The Deubiquitinase OTULIN Is an Essential Negative Regulator of Inflammation and Autoimmunity.

Methionine-1 (M1)-linked ubiquitin chains regulate the activity of NF-κB, immune homeostasis, and responses to infection. The importance of negative regulators of M1-linked chains in vivo remains poorly understood. Here, we show that the M1-specific deubiquitinase OTULIN is essential for preventing TNF-associated systemic inflammation in humans and mice. A homozygous hypomorphic mutation in human OTULIN causes a potentially fatal autoinflammatory condition termed OTULIN-related autoinflammatory syndrome (ORAS). Four independent OTULIN mouse models reveal that OTULIN deficiency in immune cells results in cell-type-specific effects, ranging from over-production of inflammatory cytokines and autoimmunity due to accumulation of M1-linked polyubiquitin and spontaneous NF-κB activation in myeloid cells to downregulation of M1-polyubiquitin signaling by degradation of LUBAC in B and T cells. Remarkably, treatment with anti-TNF neutralizing antibodies ameliorates inflammation in ORAS patients and rescues mouse phenotypes. Hence, OTULIN is critical for restraining life-threatening spontaneous inflammation and maintaining immune homeostasis.

Dual proteolytic pathways govern glycolysis and immune competence.

Proteasomes and lysosomes constitute the major cellular systems that catabolize proteins to recycle free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPPII) is a large cytosolic proteolytic complex that functions in tandem with the proteasome-ubiquitin protein degradation pathway. We found that autosomal recessive TPP2 mutations cause recurrent infections, autoimmunity, and neurodevelopmental delay in humans. We show that a major function of TPPII in mammalian cells is to maintain amino acid levels and that TPPII-deficient cells compensate by increasing lysosome number and proteolytic activity. However, the overabundant lysosomes derange cellular metabolism by consuming the key glycolytic enzyme hexokinase-2 through chaperone-mediated autophagy. This reduces glycolysis and impairs the production of effector cytokines, including IFN-γ and IL-1ß. Thus, TPPII controls the balance between intracellular amino acid availability, lysosome number, and glycolysis, which is vital for adaptive and innate immunity and neurodevelopmental health.

Waveguide-coupled single collective excitation of atomic arrays.

Considerable efforts have been recently devoted to combining ultracold atoms and nanophotonic devices1-4 to obtain not only better scalability and figures of merit than in free-space implementations, but also new paradigms for atom-photon interactions5. Dielectric waveguides offer a promising platform for such integration because they enable tight transverse confinement of the propagating light, strong photon-atom coupling in single-pass configurations and potentially long-range atom-atom interactions mediated by the guided photons. However, the preparation of non-classical quantum states in such atom-waveguide interfaces has not yet been realized. Here, by using arrays of individual caesium atoms trapped along an optical nanofibre6,7, we observe a single collective atomic excitation8,9 coupled to a nanoscale waveguide. The stored collective entangled state can be efficiently read out with an external laser pulse, leading to on-demand emission of a single photon into the guided mode. We characterize the emitted single photon via the suppression of the two-photon component and confirm the single character of the atomic excitation, which can be retrieved with an efficiency of about 25%. Our results demonstrate a capability that is essential for the emerging field of waveguide quantum electrodynamics, with applications to quantum networking, quantum nonlinear optics and quantum many-body physics10,11.

Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function.

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

Reliability of Measures of Lower-Body Strength and Speed in Academy Male Adolescent Soccer Players.

