Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions

Background. A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses in the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. Results. Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. Conclusion. Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity. © 2008 van der Walt et al; licensee BioMed Central Ltd.

Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n = 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-(13)C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1-12 h recovery (88-148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31-48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass.

Preexercise aminoacidemia and muscle protein synthesis after resistance exercise

PURPOSE We have previously shown that the aminoacidemia caused by the consumption of a rapidly digested protein after resistance exercise enhances muscle protein synthesis (MPS) more than the amino acid (AA) profile associated with a slowly digested protein. Here, we investigated whether differential feeding patterns of a whey protein mixture commencing before exercise affect postexercise intracellular signaling and MPS. METHODS Twelve resistance-trained males performed leg resistance exercise 45 min after commencing each of three volume-matched nutrition protocols: placebo (PLAC, artificially sweetened water), BOLUS (25 g of whey protein + 5 g of leucine dissolved in artificially sweetened water; 1× 500 mL), or PULSE (15× 33-mL aliquots of BOLUS drink every 15 min). RESULTS The preexercise rise in plasma AA concentration with PULSE was attenuated compared with BOLUS (P < 0.05); this effect was reversed after exercise, with two-fold greater leucine concentrations in PULSE compared with BOLUS (P < 0.05). One-hour postexercise, phosphorylation of p70 S6K and rpS6 was increased above baseline with BOLUS and PULSE, but not PLAC (P < 0.05); furthermore, PULSE > BOLUS (P < 0.05). MPS throughout 5 h of recovery was higher with protein ingestion compared with PLAC (0.037 ± 0.007), with no differences between BOLUS or PULSE (0.085 ± 0.013 vs. 0.095 ± 0.010%•h, respectively, P = 0.56). CONCLUSIONS Manipulation of aminoacidemia before resistance exercise via different patterns of intake of protein altered plasma AA profiles and postexercise intracellular signaling. However, there was no difference in the enhancement of the muscle protein synthetic response after exercise. Protein sources producing a slow AA release, when consumed before resistance exercise in sufficient amounts, are as effective as rapidly digested proteins in promoting postexercise MPS.

Low muscle glycogen concentration does not suppress the anabolic response to resistance exercise

We determined the effect of muscle glycogen concentration and postexercise nutrition on anabolic signaling and rates of myofibrillar protein synthesis after resistance exercise (REX). Sixteen young, healthy men matched for age, body mass, peak oxygen uptake (VO2peak) and strength (one repetition maximum; 1RM) were randomly assigned to either a nutrient or placebo group. After 48 h diet and exercise control, subjects undertook a glycogen-depletion protocol consisting of one-leg cycling to fatigue (LOW), whereas the other leg rested (NORM). The next morning following an overnight fast, a primed, constant infusion of L-[ring-13C6] phenylalanine was commenced and subjects completed 8 sets of 5 unilateral leg press repetitions at 80% 1RM. Immediately after REX and 2 h later, subjects consumed a 500 ml bolus of a protein/CHO (20 g whey + 40 g maltodextrin) or placebo beverage. Muscle biopsies from the vastus lateralis of both legs were taken at rest and 1 and 4 h after REX. Muscle glycogen concentration was higher in the NORM than LOW at all time points in both nutrient and placebo groups (P < 0.05). Postexercise Akt-p70S6K-rpS6 phosphorylation increased in both groups with no differences between legs (P < 0.05). mTORSer2448 phosphorylation in placebo increased 1 h after exercise in NORM (P < 0.05), whereas mTOR increased ?4-fold in LOW (P < 0.01) and ?11 fold in NORM with nutrient (P < 0.01; different between legs P < 0.05). Post-exercise rates of MPS were not different between NORM and LOW in nutrient (0.070 ± 0.022 vs. 0.068 ± 0.018 %/h) or placebo (0.045 ± 0.021 vs. 0.049 ± 0.017 %/h). We conclude that commencing high-intensity REX with low muscle glycogen availability does not compromise the anabolic signal and subsequent rates of MPS, at least during the early (4 h) postexercise recovery period.

