The polySUMOylation axis promotes nucleolar release of Tof2 for mitotic exit.

In budding yeast, the nucleolus serves as the site to sequester Cdc14, a phosphatase essential for mitotic exit. Nucleolar proteins Tof2, Net1, and Fob1 are required for this sequestration. Although it is known that these nucleolar proteins are SUMOylated, how SUMOylation regulates their activity remains unknown. Here, we show that Tof2 exhibits cell-cycle-regulated nucleolar delocalization and turnover. Depletion of the nuclear small ubiquitin-like modifier (SUMO) protease Ulp2 not only causes Tof2 polySUMOylation, nucleolar delocalization, and degradation but also leads to Cdc14 nucleolar release and activation. This outcome depends on polySUMOylation and the activity of downstream enzymes, including SUMO-targeted ubiquitin ligase and Cdc48/p97 segregase. We further developed a system to tether SUMO machinery to Tof2 and generated a SUMO-deficient tof2 mutant, and the results indicate that Tof2 polySUMOylation is necessary and sufficient for its nucleolar delocalization and degradation. Together, our work reveals a polySUMO-dependent mechanism that delocalizes Tof2 from the nucleolus to facilitate mitotic exit.

Large Particle Emissions from Human Vocalization and Playing of Wind Instruments.

Humans emit large salivary particles when talking, singing, and playing musical instruments, which have implications for respiratory disease transmission. Yet little work has been done to characterize the emission rates and size distributions of such particles. This work characterized large particle (dp > 35 µm in aerodynamic diameter) emissions from 70 volunteers of varying age and sex while vocalizing and playing wind instruments. Mitigation efficacies for face masks (while singing) and bell covers (while playing instruments) were also examined. Geometric mean particle count emission rates varied from 3.8 min-1 (geometric standard deviation [GSD] = 3.1) for brass instruments playing to 95.1 min-1 (GSD = 3.8) for talking. On average, talking produced the highest emission rates for large particles, in terms of both number and mass, followed by singing and then instrument playing. Neither age, sex, CO2 emissions, nor loudness (average dBA) were significant predictors of large particle emissions, contrary to previous findings for smaller particle sizes (i.e., for dp < 35 µm). Size distributions were similar between talking and singing (count median diameter = 53.0 µm, GSD = 1.69). Bell covers did not affect large particle emissions from most wind instruments, but face masks reduced large particle count emissions for singing by 92.5% (95% CI: 97.9%, 73.7%).

A new layer of regulation of chromosomal passenger complex (CPC) translocation in budding yeast.

The conserved chromosomal passenger complex (CPC) consists of Ipl1Aurora-B, Sli15INCENP, Bir1Survivin, and Nbl1Borealin, and localizes at the kinetochore/centromere to correct kinetochore attachment errors and to prevent checkpoint silencing. After anaphase entry, the CPC moves from the kinetochore/centromere to the spindle. In budding yeast, CPC subunit Sli15 is phosphorylated by both cyclin-dependent kinase (CDK) and Ipl1 kinase. Following anaphase onset, activated Cdc14 phosphatase reverses Sli15 phosphorylation imposed by CDK to promote CPC translocation. Although abolished Sli15 phosphorylation imposed by Ipl1 also causes CPC translocation, the regulation of Ipl1-imposed Sli15 phosphorylation remains unclear. In addition to Sli15, Cdc14 also dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. Here, we present evidence supporting the notion that kinetochore-localized Fin1-PP1 likely reverses Ipl1-imposed Sli15 phosphorylation to promote CPC translocation from the kinetochore/centromere to the spindle. Importantly, premature Fin1 kinetochore localization or phospho-deficient sli15 mutation causes checkpoint defects in response to tensionless attachments, resulting in chromosome missegregation. In addition, our data indicate that reversion of CDK- and Ipl1-imposed Sli15 phosphorylation shows an additive effect on CPC translocation. Together, these results reveal a previously unidentified pathway to regulate CPC translocation, which is important for accurate chromosome segregation.

