Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.

Habitually wearing high heels may improve user walking economy in any footwear.

The habitual use of high-heeled footwear may structurally remodel user leg muscle tendons, thereby altering their functional capabilities. High heels set users' ankles in relatively plantarflexed positions, causing calf muscle tendons to operate at relatively short lengths. Habitually operating muscle tendons at relatively short lengths induces structural remodeling that theoretically affects muscle metabolism. Because structural changes occur within the body, the user's locomotor metabolism may change in any footwear condition (e.g., conventional shoes, barefoot). Here, we studied the influence of habitual high-heel use on users' leg muscle-tendon structure and metabolism during walking in flat-soled footwear. We tested eight participants before and after 14 wk of agreeing to wear high heels as their daily shoes. Overall, participants who wore high heels >1,500 steps per day, experienced a 9% decrease in their net metabolic power during walking in flat-soled footwear (d = 1.66, P ≤ 0.049), whereas participants who took <1,000 daily steps in high heels did not (d = 0.44; P = 0.524). Across participants, for every 1,000 daily steps in high heels, net metabolic power during walking in flat-soled footwear decreased 5.3% (r = -0.73; P = 0.040). Metabolic findings were partially explained (r2 = 0.43; P = 0.478) by trending shorter medial gastrocnemius fascicle lengths (d = 0.500, P = 0.327) and increased Achilles tendon stiffness (d = 2.889, P = 0.088). The high-heel intervention did not alter user walking stride kinematics in flat-soled footwear (d ≤ 0.567, P ≥ 0.387). While our limited dataset is unable to establish the mechanisms underlying the high-heel-induced walking economy improvement, it appears that prescribing specific footwear use can be implemented to alter user muscle-tendon properties and augment their function in any shoes.NEW & NOTEWORTHY Habitually wearing high-heeled footwear structurally remodels leg muscle tendons and improves user walking economy, regardless of worn attire.

Differential contribution of sensorimotor cortex and subthalamic nucleus to unimanual and bimanual hand movements.

Why does unilateral deep brain stimulation improve motor function bilaterally? To address this clinical observation, we collected parallel neural recordings from sensorimotor cortex (SMC) and the subthalamic nucleus (STN) during repetitive ipsilateral, contralateral, and bilateral hand movements in patients with Parkinson's disease. We used a cross-validated electrode-wise encoding model to map electromyography data to the neural signals. Electrodes in the STN encoded movement at a comparable level for both hands, whereas SMC electrodes displayed a strong contralateral bias. To examine representational overlap across the two hands, we trained the model with data from one condition (contralateral hand) and used the trained weights to predict neural activity for movements produced with the other hand (ipsilateral hand). Overall, between-hand generalization was poor, and this limitation was evident in both regions. A similar method was used to probe representational overlap across different task contexts (unimanual vs. bimanual). Task context was more important for the STN compared to the SMC indicating that neural activity in the STN showed greater divergence between the unimanual and bimanual conditions. These results indicate that SMC activity is strongly lateralized and relatively context-free, whereas the STN integrates contextual information with the ongoing behavior.

Wild-type C-Raf gene dosage and dimerization drive prostate cancer metastasis.

Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

Reallocating desk workers' sitting time to standing or stepping: associations with work performance.

BACKGROUND: Studies have suggested that sitting time at work may lead to underperformance but they may underestimate the benefits to desk workers' performance of reducing occupational sitting time without considering the relative effects of the specific activities replaced. AIMS: To estimate differences in work performance (presenteeism, absenteeism and engagement) when occupational sitting time is reallocated to standing/stepping in desk workers. METHODS: Data for middle-aged desk workers were from a Japan-wide online survey (n = 2228). Self-report proportion of occupational sitting and standing/stepping, work hours and work performance indicators, including absolute (ratings relating only to self) and relative (ratings of self, compared to others) presenteeism and absenteeism, and dimensions of work engagement, were collected. Partition and isotemporal substitution models were used to investigate the associations of occupational sitting and standing/stepping time with work performance, including their reallocation effects. RESULTS: In partition models, longer occupational sitting time was associated with a lower absolute presenteeism score (i.e. less productivity), lower absolute absenteeism (i.e. longer-than-expected work hours), and lower engagement. Longer occupational standing/stepping time was associated with lower absolute absenteeism and more engagement. Isotemporal substitution models showed that each hour of occupational sitting reallocated to standing/stepping was favourably associated with overall work engagement (B = 0.087; 95% confidence interval 0.051, 0.122) and its dimensions (B ranged from 0.078 to 0.092), but was not associated with presenteeism or absenteeism. CONCLUSIONS: These findings suggest that management support and practical initiatives to encourage desk workers to replace portions of their sitting time with standing/stepping may contribute to enhanced work engagement.

Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation.

