Norstictic Acid Is a Selective Allosteric Transcriptional Regulator.

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways, and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (∼900 Å2) and have little topology, and thus, they do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprising a highly dynamic loop flanking one canonical binding surface, and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple-negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

Test-Retest Reliability and the Effects of Walking Speed on Stride Time Variability During Continuous, Overground Walking in Healthy Young Adults.

Studies have investigated the reliability and effect of walking speed on stride time variability during walking trials performed on a treadmill. The objective of this study was to investigate the reliability of stride time variability and the effect of walking speed on stride time variability, during continuous, overground walking in healthy young adults. Participants completed: (1) 2 walking trials at their preferred walking speed on 1 day and another trial 2 to 4 days later and (2) 1 trial at their preferred walking speed, 1 trial approximately 20% to 25% faster than their preferred walking speed, and 1 trial approximately 20% to 25% slower than their preferred walking speed on a separate day. Data from a waist-mounted accelerometer were used to determine the consecutive stride times for each trial. The reliability of stride time variability outcomes was generally poor (intraclass correlations: .167-.487). Although some significant differences in stride time variability were found between the preferred walking speed, fast, and slow trials, individual between-trial differences were generally below the estimated minimum difference considered to be a real difference. The development of a protocol to improve the reliability of stride time variability outcomes during continuous, overground walking would be beneficial to improve their application in research and clinical settings.

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability.

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½â€¯= 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½â€¯= 388 min).

A Synthetic Loop Replacement Peptide That Blocks Canonical NF-κB Signaling.

Aberrant canonical NF-κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non-canonical NF-κB functions. Here we show that a selective peptide-based inhibitor of canonical NF-κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non-labile bond, shows an about 10-fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF-κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein-protein interactions mediated by such structures.

An investigation of neck muscle activity in asymptomatic participants who show different lumbar spine motion patterns during prone hip extension.

OBJECTIVE: The objective of the current study was to investigate whether any differences exist in the activity of the cervical erector spinae and upper trapezius (TRA) muscles between asymptomatic participants who show "normal" and "abnormal" lumbar spine motion patterns during the prone hip extension (PHE) test. METHODS: Twenty-six asymptomatic participants recruited from a chiropractic college participated in the study. Surface electromyography was used to record the activity of the cervical erector spinae and upper TRA muscles as each participant performed a set of 4 repetitions of PHE for each leg. An examiner observed the participant perform the movement and classified him/her as "positive" or "negative" based on the presence or absence (respectively) of 1 of 3 lumbar spine motion patterns. The mean activity levels of each muscle during the positive sets of PHE were compared with those during the negative sets. RESULTS: The mean activity of the upper TRA ipsilateral to the side of hip extension was significantly higher in the positive group compared with the negative group (difference, 13.3%; 95% confidence interval, 0.2%-24.4%; P = .0465). No other significant between-group differences were noted. CONCLUSION: The results of this study indicate that the presence of abnormal lumbar spine motion patterns during the PHE test may be associated with altered cervicothoracic motor control strategies in asymptomatic individuals. Similar investigations using patients with neck pain are required to comment further on the generalizability and potential clinical importance of these findings.

Students helping students: Evaluating a pilot program of peer teaching for an undergraduate course in human anatomy.

The educational literature generally suggests that supplemental instruction (SI) is effective in improving academic performance in traditionally difficult courses. A pilot program of peer teaching based on the SI model was implemented for an undergraduate course in human anatomy. Students in the course were stratified into three groups based on the number of peer teaching sessions they attended: nonattendees (0 sessions), infrequently attended (1-3 sessions), and frequently attended (≥ 4 sessions). After controlling for academic preparedness [i.e., admission grade point average (AGPA)] using an analysis of covariance, the final grades of frequent attendees were significantly higher than those of nonattendees (P = 0.025) and infrequent attendees (P = 0.015). A multiple regression analysis was performed to estimate the relative independent contribution of several variables in predicting the final grade. The results suggest that frequent attendance (ß = 0.245, P = 0.007) and AGPA (ß = 0.555, P < 0.001) were significant positive predictors, while being a first-year student (ß = -0.217, P = 0.006) was a significant negative predictor. Collectively, these results suggest that attending a certain number of sessions may be required to gain a noticeable benefit from the program, and that first-year students (particularly those with a lower level of academic preparedness) would likely stand to benefit from maximally using the program. End-of-semester surveys and reports indicate that the program had several additional benefits, both to the students taking the course and to the students who served as program leaders.

