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We showed previously that gallic acid is produced in walnut from 3-dehydroshikimate by a shikimate dehydrogenase (JrSkDH). This study focuses on the next step in the hydrolysable tannin pathway, the formation of 1-O-galloyl-β-D-glucose from the phenolic gallic acid and UDP glucose by a glycosyltransferase. JrGGT1 (UGT84A73) and JrGGT2 (UGT84A74) are predicted to be two such glycosyltransferases, which we expressed in tobacco plants. GC-MS analysis of the transgenic tobacco revealed moderate, yet significant alterations in plant secondary metabolism, such as depleted phenolic acids, including gallic acid. We postulate that these effects are due to JrGGT1 and JrGGT2 activity, as JrGGT orthologs glycosylate these phenolic compounds in vitro. Moreover, JrGGT expression in tobacco caused upregulation of shikimic acid pathway metabolites and differing responses in phenylpropanoids, such as phenolic acids and flavonoids. In transcriptome analysis of walnut pellicle tissues, both JrGGTs showed substantial and significant expression correlations with the gallic acid-producing JrSkDHs and were highly coexpressed with the genetic circuits constituting the shikimic acid and phenylpropanoid biosynthetic pathways. Verification of JrGGT gene expression by transcriptome analysis of 20 walnut tissues revealed striking similarities with that of the pellicle data, with the greatest expression in roots, wood, buds, and leaves of Juglans regia cv. Chandler: tissues that typically accumulate hydrolysable tannins. Like the transgenic tobacco, pellicle metabolomic analyses revealed that many phenylpropanoids correlated negatively with JrGGT expression, while shikimic acid pathway metabolites correlated positively with JrGGT expression. This research supports the hypothesis that JrGGT1 and JrGGT2 play non-trivial roles in metabolism of phenolic acids, flavonoids, and ostensibly, tannins.

Walnut pellicle color is a key quality attribute that drives consumer preference and walnut sales. For the first time a high-throughput, computer vision-based phenotyping platform using a custom algorithm to quantitatively score each walnut pellicle in L* a* b* color space was deployed at large-scale. This was compared to traditional qualitative scoring by eye and was used to dissect the genetics of pellicle pigmentation. Progeny from both a bi-parental population of 168 trees ('Chandler' × 'Idaho') and a genome-wide association (GWAS) with 528 trees of the UC Davis Walnut Improvement Program were analyzed. Color phenotypes were found to have overlapping regions in the 'Chandler' genetic map on Chr01 suggesting complex genetic control. In the GWAS population, multiple, small effect QTL across Chr01, Chr07, Chr08, Chr09, Chr10, Chr12 and Chr13 were discovered. Marker trait associations were co-localized with QTL mapping on Chr01, Chr10, Chr14, and Chr16. Putative candidate genes controlling walnut pellicle pigmentation were postulated.

The transcriptome provides a functional footprint of the genome by enumerating the molecular components of cells and tissues. The field of transcript discovery has been revolutionized through high-throughput mRNA sequencing (RNA-seq). Here, we present a methodology that replicates and improves existing methodologies, and implements a workflow for error estimation and correction followed by genome annotation and transcript abundance estimation for RNA-seq derived transcriptome sequences (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). A unique feature of YeATS is the upfront determination of the errors in the sequencing or transcript assembly process by analyzing open reading frames of transcripts. YeATS identifies transcripts that have not been merged, result in broken open reading frames or contain long repeats as erroneous transcripts. We present the YeATS workflow using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut. A novel feature of the transcriptome that emerged from our analysis was the identification of a highly abundant transcript that had no known homologous genes (GenBank accession: KT023102). The amino acid composition of the longest open reading frame of this gene classifies this as a putative extensin. Also, we corroborated the transcriptional abundance of proline-rich proteins, dehydrins, senescence-associated proteins, and the DNAJ family of chaperone proteins. Thus, YeATS presents a workflow for analyzing RNA-seq data with several innovative features that differentiate it from existing software.

