Off-the-shelf CAR-T cells could prove paradigm shifting for autoimmune diseases.

Early reports suggest that chimeric antigen receptor (CAR)-T therapy has remarkable potential for treating autoimmune disease. Current approaches rely on autologous CAR-T cells, creating a bottleneck to the broad deployment of this therapy. In this issue of Cell, Wang et al.1 report the first use of allogeneic CAR-T cells in three patients with systemic autoimmune disease.

Associations between early experiences of thought interference and auditory-verbal hallucinations with first-rank symptoms and suicidality in adulthood.

BACKGROUND: Suicide is one of the major causes of premature death in patients diagnosed with a schizophrenia-spectrum psychotic disorder. However, associations between psychotic-like experiences in youth and suicidality in later life remain under-researched. AIMS: We aimed to investigate any associations between early experiences of thought interference and auditory-verbal hallucinations (AVHs) with first-rank symptoms of schizophrenia and suicidal thoughts and behaviours in adulthood. METHOD: This study used data from the Avon Longitudinal Study of Parents and Children (ALSPAC). We calculated combined thought interference score at ages 11 years 8 months, 13 years 1 month, 14 years 1 month and 16 years 6 months. We also assessed AVHs at the same age points. For outcome variables, we used specific variables measuring delusions of control, AVHs and suicidality at 24 years of age. We carried out logistic regressions and mediation analyses to assess the relationships among these variables. RESULTS: Thought interference and AVHs at all ages throughout childhood and adolescence were associated with suicidal thoughts and behaviours, and also with clinically more significant symptoms of delusions of control and AVHs at age 24. Substance use-induced psychotic-like experiences mediated a large proportion of the relationship between early psychotic-like experiences and suicidality in later life. CONCLUSIONS: Thought interference and AVHs in childhood and adolescence are associated with first-rank symptoms and suicidality in adulthood. Mental health interventions in children and adolescents need to take into account the impact of specific psychotic-like experiences and allow for the early detection of thought interference and AVH-related symptoms.

The molecular basis underlying T cell specificity towards citrullinated epitopes presented by HLA-DR4.

CD4+ T cells recognising citrullinated self-epitopes presented by HLA-DRB1 bearing the shared susceptibility epitope (SE) are implicated in rheumatoid arthritis (RA). However, the underlying T cell receptor (TCR) determinants of epitope specificity towards distinct citrullinated peptide antigens, including vimentin-64cit59-71 and α-enolase-15cit10-22 remain unclear. Using HLA-DR4-tetramers, we examine the T cell repertoire in HLA-DR4 transgenic mice and observe biased TRAV6 TCR gene usage across these two citrullinated epitopes which matches with TCR bias previously observed towards the fibrinogen ß-74cit69-81 epitope. Moreover, shared TRAV26-1 gene usage is evident in four α-enolase-15cit10-22 reactive T cells in three human samples. Crystal structures of mouse TRAV6+ and human TRAV26-1+ TCR-HLA-DR4 complexes presenting vimentin-64cit59-71 and α-enolase-15cit10-22, respectively, show three-way interactions between the TCR, SE, citrulline, and the basis for the biased selection of TRAV genes. Position 2 of the citrullinated epitope is a key determinant underpinning TCR specificity. Accordingly, we provide a molecular basis of TCR specificity towards citrullinated epitopes.

Feasibility of a new 'balanced binocular viewing' treatment for unilateral amblyopia in children aged 3-8 years (BALANCE): results of a phase 2a randomised controlled feasibility trial.