Farm animal careers and perception of 'fit' in undergraduate veterinary students: A mixed methods study.

BACKGROUND: Recruitment and retention of farm veterinarians have been the focus of recent research. Previous work suggests that a feeling of 'fit' is important for students to consider a farm career. The aim of this study was to identify whether students feel that they 'fit' in farm practice and reasons for their answer. METHODS: An online survey was distributed to students at all British and Irish veterinary schools. A mixed methods approach was considered, with thematic analysis on free text answers and regression analysis on demographic variables. RESULTS: Thematic analysis identified six themes: career opportunities, nature of farm veterinary work, relationships and interactions, individual experiences, expectations and perceptions, and no perceived barriers. Females, marginalised ethnic groups and those from an urban/suburban background were all identified as having significantly (p < 0.05) less agreement with the statement 'I feel able to pursue a career in farm practice'. LIMITATIONS: Survey limitations include those with a clear bias being likely to respond. However, alignment of the qualitative and quantitative results increased confidence in the findings of this mixed methods approach. CONCLUSION: This study confirms that biases that exist within wider society do have an influence on veterinary undergraduates' intentions to pursue a farm animal career. This is vital to consider both at a university level and when considering students' experiences on placements. Urgent action is required to improve inclusivity in the farm animal veterinary sector.

Aerosol emissions from wind instruments: effects of performer age, sex, sound pressure level, and bell covers.

Aerosol emissions from wind instruments are a suspected route of transmission for airborne infectious diseases, such as SARS-CoV-2. We evaluated aerosol number emissions (from 0.25 to 35.15 µm) from 81 volunteer performers of both sexes and varied age (12 to 63 years) while playing wind instruments (bassoon, clarinet, flute, French horn, oboe, piccolo, saxophone, trombone, trumpet, and tuba) or singing. Measured emissions spanned more than two orders of magnitude, ranging in rate from < 8 to 1,815 particles s-1, with brass instruments, on average, producing 191% (95% CI 81-367%) more aerosol than woodwinds. Being male was associated with a 70% increase in emissions (vs. female; 95% CI 9-166%). Each 1 dBA increase in sound pressure level was associated with a 28% increase (95% CI 10-40%) in emissions from brass instruments; sound pressure level was not associated with woodwind emissions. Age was not a significant predictor of emissions. The use of bell covers reduced aerosol emissions from three brass instruments tested (trombone, tuba, and trumpet), with average reductions ranging from 53 to 73%, but not for the two woodwind instruments tested (oboe and clarinet). Results from this work can facilitate infectious disease risk management for the performing arts.

Structural Basis and Kinetic Pathway of RBM39 Recruitment to DCAF15 by a Sulfonamide Molecular Glue E7820.

E7820 and indisulam are two examples of aryl sulfonamides that recruit RBM39 to Rbx-Cul4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. To understand their mechanism of action, we performed kinetic analysis on the recruitment of RBM39 to DCAF15 and solved a crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39. E7820 packs in a shallow pocket on the surface of DCAF15 and the resulting modified interface binds RBM39 through the α1 helix of the RRM2 domain. Our kinetic studies revealed that aryl sulfonamide and RBM39 bind to DCAF15 in a synergistic manner. The structural and kinetic studies confirm aryl sulfonamides as molecular glues in the recruitment of RBM39 and provide a framework for future efforts to utilize DCAF15 to degrade other proteins of interest.

Purpose and coping with adversity: A repeated measures, mixed-methods study with young adolescents.