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

Eosinophilic Fasciitis Possibly Provoked by Exercise.

A 26-year-old woman was referred to dermatology for evaluation of "lumpy skin" for 4 months. She experienced Achilles tendon pain and muscle tightness, occurring before the skin lesions had appeared. The lesions began on her arms and progressed to her legs over 1 month.

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells.

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

Dimensional structure of one-year post-COVID-19 neuropsychiatric and somatic sequelae and association with role impairment.

This study examined the latent structure of the broad range of complex neuropsychiatric morbidities occurring 1 year after COVID-19 infection. As part of the CU-COVID19 study, 248 (response rate=39.3%) of 631 adults hospitalized for COVID-19 infection in Hong Kong completed an online survey between March-2021 and January-2022. Disorder prevalence was compared against a random non-infected household sample (n=1834). 248 surveys were received on average 321 days post-infection (Mean age: 48.9, 54% female, moderate/severe/critical infection: 58.2%). 32.4% were screened to have at least one mental disorder, 78.7% of whom had concurrent fatigue/subjective cognitive impairment (SCI). Only PTSD (19.1%) was significantly more common than control (14%, p=0.047). Latent profile analysis classified individuals into P1 (12·4%)-no current neuropsychiatric morbidities, P2 (23.1%)-SCI/fatigue, P3 (45.2%)-anxiety/PTSD, P4 (19.3%)-depression. SCI and fatigue pervaded in all profiles (P2-4) with neuropsychiatric morbidities one-year post-infection. PTSD, anxiety and depressive symptoms were most important in differentiating P2-4. Past mental health and P4 independently predicted functional impairment. Neuropsychiatric morbidity was associated with past mental health, reduced resilience, financial problems, but not COVID-19 severity. Their confluence with depressive and anxiety symptoms predicted impairment and are associated with psychological and environmental factors.

The Genomic and Epigenomic Landscape of Double-Negative Metastatic Prostate Cancer.

Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease. SIGNIFICANCE: Comprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.

Towards a core outcome set for cranioplasty following traumatic brain injury and stroke 'A systematic review of reported outcomes'.

Background: There is wide-ranging published literature around cranioplasty following traumatic brain injury (TBI) and stroke, but the heterogeneity of outcomes limits the ability for meta-analysis. Consensus on appropriate outcome measures has not been reached, and given the clinical and research interest, a core outcome set (COS) would be beneficial. Objectives: To collate outcomes currently reported across the cranioplasty literature which will subsequently be used in developing a cranioplasty COS. Methods: This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. All full-text English studies with more than ten patients (prospective) or more than 20 patients (retrospective) published after 1990 examining outcomes in CP were eligible for inclusion. Results: The review included 205 studies from which 202 verbatim outcomes were extracted, grouped into 52 domains, and categorised into one or more of the OMERACT 2.0 framework core area(s). The total numbers of studies that reported outcomes in the core areas are 192 (94%) pathophysiological manifestations/ 114 (56%) resource use/economic impact/ 94 (46%) life impact/mortality 20 (10%). In addition, there are 61 outcome measures used in the 205 studies across all domains. Conclusion: This study shows considerable heterogeneity in the types of outcomes used across the cranioplasty literature, demonstrating the importance and necessity of developing a COS to help standardise reporting across the literature.

Correction to: 'Sprinting with prosthetic versus biological legs: insight from experimental data' (2023) by Beck et al.

[This corrects the article DOI: 10.1098/rsos.211799.][This corrects the article DOI: 10.1098/rsos.211799.].

Voluntary muscle coactivation in quiet standing elicits reciprocal rather than coactive agonist-antagonist control of reactive balance.

Muscle coactivation increases in challenging balance conditions as well as with advanced age and mobility impairments. Increased muscle coactivation can occur both in anticipation of (feedforward) and in reaction to (feedback) perturbations, however, the causal relationship between feedforward and feedback muscle coactivation remains elusive. Here, we hypothesized that feedforward muscle coactivation would increase both the body's initial mechanical resistance due to muscle intrinsic properties and the later feedback-mediated muscle coactivation in response to postural perturbations. Young adults voluntarily increased leg muscle coactivation using visual biofeedback before support-surface perturbations. In contrast to our hypothesis, feedforward muscle coactivation did not increase the body's initial intrinsic resistance to perturbations, nor did it increase feedback muscle coactivation. Rather, perturbations with feedforward muscle coactivation elicited a medium- to long-latency increase of feedback-mediated agonist activity but a decrease of feedback-mediated antagonist activity. This reciprocal rather than coactivation effect on ankle agonist and antagonist muscles enabled faster reactive ankle torque generation, reduced ankle dorsiflexion, and reduced center of mass (CoM) motion. We conclude that in young adults, voluntary feedforward muscle coactivation can be independently modulated with respect to feedback-mediated muscle coactivation. Furthermore, our findings suggest feedforward muscle coactivation may be useful for enabling quicker joint torque generation through reciprocal, rather than coactivated, agonist-antagonist feedback muscle activity. As such our results suggest that behavioral context is critical to whether muscle coactivation functions to increase agility versus stability.NEW & NOTEWORTHY Feedforward and feedback muscle coactivation are commonly observed in older and mobility impaired adults and are considered strategies to improve stability by increasing body stiffness prior to and in response to perturbations. In young adults, voluntary feedforward coactivation does not necessarily increase feedback coactivation in response to perturbations. Instead, feedforward coactivation enabled faster ankle torques through reciprocal agonist-antagonist muscle activity. As such, coactivation may promote either agility or stability depending on the behavioral context.