Inter-rater agreement, sensitivity, and specificity of the prone hip extension test and active straight leg raise test.

BACKGROUND: Two clinical tests used to assess for neuromuscular control deficits in low back pain (LBP) patients are the prone hip extension (PHE) test and active straight leg raise (ASLR) test. For these tests, it has been suggested examiners classify patients as "positive" or "negative" based on the presence or absence (respectively) of specific "abnormal" lumbopelvic motion patterns. The inter-rater agreement of such a classification scheme has been reported for the PHE test, but not for the ASLR test. In addition, the sensitivity and specificity of such classification schemes have not been reported for either test. The primary objectives of the current study were to investigate: 1) the inter-rater agreement of the examiner-reported classification schemes for these two tests, and 2) the sensitivity and specificity of the classification schemes. METHODS: Thirty participants with LBP and 40 asymptomatic controls took part in this cross-sectional observational study. Participants performed 3-4 repetitions of each test whilst two examiners classified them as "positive" or "negative" based on the presence or absence (respectively) of specific "abnormal" lumbopelvic motion patterns. The inter-rater agreement (Kappa statistic), sensitivity (LBP patients), and specificity (controls) were calculated for each test. RESULTS: Both tests demonstrated substantial inter-rater agreement (PHE test: Kappa = 0.76, 95% CI = 0.57-0.95, p < 0.001; ASLR test: Kappa = 0.76, 95% CI = 0.57-0.96, p < 0.001). For the PHE test, the sensitivity was 0.18-0.27 and the specificity was 0.63-0.78; the odds ratio (OR) of "positive" classifications in the LBP group was 1.25 (95% CI = 0.58-2.72; Examiner 1) and 1.27 (95% CI = 0.52-3.12; Examiner 2). For the ASLR test, the sensitivity was 0.20-0.25 and the specificity was 0.84-0.86; the OR of "positive" classifications in the LBP group was 1.72 (95% CI = 0.75-3.95; Examiner 1) and 1.57 (95% CI = 0.64-3.85; Examiner 2). CONCLUSION: Classification schemes for the PHE test and ASLR test based on the presence or absence of specific "abnormal" lumbopelvic motion patterns demonstrated substantial inter-rater agreement. However, additional investigation is required to further comment on the clinical usefulness of the motion patterns demonstrated by LBP patients during these tests as a diagnostic tool or treatment outcome.

Patient-reported perception of difficulty as a clinical indicator of dysfunctional neuromuscular control during the prone hip extension test and active straight leg raise test.

Two clinical tests used to assess for neuromuscular control deficits in patients with low back pain (LBP) are the prone hip extension (PHE) test and active straight leg raise (ASLR) test. For these tests, it has been suggested that patients be classified as "positive" if they demonstrate specific "abnormal" lumbopelvic motion patterns. For the ASLR test, the use of patient-reported perception of difficulty is also used to assess neuromuscular control. Thirty participants with LBP and 40 asymptomatic controls took part in this cross-sectional observational study. Participants performed both tests and were classified as "positive" or "negative" based on the presence or absence (respectively) of specific "abnormal" motion patterns. The participants also rated their perceived difficulty in performing the tests using a six-point scale. A two-way analysis of covariance (ANCOVA) was used to examine the effects of group (LBP/control) and examiner classification (positive/negative) on the perceived difficulty scores for each test. The LBP group perceived greater difficulty in performing both tests compared to the control group. Conversely, there was no significant difference in the perceived difficulties of the positive and negative examiner classifications for either test. Additional investigation is required to comment further on the relative usefulness of the perceived difficulty and observable motion patterns during these tests in assessing the neuromuscular control strategies of the lumbopelvic region, and their potential as a diagnostic tool or treatment outcome.

Genetic and chemical targeting of epithelial-restricted with serine box reduces EGF receptor and potentiates the efficacy of afatinib.