Chronic inflammation is one of the major causes of cartilage destruction in osteoarthritis. Here, we systematically analyzed the changes in gene expression associated with the progression of cartilage destruction in monoiodoacetate-induced arthritis (MIA) of the rat knee. Sprague Dawley female rats were given intra-articular injection of monoiodoacetate in the knee. The progression of MIA was monitored macroscopically, microscopically and by micro-computed tomography. Grade 1 damage was observed by day 5 post-monoiodoacetate injection, progressively increasing to Grade 2 by day 9, and to Grade 3-3.5 by day 21. Affymetrix GeneChip was utilized to analyze the transcriptome-wide changes in gene expression, and the expression of salient genes was confirmed by real-time-PCR. Functional networks generated by Ingenuity Pathways Analysis (IPA) from the microarray data correlated the macroscopic/histologic findings with molecular interactions of genes/gene products. Temporal changes in gene expression during the progression of MIA were categorized into five major gene clusters. IPA revealed that Grade 1 damage was associated with upregulation of acute/innate inflammatory responsive genes (Cluster I) and suppression of genes associated with musculoskeletal development and function (Cluster IV). Grade 2 damage was associated with upregulation of chronic inflammatory and immune trafficking genes (Cluster II) and downregulation of genes associated with musculoskeletal disorders (Cluster IV). The Grade 3 to 3.5 cartilage damage was associated with chronic inflammatory and immune adaptation genes (Cluster III). These findings suggest that temporal regulation of discrete gene clusters involving inflammatory mediators, receptors, and proteases may control the progression of cartilage destruction. In this process, IL-1β, TNF-α, IL-15, IL-12, chemokines, and NF-κB act as central nodes of the inflammatory networks, regulating catabolic processes. Simultaneously, upregulation of asporin, and downregulation of TGF-β complex, SOX-9, IGF and CTGF may be central to suppress matrix synthesis and chondrocytic anabolic activities, collectively contributing to the progression of cartilage destruction in MIA.

Clinical Scenario: Anterior cruciate ligament (ACL) ruptures are one of the most common injuries in young athletic populations. The leading treatment for these injuries is ACL reconstruction (ACL-r); however, nonoperative treatments are also utilized. Following ACL-r, patients experience prolonged muscle weakness and atrophy of the quadriceps muscle group, regardless of rehabilitation. Nonoperative treatment plans following ACL injury exist, but their outcomes are less familiar, in spite of providing insight as a nonsurgical “control” for postsurgical rehabilitation outcomes. Therefore, the purpose of this critically appraised topic was to evaluate quadriceps strength and function following nonoperative ACL rehabilitation using objective and subjective measures including isokinetic dynamometry, the single-leg hop test, and the International Knee Documentation Committee (IKDC) subjective knee form. Focused Clinical Question: What are the effects of nonoperative treatment on peak isokinetic knee-extensor torque, the single-leg hop tests, and the IKDC in patients who have sustained an ACL rupture? Summary of Key Findings: Patients who underwent nonsurgical ACL treatment produced limb symmetry index, with the side-to-side torque difference expressed as a percentage, and values at or above 90% for all 4 single-leg hop tests and strength tests similar to ACL-r patients. All studies showed individuals had higher IKDC scores at baseline collection when compared with patients who underwent ACL-r but showed lower IKDC scores at long-term followup compared with ACL-r patients. Clinical Bottom Line: Nonoperative treatments of ACL injuries yield similar long-term results in quadriceps strength as ACL-r. Due to the quality of evidence and the absence of randomized controlled trials on this topic, these outcomes should be considered with caution. Strength of Recommendation: The Oxford Centre for Evidence-Based Medicine taxonomy recommends a grade of B for level 2 evidence with consistent findings. [ABSTRACT FROM AUTHOR]

Purpose: Therapies for certain voice disorders purport principles of skeletal muscle rehabilitation to increase muscle mass, strength, and endurance. However, applicability of limb muscle rehabilitation to the laryngeal muscles has not been tested. In this study, the authors examined the feasibility of the rat thyroarytenoid muscle to remodel as a consequence of increased activity instantiated through chronic electrical stimulation. Method: Twenty adult Sprague-Dawley rats ("Rattus norvegicus"), assigned to a 1-week or 2-week stimulation group, were implanted with a nerve cuff electrode placed around the right recurrent laryngeal nerve and were fitted with a head connector. All animals were placed under anesthesia twice a day for 1 hr each time. Following the training, rats were killed, and thyroarytenoid muscles were isolated for histology and immunohistochemistry. Results: Mean muscle fiber area decreased, neuromuscular junction density increased, mitochondrial content increased qualitatively, and glycogen-positive fibers increased, demonstrating exercise-induced changes similar to those seen in limb muscles after endurance training. Conclusion: Rat thyroarytenoid muscles are capable of remodeling in response to chronic electrical stimulation.