ABSTRACT: Ferguson, J, Gibson, NV, Weston, M, and McCunn, R. Reliability of measures of lower body strength and speed in academy male adolescent soccer players. J Strength Cond Res 38(3): e96-e103, 2024-The Nordbord and ForceFrame represent a practical and time efficient means of assessing eccentric hamstring and isometric adductor strength in the large number of squads and players associated with youth soccer academies, yet measurement reliability in this population is unexamined. Therefore, over a period of 4 days, with no less than 24 hours and no more than 48 hours between trials, 37 players (age: 14.7 ± 0.8 years, stature: 168.7 ± 7.8 cm, mass: 57.7 ± 9.1 kg, and maturity offset: 0.8 ± 0.9 years) were assessed for eccentric hamstring strength (force, torque), isometric adductor strength (long and short lever positions), and 30-m sprint (5, 10, and 20-m splits), using the Nordbord, ForceFrame, and electronic timing gates, respectively, on 3 separate occasions. Relative reliability (intraclass correlation coefficient) was rated as good for all Nordbord (range: 0.86-0.89) and ForceFrame (0.78-0.85) measures and ranged from moderate (0.53) to excellent (0.93) for the speed measures, improving with increased distance. Absolute reliability (standard error of the measurement [%SEM]) ranged from 7 to 8% (Nordbord), 3 to 11% (ForceFrame), and 1 to 4% (sprints). Our data provide the first Nordbord and ForceFrame reliability estimates in adolescent soccer academy players. To interpret test sensitivity, practitioners are encouraged to interpret our estimates of absolute reliability against meaningful change values derived from personal experience and evidence-based knowledge and not against absolute or standardized thresholds.

The impact of asymptomatic human immunodeficiency virus (HIV)-positive disease status on inpatient complications following total joint arthroplasty: a propensity score-matched analysis.

PURPOSE: The number of patients with asymptomatic human immunodeficiency virus (AHIV) is increasing as the efficacy of antiretroviral therapy improves. While there is research on operative risks associated with having HIV, there is a lack of literature describing the impact of well-controlled HIV on postoperative complications. This study seeks to elucidate the impact of AHIV on postoperative outcomes after total hip (THA) and knee (TKA) arthroplasty. METHODS: The Nationwide Inpatient Sample was retrospectively reviewed for patients undergoing TKA and THA from 2005 to 2013. Subjects were subdivided into those with AHIV and those without HIV (non-HIV). Patient demographics, hospital-related parameters, and postoperative complications were all collected. One-to-one propensity score-matching, Chi-square analysis, and multivariate logistical regressions were performed to compare both cohorts. RESULTS: There were no significant differences between AHIV and non-HIV patients undergoing TKA or THA in terms of sex, age, insurance status, or total costs (all, p ≥ 0.081). AHIV patients had longer lengths of stay (4.0 days) than non-HIV patients after both TKA (3.3 days) and THA (3.1 days) (p ≤ 0.011). Both TKA groups had similar postoperative complication rates (p > 0.081). AHIV patients undergoing THA exhibited an increased rate of overall surgical complications compared non-HIV patients (0 vs. 4.5%, p = 0.043). AHIV was not associated with increased complications following both procedures. CONCLUSION: Despite lengthier hospital stays among AHIV patients, baseline AHIV was not associated with adverse outcomes following TKA and THA. This adds to the literature and warrants further research into the impact of asymptomatic, well-controlled HIV infection on postoperative outcomes following total joint arthroplasty.

Gating kinetics and pharmacological properties of small-conductance Ca2+-activated potassium channels.

Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τc,vl = 11.8 ± 7.1 s, and AP14145: τc,vl = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τc,s = 3.50 ± 0.81 ms, and τc,i = 32.0 ± 6.76 ms versus control: τc,s = 1.59 ± 0.19 ms, and τc,i = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τc,s = 0.75 ± 0.10 ms and τc,i = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: Ahigh = 0.76 ± 0.03 pA, and Alow = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca2+-dependent channel activation in vivo.

Direct Evidence of a Light-Dependent Sink of Superoxide within Chromophoric Dissolved Organic Matter.

Superoxide (O2• -) is produced photochemically in natural waters by chromophoric dissolved organic matter (CDOM) via the reaction of molecular oxygen with photoproduced one-electron reductants (OERs) within CDOM. In the absence of other sinks (metals or organic radicals), O2• - is believed to undergo primarily dismutation to produce hydrogen peroxide (H2O2). However, past studies have implicated the presence of an additional light-dependent sink of O2• - that does not lead to H2O2 production. Here, we provide direct evidence of this sink through O2• - injection experiments. During irradiations, spikes of O2• - are consumed to a greater extent (∼85-30% loss) and are lost much faster (up to ∼0.09 s-1) than spikes introduced post-irradiation (∼50-0% loss and ∼0.03 s-1 rate constant). The magnitude of the loss during irradiation and the rate constant are wavelength-dependent. Analysis of the H2O2 concentration post-spike indicates that this light-dependent sink does not produce H2O2 at low spike concentrations. This work further demonstrates that simply assuming that the O2• - production is twice the H2O2 production is not accurate, as previously believed.