OBJECTIVES: This study aimed to evaluate the safety of dichoptic balanced binocular viewing (BBV) for amblyopia in children, plus feasibility, adherence, acceptability, trial methodology and clinical measures of visual function. DESIGN: We carried out an observer-masked parallel-group phase 2a feasibility randomised controlled trial. SETTING: Two study sites, a secondary/tertiary and a community site. PARTICIPANTS: We enrolled 32 children aged 3-8 years with unilateral amblyopia who had completed optical adaptation where indicated. 20 children attended the 16-week exit visit (retention 63%). INTERVENTIONS: Children were randomised to BBV (movies customised to interocular acuity difference at baseline) for 1 hour a day (active intervention) or standard management as per parental choice (part-time occlusion or atropine blurring, control). All interventions were used at home, daily for 16 weeks. PRIMARY OUTCOME MEASURE: 'VacMan suppression test' of interocular balance at 16 weeks from randomisation. SECONDARY OUTCOME MEASURES: feasibility outcomes (recruitment and retention ratios, adherence with the allocated intervention); safety outcomes at other time points (changes in prevalence of diplopia, manifest strabismus, suppression/interocular balance on a range of tests); efficacy outcomes (clinical measures of visual function, such as best-corrected visual acuity, BCVA). Outcome measures were identical to those planned in the protocol. RESULTS: Primary outcome: At baseline, values for the interocular balance point were higher (indicating greater suppression of the amblyopic eye) in the occlusion group than in the BBV group. These values shifted downwards on average for the occlusion group, significantly decreasing from baseline to week 16 (t8=4.49, p=0.002). Balance values did not change between baseline and week 16 for the BBV group (t9=-0.82, p=0.435). At 16 weeks, there was no statistical difference in interocular balance/suppression change over time between the two arms. The difference at follow-up between the arms, adjusted for baseline, was -0.02 (95% CI -0.28 to 0.23, p=0.87). FEASIBILITY: We prescreened 144 records of potentially eligible children. Between 28 October 2019 and 31 July 2021, including an interruption due to the COVID-19 pandemic, 32 children were screened and randomised (recruitment rate 22%), 16 to BBV and 16 to standard treatment. 20 children attended the 16-week exit visit (retention 63%). Mean adherence with BBV as proportion of viewing time prescribed was 56.1% (SD36) at 8 and 57.9% (SD 30.2) at 16 weeks. Mean adherence with prescribed occlusion time was 90.1% (SD 19.7) at 8 and 59.2% (SD 24.8) at 16 weeks. SECONDARY SAFETY/EFFICACY OUTCOMES: One child in the BBV arm reported transient double vision, which resolved; two reported headaches, which led to withdrawal. BCVA improved from mean 0.47 (SD0.18) logMAR at randomisation to 0.26 (0.14) with standard treatment, and from 0.55 (0.28) to 0.32 (0.26) with BBV. Outcomes at 16 weeks did not differ between treatments. PARTICIPANT EXPERIENCE: Families were generally positive about BBV, but families found both patching and BBV difficult to integrate into family routines. CONCLUSIONS: Recruitment rates indicate that a future phase 3 trial will require multiple sites or a longer enrolment period. Retention and adherence rates were lower than anticipated, which will influence future study designs. Dichoptic treatment may be equal to occlusion treatment in safety and efficacy; headaches may lead to discontinuation. Integration into family routines may constitute a barrier to implementation. TRIAL REGISTRATION NUMBER: NCT03754153.

G Protein-Coupled Receptor-Ligand Pose and Functional Class Prediction.

G protein-coupled receptor (GPCR) transmembrane protein family members play essential roles in physiology. Numerous pharmaceuticals target GPCRs, and many drug discovery programs utilize virtual screening (VS) against GPCR targets. Improvements in the accuracy of predicting new molecules that bind to and either activate or inhibit GPCR function would accelerate such drug discovery programs. This work addresses two significant research questions. First, do ligand interaction fingerprints provide a substantial advantage over automated methods of binding site selection for classical docking? Second, can the functional status of prospective screening candidates be predicted from ligand interaction fingerprints using a random forest classifier? Ligand interaction fingerprints were found to offer modest advantages in sampling accurate poses, but no substantial advantage in the final set of top-ranked poses after scoring, and, thus, were not used in the generation of the ligand-receptor complexes used to train and test the random forest classifier. A binary classifier which treated agonists, antagonists, and inverse agonists as active and all other ligands as inactive proved highly effective in ligand function prediction in an external test set of GPR31 and TAAR2 candidate ligands with a hit rate of 82.6% actual actives within the set of predicted actives.

Manufactured housing communities and climate change: Understanding key vulnerabilities and recommendations for emergency managers.

