TRIM71 reactivation enhances the mitotic and hair cell-forming potential of cochlear supporting cells.

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming; however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 re-expression increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71's RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.

Unmasking Verbal Defensiveness: The Role of Psychological Threat in Sentence Completion Tests.

Shedding light on the validity of sentence completion test (SCT) verbal defensiveness as an index of defensive behavior, the current two-part study examined the relationship between psychological threat and verbal defensiveness among military security and mission-critical team candidates using SCTs. Our study showed that as the threatening nature of SCT stems increased, defensive responses also increased, substantiating the link between psychological threat and defensive behavior. In addition, expert ratings of stem content revealed moderately strong relationships with defensive responses across two different SCTs, irrespective of their structural characteristics. In contrast to previous studies using total verbal defensiveness scores, we examined specific defensive response types and their associations with stem threat ratings, finding that omissions, denial, and comments about the test were linked to stem threat levels. Lastly, our study extends the application of the SCT verbal defensiveness index beyond specialized personnel selection, finding no significant differences in verbal defensiveness based on gender or military status. Overall, these findings contribute to a comprehensive understanding of defensive behavior and its contextual variations.

Selective CRAF Inhibition Elicits Transactivation.

Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a "paradoxical activation" of RAF kinase activity through transactivation of the CRAF protomer; this leads to resistance mechanisms and secondary tumors. It has been hypothesized that CRAF-selective inhibition might bypass paradoxical activation, but no CRAF-selective inhibitor has been reported and the consequences of pharmacologically inhibiting CRAF have remained unknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectively target inhibitors to CRAF. Our results suggest that selective CRAF inhibition promotes paradoxical activation and exemplify how BOLT may be used to triage potential targets for drug discovery before any target-selective small molecules are known.

Allometric Grip Strength Norms for American Children.

Kocher, MH, Oba, Y, Kimura, IF, Stickley, CD, Morgan, CF, and Hetzler, RK. Allometric grip strength norms for American children. J Strength Cond Res 33(8): 2251-2261, 2019-To develop normative data from a large cohort of American school children (ages 6-18) for unscaled and allometrically scaled handgrip strength data that are uninfluenced by body size (body mass [BM] and stature [Ht]). Data (age, handgrip strength, BM, and Ht) were collected from the 2011-2012 and 2013-2014 National Health and Nutrition Examination Survey databases, resulting in 4,665 cases (2,384 boys and 2,281 girls). Multiple log-linear regressions were used to determine allometric exponents for BM and Ht separately for each age and sex to satisfy the common exponent and group difference principles described by Vanderburgh. Appropriateness of the allometric model was assessed through regression diagnostics, including normality and homoscedasticity of residuals. Allometrically scaled, ratio-scaled, and unscaled grip strength were then correlated with BM and Ht to examine the effectiveness of the procedure in controlling for body size. The data did not allow for development of a common exponent across age and sex that did not violate the common exponent and group difference principles. Correlations between allometrically scaled handgrip strength with BM and Ht were not significant (p ≤ 0.479) and approached zero, unlike correlations of unscaled handgrip strength with BM and Ht (p < 0.001 for all), indicating that allometric scaling was successful in removing the influence of body size. Allometric scaling handgrip strength by age and sex effectively controls for body size (Ht and BM) and perhaps maturation (Ht). The allometric exponents and normative values developed can be used to compare handgrip strength within age and sex while controlling for body size.

A reactivity-based probe of the intracellular labile ferrous iron pool.

Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.

Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling.

Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies.

A Mechanistic Model for Cooperative Behavior of Co-transcribing RNA Polymerases.

In fast-transcribing prokaryotic genes, such as an rrn gene in Escherichia coli, many RNA polymerases (RNAPs) transcribe the DNA simultaneously. Active elongation of RNAPs is often interrupted by pauses, which has been observed to cause RNAP traffic jams; yet some studies indicate that elongation seems to be faster in the presence of multiple RNAPs than elongation by a single RNAP. We propose that an interaction between RNAPs via the torque produced by RNAP motion on helically twisted DNA can explain this apparent paradox. We have incorporated the torque mechanism into a stochastic model and simulated transcription both with and without torque. Simulation results illustrate that the torque causes shorter pause durations and fewer collisions between polymerases. Our results suggest that the torsional interaction of RNAPs is an important mechanism in maintaining fast transcription times, and that transcription should be viewed as a cooperative group effort by multiple polymerases.