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 112: 1805-1813, 2012. First published March 1, 2012; doi:10.1152/japplphysiol.00170.2012.- We made sex-based comparisons of rates of myofibrillar protein synthesis (MPS) and anabolic signaling after a single bout of high-intensity resistance exercise. Eight men (20 ± 10 yr, BMI = 24.3 ± 2.4) and eight women (22 ± 1.8 yr, BMI = 23.0 ± 1.9) underwent primed constant infusions of L-[ring-13C6]phenylalanine on consecutive days with serial muscle biopsies. Biopsies were taken from the vastus lateralis at rest and 1, 3, 5, 24, 26, and 28 h after exercise. Twenty-five grams of whey protein was ingested immediately and 26 h after exercise. We also measured exercise-induced serum testosterone because it is purported to contribute to increases in myofibrillar protein synthesis (MPS) postexercise and its absence has been hypothesized to attenuate adaptative responses to resistance exercise in women. The exercise-induced area under the testosterone curve was 45-fold greater in men than women in the early (1 h) recovery period following exercise (P < 0.001). MPS was elevated similarly in men and women (2.3- and 2.7-fold, respectively) 1-5 h postexercise and after protein ingestion following 24 h recovery. Phosphorylation of mTORSer2448 was elevated to a greater extent in men than women acutely after exercise (P = 0.003), whereas increased phosphorylation of p70S6K1Thr389 was not different between sexes. Androgen receptor content was greater in men (main effect for sex, P = 0.049). Atrogin-1 mRNA abundance was decreased after 5 h recovery in both men and women (P < 0.001), and MuRF-1 expression was elevated in men after protein ingestion following 24 h recovery (P = 0.003). These results demonstrate minor sex-based differences in signaling responses and no difference in the MPS response to resistance exercise in the fed state. Interestingly, our data demonstrate that exerciseinduced increases in MPS are dissociated from postexercise testosteronemia and that stimulation of MPS occurs effectively with low systemic testosterone concentrations in women.

Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise

Background: Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Objective: Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. Design: In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. Results: BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P < 0.001) 60 min after exercise, whereas PULSE resulted in a smaller but sustained increase in aminoacidemia that remained elevated above BOLUS amounts later (180-220 min after exercise, P < 0.05). Despite an identical net area under the EAA curve, MPS was elevated to a greater extent after BOLUS than after PULSE early (1-3 h: 95% compared with 42%) and later (3-5 h: 193% compared with 121%) (both P < 0.05). There were greater changes in the phosphorylation of the Akt-mammalian target of rapamycin pathway after BOLUS than after PULSE. Conclusions: Rapid aminoacidemia in the postexercise period enhances MPS and anabolic signaling to a greater extent than an identical amount of protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis.

Alcohol ingestion impairs maximal post-exercise rates of myofibrillar protein synthesis following a single bout of concurrent training

Introduction The culture in many team sports involves consumption of large amounts of alcohol after training/competition. The effect of such a practice on recovery processes underlying protein turnover in human skeletal muscle are unknown. We determined the effect of alcohol intake on rates of myofibrillar protein synthesis (MPS) following strenuous exercise with carbohydrate (CHO) or protein ingestion. Methods In a randomized cross-over design, 8 physically active males completed three experimental trials comprising resistance exercise (8×5 reps leg extension, 80% 1 repetition maximum) followed by continuous (30 min, 63% peak power output (PPO)) and high intensity interval (10×30 s, 110% PPO) cycling. Immediately, and 4 h post-exercise, subjects consumed either 500 mL of whey protein (25 g; PRO), alcohol (1.5 g·kg body mass−1, 12±2 standard drinks) co-ingested with protein (ALC-PRO), or an energy-matched quantity of carbohydrate also with alcohol (25 g maltodextrin; ALC-CHO). Subjects also consumed a CHO meal (1.5 g CHO·kg body mass−1) 2 h post-exercise. Muscle biopsies were taken at rest, 2 and 8 h post-exercise. Results Blood alcohol concentration was elevated above baseline with ALC-CHO and ALC-PRO throughout recovery (P<0.05). Phosphorylation of mTORSer2448 2 h after exercise was higher with PRO compared to ALC-PRO and ALC-CHO (P<0.05), while p70S6K phosphorylation was higher 2 h post-exercise with ALC-PRO and PRO compared to ALC-CHO (P<0.05). Rates of MPS increased above rest for all conditions (~29–109%, P<0.05). However, compared to PRO, there was a hierarchical reduction in MPS with ALC-PRO (24%, P<0.05) and with ALC-CHO (37%, P<0.05). Conclusion We provide novel data demonstrating that alcohol consumption reduces rates of MPS following a bout of concurrent exercise, even when co-ingested with protein. We conclude that alcohol ingestion suppresses the anabolic response in skeletal muscle and may therefore impair recovery and adaptation to training and/or subsequent performance.