Manufactured housing communities (MHCs), commonly referred to as mobile home parks, provide an estimated 2.7 million American households with largely unsubsidized, affordable housing. Climate change threatens those who call these communities home by exacerbating known structural and social vulnerabilities associated with this housing type-including but not limited to increased risks to flooding, extreme temperatures, high winds, and wildfires. Climate change requires emergency managers to understand the diverse, integrated, and complex vulnerabilities of MHCs that affect their exposure to climate change risk. This article presents findings from an integrative literature review focused on the climate-related vulnerabilities of these communities described at three levels of scale: household, housing structure, and park community. It then draws on 15 years of engagement and action research with MHC residents and stakeholders in Vermont, including several federally declared flooding disasters, to distill key recommendations for emergency managers for assisting MHCs to prepare for and respond to emergencies. As climate change accelerates, emergency managers can increase efficacy by learning about the MHCs in their jurisdictions by leveraging the best available data to characterize risks, integrating MHCs into planning and mitigation activities, and engaging in conversations with stakeholders, including MHC residents and their trusted partners.

Enhancing chimeric antigen receptor T cell therapy by modulating the p53 signaling network with Δ133p53α.

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.

From Monologue to Dialogue: The Pursuit of Relationship Centered Communication Across a Large, Integrated Healthcare System.

By listening to the "voice" of patients, Northwell Health, New York's largest healthcare organization, took an evidence-based approach to empowering physicians and advanced care providers. The Relationship Centered Communication course utilizes experiential learning to strengthen patient-centered communication and empathy skills to elicit provider-patient relationships grounded in partnership. This case study highlights (1) The pragmatic cultural journey requiring visionary leadership, strong collaborations, and an evolving educational strategy. (2) Over the course of 6 years, 3300+ providers were educated in this evidence-based communication course. (3) As a result, Northwell's Hospital Consumer Assessment of Healthcare Providers and Systems "Communication with Doctors" domain has increased by 22 percentile rank points, when compared nationally to peers, in addition to other notable patient experience metric improvements within ambulatory medical practice.

Multisensory processing and proprioceptive plasticity during resizing illusions.

Bodily resizing illusions typically use visual and/or tactile inputs to produce a vivid experience of one's body changing size. Naturalistic auditory input (an input that reflects the natural sounds of a stimulus) has been used to increase illusory experience during the rubber hand illusion, whilst non-naturalistic auditory input can influence estimations of finger length. We aimed to use a non-naturalistic auditory input during a hand-based resizing illusion using augmented reality, to assess whether the addition of an auditory input would increase both subjective illusion strength and measures of performance-based tasks. Forty-four participants completed the following three conditions: no finger stretching, finger stretching without tactile feedback and finger stretching with tactile feedback. Half of the participants had an auditory input throughout all the conditions, whilst the other half did not. After each condition, the participants were given one of the following three performance tasks: stimulated (right) hand dot touch task, non-stimulated (left) hand dot touch task, and a ruler judgement task. Dot tasks involved participants reaching for the location of a virtual dot, whereas the ruler task concerned estimates of the participant's own finger on a ruler whilst the hand was hidden from view. After all trials, the participants completed a questionnaire capturing subjective illusion strength. The addition of auditory input increased subjective illusion strength for manipulations without tactile feedback but not those with tactile feedback. No facilitatory effects of audio were found for any performance task. We conclude that adding auditory input to illusory finger stretching increased subjective illusory experience in the absence of tactile feedback but did not affect performance-based measures.

H. Ralph Schumacher.

Dr Schumacher was a force in rheumatology for more than half a century through his multiple roles as a researcher, clinician, mentor, and educator. He is not likely to be soon forgotten by the rheumatology community; however, it is hoped that this chapter can provide a faithful recollection that will help bring his memory to life for some and that rings true to those who knew him and learned from him.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Temporal dynamics of normalization reweighting.

For decades, neural suppression in early visual cortex has been thought to be fixed. But recent work has challenged this assumption by showing that suppression can be reweighted based on recent history; when pairs of stimuli are repeatedly presented together, suppression between them strengthens. Here we investigate the temporal dynamics of this process using a steady-state visual evoked potential (SSVEP) paradigm that provides a time-resolved, direct index of suppression between pairs of stimuli flickering at different frequencies (5 and 7 Hz). Our initial analysis of an existing electroencephalography (EEG) dataset (N = 100) indicated that suppression increases substantially during the first 2-5 seconds of stimulus presentation (with some variation across stimulation frequency). We then collected new EEG data (N = 100) replicating this finding for both monocular and dichoptic mask arrangements in a preregistered study designed to measure reweighting. A third experiment (N = 20) used source-localized magnetoencephalography and found that these effects are apparent in primary visual cortex (V1), consistent with results from neurophysiological work. Because long-standing theories propose inhibition/excitation differences in autism, we also compared reweighting between individuals with high versus low autistic traits, and with and without an autism diagnosis, across our three datasets (total N = 220). We find no compelling differences in reweighting that are associated with autism. Our results support the normalization reweighting model and indicate that for prolonged stimulation, increases in suppression occur on the order of 2-5 seconds after stimulus onset.