Inherited ADAMTS13 mutations associated with Thrombotic Thrombocytopenic Purpura: a short review and update.

ADAMTS13 is a plasma metalloprotease with the primary function of cleaving VWF to maintain hemostasis. Circulating ADAMTS13 is in the closed conformation until blood vessel injury triggers a VWF-dependant activation to the open active form of the protein. ADAMTS13 is a multi-domain protein with the domains broadly functioning to interact and cleave VWF or maintain global latency of ADAMTS13. Thrombotic Thrombocytopenic Purpura is a disease characterized by excessive thrombi formation in the microvasculature, diagnosis is made when ADAMTS13 activity is <10%. In the hereditary form, a variety of mutations are found throughout all domains of ADAMTS13, examples are given alongside details of each domain in this article. ADAMTS13 mutations can inhibit the binding and cleavage of VWF directly or indirectly through reduced secretion, leading to increased size of VWF multimers and platelet recruitment. Molecular characterization of ADAMTS13 may provide insight into the mechanisms of TTP to aid in both scientific and clinical research.

Surgical considerations for spinal epidural hematoma evacuation in the setting of blue rubber bleb nevus syndrome in a child.

Blue rubber bleb nevus syndrome (BRBNS) is a rare condition that presents with venous malformation blebs throughout the body, most commonly on the skin and gastrointestinal tract. There have only been a limited number of reports of benign BRBNS lesions involving the spine in children, which were detected after chronic symptomatology. We herein present a unique case of a ruptured BRBNS venous malformation into the epidural space of the lumbar spine in a child presenting with acute neurologic deficit and discuss the relevant surgical considerations for operating in the setting of BRBNS.

Perioperative management of acetabular and pelvic fractures: evidence-based recommendations.

PURPOSE: The American Academy of Orthopaedic Surgeons does not currently provide clinical practice guidelines for management of PAF. Accordingly, this article aims to review and consolidate the relevant historical and recent literature in important topics pertaining to perioperative management of PAF. METHODS: A thorough literature review using PubMed, Cochrane and Embase databases was performed to assess preoperative, intraoperative and postoperative management of PAF fracture. Topics reviewed included: time from injury to definitive fixation, the role of inferior vena cava filters (IVCF), tranexamic acid (TXA) use, intraopoperative cell salvage, incisional negative pressure wound therapy (NPWT), intraoperative antibiotic powder use, heterotopic ossification prophylaxis, and pre- and postoperative venous thromboembolism (VTE) prophylaxis. RESULTS: A total of 126 articles pertaining to the preoperative, intraoperative and postoperative management of PAF were reviewed. Articles reviewed by topic include 13 articles pertaining to time to fixation, 23 on IVCF use, 14 on VTE prophylaxis, 20 on TXA use, 10 on cell salvage, 10 on iNPWT 14 on intraoperative antibiotic powder and 20 on HO prophylaxis. An additional eight articles were reviewed to describe background information. Five articles provided information for two or more treatment modalities and were therefore included in multiple categories when tabulating the number of articles reviewed per topic. CONCLUSION: The literature supports the use of radiation therapy for HO prophylaxis, early (< 5 days from injury) surgical intervention and the routine use of intraoperative TXA. The literature does not support the routine use of iNPWT or IVCF. There is inadequate information to make a recommendation regarding the use of cell salvage and wound infiltration with antibiotic powder. While the routine use of chemical VTE prophylaxis is recommended, there is insufficient evidence to recommend the optimal agent and duration of therapy.

The impact of surgical trainee involvement in total knee arthroplasty: a systematic review of surgical efficacy, patient safety, and outcomes.