Allometric Grip Strength Norms for Children of Hawaiian Lineage.

The purpose of the study was to determine allometric exponents for scaling grip strength in children that effectively control for body mass (BM) and stature (Ht) and to develop normative grip strength data for Hawaiian children. One thousand, four hundred thirty-seven students (754 boys) from a rural community in Hawaii participated in this 5-year study, resulting in 2,567 data points. Handgrip strength, BM, and Ht were collected every year. Multiple log-linear regression was used to determine allometric exponents for BM and Ht. Appropriateness of the allometric model was assessed through regression diagnostics, including normality of residuals and homoscedasticity. Allometrically scaled, ratio-scaled, and unscaled grip strength were then correlated with BM and Ht to examine the effectiveness of the procedure in controlling for body size. Allometric exponents for BM and Ht were calculated separately for each age group of boys and girls to satisfy the common exponent and group difference principles described by Vanderburgh. Unscaled grip strength had moderate to strong positive correlations with BM and Ht (p ≤ 0.05 for all) for all age groups. Ratio-scaled handgrip strength had significant moderate to strong negative correlations with BM (p ≤ 0.05 for all) and, to a lesser extent, Ht (p ≤ 0.05 for 8- to 12-year-old boys; p ≤ 0.05 for 8- to 12- and 14-year-old girls). Correlations between allometrically scaled handgrip strength and BM and Ht were not significant and approached zero. This study was the first to allometrically scale handgrip strength for BM and Ht in Hawaiian children. Allometric scaling applied to grip strength provides a useful expression of grip strength free of the confounding influence of body size.

Perspective: I believe what I remember, but it may not be true.

A growing number of research findings have challenged the conception that memory for traumatic events is highly accurate or even indelible in nature. Research involving soldiers indicates that realistic levels of high stress decrease the accuracy of eyewitness memory. In addition, recent findings from several studies show quite clearly that memories for stressful events - including those from combat trauma - are malleable and vulnerable to alteration by exposure to misinformation. Under high stress, our brains facilitate the formation of "gist" memories that allow us to avoid future dangers but which may not contain the detail and precision demanded by the judicial system. Although mental health professionals ought to play a role in educating the courts about mental illness and trauma, it is unwise for them to become advocates for the idea that traumatic memories are indelible, factual accounts of events.

Sex differences in cardiovascular and subjective stress reactions: prospective evidence in a realistic military setting.

Evidence points to heightened physiological arousal in response to acute stress exposure as both a prospective indicator and a core characteristic of posttraumatic stress disorder (PTSD). Because females may be at higher risk for PTSD development, it is important to evaluate sex differences in acute stress reactions. This study characterized sex differences in cardiovascular and subjective stress reactions among military survival trainees. One hundred and eighty-five military members (78% males) were studied before, during, and 24 h after stressful mock captivity. Cardiovascular (heart rate [HR], systolic blood pressure [SBP], diastolic blood pressure [DBP]) and dissociative states were measured at all three time points. Psychological impact of mock captivity was assessed during recovery. General linear modeling with repeated measures evaluated sex differences for each cardiovascular endpoint, and causal steps modeling was used to explore interrelationships among sex, cardiovascular reactions and psychological impact of mock captivity. Although females had lower SBP than males at all three time points, the difference was most pronounced at baseline and during stress. Accordingly, females showed greater residual elevation in SBP during recovery. Females had lower DBP at all three time points. In addition, females reported greater psychological impact of mock captivity than males. Exploratory causal steps modeling suggested that stress-induced HR may partially mediate the effect of sex on psychological impact of mock captivity. In conclusion, this study demonstrated sex-specific cardiovascular stress reactions in military personnel, along with greater psychological impact of stress exposure in females. This research may elucidate sex differences in PTSD development.

