Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection.

Antibiotic-resistant bacteria are a significant threat to human health, with one estimate suggesting they will cause 10 million worldwide deaths per year by 2050, surpassing deaths due to cancer1. Because new antibiotic development can take a decade or longer, it is imperative to effectively use currently available drugs. Antibiotic combination therapy offers promise for treating highly resistant bacterial infections, but the factors governing the sporadic efficacy of such regimens have remained unclear. Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination2. Here, using carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates, we reveal the underlying basis for the majority of effective combinations to be heteroresistance. Heteroresistance is a poorly understood mechanism of resistance reported for different classes of antibiotics3-6 in which only a subset of cells are phenotypically resistant7. Within an isolate, the subpopulations resistant to different antibiotics were distinct, and over 88% of CRE isolates exhibited heteroresistance to multiple antibiotics ('multiple heteroresistance'). Combinations targeting multiple heteroresistance were efficacious, whereas those targeting homogenous resistance were ineffective. Two pan-resistant Klebsiella isolates were eradicated by combinations targeting multiple heteroresistance, highlighting a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.

Early Impact of the Patient Protection and Affordable Care Act on People Living With HIV: A Systematic Review.

The US Patient Protection and Affordable Care Act (ACA) was the most influential policy-related change to the care of people living with HIV in decades. We systematically searched and analyzed peer-reviewed, empirical research reporting on ACA-related aspects of HIV care post-ACA full implementation, finding 12 articles that met search criteria. The results revealed largely positive evidence regarding the ACA impact on people living with HIV, particularly on their health care coverage in Medicaid expansion states. More recent reporting included improvement in virologic suppression. However, early evidence has been somewhat fragmented, and important questions concerning the impact of the ACA on HIV care quality, patients, and providers remain unanswered. As the political struggle over the ACA continues, future analyses should use national- and state-level data to examine ACA impact on HIV care quality and patient-centered health outcomes to provide in-depth, holistic understanding of HIV care in the wake of this policy change.

Analysis of green tea extract dietary supplements by micellar electrokinetic chromatography.

Dietary supplements are growing in popularity as a source of catechins such as epigallocatechin gallate (EGCG). The first determination of five catechins in green tea extract dietary supplements using an extraction followed by micellar electrokinetic chromatography (MEKC) with UV detection is presented here. The optimum run buffer is 5 mM borate-60 mM phosphate with 50 mM SDS at pH 7.00 with detection at 210 nm. The limit of detection is 2-3 microg/mL (S/N=3) and the limit of quantitation is 6-8 microg/mL (S/N = 10). Results indicate that the amount of catechins varies greatly among manufacturers, between capsules of the same manufacturers, and between batches.

Active anthocyanin degradation in Brunfelsia calycina (yesterday--today--tomorrow) flowers.

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.

Analysis of natural flavonoids by microchip-micellar electrokinetic chromatography with pulsed amperometric detection.

Catechins (catechin and other derivatives) are naturally occurring flavonoids present in a number of plants and foods. They are also part of numerous nutraceutical formulations because they are believed to have antioxidant, cancer chemo-preventative, anti-inflammatory and antimicrobial properties. The determination of catechins has traditionally been performed by HPLC. However, this methodology is both time and sample intensive and generates large amounts of organic solvent waste. In the current report, an application of MEKC using a PDMS microchip is presented for the analysis of catechins. The system uses pulsed amperometric detection for direct analysis of important naturally occurring catechins. The effect of pH, surfactant concentration, detection potential and signal stability were analyzed. Linear relationships were found between the concentration and peak current, with good stability and limits of detection of 8 [micro sign]M for catechin, epigallocatechin gallate and epicatechin, and 14 [micro sign]M for epicatechin gallate. Optimum conditions were applied to the detection of selected catechins in a commercially available green tea extract nutraceutical and the results were compared to HPLC analysis. The analysis using microchip micellar electrokinetic chromatography and pulsed amperometric detection was completed in 4.5 min, 10 times faster than the HPLC analysis.

Determination of catechins in matcha green tea by micellar electrokinetic chromatography.