PURPOSE: Trainee involvement in patient care has raised concerns about the potential risk of adverse outcomes and harming patients. We sought to analyze the impact and potential consequence of surgical trainee involvement in total knee arthroplasty (TKA) procedures in terms of surgical efficacy, patient safety, and functional outcomes. METHODS: We systematically reviewed Medline/PubMed, EMBASE, the Cochrane library, and Scopus databases in April 2021. Eligible studies reported on the impact of trainee participation in TKA procedures performed with and without such involvement. RESULTS: Twenty-three publications met our eligibility criteria and were included in our study. These studies reported on 132,624 surgeries completed on 132,416 patients. Specifically, 23,988 and 108,636 TKAs were performed with and without trainee involvement, respectively. The mean operative times for procedures with (n = 19,573) and without (n = 94,581) trainee involvement were 99.77 and 85.05 min, respectively. Both studies that reported data on cost of TKAs indicated a significant increase (p < 0.001) associated with procedures completed by teaching hospitals compared to private practices. Mean overall complication rates were 7.20% and 7.36% for TKAs performed with (n = 9,386) and without (n = 31,406) trainees. Lastly, the mean Knee Society Scale (KSS) knee scores for TKAs with (n = 478) and without (n = 806) trainee involvement were similar; 82.81 and 82.71, respectively. CONCLUSION: Our systematic review concurred with previous studies that reported trainee involvement during TKAs increases the mean operative time. However, the overall complication rates and functional outcomes were similar. Larger studies with a better methodology and higher level of evidence are still needed for a resolute conclusion.

The impact of surgical trainee involvement in total hip arthroplasty: a systematic review of surgical efficacy, patient safety, and outcomes.

PURPOSE: Concerns persist that trainee participation in surgical procedures may compromise patient care and potentiate adverse events and costs. We aimed to analyse the potential impact and consequences of surgical trainee involvement in total hip arthroplasty (THA) procedures in terms of surgical efficacy, patient safety, and functional outcomes. METHODS: We systematically reviewed Medline/PubMed, EMBASE, the Cochrane library, and Scopus databases in October 2021. Eligible studies reported a direct comparison between THA cases performed with and without trainee involvement. RESULTS: Eighteen publications met our eligibility criteria and were included in our study. The included studies reported on 142,450 THAs completed on 142,417 patients. Specifically, 48,155 and 94,295 surgeries were completed with and without trainee involvement, respectively. The mean operative times for procedures with (n = 5,662) and without (n = 14,763) trainee involvement were 106.20 and 91.41 min, respectively. Mean overall complication rates were 6.43% and 5.93% for THAs performed with (n = 4842) and without (n = 12,731) trainees. Lastly, the mean Harris Hip Scores (HHS) for THAs performed with (n = 442) and without (n = 750) trainee participation were 89.61 and 86.97, respectively. CONCLUSION: Our systematic review confirmed previous studies' reports of increased operative time for THA cases with trainee involvement. However, based on the overall similar complication rates and functional hip scores obtained, patients should be reassured concerning the relative safety of trainee involvement in THA. Future prospective studies with higher levels of evidence are still needed to reinforce the existing evidence.

A call to "own the bone": osteoporosis is a predictor for adverse two-year outcomes following total hip and knee arthroplasty.

