There is a shift in thinking about dietary protein requirements from daily requirements to individual meal requirements. Per meal, stimulation of muscle protein synthesis has a saturable dose ...relationship with the quantity of dietary protein consumed. Protein intake above the saturable dose does not further contribute to the synthetic response; the "excess" amino acids are predominantly oxidized. Given that daily dietary protein intake is finite, finding protein distribution patterns that both reduce amino acid oxidation and maximize their contribution towards protein synthesis (in theory improving net balance) could be "optimal" and is of practical scientific interest to promote beneficial changes in skeletal muscle-related outcomes. This article reviews both observational and randomized controlled trial research on the protein distribution concept. The current evidence on the efficacy of consuming an "optimal" protein distribution to favorably influence skeletal muscle-related changes is limited and inconsistent. The effect of protein distribution cannot be sufficiently disentangled from the effect of protein quantity. Consuming a more balanced protein distribution may be a practical way for adults with marginal or inadequate protein intakes (<0.80 g·kg
·d
) to achieve a moderately higher total protein intake. However, for adults already consuming 0.8-1.3 g·kg
·d
, the preponderance of evidence supports that consuming at least one meal that contains sufficient protein quantity to maximally stimulate muscle protein synthesis, independent of daily distribution, is helpful to promote skeletal muscle health.
Genetically-encoded biosensors based on FRET between fluorescent proteins of different hues enable quantitative measurement of intracellular enzyme activities and small molecule concentrations. (To ...listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.acs.org/journal/ancham.).
Potassium ion (K
+
) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K
+
biosensors are promising tools to further improve our ...understanding of K
+
-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K
+
biosensor, GINKO1, in the K
+
-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K
+
biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K
+
dynamics in multiple model organisms, including bacteria, plants, and mice.
Global mortality in children younger than 5 years has fallen substantially in the past two decades from more than 12 million in 1990, to 6·9 million in 2011, but progress is inconsistent between ...countries. Pneumonia and diarrhoea are the two leading causes of death in this age group and have overlapping risk factors. Several interventions can effectively address these problems, but are not available to those in need. We systematically reviewed evidence showing the effectiveness of various potential preventive and therapeutic interventions against childhood diarrhoea and pneumonia, and relevant delivery strategies. We used the Lives Saved Tool model to assess the effect on mortality when these interventions are applied. We estimate that if implemented at present annual rates of increase in each of the 75 Countdown countries, these interventions and packages of care could save 54% of diarrhoea and 51% of pneumonia deaths by 2025 at a cost of US$3·8 billion. However, if coverage of these key evidence-based interventions were scaled up to at least 80%, and that for immunisations to at least 90%, 95% of diarrhoea and 67% of pneumonia deaths in children younger than 5 years could be eliminated by 2025 at a cost of $6·715 billion. New delivery platforms could promote equitable access and community platforms are important catalysts in this respect. Furthermore, several of these interventions could reduce morbidity and overall burden of disease, with possible benefits for developmental outcomes.
The arsenal of engineered variants of the GFP green FP (fluorescent protein) from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology ...research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 monomeric TFP1 (teal FP 1). The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
For over 20 years, genetically encoded Ca2+ indicators have illuminated dynamic Ca2+ signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand ...how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a red fluorescent protein that is circularly permuted and fused to a Ca2+-sensing domain. When Ca2+ binds, a conformational change in the sensing domain causes a change in fluorescence. Three factors can contribute to this fluorescence change: 1) a shift in the protonation equilibrium of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient or the two-photon cross section, depending on if it is excited with one or two photons. Here, we conduct a systematic study of the photophysical properties of a range of red Ca2+ indicators to determine which factors are the most important. In total, we analyzed nine indicators, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We find that these could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases, the magnitude of the change in fluorescence was larger with two-photon excitation compared to one-photon because of a change in the two-photon cross section, by up to a factor of two.
As the technological hurdles are overcome and optogenetic techniques advance to have more control over neurons, therapies based on these approaches will begin to emerge in the clinic. Here we ...consider the technical challenges surrounding the transition of this breakthrough technology from an investigative tool to a true therapeutic avenue. The emerging strategies and remaining tasks surrounding genetically encoded molecules which respond to light as well as the vehicles required to deliver them are discussed.The use of optogenetics in humans would represent a completely new paradigm in medicine and would be associated with unprecedented technical considerations. To be applied for stimulation of neurons in humans, an ideal optogenetic tool would need to be non-immunogenic, highly sensitive, and activatable with red light or near-infrared light (to maximize light penetration while minimizing photodamage). To enable sophisticated levels of neuronal control, the combined use of optogenetic actuators and indicators could enable closed-loop all-optical neuromodulation. Such systems would introduce additional challenges related to spectral orthogonality between actuator and indicator, the need for decision making computational algorithms and requirements for large gene cassettes. As in any gene therapy, the therapeutic efficiency of optogenetics will rely on vector delivery and expression in the appropriate cell type. Although viral vectors such as those based on AAVs are showing great potential in human trials, barriers to their general use remain, including immune responses, delivery/transport, and liver clearance. Limitations associated with the gene cassette size which can be packaged in currently approved vectors also need to be addressed.
Emerging fluorescent protein technologies Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E
Current opinion in chemical biology,
08/2015, Letnik:
27
Journal Article
Recenzirano
•Established applications of fluorescent proteins are fundamental to modern research.•A number fluorescent protein-based biosensing technologies have emerged in recent years.•Fluorescent proteins ...have now been used to control protein function and gene expression.•Emerging fluorescent protein technologies are expanding the range of possible applications.
Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression.
L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of ...intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.
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