Purine-rich element binding protein B attenuates the coactivator function of myocardin by a novel molecular mechanism of smooth muscle gene repression.

Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purß) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purß to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purß specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purß homodimer. DNA binding analyses demonstrated that Purß interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purß associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purß point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purß repressor and the myocardin-serum response factor-CArG complex.

Review of the Indian Ocean spikefish genus Mephisto (Tetraodontiformes: Triacanthodidae).

We redescribe the triacanthodid spikefish Mephisto fraserbrunneri Tyler 1966 based upon eight specimens (five newly reported herein) and the first color photographs of freshly collected specimens; these data are compared with that of the single specimen of the recently described M. albomaculosus Matsuura, Psomadakis, and Mya Than Tun 2018. Both species are found in the Indian Ocean, with M. fraserbrunneri known from the Arabian Sea off the east coast of Africa to the eastern Bay of Bengal, and M. albomaculosus confirmed only from the type locality in the Andaman Sea (a color photograph of an individual M. cf. albomaculosus from the Bay of Bengal that was not retained is also presented). We describe and diagnose the genus Mephisto and provide a key to the two species based upon all available specimens. We also provide a distribution map for both species and summarize literature records. Using micro-CT data, we show that Mephisto fraserbrunneri replaces teeth intraosseously, which suggests this tooth replacement pattern is plesiomorphic for Tetraodontiformes.

Structural and functional analysis of single-nucleotide polymorphic variants of purine-rich element-binding protein B.

Purine-rich element-binding protein B (Purß) inhibits myofibroblast differentiation by repressing the expression of the smooth muscle α-actin gene (Acta2). Several reports have identified the structural domains in Purß that enable its characteristic interaction with purine-rich single-stranded DNA (ssDNA) sequences in the Acta2 promoter. However, little is known about the physical and functional effects of single-nucleotide polymorphisms that alter individual amino acid residues in Purß. This study evaluated seven rare single amino acid variants of human PURB engineered into the homologous mouse Purß protein. Mapping the location of variant residues on a homology model of the Purß homodimer suggested that most of the altered residues are remote from the predicted ssDNA-binding regions of the protein. The repressor activity of each Purß variant was assessed in transfected fibroblasts and smooth muscle cells via Acta2 promoter-reporter assays. A Q64* nonsense variant was completely inactive while missense variants exhibited repressor activity that ranged from ~1.5-fold greater to ~2-fold less than wild-type Purß. Lower activity variants P223L and R297Q were expressed in bacteria and purified to homogeneity. Each variant was physically indistinguishable from wild-type Purß in terms of quaternary structure and thermostability. Results of DNA and protein-binding assays indicated that the P223L and R297Q variants retained high affinity and specificity for purine-rich ssDNA sequences but differed in their interaction with other Acta2 regulatory proteins. These findings suggest that the presence of certain variant residues affects the Acta2 repressor activity of Purß by altering its interaction with other transcription factors but not with ssDNA.

Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element-Binding Protein B.

Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5' promoter-enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purß) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5' polypurine sequence containing an inverted MCAT cis element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purß repressor and the purine-rich strand of the MCAT element in the mouse Acta2 promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purß transcriptional repressor function in Acta2 promoter-reporter assays. In keeping with their diminished Acta2 repressor activity in fibroblasts, purified Purß variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the Acta2 corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purß.

Mapping of contributions from collateral ligaments to overall knee joint constraint: an experimental cadaveric study.

Understanding the contribution of the soft-tissues to total joint constraint (TJC) is important for predicting joint kinematics, developing surgical procedures, and increasing accuracy of computational models. Previous studies on the collateral ligaments have focused on quantifying strain and tension properties under discrete loads or kinematic paths; however, there has been little work to quantify collateral ligament contribution over a broad range of applied loads and range of motion (ROM) in passive constraint. To accomplish this, passive envelopes were collected from nine cadaveric knees instrumented with implantable pressure transducers (IPT) in the collateral ligaments. The contributions from medial and lateral collateral ligaments (LCL) were quantified by the relative contribution of each structure at various flexion angles (0-120 deg) and compound external loads (±10 N m valgus, ±8 N m external, and ±40 N anterior). Average medial collateral ligament (MCL) contributions were highest under external and valgus torques from 60 deg to 120 deg flexion. The MCL showed significant contributions to TJC under external torques throughout the flexion range. Average LCL contributions were highest from 0 deg to 60 deg flexion under external and varus torques, as well as internal torques from 60 deg to 110 deg flexion. Similarly, these regions were found to have statistically significant LCL contributions. Anterior and posterior loads generally reduced collateral contribution to TJC; however, posterior loads further reduced MCL contribution, while anterior loads further reduced LCL contribution. These results provide insight to the functional role of the collaterals over a broad range of passive constraint. Developing a map of collateral ligament contribution to TJC may be used to identify the effects of injury or surgical intervention on soft-tissue, and how collateral ligament contributions to constraint correlate with activities of daily living.