The science behind the “stain”

It has been almost fi ve years since I fi rst published the article entitled “Much Ado About Staining” in Review of Optometry, which explored what we really knew in 2006 about the relationship between “corneal staining” and contact lens multipurpose solutions (MPS). This was published just prior to the controversial “staining grid.” While the Grid showed MPS-associated hyperfl uorescence under the slitlamp at two hours, it did not explain the “what” or “why” behind it; even so, many proponents of the Grid continue to suggest that it shows us which solution/lens combinations are “biocompatible” and which are not. New evidence suggests that the preservative-associated transient hyperfl uorescence (or PATH) observed at two hours after lens insertion is a benign phenomenon due to an interaction between fl uorescein, MPS preservatives, and corneal cell membranes. The misinterpretation of PATH as “real” corneal staining, like that observed in pathological conditions, may be due in part to the fact that there is not a lot of teaching regarding the true properties of fl uorescein and what is actually occurring when we see either PATH or corneal staining. To discuss the science of fl uorescein, corneal staining, and PATH, I have asked some of the preeminent research experts in the study of fl uorescence spectroscopy and corneal staining from around the world to share their new research and personal opinions on these topics...

Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit

The myofibrillar protein synthesis (MPS) response to resistance exercise (REX) and protein ingestion during energy deficit (ED) is unknown. We determined, in young men (n=8) and women (n=7), protein signaling, resting post-absorptive MPS during energy balance [EB: 45 kcal∙(kg FFM∙d)-1] and after 5d of ED [30 kcal∙(kg FFM∙d)-1] as well as MPS while in ED after acute REX in the fasted state and with the ingestion of whey protein (15 and 30 g). Post-absorptive rates of MPS were 27% lower in ED than EB (P<0.001), but REX stimulated MPS to rates equal to EB. Ingestion of 15 and 30 g of protein after REX in ED increased MPS ~16 and ~34% above resting EB, (P<0.02). p70 S6Kthr389 phosphorylation increased above EB only with combined exercise and protein intake (~2-7 fold; P<0.05). In conclusion, short-term ED reduces post-absorptive MPS, however, a bout of REX in ED restores MPS to values observed at rest in EB. The ingestion of protein after REX further increases MPS above resting EB in a dose-dependent manner. We conclude that combining REX with increased protein availability after exercise enhances rates of skeletal muscle protein synthesis during short term ED and could, in the long term, preserve muscle mass.

Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(d,l-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 μm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Protein loaded mesoporous silica spheres as a controlled delivery platform

Background The adsorption of bovine serum albumin (BSA) onto mesoporous silica spheres (MPS) synthesized from silica colloids was studied employing real time in situ measurements. The stabilities of the BSA at different pH values, their isoelectric points and zeta potentials were determined in order to probe the interactions between the protein and the mesoporous silica. Results The pore size of MPS was designed for protein, and this, coupled with an in depth understanding of the physico-chemical characteristics of the protein and MPS has yielded a better binding capacity and delivery profile. The adsorption isotherm at pH 4.2 fitted the Langmuir model and displayed the highest adsorption capacity (71.43 mg mL-1 MPS). Furthermore, the delivery rates of BSA from the MPS under physiological conditions were shown to be dependent on the ionic strength of the buffer and protein loading concentration. Conclusion Economics and scale-up considerations of mesoporous material synthesized via destabilization of colloids by electrolyte indicate the scaleability and commercial viability of this technology as a delivery platform for biopharmaceutical applications.

Transmittance properties of contact lens multipurpose solutions and their effects on a hydrogel lens

