Variables associated with successful outcome after anterior cruciate ligament reconstruction in recreational athletes: A prospective cohort study.

BACKGROUND: Anterior cruciate ligament (ACL) injury and subsequent reconstruction is common and has a profound effect on health-related quality of life. There is currently limited understanding as to which variables are associated with a successful outcome post-ACL reconstruction (ACLR) in recreational athletes. PURPOSE: Explore the association between both patient-reported and performance-based measures, and successful outcome, post-ACLR in recreational athletes. PROCEDURES: We sought to recruit recreational athletes within one month of a primary-ACLR for a prospective cohort study. A dichotomised patient specific functional scale of ≥9 points determined a successful outcome at nine-months post-operative. Secondary patient-reported and performance-based data were collected at baseline, three-, six-, and nine-months post-operative. The association between secondary data and the primary outcome was determined using binomial logistic regression, expressed using odds ratio (OR) and 95% confidence intervals (CI). MAIN RESULTS: 90 participants were recruited (males: 58, females: 32, mean age 32.8 years [±7.9], mean height 173.5 [±10.0], mean body mass 74.0 kg [±15.8]), 87 consented to baseline measures. 47 participants completed full data collection and 21 (45%) reported a successful outcome. Higher knee osteoarthritis outcome score (OR range 1.07-1.12) and anterior cruciate ligament quality of life (ACL-QoL) scores (OR range 1.06-1.10) were associated with a successful outcome post-ACLR at various timepoints. CONCLUSIONS: Patient-reported, rather than performance-based, measures were associated with successful outcome nine-months post-ACLR in recreational athletes. Both patient-reported and performance-based characteristics are advocated to guide optimal return to function in clinical practice.

Stimulation of Distinct Rhizosphere Bacteria Drives Phosphorus and Nitrogen Mineralization in Oilseed Rape under Field Conditions.

Advances in DNA sequencing technologies have drastically changed our perception of the structure and complexity of the plant microbiome. By comparison, our ability to accurately identify the metabolically active fraction of soil microbiota and its specific functional role in augmenting plant health is relatively limited. Important ecological interactions being performed by microbes can be investigated by analyzing the extracellular protein fraction. Here, we combined a unique protein extraction method and an iterative bioinformatics pipeline to capture and identify extracellular proteins (metaexoproteomics) synthesized in the rhizosphere of Brassica spp. We first validated our method in the laboratory by successfully identifying proteins related to a host plant (Brassica rapa) and its bacterial inoculant, Pseudomonas putida BIRD-1. This identified numerous rhizosphere specific proteins linked to the acquisition of plant-derived nutrients in P. putida. Next, we analyzed natural field-soil microbial communities associated with Brassica napus L. (oilseed rape). By combining metagenomics with metaexoproteomics, 1,885 plant, insect, and microbial proteins were identified across bulk and rhizosphere samples. Metaexoproteomics identified a significant shift in the metabolically active fraction of the soil microbiota responding to the presence of B. napus roots that was not apparent in the composition of the total microbial community (metagenome). This included stimulation of rhizosphere-specialized bacteria, such as Gammaproteobacteria, Betaproteobacteria, and Flavobacteriia, and the upregulation of plant beneficial functions related to phosphorus and nitrogen mineralization. Our metaproteomic assessment of the "active" plant microbiome at the field-scale demonstrates the importance of moving beyond metagenomics to determine ecologically important plant-microbe interactions underpinning plant health. IMPORTANCE Plant-microbe interactions are critical to ecosystem function and crop production. While significant advances have been made toward understanding the structure of the plant microbiome, learning about its full functional role is still in its infancy. This is primarily due to an incomplete ability to determine in situ plant-microbe interactions actively operating under field conditions. Proteins are the functional entities of the cell. Therefore, their identification and relative quantification within a microbial community provide the best proxy for which microbes are the most metabolically active and which are driving important plant-microbe interactions. Here, we provide the first metaexoproteomics assessment of the plant microbiome using field-grown oilseed rape as the model crop species, identifying key taxa responsible for specific ecological interactions. Gaining a mechanistic understanding of the plant microbiome is central to developing engineered plant microbiomes to improve sustainable agricultural approaches and reduce our reliance on nonrenewable resources.

Identification of extracellular glycerophosphodiesterases in Pseudomonas and their role in soil organic phosphorus remineralisation.

In soils, phosphorus (P) exists in numerous organic and inorganic forms. However, plants can only acquire inorganic orthophosphate (Pi), meaning global crop production is frequently limited by P availability. To overcome this problem, rock phosphate fertilisers are heavily applied, often with negative environmental and socio-economic consequences. The organic P fraction of soil contains phospholipids that are rapidly degraded resulting in the release of bioavailable Pi. However, the mechanisms behind this process remain unknown. We identified and experimentally confirmed the function of two secreted glycerolphosphodiesterases, GlpQI and GlpQII, found in Pseudomonas stutzeri DSM4166 and Pseudomonas fluorescens SBW25, respectively. A series of co-cultivation experiments revealed that in these Pseudomonas strains, cleavage of glycerolphosphorylcholine and its breakdown product G3P occurs extracellularly allowing other bacteria to benefit from this metabolism. Analyses of metagenomic and metatranscriptomic datasets revealed that this trait is widespread among soil bacteria with Actinobacteria and Proteobacteria, specifically Betaproteobacteria and Gammaproteobacteria, the likely major players.