Purpose: Therapies for certain voice disorders purport principles of skeletal muscle rehabilitation to increase muscle mass, strength, and endurance. However, applicability of limb muscle rehabilitation to the laryngeal muscles has not been tested. In this study, the authors examined the feasibility of the rat thyroarytenoid muscle to remodel as a consequence of increased activity instantiated through chronic electrical stimulation. Method: Twenty adult Sprague-Dawley rats (Rattus norvegicus), assigned to a 1-week or 2-week stimulation group, were implanted with a nerve cuff electrode placed around the right recurrent laryngeal nerve and were fitted with a head connector. All animals were placed under anesthesia twice a day for 1 hr each time. Following the training, rats were killed, and thyroarytenoid muscles were isolated for histology and immunohistochemistry. Results: Mean muscle fiber area decreased, neuromuscular junction density increased, mitochondrial content increased qualitatively, and glycogen-positive fibers increased, demonstrating exercise-induced changes similar to those seen in limb muscles after endurance training. Conclusion: Rat thyroarytenoid muscles are capable of remodeling in response to chronic electrical stimulation. Key Words: chronic nerve stimulation, thyroarytenoid, mitochondria, neuromuscular junction, fiber size, glycogen, Voice production is the result of a balanced and highly refined interplay among the intrinsic and extrinsic laryngeal muscles, the respiratory tract, and the supraglottal resonators (Stemple, Lee, D'Amico, & [...]

Eccentric exercise is able to mechanically engage muscle, initiating strain-sensing molecules that promote muscle recovery by inducing beneficial adaptations in neural activity and muscle morphology, 2 critical components of muscle function that are negatively altered after injury. However, due to misinterpreted mathematic modeling and in situ and in vitro stretch protocols, a dogma that exposing muscle to eccentric exercise is associated with injury has been perpetuated in the literature. In response, clinicians have been biased toward using concentric exercise postinjury to improve the recovery of muscle function. Unfortunately, this conventional approach to rehabilitation does not restore muscle function, and reinjury rates remain high. Here, the authors present experimental evidence and theoretical support for the idea that isolated eccentric exercise is ideally suited to combat muscle inhibition and muscle strains and is an attractive alternative to concentric exercise. [ABSTRACT FROM AUTHOR]

Context: The role of the rotator cuff is to provide dynamic stability to the glenohumeral joint. Human and animal studies have identified sarcomerogenesis as an outcome of eccentric training indicated by more torque generation with the muscle in a lengthened position. Objective: The authors hypothesized that a home-based eccentric-exercise program could increase the shoulder external rotators' eccentric strength at terminal internal rotation (IR). Design: Prospective case series. Setting: Clinical laboratory and home exercising. Participants: 10 healthy subjects (age 30 ± 10 y). Intervention: All participants performed 2 eccentric exercises targeting the posterior shoulder for 6 wk using a home-based intervention program using side-lying external rotation (ER) and horizontal abduction. Main Outcome Measures: Dynamic eccentric shoulder strength measured at 60°/s through a 100° arc divided into 4 equal 25° arcs (ER 50-25°, ER 25-0°, IR 0-25°, IR 25-50°) to measure angular impulse to represent the work performed. In addition, isometric shoulder ER was measured at 5 points throughout the arc of motion (45° IR, 30° IR, 15° IR, 0°, and 15° ER). Comparison of isometric and dynamic strength from pre- to posttesting was evaluated with a repeated-measure ANOVA using time and arc or positions as within factors. Results: The isometric force measures revealed no significant differences between the 5 positions (P = .56). Analysis of the dynamic eccentric data revealed a significant difference between arcs (P = .02). The percentage-change score of the arc of IR 25-50° was found to be significantly greater than that of the arc of IR 0-25° (P = .007). Conclusion: After eccentric training the only arc of motion that had a positive improvement in the capacity to absorb eccentric loads was the arc of motion that represented eccentric contractions at the longest muscle length. [ABSTRACT FROM AUTHOR]