Purpose The aim was to assess the compatibility of different multipurpose solutions (MPSs) with one type of silicone hydrogel (SiH) contact lens by, assessing the changes in both ultraviolet (UV) and visible light transmissibility of the hydrogel lens caused by the MPSs. Methods The light transmittance from 200-700 nm were measured for the lotrafilcon B blister pack solution (BPS), six MPSs namely, ReNuMultiPlus Multi-Purpose Solution (Bausch and Lomb Inc., Rochester NY, USA.); Complete RevitaLens Multi-Purpose (Abbott Medical Optics Inc., Quarryvale Co. Dublin, Ireland); All In One Light (Sauflon Pharmaceuticals Ltd., Twickenham, England); SOLO-care AQUA™ (Ciba Vision Corporation Duluth, Georgia, USA.); Biomedics All-in-one solution (CooperVision, Hamble, UK); and HippiaMultiPlus All-in-one solution (Interojo Inc., Kyeonggi-do, Korea), and a lotrafilcon B SiH lens (before and after storage), using a spectrophotometer. Results The UV transmitted through the BPS and the MPS were similar (p >.05, for all), except for the HippiaMultiPlus which was lower (p < 0.001) by 19.8%. Mean transparency values were statistically (p<.001) significantly different between the BPS and the MPSs. All MP solution/SiH lens combinations resulted in relatively high UV transmittance values especially in the UVC spectrum, and significantly increased (p <.001) the visible light transmittance values of the SiH lens. Greater changes in transparency were observed in the ReNu/SiH lens (28.5%) and the Complete RevitaLens/SiH lens (24.9%) combinations. Conclusion The six MPSs showed significant variations in the transmitted UV and visible light. Similar to the BPS, all MPSs were equally transparent, but showed very poor UVA & UVB attenuation, except for the Hippia MultiPlus. The MPS/SiH lens combinations did not significantly affect the lens transparency but it significant increased the lens transmittance of UV radiation, after storage. Further in-vivo studies are needed to validate if this effect is constant.

A new method of detecting changes in corneal health in response to toxic insults

The size and arrangement of stromal collagen fibrils (CFs) influence the optical properties of the cornea and hence its function. The spatial arrangement of the collagen is still questionable in relation to the diameter of collagen fibril. In the present study, we introduce a new parameter, edge-fibrillar distance (EFD) to measure how two collagen fibrils are spaced with respect to their closest edges and their spatial distribution through normalized standard deviation of EFD (NSDEFD) accessed through the application of two commercially available multipurpose solutions (MPS): ReNu and Hippia. The corneal buttons were soaked separately in ReNu and Hippia MPS for five hours, fixed overnight in 2.5% glutaraldehyde containing cuprolinic blue and processed for transmission electron microscopy. The electron micrographs were processed using ImageJ user-coded plugin. Statistical analysis was performed to compare the image processed equivalent diameter (ED), inter-fibrillar distance (IFD), and EFD of the CFs of treated versus normal corneas. The ReNu-soaked cornea resulted in partly degenerated epithelium with loose hemidesmosomes and Bowman’s collagen. In contrast, the epithelium of the cornea soaked in Hippia was degenerated or lost but showed closely packed Bowman’s collagen. Soaking the corneas in both MPS caused a statistically significant decrease in the anterior collagen fibril, ED and a significant change in IFD, and EFD than those of the untreated corneas (p < 0.05, for all comparisons). The introduction of EFD measurement in the study directly provided a sense of gap between periphery of the collagen bundles, their spatial distribution; and in combination with ED, they showed how the corneal collagen bundles are spaced in relation to their diameters. The spatial distribution parameter NSDEFD indicated that ReNu treated cornea fibrils were uniformly distributed spatially, followed by normal and Hippia. The EFD measurement with relatively lower standard deviation and NSDEFD, a characteristic of uniform CFs distribution, can be an additional parameter used in evaluating collagen organization and accessing the effects of various treatments on corneal health and transparency.

The revolution in human monogenic disease mapping

The successful completion of the Human Genome Project (HGP) was an unprecedented scientific advance that has become an invaluable resource in the search for genes that cause monogenic and common (polygenic) diseases. Prior to the HGP, linkage analysis had successfully mapped many disease genes for monogenic disorders; however, the limitations of this approach were particularly evident for identifying causative genes in rare genetic disorders affecting lifespan and/or reproductive fitness, such as skeletal dysplasias. In this review, we illustrate the challenges of mapping disease genes in such conditions through the ultra-rare disorder fibrodysplasia ossificans progressiva (FOP) and we discuss the advances that are being made through current massively parallel (“next generation”) sequencing (MPS) technologies.

Microplastic ingestion by scleractinian corals

We report for the first time the ingestion of microplastics by scleractinian corals, and the presence of microplastics in coral reef waters adjacent to inshore reefs on Australia’s Great Barrier Reef (GRE, 18°31′S 146°23′E). Analysis of samples from sub-surface plankton tows conducted in close proximity to inshore reefs on the central GBR revealed microplastics, similar to those used in marine paints and fishing floats, were present in low concentrations at all water sampling locations. Experimental feeding trials revealed that corals mistake microplastics for prey and can consume up to ~50 μg plastic cm−2 h−1, rates similar to their consumption of plankton and Artemia nauplii in experimental feeding assays. Ingested microplastics were found wrapped in mesenterial tissue within the coral gut cavity, suggesting that ingestion of high concentrations of microplastic debris could potentially impair the health of corals.