongst new entries to the cohort. At month 18, scabies prevalence fell to 2%. Scabies acquisitions six months after each MDA were 1% and 2% whilst treatment failures were 6% and 5% respectively.

Conclusion

Scabies prevalence reduced in the six months after each MDA with a low risk of acquisition (1–2%). However, in a setting where living conditions are conducive to high scabies transmissibility, exposure to presumptive crusted scabies and population mobility, a sustained reduction in prevalence was not achieved.

Clinical Trial Registration

Australian New Zealand Clinical Trial Register (ACTRN—12609000654257).     

Breastfeeding in Saudi Arabia: a review

Background

Breastfeeding is viewed as the optimal method of infant feeding that provides many benefits to both the infant and the mother. The monitoring and reporting of breastfeeding indicators are essential for any country to plan and implement effective promotion programs for sustainable breastfeeding. The aim of this review is to examine the available studies and data on breastfeeding in Saudi Arabia, and determine the potential factors that affect breastfeeding practices and duration in this country.

Methods

The databases of Web of Knowledge, Science Direct and PubMed were searched using the relevant key words. Only studies that reported breastfeeding practices, rates and indicators in Saudi Arabia were included. Standard WHO definitions for breastfeeding categories were used in this review.

Results

Seventeen cross-sectional studies were identified and reviewed and five stated they used standard definitions. The self-administered questionnaire as a measurement tool was the predominant method of data collection. Infants' ages range from less than six months up to five years. Initiation rates were high (mostly above 90%), but a few studies reported low rates of timely initiation (within the first hour). The exclusive breastfeeding rate could not be accurately determined as rates range from 0.8% to 43.9% among studies due to the lack of clear definitions and the nature of study design. The partial (mixed) feeding method was common and the category of 'any breastfeeding' has generally high rates. The mean duration of breastfeeding has showed a progressive decline over time from 13.4 months in 1987 to 8.5 months in 2010. Factors associated with a high prevalence of breastfeeding and longer duration include increased maternal age, low educational levels, rural residence, low income, multiparity and avoiding contraceptives. The most common reason for breastfeeding cessation was insufficient breast milk. Other reasons include sickness, new pregnancy and breastfeeding problems.

Conclusions

Breastfeeding indicators in Saudi Arabia could not be monitored or compared relying on the available data because no longitudinal studies have been conducted in this country. A cohort study design would be the most appropriate procedure to rigorously assess and report valid results on breastfeeding practices and patterns in the Saudi society.     

Almost being there: video communication with young children

Background

Video communication is increasingly used to connect people around the world. This includes connecting young children with their parents and other relatives during times of separation. An important issue is the extent to which video communication with children can approximate a physical presence such that familial relationships can be truly maintained by this means.

Methodology/Principal Findings

The current study employed an adaptation of the Separation and Reunion Paradigm with children (17 months to 5 years) to investigate the potential for video communication with a parent to afford a sense of proximity and security to children. The protocol involved a free-play session with the parent, followed by two separation-reunion episodes. During one of the separation episodes the parent was ‘virtually available’ to the child via a video link. Our results revealed three important differences. First, children left alone played longer in a strange room when their parent was virtually available to them compared to when the children were left alone with neither physical nor video contact with their parent. Second, younger participants sought physical contact with their parent less at the end of the video separation episode compared to when they were left entirely alone. Finally, the comparison between free play with video and free play with parent, revealed that the children exhibit a similar level of interactivity with their parent by video as they did in person.

Conclusions/Significance

For young children a video connection can have many of the same effects as a physical presence. This is a significant finding as it is the first such empirical demonstration and indicates considerable promise in video communication as a tool to maintain family relationships when physical presence is not possible.