Efficacy of combining interview techniques in detecting deception related to bio-threat issues.

The purpose of this project was to assess the detecting deception efficacy of three well-validated "detecting deception" methods - i.e., forced choice testing (FCT), modified cognitive interviewing (MCI) and autobiographical implicit association testing (aIAT) - when applied to the issue of bio-threat. The detecting deception accuracies of FCT and MCI were 81% and 75%, respectively. Although the aIAT mean response times in block 5 differed significantly between deceptive and truthful persons, the classification accuracy was low. FCT alone reduced the group of 64 persons to 11 and detected 50% of the liars; the false positive rate was 9%. MCI alone reduced the group of 64 to 24 and detected 92% of the liars; the false positive rate was 54%. When FCT was paired with MCI, 75% of liars were detected and the false positive rate was 13%. Forced choice testing and MCI show promise as methods for detecting deception related to bio-threat under low-base-rate conditions. These methods took little time, enhanced the odds of detecting deceptive individuals and exhibited high positive likelihood ratios, suggesting that they have merit as screening tools. The aIAT required more time and was less accurate but may still serve as a useful screening tool.

Spontaneous and deliberate dissociative states in military personnel: are such states helpful?

This study explored distinctions between spontaneous and deliberate dissociative states in 335 military personnel exposed to stressful survival training. Participants completed the Clinician-Administered Dissociative States Scale (CADSS) after a stressful mock-captivity event. They were also asked to indicate whether the dissociative experiences just happened (i.e., spontaneous), or whether they chose to have them happen (i.e., deliberate); and whether they appraised the dissociative experience as helpful (i.e., facilitative) or hurtful (i.e., debilitative) to their ability to cope with the stressful event. A majority (95.4%) endorsed dissociative states during stress. More than half (57.4%) described dissociative experiences as spontaneous, 13.0% as deliberate, and 29.5% endorsed neither. In Special Forces soldiers only, those who endorsed facilitative dissociation exhibited higher total CADSS scores than those who endorsed debilitative dissociation. Seventy-three percent of spontaneous dissociators described the experience as debilitative to coping with stress; conversely, 76% of deliberate dissociators said these experiences facilitated coping with stress. Individuals with prior trauma exposure tended to appraise dissociative states as more debilitative to coping. This research may enhance the fidelity of studies of dissociation constructs and may offer pivot points for prevention and treatment of stress-related disorders.

Encoding Noncanonical Amino Acids into Phage Displayed Proteins.

Phage display facilitates the evolution of peptides and proteins for affinity selection against targets, but it is mostly limited to the chemical diversity provided by the naturally encoded amino acids. The combination of phage display with genetic code expansion allows the incorporation of noncanonical amino acids (ncAAs) into proteins expressed on the phage. In this method, we describe incorporation of one or two ncAAs in a single-chain fragment variable (scFv) antibody in response to amber or quadruplet codon. We take advantage of the pyrrolysyl-tRNA synthetase/tRNA pair to incorporate a lysine derivative and an orthogonal tyrosyl-tRNA synthetase/tRNA pair to incorporate a phenylalanine derivative. The encoding of novel chemical functionalities and building blocks in proteins displayed on phage provides the foundation for further phage display applications in fields such as imaging, protein targeting, and the production of new materials.

Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells.

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming, however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 reactivation increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71’s RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.

CMC Regulatory Considerations for Antibody-Drug Conjugates.

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included.

The Benefits of School Recess: A Systematic Review.

BACKGROUND: Recess provides many physical, mental, and social benefits for students; however, the most recent systematic review examining the benefits of recess was conducted over a decade ago. The purpose of this paper was to determine the current benefits of school recess by conducting an updated systematic review of the literature. METHODS: Multiple databases were systematically examined to find articles fitting the following inclusionary criteria: (a) school recess, (b) all schooling before college, and (c) recess benefits of any kind. Research was limited to literature published between June 2009 and July 2020. RESULTS: Nine studies were included in this review. The majority of studies examined elementary-aged students. Seven studies were conducted in the United States, and 2 studies were conducted internationally. Recess provided school-aged children academic and cognitive benefits, behavioral and emotional benefits, physical benefits, and social benefits. IMPLICATIONS FOR SCHOOL HEALTH POLICY, PRACTICE, AND EQUITY: Providing recess for all school levels positively impacts the whole child; there was no literature implicating negative impacts of recess. CONCLUSIONS: Schools can improve overall student health and belonging by redesigning and/or implementing daily recess.

