A Three-Dimensional FRET Analysis to Construct an Atomic Model of the Actin–Tropomyosin–Troponin Core Domain Complex on a Muscle Thin Filament

It is essential to knowthe detailed structure of the thin filament to understand the regulation mechanism of striated muscle contraction. Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm)–troponin (Tn) core domain complex. We generated single-cysteine mutants in the 167–195 region of Tm and in TnC, TnI, and the β-TnT 25-kDa fragment, and each was attached with an energy donor probe. An energy acceptor probe was located at actin Gln41, actin Cys374, or the actin nucleotide-binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca2+. Using the atomic coordinates for F-actin, Tm, and the Tn core domain, we searched all possible arrangements for Tm or the Tn core domain on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of Tm segment 167– 195 and the Tn core domain on F-actin with andwithout Ca2+. The bulk of the Tn core domain is located near actin subdomains 3 and 4. The central helix of TnC is nearly perpendicular to the F-actin axis, directing the N-terminal domain of TnC toward the actin outer domain. The C-terminal region in the I–T arm forms a four-helix-bundle structure with the Tm 175–185 region. After Ca2+ release, the Tn core domainmoves toward the actin outer domain and closer to the center of the F-actin axis

Cynomolgus macaque TRIMCyp-resistant HIV-1

Old World monkey TRIM5α strongly suppresses human immunodeficiency virus type 1 (HIV-1) replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophilin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between α-helices 4 and 5 (L4/5). There are seven amino acid differences between L4/5 of HIV-1 and SIVmac239. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCyp-mediated suppression. We performed random PCR mutagenesis to construct a library of HIV-1 variants containing mutations in L4/5, and then we recovered replication-competent viruses from CD4+ MT4 cells that expressed high levels of CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells expressing CM TRIMCyp and these were found to contain four amino acid substitutions (H87R, A88G, P90D and P93A) in L4/5. We then confirmed that these substitutions were sufficient to confer CM TRIMCyp resistance to HIV-1. In a separate experiment using a similar method, we obtained novel CM TRIM5α-resistant HIV-1 strains after six rounds of selection and rescue. Analysis of these mutants revealed that V86A and G116E mutations in the capsid region conferred partial resistance to CM TRIM5α without substantial fitness cost when propagated in MT4 cells expressing CM TRIM5α. These results confirmed and further extended the previous notion that CM TRIMCyp and CM TRIM5α recognize the HIV-1 capsid in different manners

Enlisting wild grass genes to combat nitrification in wheat farming: A nature-based solution

Active nitrifiers and rapid nitrification are major contributing factors to nitrogen losses in global wheat production. Suppressing nitrifier activity is an effective strategy to limit N losses from agriculture. Production and release of nitrification inhibitors from plant roots is termed "biological nitrification inhibition" (BNI). Here, we report the discovery of a chromosome region that controls BNI production in "wheat grass" Leymus racemosus (Lam.) Tzvelev, located on the short arm of the "Lr#3Ns(b)" (Lr#n), which can be transferred to wheat as T3BL.3Ns(b)S (denoted Lr#n-SA), where 3BS arm of chromosome 3B of wheat was replaced by 3Ns(b)S of L. racemosus. We successfully introduced T3BL.3Ns(b)S into the wheat cultivar "Chinese Spring" (CS-Lr#n-SA, referred to as "BNI-CS"), which resulted in the doubling of its BNI capacity. T3BL.3Ns(b)S from BNI-CS was then transferred to several elite high-yielding hexaploid wheat cultivars, leading to near doubling of BNI production in "BNI-MUNAL" and "BNI-ROELFS." Laboratory incubation studies with root-zone soil from field-grown BNI-MUNAL confirmed BNI trait expression, evident from suppression of soil nitrifier activity, reduced nitrification potential, and N2O emissions. Changes in N metabolism included reductions in both leaf nitrate, nitrate reductase activity, and enhanced glutamine synthetase activity, indicating a shift toward ammonium nutrition. Nitrogen uptake from soil organic matter mineralization improved under low N conditions. Biomass production, grain yields, and N uptake were significantly higher in BNI-MUNAL across N treatments. Grain protein levels and breadmaking attributes were not negatively impacted. Wide use of BNI functions in wheat breeding may combat nitrification in high N input-intensive farming but also can improve adaptation to low N input marginal areas.We gratefully acknowledge funding support from Japanese Ministry of Agriculture, Forestry and Fisheries, CGIAR Research Program on WHEAT during the execution of the research presented in this study

Genetic mitigation strategies to tackle agricultural GHG emissions: The case for biological nitrification inhibition technology

Accelerated soil-nitrifier activity and rapid nitrification are the cause of declining nitrogen-use efficiency (NUE) and enhanced nitrous oxide (N2O) emissions from farming. Biological nitrification inhibition (BNI) is the ability of certain plant roots to suppress soil-nitrifier activity through production and release of nitrification inhibitors. The power of phytochemicals with BNI-function needs to be harnessed to control soil-nitrifier activity and improve nitrogen-cycling in agricultural systems. Transformative biological technologies designed for genetic mitigation are needed so that BNIenabled crop-livestock and cropping systems can rein in soil-nitrifier activity to help reduce greenhouse gas (GHG) emissions and globally make farming nitrogen efficient and less harmful to environment. This will reinforce the adaptation or mitigation impact of other climate-smart agriculture technologies

Current status of bone regeneration using adipose-derived stem cells

Many bone regeneration therapies have been developed for clinical use and have variable outcomes and serious limitations. The goal of bone regeneration is to repair a bone defect in a stable and durable manner. Cellular strategies play an important role in bone tissue engineering. Clinical factors important for successful bone regeneration are the recruitment of cells to the defect site and the production of a suitable extracellular matrix consistent with bone tissues. Adipose-derived stem cells (ASCs) can be obtained in large quantities with little donor site morbidity or patient discomfort. They are multipotent somatic stem cells and have a strong potential to differentiate and secrete growth factors. In this review, we discuss the osteogenic potential of ASCs with/without several types of scaffolds in vivo and their clinical application for bone regeneration