Catechins in green tea are known to have many beneficial health properties. Recently, it has been suggested that matcha has greater potential health benefits than other green teas. Matcha is a special powdered green tea used in the Japanese tea ceremony. However, there has been no investigation to quantitate the catechin intake from matcha compared to common green teas. We have developed a rapid method of analysis of five catechins and caffeine in matcha using micellar electrokinetic chromatography. Results are presented for water and methanol extractions of matcha compared with water extraction of a popular green tea. Using a mg catechin/g of dry leaf comparison, results indicate that the concentration of epigallocatechin gallate (EGCG) available from drinking matcha is 137 times greater than the amount of EGCG available from China Green Tips green tea, and at least three times higher than the largest literature value for other green teas.

Sugars regulate sucrose transporter gene expression in citrus.

We report the isolation and characterization of two sucrose transporter cDNAs (CitSUT1 and CitSUT2) from citrus. CitSUT1 and CitSUT2 encode putative proteins (CitSUT1 and CitSUT2) of 528 and 607 amino acids, respectively. CitSUT1 and CitSUT2 share high similarities with sucrose transporters isolated from other plants. The expression of CitSUT1 in mature leaf discs is repressed by exogenous sucrose, glucose, mannose, and the glucose analog 2-deoxyglucose but not by another glucose analog 3-O-methylglucose, indicating a hexokinase (HXK)-mediated signaling pathway. CitSUT2 expression is not affected by exogenous sugars. Whereas CitSUT1 expresses strongly in source, sugar exporting organs, CitSUT2 expresses more strongly in sink, sugar importing organs, suggesting different physiological roles for these sucrose transporters.

Identification of a tyrosine in the agonist binding site of the homomeric rho1 gamma-aminobutyric acid (GABA) receptor that, when mutated, produces spontaneous opening.

Mutagenesis of recombinant rho1 gamma-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the rho1 receptor is also associated with the agonist binding site. Wild-type and mutant rho1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC(50) increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC(50) was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.

O-methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals.

Rose (Rosa hybrida) flowers produce and emit a diverse array of volatiles, characteristic to their unique scent. One of the most prominent compounds in the floral volatiles of many rose varieties is the methoxylated phenolic derivative 3,5-dimethoxytoluene (orcinol dimethyl ether). Cell-free extracts derived from developing rose petals displayed O-methyltransferase (OMT) activities toward several phenolic substrates, including 3,5-dihydroxytoluene (orcinol), 3-methoxy,5-hydroxytoluene (orcinol monomethyl ether), 1-methoxy, 2-hydroxy benezene (guaiacol), and eugenol. The activity was most prominent in rose cv Golden Gate, a variety that produces relatively high levels of orcinol dimethyl ether, as compared with rose cv Fragrant Cloud, an otherwise scented variety but which emits almost no orcinol dimethyl ether. Using a functional genomics approach, we have identified and characterized two closely related cDNAs from a rose petal library that each encode a protein capable of methylating the penultimate and immediate precursors (orcinol and orcinol monomethyl ether, respectively) to give the final orcinol dimethyl ether product. The enzymes, designated orcinol OMTs (OOMT1 and OOMT2), are closely related to other plant methyltransferases whose substrates range from isoflavones to phenylpropenes. The peak in the levels of OOMT1 and OOMT2 transcripts in the flowers coincides with peak OMT activity and with the emission of orcinol dimethyl ether.

Increased anthocyanin accumulation in aster flowers at elevated temperatures due to magnesium treatment.

Temperature is one of the main external factors affecting anthocyanin accumulation in plant tissues: low temperatures cause an increase and elevated temperatures cause a decrease in anthocyanin concentration. Several metals have been shown to increase the half-life time of anthocyanins, by forming complexes with them. We studied the combined effect of elevated temperatures and increased metal concentrations on the accumulation of anthocyanins in aster 'Sungal' flowers. It has been found that magnesium treatment of aster plants or detached flower buds, partially prevents colour fading at elevated temperatures. Anthocyanin concentration of aster 'Sungal' flowers grown at 29 degrees C/21 degrees C day/night, respectively, was about half that of flowers grown at 17 degrees C/9 degrees C. The activity of phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI) decreased as the temperature increased. Treatment of both whole plants and detached flower buds grown at elevated temperatures in the presence of magnesium salts, increased flower anthocyanin concentration by up to 80%. Measurement of magnesium following these treatments revealed an increased level of the metal in the petals, suggesting a direct effect. Magnesium treatment does not seem to cause increased synthesis of anthocyanin through a stress-related reaction, since the activities of both PAL and CHI did not increase due to this treatment. The results of this study show that increasing magnesium levels in aster petals prevents the deleterious effect of elevated temperatures on anthocyanin accumulation, thus enhancing flower colour.