EGF receptor (EGFR) is elevated in more than 90% of head and neck squamous cell carcinoma (HNSCC). However, a majority of patients with HNSCC do not respond to anti-EGFR therapeutics. Insensitivity to EGFR inhibitors may be due to kinase-independent actions of EGFR and/or activation of Her2. Strategies to reduce EGFR and Her2 protein levels in concert may be an optimal approach to enhance the efficacy of current anti-EGFR molecules. In this study, knockdown of epithelial-restricted with serine box (ESX) decreased EGFR and Her2 promoter activity, expression, and levels. ESX was elevated in primary HNSCC tumors and associated with increased EGFR and Her2. Genetic ablation of ESX decreased EGFR and Her2 levels and enhanced the antiproliferative effects of EGFR/Her2 tyrosine kinase inhibitors (TKI), lapatinib and afatinib. Biphenyl isoxazolidine, a novel small-molecule ESX inhibitor, reduced EGFR and Her2 levels and potentiated the antiproliferative efficacy of afatinib. Single-agent biphenyl isoxazolidine retarded the in vivo tumorigenicity of CAL27 cells. Importantly, the combination of biphenyl isoxazolidine and afatinib was significantly superior in vivo and resulted in a 100% response rate with a 94% reduction in tumor volume. Targeting EGFR/Her2 levels with an ESX inhibitor and EGFR/Her2 kinase activity with a TKI simultaneously is a highly active therapeutic approach to manage HNSCC. Our work provides evidence to support the further development of ESX inhibitors as an adjuvant to enhance the response rate of patients with HNSCC to current anti-EGFR/Her2 therapeutics.

Anatomically remote muscle contraction facilitates patellar tendon reflex reinforcement while mental activity does not: a within-participants experimental trial.

BACKGROUND: The Jendrassik maneuver (JM) is a remote facilitation muscular contraction shown to affect amplitude and temporal components of the human stretch reflex. Conflicting theoretical models exist regarding the neurological mechanism related to its ability to reinforce reflex parameters. One mechanism involves the gamma motoneurons of the fusimotor system, which are subject to both physical and mental activity. A second mechanism describes reduced alpha motoneuron presynaptic inhibition, which is not subject to mental activity. In the current study, we determined if mental activity could be used to create a reflex facilitation comparable to a remote muscle contraction. METHOD: Using a within-participants design, we investigated the relative effect of the JM and a successfully employed mental task (Stroop task) on the amplitude and temporal components of the patellar tendon reflex. RESULTS: We found that the addition of mental activity had no influence on the patellar tendon reflex parameters measured, while the JM provided facilitation (increased reflex amplitude, decreased total reflex time). CONCLUSION: The findings from this study support the view that the mechanism for the JM is a reduction in presynaptic inhibition of alpha motoneurons as it is influenced by physical and not mental activity.

Use of the environmentally sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide to study peptoid helix structures.

Peptoids, oligomers of N-substituted glycine, have been valuable targets for study and diverse application as peptidomimetics and as nanomaterials. Their conformational heterogeneity has made the study of peptoid structures using high-resolution analyses challenging, limiting our understanding of the physiochemical features that mediate peptoid folding. Here, we introduce a new method for the study of peptoid structure that relies on the environmentally sensitive fluorescence properties of 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN). We have prepared a 4-DMN-functionalized primary amine that is compatible with the traditional submonomer peptoid synthesis methods and incorporated it sequence-specifically into 11 of 13 new peptoids. When included as a peptoid side chain modification, the fluorescence emission intensity of 4-DMN correlates with predictions of the fluorophore's local polarity within a putative structure. 4-DMN fluorescence is maximized when the fluorophore is placed in the middle of the hydrophobic face of an amphiphilic helical peptoid. When the fluorophore is placed near the peptoid terminus or on a polar face of an amphiphilic sequence, 4-DMN fluorescence is diminished. Disruption of the peptoid secondary structure or amphiphilicity also modulates 4-DMN fluorescence. The peptoids' helical secondary structures are moderately disrupted by inclusion of a 4-DMN-modified side chain as evaluated by changes in the peptoids' CD spectral features. This new method for peptoid structure evaluation should be a valuable complement to existing peptoid structural analysis tools.