Building block aspect ratio controls assembly, architecture, and mechanics of synthetic and natural protein networks.

Fibrous networks constructed from high aspect ratio protein building blocks are ubiquitous in nature. Despite this ubiquity, the functional advantage of such building blocks over globular proteins is not understood. To answer this question, we engineered hydrogel network building blocks with varying numbers of protein L domains to control the aspect ratio. The mechanical and structural properties of photochemically crosslinked protein L networks were then characterised using shear rheology and small angle neutron scattering. We show that aspect ratio is a crucial property that defines network architecture and mechanics, by shifting the formation from translationally diffusion dominated to rotationally diffusion dominated. Additionally, we demonstrate that a similar transition is observed in the model living system: fibrin blood clot networks. The functional advantages of this transition are increased mechanical strength and the rapid assembly of homogenous networks above a critical protein concentration, crucial for in vivo biological processes such as blood clotting. In addition, manipulating aspect ratio also provides a parameter in the design of future bio-mimetic and bio-inspired materials.

Heatwave resilience of juvenile white sturgeon is associated with epigenetic and transcriptional alterations.

Heatwaves are increasing in frequency and severity, posing a significant threat to organisms globally. In aquatic environments heatwaves are often associated with low environmental oxygen, which is a deadly combination for fish. However, surprisingly little is known about the capacity of fishes to withstand these interacting stressors. This issue is particularly critical for species of extreme conservation concern such as sturgeon. We assessed the tolerance of juvenile white sturgeon from an endangered population to heatwave exposure and investigated how this exposure affects tolerance to additional acute stressors. We measured whole-animal thermal and hypoxic performance and underlying epigenetic and transcriptional mechanisms. Sturgeon exposed to a simulated heatwave had increased thermal tolerance and exhibited complete compensation for the effects of acute hypoxia. These changes were associated with an increase in mRNA levels involved in thermal and hypoxic stress (hsp90a, hsp90b, hsp70 and hif1a) following these stressors. Global DNA methylation was sensitive to heatwave exposure and rapidly responded to acute thermal and hypoxia stress over the course of an hour. These data demonstrate that juvenile white sturgeon exhibit substantial resilience to heatwaves, associated with improved cross-tolerance to additional acute stressors and involving rapid responses in both epigenetic and transcriptional mechanisms.

Different rules for binocular combination of luminance flicker in cortical and subcortical pathways.

How does the human brain combine information across the eyes? It has been known for many years that cortical normalization mechanisms implement 'ocularity invariance': equalizing neural responses to spatial patterns presented either monocularly or binocularly. Here, we used a novel combination of electrophysiology, psychophysics, pupillometry, and computational modeling to ask whether this invariance also holds for flickering luminance stimuli with no spatial contrast. We find dramatic violations of ocularity invariance for these stimuli, both in the cortex and also in the subcortical pathways that govern pupil diameter. Specifically, we find substantial binocular facilitation in both pathways with the effect being strongest in the cortex. Near-linear binocular additivity (instead of ocularity invariance) was also found using a perceptual luminance matching task. Ocularity invariance is, therefore, not a ubiquitous feature of visual processing, and the brain appears to repurpose a generic normalization algorithm for different visual functions by adjusting the amount of interocular suppression.

Enhancing precision in human neuroscience.

Human neuroscience has always been pushing the boundary of what is measurable. During the last decade, concerns about statistical power and replicability - in science in general, but also specifically in human neuroscience - have fueled an extensive debate. One important insight from this discourse is the need for larger samples, which naturally increases statistical power. An alternative is to increase the precision of measurements, which is the focus of this review. This option is often overlooked, even though statistical power benefits from increasing precision as much as from increasing sample size. Nonetheless, precision has always been at the heart of good scientific practice in human neuroscience, with researchers relying on lab traditions or rules of thumb to ensure sufficient precision for their studies. In this review, we encourage a more systematic approach to precision. We start by introducing measurement precision and its importance for well-powered studies in human neuroscience. Then, determinants for precision in a range of neuroscientific methods (MRI, M/EEG, EDA, Eye-Tracking, and Endocrinology) are elaborated. We end by discussing how a more systematic evaluation of precision and the application of respective insights can lead to an increase in reproducibility in human neuroscience.