Equivalent running leg lengths require prosthetic legs to be longer than biological legs during standing.

We aimed to determine a method for prescribing a standing prosthetic leg length (ProsL) that results in an equivalent running biological leg length (BioL) for athletes with unilateral (UTTA) and bilateral transtibial amputations (BTTA). We measured standing leg length of ten non-amputee (NA) athletes, ten athletes with UTTA, and five athletes with BTTA. All athletes performed treadmill running trials from 3 m/s to their maximum speed. We calculated standing and running BioL and ProsL lengths and assessed the running-to-standing leg length ratio (Lratio) at three instances during ground contact: touchdown, mid-stance, and take-off. Athletes with UTTA had 2.4 cm longer standing ProsL than BioL length (p = 0.030), but their ProsL length were up to 3.3 cm shorter at touchdown and 4.1 cm shorter at mid-stance than BioL, at speed 3-11.5 m/s. At touchdown, mid-stance, and take-off, athletes with BTTA had 0.01-0.05 lower Lratio at 3 m/s (p < 0.001) and 0.03-0.07 lower Lratio at 10 m/s (p < 0.001) in their ProsL compared to the BioL of NA athletes. During running, ProsL were consistently shorter than BioL. To achieve equivalent running leg lengths at touchdown and take-off, athletes with UTTA should set their running-specific prosthesis height so that their standing ProsL length is 2.8-4.5% longer than their BioL length, and athletes with BTTA should set their running-specific prosthesis height so that their standing ProsL lengths are at least 2.1-3.9% longer than their presumed BioL length. Setting ProsL length to match presumed biological dimensions during standing results in shorter legs during running.

IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing.

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

Conservation measures or hotspots of disease transmission? Agri-environment schemes can reduce disease prevalence in pollinator communities.

Insects are under pressure from agricultural intensification. To protect pollinators, conservation measures such as the EU agri-environment schemes (AES) promote planting wildflowers along fields. However, this can potentially alter disease ecology by serving as transmission hubs or by diluting infections. We tested this by measuring plant-pollinator interactions and virus infections (DWV-A, DWV-B and ABPV) across pollinator communities in agricultural landscapes over a year. AES had a direct effect on DWV-B, reducing prevalence and load in honeybees, with a tentative general dilution effect on load in early summer. DWV-A prevalence was reduced both under AES and with increasing niche overlap between competent hosts, likely via a dilution effect. By contrast, AES had no impact on ABPV, its prevalence driven by the proportion of bumblebees in the community. Epidemiological differences were also reflected in the virus phylogenies, with DWV-B showing recent rapid expansion, while DWV-A and ABPV showed slower growth rates and geographical population structure. Phylogenies indicate that all three viruses freely circulate across their host populations. Our study illustrates how complex interactions between environmental, ecological and evolutionary factors may influence wildlife disease dynamics. Supporting pollinator nutrition can mitigate the transmission of important bee diseases, providing an unexpected boost to pollinator conservation. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

It is time to abandon single-value oxygen uptake energy equivalents.

Physiologists commonly use single-value energy equivalents (e.g., 20.1 kJ/LO2 and 20.9 kJ/LO2) to convert oxygen uptake (V̇o2) to energy, but doing so ignores how the substrate oxidation ratio (carbohydrate:fat) changes across aerobic intensities. Using either 20.1 kJ/LO2 or 20.9 kJ/LO2 can incur systematic errors of up to 7%. In most circumstances, the best approach for estimating energy expenditure is to measure both V̇o2 and V̇co2 and use accurate, species-appropriate stoichiometry. However, there are circumstances when V̇co2 measurements may be unreliable. In those circumstances, we recommend that the research report or compare only V̇o2.NEW & NOTEWORTHY We quantify that the common practice of using single-value oxygen uptake energy equivalents for exercising subjects can incur systematic errors of up to 7%. We argue that such errors can be greatly reduced if researchers measure both V̇o2 and V̇co2 and adopt appropriate stoichiometry equations.