INTRODUCTION: Purpose in life is associated with positive outcomes following adversity, but the mechanisms of this relationship are unclear. In this repeated measures, mixed-methods study, we examined the relationship of purpose to coping with adversity among young adolescents. METHODS: A sample of 1357 adolescents completed a survey about purpose, negative life events, and positive reframing coping four times at six month intervals. A sub-sample of 91 survey respondents participated in an interview. Surveys were analyzed using fixed-effects and mixed-effects modeling to test the relationship between purpose and coping over time. Interviews were analyzed for purpose, adversity, and coping to understand this relationship as perceived by the participant. RESULTS: Survey results indicate that purpose and positive reframing coping are related and change together over time. Interview results suggest that purpose can be a response to negative experiences. CONCLUSIONS: There are likely underlying factors or processes driving the relationship between purpose and positive reframing coping. Some of the potential underlying factors and processes are discussed.

Transcriptional Circuit Fragility Influences HIV Proviral Fate.

Transcriptional circuit architectures in several organisms have been evolutionarily selected to dictate precise given responses. Unlike these cellular systems, HIV is regulated through a complex circuit composed of two successive phases (host and viral), which create a positive feedback loop facilitating viral replication. However, it has long remained unclear whether both phases operate identically and to what extent the host phase influences the entire circuit. Here, we report that, although the host phase is regulated by a checkpoint whereby KAP1 mediates transcription activation, the virus evolved a minimalist system bypassing KAP1. Given the complex circuit's architecture, cell-to-cell KAP1 fluctuations impart heterogeneity in the host transcriptional responses, thus affecting the feedback loop. Mathematical modeling of a complete circuit reveals how these oscillations ultimately influence homogeneous reactivation potential of a latent virus. Thus, although HIV drives molecular innovation to fuel robust gene activation, it experiences transcriptional fragility, thereby influencing viral fate and cure efforts.

Design and Activity of Specific Hypoxia-Inducible Factor-2α (HIF-2α) Inhibitors for the Treatment of Clear Cell Renal Cell Carcinoma: Discovery of Clinical Candidate ( S)-3-((2,2-Difluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1 H-inden-4-yl)oxy)-5-fluorobenzonitrile (PT2385).

HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel-Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π*Ar interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.

PPM1G Binds 7SK RNA and Hexim1 To Block P-TEFb Assembly into the 7SK snRNP and Sustain Transcription Elongation.

Transcription elongation programs are vital for the precise regulation of several biological processes. One key regulator of such programs is the P-TEFb kinase, which phosphorylates RNA polymerase II (Pol II) once released from the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. Although mechanisms of P-TEFb release from the snRNP are becoming clearer, how P-TEFb remains in the 7SK-unbound state to sustain transcription elongation programs remains unknown. Here we report that the PPM1G phosphatase (inducibly recruited by nuclear factor κB [NF-κB] to target promoters) directly binds 7SK RNA and the kinase inhibitor Hexim1 once P-TEFb has been released from the 7SK snRNP. This dual binding activity of PPM1G blocks P-TEFb reassembly onto the snRNP to sustain NF-κB-mediated Pol II transcription in response to DNA damage. Notably, the PPM1G-7SK RNA interaction is direct, kinetically follows the recruitment of PPM1G to promoters to activate NF-κB transcription, and is reversible, since the complex disassembles before resolution of the program. Strikingly, we found that the ataxia telangiectasia mutated (ATM) kinase regulates the interaction between PPM1G and the 7SK snRNP through site-specific PPM1G phosphorylation. The precise and temporally regulated interaction of a cellular enzyme and a noncoding RNA provides a new paradigm for simultaneously controlling the activation and maintenance of inducible transcription elongation programs.

Extracting changes in air temperature using acoustic coda phase delays.

Blast waves produced by 60 high-explosive detonations were recorded at short distances (few hundreds of meters); the corresponding waveforms show charge-configuration independent coda-like features (i.e., similar shapes, amplitudes, and phases) lasting several seconds. These features are modeled as reflected and/or scattered waves by acoustic reflectors/scatters surrounding the explosions. Using explosion pairs, relative coda phase delays are extracted and modeled as changes in sound speed due to changes in air temperature. Measurements from nearby weather towers are used for validation.

Current management of tibial shaft fractures: a survey of 450 Canadian orthopedic trauma surgeons.