Analysis of a rotary task following total knee arthroplasty: Stair descent with a cross-over turn.

Leg loading and knee angle differences have been reported in total knee replacement individuals during straight gait; however, little is known about the impact on the knee during turning. Rotary motions may be difficult following total knee replacement surgery; therefore, some individuals may develop strategies or utilize pre-surgical strategies in order to maintain function. The primary aim of this study was to identify differences in individuals with a total knee replacement as compared to their healthy counterparts during stair descent followed by a cross-over turn. Ground reaction force, knee angle and moments were recorded on 10 total knee replacement and 12 healthy individuals during stair descent followed by a turn and compared to walking straight. Variables were analyzed for the affected, unaffected and healthy knees during the gait cycle. On initial contact, the total knee replacement group had less ground reaction force on the affected leg compared to the unaffected leg (p = 0.021) and had delayed contact (p = 0.044) and a slower loading rate (p = 0.020) compared to healthy group. During mid-stance, the affected leg had less ground reaction force compared to the healthy leg (p = 0.049). The affected stance leg had less knee flexion during mid-stance in both the straight trial (p = 0.002) and turn (p = 0.010). Moment differed between straight and turn trials but not between groups. Stair descent with or without a turn was approached in a precautionary manner by individuals with a total knee replacement. Slow approach, reduced impact and weight-bearing with a more extended knee on the affected leg may suggest a protective strategy to avoid risk of fall.

Structural basis of multisite single-stranded DNA recognition and ACTA2 repression by purine-rich element binding protein B (Purß).

A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purß) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purß as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purß in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purß knockdown MEFs confirmed that these changes were attributable, in part, to derepression of ACTA2 transcription. To map the domains in Purß responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally derived models of the tertiary and quaternary structure of Purß. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purß homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purß repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.

Strategies utilized to transfer weight during knee flexion and extension with rotation for individuals with a total knee replacement.

Functional activities in daily life can require squatting and shifting body weight during transverse plane rotations. Stability of the knee can be challenging for people with a total knee replacement (TKR) due to reduced proprioception, nonconforming articular geometry, muscle strength, and soft tissue weakness. The objective of this study was to identify strategies utilized by individuals with TKR in double-stance transferring load during rotation and flexion. Twenty-three subjects were recruited for this study: 11 TKR subjects (age: 65 ± 6 years; BMI 27.4 ± 4.1) and 12 healthy subjects (age: 63 ± 7; BMI 24.6 ± 3.8). Each subject completed a novel crossover button push task where rotation, flexion, and extension of the knee were utilized. Each subject performed two crossover reaching tasks where the subject used the opposite hand to cross over their body and press a button next to either their shoulder (high) or knee (low), then switched hands and rotated to press the opposite button, either low or high. The two tasks related to the order they pressed the buttons while crossing over, either low-to-high (L2H) or high-to-low (H2L). Force platforms measured ground reaction forces under each foot, which were then converted to lead force ratios (LFRs) based on the total force. Knee flexion angles were also measured. No statistical differences were found in the LFRs during the H2L and L2H tasks for the different groups, although differences in the variation of the loading within subjects were noted. A significant difference was found between healthy and unaffected knee angles and a strong trend between healthy and affected subject's knee angles in both H2L and L2H tasks. Large variations in the LFR at mid-task in the TKR subjects suggested possible difficulties in maintaining positional stability during these tasks. The TKR subjects maintained more of an extended knee, which is a consistent quadriceps avoidance strategy seen by other researchers in different tasks. These outcomes suggest that individuals with a TKR utilize strategies, such as keeping an extended knee, to achieve rotary tasks during knee flexion and extension. Repeated compensatory movements could result in forces that may cause difficulty over time in the hip joints or low back. Early identification of these strategies could improve TKR success and the return to activities of daily living that involve flexion and rotation.