Formation of a conceptual framework during the development of a patient-reported outcome measure for early gastrointestinal recovery: phase I of the PRO-diGi study.

AIM: Patients admitted to hospital for abdominal surgery often experience gastrointestinal dysfunction. Many studies have reported outcomes following gastrointestinal dysfunction, yet there is no unified definition of recovery or a validated patient-reported outcome measure (PROM). The first stage of PROM development requires formation of a conceptual framework to identify key themes to patients. The aim of this study was to utilize semistructured interviews to identify core themes and concepts relevant to patients to facilitate development of a conceptual framework. METHOD: Adult patients admitted to hospital for major gastrointestinal, urological or gynaecological surgery, in an emergency or elective setting, were eligible to participate. Patients treated nonoperatively for small bowel obstruction were also eligible. Interviews were conducted by telephone, audio-recorded, transcribed, coded and analysed using NVivo software by two researchers and reviewed by lay members of the steering group. Interviews continued until data saturation was reached. Ethical approval was gained prior to interviews (21/WA/0231). RESULTS: Twenty nine interviews were completed (17 men, median age 64 years) across three specialties (20 gastrointestinal, six gynaecological, three urological). Two overarching themes of 'general recovery' and 'gastrointestinal symptoms' were identified. General recovery included three themes: 'life impact', 'mental impact', including anxiety, and 'physical impact', including fatigue. Gastrointestinal symptoms included three themes: 'abdominal symptoms' such as pain, 'diet and appetite' and 'expulsory function', such as stool frequency. A total of 18 gastrointestinal symptoms were identified during patient recovery-many of which lasted several weeks following discharge. CONCLUSION: This study reports a range of gastrointestinal and nongastrointestinal symptoms experienced by patients during early gastrointestinal recovery. Identified symptoms have been synthesized into a conceptual framework to enable development of a definitive PROM for early gastrointestinal recovery.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

CAR T therapy beyond cancer: the evolution of a living drug.

Engineering a patient's own T cells to selectively target and eliminate tumour cells has cured patients with untreatable haematologic cancers. These results have energized the field to apply chimaeric antigen receptor (CAR) T therapy throughout oncology. However, evidence from clinical and preclinical studies underscores the potential of CAR T therapy beyond oncology in treating autoimmunity, chronic infections, cardiac fibrosis, senescence-associated disease and other conditions. Concurrently, the deployment of new technologies and platforms provides further opportunity for the application of CAR T therapy to noncancerous pathologies. Here we review the rationale behind CAR T therapy, current challenges faced in oncology, a synopsis of preliminary reports in noncancerous diseases, and a discussion of relevant emerging technologies. We examine potential applications for this therapy in a wide range of contexts. Last, we highlight concerns regarding specificity and safety and outline the path forward for CAR T therapy beyond cancer.

PySilSub: An open-source Python toolbox for implementing the method of silent substitution in vision and nonvisual photoreception research.

The normal human retina contains several classes of photosensitive cell-rods for low-light vision, three cone classes for daylight vision, and intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing melanopsin for non-image-forming functions, including pupil control, melatonin suppression, and circadian photoentrainment. The spectral sensitivities of the photoreceptors overlap significantly, which means that most lights will stimulate all photoreceptors to varying degrees. The method of silent substitution is a powerful tool for stimulating individual photoreceptor classes selectively and has found much use in research and clinical settings. The main hardware requirement for silent substitution is a spectrally calibrated light stimulation system with at least as many primaries as there are photoreceptors under consideration. Device settings that will produce lights to selectively stimulate the photoreceptor(s) of interest can be found using a variety of analytic and algorithmic approaches. Here we present PySilSub (https://github.com/PySilentSubstitution/pysilsub), a novel Python package for silent substitution featuring flexible support for individual colorimetric observer models (including human and mouse observers), multiprimary stimulation devices, and solving silent substitution problems with linear algebra and constrained numerical optimization. The toolbox is registered with the Python Package Index and includes example data sets from various multiprimary systems. We hope that PySilSub will facilitate the application of silent substitution in research and clinical settings.