PURPOSE: While bone health is instrumental in orthopedic surgery, few studies have described the long-term outcomes of osteoporosis (OP) in patients undergoing total hip (THA) or knee (TKA) arthroplasties. METHODS: Using the New York State statewide planning and research cooperative system database, all patients who underwent primary TKA or THA for osteoarthritis from 2009 to 2011 with minimum 2-year follow-up were identified. They were divided based on their OP status (OP and non-OP) and 1:1 propensity score matched for age, sex, race, and Charlson/Deyo index. Cohorts were compared for demographics, hospital-related parameters, and 2-year postoperative complications and reoperations. Multivariate binary logistic regression was utilized to identify significant independent associations with 2-year medical and surgical complications and revisions. RESULTS: A total of 11,288 TKA and 8248 THA patients were identified. OP and non-OP TKA patients incurred comparable overall hospital charges for their surgical visit and hospital length of stay (LOS) (both, p ≥ 0.125). Though OP and non-OP THA patients incurred similar mean hospital charges for their surgical visit, they experienced longer hospital LOS (4.3 vs. 4.1 days, p = 0.035). For both TKA and THA, OP patients had higher rates of overall and individual medical and surgical complications (all, p < 0.05). OP was independently associated with the 2-year occurrence of any overall, surgical, and medical complications, and any revision in TKA and THA patients (all, OR ≥ 1.42, p < 0.001). CONCLUSION: Our study found OP was associated with a greater risk of 2-year adverse outcomes following TKA or THA, including medical, surgical, and overall complications as well as revision operations compared to non-OP patients.

Electrochemical Metal Recycling: Recovery of Palladium from Solution and In Situ Fabrication of Palladium-Carbon Catalysts via Impact Electrochemistry.

Recycling of critical materials, regeneration of waste, and responsible catalyst manufacture have been repeatedly documented as essential for a sustainable future with respect to the environment and energy production. Electrochemical methods have become increasingly recognized as capable of achieving these goals, and "impact" electrochemistry, with the advantages associated with dynamic nanoelectrodes, has recently emerged as a prime candidate for the recovery of metals from solution. In this report, the nanoimpact technique is used to generate carbon-supported palladium catalysts from low-concentration palladium(II) chloride solutions (i.e., a waste stream mimic) as a proof of concept. Subsequently, the catalytic properties of this material in both synthesis (Suzuki coupling reaction) and electrocatalysis (hydrogen evolution) are demonstrated. Transient reductive impact signals are shown and analyzed at potentials negative of +0.4 V (vs SCE) corresponding to the onset of palladium deposition in traditional voltammetry. Direct evidence of Pd modification was obtained through characterization by environmental scanning electron microscopy/energy-dispersive X-ray spectroscopy, inductively coupled plasma mass spectrometry, X-ray photoelectron spectroscopy, transmission electron microscopy, and thermogravimetric analysis of impacted particles. This showed the formation of deposits of Pd0 partially covering the 50 nm carbon black particles with approximately 14% Pd (wt %) under the conditions used. This material was then used to demonstrate the conversion of iodobenzene into its biphenyl product (confirmed through nuclear magnetic resonance) and the successful production of hydrogen as an electrocatalyst under acidic conditions (under cyclic voltammetry).

Germline TET2 loss of function causes childhood immunodeficiency and lymphoma.

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Post-translational polymodification of ß1-tubulin regulates motor protein localisation in platelet production and function.

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which ß1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Relatively rapid effects of testosterone on men's ratings of female attractiveness depend on relationship status and the attractiveness of stimulus faces.

Attractiveness judgements influence desires to initiate and maintain romantic relationships. Testosterone also predicts relationship initiation and maintenance; such effects may be driven by the hormone's modulation of attractiveness judgements, but no studies have investigated causal (and situation-dependent) effects of the hormone on these judgements. Using a placebo-controlled cross-over design, our preregistered analyses revealed order- and relationship- dependent effects: single heterosexual men judged the women as more appealing when testosterone was administered first (and placebo second), but marginally less appealing when placebo was administered first (and testosterone second). In a more complex model incorporating the women's attractiveness (as rated by an independent set of observers), however, we show that testosterone increases the appeal of women -but this effect depends upon the men's relationship status and the women's attractiveness. In partnered men (n = 53) who tend to derogate attractive alternatives (by rating them as less appealing), testosterone countered this effect, boosting the appeal of these attractive alternatives. In single men (n = 53), conversely, testosterone increased the appeal of low-attractive women. These differential effects highlight the possibility of a newly discovered mechanism whereby testosterone promotes male sexual reproduction through different routes depending on relationship status, promoting partner up- rather than down-grading when partnered and reducing choosiness when single. Further, such effects were relatively rapid [within 85 (±5) minutes], suggesting a potential non-genomic mechanism of action.