Follistatin promotes LIN28B-mediated supporting cell reprogramming and hair cell regeneration in the murine cochlea.

Hair cell (HC) loss within the inner ear cochlea is a leading cause for deafness in humans. Before the onset of hearing, immature supporting cells (SCs) in neonatal mice have some limited capacity for HC regeneration. Here, we show that in organoid culture, transient activation of the progenitor-specific RNA binding protein LIN28B and Activin antagonist follistatin (FST) enhances regenerative competence of maturing/mature cochlear SCs by reprogramming them into progenitor-like cells. Transcriptome profiling and mechanistic studies reveal that LIN28B drives SC reprogramming, while FST is required to counterbalance hyperactivation of transforming growth factor-ß-type signaling by LIN28B. Last, we show that LIN28B and FST coactivation enhances spontaneous cochlear HC regeneration in neonatal mice and that LIN28B may be part of an endogenous repair mechanism that primes SCs for HC regeneration. These findings indicate that SC dedifferentiation is critical for HC regeneration and identify LIN28B and FST as main regulators.

Baseline burnout symptoms predict visuospatial executive function during survival school training in special operations military personnel.

Burnout symptoms, which are characterized by exhaustion, cynicism, and a reduced sense of professional efficacy, may deleteriously affect cognitive function in military personnel. A total of 32 U.S. Military Special Operations personnel enrolled in Survival School completed measures of trauma history, dissociation, and burnout before training. They then completed the Groton Maze Learning Test (GMLT), a neuropsychological measure of integrative visuospatial executive function during three field-based phases of Survival School-enemy evasion, captivity/interrogation, and escape/release from captivity. Lower pre-training perceptions of professional efficacy were associated with reduced executive function during all of the field-based phases of Survival School, even after adjustment for years of education, cynicism, and baseline GMLT scores. Magnitudes of decrements in executive function in Marines with low efficacy relative to those with high efficacy increased as training progressed and ranged from .58 during enemy evasion to .99 during escape/release from captivity. Pre-training perceptions of burnout may predict visuospatial executive function during naturalistic training-related stress in military personnel. Assessment of burnout symptoms, particularly perceptions of professional efficacy, may help identify military personnel at risk for stress-related executive dysfunction.

Effects of preload 4 repetition maximum on 100-m sprint times in collegiate women.

The purpose of this study was to determine the effects of postactivation potentiation (PAP) on track-sprint performance after a preload set of 4 repetition maximum (4RM) parallel back half-squat exercises in collegiate women. All subjects (n = 12) participated in 2 testing sessions over a 3-week period. During the first testing session, subjects performed the Controlled protocol consisting of a 4-minute standardized warm-up, followed by a 4-minute active rest, a 100-m track sprint, a second 4-minute active rest, finalized with a second 100-m sprint. The second testing session, the Treatment protocol, consisted of a 4-minute standardized warm-up, followed by 4-minute active rest, sprint, a second 4-minute active rest, a warm-up of 4RM parallel back half-squat, a third 9-minute active rest, finalized with a second sprint. The results indicated that there was a significant improvement of 0.19 seconds (p < 0.05), when the second sprint was preceded by a 4RM back-squat protocol during Treatment. The standardized effect size, d, was 0.82, indicating a large effect size. Additionally, the results indicated that it would be expected that mean sprint times would increase 0.04-0.34 seconds (p < 0.05), when using a preload 4RM squat protocol. There were no significant differences between Control pre and posttests (p > 0.05). The findings suggest that performing a 4RM parallel back half-squat warm-up before a track sprint will have a positive PAP affect on decreased track-sprint times. Track coaches, looking for the "competitive edge" (PAP effect) may re-warm up their sprinters during meets.