BACKGROUND AND PURPOSE: Strategies to manage tibial fractures include nonoperative and operative approaches. Strategies to enhance healing include a variety of bone stimulators. It is not known what forms of management for tibial fractures predominate among Canadian orthopedic surgeons. We therefore asked a representative sample of orthopedic trauma surgeons about their management of tibial fracture patients. METHODS: This was a cross-sectional survey of 450 Canadian orthopedic trauma surgeons. We inquired about demographic variables and current tibial shaft fracture management strategies. RESULTS: 268 surgeons completed the survey, a response rate of 60%. Most respondents (80%) managed closed tibial shaft fracture operatively; 47% preferred reamed intramedullary nailing and 40% preferred unreamed. For open tibial shaft fractures, 59% of surgeons preferred reamed intramedullary nailing. Some surgeons (16%) reported use of bone stimulators for management of uncomplicated open and closed tibial shaft fractures, and almost half (45%) made use of this adjunctive modality for complicated tibial shaft fractures. Low-intensity pulsed ultrasound and electrical stimulation proved equally popular (21% each) and 80% of respondents felt that a reduction in healing time of 6 weeks or more, attributed to a bone stimulator, would be clinically important. INTERPRETATION: Current practice regarding orthopedic management of tibial shaft fractures in Canada strongly favors operative treatment with intramedullary nailing, although respondents were divided in their preference for reamed and unreamed nailing. Use of bone stimulators is common as an adjunctive modality in this injury population. Large randomized trials are needed to provide better evidence to guide clinical decision making regarding the choice of reamed or unreamed nailing for tibial shaft fractures, and to inform surgeons about the actual effect of bone stimulators.

Musculoskeletal injuries and pain in dancers: a systematic review.

OBJECTIVE: To assemble and synthesize the best evidence on the epidemiology, diagnosis, prognosis, treatment, and prevention of musculoskeletal injuries and pain in dancers. DATA SOURCES: Medline, CINAHL, PsycINFO, Embase, and other electronic databases were searched from 1966 to 2004 using key words such as dance, dancer, dancing, athletic injuries, occupational injuries, sprains and strains, and musculoskeletal diseases. In addition, the reference lists of relevant studies were examined, specialized journals were hand-searched, and the websites of major dance associations were scanned for relevant information. STUDY SELECTION: Citations were screened for relevance using a priori criteria, and relevant studies were critically reviewed for scientific merit by the best evidence synthesis method. After 1865 abstracts were screened, 103 articles were reviewed, and 32 (31%) of these were accepted as scientifically admissible (representing 29 unique studies). DATA EXTRACTION: Data from accepted studies were abstracted into evidence tables relating to the prevalence and associated factors, incidence and risk factors, diagnosis, treatment, economic costs, and prevention of musculoskeletal injuries and pain in dancers. DATA SYNTHESIS: The scientifically admissible studies consisted of 15 (52%) cohort studies, 13 (45%) cross-sectional studies, and 1 (3%) validation study of a diagnostic assessment tool. There is a high prevalence and incidence of lower extremity and back injuries, with soft tissue and overuse injuries predominating. For example, lifetime prevalence estimates for injury in professional ballet dancers ranged between 40% and 84%, while the point prevalence of minor injury in a diverse group of university and professional ballet and modern dancers was 74%. Several potential risk factors for injury are suggested by the literature, but conclusive evidence for any of these is lacking. There is preliminary evidence that comprehensive injury prevention and management strategies may help decrease the incidence of future injury. CONCLUSIONS: The dance medicine literature is young and heterogeneous, limiting our ability to draw consistent conclusions. Nonetheless, the best available evidence suggests that musculoskeletal injury is an important health issue for dancers at all skill levels. Better quality research is needed in this specialized area. Future research would benefit from clear and relevant research questions being addressed with appropriate study designs, use of conceptually valid and clinically meaningful case definitions of injury and pain, and better reporting of studies in line with current scientific standards.