The 'known' genetic potential for microbial communities to degrade organic phosphorus is reduced in low-pH soils.

In soil, bioavailable inorganic orthophosphate is found at low concentrations and thus limits biological growth. To overcome this phosphorus scarcity, plants and bacteria secrete numerous enzymes, namely acid and alkaline phosphatases, which cleave orthophosphate from various organic phosphorus substrates. Using profile hidden Markov modeling approaches, we investigated the abundance of various non specific phosphatases, both acid and alkaline, in metagenomes retrieved from soils with contrasting pH regimes. This analysis uncovered a marked reduction in the abundance and diversity of various alkaline phosphatases in low-pH soils that was not counterbalanced by an increase in acid phosphatases. Furthermore, it was also discovered that only half of the bacterial strains from different phyla deposited in the Integrated Microbial Genomes database harbor alkaline phosphatases. Taken together, our data suggests that these 'phosphatase lacking' isolates likely increase in low-pH soils and future research should ascertain how these bacteria overcome phosphorus scarcity.

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria.

Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial-driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole-cell proteomic analysis of BIRD-1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well-characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD-1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO-dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.

Truncated MEK1 is required for transient activation of MAPK signalling in G2 phase cells.

The primary endpoint of signalling through the canonical Raf-MEK-ERK MAP kinase cascade is ERK activation. Here we report a novel signalling outcome for this pathway. Activation of the MAP kinase pathway by growth factors or phorbol esters during G2 phase results in only transient activations of ERK and p90RSK, then suppression to below control levels. A small peak of ERK and p90RSK activation in early G2 phase cells was identified, and inhibition of this delayed entry into mitosis. The previously identified, proteolytically cleaved form of MEK1 termed tMEK (truncated MEK1), is also induced with G2 phase MAPK pathway activation. We demonstrate that addition of recombinant mutants of MEK1 with an N-terminal truncation similar to that of tMEK also inhibited ERK and p90RSK activations and delayed progression into mitosis. Only catalytically inactive forms of tMEK were capable of these effects, but surprisingly, phosphorylation on the activating Ser218/222 sites was also required. A lack of MEK1 or ability to accumulate tMEK resulted in the absence of the feedback inhibition of ERK and p90RSK activations. tMEK is a novel output from the canonical MAP kinase signalling pathway, acting in a MAPK signalling-regulated dominant negative manner to inhibit ERK and p90RSK activations, acting as a dampening mechanism to reduce the magnitude or duration of MAPK pathway signalling in G2/M phase.

Identification and expression analyses of cytosolic glutamine synthetase genes in barley (Hordeum vulgare L.).

Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, particularly during seed development. Three cytosolic GS isoforms (HvGS1) were identified in barley (Hordeum vulgare L. cv Golden Promise). Quantitation of gene expression, localization and response to N supply revealed that each gene plays a non-redundant role in different tissues and during development. Localization of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that it is important in N transport and remobilization. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localized to the leaf mesophyll cells, in the cortex and pericycle of roots, and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in leaves with an increasing supply of N, suggesting its role in the primary assimilation of N. HvGS1_3 was specifically and predominantly localized in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH(+)4, suggesting that it has a primary role in grain N assimilation and also in the protection against ammonium toxicity in roots. The expression of HvGS1 genes is directly correlated with protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley.

Analysis of protein prenylation in vitro and in vivo using functionalized phosphoisoprenoids.

Post-translational modifications (PTMs) expand the number of protein isoforms in eukaryotic proteome by orders of magnitude. Protein modification with isoprenoid lipids is a common PTM affecting hundreds of proteins controlling the transport of information and materials into, through, and out of the eukaryotic cell. In this modification, a soluble phosphoisoprenoid such as farnesyl (C15) or geranylgeranyl (C20) pyrophosphate moiety is recruited by one of three protein prenyltransferases to covalently modify a C-terminal cysteine(s) in a target protein. The three mammalian prenyltransferases are farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase I), and Rab geranylgeranyl transferase (also termed geranylgeranyltransferase type II - GGTase II). In this unit, synthetic isoprenoids conjugated to either a fluorophore or biotin group are used to assay the activity of protein prenyltransferases in vitro or to affinity tag prenylatable proteins in cell lysates. These protocols and their modifications can be used to study the mechanisms of protein prenylation, identify prenylation targets, and characterize inhibitors of protein prenyltransferases in vitro and in vivo.

Nucleophosmin and nucleolin regulate K-Ras plasma membrane interactions and MAPK signal transduction.

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.

Electrostatic interactions positively regulate K-Ras nanocluster formation and function.

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale environments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades.